IN VIVO EFFECTS OF DELTAMETHRIN ON SOME BIOCHEMICAL PARAMETERS OF CARP (CYPRINUS CARPIO L.) T. SZEGLETES~* CS. POLYHOS, T. BALINT, A. A. RADY, G. L./~NG, O. K U F C S A K and J. N E M C S O K
Department of Biochemistry, Attila J6zsef University, P.O. Box 533, H-6701, Szeged, Hungary. (Received: September 1993; revised: September 1994)
Abstract. The in vivo effects of deltamethrin (DM) on the blood sugar level, the acetylcholinesterase (ACHE, EC 3.1.1.7) activities of the blood serum and various organs (heart, liver and intestine), the lactate dehydrogenase (LDH, EC 1.1.2.3), glutamic-oxaloacetic transaminase (GOT, EC 2.6.1.1), and glutamic-pyruvic transaminase (GPT, EC 2.6.1.2) activities of the blood serum, the adenosine triphosphatases (EC 3.6.1.3; Na +/K + -ATPase and Mg2+-ATPase) activities of the erythrocyte plasma membrane and the catalase (EC 1.11.1.6) activity of the liver were examined throughout 96 h in adult carp (Cyprinus carpio L.) Two sublethal concentrations, 1.0 and 1.5 #g/1 of deltamethrin, were used. All fish survived the experiment except one, in an aquarium containing 1.5 ppb of DM, which died after 72 h. The AChE specific activity was significantly inhibited in the heart and intestine after 96 h at both concentrations compared to that in the control animals (P < 0.05, Student's t-test), while there was no detectable difference between the two treatment. At the same time there was no detectable change in the liver. In the serum, the AChE activity almost remained unchanged; the only significant decrease could be measured after 96 h at 1.5 #g/1 deltamethrin concentration. The blood glucose content exhibited interesting changes: after 24 h fish exposed at 1 #g/1 DM seemed to be stressed, although this increase was not significant. When these fish became used to the new conditions (in practice this meant the presence of DM), the glucose level decreased, especially after 72 h. At the same time the control animals kept in similar circumstances showed a small insignificantdecrease. Meanwhile fish in aquaria containing 1.5/~g/1 DM reacted to the treatment with an increased blood glucose level after 48 h, and this did not change until the end of the treatment. The Na+/K +-ATPase activity decreased in a dose-dependant manner, while Mg2+-ATPase was less affected. A small increase in LDH level was observed, indicating damage of different muscle tissues. However, this phenomenon appeared only with the small dosage after 24 h (P < 0.05). It has to be mentioned that the individual values varied to a large extent among of the eight fish. The GOT activities of the serum increased during the treatment. However, significant changes were only expressed after 72 and 96 h at 1 #g/1 DM concentrations (P < 0.01 and P < 0.05), and after a similar long treatment at the high dosage (P < 0.05, 72 and 96 h). The GPT did not change significantly in aquaria containing 1 /~g/1DM. The only larger increase was measured after 96 h at 1.5 #g/1 DM concentration (P < 0.05). The catalase activity in the liver of treated carp remained practically at the same level compared to that in control fish. All these changes (concerning the primary effects of this compound) demonstrate the effect of DM on different fish enzymes, at low concentrations under laboratory conditions, which might be useful in practice for biomonitoring using fish.
1. Introduction
Deltamethrin (or decamethrin, DM; type II pyrethroid; (S)-c~-cyano-3-phenoxy-
benzyl-(1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-carboxylate, * To whom all correspondence should be addressed.
Environmental Monitoring and Assessment 35:97-111, 1995. (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands.
98
T. SZEGLETES ET AL
H3C CH3
B
c:ci
B/r
/k ,c
\.
o
N-C-CHScheme 1 Formula ofdeltamethrin.
C22HI9Br2NO3; Scheme 1) is considered to be among the safest classes of insecticides available. However, under cleanwater laboratory conditions, exposure of this pyrethroid to fish seem to be toxic. The 96 h LCs0 values range between 0.4 and 2.0 #g/1 (L'Hotellier and Vincent, 1986). Extensive field studies, in experimental ponds, have shown that this high potential toxicity is not realized (World Health Organization, 1990). There is no significant difference between the acute toxicities to cold and warm water species of fish (Stephenson, 1982). It seems that DM affect fish at both high and low temperatures, although a negative temperature coefficient has been claimed for certain pyrethroids (Kumaragum and Beamish, 1981). Furthermore, the toxicity of pyrethroids (including DM) for fish is not influenced by the hardness or pH of the water (Mauck et al., 1976). DM has been observed to cause neuromuscular dysfunctions in organisms (Rose and Dewar, 1983). Its primary effect has been attributed to slowing the sodium channels of nerve cells. GABA receptors appear to be inhibited as well. Additionally, there are many secondary effects on the whole organism. According to Rose and Dewar (1983), DM has at least two distinct actions - a short term pharmacological effect at near-lethal dose levels and a more long-term neurotoxic effects that results in sparse axonal nerve damage. In the literature one can find many extensive references to the secondary effects of DM on various parameters, such as ACHE, ATPases (Shaker et al., 1988), noradrenaline and adrenaline contents in rats (de Boer et al., 1988), choline transport (Matsumura, 1988), induction of chromosome aberrations in mice (Bhunya and Pati, 1990), and the subpopulations of muscarinic and nicotinic ACh receptors (Eriksson and Nordberg, 1990). However, few investigations have been made on the mechanism of acute toxicity on fish (L'Hotellier and Vincent, 1986; Zitko et al., 1979). The present study involves the biochemical characterization of some enzymes in specific tissues of carp, the most populous teleostean species in Hungary. The
BIOCHEMICAL PARAMETERS OF CARP
99
blood serum activities of ACHE, LDH, GOT and GPT and blood glucose levels were determined in control and treated animals. The changes in these enzyme activities have been used to demonstrate tissue damage in fish (Wrobelski and La Due, 1955; Kristoffersson et al., 1974; Nemcs6k and Boross, 1982; Asztalos and Nemcs6k, 1985). Similarly, the increase in blood glucose level has been used to demonstrate metabolic stress (Wedemeyer, 1970; Nemcs6k and Boross, 1982). Changes in the AChE activity of various organs, the catalase activity of the liver and the ATPase activities of the erythrocyte membrane after DM treatment were also studied. Our study was initiated by a massive devastation of the fish population in Lake Balaton, Hungary; although most of those which died were common eels (Anguilla anguilla). Brain and liver from these fish contained residues of DM at 0.02 mg/1000 g wet tissue concentration (Gt~nczy, 1992). G6nczy suspected that one of the causes of the massive fish kill could be the presence of DM in fish. This study was conducted to evaluate the in vivo effect of two sublethal concentrations of DM on several enzymatic pathways, in four tissues of carp under cleanwater laboratory conditions at 25 °C, imitating summer water temperature.
2. Materials and Methods
Common carp (Cyprinus carpio L.) of both sexes, weighing 800-1000 g, were used in the experiments. Fish were kept individually in O2-saturated water at 25 ± 1 °C in a 100 1 aquarium. The animals were not fed during the experiments. The DM concentration in the aquarium was 1 or 1.5 #g/1. The duration of exposure was 96 h. Samples of carp blood were taken from the tail vien of all fish every 24 h. The blood was centrifuged at 4 °C and the ACHE, GOT, GPT and LDH activities and the blood glucose level was measured in a heamolysis-free serum. The pellet was used to measure the ATPase activities. The values are expressed as the average of 6-8 individuals (+ S.E.M.). The differences between the data of control and treated animals were analysed by Student's t-test. The AChE and catalase activities in the organs (heart, liver and midgut) were measured by removing the respective organ after 96 h, homogenizing it in 0.65% NaCI containing 0.5% Triton X-100 and 12.5 mM sodium phosphate with a Braun 853302/4 homogenizer (to give a final concentration of 200 mg wet weight of organ per ml of homogenate), followed by centrifugation and activity measurement in the supematant. AChE was determined with acetylthiocholine as substrate using the method of Ellman et al. (1961). The GOT and GPT activities were assayed according to Reitman and Frankel (1957), and the LDH activity according to Annon (1971), with Reanal kits (Hungary). The catalase activity in the supernatant of the liver homogenate was measured spectrophotometrically by monitoring the decrease of H202. The reaction mixture was 3.0 ml phosphate buffer [50 mM, pH 7.0; containing 1.2 #1 of 30% H202
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T. SZEGLETES ET AL
per 1.0 ml buffer (Reanal, Hungary)] + 0.025 ml supernatant. Absorbance was measured at 240 nm. The protein content of all samples was determined by the method of Lowry et al. (1951). Enzyme activities are given in mU mg -1 protein (1 U = 1 #mol substrate min- ~). Blood sugar level was determined by the glucose oxidase-peroxidase method. The reaction mixture was 2.5 ml glucose reagent [2 ml peroxidase (Reanal, Hungary) + 12.5 mg glucose oxidase (Reanal, Hungary) diluted in 50 ml 0.1 M phosphate buffer (pH 7.0) + 3.3 mg o-dianisidine (Reanal, Hungary) diluted in 1.0 ml distiled water] + 0.1 ml deproteinized blood serum. After a 35-min incubation at room temperature, the absorbance was measured at 450 nm. Erythrocyte plasma membrane was prepared according to Sorensen (1983). Erythrocytes were collected by centrifugation and washed with 0.16 M NaC1. After washing, the cells were resuspended in 0.15 M NaC1, 3 mM MgC12 and 0.01 M Tris-HC1, pH 7.2, and homogenized in a Virtis 45 homogenizer fitted with knives. Nuclei and unbroken cells were removed from the homogenate by low-speed differential centrifugation (500-1200 g for 5 min). The broken plasma membranes were collected from the supernatant by centrifugation at 25 000 g for 15 min. The membranes were then washed several times with 0.1 M choline chloride, 0.01 M Tris-HC1, pH 7.2. Finally, part of the washed membranes was resuspended in a small volume of washing medium and stored at -60 °C for the measurement of Na+/K+-ATPase activity and total protein. Proteins were again determined using the method of Lowry et al. (1951). The ATPase activity was measured in a basic incubation medium containing 40 mM Tris-HC1, pH 7.2, 6 mM MgC12, 20 mM KC1 and 150 mM NaC1 in certain cases with the addition of the inhibitor oubain (0.7 mM) (Bourre et al., 1982). The reaction was started after a 5-min temperature equilibration by the addition of 4 mM ATP (Tris salt). The incubation was carried out in a shaking bath at 22 °C for 15 min and was stopped by the addition of 1 ml of ice-cold 10% v/v TCA. The precipitated protein was centrifuged and the supernatant was analysed for inorganic phosphate as in the method of Line and Way (1984). The difference between total ATPase and Mg2+-ATPase is considered to be Na+/K +-ATPase. The specific activity of ATPase was determined in units of nmol of Pi/mg protein/h.
