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RETRACTED: Fluctuations of certain biochemical constituents and markers enzymes as a consequence of monocrotophos toxicity in the edible freshwater fish, Channa punctatus
Pesticide Biochemistry and Physiology 94 (2009) 5–9
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Shweta Agrahari a, Krishna Gopal b,* b
Department of Zoology, University of Lucknow, Lucknow 226007, India Head Aquatic Toxicology, Indian Institute of Toxicology Research, Post Box-80, MG Marg, Lucknow 226 001, India
a r t i c l e
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Article history: Received 24 June 2008 Accepted 4 February 2009 Available online 11 February 2009
a b s t r a c t
The effect of exposure to two sublethal concentrations of an organophosphorus insecticide, monocrotophos (MCP) (0.96 and 1.86 mg l 1), on biochemical constituents and the activity of some marker enzymes such as acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), and lactate dehydrogenase (LDH) in liver, kidney, gills, and muscles of the edible freshwater fish, Channa punctatus, was studied after 30 days. The results revealed that there was overall decrease in total protein, carbohydrates, glycogen, free amino acid, and total lipid in the test samples compared to control. The AAT and ALAT activities were increased in kidney, where as liver, gills and muscles showed decrease. Increase in ACP and ALP activities were observed in gills, muscles and kidney, and reduction was observed in liver. Glycogen was depleted in liver, gills and kidney indicates of typical stress related response of the fish with pesticide. LDH activity was decreased in liver, muscles and kidney, but a significant increase in LDH activity in gills was observed. The results revealed that MCP induced impairment of metabolism in C. punctatus and that the assayed enzymes can work as good biomarkers of organophosphorus contamination. Ó 2009 Elsevier Inc. All rights reserved.
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Keywords: Organophosphorus insecticide Monocrotophos Biochemical constituents Marker enzymes Channa punctatus Tissues
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Fluctuations of certain biochemical constituents and markers enzymes as a consequence of monocrotophos toxicity in the edible freshwater fish, Channa punctatus
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The study was performed with financial support of the institutional fund. The experiments did not involve human beings or experimental animals. The fish used were collected on site from a river through local resources. The study has been conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. 1. Introduction
For centuries, pesticides have been used in agriculture to enhance food production by eradicating unwanted insects and controlling disease vectors [5]. Among common pesticides, organophosphorus (OP) compounds are widely used in agriculture, medicine and industry [17]. OP pesticides, in addition to their intended effects like control of insects or other pests, are sometimes found even to affect nontarget organisms including human beings [13,21]. Monocrotophos (MCP) is an organophosphorus insecticide with a contact and systemic action. The extensive use of MCP poses a health hazard to animals and humans because of its persistence in soil and crops [42]. In humans, the main risk groups of higher-dose MCP
* Corresponding author. Fax: +91 522 2628227. E-mail address: [email protected] (K. Gopal). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.02.001
exposure are its producers, workers in pesticide industries and farm owners. Majority of population is exposed to lower doses of MCP via food, contaminated drinking water, or by application of household insecticides containing MCP [42]. Exposure to a low level of organophosphate pesticides is known to produce a variety of biochemical changes, some of which may be responsible for the adverse biological effects reported in humans and experimental animals [24]. Fishes especially Channa punctatus are often used as indicator organisms to investigate the biological effects of pollutants in the aquatic environment. In recent years, fishes have served as useful bioindicators and integrators of contaminants because of various reasons, viz., their wide distribution in the freshwater environment, the fact of being free swimmers, their ability to respond against environmental pollution and their importance as an economic food source for human beings [2]. Biochemical constituents and certain enzymes have been explored as potential biomarkers for variety of different organisms because these parameters are highly sensitive and conserved between species and less variable. Their advantages are that biochemical and enzymes activities tend to be more sensitive, less variable, more highly conserved between species, and often easier to measure as stress indices [38]. Generally freshwater fishes, have been found to be useful as indicators and integrators of certain contaminants due to their wide geographical distribution, domi-
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nant presence in freshwater water ecosystem, bottom-dwelling habits, ability to respond to environmental pollutants, importance as an economic food source for humans, and ease in being readily transported and maintained under laboratory conditions. Hence, it is of interest to investigate the impact of MCP on some biochemical constituents and the activities of certain key enzymes in different organs of the edible fresh water fish, C. punctatus. 2. Materials and methods 2.1. Chemicals All the reagents used in the present study were of analytical grade and were used without further purification. The test compound monocrotophos (MCP) was a gift from the National Organic Chemical Industry Limited (NOCIL), Bombay, India.
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2.2. Animal maintenance The investigations were carried out in the freshwater fish, C. punctatus (Bloch), procured from pollution-free water bodies and were brought to the laboratory in large aerated drums. Later, they were acclimatized for 30 days in a huge cement tank (8 6 4 ft.) and fed with commercially available dry prawn. Fish weighing 25 ± 30 g were transferred to a glass aquarium (60 30 30 cm) of 40 1 water capacity for a further period of seven days and were fed with dry prawn for conditioning. The water in the aquarium was renewed daily and was aerated mechanically. The natural photoperiod of 13:11 light:day h was maintained. The average values for water quality parameters were as following temperature 26 ± 2 °C, pH 7.2 ± 0.2, dissolved oxygen 8.10 ± 0.021 mg 1 1, total hardness 120 ± 1.9 mg 1 1 as CaCO3, alkalinity 360 ± 1.4 mg 1 1 as CaCO3, and chlorides 275.567 ± 1.24 mg 1 1 [4].
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2.3. Sub-acute studies
et al. [3] and the glycogen by Caroll et al. [43]. For estimation of the carbohydrate and glycogen, glucose was used as standard. The free amino acids in the tissues were determined as per the procedure of Ishida et al. [44]. The total lipids were extracted based on the procedures of Folch et al. [15]. Acid phosphatase (ACP) (EC 3.1.3.2) activity in gills, liver, kidney, and muscles was measured by the method of Jabeen [22], and the inorganic phosphate (Pi) liberated was measured by the method of Fiske and Subbarow [9]. The enzyme activity was expressed as lmol Pi liberated/mg protein/h. The activity of alkaline phosphatase (ALP) (EC 3.1.3.1) in gills, liver, kidney, and muscles was estimated by the method of Moss et al. [11]. The activities of’ aminotransferase such as aspartate aminotransferase (AAT) (EC 2.6.1.1) and alanine aminotransferase (ALAT) (EC 2.6.1.2) activity in liver, gills, kidney, and muscles were estimated according to the standard procedure of Yatzidis [40]. The enzymes activity was expressed as lmol of phenol formed/mg protein/h. Lactate dehydrogenase (LDH) (EC 1.1.1.27) activity was determined by the method of McQueen [26], with NADH oxidation recorded at 340 nm and expressed as lmol formazan formed/mg/h.
