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Effects of metal ions on the antioxidant enzyme activities, protein contents and lipid peroxidation of carp tissues
Camp. Biochem. Physiol. Vol. 9OC, No. 1, pp. 69-72, 1988 Printed in Great Britain
0
0306~4492/88 $3.00 + 0.00 1988 Pcrgamon Press plc
EFFECTS OF METAL IONS ON THE ANTIOXIDANT ENZYME ACTIVITIES, PROTEIN CONTENTS AND LIPID PEROXIDATION OF CARP TISSUES AKG. ALI RAGEB RADI and B. MATKOVICS* Biological Isotope Laboratory “A.J.“, University of Szeged, Hungary (Received
27 April 1987)
Abstract-l. Studies were performed regarding the effects of CuSO, in concentrations of 5, IO, 25 and 50ppm and ZnSO, in concentrations of 10 and IOOppm on the antioxidant enzyme activities, lipid peroxidation and protein contents of tissues of common carp (Cyprinus carpio morpha L.) exposed to these pollutants for 24 hr. 2. The results demonstrated that CuSO, was more toxic than ZnSO, and that both treatments brought about significant changes in these parameters in carp hepatopancreas (liver), gill and white muscle. 3. An increase of the CuSO, concentration led to significant decreases in the antioxidant enzyme activities, except that of glutathione peroxidase, which was increased significantly, and significant increases in the lipid peroxidation and protein contents. 4. An increase of the ZnSO, concentration led to slight changes in the antioxidant enzyme activities, lipid peroxidation and protein contents of carp tissues.
INTRODUCTION Copper and zinc are essential trace elements in biological systems. They primarily act as components of superoxide dismutase and other enzymes. It has been reported that the use of 100 PM copper sulphate resulted in a 90% inactivation of hexokinase and in disruption of the membranes or haemolysis (Hochstein et al., 1980). Bioconcentrations of these trace elements cause marked decreases in superoxide dismutase in cod liver, and morphological and biochemical changes related to the fish body weight (Vinikour et al., 1980; Bartkowiak et al., 1981; Nemcsok et al., 1982; Rojik et al., 1983). Since both ions are superoxide dismutase active site components, we investigated how different concentrations of these ions influence the oxidative metabolism in general, the lipid peroxidation and the protein content. MATERIALS AND METHODS The experiments were performed with common carp (Cyprinus carpio L.) weighing 350-450 g, maintained for at least 7 days before the experiments in a large tank filled with well-aerated tap water at 18 + 1°C. Stock solutions of pollutants were prepared from CuSO, .5H,O in concentrations of 5, lo,25 and 50 ppm, and from ZnSO, .7H,O in concentrations of 10 and 100 ppm. The animals were transported in well-aerated water. Twenty-four hours after treatment with CuSO, or ZnSO,, the animals were killed. The liver (hepatopancreas), gill and white muscles were removed for determination of antioxidant enzyme activities, lipid peroxidation and protein content.
*Correspondence and reprint requests should be addressed to B. Matkovics, PO Box 539, Szeged 1, H-6701, Hungary.
Enzymatic
activities
in tissues
(a) Superoxide dismutase (SOD; EC 1.15.1. I) activity was determined on the basis of inhibition of the epinephrine-adrenochrome autocatalytic transformation under basic conditions (Misra and Fridovich, 1972; Matkovics et al., 1977a). The mitochondrial manganaseSOD (Mn-SOD) activity was determined in the presence of, 5 x IO-’ M KCN (Beaucham et al., 1971). (b) Glutathione peroxidase (GP-ase; EC 1.11.1.9) activity was determined by the method of Chiu et al. (1976) with cumene hydroperoxide as substrate. The reduced glutathione (GSH) residue after the action of the enzyme was measured by the method of Sedlak et al. (1968) using the Ellman reagent. (c) Catalase (C-ase; EC 1.11.1.6) activity was determined by the spectrophotometric method of Beers and Sizer (1952). Other parameters (a) Lipidperoxidation. The method of Placer et al. (1966), based on a calorimetric thiobarbituric acid (TBA) test, was used for quantitative measurement of the tissue LP, i.e. for quantitative assay of the amount of TBA-reactive substances. (b) Protein. The quantity of protein in the homogenate supernatants was estimated by the method of Lowry et al. (1951). The results were subjected to statistical evaluation with Student’s r-test and correlation coefficients. All numerical data are given as means k SD.