3. Results and Discussion
3.1. ACHE The AChE specific activity in the heart and the midgut section was strongly inhibited by DM (50-60% decrease), while in the liver the level of this enzyme did not change. This could be observed after 96 h of treatment (Figure 1a). After 24, 48 and 72 h the blood serum AChE activity remained at a level similar to that in
BIOCHEMICALPARAMETERSOFCARP
101
mU/mg protein 120 100 8060-
[ ] Control [ ] 1.0/~g/l DM [ ] 1.5/~g/! DM [
iI !l
4020-
0 Heart
Liver
Intestine
Fig. la.
the control fish. After 96 h of treatment in 1.5 #g/l dose a significant (P < 0.05) decrease (27% decrease compared to that in the control fish) was observed. No significant difference was detected between the two applied doses (Figure lb). The LCs0 of DM is 1.84 #g/1 (96 h) for carp (L'Hotellier and Vincent, 1986). We observed a similar lethal concentration, although in one of our preliminary experiments a lower DM concentration killed all of the studied fish. It is well known that a-cyanopyrethroids, such as DM, cause a long-lasting prolongation of the normally transient increase in sodium permeability of the nerve membrane during excitation, resulting in long-lasting trains of repetitive impulses in the sense organs and a frequency-dependant depression of the nerve impulses in the nerve fibres. Since the mechanisms responsible for nerve impulse generation and conduction are basically the same throughout the entire nervous system, pyrethroids may well act in a similar way in various parts of the central nervous system. The clinical signs of DM toxicity are tremor and salivation in animals (Kavlock et al., 1979; Ray and Cremer, 1979). In fish (especially at higher dosages of DM), signs of toxicity are irregular movement of the operculum and disturbances in the coordination of swimming. In our study we observed that the surface of the skin/scales became infected after the second day of exposure at both concentrations. This is the connection between the effects of DM on the Na+-channel permeability and on the inhibition of ACHE. This inhibition is not as large as is the case
102
T. SZEGLETES ET AL
mU/mg protein ~-~Control E 1.0 #g/l DM [ ] 1.5 #g/I DM
6.0 -1
5.0
-
4.0
--
3.0' 2.0 1.0
0.0
t
t
-
24
48
72
96
Time of treatment (hrs) Fig. lb. Figs. 1(a)-(b). Effect of deltamethrin treatment on AChE specific activity of heart, liver and intestine (a) and blood serum (b) of carp at a water temperature of 25 4- 1 °C. Heart, liver and intestine were taken out from the fish after 96 h treatment. Activities, given in mU mg-~ protein, are the averages ± S.E.M. for 6 to 8 specimens. Note: Values significantly different by Student's t-test from controls are indicated: *P < 0.05. For experimental conditions, see Materials and Methods in text.
with some organophorous esters, nevertheless in fishes pyrethroids are 1000 times more toxic than other insecticides, at least in laboratory aquaria (Nemcs6k et al., 1990). A near to lethal dose of DM produces biochemical changes in the peripheral nerves, consistent with sparse axonal degeneration. Swelling and disintegration of the axons of the sciatic nerve may be observed. This destruction could be the cause of, among others, the decrease in AChE activity in the heart and intestine. These tissues are perhaps more sensitive than the tissue of the liver. AChE inhibition is particularly dangerous in the heart, since the cholinergic system has a decisive role in the innervation of the heart in fish (Pennec and Le Bras, 1984): AChE inhibition potentiates the vagal tone, which may cause adverse effects in the metabolic processes related to circulation. In this case, inhibition of the heart function interferes with the uptake of 02 and the release of CO2 at the gills, which may result in hypoxia at the tissue level (Hughes, 1976).
103
B I O C H E M I C A L P A R A M E T E R S OF CARP
TABLE I Effect of deltamethrin treatment on plasma GOT specific activity of carp at a water temperature of 25 4- 1 °C. Activities are expressed as x 10 - 4 U mg-1 protein (1 U: l#mol substrate rain-l).
1 #g/1DM 1.5 #g/1DM Control
Exposure time (h) 24 48
72
96
4.60 4- 2.69 8.594- 4.56 2.56 4- 1.64
9.07 4- 2.83** 11.204- 6.84* 3.68 4- 1.85
10.80 -4- 5.62* 13.10 4- 5.25* 3.75 4- 2.51
8.68 4- 1.56 10.404- 4.99 4.43 4- 2.95
Note: Data are means 4- S.E.M. of results for 6 to 8 specimens. Values significantly different by Student's t-test from controls are indicated: *P < 0.05; **P < 0.01. For experimental conditions, see Materials and Methods in text.
TABLE II Effect of deltamethrin treatment on plasma GPT specific activity of carp at a water temperature of 25 4- 1 o C. Activities are expressed as x l 0 - 4 U mg-I protein. Exposure time (h) 24 48 1 #g/1 DM 1.5#g/1DM Control
72
96
2.72 4- 0.43 3.47-t- 1.02 3.08+ 0.72 3.93 4- 1.68 2.644- 1.86 4.59-4- 1.54 5.094- 1.71 5.574- 0.90* 2.35 4- 1.31 2.81 4- 1.54 3.63 4- 1.74 3.60 4- 2.47
Note: Data are means 4- S.E.M. of results for 6 to 8 specimens. Values significantly different by Student's t-test from controls are indicated: *P < 0.05. For experimental conditions, see Materials and Methods in text.
3.2.
G O T AND G P T
G O T activities in the blood serum were significantly higher than those in control animals after 72 and 96 h at both concentrations (72 h 1 #g/l: P < 0.01; 72 h 1.5 #g/1 and 96 h 1 and 1.5 #g/l: P < 0.05; Table I). G P T did not change significantly at the low dosage, and even in the high dosage after shorter treatment. The only significant increase was observed after 96 h at 1.5 #g/1 D M level (P < 0.05; Table II). The d a m a g e to tissue containing considerable amounts o f G O T and G P T is easily detectable through the increasing activities o f these enzymes in the blood serum (Karmen et al., 1955). The distributions of G O T and G P T vary in the different organs ( N e m c s 6 k et al., 1981), and the ratio of the activities o f the two e n z y m e s permits more exact conclusions of the injured tissues. Carp liver contains more G P T and less GOT.
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T. SZEGLETES ET AL
U/mg protein 20-
T
1612_
4 0 Control
1.0/ g/l DM
1.5/ g/l DM
Fig. 2. Effect of deltamethrin exposure on catalase specific activity in liver of carp at a water temperature of 25 4- 1 °C after 96 h treatment. Activities, given in U mg-1 protein, are the averages 4- S.E.M. for 6 to 8 specimens. Note: For experimental conditions, see Materials and Methods in text.
