ECOTOXICOLOGY
AND
ENVIRONMENTAL
23,39-45 ( 1992)
SAFETY
The Effect of Pesticides on Carp (Cyprinus carpio L). Acetylcholinesterase and Its Biochemical Characterization A. SZAB~,* J. NEMCS~K,* B. ASZTALOS,* Z. RAKONCZAY,~ P. K.&A,?
AND LE Huu HIEU*
*Department of Biochemistry, J&sef Attila University, P.O. Box 533, 6701 Szeged, Hungary; tCentra1 Research Laboratory, Szent-Gyiirgyi Albert University School qf Medicine. Somogyi B. u. 4, 6720 Szeged* Hungary Received
March
and
5, 1990
The activity and molecular forms of acetylcholinesterase (AChE) were characterized in tissues of the carp (Cyprinus carpio). Tissue AChE activity was determined in response to specific inhibitors (ethopropazine, BW 284 C5 1) or pesticides (CuS04, paraquat (PQ), methidathion (MD)). The highest AChE activity was found in the serum (878 + 100 U/liter), followed by the brain (113 + 12 U/liter), heart (89 f 6 U/liter), and trunk muscle (35 + 5 U/liter). Experiments with specific choline esteraseinhibitors revealed a very low amount of pseudocholinesterase in all tissuesstudied. The ratio of the membrane-bound to the cytoplasmic-free AChE molecular forms was increased in the order of brain, trunk muscle, and heart. In sera of fish treated with MD (2 ppm) there was an 80% inhibition of AChE lasting for 2 weeks. Treatment with CuS04 or PQ (both 5 ppm) led to a 50% decrease in the serum AChE activity followed by a transient increase over the control level. After 2 weeks of chronic treatment, AChE activity in fish exposed to CuS04 returned to the control level, whereas in fish treated with PQ an elevated level (130% when compared to the control level) of enzyme activity was found. Our present experimental data indicate that pesticides occurring in natural waters not only inhibit AChE activity in fish but may influence the resynthesis of the enzyme as well. 0 1992 Academic Prw, Inc.
INTRODUCTION Molecular forms of acetylcholinesterase (AChE) (EC 3.1.1.7) occurring in various organs of fish have been extensively studied (Augustinsson, 1959a, 1959b; Gaal et al., 1980; Nemcsok et al., 1990). Whereas several biochemical parameters of AChE were described in these papers, the toxic effects of chemical pollutants on AChE activity in fish have not been fully revealed. Therefore, in our present study biochemical and pharmacological characterization of AChE activity in tissues of carp, the most populous teleostean species in Hungary, was performed. First, the activity and molecular forms of choline esterases in various organs of carp were determined. Second, efforts were taken to study the effects of the fungicide CuS04, the herbicide paraquat (PQ) (l,l’dimethyl-4,4’-bipyridinium), and the insecticide methidathion (MD), (O,O’dimethyl4[(2-methoxy-1,3,4-thiadiazole-5(4H)-one-4-yl)methyl] dithiophosphate) on the carp AChE under in viva conditions. Fish were exposed to combinations of these compounds to explore the possible synergism or antagonism between the various chemical pollutants which may occur in natural waters. MATERIALS
AND
METHODS
Biochemical Characterization of Carp AChE Brain, trunk muscle, heart, and blood tissue samples from carp (Cyprinus curpio L.) weighing 850 to 1000 g (n = 6-10) were homogenized in 5% (v/v) Triton X-100 39
0147-6513/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
40
SZAB6
ET AL.
containing 0.62% (m/v) NaCl solution and 0.4 M sodium phosphate buffer (PI-I 7.2) and then centrifuged at 6000 rpm for 20 min. Molecular forms of AChE, i.e., the ratio of “soluble”. to membrane-bound fractions, were determined as described previously (Rakonczay et al., 198 1). The activity of AChE was determined by the Ellman method (Ellman and Courtney, 196 1) using 20- to 50-~1 aliquots of the supematants. Activities of the two major forms of cholineesterases, pseudocholinesterase (butyrylcholineesterase) and acetylcholinesterase, were specifically inhibited by ethopropazine and BW 284 C5 1, respectively. The Efect of Pesticides on Carp AChE Activity in Vivo Carp (Cyprinus curpio L.) of both sexes weighing 1000-2000 g were used in the static aquarium experiments. The Iish were kept in 02-saturated water in 100~liter aquaria at 10 + 1“C and exposed to CuS04, paraquat or methidation by themselves or in combination with one another for 1, 4, and 6 days or 2 weeks. Final pesticide concentrations in the aquaria were as follows: CuS04, 5 ppm; PQ, 5 ppm, and MD, 2 ppm, or halves of these concentrations when effects of more than one pesticide were tested. Blood samples were taken from both the control (untreated) and the treated fish and centrifuged, and AChE activities were measured from the nonhemolyzed sera. Results are expressed as percentages of the pretreatment values. The reported data are the means + SD. of the results for samples from 6-18 fishes. RESULTS Biochemical
Characterization
AND
DISCUSSION
of Carp AChE
The activities of AChE in the carp brain, heart, and trunk muscle were 113 f 12, 89 f 6, and 35 f 5 U/liter, respectively. The ratio of the membrane-bound to the cytoplasmic-free AChE molecular forms was increased in the order of brain, trunk muscle, and heart (Fig. 1). These findings along with previous ones (Serebrenikova et al., 197 1; Nemcsok et al., 1990) provide further evidence for the different distribution pattern of the AChE molecular forms in organs of teleosts.