RESULTS C&O, and ZNSO, treatments led to marked changes in the activities of the antioxidant enzymes, the lipid peroxidation and the protein content of the carp tissues. Effects
of CuSO,
treatment
The results in Table 69
on antioxidant
1 demonstrate
enzymes
that high
AMALALI RAGEBRADIand B. MATKOVICS
70
Table I. EBcctsof GP-ase
Organs # Liver Gill MUSCk
5 PPm
35.2 k2.l 24.9 f 2.7 0.3 f 0.0
IOppm
23.2 * 2.0 24.5 f 4.4 0.4 +0.1
U/g w.t.w. 25ppm
32.4 + 0.8 39.0 f 4.2 0.4 kO.1
The given values are the means of 65
50ppm
36.8 f 0.6 43.2 f 7.3 0.5 f 0.0 fishes
4
55. I f 2.6 51.3 + 6.0 0.5 + 0.0
47 f 5 f 2 i 0.2 + 0.5 * 0.0
treatment on other parameters
The data in Table 2 indicate the effects of different concentrations of CuSO, on the protein content and lipid peroxidation. At the higher concentration, the protein content decreased compared to the control in the liver and gill, but the change was not significant in the white muscle. The lipid peroxidation revealed marked differences between the tissues of the treated and the control fish; the level increased significantly and correlated positively in the liver, gill and white muscle (r = 0.81, r = 0.88 and r = 0.87) on increase of the CuSO, concentration. Effects of ZnSO,
treatment
on antioxidant
enzymes
The results in Table 3 show that the most significant changes were observed in the liver and gill. GP-ase. This enzyme was significantly higher in the treated liver than in the control and correlated positively (r = 0.85), while in the gill and white muscle it was significantly decreased and correlated negatively (r = -0.97 and r = -0.96). C-ase. The lowest C-ase activity was measured in the gill and muscle tissue, where it exhibited a significant, negatively correlated decrease (r = -0.76 and r = - 0.93) with the treatment. In contrast, in the liver the changes were not significant. SOD. The highest activity of total-SOD and its fractions (Mn-SOD and Cu, Zn-SOD) were measured in the liver, where the treatment correlated positively (r = 0.59, r = 0.67 and r = 0.48). The lowest activities of these enzymes were found in the gill, where they
Table 2. Etkts
of different
concentrations
of CuSo, on the protein
Protein
Organs 0
5 ppm
IOppm 104.6~10.4
Gill
70.9 f 3.5
74.4 * 3.1
14.2 f 4.9
55.9*
White muscle
40.2 f 4.1
43.7 * 5.3
33.6 f 3.9
43.7f7.0
as the means
51 * 2 + 1.7 + 0.5 f 0.4 f 0.0
x x x x x x
10 ppm 10-Z 10-J 10-Z 10-Z 10-Z 10-l
49 *4x f 1.7 f 0.5 f 0.4 kO.1
25 PP~
x 10-Z 10-J x 10-2 x 10-2 x 10-Z x 10-Z
C&O,
on
w.t.w. 27 + 5 +0.6 +0.4 f 0.3 fO.l
x x x x x x
50 ppm IO-’ 10-Z 10m2 10-Z lo-* lo-’
24 f 2 *06x kO.1 + 0.2 f 0.0
x IO-* x 10~ 2 IO ’ x 10-I x 10-Z x 10-Z
correlated negatively with the treatment (r = -0.99, r = -0.98 and r = -0.66). In contrast, in the white muscle no significant changes were observed. Effects of ZnSO, lipid peroxidation
treatment
on protein
25 ppm 79.3k4.5
DISCUSSION
In agreement with the previous studies, our results demonstrated that an increase of the CuSO, concentration is accompanied by an increase in GP-ase activity. This is due to the formation of superoxide radical, which in turn undergoes dismutation to form HzOz (Hochstein et aI., 1980). There was no significant change in C-ase activity in the liver and gill. This is due to the high activity of GP-ase, which acts as a defence against the formation of H202. The SODS displayed a significant decrease in activity in the carp tissues in response to an increase of the 0; radical concentration. This radical itself, or after transformation to H,Or , causes a strong oxidation of the cysteine in the enzyme and decreases its activity, as demonstrated by Bartkowiak et al. (1981). The reaction of 0; and H,Or is also dangerous as a source of OH’ radical donor, due to the Haber-Weiss reaction. The results further show that the copper ion has a decreasing effect on the protein content, because the lipid peroxidation initiates the formation of malondialdehyde. This agent has the capacity to cross-link the amino groups of lipid and protein by the formation of Schiff bases, and the presence of
content
50 ppm 73.41t2.0
10.2 48.824.9
f SD of the results for 45
content and
The data in Table 4 indicate the changes in the protein content and lipid peroxidation. It was found that an increase of the ZnSO, concentration caused only slight changes in the protein contents of the liver and gill, but significantly increased the protein content of the white muscle. The lipid peroxidation was significantly changed in the muscle (P < 0.1% and P < 0.01% at 10 ppm and 100 ppm ZnSO, , respectively), while the lipid peroxidation in the gill and liver was not changed significantly.
and lipid peroxidation
in the three organs in common
LP nM MDA/g
97.9fI2.0
The values are expressed
10-2 10-l 10-Z 10-2 10-2 10-I
mg/g w.t.w.
