INFLUENCE OF QUINALPHOS, AN ORGANOPHOSPHOROUS PESTICIDE, ON THE ANTIOXIDANT DEFENSE SYSTEM IN HEPATIC SUBCELLULAR

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1 International Journal of Zoology and Research (IJZR) ISSN(P): ; ISSN(E): Vol. 5, Issue 6, Dec 2015, 7-16 TJPRC Pvt. Ltd. INFLUENCE OF QUINALPHOS, AN ORGANOPHOSPHOROUS PESTICIDE, ON THE ANTIOXIDANT DEFENSE SYSTEM IN HEPATIC SUBCELLULAR FRACTIONS OF FISH, OREOCHROMIS MOSSAMBICUS (PETERS, 1852) ABSTRACT P. SURYA, P. V. VIDYA & K. C. CHITRA Department of Zoology, University of Calicut, Thenhipalam, Kerala, India The freshwater fish, Oreochromis mossambicus were exposed to quinalphos at sub lethal concentration, i.e., 0.5 µg/ L for 24, 72 and 96 h. Treatment groups showed no significant (P<0.05) changes in the body weight of animal, however, a slight decrease in the hepatosomatic index were observed in treated fishes. Mucous deposition was significantly increased at 72 and 96 h of quinalphos exposure which indicate the first line of defensive mechanism of the exposed fishes to quinalphos. Activity of antioxidant enzymes as superoxide dismutase, catalase, glutathione reductase and the level of lipid peroxidation was observed in mitochondrial, nuclear and microsomal fractions of both control and treated groups. The activity of superoxide dismutase, catalase and glutathione reductase decreased significantly in all subcellular fractions of treatment groups with concomitant increase in the level of lipid peroxidation. These findings indicate that quinalphos induced oxidative stress and altered antioxidant defense system in subcellular fractions of fish hepatocytes. In addition, one of the marker enzymes in liver, alanine aminotransferase was found to be increased in all subcellular fractions of every treatment groups which reveal that the increase is to meet the energy demand by the additional supply of glucose under the toxic condition. Therefore, acute toxicity of quinalphos at sub lethal concentration imbalance antioxidant defense system in hepatic subcellular fractions of fish, Oreochromis mossambicus. Original Article KEYWORDS: Quinalphos, Oreochromis Mossambicus, Liver, Subcellular Fractions, Oxidative Stress, Antioxidant Enzymes, Lipid Peroxidation, Alanine Aminotransferase Received: Nov 17, 2015; Accepted: Nov 25, 2015; Published: Nov 28, 2015; Paper Id.: IJZRDEC20152 INTRODUCTION In recent years, human intervention has brought major changes in the aquatic ecosystem. One of the important such intervention is the application of pesticides in agriculture in order to enhance the food production. However, pesticides are found to reach the aquatic ecosystems and their deleterious effects are often noticed in non-target organisms including fish. Fish contribute to the economy of many nations and provide recreational and psychological value to the naturalists, sports, enthusiast and aquarist, which also serve as a direct link to human through food chain. Fish are able to uptake and retain different environmental contaminants dissolved in water via active or passive processes. The interest in understanding the physiological mechanisms associated with fish responding to environmental contaminants has been growing in recent years. Sub-lethal concentrations of pesticides in aquatic environments have been shown to cause structural and functional changes in aquatic organisms and this is more common than the mortality of the animals (Sancho et al., 2003). Organophosphates has been shown to inhibit oxidative phosphorylation coupled with high-energy editor@tjprc.org

2 8 P. Surya, P. V. Vidya & K. C. Chitra consumption, which widely affects the metabolism of cell/ tissues by altering cytochrome P450s and generation of reactive oxygen species in large amount, which eventually leads to oxidative stress. Quinalphos, an organophosphorous pesticide has been extensively applied in Indian agriculture for pest eradication, as well it is pertinent to study its hazardous effect on the aquatic system as it is assumed that its residue might affect the health status of the fish. It is well documented that the fish may serve as good biomonitoring tools, and, in addition to mammals, may provide useful models for further research in understanding fish antioxidant system. Researches in fish demonstrated that mammalian and piscine systems exhibit similar toxicological and adaptive responses to oxidative