The elevated GOT and the lesser increased GPT activities observed from our studies were presumably due to liver damage. However, other organs may also have been damaged (e.g. the kidney or/and the gill). In the case of GOT, a dosedependant trend cannot be clearly concluded: if there is a significant increase at all, the GOT values in carps treated with the two concentrations of DM are similar. Time-related, there is a small increase, but the only significant change is between 24 and 96 h at the low dosage (Table I). In the case of GPT, the only significant time-dependant difference is between 24 and 96 h at the high dosage (P < 0.05). The activity of GPT already increased after 48 h, but due to its high standard error, this change is not significant. 1 #g/1 DM exposure did not cause significant time-dependant differences, although a small (but still insignificant) increase was marked by the values (Table II). 3.3. CATALASE The catalase activity did not change significantly, only a small increase can be suspected from our data relative to the level in the control carp (Figure 2). During the metabolism of DM in fish, in contrast with other animals (mice and quail),
BIOCHEMICAL PARAMETERSOF CARP
105
mg/100 ml serum I ~---]Control ~ 1.0/tg/l DM ~ 1.5/tg/! DM
180 150 120 -
T
90-
@
60300 24
48
72
96
Time of treatment (hrs) Fig. 3. Effectof deltamethrin exposure on blood glucose level of carp at a water temperature of 25 :t: 1 °C. Concentrations,given in mg glucose/100 ml blood serum, are the averages =kS.E.M. for 6 to 8 specimens.Note: Valuessignificantlydifferentby Student'st-test from controls are indicated: *P < 0.05. For experimentalconditions, see Materials and Methodsin text.
the ester cleavage of this compound is very slow; the main metabolic pathway is hydroxylation to give the 4-hydroxyphenoxy derivative, which is excreted in the bile as the glucuronide (Edwards and Millburn, 1985). The presence of the derivatives is generally connected with the increased activity of enzymes, whose main role is to eliminate the multiplied radicals or derivatives during the metabolism of similar chemicals. The liver ultrastructure shows marked changes as a result from this metabolism pathway. DM increases the number of mitochondria and alters their shape, which become irregular (Catinot et al., 1989). However, in our case the two applied doses did not result in the large expected increase of catalase activity, probably due to the slow metabolism in fish, even at high water temperature (25 °C), and because of the presence of other eliminating enzyme systems. We can conclude that DM-treated fish have different enzymatic levels after 96 h in both serum GOT and GPT activities, at least at 1.5 #g/1 DM concentration compared with those in the control animals. At the same time, we could not reveal similar significant changes in catalase activities in the liver tissue.
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T. SZEGLETESET AL
TABLE III Effect of deltamethrin treatment on plasma LDH specific activity of carp at a water temperature of 25 4- 1 °C. Activities are expressed as U rag-1 protein. Exposure time (h) 24 48
72
96
1/zg/1 DM 6.01 4- 1.66" 6.794- 1.46" 5.494- 3.30 5.904- 2.98 1.5 #g/1DM 3.624- 2.05 4.09 4- 2.97 4.08 4- 2.15 4.524- 1.80 Control 3.54 4- 1.75 3.43 4- 0.98 3.09 4- 0.75 3.304- 1.63 Note: Data are means 4- S.E.M. of results for 6 to 8 specimens. Values signif-
icantly different by Student's t-test from controls are indicated: *P < 0.05. For experimental conditions, see Materials and Methods in text. 3.4. BLOOD SUGAR LEVEL Pyrethroid poisoning in fish produced a syndrome characterized by tremors, abnormal operculum movement (particularly at 1.5 /zg/1 or higher dose), ataxia and hyperactivity. The treated carp were very nervous throughout the experiment, swimming erratically from wall to wall of the aquarium, often striking against it causing intensive internal bleeding and injury to the structure of the epithelium and other tissues. This stressed stage was marked by a higher blood sugar level in the 48 h, 72 h and 96 h samples at 1.5 #g/1 dose (Figure 3). After 24 h of treatment fish kept at 1 #g/1 concentrations seemed to be stressed, but the increase of blood glucose level was not significant. When these fish became acclimatized to the new conditions, the glucose level decreased in the 72 h and 96 h samples compared to the 24 h value (Figure 3). Surprisingly, the fish exposed to 1.5 #g/1 DM reacted later to the treatment (48 h value) with a remaining level throughout the entire experiment. However, the changes in the glucose level of 24 h samples from the fish treated with 1 #g/1 DM were not significant (P > 0.05). It is possible that the higher applied dose caused a longer-lasting effect, while the smaller concentration caused a shorter one. The DM induced 'non-motor' symptoms, i.e. the increases in brain blood flow and blood glucose level, may result from a supraspinal component of DM activity (Bradbury et aI., 1983). Permanent stress includes impaired 7-globulin formation and depressed interferon production, which play important roles in the resistance of fish to various bacterial and viral diseases (Wedemeyer, 1970; Nemcs6k et al., 1982). 3.5. L D H There was a significant increase in LDH activity after 24 h of treatment at the low dose (P < 0.05). However, after 72 h and 96 h of treatment LDH activity decreased to control levels (Table III). No time-dependant difference was observed among
107
BIOCHEMICAL PARAMETERS OF CARP
[ ] 1.S ~tg/l DM nmol Pi/mg protein/hr
1.0 p.g/! DM
12.0 -
[--] Control 10.0 8.0
--
6.0
-
4.0
-
2.0
-
T
i
0.0
24
48
72
96
1
Time of treatment (hrs) Fig. 4a. the parameters at low dose. High dose yielded no significant change over time, except after the 96 h treatment. A slight increase in glucose level was observed after 24 h of treatment at low dose. In fish treated with 1.5 #g/1 DM this increase was detected after 48 h of treatment. In both cases glucose levels seemed to have increased concomitantly with LDH levels. The increased LDH activity could indicate metabolic changes in the stressed fish, primarily in the muscle cells: the catabolism of glycogen and glucose shifted towards the formation of lactate, which can be lethal for fish (Nakono and Tomlinson, 1967; Simon et al., 1983). 3.6. ATPAsES Mg2+-dependant ATPases showed a significant decrease after 48 h of treatment at high dose, while 1 #g/1 DM exposure resulted in a significant difference only after 72 h (Figure 4a). At 96 h both exposures resulted in a 60% decrease in MgZ+-ATPase activity. The Na+/K+-dependant ATPases from the erythrocyte plasma membrane decreased markedly in a dose-dependant manner (Figure 4b).
108
T. SZEGLETESET AL
nmol Pi/mg protein/hr
[ ] 1.5/~g/l DM
8.0 -
@ 1.o 7.0-
DM
D Control
6.05.0-
T
± +
4.0-
+
+ +
3.0-
+
2.01.00.0
24
48
72
96
Time of treatment (hrs) Fig. 4b. Figs. 4(a)-(b). Effect of deltamethrin exposure on Mg 2+ (a) and Na+/K+ (b) dependant ATPases specific activities of erythrocyte plasma membrane of carp at a water temperature of 25 ± 1 °C. Activities, given in nmol of Pi/mg protein/h, are the averages -4- S.E.M. for 6 to 8 specimens. Note: Values significantly different by Student's t-test from controls are indicated: *P < 0.05; +P < 0.005. For experimental conditions, see Materials and Methods in text.
The pyrethroid insecticides have been postulated to involve interactions not only with Na + channels, but also with neurotransmitters, receptor-ionophore complexes and ATPases (Dorman and Beasley, 1991). It has already been reported that ATPase activities depend strongly on pH, temperature and water pressure (Zaugg and McLain, 1976; Gibbs and Somero, 1989), because these environmental parameters may cause changes in composition of the membrane lipids. However, a similar effect may not be assumed in the present experiment. We suspect that D M directly affects ATPases function. This effect could be associated with prolonging the closure of Na + channels and thus changing ion distribution.
BIOCHEMICAL PARAMETERSOF CARP
109
4. Conclusion Our results suggest that doses of DM have significant effect on ATPases. A clear dose-dependant trend could be observed only in the case of Na+/K+-dependant ATPases. AChE decreased in heart and intestine, showing a neuronal dysfunction. In the blood AChE level a small decrease was measured, which implies that only higher dose (> 1.5 #g/l) and longer exposure (> 72 h) is detectable through AChE without killing the fish. The damage in liver (and kidney/gill) can be observed through blood transaminase level; GOT is more suitable for these purposes. Blood glucose and LDH levels in sera also might be useful as a biomonitoring tool, but in our case a dose-dependant trend could not be realized. The massive fish kill in Hungary in 1991 made our studies timely. Residues of DM were detectable in tissues of eel (Anguilla anguilla). Brain and liver from these fish contained residues of DM at 0.02 mg/1000 g wet tissue concentration (G6nczy, 1992). GOnczy suspected that one of the causes of the massive fish kill could be the presence of DM in fish. At the same time we measured, amongst others, a significant AChE decrease in the blood serum and other organs (Szegletes and Nemcs6k, 1992). Obviously, it does not imply that the main reason of the devastation in the fish populations was the use of DM-containing insecticides around the lake. However, it was a point to discuss, that the presence of DM was accompanied by a decreased AChE level in fish. We also made some in vitro experiments with this enzyme, and it showed that DM causes AChE inhibition. Transaminases also increased, and extreme high blood glucose level was detected. The present study shows the possibility of using certain biomarkers to assess chemical effects and their potential hazards in aquatic environment. These results strongly suggest wide-ranging toxic effects of deltamethrin on fish.
