Effect of Deltamethrin on glycogen phosphorylase and glucose-6-phosphatase activity in freshwater fish Labeo rohita.
Globally agriculture accounts for nearly 70 percent of all fresh water resources withdrawn from rivers, lakes and underground water etc. In addition, increasing population and industries have put further pressure on these limited resources. The problem associated with monoculture practices such as use of synthetic fertilizers, pesticides have become integral part of intensive agriculture. The aquatic organisms are subjected to stress by the dissolved pesticides which enter into the aquatic medium by the surface runoff. Alteration in the chemical composition of a natural aquatic environment usually induces changes in the behavior and physiological aspects of the inhabitants. Particularly the fish (O'Brien, 1967). Studies have revealed the importance of principal tissue metabolites viz carbohydrates, proteins and fats as energy sources as well as biomass builders. Carbohydrates serve as a reservoir of chemical energy, which is utilized according to the requirement of organisms. Animal coming in contact with pesticide experience stress and this stressful situation elicit neuroendocrine response, which in turn induces disturbance in carbohydrate metabolism. Administration of chemicals has been shown to alter the above metabolites (Murthy and Priyamvadadevi 1982; Palanichamy et al., 1986; Rajalakshmi and Reddi., 1988).Alongwith principal metabolites, intermediate metabolites may also provide information regarding energy metabolism and path way. But their in tissues are in turn influenced by the metabolic state of the organism. In the present study, an attempt has been made to study the effect of deltamethrin on enzymatic changes in carbohydrate metabolism in the freshwater fish Labeo rohita.
MATERIALS AND METHODS
The freshwater fish Labeo rohita of weight 10 [+ or -] 2 g and length 8-12 cm were collected from the unpolluted ponds in and around Anantapur, Andhra Pradesh. The fish were fed daily with commercial fish pellets Ad libitum having around 50% protein content and chopped sheep liver once in two days, and allowed to acclimatize for 15 days. Water was renewed every day to provide freshwater, rich in oxygen. Commercial grade deltamethrin (2.8% EC) of liquid formations manufactured by Agrevo India Ltd. (Trade name Decis) was purchased from local agro-chemical stores and the 96 h LC50 values were determined following the method of Finney Probit Kill-theory (1971). The fish were exposed to lethal (96 h LC50 i.e., 1 ppm) and sublethal (1/10th 96 h LC50 i.e., 0.1 ppm) concentrations of deltamethrin and at the end of the exposure periods, the fish were stunned to death and target organs like gill, liver and muscle were dissected out and glycogen phosphorylase was estimated by method of Sutherland (1955) and glucose-6-phosphatase was estimated by method of Yeung et al,. (1967). The animals were starved for 24 hours prior to each estimation so as to eliminate the possibility of differential feeding if any influencing the estimations. Statistical analysis was done according to Duncan's multiple range (DMR) test .
From the data presented in the table 1, it was noticed that there is significant elevation in the glycogen phosphorylase level in the gill, muscle and liver of fish exposed to lethal and sublethal concentration of deltamethrin. The glycogen phosphorylase content was very high in the liver of control fish than in muscle and gill. The percent increase in both lethal and sublethal concentrations was also more in liver compared to muscle and gill. Contrasting trends of elevation was noticed in lethal and sublethal exposure periods. In the tissues of fish exposed to lethal concentration, there was a progressive increase from day 1 to day 4, but in tissues of fish exposed to sublethal concentration there was a progressive reduction in the elevation of this enzyme from day 1 to day 8.
Glucose-6-phosphatase activity showed a significant increase in the liver tissue of fish exposed to lethal and sublethal concentration of deltamethrin (table 2). The elevation increased with increase in the lethal exposure period from day 1 to day 4. But the elevation was reduced with the increase in sublethal exposure periods of day 1, day 4 and day 8.