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The acute LC50 value of monocrotophos was determined in the laboratory using the semi-static method [4]. The control experiments were also performed with addition of carrier solvent alone. During sub-acute studies a total of forty fish (20 fish per aquarium) were exposed to two sublethal concentrations of MCP viz. 0.96 and 1.86 mg l 1 (corresponding to 1/20th and 1/10th of 96 h LC50 = 18.56 mg l 1) for a period of 30 days. The required concentration was maintained by adding the toxicant directly in 40 l of water and renewed daily. The experiment was repeated thrice for each exposure and control. Feeding was disrupted during experimentation; i.e., fish were starved 24 h prior to dissection. 2.4. Tissue samples
Fish were anesthetized by placing them for 10 min in benzocaine hydrochloride (200 mg l 1) and the fish tissues such as liver, gills, kidney and muscles were excised. Tissues were homogenized (10% w/v) in 0.1 M Tris–HCL buffer (pH 8.0) using Poter-Elvehjam homogenizer fitted with a Teflone pestle. The homogenates were centrifuged at 5000g for 10 min and the supernatant was further centrifuged at 5000g for 10 min. The resultant supernatant was used as the enzyme source for the estimation of all the enzyme activities. All the enzyme preparations were carried out at 4 °C. 2.5. Biochemical studies The protein content of the samples was determined according to the method of Lowry et al. [30] using crystalline bovine serum albumin standard. The total carbohydrate was estimated by Kemp
2.6. Statistical analysis
The experiments were repeated on three different occasions in triplicate and that data were analyzed by Student’s t-test. Statistical comparisons were done between control and experimental data from the same species. Significant differences from control values P < 0.05, P < 0.02, P < 0.01, P < 0.005 and P < 0.001 were accepted as levels of statistical significance [16]. 3. Results and discussion
In general, the edible freshwater fish constitute one of the major sources of nutritious foods for humans. The nutritive values of fish depend upon their biochemical composition. Biochemical changes induced by pesticide stress is due to disturbed metabolism manifested by inhibition of enzymes, retardation of growth and reduction in the fecundity and longevity of the organism. Even though, the pesticides have their own target site of action, most of them are metabolic depressors. They generally affect the activity of biologically active molecules such as proteins, carbohydrates and lipids [34]. The protein is the major intake of energy source. Diana [20] has observed the ‘‘proximate composition of aquatic animals under various feeding regimes” and indicated that the energy gain or depletion from the body is due to the changes in the amount of whole-body tissue to its depletion from the body. Hence, the protein content of the cell is considered to be an important tool for the evaluation of physiological standards. The tissue protein is metabolized to produce glucose by the process of gluconeogenesis and it is utilized for energy production under stress conditions [25]. The results of the present investigation as presented in Tables 1 and 2 indicated that, the protein content of the liver, kidney, gills and muscles decreased by 57%, 61%, 62% and 70%, respectively, as compared to the control when C. punctatus were exposed to MCP. The decrease of protein content under MCP stress may be attributed to the utilization of amino acids in various catabolic reactions. Amino acids activity in the liver, gills, kidney and muscles showed a continuous decrease as the concentration increased (Tables 1 and 2). The maximum reduction in amino acids activity of the kidney, gills, liver and muscles of exposed fish were 58%, 59%, 66% and 74%, respectively, at sublethal concentration of 1.86 mg l 1. The amino acids through transamination and deamination reactions might have supplied necessary keto acids to act as precursors for the maintenance of carbohydrate metabolism to
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S. Agrahari, K. Gopal / Pesticide Biochemistry and Physiology 94 (2009) 5–9 Table 1 Variation in the biochemical constituents of liver, kidney, gills, and muscles in the control and experimental Channa punctatus.
Protein Carbohydrates Free amino acids Glycogen Total lipid Enzymes LDH ACP ALP AAT ALAT
Liver
Kidney 1
Control
0.96 mg l
135.50 ± 0.05 5.52 ± 0.10 9.84 ± 0.02 34.10 ± 0.15 67.15 ± 2.85
98.6 ± 2.5 4.20 ± 0.3b 7.32 ± 0.02 25.45 ± 0.5 52.16 ± 0.9c
81.09 ± 0.85 0.996 ± 0.02 0.358 ± 0.05 0.702 ± 0.03 0.312 ± 0.002
64.34 ± 1.0 0.745 ± 0.05c 0.212 ± 0.034 0.593 ± 0.01a 0.258 ± 0.01c
Gills 1
Control
0.96 mg l
123.30 ± 0.08 3.63 ± 0.03 5.32 ± 0.04 13.04 ± 0.03 63.01 ± 3.12
84.02 ± 0.92 3.01 ± 0.01 4.49 ± 0.1d 10.31 ± 0.03 55.35 ± 2.12
28.46 ± 0.23 2.81 ± 0.02 6.29 ± 0.02 5.05 ± 0.04 59.25 ± 6.50
90.43 ± 0.9c 0.756 ± 0.032d 0.697 ± 0.2 0.140 ± 0.02 0.063 ± 0.04
96.25 ± 3.42 0.628 ± 0.034 0.539 ± 0.03 0.419 ± 0.005 0.228 ± 0.004
101.7 ± 2.03 0.583 ± 0.03 0.456 ± 0.018 0.109 ± 0.003 0.043 ± 0.004
Muscles
Control
1
Control
0.96 mg l
20.2 ± 0.10 2.69 ± 0.13 5.33 ± 0.01 4.23 ± 0.18b 50.80 ± 0.13
99.16 ± 0.81 0.80 ± 0.02 7.49 ± 0.09 17.41 ± 0.026 135.78 ± 5.01
76.63 ± 2.8d 0.74 ± 0.04 6.86 ± 0.13 14.92 ± 0.1 123.50 ± 2.0
112.62 ± 1.2b 0.964 ± 0.022 0.767 ± 0.06a 0.309 ± 0.001 0.178 ± 0.006d
139.75 ± 3.54 0.353 ± 0.02 0.412 ± 0.03 0.246 ± 0.002 0.509 ± 0.005
109.32 ± 4.7d 0.626 ± 0.02a 0.654 ± 0.07a 0.129 ± 0.005 0.382 ± 0.003
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Data represent means ± SE of three individuals values. a P 6 0.05 denotes significant when compared with control. b P 6 0.02 denotes significant when compared with control. c P 6 0.01 denotes significant when compared with control. d P 6 0.005 denotes significant when compared with control.
