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A cadmium-binding protein in rainbow trout
Toxicology Letters, 4 (1979) 241-246 0 Elsevier/North-Holland Biomedical Press
A MADRID-BINDING
241
PROTEIN IN RAINBOW TROUT
J.H. BEATTIE and D. PASCOE Department of Applied Biology, University of Wales institute of Science and Technology, Cathays Park, Cardiff CFI 3NU, U.K. (Received May l&h, 1979) (Accepted May 22nd, 1979)
SUMMARY
Rainbow trout, Sulmo ga~rdneri Rich were treated, by intrape~tone~ injection, with about 3.0 mg cadmium kg’ body weight over a 4day period. An extract prepared from the liver was found to contain a cadmium-binding protein of about 6700 daltons and with greater absorbance in the ultraviolet at 250 nm than at 280 nm. This protein is similar to metallothionein, isolated from a number of other species and may be responsible for the increased resistance to cadmium of rainbow trout pretreated with low levels of the same metal.
INTRODUCTION
There is now considerable evidence that pretreatment of fish with a poison may induce some degree of resistance during subsequent exposure to the same poison e.g. ammonia [l] , phenol [ 21, linear alkylate sulphonate detergent [ 31. and heavy metals such as zinc [4,5] and cadmium [6,7]. In mammals, where the same phenomenon occurs [8,9] it has been shown that pretreatment with low doses of metal stimulates the liver and other organs to synthesize a protein which is able to bind with and thereby inactivate further doses of the same metal. The purpose of this investigation was to determine whether the increased resistance to cadmium, shown by pretreated rainbow trout, Sulmo gairdneri Rich [7] could be attributed to the production of a similar metalbinding protein. MATERIALS AND METHODS
Rainbow trout (CT200 g each) obtained from a fish farm in the Wye Valley were acclimated to laboratory water conditions (temperature 15 + 1°C; total hardness 66.0 mg 1’ as CaCO,; dissolved oxygen 90-100% air saturation value) for two days. Four fish were randomly selected for cadmium treatment
242
and four for control purposes. Each test fish received an in~ape~tone~ injection of 0.5 ml of a 300 mg Cd lq saline solution on 4 successive days, i.e. a total of about 3.0 mg Cd kg-’ body weight. Fish were treated by intraperitoneal injection rather than by exposure to cadmium in the water, to ensure that a known amount of cadmium entered the body. On the seventh day, test and control fish were killed by immersion in a lethal concentration of the anaesthetic benzocaine (ethyl-4-amino benzoate) and the livers (2.1-3.7 g) removed, washed in saline and stored on ice, 5 g of liver (1.25 g from each treated fish) was then homogenized in 25 ml (20% w/v) of buffer (0.05 M Tris-HCl and 0.25 M sucrose) at pH 8.6. The homogenate was centrifuged at 10 000 X g for 15 min and the supernatant recentrifuged at 105 000 X g for 1 h to obtain the soluble fraction, Tissues were maint~ned at 4°C throughout the extraction, An extract from livers of the control fish was prepared in the same way. The particle-free, soluble fraction from each preparation was filtered (Whatman No. 1) and 3 ml applied to the surface of a G-75 Sephadex column (40 X 2.6 cm) packed with particles of standard size prepared by dry elutriation. The column was calibrated for molecular weight determination with bacitracin, molecular weight 1400; cytochrome C, 12 400; chymotrypsinogen, 25 000 and bovine serum albumin 67 000. Samples were eluted with Tris buffer and the ultraviolet absorption of the eluant continuously monitored at 250 nm using a Unicam SP1800 spectrophotometer. Individual 5 ml fractions were examined for UV absorbance at 250 and 280 nm and for cadmium using a Pye-Unicam SP191 atomic absorption spectrophotometer. Those fractions absorbing strongly at 250 nm and coinciding with the presence of cadmium were pooled and a spectrum scan carried out over the range 200300 nm, The protein concentration was determined spectrophotometrically and by the Biuret method. RESULTS
All fish appeared healthy during treatment, but on dissection some inflammation was noted at the cadmium injection site, and the livers of two fish and the kidneys of one were pale in appearance compared with controls. The elution profiles showing UV absorbance (250 nm and 280 nm) and cadmium concent~tio~ of the fractionated extracts of control and cadmiumtreated trout livers are shown in Figs. 1 and 2, respectively. Both profiles show an initial absorbance peak (elution volume 70-90 ml) and a final peak (180-220 ml) .representing high molecular weight proteins and low molecular weight materials respectively. However, an additional protein with greater absorbance at 250 nm (elution volume 120---150 ml) is present in the extract prepared from the livers of cadmium treated fish, and coincides with the occurrence of cadmium. From the concentrations of protein (molecular weight about 6700) and cadmium it can be calculated that 6.4 pg cadmium is associated with each mg protein. The UV absorbance spectrum (Fig. 3) of this
243
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1 5-
B
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O-
0 5-
O-
I
50
150
100
ELUTION
VOLUME
200
(ml)
Fig. 1. Sephadex G-75 elution profile of liver from control rainbow trout. Absorption at absorption at 250 nm (-------); cadmium (. . . . .) rg ml-‘. 280 nm ( -);
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Fig. 2. Sephadex G-75 elution profile of liver extract from cadmium treated rainbow trout. ); absorption at 250 nm (-------); cadmium (. . . . .) 4ml-‘. Absorption at 280 nm (-
244
Fig. 3. Ultraviolet absorption spectrum of cadmium-binding protein.