The control of glycogen metabolism by the enzymes phosphorylase and glycogen synthetase is effected in animal tissues mainly through the inter conversions of the active and inactive forms of these enzymes. Glycogenolysis is initiated by an enzyme, active phosphorylase 'a' ultimately producing glucose. The glucose so produced or the glucose absorbed from diet undergoes a series of reactions through anaerobic glycolytic pathway in the tissues to produce pyruvic acid and then lactic acid or the pyruvic acid undergoes oxidative decarboxylation to form acetyl Co-A which is channelised into aerobic Kreb's cycle, which in its cycle generates reduced nucleotides (NADH and FAD) for the ultimate generation of biological currency, ATP through electron transport system. Glycogen phosphorylase is an enzyme that exists in two forms, one activated with adenosine mono phosphate (AMP) and the other activated without it, which are interconversible by phosphorylation and dephosphorylation mechanism (Cori and Cori 1945; Cori et al., 1955). This, being an enzyme concerned with the metabolic breakdown of glycogen, assumes considerable importance in studies involving liver and muscle glycogen levels. In addition to phosphorylase activity, in the liver, glucose to phosphatase acts as a catalyst in the conversion of glucose-6-phosphate to glucose. Hydrolysis of glucose-6-phosphate is a key step in the process of gluconeogenesis. During stress conditions, the alterations in blood glucose glycogen levels in liver and muscle in fishes affect the rate of activity of these two enzymes.
The phosphorylation of phosphorylase 'b' to phosphorylase 'a' catalyzed by phosphorylase 'b' kinase in turn also exists in active (phosphorylated) and an inactive (non-phosphorylation) form. The latter is converted to the former in the presence of ATP and another enzyme called protein kinase. Protein kinase exists in an inactive form, which in turn activated by cyclic AMP. Epinephrine, a primary messenger acts by activating the adenyl cyclase system which catalyzes the synthesis of cyclic AMP from ATP. A single stimulus, the release of epinephrine, results not only in the increased glycogen break down by phosphorylase but also, decreased glycogen synthesis through the formation of glycogen synthetase D (an inactive form). Glycogen is the main catalytic force in the chain of chemical events which inclined towards the using up of glycogen. Because of this reason alone phosphorylase enjoys the strategic position in the glycolytic sequence (Villara Palace and Larner, 1970; Lehninger, 1978; Harper et al., 1979).
In the present investigation phosphorylase activity increased in all exposure tissues. It suggests the active break down of the tissue glycogen to enhance the energy output to meet the augmented energy demand under pesticidal stress. It indicates active break down of tissue glycogen for energy production. This is necessary for the induction and synthesis of detoxifying enzymes and transportation of necessary metabolites under stress condition. Aquatic animals mostly gill breathers suffer from hypoxia in tissues under pesticide exposure breakdown through activation of tissue phosphorylase 'a' enzyme. The boosting up of phosphorylase 'a' activity may be the result of hormonal imbalance caused by the deltamethrin treatment since it is known that pesticide administration leads to increased glycogen production which activates cyclic AMP.
The studies in Killi fish and brown bull head have indicated that epinephrine induced hepatic glycogenolysis and increased the specific activity of hepatic glycogen phosphorylase along with cyclic AMP (Umminger and Benziger, 1975). Apart from epinephrine, excess in divalent cations like Ca++ is also known to convert inactive phosphorylase 'b' to active 'a' form (Siva Prasada Rao, 1980 and Rafat Yasmeen, 1986). The release of free intra cellular calcium by hormones or by muscle depolarization provides a secondary mechanism of regulating the phosphorylase system (Rafat Yasmeen, 1986). Prevalence of anoxic/hypoxic conditions (Dezwan and Zandee, 1972; Siva Prasada Rao, 1980) due to pesticide stress in the tissues may also be responsible for the enhanced phosphorylase 'a' activity. Ultimately resulting in the depletion of glycogen reserves supports the results obtained in the present study. Ghousia Begum and Vasantha Vijaya Raghavan (1999) observed that the enzyme glycogen phosphorylase is involved in glycolytic pathway in the initial catalysis of glycogen to glucose-1-phosphate, by which the glycogen is made available for energy releases under stress. The increased phosphorylase 'a' activity in muscle tissue of C. batrachus exposed to Rogar confirm the active breakdown of tissue glycogen for metabolic processes to meet the augmented stress conditions. This is also supported by the observed decrease in phosphorylase 'a' reported in muscle of T. mossambica after exposure to sumithion (Koundinya and Ramamurthy, 1979). Rogar reduced oxidative metabolism in the muscle tissue of C. batrachus. Consequently these fish switch over to anaerobiosis 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 (Begum and Raghavan, 1995).