89.2i15.9
Liver
5 PPm x x x x x x
concentrationsof
+ SD.
GP-ase activity was observed in the liver and gill. On increase in the CuSO, concentration, this enzyme increased significantly in these tissues and correlated positively: r = 0.12, r = 0.89 and r = 0.63, respectively. A low CuSO, concentration caused more significant changes than a higher one in the carp tissues. SODS. The total -SOD and its fractions Mn-SOD and Cu, Zn-SOD displayed significant decreases in activity and correlated negatively in the liver, gill and muscle (r = -0.89, r = -0.75 and r = -0.52) on increase of the CuSO, concentration. Effects of &SO,
different
C-ase BU/g
38.2k2.0 animals.
0
5 ppm
lOPPm
34.951.6
27.4k2.5
29.lk4.0
26.Ok4.5
35.8k2.3
12.2f2.1
15.4&1.0
carp
w.t.w. 25 ppm
50 ppm
59.lk3.8
53.9k6.3
42.5k9.0
50.9k4.3
52.2k3.7
14.6k2.3
37.Ok4.5
37.1 f1.8
71
Effects of metal ions in carp tissues antioxidant enzymes in the three organs in the common carp Total-SOD U/g w.t.w. Mn-SOD U/g w.t.w. #J 1283.9 k214.9 208.2 f 15.1 32.2 f 3.4
5ppm 1173.7 k280.9 229.2 f 32.7 34.1 f 4.6
IOppm
25ppm
50ppm
869.6 787.6 F74.0 +93.5 89.2 277.7 f 15.6 f 7.0 28.1 38.6 +3.0 fl.7
Q
306.8 96. I k10.5 f16.1 29.6 8.6 + 5.9 f 1.0 24.4 4.5 k4.2 kO.4
Sppm
IOppm
49.6 +9.2 18.5 +2.7 4.4 f0.5
malondialydehyde is also associated with the polymerization of specific membrane proteins. The lipid peroxidation is increased significantly due to the high formation of 0, and HzOz, which initiate the peroxidation of unsaturated fatty acids in the membrane phospholipids. The degradation of peroxidized fatty acids may lead to the formation of malondialdehyde. Our results on ZnSO, revealed that an increase in the ZnSO, concentration led to slight changes in the antioxidant enzyme activities. The data on GP-ase demonstrated that the activity of this enzyme increased in the liver due to the ZnSO, enhancement of the glucose metabolism (Nemcs6k and Boross, 1982), which permits the cells to maintain the glutathione level. The lowest activity of C-ase was measured in the gill tissue; this was explained by the increased generation of H,O,, which led to a decreased C-ase activity. The changes in SOD activities varied between increases and decreases in the liver and gill, this is because ZnSO, is less toxic than CuSO, and its generation of 0, is slight enough not to cause oxidation of the SODS. The changes in the protein content in the liver and gill were very slight. However, there was a significant increase in the white muscle, where large amounts of ZnSO, are accumulated and cause polymerization of the specific membrane proteins. Similarly, a high level of lipid peroxidation was measured only in the white muscle. This may be because ZnSO, oxidized molecular oxygen to form the superoxide radical as a result of dismutation; this reaction would act as a source of HzOz, which initiates the peroxidation of polyunsaturated fatty acids in the membrane and may lead to the formation of malondialdehyde. An increased lipid peroxidation in other organs may also be explained by the very rich lipid contents.
42.4 f12.1 40.4 k2.5 4.8 fO.l
Cu,
25ppm 50ppm 89.8 rfr6.3 9.6 +2.3 2.5 50.5
65.7 f5.3 5.7 f0.7 2.0 50.2
Q 1183.4 k213.7 199.6 * 15.5 27.7 f 3.5
h-SOD U/g w.t.w. IOppm 25ppm
5 PPm
1165.3 f262.9 210.8 f 30.9 29.7 f 4.2
826.4 574.7 237.5 f 16.2 33.8 It 3.0
697.8 f98.9 79.7 f 5.5 25.6 f 1.6
Bartkowiak
A., Grzelinska
E., Varga I. Sz. and Leyko W.
