Turkish Journal of Fisheries and Aquatic Sciences
www.trjfas.org ISSN 1303-2712 DOI: 10.4194/1303-2712-v17_3_05
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Assessment of Antioxidant Biomarkers and Protein Levels in Tissues of
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Oreochromis mossambicus and Channa punctatus Exposed to Toxicity by
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Fungicides
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Neelanjana Choudhurya, *, Jayanta Tarafdarb and Ashis Kumar Panigrahic
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a
Department of Plant Pathology, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia (West
Bengal)-741235. India. b
Officer-in-Charge, AICRP- Tuber Crops, Kalyani Centre, Directorate of Research, Bidhan Chandra Krishi Viswavidyalaya,
Kalyani, Nadia (West Bengal)-741235. India. c
University of Kalyani, Fishery Extension Laboratory, Department of Zoology, Kalyani, Nadia (West Bengal)-741235.
India. Email:
[email protected] Phone:- 09163224805
Abstract
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The present investigation is about change in antioxidant enzymes and protein profile in Oreochromis mossambicus and
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Keywords: Oreochromis mossambicus, Channa punctatus, fungicides, Antioxidant enzymes, Protein profile.
Channa punctatus due to toxicity induced by application of fungicides in paddy-cum-fish ecosystem in India. Antioxidant enzymes like Lactate dehydrogenase (LDH), Malate dehydrogenase (MDH) and Peroxidase (Pox) are used as biomarkers for toxicological study. Oreochromis mossambicus and Channa punctatus were exposed to different concentration of Azoxystrobin 23% SC and Hexaconazole 5% SC respectively and LC 50 was determined. LC50 for Amister was detected to be 0.008 ml/lit and that for Contaf plus was found to be 0.025 ml/lit. In case of Oreochromis mossambicus 1/2.5th, 1/5th and 1/7.5th of LC50 (0.0032 ml/lit, 0.0016 ml/lit and 0.001 ml/lit) were selected for chronic sub-lethal study. For Channa punctatus ½nd, ¼th and 1/6th of LC50 value (0.0125 ml/lit, 0.0062 ml/lit and 0.0041 ml/lit) were chosen. After 3 months of exposure, the fish exposed to toxicity were sacrificed and stress enzymes and protein profile were checked in gill, heart, liver, kidney, muscle and spleen. Overall result revealed a gradual increase in LDH and Peroxidase but decrement in level of MDH and protein profile was also changed due to toxicity by fungicides.
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Introduction
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Stressor factors including water pollution, viral and bacterial infections, parasitic invasions,
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malnutrition, severe bleeding may induce to the perturbation of many haematochemical parameters in fish
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(Fazio et al., 2013, 2014, 2015). Several researches have been carried out with the characterization of tissue and
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organ specific isoenzyme patterns (Mo et al., 1975; Seimiya et al., 1997) among which little were concerned
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with fishes. Few studies have concerned with the isoenzymatic profiles in the Nile tilapia (Li & Zhao, 2001;
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Shahjahan et al., 2008). Chaudhuri and Krishna (1998) reported the tissue specificity and variation in the degree
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of expression of five enzyme systems in Labeo rohita from river Yamuna in liver, muscle, heart and brain
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tissues. Certain antioxidant enzymes like LDH (lactate dehydrogenase), MDH (malate dehydrogenase), ALT
Turkish Journal of Fisheries and Aquatic Sciences
www.trjfas.org ISSN 1303-2712 DOI: 10.4194/1303-2712-v17_3_05
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(Alanine transaminase), AST (Aspartate transaminase), SDH (succinic dehydrogenase), and Peroxidase are
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being extensively used as potential biomarkers for measurement of tissue and organ damage due to pesticidal
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toxicity. LDH and MDH isoenzymes are major stress related enzyme system found in fishes.