References Annon, L.: 1971, 'Photometric Determination of LDH Activity in Blood Serum', Zeitungfar Klinischer Chemistrie und Klinischer Biochemistrie 8, 658. Asztalos, B. and Nemcs6k, J.: 1985, 'Effect of Pesticides on the LDH Activity and Isoenzyme Pattern of Carp (Cyprinus carpio L.) Serum', Comp. Biochem. Physiol. 82C, 217-219. Bhunya, S. P. and Pati, P. C.: 1990 'Effect of Deltamethrin, a Synthetic Pyrethroid, on the Induction of Chromosome Aberrations, Micronuclei and Sperm Abnormalities in Mice', Mutagenesis 5, 229-232. Bourre, J. M., Chanez, C., Dumont, O. and Flexor, M. A.: 1982, 'Alteration of 5-Nucleotidase and Na+.K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (Jumpy, Quaking, Trembler, Shivered and Mid). Comparison with CNPase in the Developing Sciatic Nerve from Trembler', J. Neurochem. 38, 643-649. Bradbury, J. E., Forshaw, P. J., Gray, A. J. and Ray, D. E.: 1983, 'The Action of Mephenesin and Other Agents on the Effects Produced by Two Neurotoxic Pyrethroids in the Intact and Spinal Rat', Neuropharmacol. 22(7), 907-914. Catinot, R., Hoellinger, H., Pfister, A., Sonnier, M. and Simon, M. T.: 1989, 'Effects on Rats of Subacute Intoxication with Deltamethrin via an Osmotic Pump', Drug Chem. Toxicol. 12, 173196.
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de Boer, S. F., van der Gugten, J., Slangen, J. L. and Hijzen, T. H.: 1988, 'Changes in Plasma Corticosterone and Catecholamine Contents Induced by Low Doses of Deltamethrin in Rats', Toxicology 49, 263-270. Dorman, D. C. and Beasley, V. R.: 1991, 'Neurotoxicology of Pyrethrin and the Pyrethroid Insecticides', Veter. Human Toxicol. 33, 238-243. Edwards, R. and Millburn, P.: 1985, 'Toxicity and Metabolism of Cypermethrin in Fish Compared with Other Vertebrates' Pesticide Sci. 16, 201-202. Ellman, G. L., Courtney, D. D., Andres, V. and Featherstone, R. M.: 1961, 'A New and Rapid Colorimetric Determination of AChE Activity', Biochem. Pharmacol. 7, 88-95. Eriksson, P. and Nordberg, A.: 1990, 'Effects of two Pyrethroids, Bioallethrin and Deltamethrin, on Subpopulations of Muscarinic and Nicotinic Receptors in the Neonatal Mouse Brain', Toxicol. Appl. Pharmacol. 102, 456-463. Gibbs, A. and Somero, G. N.: 1989, 'Pressure Adaptation of Na+/K+-ATPase in Gills of Marine Teleost', J. Exper. Biol. 143, 475-492. Gt~nczy, J.: 1992, 'Data and Ideas to Clear the Question of the Massive Fish Kill', Fishing, 1, 21-23. (In Hungarian) Hughes, G, M.: 1976, 'Polluted Fish Respiratory Physiology', in: Lockwood, A. P. M. (Ed.) Effects of Pollutants on Aquatic Organism, Cambridge University Press, London, 121-146. Karmen, A., Wrobelski, F. and La Due, J. A.: 1955, 'Transaminase Activity in Human Blood', J. Clin. Invest. 34, 126. Kavlock, R., Chernoff, N., Baron, R., Linder, R., Rogers, E., Carver, B., Dilley, J. and Simmon, V.: 1979, 'Toxicity Studies with Decamethrin, a Synthetic Pyrethroid Insecticide', J. Environ. Path. Toxicol. 2, 751-765. Kristoffersson, R., Broberg, S., Oskari, A. and Pekkarinen, M.: 1974 'Effect of a Sublethal Concentration of Phenol on Some Blood Plasma Enzyme Activities in the Pike (Esox lucius L.) in Brackish Water', Ann. Zoologic Fennici 11, 220-223. Kumaraguru, A. K. and Beamish, F. W. H.: 1981, 'Lethal Toxicity of Permethrin (NRDC-143) to Rainbow Trout, Salmo gairdneri, in Relation to Body Weight and Temperature', Water Res. 15, 503-505. L'Hotellier, M. and Vincent, P.: 1986, 'Assessment of the Impact of Deltamethrin on Aquatic Species', in British Crop Protection Conference - Pests and Disease, pp. 1109-1116. Line, S. C. and Way, E. L.: 1984, 'Characterization of Calcium-activated and Magnesium-activated ATPase of Brain Nerve Endings', J. Neurochem. 42, 1697-1706. Lowry, O. H., Rosenbrough, N. J., Forr, A. L. and Randall, R. J.: 1951, 'Protein Measurement with the Folin Phenol Reagent', J. Biol. Chem. 193, 269-275. Matsumura, F.: 1988, 'Deltamethrin-induced Changes in Choline Transport and Phosphorylation Activities in Synaptosomes from the Optic Lobe of Squid', 'Loligo pealei', Comp. Biochem. Physiol. 89C, 179-183. Mauck, W. L., Olsen, L. E. and Marking, L. L.: 1976, 'Toxicity of Natural Pyrethrins and Five Pyrethroids to Fish', Arch. Environ. Contamin. Toxicol. 4, 18-29. Nakono, T. and Tomlinson, N.: 1967, 'Catecholamine and Carbohydrate Concentrations in Rainbow Trout (Salmo gairdnert) in Relation to Physical Disturbances', J. Fisheries Res. Board Canada 24, 1701-1715. Nemcs6k, J. and Boross, L.: 1982, 'Comparative Studies on the Sensitivity of Different Fish Species to Metal Pollution', Acta Biologica Academ. Scientiar. Hungariae 33, 23-27. Nemcs6k, J., Benedeczky, I., Boross, B., Asztalos, B. and Orb~n, L.: 1981, 'Subcellular Localization of Transaminase Enzymes in Fishes and their Significance in the Detection of Water Pollution', Acta Biologica Szegediensis 27, 9-15. Nemcs6k, J., Ol~h, L. and Boross, L.: 1982, 'Studies on Stress Effect caused by Malachite Green and Formalin Treatments of Common Carp (Cyprinus carpio L.)', Aquacultura Hungarica (Szarvas) III, 57-61. Nemcs6k, J., Rakonczay, Z., K~isa, P., Asztalos, B. and Szab6, A.: 1990, 'Effects of Methidathion, on Distribution of Molecular Forms of Acetylcholinesterase in Carp, as Revealed by Density Gradient Centrifugation', Pesticide Biochem. Physiol. 37, 140-144.
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Pennec, J. P. and Le Bras, Y. M.: 1984, 'Storage and Release of Catecholamines by Nervous Endings in the Isolated Heart of the Eel (Anguilla Anguilla L.)', Comp. Biochem. Physiol 1, 167-171. Ray, D. E. and Cremer, J. E.: 1969, 'The Action of Decamethrin (a Synthetic Pyrethroid) on the Rat', Pesticide Biochem. Physiol. 10, 333-340. Reitman, S. and Frankel, S.: 1957, 'A Colorimetric Method for the Determination of Serum Glutamic Oxaloacetic and Glutamic Pyruvic Acid Transaminase', Amer. J. Clin. Pathol. 28, 56. Rose, G. P. and Dewar, A. J.: 1983, 'Intoxication with Four Synthetic Pyrethroids Fail to Show any Correlation Between Neuromuscular Dysfunction and Neurobiochemical Abnormalities', Arch. ToxicoL 53, 297-316. Shaker, N., Hassan, G. A., el Nouty, E D., Abo Elezz, Z. and Abd Allah, G. A.: 1988, 'In vivo Chronic Effect of Dimethoate and Deltamethrin on Rabbits', J. Environ. Scien. Health B. 23, 387-399. Simon, L. M., Nemcs6k, J. and Boross, L..: 1983, 'Studies on the Effect of Paraquat on Glycogen Mobilization in Liver of Common Carp (Cyprinus carpio L.)', Comp. Biochem. Physiol. 75C, 167-169. Sorensen, P. G.: 1983, 'The Mg 2+ Dependant ATPase from the Erythrocyte Plasma Membrane of the Flounder Platichthys Flesus L. General Properties and Some Observations on the Steady State Kinetics', Comp. Biochem. Physiol. 79B. 153-161. Stephenson, R. R.: 1982, 'Aquatic Toxicology of Cypermethrin. I. Acute Toxicity to Some Freshwater Fish and Invertebrates in Laboratory Tests', Aquatic Toxicol. 2, 175-185. Szegletes, T. and Nemcs6k, J.: 1992, 'The Role of Ecological Factors in a Massive Devastation of the Fish Population in Lake Balaton', Fishing 1, 19-21. (In Hungarian.) Wedemeyer, G. A.: 1970, 'The Role of Stress in the Disease Resistance of Fishes', in: Sniesko, S. F. (Ed.) A Symposium on Diseases of Fishes and Shellfishes', Amer. Fish. Soc. Spec. 5, 30-35. World Health Organization: 1990, Environmental Health Criteria 97." Deltamethrin, WHO, Geneva (NLM Classification: WA 240). Wrobelski, E and La Due, J. S.: 1955, 'Serum Glutamate Oxaloacetate Transaminase as an Index of Liver Celt Injury', Proc. Soc. Experimental Biol. 90, 210. Zaugg, W. S. and McLain, L. R.: 1976, 'Influence of Water Temperature on Gill Sodium, Potassiumstimulated ATPase Activity in Juvenile Coho Salmon (Onhorhynchus kisutch)', Comp. Biochem. PhysioL 54A, 419-421. Zitko, V., McLeese, D. W., Metcalfe, C. D. and Carson, W. G.: 1979, 'Toxicity of Permethrin, Decamethrin, and Related Pyrethroids to Salmon and Lobster', Bull. Environ. Contamin. Toxicol. 21, 338-343.
