A study on Transaminases in Lindane Exposed Fish C. punctatus

The transaminases such as Glutamate Oxaloacetate Transaminase (GOT or AST) and Glutamate Pyruvate Transaminase (GPT or ALT) are the key enzymes known for their roles in utilization of protein and carbohydrates. The activities of these two enzymes have been shown to alter in fish and other organisms due to pesticide stress. Therefore, the study was initiated to monitor the levels of these enzymes in C. punctatus exposed to 3 subacute concentrations of lindane (0.025, 0.05, 0.1 mg/l) for 96h. The results showed increase in activity of AST and ALT in all the tissue of fish at highest concentration; the values being in following order: gills>kidney>brain>heart>muscle>liver for AST and for ALT the values were in following order: brain>muscles=kidney>gills>liver>heart, after 96hr treatment period. At highest concentration of lindane (0.1 mg/l) tested the maximum rise in AST activity was found in gills (50.84%) and minimum in liver (27.99%). The other organs such as kidney, brain, heart and muscle exhibited 49.42, 45.14, 39.70 and 33.50% rise, respectively. The activity of ALT, however, showed maximum increase in brain (59.60%) and minimum in heart (30.26%) at highest concentration of lindane (0.1 mg/l) tested. The kidney, muscle, gills and liver registered 57.00, 57.02, 52.63 and 38.18% rise, respectively, under this condition.

The transaminases or aminotransferases function as a link between carbohydrate and protein metabolism and frequently used as markers for the aquatic pollution [1,2]. These marker enzymes catalyse the transamination reactions between an amino acid and an α-keto acid for synthesis of amino acids and proteins. In this context, the amino (NH2) group of one molecule is transferred to the keto (C = O) group of the other molecule, thereby turning the former amino acid into a keto acid, and the later into an amino acid. It requires vitamin B6 (pyridoxal phosphate, PLP) as a coenzyme which is converted to Pyridoxamine Phosphate (PMP) when an amino acid forms keto acid. It follows ping-pong mechanism of enzyme action. The half-life of ALT is around 47h and AST is 18h, destroyed by sinusoidal cells in liver. Fluctuating level of activity of this enzyme indicates liver or cardiac damage. The ALT and AST catalyse the following reactions:

Alanine + α- ketoglutaric acid ALT pyruvic acid + glutamic acid

Aspartic acid + α- ketoglutaric acid AST oxaloacetate + glutamic acid

The glutamate can undergo oxidative deamination to remove ammonia as urea in liver by ornithine cycle. Oxaloacetate can also be obtained from Krebs cycle which can again form aspartae.

Lindane is a hydrophobic, highly persistent, resistant to photodegradation and non-biodegradable organochlorine pesticide. It is used as insecticides, weedicides, fungicides, pesticides, scabicides, and pediculicides. It gets highly bioaccumulated in aquatic organisms and cause perturbations in their biochemical pathways. It is bioaccumulated in the fatty tissues of fish at ratio of 500:1200 due to their hydrophobic/lipophillic nature [3]. Several studies have reported about the alterations caused in aquatic biota due to lindane and many other toxicants. In our earlier study of acute toxicity tests, experimentally calculated LC50 of lindane was 0.15 mg/l, 100% mortality was recorded within 96h at 0.3 mg/l in lindane exposed fish C. punctatus [4]. Its exposure has affected the activities of enzyme like lactate dehydrogenase, phosphatases, acetylcholinesterases [5-7]. Its use has been banned since 2009 under Stockholm convention for persistent organo chlorine pesticide [8,9]. In humans its chronic exposure has resulted in anemia, hepatic, renal, and reproductive organs diseases [10]. It is an important part of food chain, so studies on fish will always show their direct impact on humans. In the present study, we have evaluated the impact of lindane on the activities of transaminases in different tissues of the fish, C. punctatus, exposed to three different subacute concentrations of the pesticide for 96h.

Healthy Snake head fish, Channa punctatus, having mean length (10-15 cm) and mean weight (25-30 g) and checked for no external signs of injury or diseases were used for experiments. The fish were washed and treated with potassium permanganate (0.1%, w/v) for five min to remove any dermal adherent. The fish were acclimatized for 7 days in aerated tap water at 18-22°C in separate glass tanks (1’x1’) each containing 10 fish under standard laboratory conditions. Tanks were cleaned and water was exchanged every 24h by siphoning and replacing it with fresh water. Test media were kept well aerated. All tests were one in triplets at room temperature in filtered and aerated water. Lindane used in study was procured from Rallis India Ltd. Bangalore, India and was dissolved in acetone (AR) grade for use. All other reagents used in this study were analytical grade.