stress (Kelly et al., 1998). There are several advantages in using piscine model to study oxidative stress, such as the reduction in the number of mammals used in research, the potential for a reduction in the cost of animal maintenance, and the ability to increase the power of the experiment by increasing the number of organisms per study. Furthermore, the use of piscine models to study oxidative stress allows for the evaluation of environmental issues from both a human health and ecological perspective. Therefore, the main objective of the present study is to evaluate the sublethal effect of quinalphos on the antioxidant defense system in subcellular organelles such as mitochondrial, nuclear and microsomal fractions, which provide an effective tool to diagnose the acute toxic effect of the organophosphorous compound in freshwater fish, Oreochromis mossambicus. MATERIALS AND METHODS Standardization Procedures Fresh water fish, Oreochromis mossambicus weighing 3.5 ± 0.5 g and length 4.5 ± 1 cm were collected from a fish farm, Kaloos Aquarium, Kottakal, Malappuram District, Kerala. Fishes were acclimatized to the laboratory conditions in dechlorinated and well-aerated cement tanks of 40 L capacity for four weeks prior to experiments with constant supply of water and good lighting system. The physico-chemical features of the tap water were estimated as per APHA (1998) by maintaining water temperature in the test ranged from 28 ± 2 C, oxygen saturation of water between 70 and 100 % and ph between 6.5 to 7.0. Median Lethal Concentration The LC 50 values of quinalphos for 96 h were determined by probit analysis, with a confident limit of 5 % level (Finney, 1971). The concentration of any toxicant which kills 50 percentage of the test animals during a specific period is referred to as median lethal concentration (LC 50 ) or median tolerance limit. For determining the median lethal concentration, 10 animals were exposed to different concentrations (1, 2, 3, 4, 5, 6 and 7 µg/ L) of quinalphos for 96 h along with separate control group, without toxicant. LC h that kills 50 % of exposed animals was computed on the basis of probit analysis, which was 5 µg/ L. One-tenth of quinalphos concentration (0.5 µg/ L) was chosen as sublethal concentration and it was used in the present study. Treatments Quinalphos - O, O-Diethyl O-2-quinoxalinyl phosphorothioate of 97% purity was used in the experiment. Animals were grouped into four, three tanks with toxicant and a control tank, each group with ten fishes were exposed to single sublethal concentration (0.5 µg/ L) for three durations (24, 72 and 96 h) and were maintained separately. Impact Factor (JCC): NAAS Rating: 2.59

3 Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System 9 in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852) Group I: Control group (without toxicant) Group II: Quinalphos at 0.5 µg/ L for 24 h Group III: Quinalphos at 0.5 µg/ L for 72 h Group IV: Quinalphos at 0.5 µg/ L for 96 h Killing of Animals and Tissue Processing The fish was caught very gently using a small dip net, one at a time with least disturbance. At the end of each exposure time, fishes were decapitated and liver tissue was dissected. Different sub-cellular fractions were obtained by the differential centrifugation method as described by Palade and Siekevitz (1956). A 1% (w/v) homogenate of liver tissue was prepared in ice-cold 0.25 M sucrose solution with the help of a motor-driven glass Teflon homogenizer on crushed ice for a minute. The homogenate was centrifuged at 1000g for 10 minutes at 4 C to obtain the nuclear pellet. Mitochondrial pellet was obtained by centrifuging the post-nuclear supernatant at 10,000g for 10 minutes at 4 C. As microsomal membranes can sediment prematurely during traditional pre-clearances (6,000 10,000g), it is evidently not necessary to use ultracentrifugation (100,000g) to collect microsomes. In fact, it is possible to sediment quantitatively all major microsomal-type membranes at 21,000g in a normal bench-top microcentrifuge. The obtained nuclear, mitochondrial and microsomal fractions were used for the following biochemical estimations. Biochemical Analysis Protein was estimated by the method of Lowry et al (1951) with bovine serum albumin as the standard. Activity of antioxidant enzymes as superoxide dismutase (Marklund and Marklund, 1974), catalase (Claiborne, 1985), glutathione reductase (Carlberg and Mannervik, 1985) was estimated. Level of lipid peroxidation (Ohkawa et al., 1979) and the activity of alanine aminotransferase were measured as described by Segal and Matsuzawa (1970). Statistical Analyses All biochemical estimations were carried out in duplicate and are presented as mean ± SD for ten animals per group where the differences were set significant at p<0.05 against the control group which were denoted with asterisk (*) symbol in the figures. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Duncan s Multiple Range test using statistical package SPSS RESULTS Exposure to quinalphos at the sub lethal concentration of 0.5 µg/ L showed no significant changes in the body weight after the end of 24 h, 72 h and 96 h of treatment when compared to those of the corresponding control group (Figure 1). editor@tjprc.org

4 10 P. Surya, P. V. Vidya & K. C. Chitra Figure 1: Effect of Quinalphos on the Body Weights of Freshwater Fish, Oreochromis Mossambicus The hepatosomatic index of the quinalphos-treated fishes showed a slight decrease in all treatment groups, but the result was not significant when compared to that of the control group (Figure 2). Figure 2: Effect of Quinalphos on the Hepatosomatic Index of the Freshwater Fish, Oreochromis Mossambicus At the end of 24 h of quinalphos treatment there was no significant deposition of mucous; however, at the end of 72 h and 96 h of treatment fishes showed a remarkable deposition of mucous over the body with the percentage increased to 50% when compared with those of control fish (Figure 3). Figure 3: Effect of Quinalphos on Mucous Secretion in Freshwater Fish, Oreochromis Mossambicus Impact Factor (JCC): NAAS Rating: 2.59

5 Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System 11 in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852) The activity of superoxide dismutase remain unchanged after 24 h of quinalphos exposure in mitochondrial and microsomal fractions, but a significant (p<0.05) reduction was observed in nuclear fractions. However, there was a significant decrease in the activity of the enzyme in all duration in the nuclear fractions as well as in 72 h and 96 h of mitochondrial and microsomal fractions than that of the corresponding control group (Figure 4). Figure 4: Effect of Quinalphos on the Activity of Superoxide Dismutase in Hepatic Subcellular Fractions in the Freshwater Fish, Oreochromis Mossambicus The activity of catalase decreased significantly (P<0.05) in all duration in mitochondrial and nuclear fractions and only at 96 h in the microsomal fractions (Figure 5). Figure 5: Effect of Quinalphos on the Activity of Catalase in Hepatic Subcellular Fractions in the Freshwater Fish, Oreochromis Mossambicus On the other hand, the activity of glutathione reductase was found to be significantly (p<0.05) decreased in all durations in mitochondrial and microsomal fractions, but only at 96 h treatment group in nuclear fractions (Figure 6). editor@tjprc.org

6 12 P. Surya, P. V. Vidya & K. C. Chitra Effect of quinalphos on the activity of glutathione reductase in hepatic subcellular fractions in the freshwater fish, Oreochromis mossambicus 8 nmol NADPH oxidized/ min/ mg protein * * * * * * * Mitochondrial fractions Nuclear fractions Microsomal fractions Control 24 h 72 h 96 h Figure 6: Effect of Quinalphos on the Activity of Glutathione Reductase in Hepatic Subcellular Fractions in the Freshwater Fish, Oreochromis Mossambicus The level of lipid peroxidation was increased significantly (p<0.05) at the end of 72 h and 96 h of mitochondrial and nuclear fractions. However, no such changes were observed in all treatment durations in the microsomal fractions (Figure 7). Figure 7: Effect of Quinalphos on the Level of Lipid Peroxidation in the Hepatic Subcellular Fractions in the Freshwater Fish, Oreochromis Mossambicus There was a significant (p<0.05) increase in the activity of alanine aminotransferase in the hepatic mitochondrial, nuclear and microsomal fractions at the end of 24 h, 72 h and 96 h of quinalphos-treated fishes as compared with the control group (Figure 8). Impact Factor (JCC): NAAS Rating: 2.59