Department of Biochemistry, Attila J6zsef University, P.O. Box 533, H-6701, Szeged, Hungary. (Received: September 1993; revised: September 1994)
Abstract. The in vivo effects of deltamethrin (DM) on the blood sugar level, the acetylcholinesterase (ACHE, EC 3.1.1.7) activities of the blood serum and various organs (heart, liver and intestine), the lactate dehydrogenase (LDH, EC 1.1.2.3), glutamic-oxaloacetic transaminase (GOT, EC 2.6.1.1), and glutamic-pyruvic transaminase (GPT, EC 2.6.1.2) activities of the blood serum, the adenosine triphosphatases (EC 3.6.1.3; Na +/K + -ATPase and Mg2+-ATPase) activities of the erythrocyte plasma membrane and the catalase (EC 1.11.1.6) activity of the liver were examined throughout 96 h in adult carp (Cyprinus carpio L.) Two sublethal concentrations, 1.0 and 1.5 #g/1 of deltamethrin, were used. All fish survived the experiment except one, in an aquarium containing 1.5 ppb of DM, which died after 72 h. The AChE specific activity was significantly inhibited in the heart and intestine after 96 h at both concentrations compared to that in the control animals (P < 0.05, Student's t-test), while there was no detectable difference between the two treatment. At the same time there was no detectable change in the liver. In the serum, the AChE activity almost remained unchanged; the only significant decrease could be measured after 96 h at 1.5 #g/1 deltamethrin concentration. The blood glucose content exhibited interesting changes: after 24 h fish exposed at 1 #g/1 DM seemed to be stressed, although this increase was not significant. When these fish became used to the new conditions (in practice this meant the presence of DM), the glucose level decreased, especially after 72 h. At the same time the control animals kept in similar circumstances showed a small insignificantdecrease. Meanwhile fish in aquaria containing 1.5/~g/1 DM reacted to the treatment with an increased blood glucose level after 48 h, and this did not change until the end of the treatment. The Na+/K +-ATPase activity decreased in a dose-dependant manner, while Mg2+-ATPase was less affected. A small increase in LDH level was observed, indicating damage of different muscle tissues. However, this phenomenon appeared only with the small dosage after 24 h (P < 0.05). It has to be mentioned that the individual values varied to a large extent among of the eight fish. The GOT activities of the serum increased during the treatment. However, significant changes were only expressed after 72 and 96 h at 1 #g/1 DM concentrations (P < 0.01 and P < 0.05), and after a similar long treatment at the high dosage (P < 0.05, 72 and 96 h). The GPT did not change significantly in aquaria containing 1 /~g/1DM. The only larger increase was measured after 96 h at 1.5 #g/1 DM concentration (P < 0.05). The catalase activity in the liver of treated carp remained practically at the same level compared to that in control fish. All these changes (concerning the primary effects of this compound) demonstrate the effect of DM on different fish enzymes, at low concentrations under laboratory conditions, which might be useful in practice for biomonitoring using fish.
1. Introduction
Deltamethrin (or decamethrin, DM; type II pyrethroid; (S)-c~-cyano-3-phenoxy-
benzyl-(1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-carboxylate, * To whom all correspondence should be addressed.
Environmental Monitoring and Assessment 35:97-111, 1995. (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands.
98
T. SZEGLETES ET AL
H3C CH3
B
c:ci
B/r
/k ,c
\.
o
N-C-CHScheme 1 Formula ofdeltamethrin.
C22HI9Br2NO3; Scheme 1) is considered to be among the safest classes of insecticides available. However, under cleanwater laboratory conditions, exposure of this pyrethroid to fish seem to be toxic. The 96 h LCs0 values range between 0.4 and 2.0 #g/1 (L'Hotellier and Vincent, 1986). Extensive field studies, in experimental ponds, have shown that this high potential toxicity is not realized (World Health Organization, 1990). There is no significant difference between the acute toxicities to cold and warm water species of fish (Stephenson, 1982). It seems that DM affect fish at both high and low temperatures, although a negative temperature coefficient has been claimed for certain pyrethroids (Kumaragum and Beamish, 1981). Furthermore, the toxicity of pyrethroids (including DM) for fish is not influenced by the hardness or pH of the water (Mauck et al., 1976). DM has been observed to cause neuromuscular dysfunctions in organisms (Rose and Dewar, 1983). Its primary effect has been attributed to slowing the sodium channels of nerve cells. GABA receptors appear to be inhibited as well. Additionally, there are many secondary effects on the whole organism. According to Rose and Dewar (1983), DM has at least two distinct actions - a short term pharmacological effect at near-lethal dose levels and a more long-term neurotoxic effects that results in sparse axonal nerve damage. In the literature one can find many extensive references to the secondary effects of DM on various parameters, such as ACHE, ATPases (Shaker et al., 1988), noradrenaline and adrenaline contents in rats (de Boer et al., 1988), choline transport (Matsumura, 1988), induction of chromosome aberrations in mice (Bhunya and Pati, 1990), and the subpopulations of muscarinic and nicotinic ACh receptors (Eriksson and Nordberg, 1990). However, few investigations have been made on the mechanism of acute toxicity on fish (L'Hotellier and Vincent, 1986; Zitko et al., 1979). The present study involves the biochemical characterization of some enzymes in specific tissues of carp, the most populous teleostean species in Hungary. The
BIOCHEMICAL PARAMETERS OF CARP
99
blood serum activities of ACHE, LDH, GOT and GPT and blood glucose levels were determined in control and treated animals. The changes in these enzyme activities have been used to demonstrate tissue damage in fish (Wrobelski and La Due, 1955; Kristoffersson et al., 1974; Nemcs6k and Boross, 1982; Asztalos and Nemcs6k, 1985). Similarly, the increase in blood glucose level has been used to demonstrate metabolic stress (Wedemeyer, 1970; Nemcs6k and Boross, 1982). Changes in the AChE activity of various organs, the catalase activity of the liver and the ATPase activities of the erythrocyte membrane after DM treatment were also studied. Our study was initiated by a massive devastation of the fish population in Lake Balaton, Hungary; although most of those which died were common eels (Anguilla anguilla). Brain and liver from these fish contained residues of DM at 0.02 mg/1000 g wet tissue concentration (Gt~nczy, 1992). G6nczy suspected that one of the causes of the massive fish kill could be the presence of DM in fish. This study was conducted to evaluate the in vivo effect of two sublethal concentrations of DM on several enzymatic pathways, in four tissues of carp under cleanwater laboratory conditions at 25 °C, imitating summer water temperature.
2. Materials and Methods
Common carp (Cyprinus carpio L.) of both sexes, weighing 800-1000 g, were used in the experiments. Fish were kept individually in O2-saturated water at 25 ± 1 °C in a 100 1 aquarium. The animals were not fed during the experiments. The DM concentration in the aquarium was 1 or 1.5 #g/1. The duration of exposure was 96 h. Samples of carp blood were taken from the tail vien of all fish every 24 h. The blood was centrifuged at 4 °C and the ACHE, GOT, GPT and LDH activities and the blood glucose level was measured in a heamolysis-free serum. The pellet was used to measure the ATPase activities. The values are expressed as the average of 6-8 individuals (+ S.E.M.). The differences between the data of control and treated animals were analysed by Student's t-test. The AChE and catalase activities in the organs (heart, liver and midgut) were measured by removing the respective organ after 96 h, homogenizing it in 0.65% NaCI containing 0.5% Triton X-100 and 12.5 mM sodium phosphate with a Braun 853302/4 homogenizer (to give a final concentration of 200 mg wet weight of organ per ml of homogenate), followed by centrifugation and activity measurement in the supematant. AChE was determined with acetylthiocholine as substrate using the method of Ellman et al. (1961). The GOT and GPT activities were assayed according to Reitman and Frankel (1957), and the LDH activity according to Annon (1971), with Reanal kits (Hungary). The catalase activity in the supernatant of the liver homogenate was measured spectrophotometrically by monitoring the decrease of H202. The reaction mixture was 3.0 ml phosphate buffer [50 mM, pH 7.0; containing 1.2 #1 of 30% H202
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T. SZEGLETES ET AL
per 1.0 ml buffer (Reanal, Hungary)] + 0.025 ml supernatant. Absorbance was measured at 240 nm. The protein content of all samples was determined by the method of Lowry et al. (1951). Enzyme activities are given in mU mg -1 protein (1 U = 1 #mol substrate min- ~). Blood sugar level was determined by the glucose oxidase-peroxidase method. The reaction mixture was 2.5 ml glucose reagent [2 ml peroxidase (Reanal, Hungary) + 12.5 mg glucose oxidase (Reanal, Hungary) diluted in 50 ml 0.1 M phosphate buffer (pH 7.0) + 3.3 mg o-dianisidine (Reanal, Hungary) diluted in 1.0 ml distiled water] + 0.1 ml deproteinized blood serum. After a 35-min incubation at room temperature, the absorbance was measured at 450 nm. Erythrocyte plasma membrane was prepared according to Sorensen (1983). Erythrocytes were collected by centrifugation and washed with 0.16 M NaC1. After washing, the cells were resuspended in 0.15 M NaC1, 3 mM MgC12 and 0.01 M Tris-HC1, pH 7.2, and homogenized in a Virtis 45 homogenizer fitted with knives. Nuclei and unbroken cells were removed from the homogenate by low-speed differential centrifugation (500-1200 g for 5 min). The broken plasma membranes were collected from the supernatant by centrifugation at 25 000 g for 15 min. The membranes were then washed several times with 0.1 M choline chloride, 0.01 M Tris-HC1, pH 7.2. Finally, part of the washed membranes was resuspended in a small volume of washing medium and stored at -60 °C for the measurement of Na+/K+-ATPase activity and total protein. Proteins were again determined using the method of Lowry et al. (1951). The ATPase activity was measured in a basic incubation medium containing 40 mM Tris-HC1, pH 7.2, 6 mM MgC12, 20 mM KC1 and 150 mM NaC1 in certain cases with the addition of the inhibitor oubain (0.7 mM) (Bourre et al., 1982). The reaction was started after a 5-min temperature equilibration by the addition of 4 mM ATP (Tris salt). The incubation was carried out in a shaking bath at 22 °C for 15 min and was stopped by the addition of 1 ml of ice-cold 10% v/v TCA. The precipitated protein was centrifuged and the supernatant was analysed for inorganic phosphate as in the method of Line and Way (1984). The difference between total ATPase and Mg2+-ATPase is considered to be Na+/K +-ATPase. The specific activity of ATPase was determined in units of nmol of Pi/mg protein/h.