241.1 +5.25 23.8 + 6.0 22.4 f 4.1
(1981) Studies on superoxide dismutase from cod (Go& morhua) liver. Int. J. Biochem. 13, 1039-1042. Beauchamp C. and Fridovich I. (1971) Superoxide dismutase: improved assay and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287. Beers R. F. Jr and Sizer I. W. (1952) Spectrophotometry for measuring the breakdown of hydrogen peroxide by catalase.. J. biol. Chem. 1%. 133-140. Chiu D. T. Y., Stults c. H. and Tappal A. L. (1976) Purification and properties of rat lung soluble glutathione peroxidase. Biochem. Biophys. Acta 445, 558-566. Hochstein P., Kumar K. S. and Forman S. J. (1980). Lipid peroxidation and cytotoxicity of copper. Ann. N. Y. Acad. Sci. 355, 24&248. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Matkovics B., NovLk R., Hoang due Hahn, Szab6 L. and Zalesna G. (1977) A comparative study of some important experimental animal peroxide metabolism enzymes. Camp. Biochem. Physiol. 56B, 31-34. Misra H. P. and Fridovich I. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. biol. Chem. 247, 3170-3175.
Nemcsbk J. and Boross L. (1982) Comparative studies on the sensitivity of different fish species to metal pollution. Acta biol. Hung. 33, 23-27.
Placer Z. A., Cushman L. and Johnson B. C. (1966) Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal. Biochem. 16, 359-364. Rojik I., Nemcsbk J. and Boross L. (1983) Morphological and biochemical studies on liver, kidney and gill of fishes affected by pesticides. Acta biol. Hung. 34, 81-92. Sedlak I. and Lindsay R. H. (1968) Estimation of total protein-bond and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192-205. Vinikour W. S.. Goldstein R. M. and Anderson R. V. (19801 Bioconcentration patterns of zinc, copper, cadmium anh lead in selected fish species from the Fox river, Illinois. Bull. environ. Contam. Toxicol. 24, 727-734.
REFERENCES
50ppm
17.93 f I .03
0.25 f 0.03
Gill
Ml&e
0.19 f 0.01 P
12.14 + 2.21 P
21.71 f 3.96 NS
lOppm
0.3 + IO_’ + 0.00
0.8 x IO_’ + 0.1 x 10-2
3 x 10-z * 0.1 x 10-Z
37.70 f I .02
29.02 f 0.92
Gill
White muscle
mg/g w.t.w. 10 PPm
36.47 f 2.74 P cO.Ol%
33.81 f 2.45 P
61.31 f 1.55 NS
Protein
32.24 f 1.82
167.98 + 8.78
f SD.
37. I8 + 3.97 P
33.79 * I.91 P
56.06 k 4.24 P< I%
100 pmm
4.23 f0.11
23.79 i I .08
35.86 k I .33
30.02 k 3.49 P
40.18 & 3.86 NS
36.85 + 1.63 NS
carp
3.48 k 0.34 P < 0.01
21.65 k 2.48 P < 0.01
94.91 f 17.97 P
lOppm
46.42 + 4.10 P
34.122 f 2.27 NS
35.35 f 3.46 NS
2.45 * 0.39 P < 0.01
10.85 5 0.67 P
206.29 + 16.97 P
100 PPm
U/g w.t.w
in the three
12.29 f 0.78
126.58 k 7.60 -
0
LP nM MDA/g w.t.w. 10 PPm 100 PPm
35.78 f 3.08
0
36.17 f I.21 P < 0.01
24.11 3.34 P < 0.01
1420.27 * 114.11 P
100 PPm
Mn-SOD
in the common
and lipid peroxidation
40.79 f 4.64 P < 0.01
91.31 7.9 I P < 0.01
1031.88 * 31.14 -
lOppm
of Z&O, on the protein content organs in common carp
The given values are the results for 4 fish, means NS = not significant.
62.44 f I .20
0
Liver
Organs
0.3 x 10-2 f 0.03 x 10-2 P
1159.97 f 102.98
0
in three organs
U/g w.t.w
enzymes
Total-SOD
on antioxidant
2.8 x 10-2 f 0.2 f IO_’ NS
‘00 PPm
of ZnSO,
0.3 x 10-2 0.1 x 10-2 * 0.01 x IO * &O.Ol x IO * NS P < 0.01
0.6 x IO * f 0.00 NS
2.9 x IO-’ + 0.1 x 10-Z NS
IOPPm
Bu/g w.t.w,
Table 4. Effects of two concentrations
f SD.
0.003 * 0.001 P < 0.01
2.61 kO.19 P < 0.01
51.49 k 3.29 P c 0.01
e
Case
Table 3. Effects of two concentrations
1OOppm
U/g w.t.w.
The given values are the means of 4 fishes NS = not significant.
21.67 f 1.06
Liver
0
GP-ase
28.01 f 1.93
149.51 f 18.04
1033.39 + 110.46 -
0
31.32 & 4.97 P
69.66 + 5.59 P < 0.01
936.91 f 26.34 NS
IOppm
33.12 f 1.43 P < 0.01
13.26 * 3.47 P
1188.94 * 120.06 NS
100 ppm
Cu Zn-SOD U/g w.t.w.