0.96 mg l
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Constituents
Table 2 Variation in the biochemical constituents of liver, kidney, gills, and muscles in the control and experimental Channa punctatus. Constituents
Liver
Kidney
Control Protein Carbohydrates Free amino acids Glycogen Total lipid Enzymes LDH ACP ALP AAT ALAT
1.86 mg l
1
Control
d
135.50 ± 0.05 5.52 ± 0.10 9.84 ± 0.02 34.10 ± 0.15 67.15 ± 2.85
52.40 ± 7.80 1.90 ± 0.51c 2.43 ± 0.82d 16.43 ± 2.24c 37.58 ± 1.2d
81.09 ± 0.85 0.996 ± 0.02 0.358 ± 0.05 0.702 ± 0.03 0.312 ± 0.002
41.32 ± 4.11d 0.523 ± 0.05d 0.142 ± 0.01a 0.095 ± 0.051d 0.109 ± 0.02d
123.30 ± 0.08 3.63 ± 0.03 5.32 ± 0.04 13.04 ± 0.03 63.01 ± 3.12
101.7 ± 2.03 0.583 ± 0.03 0.456 ± 0.018 0.109 ± 0.003 0.043 ± 0.004
Gills
1.86 mg l
1
c
Control
46.31 ± 9.0 2.10 ± 2.03c 1.76 ± 0.39d 7.73 ± 1.00b 28.40 ± 1.0d
28.46 ± 0.23 2.81 ± 0.02 6.29 ± 0.02 5.05 ± 0.04 59.25 ± 6.50
73.88 ± 2.0d 1.098 ± 0.04d 0.782 ± 0.03d 0.196 ± 0.007d 0.073 ± 0.001c
96.25 ± 3.42 0.628 ± 0.034 0.539 ± 0.03 0.419 ± 0.005 0.228 ± 0.004
Muscles
1.86 mg l
1
1
Control
1.86 mg l
8.54 ± 3.2 1.43 ± 0.18c 2.61 ± 0.59c 2.10 ± 0.31d 32.31 ± 1.26a
99.16 ± 0.81 0.80 ± 0.02 7.49 ± 0.09 17.41 ± 0.026 135.78 ± 5.01
42.25 ± 6.12d 0.26 ± 0.078c 3.10 ± 0.63c 6.80 ± 1.78c 95.69 ± 1.54c
159.23 ± 7.0c 1.162 ± 0.06c 0.954 ± 0.05c 0.221 ± 0.02d 0.054 ± 0.02d
139.75 ± 3.54 0.353 ± 0.02 0.412 ± 0.03 0.246 ± 0.002 0.509 ± 0.005
86.56 ± 5.02c 0.889 ± 0.05d 0.923 ± 0.06c 0.078 ± 0.023c 0.158 ± 0.05c
c
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Data represent means ± SE of three individuals values. a P 6 0.02 denotes significant when compared with control. b P 6 0.01 denotes significant when compared with control. c P 6 0.005 denotes significant when compared with control. d P 6 0.001 denotes significant when compared with control.
meet the energy requirements during pollutants stress. Another possibility for the observed protein reduction should have been due to the blocking of protein synthesis or protein denaturation or interruption in the amino acid synthesis [8]. The depletion of protein content may also be due to the rapid utilization of tissue protein as the food utilization decreases when the animals are under stress conditions. Moreover, the chemical-exposed animals obtain extra energy requirement from the tissue protein [6]. The reduction in protein content indicates that the tissue protein may undergo proteolysis, which results in the production of free amino acids and is used in the trichloroacetic acid cycle for energy production under stress conditions. The carbohydrate level was depleted in liver, kidney, gills and muscles during the entire period of experiment (Tables 1 and 2). Carbohydrates in the tissues of C. punctatus exist as protein-bound sugars and glycogen. It is well known that the sugar serves as energy reserve for the metabolic process. Carbohydrates are considered to be the first among the organic nutrients degraded in response to stress conditions imposed on an animal. Chemical stress causes rapid depletion of stored carbohydrates primarily in liver and other tissues [7]. The glycogen level of different tissues showed a decreasing trend as MCP concentration increased (Tables 1 and 2). Depletion of glycogen may be due to utilization of stored carbohydrates in li-
ver for energy production as a result of pesticide-induced hypoxia. Glycogen depletion in liver and muscles after toxic stress has been reported in several studies with aquatic animals [23,31]. The depletion of glycogen in the tissues is indication of typical stress response in fish challenged with pesticides. The C. punctatus after exposure to two different concentrations of MCP showed over all decrease in the levels of carbohydrate and glycogen into all the fish organs. The maximum decrease (52%) was observed for glycogen in liver, whereas only slight changes were recorded in kidney and gills. Dhavale and Masurekar [10] are of the opinion that the decreased level of carbohydrate and glycogen in tissues of toxicant-exposed animals may be due to the prevalence of hypoxic condition in the tissues as a result of pollutant stress. Normally during hypoxic conditions, carbohydrate metabolism is increased to release energy, resulting in the extra expenditure of carbohydrate. The depletion of glycogen observed in all tissues of C. punctatus is, an indication of typical stress related response of the fish to the pesticide. These results are in accordance with the finding of Ghosh and Shrotri [32], who have suggested that decreased level of carbohydrates in the toxicant-exposed animals either induces the glycogenolysis, possibly by increasing the activity of glycogen phosphorylase to meet the energy demand under stress conditions or the toxicant has inhibitory effect on glycogenesis pathway.
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the assayed enzymes can work as good biomarkers of organophosphorus contamination. Acknowledgments The authors thank to Director, Indian Institute of Toxicology Research, Lucknow, for providing the facilities and constant encouragement. References [2] A. Gupta, D.K. Rai, R.S. Pandey, B. Sharma, Analysis of some heavy metals in the riverine water, sediments and fish from river Ganges at Allahabad, Environ. Monit. Assessment, (2008), in press. [3] A. Kemp, J.M. Adrienne, Kits Van Heijningen, A colorimetric method for the determination of glycogen in tissues, The Biochem. J. 56 (1954) 640–648. [4] APHA, Standard Methods for the Examination of Water and Wastewater, 21st ed., APHA, Washington, DC, 2005. [5] A. Prakasam, S. Sethupathy, S. Lalitha, Plasma and RBCs antioxidant status in occupational male pesticide sprayers, Clin. Chim. Acta 310 (2001) 107–112. [6] A. Yadav, A. Gopesh, R.S. Pandey, D.K. Rai, B. Sharma, Fertilizer industry effluent induced biochemical changes in fresh water teleost, Channa striatus (Bloch), Bull. Environ. Contam. Toxicol. 79 (6) (2007) 588–595. [7] B. Jyothi, G. Narayan, Pesticide induced alterations of non-protein nitrogenous constituents in the serum of a freshwater catfish, Clarias batrachus (Linn.), Indian J. Exp. Biol. 38 (2000) 1058–1061. [8] B.S. Jha, Alterations in the protein and lipid contents of intestine, liver and gonads in the lead exposed freshwater murrel Channa punctatus (Bloch), J. Ecobiol. 3 (1991) 29–34. [9] C.H. Fiske, Y. Subbarow, The colorimetric determination of phosphorous, J. Biol. Chem. 66 (1925) 375–400. [10] D.M. Dhavale, V.B. Masurekar, Variations in the glucose and glycogen content in the tissues of Scylla serrata (Forskal) under the influence of cadmium toxicity, Geobios 13 (1986) 139–142. [11] D.W. Moss, D.N. Baron, P.G. Walker, J.H. Wilkinson, Standardization of clinical enzyme assays, J. Clin. Pathol. 24 (1971) 740–743. [12] D.W. Peakall, Biomarkers, the way forward in environmental assessment, Toxicol. Ecotoxicol. News 1 (1994) 55–60. [13] G. Chantelli-Forti, M. Paolini, P. Hrelia, Multiple end point procedure to evaluate risk from pesticides, Environ. Hlth. Perspect. 