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Effect of Acute Exposure of the Organophosphate Insecticide Rogor on Some Biochemical Aspects ofClarias batrachus(Linnaeus)
Environmental Research Section A 80, 80 — 83 (1999) Article ID enrs.1998.3871, available online at http://www.idealibrary.com on
Effect of Acute Exposure of the Organophosphate Insecticide Rogor on Some Biochemical Aspects of Clarias batrachus (Linnaeus) Ghousia Begum1 and Shantha Vijayaraghavan Department of Zoology, Osmania University, Hyderabad, 500 007, Andhra Pradesh, India Received August 22, 1997
to explain the increased damage to aquaculture due to pollution. Today, pesticides have occupied all segments of the environment due to their use in field operations as potent and economically useful poisons. This in turn affects the nontarget organisms. It is known that in fishes organophosphate (OP) pesticides are neurotoxic and they inhibit acetylcholinesterase activity with subsequent disruption of nervous functions, thereby interfering with some of the vital physiological functions (Prasada Rao and Ramana Rao, 1983). Energy metabolism plays a key role as the animal is forced to expend more energy to overcome toxic stress. Based on this, an attempt was made to study the sublethal toxicity of Rogor (an OP insecticide) on the carbohydrate metabolism taking key metabolites and enzymes in the muscle tissue of Clarias batrachus. This freshwater, air-breathing teleost fish was selected because of its wide availability and edibility in India. Muscle tissue of fish is the main consumable part and bioanalysis of this tissue is very essential in order to monitor fish quality for human consumption and health.
Indian catfish (Clarias batrachus), a common fish of commercial importance, was exposed to a sublethal concentration (1/3 of 96 h LC50 value) of Rogor for periods ranging from 24 to 192 h. The changes in glycogen, lactate, lactate dehydrogenase, and glycogen phosphorylase (a, ab) contents of muscle tissue were studied. A gradual decrease in muscle glycogen and an increase in lactate contents were observed. The activity level of lactate dehydrogenase showed a sharp rise initially for 48 h followed by a decline after 96 and 192 h. The glycogen phosphorylase a and ab in muscle tissue enhanced in response to Rogor exposure. 1999 Academic Press
Key Words : food fish; pesticide concentration; environmental pollution.
INTRODUCTION
Investigations have shown that changes in carbohydrate metabolism in fish induced by the stress of severe muscular exercise resemble the changes displayed by higher vertebrates like mammals. These changes include depletion of glycogen stores from fish tissues like liver and muscle (Laul et al., 1974). Black (1958) reported an elevation in lactic acid levels in liver, muscle, and blood and suggested that an uncontrolled entry of lactic acid into the tissues interferes with internal mechanisms that maintain the acid base balance. Lactic acid may also reduce the affinity of hemoglobin for both oxygen and carbondioxide, diminishing the oxygen-carrying capacity of blood. He stated that in these cases fish deaths can be due to lack of sufficient oxygen for energy production from lactic acid. These ideas have recently been extended to studies on aquatic pollution
MATERIALS AND METHODS
Only healthy C. batrachus, averaging 40 g in weight and 22 cm in length, were chosen as experimental animals. Fish were acclimated 2 weeks prior to experimentation. They were supplied daily with commercial fish feed at a rate of 2.5% body weight. The insecticide used in this study was a commercial formulation of Rogor (‘‘dimethoate’’ 300 g/liter EC emulsion, Rallis India Ltd.,, Bombay, India). Solution of the test substance was made by dissolving the insecticide in a small quantity of ethanol and diluting with test water to obtain the desired concentration 1.0 mL/liter. The sublethal level of Rogor (21.66 mg/liter) is one-third of the LC50 value (65 mg/liter) for 96 h. Physicochemical characteristics of the laboratory water and the method of
To whom correspondence should be addressed at H. No. 16-3380, Chanchalguda, Hyderabad-500 024. Andhra Pradesh, India. E-mail: [email protected]. 80 0013-9351/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.