Exposure to lindane

The three subacute concentrations of lindane prepared were 0.025, 0.05, 0.1 mg/l for the exposure of C. punctatus for 96h. The equal volume of pesticide free, acetone was maintained in control aquaria as lindane added to the experimental aquaria was dissolved in acetone. In another group of fish treated with the corresponding concentration of acetone has been used in our earlier studies and it did not exert any adverse effect and the results were comparable to that of control and also acetone has low toxicity to fish and reported 24h LC50 values being in the range of 6000 mg/l [11,12]. All aquaria were constantly aerated during the period of exposure by aerator and the fish were fed properly. The water was changed after 24h each and replenished with fresh lindane.

Preparation of tissue homogenate

The fish tissues were homogenized in 10% (w/v) in cold 0.05 M sodium phosphate buffer, pH 7.4 and the homogenates were centrifuged at 10,000 xg for 10 min in cold (4°C) condition. These supernatants were either used for biochemical assays or stored frozen at -20°C for further use.

Protein determination: The supernatants were used for protein determination using Folin-Ciocalteau reagent [13]. The Bovine Serum Albumin (BSA) was used as a standard. The intensity of blue color was measured colorimetrically at 620 nm. The total protein in the tissue’s extracts were determined and used for calculating specific activities of the transaminases.

Assays of the activities of glutamate-pyruvate-transaminase (ALT) and glutamate-oxaloacetate-transaminase (AST)

The activities of enzymes were measured in cell-free homogenate of each tissue by the method of Reitman and Frankel [14]. The enzymes activities were directly proportional to the amount of pyruvic acid for ALT and oxaloacetate for AST, as determined colorimetrically by the formation of hydrazine with Dinitro-Phenyl Hydrazine Reagent (DNPH), which develops coloured complex in alkaline medium. The cell- free extract (100-200 µg protein) of each organ homogenate was mixed with substrate either for ALT (200 mM alanine and 2 mM α-ketoglutarate prepared in 100 mM sodium phosphate buffer pH 7.4) or AST (200 mM aspartic acid and 2 mM α-ketoglutarate prepared in 100 mM sodium phosphate buffer pH 7.4). The reaction mixture was incubated at 37°C for 30 min with intermittent shaking. The reaction was stopped by the addition of DNPH (0.5 ml, 1 mM) and was further incubated for 20 min. The color was developed by the addition of NaOH (5.0 ml, 0.1 N). The intensity of color was measured after 10min of addition of NaOH at λ_max 510 nm.

Calculation of enzyme activity

The activities of ALT and AST were calculated as follows:

∆OD x 200/ 0.2 x incubation time (min) x protein concentration (mg) and the unit was expressed as µM pyruvate formed min-1 mg-1 protein, where 200nm sodium pyruvate corresponds to 0.2 OD at λ_max 510 nm.

Effect of lindane on the transamination and oxidative deamination reaction in different organs of the fish exposed to subacute concentrations of lindane.

The effect of sub lethal concentrations of lindane (0.025, 0.05 and 0.1 mg/l) on the activities of AST and ALT was evaluated in different tissues of C. punctatus exposed to pesticide for 96h are shown in tables 1,2.

The data demonstrated that the liver of control fish contained maximum activity of AST followed by other organs such as muscle, heart, kidney, brain and gills of unexposed snake head fish; the values being 112.93 ± 1.16, 110.03 ± 1.17, 108.04 ± 1.13, 92.75 ± 1.16, 86.61 ± 1.72 and 84.05 ± 2.3 units/mg protein, respectively. The treatment of the fish with lindane resulted into significant increase in the activity of AST in all the tissues tested. At the lowest concentration (0.025 mg/l), kidney was maximally affected showing about 22% increase in AST activity whereas muscle was least affected with about 4% increase in activity. Under this condition, gills and heart showed similar level of influence of pesticide exposure causing rise in AST activity by about 16%. The effect of lindane was more pronounced when the fish was treated with high concentration (0.1 mg/l) for 96h. At highest concentration of lindane (0.1 mg/l), the maximum rise in AST activity was found in gills (50.84%) and minimum in liver (27.99%). The other organs such as kidney, brain, heart and muscle exhibited 49.42, 45.14, 39.70 and 33.50% rise in AST activity, respectively. The order of influence of the pesticide at highest concentration (0.1 mg/l) in the activity of AST in different fish tissues tested was as following: gills>kidney>brain>heart>muscle> liver. These results suggested that gills of C. punctatus were highly affected at 0.1 mg/l concentration of lindane, whereas kidney exhibited greatest sensitivity to lindane at lowest concentration (0.025 mg/l) (Table 1).