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As evidenced from previous studies, LDH is an important glycolytic enzyme present in all cells of
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almost all body tissues and changes in the enzyme activity may provide direct or indirect role to indicate the
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toxicity. Amacher (2002) said LDH is a terminal enzyme of anaerobic glycolysis and it mediates inter-
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converson of lactate to pyruvate depending on the availability of co-enzyme NAD. Therefore, being of crucial
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importance for muscular physiology, where under chemical stress more amount of energy is needed in short
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span of time (Coppo et al., 2002; Baghi et al., 1995). The significant changes to enzyme activity indicates
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damage to any or all organs like liver, kidney or muscle injuries (Young et al., 1999; De Coen et al., 2001).
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Exposure to fungicides and insecticides causes decline in MDH and elevation in Peroxidase in all fish
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species. This enzyme not only converts malate to oxaloacetate but also plays a significant role in CO2 fixation
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besides gluconeogenesis (Lehninger principles of biochemistry 5th edition).
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Malate + NAD+ → Oxaloacetate + NADH+ + H+
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Peroxidase enzyme is involved in phagocytosis and immune cell function (Rodriguez et al., 2003;
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Soares-da-Silva et al., 2002), cell adhesion (Holmblad & Soderhall, 1999), antioxidant function (Gamble et al.,
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1995; Galloway & Depledge, 2001) and helps in formation of melanin by oxidative polymerization of
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hydroquinones (D’Ischa et al., 1991). Previous investigations confirmed that due to pesticidal impact on fish
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reveals changes in protein profile. It is also found that a high proteolytic activity or increased production of
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protease enzyme causes low levels of protein content in tissues of fish under stress (Baise et al., 2012; Nagaraju
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et al., 2013). In this investigation, a comparative study for antioxidant biomarkers and protein profile has been
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presented due to toxicity induced by Amister (Azoxystrobin 23% SC) in Oreochromis mossambicus and Contaf
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plus (Hexaconazole 5% SC) applied to Channa punctatus.
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Materials and Methods
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Sample collection and acclimatization
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The samples were collected from a local market of length 30±5 cm and weight 52±5 g. Prior to the
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experimentation the normal uninfected healthy fish were selected for experiment. The fish were cleaned under
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running tap water and disinfected using 0.02% KMNO4 and 0.004% formalin solution to remove external
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infection of fungi, algae, etc. The samples were acclimatized for 15 days to laboratory conditions and kept in
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aquariums containing 35 litre of water and regularly fed. The changes in physiochemical characteristics of
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water, such as temperature, pH, hardness, total alkalinity and DO (dissolved oxygen) of experimental water
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were recorded throughout the experimental period (Table 1).
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Experimental design
Turkish Journal of Fisheries and Aquatic Sciences
www.trjfas.org ISSN 1303-2712 DOI: 10.4194/1303-2712-v17_3_05
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10 samples of each Oreochromis mossambicus and Channa punctatus were randomly selected from the
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stock and were exposed to ten different concentrations of fungicides Amister and Contaf plus respectively for 96
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hr to determine mean lethal concentration (LC50). Different concentrations of Amister (Azoxystrobin 23% SC)
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application was restricted from 0.001-0.010 ml/lit (0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009,
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0.01 ml/lit) and Contaf plus (Hexaconazole 5% SC) were ranged from 0.005-0.05 ml/lit (0.005, 0.01, 0.015,
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0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05 ml/lit). LC50 for Amister was detected to be 0.008 ml/lit and that for
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Contaf plus was found to be 0.025 ml/lit. In case of Oreochromis mossambicus 1/2.5th, 1/5th and 1/7.5th of LC50
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(0.0032 ml/lit, 0.0016 ml/lit and 0.001 ml/lit) were selected for chronic sub-lethal study. For Channa punctatus
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½nd, ¼th and 1/6th of LC50 value (0.0125 ml/lit, 0.0062 ml/lit and 0.0041 ml/lit) were chosen. A control group
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was maintained simultaneously in both the experiments. All these experiments were performed in triplicates.
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The toxicated fish samples were subjected to Native PAGE for stress related enzymatic study and also
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protein profile was checked.