81
EFFECT OF ROGOR ON MUSCLE OF Clarias batrachus
lethal concentration determination, which is based on the procedure of Finney (1964), have been described earlier (Begum and Vijayaraghavan, 1994). Experimental fish were divided into two groups of 24 individuals each and placed in separate glass aquaria (capacity: 30 liters each). Group I fish reared in tap water served as control. Group II exposed to 21.66 mg/liter for 192 h served as experimental. The aquaria were cleaned daily and the water, along with the Rogor, was renewed to keep the concentration constant throughout the test period of 24, 48, 96, and 192 h. After the stipulated exposure periods, 6 individuals from each group were removed and sacrificed for muscle tissue sampling. Muscle tissue was immediately removed and frozen and used within an hour for the assay of metabolites and enzymes. Glycogen of muscle tissue was quantitatively estimated by the methods of Kemp and Van Heijninger (1954) using 10% homogen´ ate, w/v, prepared with 80% methanol. Lactate was estimated by the method of Barker and Summerson (1941) using 10% muscle homogenate, w/v. For the assay of enzyme activities 10%, w/v, tissue homogenate was prepared in required media at 4°C in an ice-jacketed glass homogenizer with a motordriven Teflon-coated pestle. The homogenate was centrifuged at 3000 rpm for 15 min at 4°C to remove cell debris. A clear cell-free extract was used for the assay. Homogenate medium containing 100 M phosphate buffer, 0.03 M sucrose, adjusted to pH 7.5, was used for the estimation of lactate dehydrogenase (L-lactate, NAD oxidoreductase EC 1.1.1.27) (King, 1995). For phosphorylase assay, the homogenate was prepared in a medium containing sodium fluoride (0.1 M) and EDTA (0.037 M) at pH 6.8 as suggested by Guillory and Mammaerts (1962) to prevent enzymatic interconversion of the two phosphorylases. The glycogen phosphorylase (1, 4-glucon, orthophosphate glucosyl transferase EC 2.4.1.1.) activity was estimated by the method of Cori et al. (1955) in the direction of glycogen synthesis by determining the amount of inorganic phosphate (P ) formed from glucose 1-phosphate. The P liberated was estimated by the method of Tussaky and Shorr (1953). Protein was estimated by the method of Lowry et al. (1951), using bovine serum albumin as standard. For each parameter, six observations were made. The data were statistically analyzed using Student’s t test. RESULTS
During the exposure to a sublethal dose of Rogor, observations were made to detect the external signs
of poisoning on the exposed C. batrachus. No mortality and visible symptoms by toxic reaction were observed in the fish exposed to 21.66 mg/liter. Rogor exerted an adverse effect on the glycogen content of muscle tissue. A relatively rapid reduction up to 96 h and a slow deceleration of this rate after 192 h of exposure occurred in muscle tissue. There was a significant elevation in the muscle lactate content. The rise was significant throughout the exposure (Table 1). The activity of LDH was elevated in muscle at all treatment periods. Initially elevation was slow and reached a maximum after 48 h and then declined after 96 and 192 h. A significant increase in phosphorylase (a and ab) was observed in muscle tissue
TABLE 1 Biochemical Alterations in Muscle Tissue of C. batrachus exposed to Rogor Exposure Period (in h) Parameters
24
48
96
192
Glycogen (mg/g wet wt)
Con SE Exp SE
1.75 1.98 1.70 1.49 $0.08 $0.02 $0.06 $0.04 1.32A 1.06A 0.63A 0.80,1 $0.08 $0.02 $0.01 $0.40 (!24) (!46) (!63) (!46)
Lactate (mg/m wet wt)
Con SE Exp SE
0.48 0.50 0.53 0.52 $0.01 $0.01 $0.01 $0.01 0.87A 1.01A 1.11A 0.98@ $0.03 $0.02 $0.03 $0.09 (#81) (#99) (#107) (#87)
Lactate dehydrogenase (m mol of formazan/ mg protein/h)
Con SE
0.43 $0.18
Exp SE
0.77,1 0.93A 0.87A 0.80A $0.29 $0.02 $0.03 $0.02 (#77) (#123) (#97) (#73)
Glycogen phosphorylase (m mol of P /mg protein/h) a
Con SE
7.92 $0.80
Exp SE
13.13@ 15.93A 16.00A 13.96? $0.53 $0.34 $0.59 $0.67 (#65) (#68) (#99) (#96)
ab
Con SE Exp SE
15.43 13.73 12.16 15.00 $0.79 $0.53 $1.13 $0.53 22.15@ 26.32A 25.00A 27.92? $1.39 $0.81 $0.72 $0.92 (#43) (#91) (#105) (#86)
0.42 $0.02
9.44 $0.19
0.44 $0.01
8.03 $0.93
0.46 $0.01
7.10 $1.78
Note. Con control; Exp experimental. Values are means$SE of six observations. Values in parentheses are percentage changes over controls. Statistical significance: ?P:0.05, @P:0.01, AP:0.001, ,1 not significant.