0
0306~4492/88 $3.00 + 0.00 1988 Pcrgamon Press plc
EFFECTS OF METAL IONS ON THE ANTIOXIDANT ENZYME ACTIVITIES, PROTEIN CONTENTS AND LIPID PEROXIDATION OF CARP TISSUES AKG. ALI RAGEB RADI and B. MATKOVICS* Biological Isotope Laboratory “A.J.“, University of Szeged, Hungary (Received
27 April 1987)
Abstract-l. Studies were performed regarding the effects of CuSO, in concentrations of 5, IO, 25 and 50ppm and ZnSO, in concentrations of 10 and IOOppm on the antioxidant enzyme activities, lipid peroxidation and protein contents of tissues of common carp (Cyprinus carpio morpha L.) exposed to these pollutants for 24 hr. 2. The results demonstrated that CuSO, was more toxic than ZnSO, and that both treatments brought about significant changes in these parameters in carp hepatopancreas (liver), gill and white muscle. 3. An increase of the CuSO, concentration led to significant decreases in the antioxidant enzyme activities, except that of glutathione peroxidase, which was increased significantly, and significant increases in the lipid peroxidation and protein contents. 4. An increase of the ZnSO, concentration led to slight changes in the antioxidant enzyme activities, lipid peroxidation and protein contents of carp tissues.
INTRODUCTION Copper and zinc are essential trace elements in biological systems. They primarily act as components of superoxide dismutase and other enzymes. It has been reported that the use of 100 PM copper sulphate resulted in a 90% inactivation of hexokinase and in disruption of the membranes or haemolysis (Hochstein et al., 1980). Bioconcentrations of these trace elements cause marked decreases in superoxide dismutase in cod liver, and morphological and biochemical changes related to the fish body weight (Vinikour et al., 1980; Bartkowiak et al., 1981; Nemcsok et al., 1982; Rojik et al., 1983). Since both ions are superoxide dismutase active site components, we investigated how different concentrations of these ions influence the oxidative metabolism in general, the lipid peroxidation and the protein content. MATERIALS AND METHODS The experiments were performed with common carp (Cyprinus carpio L.) weighing 350-450 g, maintained for at least 7 days before the experiments in a large tank filled with well-aerated tap water at 18 + 1°C. Stock solutions of pollutants were prepared from CuSO, .5H,O in concentrations of 5, lo,25 and 50 ppm, and from ZnSO, .7H,O in concentrations of 10 and 100 ppm. The animals were transported in well-aerated water. Twenty-four hours after treatment with CuSO, or ZnSO,, the animals were killed. The liver (hepatopancreas), gill and white muscles were removed for determination of antioxidant enzyme activities, lipid peroxidation and protein content.
*Correspondence and reprint requests should be addressed to B. Matkovics, PO Box 539, Szeged 1, H-6701, Hungary.
Enzymatic
activities
in tissues
(a) Superoxide dismutase (SOD; EC 1.15.1. I) activity was determined on the basis of inhibition of the epinephrine-adrenochrome autocatalytic transformation under basic conditions (Misra and Fridovich, 1972; Matkovics et al., 1977a). The mitochondrial manganaseSOD (Mn-SOD) activity was determined in the presence of, 5 x IO-’ M KCN (Beaucham et al., 1971). (b) Glutathione peroxidase (GP-ase; EC 1.11.1.9) activity was determined by the method of Chiu et al. (1976) with cumene hydroperoxide as substrate. The reduced glutathione (GSH) residue after the action of the enzyme was measured by the method of Sedlak et al. (1968) using the Ellman reagent. (c) Catalase (C-ase; EC 1.11.1.6) activity was determined by the spectrophotometric method of Beers and Sizer (1952). Other parameters (a) Lipidperoxidation. The method of Placer et al. (1966), based on a calorimetric thiobarbituric acid (TBA) test, was used for quantitative measurement of the tissue LP, i.e. for quantitative assay of the amount of TBA-reactive substances. (b) Protein. The quantity of protein in the homogenate supernatants was estimated by the method of Lowry et al. (1951). The results were subjected to statistical evaluation with Student’s r-test and correlation coefficients. All numerical data are given as means k SD.
