CHAPTER-IV. Please purchase PDF Split-Merge on to remove this watermark.

Transcription

1 CHAPTER-IV

2 INTRODUCTION Effects of xenobiotic contamination in an ecosystem can be estimated through analysis of enzymological and biochemical changes in organisms inhabiting that region (Siroka and Drastichova, 2004; Havelková et al., 2007). Kori-Siakpere (2008) has stated that biochemical effects are mostly sublethal in origin as most of the toxicants exert their effects by reacting with enzymes or metabolites and other functional components of the cell in an organism. These effects might lead to irreversible and detrimental disturbances of integrated functions such as behavior, growth as well as survival (El-Naga et al., 2005). Enzyme activities have also been used as sensitive indicator of stress in fish exposed to diverse group of water pollutants and also to predict the possible level of threat to life (Kavitha et al., 2010). Foster (1980) suggested that alterations in enzyme activities can be easily measured even at low levels making it an important contributor of disease diagnosis. Several authors have reported that changes in enzyme activities reveal tissue damage in fish (Wroblewski and Ladue, 1995; Mazorra et al., 2002; Ozmen et al., 2006; Adamu and Iloba, 2008; Gabriel et al., 2011b; Nte et al., 2012). Gabriel and Akinrotimi (2011a) noted that biomarker can also be used to confirm and assess fish exposure to toxicants, providing a link between external exposure and internal structure and degree of responses to toxicant exposure observed between different individuals. Certain enzymes have been explored as potential biomarkers for a variety of organisms because of their highly sensitivity, less variability, and conserved nature between species (Vijayavel and Balasubramanian, 2006; Bláhová et al., 2009; Malarvizhi et al., 2012). These biomarkers may be sensitive and specific early warning signs for aquatic pollution (Strmac and Braunbeck, 2000). Akinrotimi et al. (2009) have reported that changes in enzyme activities in the treated fish is one of the major biomarkers specifying the consequences of pollutants in tissues, organs and body fluids of fish. Physiological indicators, such as enzymes, could be used as biomarkers of effect in possible environmental contamination, even before the health of aquatic organism get affected and also used as important parameters for testing water for the presence of toxicants (Zollner, 1993; Barnhoorn and van Vuren, 2004; Mekkawy et al., 2011). 77

3 Enzyme enticement or depression in fish or other organisms have been proposed for pollution monitoring studies. Fish respond to toxicants by altering their enzyme activities and the inhibition or induction of these enzyme activities has been used to indicate tissue damage (Nemcsok and Boross, 1982; Webb et al., 2005). Agrahari et al. (2007) have suggested that an increase of enzymes activity in the extracellular fluid or organs is a sensitive indicator of minor cellular damage.tissue specific enzymes may be released into the circulation on cellular damage in certain organs (Heath, 1987; Osman et al., 2007). Changes in plasma enzymes are used as possible tool for indicating tissue injury, environmental stress, or a diseased condition especially in aquatic environment (Wiseman et al., 2007; Kori-Siakpere et al., 2010; Inyang et al., 2011). Boyd (1983) noted that the rate of increase of plasma enzyme activity depends on the concentration of an enzyme in cells, the rate of leakage caused by injury and the rate of clearance of the enzyme from plasma. Fish cellular enzymes are an indicator of its health condition and its measurement has used as diagnostic tool in monitoring programs of aquatic pollution (Bernet et al., 2001; Fernandes et al., 2008; Orrego et al., 2011). The variations in the activity of cellular enzymes depend primarily on the magnitude and severity of cell damage (Szegletes et al., 1995; Napierska et al., 2009; Mesquita et al., 2011). Many enzymes such as carboxyl esterase (CE), lactate dehydrogenase (LDH), alkaline and acid phosphates (ALP, ACP), glutamate oxaloacetate transaminase and glutamate pyruvate transaminase (GOT and GPT) are measured as useful biomarkers to determine cellular impairment and cell rupture (Malarvizhi et al., 2012). Estimation of enzymes likes alkaline and aspartate aminotransferase (ALT, AST) and lactate dehydrogenase (LDH) are considered useful biomarkers to determine pollution level during chronic exposure (Asztalos et al., 1990; Basaglia, 2000; Ozmen et al., 2006). They may also used to biomarkers of acute hepatic damage (Rajamanickam and Muthuswamy, 2008; Aydin-Sinan et al., 2012). Nemcsok and Boross (1982) reported that among the enzymes, transaminases like glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) play a vital role in protein and carbohydrate metabolism and act as an indicator for tissue 78

