Oxidative Stress in Liver and White Muscles of Nile ... - eJManager

Egypt. J. Exp. Biol. (Zool.), 11(1): 23 – 36 (2015)

© The E gypt ian S oc iet y of E xper im ent al B iology

RESEARCH ARTICLE Moha m e d A. M. H e ga zi* S om ya Z. Abd E l H ale im * Mahm oud M. S . S ir da h* * S a le h N . Mw af y* *

O XID A T IV E S T RE SS I N LI V ER A N D WH I T E M U S C LE S R E SP O N SE S TO P O L LU TE D A RE A I N T H E NILE RI VE R

ABSTRACT: Oxidative stress in liver and white muscles of Nile tilapia, Oeochromis niloticus captured in Rosetta branch of Nile River at Kafr El-Zayat area, AlGharbiyah Governorate, Egypt was studied. The toxic effects of water in this area that, receive a lot of sewage and other industrial pollutants discharge, on the antioxidant system of Nile tilapia (62.4 ± 6.9 g) were investigated. Water samples were analyzed for physicochemical parameters including pH, turbidity, total dissolved solids, ammonia, nitrite and nitrate. The activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR) and the level of lipid peroxidation (LPO) in liver and white muscles were assayed. The examined physicochemical parameters found to be in excess than the maximum standard limits in water samples of the examined areas. The increase was more detectable during autumn and winter than other seasons. The alterations in the physicochemical parameters of water are consistent with the alteration reported in the enzymes activity of SOD, GST, GR, and GPx, as well as LPO, concentration which increased significantly (P ≤ 0.05) as compared to the control. However, the increase was more detectable during autumn and winter than in summer and spring seasons. The physicochemical parameters considered as the most important principles in the identification of the nature, quality and type of the water and/or any aquatic system. Antioxidant enzymes and the oxidative stress biomarkers lipid peroxidation considered as the most important biomarkers used to identify polluted marine sites. This can be taken as a useful biomarker for environmental managers in investigating the exposure of fish to contaminated freshwaters. This study could be beneficial for ecotoxicological researches in freshwaters as it provides data about antioxidant system response for fish exposed to different water pollutants. KEY WO RDS : Superoxide dismutase, catal ase, glut athioneS-transferase, glutathione reductase, glutathione peroxidase, li pid peroxidat ion ISSN: 2090 - 0511

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CO RRESP ON DENCE: Moha m ed A. M. H eg a zi * Department of Zoology, Faculty of Science, Tanta, Egypt E-m ail: m _a_m_hegazi@hotm ai l.com S om y a Z . A bd El H al eim* Mahm oud M oh am ed S. Si r dah * * S al eh N. Mw afy ** *Department of Zoology, Faculty of Science, Tanta, Egypt **Departm ent of Biology, Faculty of Sci ence, Al-Azhar University of G aza, Gaza Strip, P alestine

ARTIC LE CO DE : 03.01.15 INTRO DU CTIO N: Pollution is one of the biggest global killers. Over the last three decades there has been increasing global concern over the public health impacts attributed to environmental pollution, in particular, the global burden of disease. W ater pollution is one of the principal environmental and public health problems, of which Egypt and the Middle East region are facing (Anwar, 2003). Water pollution does not only greatly damage and threatened the aquatic ecosystems and the terrestrial organisms (Authman et al., 2013). The natural resources of water polluted with a variety of solid and liquid wastes. Every waste ultimately dumped or emptied in natural water bodies (Garg et al., 2009). Contamination of water can result from industrial and agricultural and sewage sources. Deficiencies in the treatment of wastewater, the disposal of untreated sewage, and inadequate operation and maintenance of treatment plants result in health risks (Anwar, 2003). About a quarter of the diseases facing humankind today occur due to prolonged exposure to environmental pollution (EPH, 2010; Azimi and Moghaddam, 2013). It was reported that the two biggest components of the global water crisis are the contamination of drinking supplies with

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human discharge and the massive wastage of water that is inherent in prevailing agricultural practices (WHO, 1998). River Nile from Aswan to El-Kanater Barrage receives wastewater discharge from 124-point sources, of which 67 are agricultural drains and the remainders are industrial sources (NWRC, 2000). Rosetta Branch, starting downstream of Delta Barrage receives relatively high concentrations of organic compounds, nutrients and oil and grease. Major sources of pollution are Rahawy drain (which receives part of Greater Cairo wastewater), Sabal drain, El-Tahrrer drain; Zawiet El-Bahr drain and Tala drain (Fig. 1).