101 (1993) 15–20. [14] G. Tripathi, S.P. Shukla, Malate and lactate dehydrogenases of a freshwater cat fish, impact of endosulfan, Biomed. Environ. Sci. 3 (1990) 52–58. [15] J. Folch, M. Lees, G.H. Bloane-Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 26 (1957) 497– 509. [16] J.H. Zar, Biostatistical Analysis, second ed., Prentice-Hall, Inc., Englewood Cliffs, NJ, 1984. pp. 401–403. [17] J. Storm, K. Rozman, J. Doull, Occupational exposure limits for 30 organophosphates pesticide based on inhibition of red blood cell acetylcholinesterase, Toxicology 150 (2000) 1–29. [18] J.V. Rao, Biochemical alterations in euryhaline fish, Oreochromis mossambicus exposed to sub-lethal concentrations of an organophosphorus insecticide, monocrotophos, Chemosphere 65 (2006) 1814–1820. [19] J.V. Rao, Sublethal effects of an organophosphorus insecticide (RPR-II) on biochemical parameters of tilapia, Oreochromis mossambicus, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 143 (2006) 492–498. [20] J.S. Diana, An experiment analysis of the metabolic rate and food utilized of northern pike, Comp. Biochem. Physiol. A 71 (1982) 395. [21] K. Chaudhuri, S. Selvaraj, A.K. Pal, Studies on the genotoxicity of endosulfan in bacterial systems, Mutat. Res. 439 (1999) 63–67. [22] K. Jabeen, Toxicological evaluations of some pesticides against chicken (Gallus gallus domesticus and rat (Rattus rattus norvegius) with special reference to hematology, blood and urine biochemistry, Ph.D. Thesis, Osmania University, Hyderabad, India, 1984. [23] L.H. Aguiar, G. Moraes, I.M. Avilez, A.E. Altran, C.F. Correˆa,, Metabolical effects of folidol 600 on the neotropical freshwater fish matrinxa~, Brycon cephalus, Environ. Res. 9 (2004) 224–230. [24] L.G. Sultatos, Mammalian toxicology of organophosphorus pesticides, J. Toxicol. Environ. Hlth. 43 (1994) 271–289. [25] M. Elumalai, M.P. Balasubramanian, Influence of naphthalene on esterase activity during vitellogenesis of marine edible crab, Scylla serrata, Bull. Environ. Contam. Toxicol. 62 (1999) 743–748. [26] M.J. McQueen, True Arrhenius relationships of human lactate dehydrogenase, Z. Klin. Chem. Klin. Biochem. 13 (1975) 17–19. [27] M. Rajyasree, P. Neeraja, Aspartate and alanine aminotransferase activities in fish tissue subcellular fractionation on exposure to ambient urea, Indian, J. Fish. 3 (1989) 88–91. [28] N.S. Oluah, Effect of sublethal copper (II) ions on the serum transaminase activity in catfish Clarias albopunctatus, J. Aquat. Sci. 13 (1998) 45–47. [29] N.S. Oluah, Plasma aspartate aminotransferase activity in the catfish Clarias albopunctatus exposed to sublethal zinc and mercury, Bull. Environ. Contam. Toxicol. 63 (1999) 343–349.
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In the low availability of carbohydrates, lipids serve as source of energy for supporting physiological activities of the body. The lipid content of liver, kidney, gills and muscles showed decreased levels in the C. punctatus exposed to monocrotophos for 30 days at both MCP concentrations (Tables 1 and 2). The decline in the lipid content might be due to the utilization of lipids for meeting the energy demand under the stress. The term biomarker is defined as a change in a biological system due to external stimuli. This ranges from molecular through cellular and physiological responses to behavioral changes, which are related mainly to the toxic effects of environmental pollutants [12]. The analysis of marker enzymes such as lactate dehydrogenase, aminotransaminases, and phosphatases serves as specific indications of water-pollution-induced changes in the enzyme activity of fish. Significant inhibition in the activity levels of these enzymes were observed in the liver. On the contrary, these were observed elevated levels in the kidney, gills and muscles in relation to MCP exposure. LDH is an enzyme recognize as a potential marker for assessing the toxicity of a chemical. The level of LDH was found to be increased in the gills and decreased in the liver, kidney and muscles in the MCP-exposed fish. The result of the present finding study is in accordance with the findings of Rao [18,19]. Several reports revealed decreased LDH activity in tissues under various toxic conditions [14,35]. This might be due to the higher glycolysis rate, which is the only energy-producing pathway for the animal when it is under stress conditions. LDH is an important glycolytic enzyme in biological systems and is inducible by oxygen stress. Phosphatases play major roles in the molting physiology of many fishes [39]. Acid phosphatase is a lysosomal enzyme that hydrolyses the phospho-esters in acidic medium. Alkaline phosphatase catalyses dephosphorylation of many molecules including nucleotides, proteins, and alkaloids at alkaline pH. It is well known that alkaline phosphatase is involved in carbohydrate metabolism, growth and differentiation, protein synthesis, synthesis of certain enzymes, secretion activity, and transport to phosphorylated intermediates across the cell membranes. In the present investigation rise in the activities of phosphatases in the gills, kidney and muscles, and a decline in liver in MCP-exposed C. punctatus were observed. Alterations in alkaline phosphatase (ALP) and acid phosphatase (ACP) activities in tissues and serum have been reported in pesticide treated fish [33]. The alanine aminotransferase (ALAT) and aspartate aminotransferase (AAT) are liver specific enzymes and they are sensitive markers of hepatotoxicity and histopathologic changes and can be assessed within a shorter time [41]. The increase in ALAT and AAT indicate the tissue damage in liver, kidney and gills of Clarias albopunctatus and C. punctatus [27,29]. In the present study the increase in ALAT and AAT activities was observed only in the kidney of C. punctatus. Similar results have also been reported by other workers [28,29,37] in different fish species such as C. albopunctatus and Carassius auratus gibelio. The results presented in Tables 1 and 2 indicated that the alanine and aspartate aminotransferase activities increased in kidney and decreased in liver, gills and muscles decreased in liver, gills and muscles of C. punctatus exposed to MCP. This is in accordance with the findings of Rao [18,19] who has reported similar enzymatic changes in Oreochromis mossambicus due to monocrotophos stress. The effects of MCP at higher degree are always preceded by fluctuations in the biological processes such as biochemical and enzyme systems allowing the identification of suitable biomarker signals. It is therefore of major significance to integrate the biochemical biomarkers in assessing the potential hazards and risks due to toxic environmental pollutants. This study showed that MCP induced impairment of metabolism in C. punctatus and that
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[30] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [31] P.S. Bhavan, P. Geraldine, Alterations in concentrations of protein, carbohydrate, glycogen, free sugar, and lipid in the prawn Macrobrachium malcolmsonii on exposure to sublethal concentrations of endosulfan, Pestic. Biochem. Physiol. 58 (1997) 89–101. [32] R. Ghosh, R.V. Shrotri, Blood glucose and tissue glycogen interrelationship in Scylla serrata (Forskal) chronically exposed to thiodan, Environ. Biol. 13 (1992) 233–237. [33] V. Palanivelu, K. Vijayavel, S. Ezhilarasibalasubramanian, M.P. Balasubramanian, Influence of insecticidal derivative (Cartap Hydrochloride) from the marine polychaete in certain enzyme systems of the fresh water fish Oreochromis mossambicus, J. Environ. Biol. 26 (2005) 191–196. [34] R.K. Singh, B. Sharma, Carbofuran induced biochemical changes in Clarias batrachus, Pesticide Sci. 53 (1998) 285–290. [35] R. Mishra, S.P. Shukla, Endosulfan effects on muscles malate dehydrogenase of the freshwater catfish Clarias batrachus, Ecotoxicol. Environ. Saf. 56 (2003) 425–433. [37] R.V. Zikic, A.S. Stajn, S.Z. Pavlovic, B.I. Ognjanovic, Z.S. Saicic, Activities of superoxide dismutase and catalase in erythrocytes and plasma transaminase of gold fish (Carassius auratus gibelio Bloch.) exposed to cadmium, Physiol. Res. 50 (2001) 105–111.