82
BEGUM AND VIJAYARAGHAVAN
of exposed fish throughout the exposure period of 192 h (Table 1). DISCUSSION
Results obtained in this investigation are clearly suggestive of disrupted carbohydrate metabolism in C. batrachus exposed to sublethal levels of Rogor. The decrement in glycogen content in the tissue suggests its mobilization to meet energy demands warranted by the toxic environment. Short-term exposure resulted in glycogenolysis in muscle tissue which indicates a typical stress response to Rogor. In order to meet the heightened energy demands of such stressed animals, glycogen may be rapidly catabolized, resulting in huge loses of its reserves. Similar depletion in muscle and liver glycogen of C. batrachus exposed to dichlorvos, an OP pesticide, was observed by Verma et al, (1983). The exact mechanism of stimulation of glycogen breakdown in fish affected by rogor is not yet understood. However, Nakano and Tomlinson (1967) have suggested that catecholamines, whose levels in fish blood rise under stressful environmental conditions, enhance the utilization of glycogen for energy production. This effect on glycogen levels appears to be related, at least to some extent, to the detoxication mechanisms, essential for metabolism or degradation and elimination of the pesticides from the body. The marked depletion in glycogen stores accompanied by an increase in blood glucose levels in C. batrachus exposed to Rogor was reported by Begum and Vijayaraghavan (1994). Although the plasma levels of catecholamines were not monitored in this study, their involvement is likely, for it has been demonstrated that, an enhanced secretion of catecholamines occurs under stress conditions in the rainbow trout (Nakano and Tomlinson, 1967). However, the nature of toxicant, exposure time, and strength of stimulus are important factors in the hormonally mediated changes in tissue glycogen reserves. Thus, the possible mechanism involved in the glycogen depletion may be diverse and could also be due to the modification in other parts of carbohydrate metabolism. Lactate is the end product of glycolysis under hypoxic conditions. The changes in lactate level indicate metabolic disorders. Elevated lactic acid content suggests a severe respiratory stress in the fish tissue. According to Huckabee (1958) an upward trend in lactate in the tissues may be taken to indicate that oxygen supply to the tissues is not adequate for the normal metabolic function. In the present study this condition may be ascribed to the
inability of the stressed C. batrachus to derive enough oxygen from surrounding Rogor-polluted water. The inadequate supply of oxygen in the tissue has facilitated the conversion of pyruvate to lactate, due to enhanced glycolysis for an increased supply of energy. The elevation of tissue lactate in liver and muscle of Sarotherodon mossambicus subjected to the OP malathion reported by Ramalingam (1984) is drawn in support of the present findings. Basha et al. (1984) have shown that though initially there is an increase in the respiratory rate of fish exposed to malathion, it is soon followed by a decreased rate. They ascribed this to the onset of symptoms of poisoning. According to Gill et al. (1988) respiratory distress in P. conchonius exposed to dimethoate is due to degeneration of secondary lamellae, edematous separation of respiratory epithelium, and degenerated chloride cells in the interlamellar crypts of gill tissue. It may be assumed that as a result of reduced efficiency of the damaged gills to function as respiratory organs, other metabolically active tissues like liver and muscle receive less oxygen, leading to severe tissue hypoxia. Development of such internal hypoxic conditions may be ultimately responsible for the shift to the less efficient anerobic metabolism, indicated by the change in lactate content observed during the present study. Lactate dehydrogenase forms the center of a delicately balanced equilibrium between catabolism and anabolism of carbohydrates (Everse and Kaplan, 1973). Stimulation of LDH and the rapid rate of glycolysis observed in the present study indicate that the end product of glycolysis, pyruvate, was not routed through Kreb’s cycle but through the lactic acid cycle under hypoxic conditions, leading to the accumulation of lactic acid. Similar observations were reported by Ghosh (1987) in liver and muscle of C. batrachus exposed to three OP pesticides, adding substantial support to the present observations. The enzyme glycogen phosphorylase is involved in the glycolytic pathway in the initial catalysis of glycogen to glucose 1-phosphate, by which the glycogen is made available for energy release. The increased phosphorylase a and ab activities in muscle tissue of C. batrachus exposed to Rogor confirm the active breakdown of tissue glycogen for metabolic processes to meet the augmented stress conditions. This is also corroborated by the observed decrease in levels of glycogen. Similar increases in phosphorylase a and ab activities were reported in muscle of T. mossambica after exposure to sumithion (Koundinya and Rama Murthy, 1979). The present study shows that Rogor reduces oxidative metabolism in the muscle tissue of C. batrachus.