4 damage and cell rupture. Nelson and Cox (2000) reported that serum GOT and GPT are important diagnostic tools in medicine and clinics, and are used to detect the toxic effects of various pollutants. Transaminases like aspartate aminotransferase (AST) and alanineaminotransferase (ALT) can be used to detect tissue damage caused by the toxicants and also used for aquatic monitoring (Nemcsók and Benedeczky, 1990). Kopecka and Pempkowiak (2008) has pointed out that determinations of ALT and AST activities in blood plasma and organs have incidentally been applied in fish research to indicate bacterial, viral and parasitic infections, intoxications and water pollution. Transaminase can be used for the detection of tissue damage caused by pollution (Öner et al., 2009; Gabriel et al., 2012). These are liver specific enzymes, thus are more sensitive measure of hepatotoxicity as well as histopathologic changes and even used for diagnosis of heart damage and can be assessed within a shorter time (Balint et al., 1997; Ozer et al., 2008). Transaminases are located in the cytoplasm and mitochondria playing a vital role in protein metabolism (Nemcsok et al., 1981). During the catabolism of most of the L-amino acids, the first step is the removal of the alpha amino groups which is promoted by aminotransferase or transaminases. The enzymes aminotransaminases are the strategic link between carbohydrate and protein metabolism interconverting the metabolites such as alpha ketoglutarate pyruvate and oxaloacetate on one hand and alanine aspartate and glutamate on the other hand (Höhne and Bornscheuer, 2012). GOT, also called aspartate aminotransferase (ASAT), catalyzes an important reaction of the molecular rearrangement involving amino acids linked to the citric acid cycle at two points (oxaloacetic and ketoglutaric acids), being the most important mechanism for introducing reduction equivalents into mitochondria (Urich, 1994). GPT, also called alanine aminotransferase (ALAT), predominates in organs with intensive glycogenesis, such as the liver (Urich, 1994; de la Torre et al., 2000). These enzymes are found predominantly in liver, cardiac cells and striated muscle tissue actively participate in transamination reactions. Samsonova et al. (2005) noted that aminotransaminases plays an important role in the utilization of amino acids for oxidation and /or for gluconeogenesis. 79

5 Glutamate oxaloacetate and pyruvate transaminases not only function as link enzymes between the protein and carbohydrate metabolism, but also serve as an indicator of altered physiological or stress condition (Knox and Greengard, 1965). The activities of transaminases may be used as a sensitive marker in teleost fish exposed to insecticides (Svoboda, 2001; Li et al., 2008; Dogan and Can, 2011). Alterations of these enzymes involved in the metabolism of amino acids allow the identification of tissue damage in organs such as the liver, muscle, gill and kidney (Karan et al., 1998; Asagba et al., 2004; Borges et al., 2007; Bacchetta et al., 2011). Increase in the activities of plasma GOT and GPT can be used as a sensitive indicator to assess even very minute cellular damage (van der Oost et al., 2003). Increases in aspartate aminotransferase and alanine aminotransferase enzymes were recorded in fish rainbow trout (Oncorhynchus mykiss) exposed to diazinon (Banaee et al., 2011), in C. carpio, Channa punctatus and Clarias batrachus exposed to cypermethrin (David et al., 2004; Kumar et al., 2011), in silver catfish, Rhamdia quelen exposed to clomazone herbicide (Crestani et al., 2007), in European eel Anguilla anguilla exposed to propanil (Sancho et al., 2009) and in catfish (Clarias gariepinus) exposed to deltamethrin (Amin and Hashem, 2012). Increased GOT and GPT level was also recorded in Cyprinus carpio exposed to simazine (Velisek et al., 2012), in Labeo rohita (Hamilton) exposed to fenvalerate (Prusty et al., 2011), in tilapia and common carp after exposure to deltamethrin (Velíšek et al., 2006b; El-Sayed et al., 2007), in Cyprinus carpio exposed to arsenic (Lavanya et al., 2011). On the other hand toxicants can also inhibit the activity or synthesis of these enzymes, resulting in decreased activities in the organs (Jung et al.,2003; de Aguiar et al., 2004; Gabriel et al., 2012). Significant decrease in GOT activity was noted in Cyprinus carpio exposed to arsenic (Lavanya et al., 2011), in Catla catla exposed to arsenic (Kavitha et al., 2010), in Cyprinus carpio exposed to pharmaceutical drugs such as clofibric acid, diclofenacand carbamazepine (Saravanan et al., 2011c; Malarvizhi et al., 2012), in Cyprinus carpio exposed to cypermethrin (Sivakumari et al., 1997). Lactate dehydrogenase (LDH) (EC ) is an enzyme found in many cells, in cardiac and skeletal muscle, liver, kidney and the red blood cells (Kaplan and Pesce, 1996) 80