7 Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System 13 in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852) DISCUSSIONS Figure 8: Effect of Quinalphos on the Activity of Alanine Aminotransferase in Hepatic Subcellular fractions in the Freshwater Fish, Oreochromis Mossambicus The aquatic environment is exposed to substantial amount of environmental pollutants that have the potential impact to cause oxidative stress in aquatic organisms through free radical generation. Ecotoxicological studies on aquatic organisms have focused primarily on redox cycling compounds and their effects on the major organs of biotransformation using the adult or larval stages of fishes. In the present study quinalphos was selected as the toxicant and exposed at sub lethal concentration in order to determine its effect on the fish antioxidant system in hepatic subcellular fractions. Some of our laboratory studies in the past few years provided evidence of reactive oxygen species (ROS) production in various vital organs as liver, gill, muscle and brain of fish exposed to toxic pollutants (Chitra et al., 2012; 2013; Asifa et al., 2014; Thulasi and Chitra, 2015). Also, the hepatic marker enzymes as well lipid peroxidation was also estimated to assess the toxic effect of quinalphos on hepatic subcellular fractions as mitochondrial, nuclear and microsomal fractions in the freshwater teleost fish, Oreochromis mossambicus. Several investigations have reported that the changes in the antioxidant defense system can be used as biomarkers of oxidative stress due to exposure to various pollutants in aquatic organisms. The antioxidant defense system of an organism can be subdivided into enzymatic antioxidants, such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase; and nonenzymatic antioxidants, such as glutathione, vitamin E, ascorbate, beta carotene, and urate (de Zwart et al., 1999). The alterations in the enzymatic activities could directly reflect the metabolic disturbances and cell damage in specific organs. The body weights of fishes were monitored throughout the experiment and it was observed that there was no significant change in the weight of the animal. Measures of animal growth are routinely evaluated in toxicology studies and are keys to interpret the compound-related effects. The present results suggest that quinalphos exposure did not showed toxicant-related effect on the body weight of the treated fishes. However, mucous deposition was significantly increased at 72 h and 96 h of quinalphos treatment than that of control group. Mucous cells are considered efficient in seizing the toxic agents and thus help to prevent the entrance of these agents into the gills (Perry and Laurent, 1993). Hypersecretion of mucous may be the consequence of a chronic defensive mechanism of the fish against the exposure to the environmental toxicant quinalphos. Tissue somatic indices are commonly reported in fisheries studies because it is easily determined and some indices, such as the hepatosomatic index can be an excellent predictor of adverse health status of fish. In the present study, editor@tjprc.org

8 14 P. Surya, P. V. Vidya & K. C. Chitra the hepatosomatic index of the quinalphos-treated fishes showed a slight decrease in all treatment groups, but the result was not significant when compared to that of the control group. Regulated production of free radicals in higher organisms and maintenance of redox homeostasis are essential for the physiological health of organisms. But, during these metabolic processes, a small proportion (2 3%) of free radicals may escape from the protective shield of antioxidant mechanisms, causing oxidative damage to cellular components. Biological systems have developed during their evolution an adequate enzymatic and non-enzymatic antioxidant mechanism to protect their cellular components from oxidative damage. The imbalance between the generation and the neutralization of ROS by antioxidant mechanisms within an organism is called oxidative stress (Davies, 1995). Free radicals/ ROS generated in tissues and in sub-cellular compartments are effectively scavenged by the antioxidant defence system, which constitutes antioxidant enzymes such as, superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. Superoxide dismutase (SOD) catalysis the dismutation of superoxide to hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ). The conversion of H 2 O 2 to 2H 2 O is by the enzyme glutathione peroxidase and the conversion of H 2 O 2 to O 2 and H 2 O is by the enzyme catalase. Since the reaction catalyzed by glutathione peroxidase requires glutathione (GSH) as