cadmium-binding protein (Cd-BP) is minimal at 280 nm but shows a slight shoulder at 250 nm. Neither this protein nor any significant levels of cadmium were present in the extract prepared from control fish livers. DISCUSSION
Much of the cadmium polluting freshwater systems occurs as a result of mining activities, zinc smelting and electroplating. It is extremely toxic to fish [lo-161 causing death or a variety of sublethal effects. However, it has been shown [ 71 that rainbow trout alevins exposed to a low concentration of cadmium are subsequently more resistant to cadmium than untreated alevins. It appears from the present investigation that pretreatment induces the synthesis of a protein which is able to bind with, and effectively detoxify, cadmium. This Cd-BP has a molecular weight of about 6700 and is similar to metallothionein, a protein of reported molecular weight 6000-10 000 which was first found in horse kidney [17] and later detected in the liver and kidney of a number of other species‘such as man [ 18,191, rat [ 20-221, chicken [ 231. The UV absorption spectrum of the Cd-BP shows minimal absorbance at 280 nm, indicating a deficiency of aromatic amino acids, and a slight shoulder at 250 nm. Recent investigations carried out in this laboratory have shown that removal of cadmium from the protein by extraction at pH 2.0 leads to a reduction in absorbance at 250 nm. Although these features all suggest that the Cd-BP is metallothionein [ 22-261, confirmation depends upon
245
the demonstration of other characteristics such as a high cysteine content. There have been few previous studies of this kind on fish; however, it has been reported [ 271 that a low molecular weight mercury-binding protein is found in the liver, kidneys and gills of freshwater eels exposed to 0.4 mg Hg l-“. In another investigation [ 281 it has been shown that, following the injection of a mixture of CdCl*, HgCl, and ZnClz into goldfish Curassius aurutus, much of the three metals was found to be associated with a protein in the liver and kidney. A low molecular weight cadmium-binding glycoprotein has been isolated from the liver of plaice follo~ng intraperitone~ exposure to CdClz [29,30] _ REFERENCES 1 R. Lloyd and L.D. Orr, The diuretic response by rainbow trout to sub-lethal concentrations of ammonia, Wat. Res., 3 (1969) 335. 2 I. Malacea, Untersuchungen iiber die Gewohnung der Fische an hohe Konzentrationen toxischer Substanzen, Arch. Hydrobiol., 65 (1968) 74. 3 K.E.F. Hokanson and L.L. Smith, Some factors influencing toxicity of linear alkylate sulfonate (LAS) to the bluegill, Trans. Am, Fish. Sot., 100 (1971) 1. 4 R. Lloyd, The toxicity of zinc sulphate to rainbow trout, Ann. App. Biol., 48 (1960) 84. 5 R.W. Edwards and V.M. Brown, Pollution and fisheries: A progress report, J. Inst. Wat. Poll. Con., 66 (1967) 63. 6 J.H. Beattie and D. Pascoe, Cadmium uptake by rainbow trout, Salmo gairdneri Richardson, eggs and alevins, J. Fish Biol., 13 (1978) 631. 7 D. Pascoe and J.H. Beattie, Resistance to cadmium by pretreated rainbow trout alevins, J. Fish Biol., 14 (1979) 303. 8 G. Gabbiani, D. Bait and C. Deziel, Studies on tolerance and ionic antagonism for cadmium or mercury, Can. J. Physiol. Pharmacol., 45 (1967) 443. 