6 It is involved in the interconversion of pyruvic acid and lactic acid and also serves as a pivotal enzyme between the glycolytic pathway and the tricarboxylic acid cycle (Tripathi and Singh, 2004). It is a source of the oxidised coenzyme during the period of transient anaerobiosis or a reduced form of such coenzyme during aerobiosis (Coppes, 1992). LDH is the terminal enzyme of anaerobic glycolysis, therefore, being of crucial importance to the muscular physiology, particularly in conditions of chemical stress when high levels of energy may be required in a short period of time (Ribeiro et al., 1999; Rees et al., 2001; Ozmen et al., 2008; Ombres et al., 2011). LDH is a tetrameric and important glycolytic enzyme which has been recognized as a potential marker for assessing the toxicity of a chemical and widely uses as a marker of tissue damage (Diamantino et al., 2001; Velisek et al., 2006a). It is involved in carbohydrate metabolism and has been used as indicative criteria of exposure to chemical stress (Wu and Lam, 1997; Osten et al., 2005; Narra et al., 2011). Diamantino et al. (2001) reported that lactate dehydrogenase is also used as indicative criteria of exposure due to chemical stress and anaerobic capacity of tissue. Cohen et al. (2001) stated that it may play a greater role in fish energy metabolism particularly in conditions of chemical stress than it does in mammals, when high levels of energy may be required in a short period of time. Therefore, LDH has also been used to be indicative of hypoxic conditions in the organism and plays an important role in glycolysis (Koukourakis et al., 2003; Daniela et al., 2008). It has been used as a biomarker of hypoxia in mussels (Wu and Lam, 1997) and in situations of chemical stress, as in fish (Gagnon and Holdway, 1999; Cohen et al., 2001), daphnia (Guilhermino et al., 1994; Diamantino et al., 2001) and isopods (Ribeiro et al., 1999) when organisms require additional energy. Alterations of the normal LDH activity pattern were found after exposure to a variety of different types of compounds (Ribeiro et al., 1999), including DCA (Guilhermino et al., 1994). A marked elevation in the activity of lactate dehydrogenase was observed in serum of O. niloticus, and gills and haemolymph of freshwater crab, Spiralothelphusa hydrodroma on exposure to cypermethrin, respectively (Fırat et al., 2011; Sreenivasan et al., 2011). An increase in LDH activity was also noticed in Notopterus notopterus exposed to mercuric chloride (Verma and Chand, 1986), 81

7 in Pomatoschistus microps to 3, 4-dichloroaniline (Monteiro et al., 2006b) and in Indian major carp (Catla catla, Labeo rohita and Cirrhinus mrigala) due to nitrite toxicity (Das et al., 2004b). However, Ganathy et al. (1994) documented decrease in LDH activity in different tissues of C. punctatus exposed to hexachlorocyclohexane. Similar reduction in LDH activity was reported with increasing concentration of quinphos, paddan and cypermethrin in Labeo rohita (Das and Mukherjee, 2003), 2-butenoic acid -3-(diethoxyphosphinothioyl) methyl ester (RPR-II) in Oreochromis mossambicus (Venkateswara Rao, 2006b), monocrotophos in Channa punctatus (Agrahari et al., 2007), in the serum and hepatopancreas of C. carpio exposed to different concentrations of curacron and simazine (Joseph and Raj, 2011) and in the crab Carcinus maenas exposed to Cu, Cr or a mixture of both (Elumalai et al., 2002). In contrast to the above, Osten et al. (2005) found no alterations in LDH activity was observed in the acute toxicity and the in vivo effects of commercial chlorpyrifos, carbofuran and glyphosate formulations in mosquitofish, Gambusia yucatana. From the forgoing review of literature it can be concluded that there is a lack of available information about the enzymological effect of methyl parathion, on the aquatic organisms particularly in Indian freshwater fish. Hence in the present study an attempt was made to study the effect of widely used methyl parathion on certain transaminases like GOT, GPT and LDH (in gill, liver and plasma) of a fresh water fish Catla catla in order to use these biomarkers in the field of environmental toxicology. 82

8 MATERIALS AND METHODS ESTIMATION OF GOT GOT activity was estimated by 2, 4-DNPH method (Reitman and Franckel, 1957). PRINCIPLE GOT catalyses the transamination of L- Aspartate and α- ketoglutarate (α-kg) to form Oxaloacetate and L- Glutamate. Oxaloacetate so formed is coupled with 2, 4-Dinitrophenyl hydrazine (2, 4-DNPH) to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can be measured colorimetrically. ά- Ketoglutarate +L-Aspartate L-Glutamate+ Oxaloacetate Oxaloacetate + 2, 4-DNPH Corresponding Hydrazone (brown colour) REAGENTS Reagent 1: Buffered Aspartate ά-ketoglutarate substrate, pη 7.4 Reagent 2: 2, 4-DNPH colour reagent Reagent 3: Sodium hydroxide, 4N Reagent 4: Working Pyruvate standard, 2mm. PREPARATION OF WORKING SOLUTION Solution 1: Dilute 1ml of Reagent 3 to 10ml with purified water. PROCEDURE Four test tubes were taken and marked as Blank (B), Standard (S), Test (T), and Control (C). To each test tube 0.25 ml of Reagent 1 (Buffered Aspartate ά-ketoglutarate substrate) was added. Then 0.05 ml of sample from methyl parathion treated fish and Standard (Reagent 4) were added to respective Test and Standard tube, respectively, mixed well and incubated at 37ºC for 60 minutes. Then 0.25 ml of Reagent-2 (2, 4-DNPH colour reagent) was added to all tubes and mixed well. After that 0.05 ml of deionised water and sample were added to the test tubes Blank 83