EFFECT OF ROGOR ON MUSCLE OF Clarias batrachus
Consequently these fish switch over to anerobiosis as evidenced from the increased lactate content in muscle tissue. Further it has already been reported that there is bioaccumulation of this pesticide in the muscle tissue after continuous exposure for 8 days (Begum et al., 1984). Therefore there is a chance of a residual effect of the Rogor in the muscle tissue which will affect the quality of the muscle, making it unfit for human consumption. REFERENCES Barker, S. B., and Summerson, W. H. (1941). The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138, 535—554. Basha, S. M., Prasada Rao, K. S. Sambasiva Rao, K. R. S., and Ramana Rao, K. V. (1984). Respiratory potentials of the fish (T. mossambica) under malathion carbaryl and lindane intoxication. Bull Environ. Contam. Toxicol. 32, 570—574. Begum, G., and Vijayaraghavan, S. (1994). In vivo toxicity of dimethoate on proteins and transaminases in the liver tissue of fresh water fish Clarias batrachus (Linn). Bull. Environ. Contam. Toxicol. 54, 370—375. Begum, G., and Vijayaraghavan, S. (1994). Level of blood glucose in freshwater fish, Clarias batrachus (Linn) during commercial dimethoate intoxication. J. Aquat. Biol. 9, 74—76. Begum, G. Vijayaraghavan, S., Sarma, P. N., and Husain, S. (1994). Study of dimethoate bioaccumulation in liver and Muscle tissues of Clarias batrachus and its elimination following cessation of exposure. Pest. Sci. 40, 201—205. Black, E. C. (1958). Hyperactivity as a lethal factor in fish. J. Fish. Res. Bd. Can. 15, 573—586. Cori G. T., Illingworth, G., and Keller, P. J. (1955). In ‘‘Methods in Enzymology’’ (S. P. Colowick and O. Kaplan, Eds.), Vol. 1, pp. 200—205. Academic Press, New York. Everse, T., and Kaplan, N. O. (1973). Lactate dehydrogenases: Structure and function. In ‘‘Advances in Enzymology’’ (A. Meister, Ed.), Vol. 27, pp. 61—133. Wiley, New York. Finney, D. J. (1964). ‘‘Probit Analysis,’’ p. 20. Cambridge Univ. Press, London. Ghosh, T. K. (1987). Toxic impact of three organophosphate pesticides on carbohydrate metabolism in a freshwater Indian catfish, Clarias batrachus. Proc. Indian. Natl. Sci. Acad. 53, 135—142.