Elevated phosphorylase 'a' activity was observed in the liver, muscle and tissue of Claris batrachus exposed endosulfan (Srinivas, 1993). Increase in the activity of phosphorylase throughout the exposure period suggests the increased oxidation of glucose was through glycolytic pathway. Depletion in the glucose could also be attributed to its sequestration into blood (Gopal et al., 1980). It indicate glycolysis and hyperglycemia by activation of the phospshorylase enzyme system during pesticide poisoning (Reddy et al., 1994). Glucose-6-phosphatase is localized in the endoplasmic reticulum of the cell. It catalysis the rate limiting reaction of glucose-6-phosphatase into the glucose and phosphate. Any effect on the activity of the enzyme due to pesticide impact will result in impairment of carbohydrate metabolism (Grant and Mehrle, 1970). Glucose-6-phosphatase, an enzyme present in liver but not in muscle. The Mg2+ dependent enzyme is found in the endoplasmic reticulum of hepatocytes. Glucose produced by gluconeogenesis in the liver or in the diet is delivered to brain and muscle through the blood stream. Glucose-6-phosphatase may be oxidized for energy production via glycolysis, decarboxylation of pyruvate and citric acid cycle. Glucose-6-phosphatase is the substrate for the pentose phosphate pathway, yielding, both reducing power NADH, needed for the biosynthesis of fatty acids and cholesterol.
In the present study glucose-6-phosphatase activity was elevated in liver tissue of deltamethrin exposed to Labeo rohita. It is evident from the results that the liver is organ which is most affected as site of carbohydrate metabolism. It is also the first organ to receive molecule carried through portal circulation. Pesticide treatment blocks the mobilization of liver glycogen by inhibiting the glucose-6-phosphatese activity. David (1995) observed elevated levels of glucose-6-phosphatase activity in Labeo rohita exposed to fenvalerate. Rashatwar and Ilyas (1984) studied the toxic effect of phosphomidon on glucose-6-phosphatase activity in the fish Nemachelius, Sastry et al., (1993) observed aldrin induced alterations in the activity of glucose-6-phosphatase in the freshwater teleost fish Channa punctatus. Srinivas (1993) studied the glucose-6-phosphatase in the fresh water fish Clarias batrachus which exposed to endosulfan. Sastry and Sharma (1980) observed the glucose-6-phosphatase activity in Ophiocephalus exposed to diazinon. Sastry and Sharma (1980) observed in Nemachelius denisonii exposed to phosphomidon, are in support of present results obtained and hence it is evident that deltamethrin affects the carbohydrate metabolism through inhibitory effect of an enzyme system.
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* V.Venkata Rathnamma, M. Vijaya Kumar and G.H. Philip
* Department of Zoology, Acharya Nagarjuna University, Nagarjunanagar-522510, Guntur. (A.P.), India
Table-1: Glycogen phosphorylase activity ([micro]M/mg/protein/h) in the tissues of fish Labeo rohita on exposure to lethal and sublethal concentrations of deltamethrin Name of Control Exposure period in days the Tissue Lethal Sublethal 1 4 1 4 8 Gill 0.667b 0.749e 1.155f 0.475a 0.740d 0.7122c SD [+ or -] 0.021 0.022 0.015 0.019 0.016 0.002 % change 12.3 73.21 12.03 10.94 6.74 Liver 3.970a 4.370c 7.112f 4.558e 4.482d 4.319b SD [+ or -] 0.013 0.023 0.006 0.033 0.005 0.006 % change 10.07 79.11 17.80 12.89 8.77 Muscle 2.566a 3.064e 4.336f 2.869d 2.847c 2.738b SD [+ or -] 0.022 0.001 0.023 0.023 0.037 0.018 % change +19.43 +68.98 +11.80 +10.97 +6.70 Means are [+ or -] SD (n = 6) for a tissue in a column followed by the same letter are not signiftly different (P < 0.05) from each other according to Duncan's multiple range (DMR) test. The values below the mean are per cent change over control. Table-2: Glucose-6-phosphatase activity ([micro]M/mg/protein/h) in the tissues of fish Labeo rohita on exposure to lethal and sublethal concentrations of deltamethrin Name of Exposure period in days the Control Tissue Lethal Sublethal 1 4 1 4 8 Liver 2.462a 2.809d 3.969f 2.958e 2.777c 2.743b SD [+ or -] 0.244 0.004 0.017 0.026 0.012 0.022 % change +14.09 +61.20 +20.15 +12.81 +11.39