9 and Control, respectively and mixed well. All test tubes were allowed to stand at room temperature for 20 minutes. Then to all the test tubes about 2.5 ml of Solution-1 was added, mixed well and allow to stand at room temperature for 10 minutes. The O.D values were measured against distilled water using UV Spectrophotometer at 505 nm. The same procedure was repeated for Control (untreated) sample. CALCULATION Absorbance of Test- Absorbance of Control GOT activity IU/L= Conc. of Standard Absorbance of Standard- Absorbance of Blank ESTIMATION OF GPT GPT activity was estimated by 2, 4-DNPH method (Reitman and Franckel, 1957). PRINCIPLE GPT (ALT) catalyses the transamination of L- Alanine and ά-ketoglutarate to form pyruvate and L- Glutamate. Pyruvate so formed is coupled with 2, 4- Dinitrophenyl Hydrazine to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can we measured calorimetrically. ά-ketoglutarate +L-Alanine L-Glutamate+ Pyruvate Pyruvate +2, 4- DNPH Corresponding Hydrazone (brown colour) REAGENTS Reagent 1: Buffered Alanine ά- KG substrate, PH 7.4 Reagent 2: DNPH color Reagent Reagent 3: Sodium Hydroxide, 4N Reagent 3: Working pyruvate standard, 2mM. PREPARATION OF WORKING SOLUTION Solution I: Dilution 1ml of Reagent 3 to 10 ml with purified water. 84

10 PROCEDURE Four test tubes were taken and marked as Blank (B), Standard (S), Test (T) and Control (C). To each test tube 0.25 ml of Reagent 1 (Buffered Alanine ά- KG substrate) was added. Then 0.05 ml of sample from methyl parathion treated fish and Standand (Reagent IV) were added to respective Test and Standard tubes, respectively, mixed well and incubated at 37ºC for 30 minutes. Then 0.25 ml of Reagent-2 (DNPH color reagent) was added to all tubes and mixed well. After that 0.05 ml of deionised water and sample were added to the test tubes Blank and Control, respectively and mixed well. All test tubes were allowed to stand at room temperature for 20 minutes. Then to all the test tubes about 2.5 ml of Solution-1 was added, mixed well and allow to stand at room temperature for 10 minutes. The O.D values were measured against distilled water using UV Spectrophotometer at 505 nm. The same procedure was repeated for Control (Untreated) sample. CALCULATION Absorbance of Test- Absorbance of Control GPT activity (IU/L) = Conc. of Standard Absorbance of Standard- Absorbance of Blank ESTIMATION OF LDH LDH activity was estimated by 2, H-DNPH method (Tietz, 1976). PRINCIPLE Lactate Dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate with simultaneous oxidation of reduced NADH to NAD. The rate of decrease in absorbance due to formation of NAD is measured at 340 and is proportional to the LDH activity in the sample. LDH catalyses the following reaction Pyruvate + NADH + + H LDH ---- Lactate + NAD Where, NADH =Nicotinamide adenine dinucleotide (reduced form) LDH = Lactate Dehydrogenase. 85

11 REAGENT COMPOSITION Reagent 1: Buffer Pyruvate 50 m mol / L Reagent 2: Substrate NADH 0.18 m mol / L. WORKING REAGENT PREPARATION Four parts of Reagent 1 was mixed with one part of Reagent 2 and the contents were mixed properly before use and labeled the bottle as working Reagent. PROCEDURE For the estimation of LDH, two test tubes were taken and marked as Test (T) and Control (C). To each tube 1000 µl of working reagent was added. Then 10 µl of sample from experimental and control fish was added into the respective tubes (Test and Control tube). The contents in the tubes were mixed well. The O.D values were measured against distilled water using UV Spectrophotometer. The absorbance was noted after 60 seconds at 37ºC at 340 nm. Again the absorbance was repeated for four times at every 30 seconds (i.e.) upto 120 seconds (2 minutes). CALCULATION LDH activity (IU/L) = ΔA Kinetic factor Where, ΔA/minute = Change in absorbance per minute Where, M = Molar extinction coefficient of NADH = lit/mol/cm at 340 nm TV = Sample volume + Working Reagent volume SV = Sample volume P = Optical path length 10 6 = Constant The kinetic factor K will change if the sample volume, the working reagent volume or the optical path lengths are altered. 86

12 RESULTS The effect of methyl parathion on GOT, GPT and LDH activities in the gill, liver and plasma of Catla catla during acute exposure is shown in Table 15 and Fig. 14a to 14c. GOT and GPT activities in liver and plasma was significantly (P < 0.05) increased in the fish exposed to acute concentration (0.09 ppm) of methyl parathion. In contrast to the above, GOT and GPT activity in gill was significantly (P < 0.05) decreased from that of control group showing a percent decrease of 8.51 and respectively. LDH activity was found to be significantly (P < 0.05) decreased in all the organs studied showing a percent decrease of 6.91, and in gill, liver and plasma respectively. Statistical analyses indicate that all the values were significant at 5 % level. Table 16 and Fig. 15 shows the effect of methyl parathion (0.009 ppm) at sublethal concentration on GOT activity in gill of fish, Catla catla exposed for a period of 35 days. During the above exposure period, a biphasic response in GOT activity was noted in gill. The GOT activity significantly (P < 0.05) decreased up to 21 st day with a maximum percent decrease of at the end of 21 st day. After 21 st day, a significant increase in GOT activity was observed till the end of the study period. Statistical analyses indicate that all the values were significant at 5 % level. Alterations in GOT activity in liver of fish Catla catla exposed to sublethal concentration of methyl parathion for a period of 35 days is presented in Table 17 and Fig. 16. At the end of 7 th day the GOT activity was found to be elevated showing a maximum percent increase of However, after 7 th day the significant increase in GOT activity was gradually recovered showing a percent increase of and at the end of 14 and 21 st day respectively. But after 21 st day, the enzyme activity was significantly (P < 0.05) decreased in the rest of the study period. All the values were found to be significant at 5% level. 87