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Gill, T. S., Pant, J. C., and Pant, J. (1988) Liver and kidney lesions associated with experimental exposure to carbaryl and dimethoate in the fish (Puntius conchonius Ham). Bull. Environ. Contam. Toxicol. 41, 71—78. Guillory, R. J., and Mammaerts, W. F. H. M. (1962). The state of activity of phosphorylase in frog sartorius muscle. Biochem. Biophys. Acta 65, 316—325. Huckabee, W. E. (1958). Relationship of pyruvate and lactate during anerobic metabolism. II. Exercise and formation of O2debt. J. Clin. Invest. 37, 255—263. Kemp, A., and Kitsvan Heijinger, A. J. M. (1954). A colorimetric micro method for the determination of glucose and glycogen J. Biochem. 56, 646—648. King, J. (1965). Lactate dehydrogenase. In ‘‘Practical Clinical Enzymology’’, pp. 93—91. Van Nostrand, London. Koundinya, P. R., and Ramamurthi, R. (1979). Effect of OP pesticide sumithion (fentitrothion) on some aspects of carbohydrate metabolism in a freshwater fish, Sarotherodon (Tilapia) mossombicus (Peters). Experientia 35, 1632—1633. Laul, R. T., Pradhan, P. V., and Bhagwat, A. M. (1974). Effect of muscular exercise on glycogen content in Tilapia mossambica (in captivity) J. Biol. Chem. 17, 72—77. Lowry, O. 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. Nakano, T., and Tomlinson, N. (1967). Catecholamine and carbohydrate concentrations in rainbow trout (Salmo gairdneri) in relation to physical disturbance. J. Fish Res. Bd. Can. 24, 1707—1715. Prasada Rao, K. S., and Ramana Rao, K. V. (1983). Regulation of phosphorylases and aldolases in tissues of the teleost (Tilapia mossambica) under methyl parathion impact. Bull. Environ. Contam. Toxicol. 31, 474—478. Ramalingam, K. (1984). Toxic effects of DDT, malathion and mercury on the tissue carbohydrate metabolism of Sarotherodon mossambicus (Peters) Proc. Indian. Acad. Sci. 93, 443—448. Tussaky, H. H., and Shorr, E. (1953). A micro colorimetric method for the determination of inorganic phosphate. J. Biol. Chem. 202, 675—686. Verma, S. R., Saritha Rani, Tonk, I. P., and Dalela, R. C. (1983). Pesticide induced dysfunction in carbohydrate metabolism in three freshwater fishes. Water Res., 127—133.
Effect of Acute Exposure of the Organophosphate Insecticide Rogor on Some Biochemical Aspects of Clarias batrachus (Linnaeus) Ghousia Begum1 and Shantha Vijayaraghavan Department of Zoology, Osmania University, Hyderabad, 500 007, Andhra Pradesh, India Received August 22, 1997
to explain the increased damage to aquaculture due to pollution. Today, pesticides have occupied all segments of the environment due to their use in field operations as potent and economically useful poisons. This in turn affects the nontarget organisms. It is known that in fishes organophosphate (OP) pesticides are neurotoxic and they inhibit acetylcholinesterase activity with subsequent disruption of nervous functions, thereby interfering with some of the vital physiological functions (Prasada Rao and Ramana Rao, 1983). Energy metabolism plays a key role as the animal is forced to expend more energy to overcome toxic stress. Based on this, an attempt was made to study the sublethal toxicity of Rogor (an OP insecticide) on the carbohydrate metabolism taking key metabolites and enzymes in the muscle tissue of Clarias batrachus. This freshwater, air-breathing teleost fish was selected because of its wide availability and edibility in India. Muscle tissue of fish is the main consumable part and bioanalysis of this tissue is very essential in order to monitor fish quality for human consumption and health.
Indian catfish (Clarias batrachus), a common fish of commercial importance, was exposed to a sublethal concentration (1/3 of 96 h LC50 value) of Rogor for periods ranging from 24 to 192 h. The changes in glycogen, lactate, lactate dehydrogenase, and glycogen phosphorylase (a, ab) contents of muscle tissue were studied. A gradual decrease in muscle glycogen and an increase in lactate contents were observed. The activity level of lactate dehydrogenase showed a sharp rise initially for 48 h followed by a decline after 96 and 192 h. The glycogen phosphorylase a and ab in muscle tissue enhanced in response to Rogor exposure. 1999 Academic Press
Key Words : food fish; pesticide concentration; environmental pollution.
INTRODUCTION
Investigations have shown that changes in carbohydrate metabolism in fish induced by the stress of severe muscular exercise resemble the changes displayed by higher vertebrates like mammals. These changes include depletion of glycogen stores from fish tissues like liver and muscle (Laul et al., 1974). Black (1958) reported an elevation in lactic acid levels in liver, muscle, and blood and suggested that an uncontrolled entry of lactic acid into the tissues interferes with internal mechanisms that maintain the acid base balance. Lactic acid may also reduce the affinity of hemoglobin for both oxygen and carbondioxide, diminishing the oxygen-carrying capacity of blood. He stated that in these cases fish deaths can be due to lack of sufficient oxygen for energy production from lactic acid. These ideas have recently been extended to studies on aquatic pollution
MATERIALS AND METHODS
Only healthy C. batrachus, averaging 40 g in weight and 22 cm in length, were chosen as experimental animals. Fish were acclimated 2 weeks prior to experimentation. They were supplied daily with commercial fish feed at a rate of 2.5% body weight. The insecticide used in this study was a commercial formulation of Rogor (‘‘dimethoate’’ 300 g/liter EC emulsion, Rallis India Ltd.,, Bombay, India). Solution of the test substance was made by dissolving the insecticide in a small quantity of ethanol and diluting with test water to obtain the desired concentration 1.0 mL/liter. The sublethal level of Rogor (21.66 mg/liter) is one-third of the LC50 value (65 mg/liter) for 96 h. Physicochemical characteristics of the laboratory water and the method of
To whom correspondence should be addressed at H. No. 16-3380, Chanchalguda, Hyderabad-500 024. Andhra Pradesh, India. E-mail: [email protected]. 80 0013-9351/99 $30.00 Copyright 1999 by Academic Press All rights of reproduction in any form reserved.