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Shweta Agrahari a, Krishna Gopal b,* b
Department of Zoology, University of Lucknow, Lucknow 226007, India Head Aquatic Toxicology, Indian Institute of Toxicology Research, Post Box-80, MG Marg, Lucknow 226 001, India
a r t i c l e
i n f o
Article history: Received 24 June 2008 Accepted 4 February 2009 Available online 11 February 2009
a b s t r a c t
The effect of exposure to two sublethal concentrations of an organophosphorus insecticide, monocrotophos (MCP) (0.96 and 1.86 mg l 1), on biochemical constituents and the activity of some marker enzymes such as acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), and lactate dehydrogenase (LDH) in liver, kidney, gills, and muscles of the edible freshwater fish, Channa punctatus, was studied after 30 days. The results revealed that there was overall decrease in total protein, carbohydrates, glycogen, free amino acid, and total lipid in the test samples compared to control. The AAT and ALAT activities were increased in kidney, where as liver, gills and muscles showed decrease. Increase in ACP and ALP activities were observed in gills, muscles and kidney, and reduction was observed in liver. Glycogen was depleted in liver, gills and kidney indicates of typical stress related response of the fish with pesticide. LDH activity was decreased in liver, muscles and kidney, but a significant increase in LDH activity in gills was observed. The results revealed that MCP induced impairment of metabolism in C. punctatus and that the assayed enzymes can work as good biomarkers of organophosphorus contamination. Ó 2009 Elsevier Inc. All rights reserved.
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Keywords: Organophosphorus insecticide Monocrotophos Biochemical constituents Marker enzymes Channa punctatus Tissues
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Fluctuations of certain biochemical constituents and markers enzymes as a consequence of monocrotophos toxicity in the edible freshwater fish, Channa punctatus
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The study was performed with financial support of the institutional fund. The experiments did not involve human beings or experimental animals. The fish used were collected on site from a river through local resources. The study has been conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. 1. Introduction
For centuries, pesticides have been used in agriculture to enhance food production by eradicating unwanted insects and controlling disease vectors [5]. Among common pesticides, organophosphorus (OP) compounds are widely used in agriculture, medicine and industry [17]. OP pesticides, in addition to their intended effects like control of insects or other pests, are sometimes found even to affect nontarget organisms including human beings [13,21]. Monocrotophos (MCP) is an organophosphorus insecticide with a contact and systemic action. The extensive use of MCP poses a health hazard to animals and humans because of its persistence in soil and crops [42]. In humans, the main risk groups of higher-dose MCP
* Corresponding author. Fax: +91 522 2628227. E-mail address: [email protected] (K. Gopal). 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.02.001
exposure are its producers, workers in pesticide industries and farm owners. Majority of population is exposed to lower doses of MCP via food, contaminated drinking water, or by application of household insecticides containing MCP [42]. Exposure to a low level of organophosphate pesticides is known to produce a variety of biochemical changes, some of which may be responsible for the adverse biological effects reported in humans and experimental animals [24]. Fishes especially Channa punctatus are often used as indicator organisms to investigate the biological effects of pollutants in the aquatic environment. In recent years, fishes have served as useful bioindicators and integrators of contaminants because of various reasons, viz., their wide distribution in the freshwater environment, the fact of being free swimmers, their ability to respond against environmental pollution and their importance as an economic food source for human beings [2]. Biochemical constituents and certain enzymes have been explored as potential biomarkers for variety of different organisms because these parameters are highly sensitive and conserved between species and less variable. Their advantages are that biochemical and enzymes activities tend to be more sensitive, less variable, more highly conserved between species, and often easier to measure as stress indices [38]. Generally freshwater fishes, have been found to be useful as indicators and integrators of certain contaminants due to their wide geographical distribution, domi-
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nant presence in freshwater water ecosystem, bottom-dwelling habits, ability to respond to environmental pollutants, importance as an economic food source for humans, and ease in being readily transported and maintained under laboratory conditions. Hence, it is of interest to investigate the impact of MCP on some biochemical constituents and the activities of certain key enzymes in different organs of the edible fresh water fish, C. punctatus. 2. Materials and methods 2.1. Chemicals All the reagents used in the present study were of analytical grade and were used without further purification. The test compound monocrotophos (MCP) was a gift from the National Organic Chemical Industry Limited (NOCIL), Bombay, India.
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2.2. Animal maintenance The investigations were carried out in the freshwater fish, C. punctatus (Bloch), procured from pollution-free water bodies and were brought to the laboratory in large aerated drums. Later, they were acclimatized for 30 days in a huge cement tank (8 6 4 ft.) and fed with commercially available dry prawn. Fish weighing 25 ± 30 g were transferred to a glass aquarium (60 30 30 cm) of 40 1 water capacity for a further period of seven days and were fed with dry prawn for conditioning. The water in the aquarium was renewed daily and was aerated mechanically. The natural photoperiod of 13:11 light:day h was maintained. The average values for water quality parameters were as following temperature 26 ± 2 °C, pH 7.2 ± 0.2, dissolved oxygen 8.10 ± 0.021 mg 1 1, total hardness 120 ± 1.9 mg 1 1 as CaCO3, alkalinity 360 ± 1.4 mg 1 1 as CaCO3, and chlorides 275.567 ± 1.24 mg 1 1 [4].
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2.3. Sub-acute studies
et al. [3] and the glycogen by Caroll et al. [43]. For estimation of the carbohydrate and glycogen, glucose was used as standard. The free amino acids in the tissues were determined as per the procedure of Ishida et al. [44]. The total lipids were extracted based on the procedures of Folch et al. [15]. Acid phosphatase (ACP) (EC 3.1.3.2) activity in gills, liver, kidney, and muscles was measured by the method of Jabeen [22], and the inorganic phosphate (Pi) liberated was measured by the method of Fiske and Subbarow [9]. The enzyme activity was expressed as lmol Pi liberated/mg protein/h. The activity of alkaline phosphatase (ALP) (EC 3.1.3.1) in gills, liver, kidney, and muscles was estimated by the method of Moss et al. [11]. The activities of’ aminotransferase such as aspartate aminotransferase (AAT) (EC 2.6.1.1) and alanine aminotransferase (ALAT) (EC 2.6.1.2) activity in liver, gills, kidney, and muscles were estimated according to the standard procedure of Yatzidis [40]. The enzymes activity was expressed as lmol of phenol formed/mg protein/h. Lactate dehydrogenase (LDH) (EC 1.1.1.27) activity was determined by the method of McQueen [26], with NADH oxidation recorded at 340 nm and expressed as lmol formazan formed/mg/h.