I
100
0
Soluble Membrane bound I
60
60 # 40
20
0
Brain
Muscle
HlXt
FIG. 1. The distribution of soluble and membrane-bound AChE in organs of carp. The values are averages + S.D. of 6 to 10 individual measurements.
PESTICIDE
TOXICITY
41
TO CARP AChE
TABLE
1
THE EFFECTS OF BW 254 CS 1 AND ETHOPROPAZINE ON THE AChE ACTIVITY (“70) IN ORGANS OF CARP Inhibitor (mol/liter) BW 254 CS 1 10-s 5 x 10-s 10-7 5 x 10-7 10-6 Ethopropazin 10-5 5 x 10-S 10-4 5 x 10-4 10-3
Brain
Muscle
67 -+- 1 44 f 3 26 f 5 9+3 N.D.
loo-+ 93* 69+ 43+21 26+
97 79 49 28 19
f 1 * 3 f 9 I!Z7 + 5
Plasma
Heart 7 11 9 17
62 +- 34 43 -+ 38 21 t20 9+ 10 4+ 7
65-+ 38? 21-t 9* l&
9 9 9 8
N.D. 78f 4 58 t 13 42 + 30 N.D.
N.D. 845 1 702 7 58 k 11 47+ 16
N.D. 86+ 75-t 61? 47f
6 4 6 5 1
Note. The results are expressed as means f S.D. of 6-10 measurements. Values are expressed as % of control. N.D. stands for not detected.
The presence of pseudocholinesterase (butyrylcholineesterase) in the carp brain, heart, trunk muscle, and serum were tested for by the use of specific cholineesterase inhibitors, BW 284 C51 and ethopropazine (Table 1). Half-maximal inhibition of AChE by BW 284 C5 1 was found at the concentration of 1O-7 to lo-* Min all tissues studied. Ethopropazine, known to be specific for the pseudocholinesterases, had its half-maximal inhibition at fourfold higher concentrations (Table 2). Early studies on fish cholineesterases (Augustinsson, 1959a, 1959b) reported no pseudocholinesterase activity in the brain, liver, air bladder, and trunk muscle. In contrast, others (Clos and Serfaty, 1957, 1958; Gahl et al., 1980) have shown the presence of pseudocholinesterase in fish trunk muscle and blood. Our present results show that an insignificant amount of pseudocholinesterase is present in the fish organs. This is in harmony with
TABLE
2
HALF-MAXIMAL CONCENTRATIONS FOR BW 254 C5 1 AND ETHOPROPAZINE IN INHIBITION OF AChE ACTIVITY IN ORGANS OF CARP ICsO (mol/liter) Organ Brain Skeletal muscle Heart Serum
BW 254 CS 1 3.0 3.8 2.0 3.0
x x x x
10-s 10-7 10-8 10-s
Ethopropazine 1.3 8.6 2.3 8.0
Note. Data are means + S.D. of results for 6-10 animals.
x x x x
10-4 10-4 1O-4 1O-4
42
SZAB6 ET AL.
200
&ST
-
150-
100
-
50 -
0
I
I
I
, 16
0
FIG. 2. The effects of 2 ppm MD, 5 ppm CuS04, or 1 ppm MD plus 2.5 ppm CuSO, on the serum AChE activity of carp. Results are expressed as percentages of the pretreatment (control). Data are the means + S.D. of the results for 6 to 18 individuals.
the findings of Pecot-Dechavissine esterase activity in fish muscle.
(1962) which revealed a 1.4 to 2.6% pseudocholin-
The E$^ct of Pesticideson Carp AChE Activity in Vivo Administration of 2 ppm MD to the aquaria led to an 80% decrease in the AChE activity of carp serum. This was maintained during the entire 2-week-long experimental period. CuS04, a potent fungicide, seemed to decrease the AChE inhibition evoked by MD. However, CuS04 by itself exerted a biphasic pattern effect on the AChE activity. After a transient decrease in the first day, there was a 71% increase in the
200
$2
-
150
AND
ENVIRONMENTAL
23,39-45 ( 1992)
SAFETY
The Effect of Pesticides on Carp (Cyprinus carpio L). Acetylcholinesterase and Its Biochemical Characterization A. SZAB~,* J. NEMCS~K,* B. ASZTALOS,* Z. RAKONCZAY,~ P. K.&A,?