13 LDH (IU/L) GOT (IU/L) GPT (IU/L) Enzymes Control Experiment Control Experiment Gill Liver Plasma Gill Liver Plasma Fig. 14 a. Fig. 14 b. Control Experiment Gill Liver Plasma Fig. 14 c. Fig. 14 Enzymological parameters (a. GPT, b. GOT, c. LDH) of C. catla exposed to acute concentration of methyl parathion. Bar represent the mean ± SE. The values presented in parenthesis are percentage change during exposure period. Significant at 5% level (P < 0.05) 88

14 GOT (IU/L) in Gill Enzymes 160 Control Experiment Exposure period (in Days) Fig. 15. Fig. 15. GOT activity in the gill of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level (P < 0.05) 89

15 GOT (IU/l) in Liver Enzymes 350 Control Experiment Exposure period (in Days) Fig. 16. Fig. 16. GOT activity in the liver of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level (P < 0.05) 90

16 Changes in the plasma GOT activity of C. catla exposed to sublethal toxicity of methyl parathion for 35 days are shown in Table 18 and Fig. 17. During the above exposure period, the enzyme activity was gradually increased as the exposure period increased showing a minimum percent increase of 8.22 at the end of 7 th day and a maximum percent increase of at the end of 35 th day. Statistical analyses indicate that all the values were significant at 5 % level. GPT activity in the gill of Catla catla exposed to sublethal toxicity of methyl parathion for a period of 35 days is presented in Table 19 and Fig. 18. During the study period a gradual decrease in GPT activity was observed throughout the study period showing a maximum percent decrease of 59.4 at the end of 7 th day and a minimum percent decrease of at the end of 35 th day. The statistical analysis indicated all the values to be significant at 5% level. Table 20 and Fig. 19 depict the data on the changes in GPT activity in the liver of C. catla exposed to sublethal concentration of methyl parathion. During the above exposure period the enzyme activity was increased upto 28 th day with a maximum percentage increase of at the end of 28 th day. At the end of the study period (35 th day) the GPT activity decreased by 21 percent when compared with the control group. The values were found to be statistically significant at 5%. Changes in the GPT activity in plasma of C. catla exposed to sublethal concentration of methyl parathion is presented in Table 21 and Fig. 20. A significant increase (P < 0.05) in GPT activity was observed throughout the study period showing a direct relationship with the exposure period. A minimum percent increase of 9.09 was noted at the end of 7 th day and a maximum percent increase of was noted at the end of 35 th day. The statistical analysis indicated all the values to be significant at 5% level. 91

17 GOT (IU/L) in Plasma Enzymes Control Experiment Exposure period (in Days) Fig. 17. Fig. 17. GOT activity in the plasma of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level (P < 0.05) 92

18 GPT (IU/L) in Gill Enzymes 90 Control Experiment Exposure period (in Days) Fig. 18. Fig. 18. GPT activity in gill of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level (P < 0.05) 93

19 GPT (IU/L) in Liver Enzymes Control Experiment Exposure period (in Days) Fig. 19. Fig. 19. GPT activity in the liver of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level 94

20 GPT (IU/L) in Plasma Enzymes Control Experiment Exposure period (in Days) Fig. 20. Fig. 20. GPT activity in the plasma of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level 95

21 Data on the LDH activity in the gill of C. catla exposed to sublethal concentration (0.009 ppm) of methyl parathion is presented in Table 22 and Fig 21. The enzyme activity was observed to be decrease till 21 st day with a maximum decrease percentage of on that day. After the 21 st day, elevation in LDH activity was observed till the end of the study period (35 th day). The statistical analysis reveals that all the values were significant at 5% level. Changes in LDH activity in the liver of C. catla exposed to sublethal concentration of methyl parathion is presented in Table 23 and Fig. 22. During the above exposure period a significant (P < 0.05) reduction in LDH activity was observed at the end of 7 th day. After 7 th day, a gradual increase in LDH activity was observed throughout the study period with a maximum increase percentage of at the end of 28 th day. The values were statistically significant at 5% level when compared to the control. Table 24 and Fig. 23 represent the data on alteration in LDH activity in plasma of C. catla exposed to sublethal concentration of methyl parathion carried out for a period of 35 days. A gradual reduction in LDH activity was observed throughout the study period with a maximum decrease percentage of at the end of 35 th day. The statistical analysis reveals the values to be significant at 5% level when compared to the control. 96