substrate and depends in part on the ratio of glutathione disulfide (GSSG): GSH, the concentrations of these reactants and their ratio, which is a reflection of the redox state of the cell, are important to ROS detoxification. Glutathione peroxidase/ reductase directly act as antioxidant enzymes to inhibit lipid peroxidation (Sikka, 2001). In the present study a decrease in the activity of superoxide dismutase have been shown to increase the level of superoxide anion, which is known to inactivate catalase activity (Kono and Fridovich, 1982). Similarly, catalase or glutathione peroxidase has been shown to eliminate hydrogen peroxide from the cell leading to the inactivation of superoxide dismutase and generation of lipid peroxides (Bray et al., 1974). The observed results suggest that the toxic effect of quinalphos in the generation of free radicals are more pronounced in mitochondrial and nuclear fractions than that of microsomal fractions. Molecular biomarkers are used to test oxidative damage in biomolecules and various aspects of oxidative stress by free radicals in experimental animals. The most frequently used methods to monitor lipid peroxidation are based on measuring of the end products. One of the methods is by measuring production of malondialdehyde and it is of particular interest because it is commonly used assay than measured with thiobarbituric acid-reactive substances (TBARS). Malondialdehyde is one of the end products of lipid peroxidation and it is very attractive to monitor its concentration, which is a major oxidation product of peroxidized polyunsaturated fatty acids. Therefore, increased malondialdehyde content is an important indicator of lipid peroxidation (Freeman and Crapo, 1981). There are some relatively new approaches proposed recently to measure the end products of lipid peroxidation and they are HPLC and immunotechniques, which are more specific than malondialdehyde and TBARS measurement (Claeson et al., 2001). But, in the present study the simplified method as the production of malondialdehyde as a marker of lipid peroxidation was performed and it was observed that the level of lipid peroxidation was increased significantly at the end of 72 h and 96 h in mitochondrial and nuclear fractions. However, no such changes were observed in all treatment durations in the microsomal fractions. The end products of lipid peroxidation are considered as a dynamic parameter because they can be further either catabolized, or interact with other cellular components as proteins. Increased lipid peroxidation may indicate an increased oxygen free radical generation in the hepatic sub cellular fractions (Thiele et al., 1995). Aminotransaminases play an important role in the utilization of amino acids for the oxidation and/or for gluconeogenesis (Samsonova et al., 2005). Alanine aminotransferases, is an enzyme frequently used in the diagnosis of Impact Factor (JCC): NAAS Rating: 2.59

9 Influence of Quinalphos, an Organophosphorous Pesticide, on the Antioxidant Defense System 15 in Hepatic Subcellular Fractions of Fish, Oreochromis Mossambicus (Peters, 1852) damage caused by pollutants in various tissues such as liver, muscle, and gills. This enzyme is known to play a key role in mobilizing L-amino acids for gluconeogenesis and function as links between carbohydrate and protein metabolism under altered physiological, pathological, and induced environmental conditions (Victor, 1985). The elevation of the activity of this enzyme provides the oxaloacetate required for the gluconeogenesis pathway to meet the additional supply of glucose for the production of energy under reduced phase of oxidative metabolism. Elevation in the levels of alanine aminotransferase in liver of the fish can be considered as a response to the stress induced by quinalphos to generate ketoacid-like ketoglutarate and oxaloacetate for contributing to gluconeogenesis and/or energy production necessary to meet the excess energy demand under the toxic manifestation. The increases in activities of aminotransferases as observed in the present study were agreement with earlier reports (Arshad et al., 2007). The present results revealed, significant increases in liver enzyme in mitochondrial, nuclear and microsomal fractions of quinalphos-treated fish throughout the experimental periods may appear to reflect the stress effect of quinalphos. CONCLUSIONS As the source of the above mentioned discussion it can be summarized that exposure to quinalphos induced oxidative stress in hepatic subcellular fractions of fish by inducing ROS generation thereby inhibited the activities of antioxidant enzymes in the freshwater fish, Oreochromis mossambicus. ACKNOWLEDGEMENTS REFERENCES The authors acknowledge UGC-SAP/ BSR for utilizing the equipments during this study. 