3. Results and Discussion
3.1. ACHE The AChE specific activity in the heart and the midgut section was strongly inhibited by DM (50-60% decrease), while in the liver the level of this enzyme did not change. This could be observed after 96 h of treatment (Figure 1a). After 24, 48 and 72 h the blood serum AChE activity remained at a level similar to that in
BIOCHEMICALPARAMETERSOFCARP
101
mU/mg protein 120 100 8060-
[ ] Control [ ] 1.0/~g/l DM [ ] 1.5/~g/! DM [
iI !l
4020-
0 Heart
Liver
Intestine
Fig. la.
the control fish. After 96 h of treatment in 1.5 #g/l dose a significant (P < 0.05) decrease (27% decrease compared to that in the control fish) was observed. No significant difference was detected between the two applied doses (Figure lb). The LCs0 of DM is 1.84 #g/1 (96 h) for carp (L'Hotellier and Vincent, 1986). We observed a similar lethal concentration, although in one of our preliminary experiments a lower DM concentration killed all of the studied fish. It is well known that a-cyanopyrethroids, such as DM, cause a long-lasting prolongation of the normally transient increase in sodium permeability of the nerve membrane during excitation, resulting in long-lasting trains of repetitive impulses in the sense organs and a frequency-dependant depression of the nerve impulses in the nerve fibres. Since the mechanisms responsible for nerve impulse generation and conduction are basically the same throughout the entire nervous system, pyrethroids may well act in a similar way in various parts of the central nervous system. The clinical signs of DM toxicity are tremor and salivation in animals (Kavlock et al., 1979; Ray and Cremer, 1979). In fish (especially at higher dosages of DM), signs of toxicity are irregular movement of the operculum and disturbances in the coordination of swimming. In our study we observed that the surface of the skin/scales became infected after the second day of exposure at both concentrations. This is the connection between the effects of DM on the Na+-channel permeability and on the inhibition of ACHE. This inhibition is not as large as is the case
102
T. SZEGLETES ET AL
mU/mg protein ~-~Control E 1.0 #g/l DM [ ] 1.5 #g/I DM
6.0 -1
5.0
-
4.0
--
3.0' 2.0 1.0
0.0
t
t
-
24
48
72
96
Time of treatment (hrs) Fig. lb. Figs. 1(a)-(b). Effect of deltamethrin treatment on AChE specific activity of heart, liver and intestine (a) and blood serum (b) of carp at a water temperature of 25 4- 1 °C. Heart, liver and intestine were taken out from the fish after 96 h treatment. Activities, given in mU mg-~ protein, are the averages ± S.E.M. for 6 to 8 specimens. Note: Values significantly different by Student's t-test from controls are indicated: *P < 0.05. For experimental conditions, see Materials and Methods in text.
with some organophorous esters, nevertheless in fishes pyrethroids are 1000 times more toxic than other insecticides, at least in laboratory aquaria (Nemcs6k et al., 1990). A near to lethal dose of DM produces biochemical changes in the peripheral nerves, consistent with sparse axonal degeneration. Swelling and disintegration of the axons of the sciatic nerve may be observed. This destruction could be the cause of, among others, the decrease in AChE activity in the heart and intestine. These tissues are perhaps more sensitive than the tissue of the liver. AChE inhibition is particularly dangerous in the heart, since the cholinergic system has a decisive role in the innervation of the heart in fish (Pennec and Le Bras, 1984): AChE inhibition potentiates the vagal tone, which may cause adverse effects in the metabolic processes related to circulation. In this case, inhibition of the heart function interferes with the uptake of 02 and the release of CO2 at the gills, which may result in hypoxia at the tissue level (Hughes, 1976).
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B I O C H E M I C A L P A R A M E T E R S OF CARP
TABLE I Effect of deltamethrin treatment on plasma GOT specific activity of carp at a water temperature of 25 4- 1 °C. Activities are expressed as x 10 - 4 U mg-1 protein (1 U: l#mol substrate rain-l).
1 #g/1DM 1.5 #g/1DM Control
Exposure time (h) 24 48
72
96
4.60 4- 2.69 8.594- 4.56 2.56 4- 1.64
9.07 4- 2.83** 11.204- 6.84* 3.68 4- 1.85
10.80 -4- 5.62* 13.10 4- 5.25* 3.75 4- 2.51
8.68 4- 1.56 10.404- 4.99 4.43 4- 2.95
Note: Data are means 4- S.E.M. of results for 6 to 8 specimens. Values significantly different by Student's t-test from controls are indicated: *P < 0.05; **P < 0.01. For experimental conditions, see Materials and Methods in text.
TABLE II Effect of deltamethrin treatment on plasma GPT specific activity of carp at a water temperature of 25 4- 1 o C. Activities are expressed as x l 0 - 4 U mg-I protein. Exposure time (h) 24 48 1 #g/1 DM 1.5#g/1DM Control
72
96
2.72 4- 0.43 3.47-t- 1.02 3.08+ 0.72 3.93 4- 1.68 2.644- 1.86 4.59-4- 1.54 5.094- 1.71 5.574- 0.90* 2.35 4- 1.31 2.81 4- 1.54 3.63 4- 1.74 3.60 4- 2.47
Note: Data are means 4- S.E.M. of results for 6 to 8 specimens. Values significantly different by Student's t-test from controls are indicated: *P < 0.05. For experimental conditions, see Materials and Methods in text.
3.2.
G O T AND G P T
G O T activities in the blood serum were significantly higher than those in control animals after 72 and 96 h at both concentrations (72 h 1 #g/l: P < 0.01; 72 h 1.5 #g/1 and 96 h 1 and 1.5 #g/l: P < 0.05; Table I). G P T did not change significantly at the low dosage, and even in the high dosage after shorter treatment. The only significant increase was observed after 96 h at 1.5 #g/1 D M level (P < 0.05; Table II). The d a m a g e to tissue containing considerable amounts o f G O T and G P T is easily detectable through the increasing activities o f these enzymes in the blood serum (Karmen et al., 1955). The distributions of G O T and G P T vary in the different organs ( N e m c s 6 k et al., 1981), and the ratio of the activities o f the two e n z y m e s permits more exact conclusions of the injured tissues. Carp liver contains more G P T and less GOT.
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T. SZEGLETES ET AL
U/mg protein 20-
T
1612_
4 0 Control
1.0/ g/l DM
1.5/ g/l DM
Fig. 2. Effect of deltamethrin exposure on catalase specific activity in liver of carp at a water temperature of 25 4- 1 °C after 96 h treatment. Activities, given in U mg-1 protein, are the averages 4- S.E.M. for 6 to 8 specimens. Note: For experimental conditions, see Materials and Methods in text.