22 LDH (IU/L) in Gill Enzymes 3000 Control Experiment Exposure period (in Days) Fig. 21. Fig. 21. LDH activity in the gill of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level 97

23 LDH (IU/L) in Liver Enzymes 3000 Control Experiment Exposure period (in Days) Fig. 22. Fig. 22. LDH activity in the liver of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level 98

24 LDH (IU/L) in Plasma Enzymes 1400 Control Experiment Exposure period (in Days) Fig. 23. Fig. 23. LDH activity in plasma of C. catla exposed to sublethal concentration of methyl parathion for 35 days. Bar represent the mean ± SE. - represents statistically significant at 5% level 99

25 DISCUSSION Pollution impact on ecosystem and human health is an urgent and international issue since there is an ever increasing number of an example of environmental disturbances, likely to affect the biota and humans, both natural and anthropogenic stress (Islam and Tanaka, 2004; Marcogliese et al., 2009; Smale et al., 2011). The assessment of environmental status has become an important issue in the striving for a sustainable society and use of natural resources (Rahman et al., 2002; Parvez and Raisuddin, 2006a). Huggett et al. (2003) reported that aquatic vertebrates particularly fish appear to have similar enzyme and receptor systems as in mammalian system. Moreover, fish react to environmental toxicants by changing and adapting their metabolic functions (Malarvizhi et al., 2012). Enzyme activities are considered as sensitive biochemical indicators and widely used to assess the health of the organism in aquatic toxicology (Gül et al., 2004). Mayer et al. (1992) has pointed out that basically there are four different process that may suggest the responses of enzyme to specific or non-specific chemical stress; they are 1) direct enzyme inhibition, 2) enzyme induction by specific classes of chemicals, 3) elevation of serum enzymes viz., tissue damage and 4) alterations in enzyme activities a result of changes in metabolic pathways or fluxes. The authors further added that enzyme activity is generally regulated such that specific substances or entire pathways may be homeostasically adjusted to compensate for endogenous or exogenous changes. Lavanya et al. (2011) reported several enzymes like LDH, ACP, ALP, GOT, GPT have been used to determine pollution exposure in animals and to monitoring of water pollution. These enzymes serve as a good bioindicators in animals subjected to chronic exposure of xenobiotics (den Besten et al., 2001; Ozmen et al., 2006). Among the array of enzymes used the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are widely used to detect the tissue damage caused by the toxicants (Jung et al., 2003). They may also be used as biomarkers of cellular damage in blood plasma, protein degradation and liver damage (Markovich and James, 1999). During long term exposure, toxicants may accumulate in various tissue and leads to changes in the enzymatic activities. 100

26 Variations in transaminases activities in fish exposed to toxicants generally indicate the tissue damage or liver dysfunction due to toxicant stress (Oluah, 1999; Adham, 2002; Venkateswara Rao, 2006a, c; Min and Kang, 2008). Sepici-Dinçel et al. (2009) observed that the increase in activities of transaminases activity in the muscle and liver of common carp exposed to cyfluthrin may be due to a disturbance in the Kreb s cycle. Yildirim et al. (2006) exposed Oreochromis niloticus to deltamethrin for four days and observed increase in enzyme activities (AST and ALT) in the gill, liver and kidney and assumed that the observed (enzyme elevation) is intended to increase the role of proteins in the energy production during toxicant stress. This will eventually result in a shift in biosynthesis and the energy metabolism pathway of the exposed organism (Gagneten and Paggi, 2009). Changes in protein and carbohydrate metabolism during stress conditions may also affect the activity of GOT and GPT and the elevation of transaminases can be taken as a measure of compensatory mechanism to impaired metabolism (Reddy and Venugopal, 1991). Several soluble enzymes of blood serum have been considered as indicators of the hepatic dysfunction and damage (Kavitha et al., 2010). The significant increase in GOT and GPT activity in gill, liver and muscle of fish Cyprinus carpio during acute and sublethal carbamazepine treatment indicates that the damage of the organs due to drug toxicity or the organism tries to mitigate the drug induced stress by increased rate of metabolism (Malarvizhi et al., 2012). John (2007) reported that pesticide induced induction in aminotransferase activities has been reported in fish and this elevation was directly attributed to toxic action of pesticide on liver. Patnaik (2010) noted increased values of GOT and GPT activity in C. batrachus exposed to carbaryl due to pesticide intoxication which suggest enhanced protein catabolism and probable hepatocellular damage in the organism. In the present study, the significant increase in GOT and GPT activity in gill and liver during sublethal exposure indicate the damage of the organ due to pesticide accumulation or the organism tries to mitigate the toxicant induced stress by increased rate of metabolism. Likewise, the observed increase in GOT and GPT activity in gill and liver may indicate that an increase energy demand to meet the stress caused by MP. In general, the utilization of amino acids for the oxidation or for gluconeogenesis during stress condition may leads to an increase in transaminases activities (Philip et al., 1995). 101