The data demonstrated the presence of maximum activity of ALT in the heart (87.77 ± 3.47 units/mg protein) of control fish followed by liver, kidney, muscle, brain and gills; the values being, 79.48 ± 1.77, 66.75 ± 3.47, 62.70 ± 1.16, 62.0 ± 1.21 and 57.97 ± 1.14 units/mg protein, respectively. The order of presence of activities of ALT in different fish tissues of the control fish was as following: heart>liver> kidney>muscle>brain >gills (Table 2). Similar to the influence of the pesticide on the activity of AST, the subacute concentrations of lindane resulted into significant increase in the activity of ALT in all the tissues tested.

At the lowest concentration (0.025 mg/l), muscle of the fish was maximally affected showing 24.37% rise in the enzyme activity, whereas heart exhibited least impact of pesticide with only 3.84% increase. The effect of the pesticide was concentration dependent. At the highest concentration of lindane (0.1 mg/l), brain exhibited maximum increase (59.60%) in the activity of ALT, whereas heart showed least impact of the pesticide as only 30% rise in its activity was recorded. Under this condition, the other tissues such as muscle, kidney, gills and liver registered 57.00, 57.02, 52.63 and 38.18% rise in ALT activity, respectively. The muscle and kidney displayed the influence of the pesticide to equal extent showing about 57% rise in the ALT activity in each case. The order of increase in the activity of ALT in these fish tissues due to pesticide treatment may be summarised as following: brain>muscle= kidney>gills>liver>heart (Table 2).

The comparison of the data presented in the tables 1,2 indicated that exposure of C. punctatus to the subacute concentrations of lindane caused regular increase in the activities of AST and ALT in different body organs of the fish. The effect was concentration dependent. The highest subacute concentration of lindane (0.1 mg/l) caused maximum increase in the activities of these two enzymes. However, the lowest concentration of lindane (0.025 mg/l) caused minimum induction in the activities of both transaminases. At highest concentrations of the pesticide, gills were maximally affected showing about 51% rise in activity and liver was least affected with about 28% increase in GOT activity (Table 1). Lindane at highest concentration (0.1 mg/l) exerted maximum affect in brain causing about 60% rise in GPT activity; kidney and muscles were equally affected with 57% increase in GPT activity and the heart was least affected showing about 30% induction in enzyme activity (Table 2). The data also suggested that the effect of lindane was tissue specific.

Under stress conditions the body mechanisms are altered to combat the effect of pollutants/stressors in order to maintain physiological equilibrium in the organism. The aminotransaminases (AST and ALT) in fish have been frequently used as markers for the aquatic pollution. The alterations in the activities of these enzymes have been shown to diagnose the impact of sublethal toxicity of environmental contaminants in fish [15]. Aminotransferases function as a link between protein and carbohydrate metabolism by the interconversion of strategic compounds like α-ketoglutarate and alanine to pyruvic acid and glutamic acid by process known as transamination [16].

In the present investigation lindane at sublethal concentrations (0.025, 0.05 and 0.1 mg/l) increased the level of AST and ALT in different tissues of C. punctatus exposed to pesticide for 96h. The gills and kidney of C. punctatus were most affected organs as lindane induced marked elevation in the levels of AST activity in these tissues. These findings were in agreement with that of Sharma who reported similar results in a fresh water cat fish, C. batrachus exposed to an organocarbamate pesticide, carbaryl [17].