The elevated GOT and the lesser increased GPT activities observed from our studies were presumably due to liver damage. However, other organs may also have been damaged (e.g. the kidney or/and the gill). In the case of GOT, a dosedependant trend cannot be clearly concluded: if there is a significant increase at all, the GOT values in carps treated with the two concentrations of DM are similar. Time-related, there is a small increase, but the only significant change is between 24 and 96 h at the low dosage (Table I). In the case of GPT, the only significant time-dependant difference is between 24 and 96 h at the high dosage (P < 0.05). The activity of GPT already increased after 48 h, but due to its high standard error, this change is not significant. 1 #g/1 DM exposure did not cause significant time-dependant differences, although a small (but still insignificant) increase was marked by the values (Table II). 3.3. CATALASE The catalase activity did not change significantly, only a small increase can be suspected from our data relative to the level in the control carp (Figure 2). During the metabolism of DM in fish, in contrast with other animals (mice and quail),
BIOCHEMICAL PARAMETERSOF CARP
105
mg/100 ml serum I ~---]Control ~ 1.0/tg/l DM ~ 1.5/tg/! DM
180 150 120 -
T
90-
@
60300 24
48
72
96
Time of treatment (hrs) Fig. 3. Effectof deltamethrin exposure on blood glucose level of carp at a water temperature of 25 :t: 1 °C. Concentrations,given in mg glucose/100 ml blood serum, are the averages =kS.E.M. for 6 to 8 specimens.Note: Valuessignificantlydifferentby Student'st-test from controls are indicated: *P < 0.05. For experimentalconditions, see Materials and Methodsin text.
the ester cleavage of this compound is very slow; the main metabolic pathway is hydroxylation to give the 4-hydroxyphenoxy derivative, which is excreted in the bile as the glucuronide (Edwards and Millburn, 1985). The presence of the derivatives is generally connected with the increased activity of enzymes, whose main role is to eliminate the multiplied radicals or derivatives during the metabolism of similar chemicals. The liver ultrastructure shows marked changes as a result from this metabolism pathway. DM increases the number of mitochondria and alters their shape, which become irregular (Catinot et al., 1989). However, in our case the two applied doses did not result in the large expected increase of catalase activity, probably due to the slow metabolism in fish, even at high water temperature (25 °C), and because of the presence of other eliminating enzyme systems. We can conclude that DM-treated fish have different enzymatic levels after 96 h in both serum GOT and GPT activities, at least at 1.5 #g/1 DM concentration compared with those in the control animals. At the same time, we could not reveal similar significant changes in catalase activities in the liver tissue.
106
T. SZEGLETESET AL
TABLE III Effect of deltamethrin treatment on plasma LDH specific activity of carp at a water temperature of 25 4- 1 °C. Activities are expressed as U rag-1 protein. Exposure time (h) 24 48
72
96
1/zg/1 DM 6.01 4- 1.66" 6.794- 1.46" 5.494- 3.30 5.904- 2.98 1.5 #g/1DM 3.624- 2.05 4.09 4- 2.97 4.08 4- 2.15 4.524- 1.80 Control 3.54 4- 1.75 3.43 4- 0.98 3.09 4- 0.75 3.304- 1.63 Note: Data are means 4- S.E.M. of results for 6 to 8 specimens. Values signif-
icantly different by Student's t-test from controls are indicated: *P < 0.05. For experimental conditions, see Materials and Methods in text. 3.4. BLOOD SUGAR LEVEL Pyrethroid poisoning in fish produced a syndrome characterized by tremors, abnormal operculum movement (particularly at 1.5 /zg/1 or higher dose), ataxia and hyperactivity. The treated carp were very nervous throughout the experiment, swimming erratically from wall to wall of the aquarium, often striking against it causing intensive internal bleeding and injury to the structure of the epithelium and other tissues. This stressed stage was marked by a higher blood sugar level in the 48 h, 72 h and 96 h samples at 1.5 #g/1 dose (Figure 3). After 24 h of treatment fish kept at 1 #g/1 concentrations seemed to be stressed, but the increase of blood glucose level was not significant. When these fish became acclimatized to the new conditions, the glucose level decreased in the 72 h and 96 h samples compared to the 24 h value (Figure 3). Surprisingly, the fish exposed to 1.5 #g/1 DM reacted later to the treatment (48 h value) with a remaining level throughout the entire experiment. However, the changes in the glucose level of 24 h samples from the fish treated with 1 #g/1 DM were not significant (P > 0.05). It is possible that the higher applied dose caused a longer-lasting effect, while the smaller concentration caused a shorter one. The DM induced 'non-motor' symptoms, i.e. the increases in brain blood flow and blood glucose level, may result from a supraspinal component of DM activity (Bradbury et aI., 1983). Permanent stress includes impaired 7-globulin formation and depressed interferon production, which play important roles in the resistance of fish to various bacterial and viral diseases (Wedemeyer, 1970; Nemcs6k et al., 1982). 3.5. L D H There was a significant increase in LDH activity after 24 h of treatment at the low dose (P < 0.05). However, after 72 h and 96 h of treatment LDH activity decreased to control levels (Table III). No time-dependant difference was observed among
107
BIOCHEMICAL PARAMETERS OF CARP
[ ] 1.S ~tg/l DM nmol Pi/mg protein/hr
1.0 p.g/! DM
12.0 -
[--] Control 10.0 8.0
--
6.0
-
4.0
-
2.0
-
T
i
0.0
24
48
72
96
1
Time of treatment (hrs) Fig. 4a. the parameters at low dose. High dose yielded no significant change over time, except after the 96 h treatment. A slight increase in glucose level was observed after 24 h of treatment at low dose. In fish treated with 1.5 #g/1 DM this increase was detected after 48 h of treatment. In both cases glucose levels seemed to have increased concomitantly with LDH levels. The increased LDH activity could indicate metabolic changes in the stressed fish, primarily in the muscle cells: the catabolism of glycogen and glucose shifted towards the formation of lactate, which can be lethal for fish (Nakono and Tomlinson, 1967; Simon et al., 1983). 3.6. ATPAsES Mg2+-dependant ATPases showed a significant decrease after 48 h of treatment at high dose, while 1 #g/1 DM exposure resulted in a significant difference only after 72 h (Figure 4a). At 96 h both exposures resulted in a 60% decrease in MgZ+-ATPase activity. The Na+/K+-dependant ATPases from the erythrocyte plasma membrane decreased markedly in a dose-dependant manner (Figure 4b).
108
T. SZEGLETESET AL
nmol Pi/mg protein/hr
[ ] 1.5/~g/l DM
8.0 -
@ 1.o 7.0-
DM
D Control
6.05.0-
T
± +
4.0-
+
+ +
3.0-
+
2.01.00.0
24
48
72
96
Time of treatment (hrs) Fig. 4b. Figs. 4(a)-(b). Effect of deltamethrin exposure on Mg 2+ (a) and Na+/K+ (b) dependant ATPases specific activities of erythrocyte plasma membrane of carp at a water temperature of 25 ± 1 °C. Activities, given in nmol of Pi/mg protein/h, are the averages -4- S.E.M. for 6 to 8 specimens. Note: Values significantly different by Student's t-test from controls are indicated: *P < 0.05; +P < 0.005. For experimental conditions, see Materials and Methods in text.
The pyrethroid insecticides have been postulated to involve interactions not only with Na + channels, but also with neurotransmitters, receptor-ionophore complexes and ATPases (Dorman and Beasley, 1991). It has already been reported that ATPase activities depend strongly on pH, temperature and water pressure (Zaugg and McLain, 1976; Gibbs and Somero, 1989), because these environmental parameters may cause changes in composition of the membrane lipids. However, a similar effect may not be assumed in the present experiment. We suspect that D M directly affects ATPases function. This effect could be associated with prolonging the closure of Na + channels and thus changing ion distribution.
BIOCHEMICAL PARAMETERSOF CARP
109
4. Conclusion Our results suggest that doses of DM have significant effect on ATPases. A clear dose-dependant trend could be observed only in the case of Na+/K+-dependant ATPases. AChE decreased in heart and intestine, showing a neuronal dysfunction. In the blood AChE level a small decrease was measured, which implies that only higher dose (> 1.5 #g/l) and longer exposure (> 72 h) is detectable through AChE without killing the fish. The damage in liver (and kidney/gill) can be observed through blood transaminase level; GOT is more suitable for these purposes. Blood glucose and LDH levels in sera also might be useful as a biomonitoring tool, but in our case a dose-dependant trend could not be realized. The massive fish kill in Hungary in 1991 made our studies timely. Residues of DM were detectable in tissues of eel (Anguilla anguilla). Brain and liver from these fish contained residues of DM at 0.02 mg/1000 g wet tissue concentration (G6nczy, 1992). GOnczy suspected that one of the causes of the massive fish kill could be the presence of DM in fish. At the same time we measured, amongst others, a significant AChE decrease in the blood serum and other organs (Szegletes and Nemcs6k, 1992). Obviously, it does not imply that the main reason of the devastation in the fish populations was the use of DM-containing insecticides around the lake. However, it was a point to discuss, that the presence of DM was accompanied by a decreased AChE level in fish. We also made some in vitro experiments with this enzyme, and it showed that DM causes AChE inhibition. Transaminases also increased, and extreme high blood glucose level was detected. The present study shows the possibility of using certain biomarkers to assess chemical effects and their potential hazards in aquatic environment. These results strongly suggest wide-ranging toxic effects of deltamethrin on fish.
References Annon, L.: 1971, 'Photometric Determination of LDH Activity in Blood Serum', Zeitungfar Klinischer Chemistrie und Klinischer Biochemistrie 8, 658. Asztalos, B. and Nemcs6k, J.: 1985, 'Effect of Pesticides on the LDH Activity and Isoenzyme Pattern of Carp (Cyprinus carpio L.) Serum', Comp. Biochem. Physiol. 82C, 217-219. Bhunya, S. P. and Pati, P. C.: 1990 'Effect of Deltamethrin, a Synthetic Pyrethroid, on the Induction of Chromosome Aberrations, Micronuclei and Sperm Abnormalities in Mice', Mutagenesis 5, 229-232. Bourre, J. M., Chanez, C., Dumont, O. and Flexor, M. A.: 1982, 'Alteration of 5-Nucleotidase and Na+.K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (Jumpy, Quaking, Trembler, Shivered and Mid). Comparison with CNPase in the Developing Sciatic Nerve from Trembler', J. Neurochem. 38, 643-649. Bradbury, J. E., Forshaw, P. J., Gray, A. J. and Ray, D. E.: 1983, 'The Action of Mephenesin and Other Agents on the Effects Produced by Two Neurotoxic Pyrethroids in the Intact and Spinal Rat', Neuropharmacol. 22(7), 907-914. Catinot, R., Hoellinger, H., Pfister, A., Sonnier, M. and Simon, M. T.: 1989, 'Effects on Rats of Subacute Intoxication with Deltamethrin via an Osmotic Pump', Drug Chem. Toxicol. 12, 173196.