27 However the observed decrease in GOT and GPT activity in gill and liver acute and sublethal treatment signifies that detoxification mechanism may not be sufficiently effective to prevent the action of the pesticide on the system. Moreover, the observed decrease in GOT and GPT activity in gill and liver during sublethal exposure of MP may be suggestive of damaged hepatocytes that are no longer capable of synthesizing the GOT protein. Venkateswara Rao (2006b) suggested that the decrease in the activity of aminotransferases in liver of fish Oreochromis mossambicus exposed to organophosphorus pesticide (RPR II) may be due to liver damage which results in release of these enzymes into plasma. In the present investigation also an increase in GOT and GPT activity was observed in plasma of fish both in acute and sublethal treatment. The decreased activities of GOT indicate disturbance in the structure and integrity of cell organelles (Malarvizhi et al., 2012).Such damage to cell organelles has been reported in various studies (Karatas and Kalay, 2002). Sivakumari et al. (1997) reported that the inhibition of GPT activity in fish indicate enzyme loss from soluble regions of the hepatocytes and not from the mitochondrial fractions. Humtsoe et al. (2007) reported significant decrease in liver AST and ALT in Labeo rohita exposed to arsenic which reflects significant decrease in structure and function of cell organelles like endoplasmic reticulum and membrane transport system. Li et al. (2008) observed a declining trend in the activity of liver GOT at 24 h in top mouth gudgeon, Pseudorasbor aparva exposed to methomyl. Their results may not have manifested the induction mechanism, which might indicate a correlation between exposure to pesticide, exposure time and concentration, and biological species but it may be that hepatic cells were injured, explaining the protein metabolism dysfunction. Similar reduction was also observed in transaminase activity (GPT and GOT) in liver and gill of tilapia, Oreochromis mossambicus, exposed to organophosphorus insecticides, 2-butenoic acid-3-(diethoxyphosphinothioyl) methyl ester (RPR-II), and monocrotophos, respectively (Venkateswara Rao, 2006a, b). The decrease in transaminase activity in liver reported in the present study may probably be due to disturbed permeability and integrity of cell organelles, like endoplasmic reticulum and membrane transport system by methyl 102

28 parathion, as supported by Roy (2002). de Smet and Blust (2001) reported that changes in alanine aminotransferase and aspartate aminotransferase activities may be due to change in protein metabolism in the tissues due to stress. Transaminases like GOT and GPT are usually present in tissues/organs like gill, liver, muscle, brain etc., and injuries to cells in these tissues/organs due to toxicant stress may result release of these enzymes into plasma (Patnaik, 2010). Increases in GOT and GPT levels in Channa punctatus after 15 and 60 days of exposure to monocrotophos is also indicate liver damage as suggested by Agrahari et al. (2007). The significant increase in AST and ALT levels in fish exposed to arsenic indicates hepatic damage due to arsenic accumulation which in turn releases these enzymes into blood stream (Lavanya et al., 2011). Roy and Bhattacharya (2006) noted significant changes in serum GOT and GPT in C. punctatus exposed to As 2 O 3 and indicate that the changes may be due to histopathological lesions in liver. The damage and severity of the organ is dependent on the type of toxicant and duration of exposure (Casillas et al., 1983; Jacobson-Kram and Keller, 2001). Dogan and Can (2011) noted that dimethoate exposure resulted in duration dependant induction of transaminase enzyme activities. They suggested that when hepatocytes are damaged, enzymes normally located in cytosol are liberated into the extracellular space and enter the circulation due to membrane defects causing increased permeability (Ozer et al., 2008). Shakoori et al. (1990) reported that the increase in blood enzymatic activity is either due to (i) leakage of these enzymes from hepatic cells and thus raising levels in blood, (ii) increased synthesis and (iii) enzyme induction of these enzymes. In the present study, the significant increase in GOT and GPT activity in plasma during acute as well as sublethal exposure treatment might have resulted from the organal damage due to MP accumulation. Upon MP exposure reactive oxygen species (ROS) may be generated which may damage the organs like gill, liver, muscle resulting in leakage of these enzymes into plasma. The elevation in plasma transaminases activity can be effectively uses as biomarkers of MP toxicity in fish. van der Oost et al. (2003) reported that an increase of these enzyme activities in the extracellular fluid or plasma 103