9 H. Yoshikawa, Preventive effect of pretreatment with low doses of metals on the acute toxicity of metals in mice, Ind. Health, 8 (1970) 184. 10 I.R. Ball, The toxicity of cadmium to rainbow trout Salmo gairdneri Richardson, Wat. Res., 1 (1967) 805. 11 B-E. Bengtsson, C.H. Carlin, A. Larsson and 0. Svanberg, Vertebral damage in minnows, Phoxinus phoxinus L,, exposed to cadmium, AMBIO, 4 (1975) 166. 12 G.R. Gardner and P.P. Yevich, Histological and haematological responses of an estuarine teleost to cadmium, J. Fish Res. Bd Can., 27 (1970) 2185. 13 A. Larsson, B-E. Bengtsson and 0. Svanberg, Some haematological and biochemical effects of cadmium on fish, in A.P.M. Lockwood (Ed.) Effects of Pollutants on Aquatic Organisms, Camb. University Press, Cambridge, 1976, pp. 35-46. 14 D. Pascoe and P. Cram, The effect of parasitism on the toxicity of cadmium to the three-spined stickleback, Gasterosteus aculeatus L., J. Fish Biol., 10 (1977) 467. 15 D. Pascoe and D.L. Mattey, Studies on the toxicity of cadmium to the three-spined stickleback, Gasterosteus aculeatus L., J. Fish Biol., 11 (1977) 207. 16 R.L. Spehar, Cadmium and zinc toxicity to the flagfish Jordanella floridae, J. Fish Res. Bd Can., 33 (1976) 1939. 17 M. Margoshes and B.L. Vallee, A cadmium protein from equine kidney cortex, J. Am. Chem. Sot., 79 (1957) 4813. 18 J.M. Wisniewska-Knypl, J. Jablonska and Z. Myslak, Binding of cadmium on metallothionein in man: an analysis of a fatal poisoning by cadmium iodide, Arch. Toxicol., 28 (1971) 46.
246 19 H.O. Buhler and J.H.R. Kagi, Human hepatic metallothioneins, FEBS Lett., 39 (1974) 229. 20 K. Tanaka, K. Sueda, S. Onasaka and K. Okahara, Fate of ‘09Cd-labelled metallothionein in rats, Toxicol. Appl. Pharmacol., 33 (1975) 258. 21 J.M. Wisniewska-Knypl, B.B. Trojanowska, J.K. Piotrowski and J.K. Jablonska, Binding of mercury in rat liver by metallothionein, Acta Biochim. Polon., 19 (1972) 11. 22 D.R. Winge, R. Premakumar and K.V. Rajagopalan, Metal-induced formation of metallothionein in rat liver, Arch. Biochem. Biophys., 170 (1975) 242. 23 U. Weser, F. Donay and H. Rupp, Cadmium-induced synthesis of hepatic metallothionein in chicken and rats, FEBS Lett., 32 (1973) 171. 24 J.H.R. Kagi and B.L. Vallee, Met~lothionein: a cadmium- and zinc-containing protein from equine renal cortex, J, Biol, Chem., 235 (1960) 3460. 25 A.P. Leber and T.S. Miya, A mechanism for cadmium- and zinc-induced tolerance to cadmium toxicity: involvement of metallothionein, Toxicol. Appl. Pharmacol., 37 (1976) 403. 26 M. Nordberg, B. Trojanowska and G.F. Nordberg, Studies on metal-binding proteins of low molecular weight from renal tissue of rabbits exposed to cadmium or mercury, Environ. Physiol. Biochem., 4 (1974) 11. 27 J.M. Bouquegneau, Ch. Gerday and A. Disteche, Fish mercury-binding thionein related to adaptation mechanisms, FEBS Lett., 55 (1975) 173, 28 E. Marafante, Binding of mercury and zinc to cadmium-binding protein in liver and kidney of goldfish (Curassius aurutus L.), Experientia, 32 (1976) 149. 29 J. Overnell, I.A. Davidson and T.L. Coombs, A cadmium-binding glycoprotein from the liver of the plaice (Pleuronectes ptubssu), Trans. Biochem. Sot., 5 (1977) 267. 30 T.L. Coombs in A.D. McIntyre and CF. Mills (Eds.), Ecological Toxicology Research, Plenum, New York 1974, pp. 187-195.