81
EFFECT OF ROGOR ON MUSCLE OF Clarias batrachus
lethal concentration determination, which is based on the procedure of Finney (1964), have been described earlier (Begum and Vijayaraghavan, 1994). Experimental fish were divided into two groups of 24 individuals each and placed in separate glass aquaria (capacity: 30 liters each). Group I fish reared in tap water served as control. Group II exposed to 21.66 mg/liter for 192 h served as experimental. The aquaria were cleaned daily and the water, along with the Rogor, was renewed to keep the concentration constant throughout the test period of 24, 48, 96, and 192 h. After the stipulated exposure periods, 6 individuals from each group were removed and sacrificed for muscle tissue sampling. Muscle tissue was immediately removed and frozen and used within an hour for the assay of metabolites and enzymes. Glycogen of muscle tissue was quantitatively estimated by the methods of Kemp and Van Heijninger (1954) using 10% homogen´ ate, w/v, prepared with 80% methanol. Lactate was estimated by the method of Barker and Summerson (1941) using 10% muscle homogenate, w/v. For the assay of enzyme activities 10%, w/v, tissue homogenate was prepared in required media at 4°C in an ice-jacketed glass homogenizer with a motordriven Teflon-coated pestle. The homogenate was centrifuged at 3000 rpm for 15 min at 4°C to remove cell debris. A clear cell-free extract was used for the assay. Homogenate medium containing 100 M phosphate buffer, 0.03 M sucrose, adjusted to pH 7.5, was used for the estimation of lactate dehydrogenase (L-lactate, NAD oxidoreductase EC 1.1.1.27) (King, 1995). For phosphorylase assay, the homogenate was prepared in a medium containing sodium fluoride (0.1 M) and EDTA (0.037 M) at pH 6.8 as suggested by Guillory and Mammaerts (1962) to prevent enzymatic interconversion of the two phosphorylases. The glycogen phosphorylase (1, 4-glucon, orthophosphate glucosyl transferase EC 2.4.1.1.) activity was estimated by the method of Cori et al. (1955) in the direction of glycogen synthesis by determining the amount of inorganic phosphate (P ) formed from glucose 1-phosphate. The P liberated was estimated by the method of Tussaky and Shorr (1953). Protein was estimated by the method of Lowry et al. (1951), using bovine serum albumin as standard. For each parameter, six observations were made. The data were statistically analyzed using Student’s t test. RESULTS
During the exposure to a sublethal dose of Rogor, observations were made to detect the external signs
of poisoning on the exposed C. batrachus. No mortality and visible symptoms by toxic reaction were observed in the fish exposed to 21.66 mg/liter. Rogor exerted an adverse effect on the glycogen content of muscle tissue. A relatively rapid reduction up to 96 h and a slow deceleration of this rate after 192 h of exposure occurred in muscle tissue. There was a significant elevation in the muscle lactate content. The rise was significant throughout the exposure (Table 1). The activity of LDH was elevated in muscle at all treatment periods. Initially elevation was slow and reached a maximum after 48 h and then declined after 96 and 192 h. A significant increase in phosphorylase (a and ab) was observed in muscle tissue
TABLE 1 Biochemical Alterations in Muscle Tissue of C. batrachus exposed to Rogor Exposure Period (in h) Parameters
24
48
96
192
Glycogen (mg/g wet wt)
Con SE Exp SE
1.75 1.98 1.70 1.49 $0.08 $0.02 $0.06 $0.04 1.32A 1.06A 0.63A 0.80,1 $0.08 $0.02 $0.01 $0.40 (!24) (!46) (!63) (!46)
Lactate (mg/m wet wt)
Con SE Exp SE
0.48 0.50 0.53 0.52 $0.01 $0.01 $0.01 $0.01 0.87A 1.01A 1.11A 0.98@ $0.03 $0.02 $0.03 $0.09 (#81) (#99) (#107) (#87)
Lactate dehydrogenase (m mol of formazan/ mg protein/h)
Con SE
0.43 $0.18
Exp SE
0.77,1 0.93A 0.87A 0.80A $0.29 $0.02 $0.03 $0.02 (#77) (#123) (#97) (#73)
Glycogen phosphorylase (m mol of P /mg protein/h) a
Con SE
7.92 $0.80
Exp SE