29 is a sensitive indicator of even minor cellular damage. Thus, the measurement of transaminase activities in blood plasma of fish can be used as indicator for pesticide toxicity. Decrease in serum ALT activity during arsenic exposure in fish indicates a congested condition in liver (Datta et al., 2007). The pathopathological condition in liver during arsenic exposure may leads to hepatocytes death (Limuro et al., 1998). Susan et al. (1999) observed decrease in GOT level during chronic exposure and indicate that detoxification mechanism may not be sufficiently effective to prevent the action of the arsenic trioxide on the system. The decrease in plasma GOT activity in both clofibric acid and diclofenac treatments indicated the accumulation and toxicity of these drugs in liver which might have caused the necrosis (Saravanan et al., 2011c). In the present study the observed reduction in plasma GOT and GPT activity can be attributed to pesticide accumulation in liver which in turn leads to death of liver cells. In the present study the decrease in LDH activity in gill and liver during acute and sublethal MP exposure indicate the higher glycolysis rate under pesticide stress. MP may inhibit the aerobic and anaerobic metabolism of fish resulting in a decrease in LDH activity. A decrease in biosynthetic activities and anaerobic capacity of fish due to pesticide stress may also leads to a decrease in LDH activity in tissues of fish (Tripathi and Verma, 2004; Venkateswara Rao, 2006a). Joseph and Raj (2011) reported a decrease in LDH activity in C. carpio when exposed to different concentrations of curacon. They reasoned that the reduction may be due to the functioning of intermediates into the TCA cycle. Several other reports have suggested that decreased LDH activity in tissues under various pesticide toxicity conditions might be due to the higher glycolysis rate, which is the only energy producing pathway for the animal when it is under stress conditions (Tripathi and Shukla, 1990; Mishra and Shukla, 2003; Malarvizhi et al., 2012). Inhibition in the activity of the LDH may be either due to the change in mitochondrial membrane junction or it may be due to impaired glycolysis (Sastry and Siddiqui, 1984). Yadav et al. (2007) reported that fertilizer industry effluent caused marked reduction in tissue LDH activity in Channa striatus. Decreased LDH activity may be due to lower 104

30 metabolic rate under toxic conditions (Agrahari et al., 2007). Probably, the inhibition of LDH in the present study during acute and sublethal treatment may be due to impaired carbohydrate metabolism. Thus, the measurement of alteration in the LDH activity in gill, liver and kidney can be used as a biomarker indicating stress. Inhibition of LDH activity during sublethal treatment may be due to impaired carbohydrate metabolism. LDH activity is a good indicator of the anaerobic capacity of a tissue (Dickson et al., 1993). Increased LDH activity in muscle and liver tissues indicates metabolic changes in chemically stressed fish. In the intoxicated juvenile pink snapper, the catabolism of glycogen and glucose appears to have shifted towards the formation of lactate, which may have adverse effects on the animal (Szegletes et al., 1995). Accumulation of lactate may lead to metabolic acidosis and subsequent muscle fatigue (Moose, 1980), with implications on foraging and escape capacities in wild animals. Increased LDH activity levels have been observed in conditions of chemical stress when high levels of energy are required in a short period of time (de Coen et al., 2001). Further, prevalence of anoxia during stress conditions may leads to an increase in LDH activity in tissues (Das et al., 2004a). The observed elevation of LDH activity in gill and liver during sublethal treatment might have resulted from damage of these tissues due to MP toxicity. Elevated levels of LDH activity of muscle, liver and brain tissues of C. carpio chronically exposed to distillery effluent results from damage to tissues. The conversion of pyruvate into lactate is favored resulting in increased lactic acid content in blood (Verma and Chand, 1986; Sivakumari et al., 1997). The elevation of LDH activity in the gill, liver, and muscle in the present study may have occurred due to the stress induced increase in the rate of glycolysis, as suggested by Ghosh (1987). Moreover, increased LDH activity in gill and liver tissues in the present study indicates metabolic changes in methyl parathion stressed fish. As the rate of glycolysis increases, the pyruvate (the product of glycolysis) is not routed to Kreb s cycle, rather catalyses to lactate, thereby shifting the respiratory metabolism from aerobiosis to anaerobiosis. Any changes in protein and carbohydrate metabolism may cause change in LDH activity (Abston and Yarbrough, 1976). Elevated LDH activity in gills suggests that the aerobic 105

31 catabolism of glycogen and glucose has shifted towards the formation of lactate, which may have adverse long-term effects on the organisms (Szegletes et al., 1995). The elevation of LDH activity in gill, liver and muscle of fish Cyprinus carpio exposed to carbamazepine has occurred may be due to the metabolic changes caused by the drug (Malarvizhi et al., 2012). Further disruption of respiratory epithelium might have caused tissue hypoxia resulting in a decrease in oxidative metabolism which may be responsible for increase in LDH activity in toxicant stressed animals (Gill et al., 1990). The increase in plasma LDH activity in benomyl treated fish Oreochromis niloticus indicate that the toxicity may be produced through anaerobic mechanism as a result the aerobic oxidation via the Kreb s cycle was affected (Min and Kang, 2008). Sivakumari et al. (1997) reported changes in the dehydrogenase activity in pesticide-treated fish may be due to severe cellular damage, leading to increased release of dehydrogenase that impaired carbohydrate and protein metabolism. The significant increase of these enzymes may indicate the changes in the histological structure of the hepatic and extra hepatic tissues (Svoboda, 2001; El-Sayed et al., 2007). Abston and Yarbrough (1976) suggested that any changes in protein and carbohydrate metabolism might cause change in LDH activity. From the results we can conclude that methyl parathion which is a commonly used organophosphorous pesticide in many parts of the country can alter the enzymological parameters of fish, especially when exposed to sublethal concentration. These parameters can be used as non specific biomarkers in assessing the pesticide toxicity in aquatic ecosystem. Further, it is important to evaluate the residual effects of this pesticide in different body tissues of fish as they are ultimately consumed by the human beings. 106