A MADRID-BINDING
241
PROTEIN IN RAINBOW TROUT
J.H. BEATTIE and D. PASCOE Department of Applied Biology, University of Wales institute of Science and Technology, Cathays Park, Cardiff CFI 3NU, U.K. (Received May l&h, 1979) (Accepted May 22nd, 1979)
SUMMARY
Rainbow trout, Sulmo ga~rdneri Rich were treated, by intrape~tone~ injection, with about 3.0 mg cadmium kg’ body weight over a 4day period. An extract prepared from the liver was found to contain a cadmium-binding protein of about 6700 daltons and with greater absorbance in the ultraviolet at 250 nm than at 280 nm. This protein is similar to metallothionein, isolated from a number of other species and may be responsible for the increased resistance to cadmium of rainbow trout pretreated with low levels of the same metal.
INTRODUCTION
There is now considerable evidence that pretreatment of fish with a poison may induce some degree of resistance during subsequent exposure to the same poison e.g. ammonia [l] , phenol [ 21, linear alkylate sulphonate detergent [ 31. and heavy metals such as zinc [4,5] and cadmium [6,7]. In mammals, where the same phenomenon occurs [8,9] it has been shown that pretreatment with low doses of metal stimulates the liver and other organs to synthesize a protein which is able to bind with and thereby inactivate further doses of the same metal. The purpose of this investigation was to determine whether the increased resistance to cadmium, shown by pretreated rainbow trout, Sulmo gairdneri Rich [7] could be attributed to the production of a similar metalbinding protein. MATERIALS AND METHODS
Rainbow trout (CT200 g each) obtained from a fish farm in the Wye Valley were acclimated to laboratory water conditions (temperature 15 + 1°C; total hardness 66.0 mg 1’ as CaCO,; dissolved oxygen 90-100% air saturation value) for two days. Four fish were randomly selected for cadmium treatment
242
and four for control purposes. Each test fish received an in~ape~tone~ injection of 0.5 ml of a 300 mg Cd lq saline solution on 4 successive days, i.e. a total of about 3.0 mg Cd kg-’ body weight. Fish were treated by intraperitoneal injection rather than by exposure to cadmium in the water, to ensure that a known amount of cadmium entered the body. On the seventh day, test and control fish were killed by immersion in a lethal concentration of the anaesthetic benzocaine (ethyl-4-amino benzoate) and the livers (2.1-3.7 g) removed, washed in saline and stored on ice, 5 g of liver (1.25 g from each treated fish) was then homogenized in 25 ml (20% w/v) of buffer (0.05 M Tris-HCl and 0.25 M sucrose) at pH 8.6. The homogenate was centrifuged at 10 000 X g for 15 min and the supernatant recentrifuged at 105 000 X g for 1 h to obtain the soluble fraction, Tissues were maint~ned at 4°C throughout the extraction, An extract from livers of the control fish was prepared in the same way. The particle-free, soluble fraction from each preparation was filtered (Whatman No. 1) and 3 ml applied to the surface of a G-75 Sephadex column (40 X 2.6 cm) packed with particles of standard size prepared by dry elutriation. The column was calibrated for molecular weight determination with bacitracin, molecular weight 1400; cytochrome C, 12 400; chymotrypsinogen, 25 000 and bovine serum albumin 67 000. Samples were eluted with Tris buffer and the ultraviolet absorption of the eluant continuously monitored at 250 nm using a Unicam SP1800 spectrophotometer. Individual 5 ml fractions were examined for UV absorbance at 250 and 280 nm and for cadmium using a Pye-Unicam SP191 atomic absorption spectrophotometer. Those fractions absorbing strongly at 250 nm and coinciding with the presence of cadmium were pooled and a spectrum scan carried out over the range 200300 nm, The protein concentration was determined spectrophotometrically and by the Biuret method. RESULTS
All fish appeared healthy during treatment, but on dissection some inflammation was noted at the cadmium injection site, and the livers of two fish and the kidneys of one were pale in appearance compared with controls. The elution profiles showing UV absorbance (250 nm and 280 nm) and cadmium concent~tio~ of the fractionated extracts of control and cadmiumtreated trout livers are shown in Figs. 1 and 2, respectively. Both profiles show an initial absorbance peak (elution volume 70-90 ml) and a final peak (180-220 ml) .representing high molecular weight proteins and low molecular weight materials respectively. However, an additional protein with greater absorbance at 250 nm (elution volume 120---150 ml) is present in the extract prepared from the livers of cadmium treated fish, and coincides with the occurrence of cadmium. From the concentrations of protein (molecular weight about 6700) and cadmium it can be calculated that 6.4 pg cadmium is associated with each mg protein. The UV absorbance spectrum (Fig. 3) of this
243
2
o-
1 5-
B
$7
w 2
O-
0 5-
O-
I
50
150
100
ELUTION
VOLUME
200
(ml)
Fig. 1. Sephadex G-75 elution profile of liver from control rainbow trout. Absorption at absorption at 250 nm (-------); cadmium (. . . . .) rg ml-‘. 280 nm ( -);
2c I-
1 5-
-
1 .’