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The acute LC50 value of monocrotophos was determined in the laboratory using the semi-static method [4]. The control experiments were also performed with addition of carrier solvent alone. During sub-acute studies a total of forty fish (20 fish per aquarium) were exposed to two sublethal concentrations of MCP viz. 0.96 and 1.86 mg l 1 (corresponding to 1/20th and 1/10th of 96 h LC50 = 18.56 mg l 1) for a period of 30 days. The required concentration was maintained by adding the toxicant directly in 40 l of water and renewed daily. The experiment was repeated thrice for each exposure and control. Feeding was disrupted during experimentation; i.e., fish were starved 24 h prior to dissection. 2.4. Tissue samples
Fish were anesthetized by placing them for 10 min in benzocaine hydrochloride (200 mg l 1) and the fish tissues such as liver, gills, kidney and muscles were excised. Tissues were homogenized (10% w/v) in 0.1 M Tris–HCL buffer (pH 8.0) using Poter-Elvehjam homogenizer fitted with a Teflone pestle. The homogenates were centrifuged at 5000g for 10 min and the supernatant was further centrifuged at 5000g for 10 min. The resultant supernatant was used as the enzyme source for the estimation of all the enzyme activities. All the enzyme preparations were carried out at 4 °C. 2.5. Biochemical studies The protein content of the samples was determined according to the method of Lowry et al. [30] using crystalline bovine serum albumin standard. The total carbohydrate was estimated by Kemp
2.6. Statistical analysis
The experiments were repeated on three different occasions in triplicate and that data were analyzed by Student’s t-test. Statistical comparisons were done between control and experimental data from the same species. Significant differences from control values P < 0.05, P < 0.02, P < 0.01, P < 0.005 and P < 0.001 were accepted as levels of statistical significance [16]. 3. Results and discussion
In general, the edible freshwater fish constitute one of the major sources of nutritious foods for humans. The nutritive values of fish depend upon their biochemical composition. Biochemical changes induced by pesticide stress is due to disturbed metabolism manifested by inhibition of enzymes, retardation of growth and reduction in the fecundity and longevity of the organism. Even though, the pesticides have their own target site of action, most of them are metabolic depressors. They generally affect the activity of biologically active molecules such as proteins, carbohydrates and lipids [34]. The protein is the major intake of energy source. Diana [20] has observed the ‘‘proximate composition of aquatic animals under various feeding regimes” and indicated that the energy gain or depletion from the body is due to the changes in the amount of whole-body tissue to its depletion from the body. Hence, the protein content of the cell is considered to be an important tool for the evaluation of physiological standards. The tissue protein is metabolized to produce glucose by the process of gluconeogenesis and it is utilized for energy production under stress conditions [25]. The results of the present investigation as presented in Tables 1 and 2 indicated that, the protein content of the liver, kidney, gills and muscles decreased by 57%, 61%, 62% and 70%, respectively, as compared to the control when C. punctatus were exposed to MCP. The decrease of protein content under MCP stress may be attributed to the utilization of amino acids in various catabolic reactions. Amino acids activity in the liver, gills, kidney and muscles showed a continuous decrease as the concentration increased (Tables 1 and 2). The maximum reduction in amino acids activity of the kidney, gills, liver and muscles of exposed fish were 58%, 59%, 66% and 74%, respectively, at sublethal concentration of 1.86 mg l 1. The amino acids through transamination and deamination reactions might have supplied necessary keto acids to act as precursors for the maintenance of carbohydrate metabolism to
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S. Agrahari, K. Gopal / Pesticide Biochemistry and Physiology 94 (2009) 5–9 Table 1 Variation in the biochemical constituents of liver, kidney, gills, and muscles in the control and experimental Channa punctatus.
Protein Carbohydrates Free amino acids Glycogen Total lipid Enzymes LDH ACP ALP AAT ALAT
Liver
Kidney 1
Control
0.96 mg l
135.50 ± 0.05 5.52 ± 0.10 9.84 ± 0.02 34.10 ± 0.15 67.15 ± 2.85
98.6 ± 2.5 4.20 ± 0.3b 7.32 ± 0.02 25.45 ± 0.5 52.16 ± 0.9c
81.09 ± 0.85 0.996 ± 0.02 0.358 ± 0.05 0.702 ± 0.03 0.312 ± 0.002
64.34 ± 1.0 0.745 ± 0.05c 0.212 ± 0.034 0.593 ± 0.01a 0.258 ± 0.01c
Gills 1
Control
0.96 mg l
123.30 ± 0.08 3.63 ± 0.03 5.32 ± 0.04 13.04 ± 0.03 63.01 ± 3.12
84.02 ± 0.92 3.01 ± 0.01 4.49 ± 0.1d 10.31 ± 0.03 55.35 ± 2.12
28.46 ± 0.23 2.81 ± 0.02 6.29 ± 0.02 5.05 ± 0.04 59.25 ± 6.50
90.43 ± 0.9c 0.756 ± 0.032d 0.697 ± 0.2 0.140 ± 0.02 0.063 ± 0.04
96.25 ± 3.42 0.628 ± 0.034 0.539 ± 0.03 0.419 ± 0.005 0.228 ± 0.004
101.7 ± 2.03 0.583 ± 0.03 0.456 ± 0.018 0.109 ± 0.003 0.043 ± 0.004
Muscles
Control
1
Control
0.96 mg l
20.2 ± 0.10 2.69 ± 0.13 5.33 ± 0.01 4.23 ± 0.18b 50.80 ± 0.13
99.16 ± 0.81 0.80 ± 0.02 7.49 ± 0.09 17.41 ± 0.026 135.78 ± 5.01
76.63 ± 2.8d 0.74 ± 0.04 6.86 ± 0.13 14.92 ± 0.1 123.50 ± 2.0
112.62 ± 1.2b 0.964 ± 0.022 0.767 ± 0.06a 0.309 ± 0.001 0.178 ± 0.006d
139.75 ± 3.54 0.353 ± 0.02 0.412 ± 0.03 0.246 ± 0.002 0.509 ± 0.005
109.32 ± 4.7d 0.626 ± 0.02a 0.654 ± 0.07a 0.129 ± 0.005 0.382 ± 0.003
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Data represent means ± SE of three individuals values. a P 6 0.05 denotes significant when compared with control. b P 6 0.02 denotes significant when compared with control. c P 6 0.01 denotes significant when compared with control. d P 6 0.005 denotes significant when compared with control.