AND LE Huu HIEU*
*Department of Biochemistry, J&sef Attila University, P.O. Box 533, 6701 Szeged, Hungary; tCentra1 Research Laboratory, Szent-Gyiirgyi Albert University School qf Medicine. Somogyi B. u. 4, 6720 Szeged* Hungary Received
March
and
5, 1990
The activity and molecular forms of acetylcholinesterase (AChE) were characterized in tissues of the carp (Cyprinus carpio). Tissue AChE activity was determined in response to specific inhibitors (ethopropazine, BW 284 C5 1) or pesticides (CuS04, paraquat (PQ), methidathion (MD)). The highest AChE activity was found in the serum (878 + 100 U/liter), followed by the brain (113 + 12 U/liter), heart (89 f 6 U/liter), and trunk muscle (35 + 5 U/liter). Experiments with specific choline esteraseinhibitors revealed a very low amount of pseudocholinesterase in all tissuesstudied. The ratio of the membrane-bound to the cytoplasmic-free AChE molecular forms was increased in the order of brain, trunk muscle, and heart. In sera of fish treated with MD (2 ppm) there was an 80% inhibition of AChE lasting for 2 weeks. Treatment with CuS04 or PQ (both 5 ppm) led to a 50% decrease in the serum AChE activity followed by a transient increase over the control level. After 2 weeks of chronic treatment, AChE activity in fish exposed to CuS04 returned to the control level, whereas in fish treated with PQ an elevated level (130% when compared to the control level) of enzyme activity was found. Our present experimental data indicate that pesticides occurring in natural waters not only inhibit AChE activity in fish but may influence the resynthesis of the enzyme as well. 0 1992 Academic Prw, Inc.
INTRODUCTION Molecular forms of acetylcholinesterase (AChE) (EC 3.1.1.7) occurring in various organs of fish have been extensively studied (Augustinsson, 1959a, 1959b; Gaal et al., 1980; Nemcsok et al., 1990). Whereas several biochemical parameters of AChE were described in these papers, the toxic effects of chemical pollutants on AChE activity in fish have not been fully revealed. Therefore, in our present study biochemical and pharmacological characterization of AChE activity in tissues of carp, the most populous teleostean species in Hungary, was performed. First, the activity and molecular forms of choline esterases in various organs of carp were determined. Second, efforts were taken to study the effects of the fungicide CuS04, the herbicide paraquat (PQ) (l,l’dimethyl-4,4’-bipyridinium), and the insecticide methidathion (MD), (O,O’dimethyl4[(2-methoxy-1,3,4-thiadiazole-5(4H)-one-4-yl)methyl] dithiophosphate) on the carp AChE under in viva conditions. Fish were exposed to combinations of these compounds to explore the possible synergism or antagonism between the various chemical pollutants which may occur in natural waters. MATERIALS
AND
METHODS
Biochemical Characterization of Carp AChE Brain, trunk muscle, heart, and blood tissue samples from carp (Cyprinus curpio L.) weighing 850 to 1000 g (n = 6-10) were homogenized in 5% (v/v) Triton X-100 39
0147-6513/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
40
SZAB6
ET AL.
containing 0.62% (m/v) NaCl solution and 0.4 M sodium phosphate buffer (PI-I 7.2) and then centrifuged at 6000 rpm for 20 min. Molecular forms of AChE, i.e., the ratio of “soluble”. to membrane-bound fractions, were determined as described previously (Rakonczay et al., 198 1). The activity of AChE was determined by the Ellman method (Ellman and Courtney, 196 1) using 20- to 50-~1 aliquots of the supematants. Activities of the two major forms of cholineesterases, pseudocholinesterase (butyrylcholineesterase) and acetylcholinesterase, were specifically inhibited by ethopropazine and BW 284 C5 1, respectively. The Efect of Pesticides on Carp AChE Activity in Vivo Carp (Cyprinus curpio L.) of both sexes weighing 1000-2000 g were used in the static aquarium experiments. The Iish were kept in 02-saturated water in 100~liter aquaria at 10 + 1“C and exposed to CuS04, paraquat or methidation by themselves or in combination with one another for 1, 4, and 6 days or 2 weeks. Final pesticide concentrations in the aquaria were as follows: CuS04, 5 ppm; PQ, 5 ppm, and MD, 2 ppm, or halves of these concentrations when effects of more than one pesticide were tested. Blood samples were taken from both the control (untreated) and the treated fish and centrifuged, and AChE activities were measured from the nonhemolyzed sera. Results are expressed as percentages of the pretreatment values. The reported data are the means + SD. of the results for samples from 6-18 fishes. RESULTS Biochemical
Characterization
AND
DISCUSSION
of Carp AChE
The activities of AChE in the carp brain, heart, and trunk muscle were 113 f 12, 89 f 6, and 35 f 5 U/liter, respectively. The ratio of the membrane-bound to the cytoplasmic-free AChE molecular forms was increased in the order of brain, trunk muscle, and heart (Fig. 1). These findings along with previous ones (Serebrenikova et al., 197 1; Nemcsok et al., 1990) provide further evidence for the different distribution pattern of the AChE molecular forms in organs of teleosts.