-
1.1
-
0.
P
$1 .0 8
3
0‘5-
-0
oELUTION
VOLUME
(ml)
Fig. 2. Sephadex G-75 elution profile of liver extract from cadmium treated rainbow trout. ); absorption at 250 nm (-------); cadmium (. . . . .) 4ml-‘. Absorption at 280 nm (-
244
Fig. 3. Ultraviolet absorption spectrum of cadmium-binding protein.
cadmium-binding protein (Cd-BP) is minimal at 280 nm but shows a slight shoulder at 250 nm. Neither this protein nor any significant levels of cadmium were present in the extract prepared from control fish livers. DISCUSSION
Much of the cadmium polluting freshwater systems occurs as a result of mining activities, zinc smelting and electroplating. It is extremely toxic to fish [lo-161 causing death or a variety of sublethal effects. However, it has been shown [ 71 that rainbow trout alevins exposed to a low concentration of cadmium are subsequently more resistant to cadmium than untreated alevins. It appears from the present investigation that pretreatment induces the synthesis of a protein which is able to bind with, and effectively detoxify, cadmium. This Cd-BP has a molecular weight of about 6700 and is similar to metallothionein, a protein of reported molecular weight 6000-10 000 which was first found in horse kidney [17] and later detected in the liver and kidney of a number of other species‘such as man [ 18,191, rat [ 20-221, chicken [ 231. The UV absorption spectrum of the Cd-BP shows minimal absorbance at 280 nm, indicating a deficiency of aromatic amino acids, and a slight shoulder at 250 nm. Recent investigations carried out in this laboratory have shown that removal of cadmium from the protein by extraction at pH 2.0 leads to a reduction in absorbance at 250 nm. Although these features all suggest that the Cd-BP is metallothionein [ 22-261, confirmation depends upon
245
the demonstration of other characteristics such as a high cysteine content. There have been few previous studies of this kind on fish; however, it has been reported [ 271 that a low molecular weight mercury-binding protein is found in the liver, kidneys and gills of freshwater eels exposed to 0.4 mg Hg l-“. In another investigation [ 281 it has been shown that, following the injection of a mixture of CdCl*, HgCl, and ZnClz into goldfish Curassius aurutus, much of the three metals was found to be associated with a protein in the liver and kidney. A low molecular weight cadmium-binding glycoprotein has been isolated from the liver of plaice follo~ng intraperitone~ exposure to CdClz [29,30] _ REFERENCES 1 R. Lloyd and L.D. Orr, The diuretic response by rainbow trout to sub-lethal concentrations of ammonia, Wat. Res., 3 (1969) 335. 2 I. Malacea, Untersuchungen iiber die Gewohnung der Fische an hohe Konzentrationen toxischer Substanzen, Arch. Hydrobiol., 65 (1968) 74. 3 K.E.F. Hokanson and L.L. Smith, Some factors influencing toxicity of linear alkylate sulfonate (LAS) to the bluegill, Trans. Am, Fish. Sot., 100 (1971) 1. 4 R. Lloyd, The toxicity of zinc sulphate to rainbow trout, Ann. App. Biol., 48 (1960) 84. 5 R.W. Edwards and V.M. Brown, Pollution and fisheries: A progress report, J. Inst. Wat. Poll. Con., 66 (1967) 63. 6 J.H. Beattie and D. Pascoe, Cadmium uptake by rainbow trout, Salmo gairdneri Richardson, eggs and alevins, J. Fish Biol., 13 (1978) 631. 7 D. Pascoe and J.H. Beattie, Resistance to cadmium by pretreated rainbow trout alevins, J. Fish Biol., 14 (1979) 303. 