RESULTS C&O, and ZNSO, treatments led to marked changes in the activities of the antioxidant enzymes, the lipid peroxidation and the protein content of the carp tissues. Effects
of CuSO,
treatment
The results in Table 69
on antioxidant
1 demonstrate
enzymes
that high
AMALALI RAGEBRADIand B. MATKOVICS
70
Table I. EBcctsof GP-ase
Organs # Liver Gill MUSCk
5 PPm
35.2 k2.l 24.9 f 2.7 0.3 f 0.0
IOppm
23.2 * 2.0 24.5 f 4.4 0.4 +0.1
U/g w.t.w. 25ppm
32.4 + 0.8 39.0 f 4.2 0.4 kO.1
The given values are the means of 65
50ppm
36.8 f 0.6 43.2 f 7.3 0.5 f 0.0 fishes
4
55. I f 2.6 51.3 + 6.0 0.5 + 0.0
47 f 5 f 2 i 0.2 + 0.5 * 0.0
treatment on other parameters
The data in Table 2 indicate the effects of different concentrations of CuSO, on the protein content and lipid peroxidation. At the higher concentration, the protein content decreased compared to the control in the liver and gill, but the change was not significant in the white muscle. The lipid peroxidation revealed marked differences between the tissues of the treated and the control fish; the level increased significantly and correlated positively in the liver, gill and white muscle (r = 0.81, r = 0.88 and r = 0.87) on increase of the CuSO, concentration. Effects of ZnSO,
treatment
on antioxidant
enzymes
The results in Table 3 show that the most significant changes were observed in the liver and gill. GP-ase. This enzyme was significantly higher in the treated liver than in the control and correlated positively (r = 0.85), while in the gill and white muscle it was significantly decreased and correlated negatively (r = -0.97 and r = -0.96). C-ase. The lowest C-ase activity was measured in the gill and muscle tissue, where it exhibited a significant, negatively correlated decrease (r = -0.76 and r = - 0.93) with the treatment. In contrast, in the liver the changes were not significant. SOD. The highest activity of total-SOD and its fractions (Mn-SOD and Cu, Zn-SOD) were measured in the liver, where the treatment correlated positively (r = 0.59, r = 0.67 and r = 0.48). The lowest activities of these enzymes were found in the gill, where they
Table 2. Etkts
of different
concentrations
of CuSo, on the protein
Protein
Organs 0
5 ppm
IOppm 104.6~10.4
Gill
70.9 f 3.5
74.4 * 3.1
14.2 f 4.9
55.9*
White muscle
40.2 f 4.1
43.7 * 5.3
33.6 f 3.9
43.7f7.0
as the means
51 * 2 + 1.7 + 0.5 f 0.4 f 0.0
x x x x x x
10 ppm 10-Z 10-J 10-Z 10-Z 10-Z 10-l
49 *4x f 1.7 f 0.5 f 0.4 kO.1
25 PP~
x 10-Z 10-J x 10-2 x 10-2 x 10-Z x 10-Z
C&O,
on
w.t.w. 27 + 5 +0.6 +0.4 f 0.3 fO.l
x x x x x x
50 ppm IO-’ 10-Z 10m2 10-Z lo-* lo-’
24 f 2 *06x kO.1 + 0.2 f 0.0
x IO-* x 10~ 2 IO ’ x 10-I x 10-Z x 10-Z
correlated negatively with the treatment (r = -0.99, r = -0.98 and r = -0.66). In contrast, in the white muscle no significant changes were observed. Effects of ZnSO, lipid peroxidation
treatment
on protein
25 ppm 79.3k4.5
DISCUSSION
In agreement with the previous studies, our results demonstrated that an increase of the CuSO, concentration is accompanied by an increase in GP-ase activity. This is due to the formation of superoxide radical, which in turn undergoes dismutation to form HzOz (Hochstein et aI., 1980). There was no significant change in C-ase activity in the liver and gill. This is due to the high activity of GP-ase, which acts as a defence against the formation of H202. The SODS displayed a significant decrease in activity in the carp tissues in response to an increase of the 0; radical concentration. This radical itself, or after transformation to H,Or , causes a strong oxidation of the cysteine in the enzyme and decreases its activity, as demonstrated by Bartkowiak et al. (1981). The reaction of 0; and H,Or is also dangerous as a source of OH’ radical donor, due to the Haber-Weiss reaction. The results further show that the copper ion has a decreasing effect on the protein content, because the lipid peroxidation initiates the formation of malondialdehyde. This agent has the capacity to cross-link the amino groups of lipid and protein by the formation of Schiff bases, and the presence of
content
50 ppm 73.41t2.0
10.2 48.824.9
f SD of the results for 45
content and
The data in Table 4 indicate the changes in the protein content and lipid peroxidation. It was found that an increase of the ZnSO, concentration caused only slight changes in the protein contents of the liver and gill, but significantly increased the protein content of the white muscle. The lipid peroxidation was significantly changed in the muscle (P < 0.1% and P < 0.01% at 10 ppm and 100 ppm ZnSO, , respectively), while the lipid peroxidation in the gill and liver was not changed significantly.
and lipid peroxidation
in the three organs in common
LP nM MDA/g
97.9fI2.0
The values are expressed
10-2 10-l 10-Z 10-2 10-2 10-I
mg/g w.t.w.