13.13@ 15.93A 16.00A 13.96? $0.53 $0.34 $0.59 $0.67 (#65) (#68) (#99) (#96)
ab
Con SE Exp SE
15.43 13.73 12.16 15.00 $0.79 $0.53 $1.13 $0.53 22.15@ 26.32A 25.00A 27.92? $1.39 $0.81 $0.72 $0.92 (#43) (#91) (#105) (#86)
0.42 $0.02
9.44 $0.19
0.44 $0.01
8.03 $0.93
0.46 $0.01
7.10 $1.78
Note. Con control; Exp experimental. Values are means$SE of six observations. Values in parentheses are percentage changes over controls. Statistical significance: ?P:0.05, @P:0.01, AP:0.001, ,1 not significant.
82
BEGUM AND VIJAYARAGHAVAN
of exposed fish throughout the exposure period of 192 h (Table 1). DISCUSSION
Results obtained in this investigation are clearly suggestive of disrupted carbohydrate metabolism in C. batrachus exposed to sublethal levels of Rogor. The decrement in glycogen content in the tissue suggests its mobilization to meet energy demands warranted by the toxic environment. Short-term exposure resulted in glycogenolysis in muscle tissue which indicates a typical stress response to Rogor. In order to meet the heightened energy demands of such stressed animals, glycogen may be rapidly catabolized, resulting in huge loses of its reserves. Similar depletion in muscle and liver glycogen of C. batrachus exposed to dichlorvos, an OP pesticide, was observed by Verma et al, (1983). The exact mechanism of stimulation of glycogen breakdown in fish affected by rogor is not yet understood. However, Nakano and Tomlinson (1967) have suggested that catecholamines, whose levels in fish blood rise under stressful environmental conditions, enhance the utilization of glycogen for energy production. This effect on glycogen levels appears to be related, at least to some extent, to the detoxication mechanisms, essential for metabolism or degradation and elimination of the pesticides from the body. The marked depletion in glycogen stores accompanied by an increase in blood glucose levels in C. batrachus exposed to Rogor was reported by Begum and Vijayaraghavan (1994). Although the plasma levels of catecholamines were not monitored in this study, their involvement is likely, for it has been demonstrated that, an enhanced secretion of catecholamines occurs under stress conditions in the rainbow trout (Nakano and Tomlinson, 1967). However, the nature of toxicant, exposure time, and strength of stimulus are important factors in the hormonally mediated changes in tissue glycogen reserves. Thus, the possible mechanism involved in the glycogen depletion may be diverse and could also be due to the modification in other parts of carbohydrate metabolism. Lactate is the end product of glycolysis under hypoxic conditions. The changes in lactate level indicate metabolic disorders. Elevated lactic acid content suggests a severe respiratory stress in the fish tissue. According to Huckabee (1958) an upward trend in lactate in the tissues may be taken to indicate that oxygen supply to the tissues is not adequate for the normal metabolic function. In the present study this condition may be ascribed to the
inability of the stressed C. batrachus to derive enough oxygen from surrounding Rogor-polluted water. The inadequate supply of oxygen in the tissue has facilitated the conversion of pyruvate to lactate, due to enhanced glycolysis for an increased supply of energy. The elevation of tissue lactate in liver and muscle of Sarotherodon mossambicus subjected to the OP malathion reported by Ramalingam (1984) is drawn in support of the present findings. Basha et al. (1984) have shown that though initially there is an increase in the respiratory rate of fish exposed to malathion, it is soon followed by a decreased rate. They ascribed this to the onset of symptoms of poisoning. According to Gill et al. (1988) respiratory distress in P. conchonius exposed to dimethoate is due to degeneration of secondary lamellae, edematous separation of respiratory epithelium, and degenerated chloride cells in the interlamellar crypts of gill tissue. It may be assumed that as a result of reduced efficiency