I
100
0
Soluble Membrane bound I
60
60 # 40
20
0
Brain
Muscle
HlXt
FIG. 1. The distribution of soluble and membrane-bound AChE in organs of carp. The values are averages + S.D. of 6 to 10 individual measurements.
PESTICIDE
TOXICITY
41
TO CARP AChE
TABLE
1
THE EFFECTS OF BW 254 CS 1 AND ETHOPROPAZINE ON THE AChE ACTIVITY (“70) IN ORGANS OF CARP Inhibitor (mol/liter) BW 254 CS 1 10-s 5 x 10-s 10-7 5 x 10-7 10-6 Ethopropazin 10-5 5 x 10-S 10-4 5 x 10-4 10-3
Brain
Muscle
67 -+- 1 44 f 3 26 f 5 9+3 N.D.
loo-+ 93* 69+ 43+21 26+
97 79 49 28 19
f 1 * 3 f 9 I!Z7 + 5
Plasma
Heart 7 11 9 17
62 +- 34 43 -+ 38 21 t20 9+ 10 4+ 7
65-+ 38? 21-t 9* l&
9 9 9 8
N.D. 78f 4 58 t 13 42 + 30 N.D.
N.D. 845 1 702 7 58 k 11 47+ 16
N.D. 86+ 75-t 61? 47f
6 4 6 5 1
Note. The results are expressed as means f S.D. of 6-10 measurements. Values are expressed as % of control. N.D. stands for not detected.
The presence of pseudocholinesterase (butyrylcholineesterase) in the carp brain, heart, trunk muscle, and serum were tested for by the use of specific cholineesterase inhibitors, BW 284 C51 and ethopropazine (Table 1). Half-maximal inhibition of AChE by BW 284 C5 1 was found at the concentration of 1O-7 to lo-* Min all tissues studied. Ethopropazine, known to be specific for the pseudocholinesterases, had its half-maximal inhibition at fourfold higher concentrations (Table 2). Early studies on fish cholineesterases (Augustinsson, 1959a, 1959b) reported no pseudocholinesterase activity in the brain, liver, air bladder, and trunk muscle. In contrast, others (Clos and Serfaty, 1957, 1958; Gahl et al., 1980) have shown the presence of pseudocholinesterase in fish trunk muscle and blood. Our present results show that an insignificant amount of pseudocholinesterase is present in the fish organs. This is in harmony with
TABLE
2
HALF-MAXIMAL CONCENTRATIONS FOR BW 254 C5 1 AND ETHOPROPAZINE IN INHIBITION OF AChE ACTIVITY IN ORGANS OF CARP ICsO (mol/liter) Organ Brain Skeletal muscle Heart Serum
BW 254 CS 1 3.0 3.8 2.0 3.0
x x x x
10-s 10-7 10-8 10-s
Ethopropazine 1.3 8.6 2.3 8.0
Note. Data are means + S.D. of results for 6-10 animals.
x x x x
10-4 10-4 1O-4 1O-4
42
SZAB6 ET AL.
200
&ST
-
150-
100
-
50 -
0
I
I
I
, 16
0
FIG. 2. The effects of 2 ppm MD, 5 ppm CuS04, or 1 ppm MD plus 2.5 ppm CuSO, on the serum AChE activity of carp. Results are expressed as percentages of the pretreatment (control). Data are the means + S.D. of the results for 6 to 18 individuals.
the findings of Pecot-Dechavissine esterase activity in fish muscle.
(1962) which revealed a 1.4 to 2.6% pseudocholin-
The E$^ct of Pesticideson Carp AChE Activity in Vivo Administration of 2 ppm MD to the aquaria led to an 80% decrease in the AChE activity of carp serum. This was maintained during the entire 2-week-long experimental period. CuS04, a potent fungicide, seemed to decrease the AChE inhibition evoked by MD. However, CuS04 by itself exerted a biphasic pattern effect on the AChE activity. After a transient decrease in the first day, there was a 71% increase in the
200
$2
-
150