89.2i15.9
Liver
5 PPm x x x x x x
concentrationsof
+ SD.
GP-ase activity was observed in the liver and gill. On increase in the CuSO, concentration, this enzyme increased significantly in these tissues and correlated positively: r = 0.12, r = 0.89 and r = 0.63, respectively. A low CuSO, concentration caused more significant changes than a higher one in the carp tissues. SODS. The total -SOD and its fractions Mn-SOD and Cu, Zn-SOD displayed significant decreases in activity and correlated negatively in the liver, gill and muscle (r = -0.89, r = -0.75 and r = -0.52) on increase of the CuSO, concentration. Effects of &SO,
different
C-ase BU/g
38.2k2.0 animals.
0
5 ppm
lOPPm
34.951.6
27.4k2.5
29.lk4.0
26.Ok4.5
35.8k2.3
12.2f2.1
15.4&1.0
carp
w.t.w. 25 ppm
50 ppm
59.lk3.8
53.9k6.3
42.5k9.0
50.9k4.3
52.2k3.7
14.6k2.3
37.Ok4.5
37.1 f1.8
71
Effects of metal ions in carp tissues antioxidant enzymes in the three organs in the common carp Total-SOD U/g w.t.w. Mn-SOD U/g w.t.w. #J 1283.9 k214.9 208.2 f 15.1 32.2 f 3.4
5ppm 1173.7 k280.9 229.2 f 32.7 34.1 f 4.6
IOppm
25ppm
50ppm
869.6 787.6 F74.0 +93.5 89.2 277.7 f 15.6 f 7.0 28.1 38.6 +3.0 fl.7
Q
306.8 96. I k10.5 f16.1 29.6 8.6 + 5.9 f 1.0 24.4 4.5 k4.2 kO.4
Sppm
IOppm
49.6 +9.2 18.5 +2.7 4.4 f0.5
malondialydehyde is also associated with the polymerization of specific membrane proteins. The lipid peroxidation is increased significantly due to the high formation of 0, and HzOz, which initiate the peroxidation of unsaturated fatty acids in the membrane phospholipids. The degradation of peroxidized fatty acids may lead to the formation of malondialdehyde. Our results on ZnSO, revealed that an increase in the ZnSO, concentration led to slight changes in the antioxidant enzyme activities. The data on GP-ase demonstrated that the activity of this enzyme increased in the liver due to the ZnSO, enhancement of the glucose metabolism (Nemcs6k and Boross, 1982), which permits the cells to maintain the glutathione level. The lowest activity of C-ase was measured in the gill tissue; this was explained by the increased generation of H,O,, which led to a decreased C-ase activity. The changes in SOD activities varied between increases and decreases in the liver and gill, this is because ZnSO, is less toxic than CuSO, and its generation of 0, is slight enough not to cause oxidation of the SODS. The changes in the protein content in the liver and gill were very slight. However, there was a significant increase in the white muscle, where large amounts of ZnSO, are accumulated and cause polymerization of the specific membrane proteins. Similarly, a high level of lipid peroxidation was measured only in the white muscle. This may be because ZnSO, oxidized molecular oxygen to form the superoxide radical as a result of dismutation; this reaction would act as a source of HzOz, which initiates the peroxidation of polyunsaturated fatty acids in the membrane and may lead to the formation of malondialdehyde. An increased lipid peroxidation in other organs may also be explained by the very rich lipid contents.
42.4 f12.1 40.4 k2.5 4.8 fO.l
Cu,
25ppm 50ppm 89.8 rfr6.3 9.6 +2.3 2.5 50.5
65.7 f5.3 5.7 f0.7 2.0 50.2
Q 1183.4 k213.7 199.6 * 15.5 27.7 f 3.5
h-SOD U/g w.t.w. IOppm 25ppm
5 PPm
1165.3 f262.9 210.8 f 30.9 29.7 f 4.2
826.4 574.7 237.5 f 16.2 33.8 It 3.0
697.8 f98.9 79.7 f 5.5 25.6 f 1.6
Bartkowiak
A., Grzelinska
E., Varga I. Sz. and Leyko W.