0.96 mg l
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Constituents
Table 2 Variation in the biochemical constituents of liver, kidney, gills, and muscles in the control and experimental Channa punctatus. Constituents
Liver
Kidney
Control Protein Carbohydrates Free amino acids Glycogen Total lipid Enzymes LDH ACP ALP AAT ALAT
1.86 mg l
1
Control
d
135.50 ± 0.05 5.52 ± 0.10 9.84 ± 0.02 34.10 ± 0.15 67.15 ± 2.85
52.40 ± 7.80 1.90 ± 0.51c 2.43 ± 0.82d 16.43 ± 2.24c 37.58 ± 1.2d
81.09 ± 0.85 0.996 ± 0.02 0.358 ± 0.05 0.702 ± 0.03 0.312 ± 0.002
41.32 ± 4.11d 0.523 ± 0.05d 0.142 ± 0.01a 0.095 ± 0.051d 0.109 ± 0.02d
123.30 ± 0.08 3.63 ± 0.03 5.32 ± 0.04 13.04 ± 0.03 63.01 ± 3.12
101.7 ± 2.03 0.583 ± 0.03 0.456 ± 0.018 0.109 ± 0.003 0.043 ± 0.004
Gills
1.86 mg l
1
c
Control
46.31 ± 9.0 2.10 ± 2.03c 1.76 ± 0.39d 7.73 ± 1.00b 28.40 ± 1.0d
28.46 ± 0.23 2.81 ± 0.02 6.29 ± 0.02 5.05 ± 0.04 59.25 ± 6.50
73.88 ± 2.0d 1.098 ± 0.04d 0.782 ± 0.03d 0.196 ± 0.007d 0.073 ± 0.001c
96.25 ± 3.42 0.628 ± 0.034 0.539 ± 0.03 0.419 ± 0.005 0.228 ± 0.004
Muscles
1.86 mg l
1
1
Control
1.86 mg l
8.54 ± 3.2 1.43 ± 0.18c 2.61 ± 0.59c 2.10 ± 0.31d 32.31 ± 1.26a
99.16 ± 0.81 0.80 ± 0.02 7.49 ± 0.09 17.41 ± 0.026 135.78 ± 5.01
42.25 ± 6.12d 0.26 ± 0.078c 3.10 ± 0.63c 6.80 ± 1.78c 95.69 ± 1.54c
159.23 ± 7.0c 1.162 ± 0.06c 0.954 ± 0.05c 0.221 ± 0.02d 0.054 ± 0.02d
139.75 ± 3.54 0.353 ± 0.02 0.412 ± 0.03 0.246 ± 0.002 0.509 ± 0.005
86.56 ± 5.02c 0.889 ± 0.05d 0.923 ± 0.06c 0.078 ± 0.023c 0.158 ± 0.05c
c
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Data represent means ± SE of three individuals values. a P 6 0.02 denotes significant when compared with control. b P 6 0.01 denotes significant when compared with control. c P 6 0.005 denotes significant when compared with control. d P 6 0.001 denotes significant when compared with control.
meet the energy requirements during pollutants stress. Another possibility for the observed protein reduction should have been due to the blocking of protein synthesis or protein denaturation or interruption in the amino acid synthesis [8]. The depletion of protein content may also be due to the rapid utilization of tissue protein as the food utilization decreases when the animals are under stress conditions. Moreover, the chemical-exposed animals obtain extra energy requirement from the tissue protein [6]. The reduction in protein content indicates that the tissue protein may undergo proteolysis, which results in the production of free amino acids and is used in the trichloroacetic acid cycle for energy production under stress conditions. The carbohydrate level was depleted in liver, kidney, gills and muscles during the entire period of experiment (Tables 1 and 2). Carbohydrates in the tissues of C. punctatus exist as protein-bound sugars and glycogen. It is well known that the sugar serves as energy reserve for the metabolic process. Carbohydrates are considered to be the first among the organic nutrients degraded in response to stress conditions imposed on an animal. Chemical stress causes rapid depletion of stored carbohydrates primarily in liver and other tissues [7]. The glycogen level of different tissues showed a decreasing trend as MCP concentration increased (Tables 1 and 2). Depletion of glycogen may be due to utilization of stored carbohydrates in li-
ver for energy production as a result of pesticide-induced hypoxia. Glycogen depletion in liver and muscles after toxic stress has been reported in several studies with aquatic animals [23,31]. The depletion of glycogen in the tissues is indication of typical stress response in fish challenged with pesticides. The C. punctatus after exposure to two different concentrations of MCP showed over all decrease in the levels of carbohydrate and glycogen into all the fish organs. The maximum decrease (52%) was observed for glycogen in liver, whereas only slight changes were recorded in kidney and gills. Dhavale and Masurekar [10] are of the opinion that the decreased level of carbohydrate and glycogen in tissues of toxicant-exposed animals may be due to the prevalence of hypoxic condition in the tissues as a result of pollutant stress. Normally during hypoxic conditions, carbohydrate metabolism is increased to release energy, resulting in the extra expenditure of carbohydrate. The depletion of glycogen observed in all tissues of C. punctatus is, an indication of typical stress related response of the fish to the pesticide. These results are in accordance with the finding of Ghosh and Shrotri [32], who have suggested that decreased level of carbohydrates in the toxicant-exposed animals either induces the glycogenolysis, possibly by increasing the activity of glycogen phosphorylase to meet the energy demand under stress conditions or the toxicant has inhibitory effect on glycogenesis pathway.