The effect of lindane in present investigation was found to be concentration dependent which indicates a correlation between exposure of pesticide and increased levels of these two enzymes [18,19]. The increase in the levels of these enzymes may be causing imbalances in the physiology or anatomy of fish tissues exposed to subacute concentrations of lindane for 96h. The elevated enzyme activities of AST and ALT in response to the pesticide treatment have been shown to be associated with altered carbohydrate metabolism or deamination reactions which act as source of ketoacid for Kreb’s cycle and gluconeogenesis as is also evident from the decreased protein content in the pesticide treated fish tissues [20,21]. These results also indicate towards increased level of energy demand, enhanced metabolic pathway and excessive utilization of amino acids. The liver is the metabolic centre for detoxification of chemicals. Increase in the activities of these enzymes have been reported by researches like in the muscle and liver of the fish, Cyprinus carpio, exposed to 2,4 diamin an organochlorine pesticide [22], in different tissues of the sailfin molly, Poscillia latipinna exposed to a low concentration (0.03 mg/l) of dieldrin [23], in Anabas testudineus (Bloch) exposed to lethal (10.5 mg/l) and sublethal (4 mg/l) concentration of disyston [24], in brain, liver, muscle and gills of Oreochromis mossambicus exposed to lethal (0.15 mg/l) and sublethal (0.05 mg/l) concentrations of lindane [25]. Elevated levels of AST and ALT enzymes were reported in different tissues of Tilapia mossambica exposed to parathion-methyl [26], in Channa striatus exposed to malathion and phosphomidon [27], in rosy barb Puntius conchonius exposed to aldicarb, phosphamidon and endosulfan [28] and in Cyprinus carpio exposed to paraquat [29], in liver and muscle tissues of Clarias batrachus exposed to carbofuran during the 6-day exposure period [30]. The results indicated that the enhanced activities of transaminases provided oxaloacetic, pyruvate, a-ketoglutarate, and glutaric acid to meet the increase energy demand during xenobiotic-imposed stress conditions. Ramaswamy and coworkers also found that Sarotherodon mossambicus, when exposed to sublethal and lethal concentrations of carbaryl, showed adaptive elevation in the activity levels of ALT and AST enzymes, particularly in liver and muscle, thereby probably aiding gluconeogenesis by mobilizing the glucogenic L–amino acids (like alanine and aspartic acid) through transamination of glucogenic amino acids to meet the energy demand under carbaryl toxicity [31].

The effects of other group of pesticides and toxicants on the levels of transaminases in fish tissues have also been studied. The declining effect of methomyl on the activities of enzyme in the fish, Pseudorasbora parva, tissues by exposing them to 0.043-0.142 mg/l for 24h [32], in liver and gills of tilapia, Oreochromis mossambicus, exposed to 0.017 mg/l of novel organophosphorus insecticides, 2-butenoic acid-3-(diethoxyphosphinothioyl) methylester (RPR-II), and 1.15 mg/l of monocrotophos, respectively [33,34]. Decrease in activity in the fish Clarius gariepinus, exposed to aqueous extract of Lepidhagathis alopecuroides leaves could be attributed to a fall in the synthesis of glycogen caused by lowered metabolic demands and also due to electrolytic imbalance caused by tissue overhydration [35,36]. Increased activities in Channa punctatus to sublethal concentrations (0.96 and 1.86 mg/l) of monocotrophs for 15 and 60 days in blood plasma indicated hepatic tissue damage [37].

The heavy metals have also been reported to induce perturbations in the activities of GOT and GPT [38]. The exposure of fish to cadmium also caused a decrease in Phosphofructokinase (PFK) activities in the fish muscle and liver, which showed that the glycolytic pathways in these tissues were diminished. The toxic effects of CdCl₂ exposure could be accredited to the inhibition of energy yielding processes and the metabolic changes brought on by the CdCl₂ exposure were similar to those seen in starvation [39,40]. Almeida conducted a study to evaluate the influence of exposure to sublethal concentrations of CdCl₂ on the metabolic pathways of the freshwater fish Oreo-chromis niloticus. The results indicated slower growth rate and altered liver function. The total protein concentrations of the white muscle and liver were significantly decreased. They have reported significant decrease in the activities of transaminases in the red and white muscles of the CdCl₂-exposed fish [41]. Fluctuating activities seen in Oreochromis niloticus exposed to organophosphates and pyrethtroids [42]. It has been proved that stress can not only be of toxicants, but the conditions in which fish are reared are also important. Clarias gariepinus reared in earthen ponds have shown higher activities indicating heaptic stress as compared to those reared in concrete tanks [43]. Moderate changes were seen in fish Pangasianodon hypophthalmus at high temperature [44]. Histopathological changes like necrosis, bleeding, and epithelial degeneration were seen in immature grass carp exposed to lindane [45]. Recent studies have shown increased expression of genes like abcg5 and abcg8 in zebra fish larvae; sod-5 and isp in C. elegans responsible for reduced toxicity and hence detoxification ability of fish on lindane exposure [46,47].

The results from the present study indicated that lindane at sublethal concentrations may cause significant perturbations in the activities of transaminases in almost all the tissues of the fresh water fish, C. punctatus. The effect of the pesticide was concentration dependent. The levels of activities of AST and ALT in different fish tissues may serve as potential indicators of lindane toxicity in the fish. The data may be useful to the environmentalists and policy makers for designing strategies to combat lindane contamination in water bodies to safeguard the health of aquatic organisms and finally the humans, who may consume pesticide infested fish.

The authors are thankful to the University of Allahabad for providing facilities to carry out the present work. AG is grateful to UGC-New Delhi for financial support in the form of a fellowship.

Conflicts of interest

Authors declare no conflict of interests.

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