Fig. 1. Location Map of Rosetta Branch of the Nile River

Unf ortunately, several industri al companies at Kafr El-Zayat City affect the Rosetta Branch. These industrial outfalls are El-Mobidat, El-Malyia, S alt and Soda companies, and Mashroa Eldalgamon for sewage and wastewater. Tayel et al. (2008) reported that these industrial outfalls are discharging directly at the east bank of the branch (Fig. 2). The Rosetta branch water serves for a wi de range of f uncti ons i ncluding agricultural, i ndustrial and dom esti c water supply, fisheries and recreation.

Fig. 2. Map of Nile Delta (Egypt) and Kafer El-Zayat city showing the experimental sites, 1 Firon fish farm, the reference site, 2 Binufar, 3 Industrial zone, 4 Railway ISSN: 2090 - 0511

bridge, 5 Between the bridges.

Previous studi es have reported that exposure of fish to pollutants (agricultural, industrial and sewage) evolved anti oxidative defense system which incl ude enzym es such as gl utathi one-S-transf erase, glutathi one reductase, glutathione reductase superoxide dismutase and catalase that show general hi gh acti vity in the liver, a major organ f or xenobiotic (Freitas et al., 2007; Hegazi et al., 2010). These enzym es present in all organisms metabolize m any pollutants i n order to protect t he organisms against the deleteri ous effects of xenobiotics. It suggested that som e of these enzym es can constitute good m ol ecular bioindicators f or oxidati ve stress and can indicate the magnitude of response in vertebrat e population chronically exposed to contaminants, such as m etals and other xenobiotic (G ad, 2009). Kafr El-Zayat is one of the largest industrial cities in Egypt due t o the presence of large number of factories and compani es such as chemical, pesticides, fertilizers, pestici des, paper, and oil and soap factories, so it take rank i n the global pollution. Therefore, t he present study investigated the physiol ogical changes in liver and white m uscles of Nile tilapia, O eochromis nilot icus, exposed to polluted water of Rosett a Branch i n Kafr El -Zayat City. This finding could open a new avenue of research i n identifying and designing novel strategies, whi ch could transl ate into better protection f or fish and human. M ATERIAL AND M ETHO DS: Study area: Nil e Tilapia samples were collected from Rosetta branch of Nile River at Kafr El-Zayat Al-G harbia G overnorate, Egypt. Five sampling localities in this area were s el ected according to their position rel ative to municipal sewage ef fluents and other sources of pollution (Fig. 2). The first l ocality Faraon fish farm, Q ast a (30° 53′ 26.55" N, 30° 47′ 50.49" E) was the cleanest of the four, and thus can be considered a reference locality. The second locality (30° 49′ 43" N, 30° 48′ 7" E) was located downstream from the effluent discharge from Rosetta branch, Bi nufar, Kafr El-Zayat. This locality is receiving extensive industrial wastewater discharge because it located near the fertilizer factory in Kafr ElZayat. The third locality was located in the front of Soda and Salt fact ory (30° 49 ′ 35" N, 30° 48′ 25" E), and receive extensive industrial wastewater discharge. The fourt h locality Railway bridge (30° 49′ 7" N, 30° 48′ 46" E) w as the site from whi ch we start our tour, receive extensive urban discharge. The fifth locality (30° 48′ 51" N , 30° 48′ 37" E) was located between the bridges and receive the contamination from El Dalgamon municipal ef fluents. R osetta branch of Nile River at Kafr



Hegazi et al., Oxidative Stress in Liver and White Muscles of Nile Tilapia as Responses to Polluted Area in the Nile River

El-Zayat area receives annually, sewage, agricultural, industrial drainage water without treatm ent from several drains. The m ain waste drains are shown in figures 1 & 2. Som e Fisherman catch fish from these regions, sell it in t he market and it may constitut e health hazard for consum ers. Collection of Sampl es: Water samples: W ater samples were collected from the five selected sites of the Nil e Ri ver from summ er 2012 t o spring 2013, using 500 ml sampling bottle. The sampl es for each site were com posited for all analyti cal procedures conduct in the laboratory. Fi sh Samples: Nile tilapi a samples were caught and collected at each sampling site with the help of fisherm en. The fish were coll ected from five sampling localities in Rosetta branch of Nile River in Kafr El-Zayat. After collecti on, the fish taken to laboratory using an icebox at 4 o C and 10 fish were selected for examination. The selected fish wei gh w ere from 62.4 ± 6.9 g. The fish weight and length were registered. Water samples and physicochemical parameters: The physicochemical analyses of water were done i n the w ater analysis unit in the central laboratory, Tanta University using standard m et hods. The pH was m easured at the sampling site using microprocessor based pocket pH m eter (water proof pH scan W PI). The pH values were checked again in the laboratory usi ng pH m eter (Hanna instrum ent). Conductivity and total di ssol ved solid values were registered immediately after arrival to t he l aboratory using digit al conductivity meter (Hanna instrum ent). Turbidity w ere measured using turbidity m et er, titrimetric m ethod w as used for the determination of alkalinity, whereas EDTA titrim etric method was used for total hardness analysis. The l evel of ammonia, nitrite and nitrate of the water was determined using UVVi s double beam pc scanning Spectrophotom eter UVD 2950 following standard m ethod of American Public Health Association (AP HA, 1998). Ti ssue sampling: A small piece of tissue (white muscles or right liver lobe) was carefully excised on ice, avoiding squeezing the tissue, and washed i n ice-cold isotonic NaCl saline, was blotted dry with filter paper and weighed. The tissue was homogenized in ice-cold phosphate buffer (50 mM, pH 7.4) 10% (w/v) using Omni international homogenizer (USA) at 22,000 rpm for 20 s each with 10 s intervals. The supernatant was freeze-thawed thrice to complet ely disrupt mitochondri a. Then the supernatant was centrifuged at 6000×g in cooling centrifuge at 4 o C for 15 min and the yielded supernat ant which contains the cyt osolic and mitochondrial enzym es was ISSN: 2090 - 0511