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the assayed enzymes can work as good biomarkers of organophosphorus contamination. Acknowledgments The authors thank to Director, Indian Institute of Toxicology Research, Lucknow, for providing the facilities and constant encouragement. References [2] A. Gupta, D.K. Rai, R.S. Pandey, B. Sharma, Analysis of some heavy metals in the riverine water, sediments and fish from river Ganges at Allahabad, Environ. Monit. Assessment, (2008), in press. [3] A. Kemp, J.M. Adrienne, Kits Van Heijningen, A colorimetric method for the determination of glycogen in tissues, The Biochem. J. 56 (1954) 640–648. [4] APHA, Standard Methods for the Examination of Water and Wastewater, 21st ed., APHA, Washington, DC, 2005. [5] A. Prakasam, S. Sethupathy, S. Lalitha, Plasma and RBCs antioxidant status in occupational male pesticide sprayers, Clin. Chim. Acta 310 (2001) 107–112. [6] A. Yadav, A. Gopesh, R.S. Pandey, D.K. Rai, B. Sharma, Fertilizer industry effluent induced biochemical changes in fresh water teleost, Channa striatus (Bloch), Bull. Environ. Contam. Toxicol. 79 (6) (2007) 588–595. [7] B. Jyothi, G. Narayan, Pesticide induced alterations of non-protein nitrogenous constituents in the serum of a freshwater catfish, Clarias batrachus (Linn.), Indian J. Exp. Biol. 38 (2000) 1058–1061. [8] B.S. Jha, Alterations in the protein and lipid contents of intestine, liver and gonads in the lead exposed freshwater murrel Channa punctatus (Bloch), J. Ecobiol. 3 (1991) 29–34. [9] C.H. Fiske, Y. Subbarow, The colorimetric determination of phosphorous, J. Biol. Chem. 66 (1925) 375–400. [10] D.M. Dhavale, V.B. Masurekar, Variations in the glucose and glycogen content in the tissues of Scylla serrata (Forskal) under the influence of cadmium toxicity, Geobios 13 (1986) 139–142. [11] D.W. Moss, D.N. Baron, P.G. Walker, J.H. Wilkinson, Standardization of clinical enzyme assays, J. Clin. Pathol. 24 (1971) 740–743. [12] D.W. Peakall, Biomarkers, the way forward in environmental assessment, Toxicol. Ecotoxicol. News 1 (1994) 55–60. [13] G. Chantelli-Forti, M. Paolini, P. Hrelia, Multiple end point procedure to evaluate risk from pesticides, Environ. Hlth. Perspect. 101 (1993) 15–20. [14] G. Tripathi, S.P. Shukla, Malate and lactate dehydrogenases of a freshwater cat fish, impact of endosulfan, Biomed. Environ. Sci. 3 (1990) 52–58. [15] J. Folch, M. Lees, G.H. Bloane-Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 26 (1957) 497– 509. [16] J.H. Zar, Biostatistical Analysis, second ed., Prentice-Hall, Inc., Englewood Cliffs, NJ, 1984. pp. 401–403. [17] J. Storm, K. Rozman, J. Doull, Occupational exposure limits for 30 organophosphates pesticide based on inhibition of red blood cell acetylcholinesterase, Toxicology 150 (2000) 1–29. [18] J.V. Rao, Biochemical alterations in euryhaline fish, Oreochromis mossambicus exposed to sub-lethal concentrations of an organophosphorus insecticide, monocrotophos, Chemosphere 65 (2006) 1814–1820. [19] J.V. Rao, Sublethal effects of an organophosphorus insecticide (RPR-II) on biochemical parameters of tilapia, Oreochromis mossambicus, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 143 (2006) 492–498. [20] J.S. Diana, An experiment analysis of the metabolic rate and food utilized of northern pike, Comp. Biochem. Physiol. A 71 (1982) 395. [21] K. Chaudhuri, S. Selvaraj, A.K. Pal, Studies on the genotoxicity of endosulfan in bacterial systems, Mutat. Res. 439 (1999) 63–67. [22] K. Jabeen, Toxicological evaluations of some pesticides against chicken (Gallus gallus domesticus and rat (Rattus rattus norvegius) with special reference to hematology, blood and urine biochemistry, Ph.D. Thesis, Osmania University, Hyderabad, India, 1984. [23] L.H. Aguiar, G. Moraes, I.M. Avilez, A.E. Altran, C.F. Correˆa,, Metabolical effects of folidol 600 on the neotropical freshwater fish matrinxa~, Brycon cephalus, Environ. Res. 9 (2004) 224–230. [24] L.G. Sultatos, Mammalian toxicology of organophosphorus pesticides, J. Toxicol. Environ. Hlth. 43 (1994) 271–289. [25] M. Elumalai, M.P. Balasubramanian, Influence of naphthalene on esterase activity during vitellogenesis of marine edible crab, Scylla serrata, Bull. Environ. Contam. Toxicol. 62 (1999) 743–748. [26] M.J. McQueen, True Arrhenius relationships of human lactate dehydrogenase, Z. Klin. Chem. Klin. Biochem. 13 (1975) 17–19. [27] M. Rajyasree, P. Neeraja, Aspartate and alanine aminotransferase activities in fish tissue subcellular fractionation on exposure to ambient urea, Indian, J. Fish. 3 (1989) 88–91. [28] N.S. Oluah, Effect of sublethal copper (II) ions on the serum transaminase activity in catfish Clarias albopunctatus, J. Aquat. Sci. 13 (1998) 45–47. [29] N.S. Oluah, Plasma aspartate aminotransferase activity in the catfish Clarias albopunctatus exposed to sublethal zinc and mercury, Bull. Environ. Contam. Toxicol. 63 (1999) 343–349.
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In the low availability of carbohydrates, lipids serve as source of energy for supporting physiological activities of the body. The lipid content of liver, kidney, gills and muscles showed decreased levels in the C. punctatus exposed to monocrotophos for 30 days at both MCP concentrations (Tables 1 and 2). The decline in the lipid content might be due to the utilization of lipids for meeting the energy demand under the stress. The term biomarker is defined as a change in a biological system due to external stimuli. This ranges from molecular through cellular and physiological responses to behavioral changes, which are related mainly to the toxic effects of environmental pollutants [12]. The analysis of marker enzymes such as lactate dehydrogenase, aminotransaminases, and phosphatases serves as specific indications of water-pollution-induced changes in the enzyme activity of fish. Significant inhibition in the activity levels of these enzymes were observed in the liver. On the contrary, these were observed elevated levels in the kidney, gills and muscles in relation to MCP exposure. LDH is an enzyme recognize as a potential marker for assessing the toxicity of a chemical. The level of LDH was found to be increased in the gills and decreased in the liver, kidney and muscles in the MCP-exposed fish. The result of the present finding study is in accordance with the findings of Rao [18,19]. Several reports revealed decreased LDH activity in tissues under various toxic conditions [14,35]. This might be due to the higher glycolysis rate, which is the only energy-producing pathway for the animal when it is under stress conditions. LDH is an important glycolytic enzyme in biological systems and is inducible by oxygen stress. Phosphatases play major roles in the molting physiology of many fishes [39]. Acid phosphatase is a lysosomal enzyme that hydrolyses the phospho-esters in acidic medium. Alkaline phosphatase catalyses dephosphorylation of many molecules including nucleotides, proteins, and alkaloids at alkaline pH. It is well known that alkaline phosphatase is involved in carbohydrate metabolism, growth and differentiation, protein synthesis, synthesis of certain enzymes, secretion activity, and transport to phosphorylated intermediates across the cell membranes. In the present investigation rise in the activities of phosphatases in the gills, kidney and muscles, and a decline in liver in MCP-exposed C. punctatus were observed. Alterations in alkaline phosphatase (ALP) and acid phosphatase (ACP) activities in tissues and serum have been reported in pesticide treated fish [33]. The alanine aminotransferase (ALAT) and aspartate aminotransferase (AAT) are liver specific enzymes and they are sensitive markers of hepatotoxicity and histopathologic changes and can be assessed within a shorter time [41]. The increase in ALAT and AAT indicate the tissue damage in liver, kidney and gills of Clarias albopunctatus and C. punctatus [27,29]. In the present study the increase in ALAT and AAT activities was observed only in the kidney of C. punctatus. Similar results have also been reported by other workers [28,29,37] in different fish species such as C. albopunctatus and Carassius auratus gibelio. The results presented in Tables 1 and 2 indicated that the alanine and aspartate aminotransferase activities increased in kidney and decreased in liver, gills and muscles decreased in liver, gills and muscles of C. punctatus exposed to MCP. This is in accordance with the findings of Rao [18,19] who has reported similar enzymatic changes in Oreochromis mossambicus due to monocrotophos stress. The effects of MCP at higher degree are always preceded by fluctuations in the biological processes such as biochemical and enzyme systems allowing the identification of suitable biomarker signals. It is therefore of major significance to integrate the biochemical biomarkers in assessing the potential hazards and risks due to toxic environmental pollutants. This study showed that MCP induced impairment of metabolism in C. punctatus and that
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