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de Boer, S. F., van der Gugten, J., Slangen, J. L. and Hijzen, T. H.: 1988, 'Changes in Plasma Corticosterone and Catecholamine Contents Induced by Low Doses of Deltamethrin in Rats', Toxicology 49, 263-270. Dorman, D. C. and Beasley, V. R.: 1991, 'Neurotoxicology of Pyrethrin and the Pyrethroid Insecticides', Veter. Human Toxicol. 33, 238-243. Edwards, R. and Millburn, P.: 1985, 'Toxicity and Metabolism of Cypermethrin in Fish Compared with Other Vertebrates' Pesticide Sci. 16, 201-202. Ellman, G. L., Courtney, D. D., Andres, V. and Featherstone, R. M.: 1961, 'A New and Rapid Colorimetric Determination of AChE Activity', Biochem. Pharmacol. 7, 88-95. Eriksson, P. and Nordberg, A.: 1990, 'Effects of two Pyrethroids, Bioallethrin and Deltamethrin, on Subpopulations of Muscarinic and Nicotinic Receptors in the Neonatal Mouse Brain', Toxicol. Appl. Pharmacol. 102, 456-463. Gibbs, A. and Somero, G. N.: 1989, 'Pressure Adaptation of Na+/K+-ATPase in Gills of Marine Teleost', J. Exper. Biol. 143, 475-492. Gt~nczy, J.: 1992, 'Data and Ideas to Clear the Question of the Massive Fish Kill', Fishing, 1, 21-23. (In Hungarian) Hughes, G, M.: 1976, 'Polluted Fish Respiratory Physiology', in: Lockwood, A. P. M. (Ed.) Effects of Pollutants on Aquatic Organism, Cambridge University Press, London, 121-146. Karmen, A., Wrobelski, F. and La Due, J. A.: 1955, 'Transaminase Activity in Human Blood', J. Clin. Invest. 34, 126. Kavlock, R., Chernoff, N., Baron, R., Linder, R., Rogers, E., Carver, B., Dilley, J. and Simmon, V.: 1979, 'Toxicity Studies with Decamethrin, a Synthetic Pyrethroid Insecticide', J. Environ. Path. Toxicol. 2, 751-765. Kristoffersson, R., Broberg, S., Oskari, A. and Pekkarinen, M.: 1974 'Effect of a Sublethal Concentration of Phenol on Some Blood Plasma Enzyme Activities in the Pike (Esox lucius L.) in Brackish Water', Ann. Zoologic Fennici 11, 220-223. Kumaraguru, A. K. and Beamish, F. W. H.: 1981, 'Lethal Toxicity of Permethrin (NRDC-143) to Rainbow Trout, Salmo gairdneri, in Relation to Body Weight and Temperature', Water Res. 15, 503-505. L'Hotellier, M. and Vincent, P.: 1986, 'Assessment of the Impact of Deltamethrin on Aquatic Species', in British Crop Protection Conference - Pests and Disease, pp. 1109-1116. Line, S. C. and Way, E. L.: 1984, 'Characterization of Calcium-activated and Magnesium-activated ATPase of Brain Nerve Endings', J. Neurochem. 42, 1697-1706. Lowry, O. H., Rosenbrough, N. J., Forr, A. L. and Randall, R. J.: 1951, 'Protein Measurement with the Folin Phenol Reagent', J. Biol. Chem. 193, 269-275. Matsumura, F.: 1988, 'Deltamethrin-induced Changes in Choline Transport and Phosphorylation Activities in Synaptosomes from the Optic Lobe of Squid', 'Loligo pealei', Comp. Biochem. Physiol. 89C, 179-183. Mauck, W. L., Olsen, L. E. and Marking, L. L.: 1976, 'Toxicity of Natural Pyrethrins and Five Pyrethroids to Fish', Arch. Environ. Contamin. Toxicol. 4, 18-29. Nakono, T. and Tomlinson, N.: 1967, 'Catecholamine and Carbohydrate Concentrations in Rainbow Trout (Salmo gairdnert) in Relation to Physical Disturbances', J. Fisheries Res. Board Canada 24, 1701-1715. Nemcs6k, J. and Boross, L.: 1982, 'Comparative Studies on the Sensitivity of Different Fish Species to Metal Pollution', Acta Biologica Academ. Scientiar. Hungariae 33, 23-27. Nemcs6k, J., Benedeczky, I., Boross, B., Asztalos, B. and Orb~n, L.: 1981, 'Subcellular Localization of Transaminase Enzymes in Fishes and their Significance in the Detection of Water Pollution', Acta Biologica Szegediensis 27, 9-15. Nemcs6k, J., Ol~h, L. and Boross, L.: 1982, 'Studies on Stress Effect caused by Malachite Green and Formalin Treatments of Common Carp (Cyprinus carpio L.)', Aquacultura Hungarica (Szarvas) III, 57-61. Nemcs6k, J., Rakonczay, Z., K~isa, P., Asztalos, B. and Szab6, A.: 1990, 'Effects of Methidathion, on Distribution of Molecular Forms of Acetylcholinesterase in Carp, as Revealed by Density Gradient Centrifugation', Pesticide Biochem. Physiol. 37, 140-144.
BIOCHEMICAL PARAMETERSOF CARP
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Pennec, J. P. and Le Bras, Y. M.: 1984, 'Storage and Release of Catecholamines by Nervous Endings in the Isolated Heart of the Eel (Anguilla Anguilla L.)', Comp. Biochem. Physiol 1, 167-171. Ray, D. E. and Cremer, J. E.: 1969, 'The Action of Decamethrin (a Synthetic Pyrethroid) on the Rat', Pesticide Biochem. Physiol. 10, 333-340. Reitman, S. and Frankel, S.: 1957, 'A Colorimetric Method for the Determination of Serum Glutamic Oxaloacetic and Glutamic Pyruvic Acid Transaminase', Amer. J. Clin. Pathol. 28, 56. Rose, G. P. and Dewar, A. J.: 1983, 'Intoxication with Four Synthetic Pyrethroids Fail to Show any Correlation Between Neuromuscular Dysfunction and Neurobiochemical Abnormalities', Arch. ToxicoL 53, 297-316. Shaker, N., Hassan, G. A., el Nouty, E D., Abo Elezz, Z. and Abd Allah, G. A.: 1988, 'In vivo Chronic Effect of Dimethoate and Deltamethrin on Rabbits', J. Environ. Scien. Health B. 23, 387-399. Simon, L. M., Nemcs6k, J. and Boross, L..: 1983, 'Studies on the Effect of Paraquat on Glycogen Mobilization in Liver of Common Carp (Cyprinus carpio L.)', Comp. Biochem. Physiol. 75C, 167-169. Sorensen, P. G.: 1983, 'The Mg 2+ Dependant ATPase from the Erythrocyte Plasma Membrane of the Flounder Platichthys Flesus L. General Properties and Some Observations on the Steady State Kinetics', Comp. Biochem. Physiol. 79B. 153-161. Stephenson, R. R.: 1982, 'Aquatic Toxicology of Cypermethrin. I. Acute Toxicity to Some Freshwater Fish and Invertebrates in Laboratory Tests', Aquatic Toxicol. 2, 175-185. Szegletes, T. and Nemcs6k, J.: 1992, 'The Role of Ecological Factors in a Massive Devastation of the Fish Population in Lake Balaton', Fishing 1, 19-21. (In Hungarian.) Wedemeyer, G. A.: 1970, 'The Role of Stress in the Disease Resistance of Fishes', in: Sniesko, S. F. (Ed.) A Symposium on Diseases of Fishes and Shellfishes', Amer. Fish. Soc. Spec. 5, 30-35. World Health Organization: 1990, Environmental Health Criteria 97." Deltamethrin, WHO, Geneva (NLM Classification: WA 240). Wrobelski, E and La Due, J. S.: 1955, 'Serum Glutamate Oxaloacetate Transaminase as an Index of Liver Celt Injury', Proc. Soc. Experimental Biol. 90, 210. Zaugg, W. S. and McLain, L. R.: 1976, 'Influence of Water Temperature on Gill Sodium, Potassiumstimulated ATPase Activity in Juvenile Coho Salmon (Onhorhynchus kisutch)', Comp. Biochem. PhysioL 54A, 419-421. Zitko, V., McLeese, D. W., Metcalfe, C. D. and Carson, W. G.: 1979, 'Toxicity of Permethrin, Decamethrin, and Related Pyrethroids to Salmon and Lobster', Bull. Environ. Contamin. Toxicol. 21, 338-343.