1. APHA. (1998). Standard methods for the examination of water and waste water, 20 th Edition, Washington, DC. 2. Arshad, N., Shabbir, G., Aleem, S., & Arshad, M. (2007). GOT is one of the enzymes, which gives valuable diagnostic information for a number of disease conditions. Asian J Exp Sci. 21, Asifa, K. P., Balakrishnan, V., & Chitra, K. C. (2014). Toxicity evaluation of chlordecone and its effect on oxidative imbalance in the Cichlid fish, Etroplus maculatus (Bloch). Int J Zool Res. 4, Bray, M. A., Gordon, D., & Morley, J. (1974). Role of prostaglandins in cellular immunity. British J Pharmacol. 52, pp Carlberg, I., & Mannervik, B. J. (1985). Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem. 250, Chitra, K. C., Nikhila, P., & Asifa, K. P. (2013). Short-term exposure to quinalphos induced biochemical and hematological changes in freshwater fish, Oreochromis mossambicus. J Adv Lab Res Biol. 3, Chitra, K. C., Pushpalatha, E., & Kannan, V. M. (2012). Quinalphos-induced antioxidant status and histopathological changes in the gill of the freshwater fish, Oreochromis mossambicus. J Adv Lab Res Biol. 3, Claeson, K., Thorsen, G., & Karlberg, B. (2001). Methyl malondialdehyde as an internal standard for the determination of malondialdehyde. J Chromatography. B751, Claiborne, A. (1985). Catalase activity. In: CRC Handbook of methods for oxygen radical research. R Greenwald (ed.), CRC Press, Boca Raton, Florida, Davies, K. J. A. (1995). Oxidative stress, the paradox of aerobic life. In: Rice-Evans C, Halliwell B, Land GG. (Eds.), Free Radical and Oxidative Stress: Environment, Drugs and Food Additives. London, Portland Press. pp editor@tjprc.org

10 16 P. Surya, P. V. Vidya & K. C. Chitra 11. de Zwart, L. L., Meerman, J. H. N., Commandeur, J. N. M., & Vermeulen, N. P. E. (1999). Biomarkers of free radical damage. Applications in experimental animals and in humans. Free Rad Biol Med. 26, Finney, D. J. (1971). Probit analysis, 3rd (Ed.), Cambridge University Press, London, Freeman, B. A., & Crapo, J. D. (1981). Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J Biol Chem. 256, Kelly, K. A., Havrilla, C. M., Brady, T. C., Abramo, K. H., & Levin, E. D. (1998). Oxidative stress in toxicology: established mammalian and emerging piscine model systems. Environ Health Perspect. 106, Kono, Y., & Fridovich, I. (1982). Superoxide radical inhibits catalase. J Biol Chem. 257, Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193, Marklund, S., & Marklund, G. (1974). Involvement of superoxide anion radical in antioxidation of pyrogallol and a constituent assay for superoxide dismutase. Eur J Biochem. 47, Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal Biochem. 95, Palade, G. E., & Siekevitz, P. (1956). Liver microsomes: an integrated morphological and biochemical study. J Biophys Biochem Cytol. 25, Perry, S. F., & Laurent, P. (1993). Environmental effects on fish gill structure and function. In: Rankin, J. C., Jensen, F. B. (Eds.), Fish Ecophysiology. London, pp Samsonova, M. V., Lapteva, T. I., & Filippovich, B. (2005). Aminotransferases in early development of salmonid fish. Russ J Dev Biol. 36, Sancho, E., Fernandez-Vega, C., Ferrando, M. D., & Andreu-Moliner, E. (2003). Eel ATPase activity as biomarker of thiobencarb exposure. Ecotoxicol Environ Safety. 56, Segal, H. L., & Matsuzawa, T. (1970). Alanine aminotransferase from rat liver. Methods Enzymol. 17, Sikka, S. C. (2001). Relative impact of oxidative stress on male reproductive function. Curr Med Chem. 8, Thiele, J. J., Freisleben, H. J., Fuchs, J., & Ochsendorf, F. R. (1995). Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen. Human Reprod. 10, Thulasi, K. V., Asifa, K. P., & Chitra, K. C. (2015). Acute exposure to bisphenol-a altered muscular antioxidant system in cichlid fish, Etroplus maculatus (Bloch, 1795). Global J Res Anal. 8, Victor, W. R. (1985). General Properties of Enzymes. In: Harper s Review of Biochemistry. California: Maruzen Co. pp Impact Factor (JCC): NAAS Rating: 2.59