241.1 +5.25 23.8 + 6.0 22.4 f 4.1
(1981) Studies on superoxide dismutase from cod (Go& morhua) liver. Int. J. Biochem. 13, 1039-1042. Beauchamp C. and Fridovich I. (1971) Superoxide dismutase: improved assay and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287. Beers R. F. Jr and Sizer I. W. (1952) Spectrophotometry for measuring the breakdown of hydrogen peroxide by catalase.. J. biol. Chem. 1%. 133-140. Chiu D. T. Y., Stults c. H. and Tappal A. L. (1976) Purification and properties of rat lung soluble glutathione peroxidase. Biochem. Biophys. Acta 445, 558-566. Hochstein P., Kumar K. S. and Forman S. J. (1980). Lipid peroxidation and cytotoxicity of copper. Ann. N. Y. Acad. Sci. 355, 24&248. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Matkovics B., NovLk R., Hoang due Hahn, Szab6 L. and Zalesna G. (1977) A comparative study of some important experimental animal peroxide metabolism enzymes. Camp. Biochem. Physiol. 56B, 31-34. Misra H. P. and Fridovich I. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. biol. Chem. 247, 3170-3175.
Nemcsbk J. and Boross L. (1982) Comparative studies on the sensitivity of different fish species to metal pollution. Acta biol. Hung. 33, 23-27.
Placer Z. A., Cushman L. and Johnson B. C. (1966) Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal. Biochem. 16, 359-364. Rojik I., Nemcsbk J. and Boross L. (1983) Morphological and biochemical studies on liver, kidney and gill of fishes affected by pesticides. Acta biol. Hung. 34, 81-92. Sedlak I. and Lindsay R. H. (1968) Estimation of total protein-bond and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192-205. Vinikour W. S.. Goldstein R. M. and Anderson R. V. (19801 Bioconcentration patterns of zinc, copper, cadmium anh lead in selected fish species from the Fox river, Illinois. Bull. environ. Contam. Toxicol. 24, 727-734.
REFERENCES
50ppm
17.93 f I .03
0.25 f 0.03
Gill
Ml&e
0.19 f 0.01 P
12.14 + 2.21 P
21.71 f 3.96 NS
lOppm
0.3 + IO_’ + 0.00
0.8 x IO_’ + 0.1 x 10-2
3 x 10-z * 0.1 x 10-Z
37.70 f I .02
29.02 f 0.92
Gill
White muscle
mg/g w.t.w. 10 PPm
36.47 f 2.74 P cO.Ol%
33.81 f 2.45 P
61.31 f 1.55 NS
Protein
32.24 f 1.82
167.98 + 8.78
f SD.
37. I8 + 3.97 P
33.79 * I.91 P
56.06 k 4.24 P< I%
100 pmm
4.23 f0.11
23.79 i I .08
35.86 k I .33
30.02 k 3.49 P
40.18 & 3.86 NS
36.85 + 1.63 NS
carp
3.48 k 0.34 P < 0.01
21.65 k 2.48 P < 0.01
94.91 f 17.97 P
lOppm
46.42 + 4.10 P
34.122 f 2.27 NS
35.35 f 3.46 NS
2.45 * 0.39 P < 0.01
10.85 5 0.67 P
206.29 + 16.97 P
100 PPm
U/g w.t.w
in the three
12.29 f 0.78
126.58 k 7.60 -
0
LP nM MDA/g w.t.w. 10 PPm 100 PPm
35.78 f 3.08
0
36.17 f I.21 P < 0.01
24.11 3.34 P < 0.01
1420.27 * 114.11 P
100 PPm
Mn-SOD
in the common
and lipid peroxidation
40.79 f 4.64 P < 0.01
91.31 7.9 I P < 0.01
1031.88 * 31.14 -
lOppm
of Z&O, on the protein content organs in common carp
The given values are the results for 4 fish, means NS = not significant.
62.44 f I .20
0
Liver
Organs
0.3 x 10-2 f 0.03 x 10-2 P
1159.97 f 102.98
0
in three organs
U/g w.t.w
enzymes
Total-SOD
on antioxidant
2.8 x 10-2 f 0.2 f IO_’ NS
‘00 PPm
of ZnSO,
0.3 x 10-2 0.1 x 10-2 * 0.01 x IO * &O.Ol x IO * NS P < 0.01
0.6 x IO * f 0.00 NS
2.9 x IO-’ + 0.1 x 10-Z NS
IOPPm
Bu/g w.t.w,
Table 4. Effects of two concentrations
f SD.
0.003 * 0.001 P < 0.01
2.61 kO.19 P < 0.01
51.49 k 3.29 P c 0.01
e
Case
Table 3. Effects of two concentrations
1OOppm
U/g w.t.w.
The given values are the means of 4 fishes NS = not significant.
21.67 f 1.06
Liver
0
GP-ase
28.01 f 1.93
149.51 f 18.04
1033.39 + 110.46 -
0
31.32 & 4.97 P
69.66 + 5.59 P < 0.01
936.91 f 26.34 NS
IOppm
33.12 f 1.43 P < 0.01
13.26 * 3.47 P
1188.94 * 120.06 NS
100 ppm
Cu Zn-SOD U/g w.t.w.