8 G. Gabbiani, D. Bait and C. Deziel, Studies on tolerance and ionic antagonism for cadmium or mercury, Can. J. Physiol. Pharmacol., 45 (1967) 443. 9 H. Yoshikawa, Preventive effect of pretreatment with low doses of metals on the acute toxicity of metals in mice, Ind. Health, 8 (1970) 184. 10 I.R. Ball, The toxicity of cadmium to rainbow trout Salmo gairdneri Richardson, Wat. Res., 1 (1967) 805. 11 B-E. Bengtsson, C.H. Carlin, A. Larsson and 0. Svanberg, Vertebral damage in minnows, Phoxinus phoxinus L,, exposed to cadmium, AMBIO, 4 (1975) 166. 12 G.R. Gardner and P.P. Yevich, Histological and haematological responses of an estuarine teleost to cadmium, J. Fish Res. Bd Can., 27 (1970) 2185. 13 A. Larsson, B-E. Bengtsson and 0. Svanberg, Some haematological and biochemical effects of cadmium on fish, in A.P.M. Lockwood (Ed.) Effects of Pollutants on Aquatic Organisms, Camb. University Press, Cambridge, 1976, pp. 35-46. 14 D. Pascoe and P. Cram, The effect of parasitism on the toxicity of cadmium to the three-spined stickleback, Gasterosteus aculeatus L., J. Fish Biol., 10 (1977) 467. 15 D. Pascoe and D.L. Mattey, Studies on the toxicity of cadmium to the three-spined stickleback, Gasterosteus aculeatus L., J. Fish Biol., 11 (1977) 207. 16 R.L. Spehar, Cadmium and zinc toxicity to the flagfish Jordanella floridae, J. Fish Res. Bd Can., 33 (1976) 1939. 17 M. Margoshes and B.L. Vallee, A cadmium protein from equine kidney cortex, J. Am. Chem. Sot., 79 (1957) 4813. 18 J.M. Wisniewska-Knypl, J. Jablonska and Z. Myslak, Binding of cadmium on metallothionein in man: an analysis of a fatal poisoning by cadmium iodide, Arch. Toxicol., 28 (1971) 46.
246 19 H.O. Buhler and J.H.R. Kagi, Human hepatic metallothioneins, FEBS Lett., 39 (1974) 229. 20 K. Tanaka, K. Sueda, S. Onasaka and K. Okahara, Fate of ‘09Cd-labelled metallothionein in rats, Toxicol. Appl. Pharmacol., 33 (1975) 258. 21 J.M. Wisniewska-Knypl, B.B. Trojanowska, J.K. Piotrowski and J.K. Jablonska, Binding of mercury in rat liver by metallothionein, Acta Biochim. Polon., 19 (1972) 11. 22 D.R. Winge, R. Premakumar and K.V. Rajagopalan, Metal-induced formation of metallothionein in rat liver, Arch. Biochem. Biophys., 170 (1975) 242. 23 U. Weser, F. Donay and H. Rupp, Cadmium-induced synthesis of hepatic metallothionein in chicken and rats, FEBS Lett., 32 (1973) 171. 24 J.H.R. Kagi and B.L. Vallee, Met~lothionein: a cadmium- and zinc-containing protein from equine renal cortex, J, Biol, Chem., 235 (1960) 3460. 25 A.P. Leber and T.S. Miya, A mechanism for cadmium- and zinc-induced tolerance to cadmium toxicity: involvement of metallothionein, Toxicol. Appl. Pharmacol., 37 (1976) 403. 26 M. Nordberg, B. Trojanowska and G.F. Nordberg, Studies on metal-binding proteins of low molecular weight from renal tissue of rabbits exposed to cadmium or mercury, Environ. Physiol. Biochem., 4 (1974) 11. 27 J.M. Bouquegneau, Ch. Gerday and A. Disteche, Fish mercury-binding thionein related to adaptation mechanisms, FEBS Lett., 55 (1975) 173, 28 E. Marafante, Binding of mercury and zinc to cadmium-binding protein in liver and kidney of goldfish (Curassius aurutus L.), Experientia, 32 (1976) 149. 29 J. Overnell, I.A. Davidson and T.L. Coombs, A cadmium-binding glycoprotein from the liver of the plaice (Pleuronectes ptubssu), Trans. Biochem. Sot., 5 (1977) 267. 30 T.L. Coombs in A.D. McIntyre and CF. Mills (Eds.), Ecological Toxicology Research, Plenum, New York 1974, pp. 187-195.