of the damaged gills to function as respiratory organs, other metabolically active tissues like liver and muscle receive less oxygen, leading to severe tissue hypoxia. Development of such internal hypoxic conditions may be ultimately responsible for the shift to the less efficient anerobic metabolism, indicated by the change in lactate content observed during the present study. Lactate dehydrogenase forms the center of a delicately balanced equilibrium between catabolism and anabolism of carbohydrates (Everse and Kaplan, 1973). Stimulation of LDH and the rapid rate of glycolysis observed in the present study indicate that the end product of glycolysis, pyruvate, was not routed through Kreb’s cycle but through the lactic acid cycle under hypoxic conditions, leading to the accumulation of lactic acid. Similar observations were reported by Ghosh (1987) in liver and muscle of C. batrachus exposed to three OP pesticides, adding substantial support to the present observations. The enzyme glycogen phosphorylase is involved in the glycolytic pathway in the initial catalysis of glycogen to glucose 1-phosphate, by which the glycogen is made available for energy release. The increased phosphorylase a and ab activities in muscle tissue of C. batrachus exposed to Rogor confirm the active breakdown of tissue glycogen for metabolic processes to meet the augmented stress conditions. This is also corroborated by the observed decrease in levels of glycogen. Similar increases in phosphorylase a and ab activities were reported in muscle of T. mossambica after exposure to sumithion (Koundinya and Rama Murthy, 1979). The present study shows that Rogor reduces oxidative metabolism in the muscle tissue of C. batrachus.
EFFECT OF ROGOR ON MUSCLE OF Clarias batrachus
Consequently these fish switch over to anerobiosis as evidenced from the increased lactate content in muscle tissue. Further it has already been reported that there is bioaccumulation of this pesticide in the muscle tissue after continuous exposure for 8 days (Begum et al., 1984). Therefore there is a chance of a residual effect of the Rogor in the muscle tissue which will affect the quality of the muscle, making it unfit for human consumption. REFERENCES Barker, S. B., and Summerson, W. H. (1941). The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138, 535—554. Basha, S. M., Prasada Rao, K. S. Sambasiva Rao, K. R. S., and Ramana Rao, K. V. (1984). Respiratory potentials of the fish (T. mossambica) under malathion carbaryl and lindane intoxication. Bull Environ. Contam. Toxicol. 32, 570—574. Begum, G., and Vijayaraghavan, S. (1994). In vivo toxicity of dimethoate on proteins and transaminases in the liver tissue of fresh water fish Clarias batrachus (Linn). Bull. Environ. Contam. Toxicol. 54, 370—375. Begum, G., and Vijayaraghavan, S. (1994). Level of blood glucose in freshwater fish, Clarias batrachus (Linn) during commercial dimethoate intoxication. J. Aquat. Biol. 9, 74—76. Begum, G. Vijayaraghavan, S., Sarma, P. N., and Husain, S. (1994). Study of dimethoate bioaccumulation in liver and Muscle tissues of Clarias batrachus and its elimination following cessation of exposure. Pest. Sci. 40, 201—205. Black, E. C. (1958). Hyperactivity as a lethal factor in fish. J. Fish. Res. Bd. Can. 15, 573—586. Cori G. T., Illingworth, G., and Keller, P. J. (1955). In ‘‘Methods in Enzymology’’ (S. P. Colowick and O. Kaplan, Eds.), Vol. 1, pp. 200—205. Academic Press, New York. Everse, T., and Kaplan, N. O. (1973). Lactate dehydrogenases: Structure and function. In ‘‘Advances in Enzymology’’ (A. Meister, Ed.), Vol. 27, pp. 61—133. Wiley, New York. Finney, D. J. (1964). ‘‘Probit Analysis,’’ p. 20. Cambridge Univ. Press, London. Ghosh, T. K. (1987). Toxic impact of three organophosphate pesticides on carbohydrate metabolism in a freshwater Indian catfish, Clarias batrachus. Proc. Indian. Natl. Sci. Acad. 53, 135—142.
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