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Native PAGE
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At the end of 90 days, the toxicated fish samples were collected and subjected for enzymological study.
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For sample preparation 100 mg of different organs, gill, heart, liver, kidney, muscle and spleen were taken and
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menced in 500 μl (1:5) of 0.1M Tris-HCl buffer (pH 7.4). The homogenized lysate was centrifuged at 14000
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rpm for 40 min at 4 ºC and supernatent was preserved for enzyme analysis (Native PAGE). Antioxidant
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enzymatic study for LDH, MDH and Peroxidase was done. Native PAGE (10% native polyacrylamide gel) was
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performed for qualitative study of LDH, MDH and Peroxidase as described by Stegmann et al., (1985) for
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different tissues, gill, heart, liver, kidney and spleen.
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Gel Staining The staining procedure was followed as described by Bader (1998).
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LDH: After electrophoresis, the gel was incubated in 100 ml of 0.05 M Tris HCl pH 8.5 containing 25 mg NBT,
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25 mg EDTA, 25 mg NAD, 1ml lactic acid and 3 mg PMS and kept for 15-30 min in dark.
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MDH: After electrophoresis, the native gel was placed in 0.05 M Tris HCl pH 8.5 (100 ml) containing 25 mg
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NBT, 25 mg EDTA, 25 mg NAD, 10 mg malic acid and 3 mg PMS for 15-30 min in dark.
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Peroxidase: After gel run, incubation was done in 100 ml of Tris-HCl buffer bearing 50 mg O-dianisidine
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(previously dissolved in few drops of acetic acid) and 1ml of hydrogen peroxide and left for 15-30 min in dark.
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Isolation of Protein
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500 mg fish organs (gill, heart, liver, kidney, muscle and spleen) were thawed in urea-thiourea buffer (7
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M urea, 2 M thiourea, 4% CHAPS, 45 mM Tris, 60 mM DTT and protease inhibitor- PMSF). Thawed samples
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were vortexed and kept at 4⁰C for 30 min. Mechanically disrupted and kept on ice. Samples were adjusted to
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900 µl of lysis buffer (20 mM Tris, 100 mM NaCl, 1% Triton and Protease inhibitor - PMSF) and incubated for
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15 min at 35 ⁰C. Reincubated in ice for 10 min. 100 µl of lysis buffer was added and incubated for 10 min along
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with DNAse I. The samples were centrifuged at 12000 rpm for 15 min at 4⁰C (middle aq phase bears protein).
Turkish Journal of Fisheries and Aquatic Sciences
www.trjfas.org ISSN 1303-2712 DOI: 10.4194/1303-2712-v17_3_05
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Extended delipidation was accompanied by tri-n-butylphophate-acetone-methanol precipitation. Precipitated
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proteins were estimated subjected to SDS-PAGE.
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Protein Estimation
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Protein concentration was calculated by Lowry’s method (Lowry et al., 1951). Absorbance was measured at 750
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nm by UV-VIS Elico spectrophotometer.
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SDS-PAGE of Protein profile
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SDS-PAGE was performed for protein profiling of fungicides exposed fishes. 10% polyacrylamide gel was
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casted according to Stegmann et al., (1985) and with Coomasie Brilliant Blue by incubating it overnight in
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staining solution bearing the dye followed by destaining (Methanol: 25 ml, Glacial acetic acid: 25 ml and water:
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200 ml) and photographed.
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Data analysis
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LC50 was determined using SPSS Vs. 17 and enzymological calculations were done by Student’s t-Test.
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Results
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Native PAGE of stress enzymes in Oreochromis mossambicus
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The results are summarized in Table 2, 3 and 4. The gradual increase of lactate dehydrogenase (LDH)
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and peroxidase (Pox) in gill, heart, kidney, liver, muscle and spleen exposure to 30 d, 60 d and 90 d for 0.001,
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0.0016 and 0.0032 ml/lit. The sub-lethal concentrations of Azoxystrobin caused significant depletion (P