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saved for imm ediate enzym e assays. The UV/vis Spectrophotom eter (JENW AY 6505, UK) used for the m easurem ents of enzym e activities and oxidative stress param eters at 25 o C. Antioxidant enzyme activities and lipid peroxidation assays: Total superoxide dismutase (SOD, EC 1.15.1.1): The activity assayed accordi ng to the m ethod of Paol etti and Mocali, (1990), which monitors NADH oxidation. The SOD activity was assayed by assessing the i nhibition of NADH oxidation by β-m ercaptoethanol in the presence of EDTA and Mn as substrate. NADH solution made fresh daily, the assays run by addi ng sequentially to the cuvette: 0. 8 ml of 50 mM phosphate buffer pH 7.4, 55 µl EDTA/Mn soluti on of 100/50 mM, 40 µl NADH solution of 7.5 mM and different volumes from sampl e tissue extract. The reaction t hen initiated by adding 100 μl β-m ercaptoethanol solution. The changes in ΔE of NADH followed per minute for 15 minutes (Ex = 6.22/mM/1cm at 340 nm). O ne unit of SOD activity defi ned as the am ount of cell extract required to inhibit t he rate of NADH oxi dation of the control by 50%. The specific activity was expresses as unit per gram wet weight tissue (wwt). Catalase (CAT, EC 1.11.1.6): Catal ase activity assayed according t o the method of X u et al. (1997). Assay mixtures contained 10µl sample tissue extract added to 1.0 ml of H 2 O 2 phosphate buffer pH 7.4. The changes in ΔE of H 2 O 2 followed per minute f or 15 minutes (Ex = 0.04/mM/1cm at 240 nm). Glutathione reductase (GR, EC 1.6.4.2): Glutat hione reduct ase activity assayed according to the m ethod of Smith et al. (1988). Ten µl of sample tissue extract were added to 1 ml of reacti on mixture contai ned 50 mM phosphate buff er pH 7.4 containing 2 mM EDTA and 0.15 mM NADPH. The reaction initiated by the addition of 10 µl of 1 mM oxidised glutathione (G SSG). Specific activities were determined following the changes of Δ E per minute (Ex = 6.22/mM/1cm at 340 nm). Glutathione S-transferase (GST, EC 2.5.1.18): Glutat hione-S-transferase activity assayed according to the m ethod of Habig et al. (1974). The final reaction mixture contains 1.45 ml of (1.2 ml Phosphate buffer 50 mM, pH 7.4, 100 µl of 1 mM CDNB, 100 µl of 1 mM GSH). The reaction initiated by the addition of 50 µl sample tissue extract. Specific acti vities were determi ned following the changes of ΔE per minute (Ex = 9.6 /mM/1cm at 340 nm). Glutathione peroxidase (GPx, EC 1.11.1.9): Glutat hione peroxidase activity assayed according t o the m ethod of Pagli a and Valentine (1967). One milliliter of 50 mM



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sodium phosphat e pH 7.4 containing 1 mM GSH, 4 mM sodium azide, 2 units of G R, and 0.25 mM NA DPH, add 10 µl sampl e tissue extract and 10 µl H 2 O 2 (0.2 mM H 2 O 2 in phosphate buffer pH 7.4). S pecific activities were determined f ollowi ng the changes of ΔE per minut e (E x = 6. 22/mM/1cm at 340 nm). Li pid peroxidati on (LPO): Lipid peroxidation is the degradation of lipids that occurs, as a result of oxidative damage, is a useful marker for oxidative stress. Polyunsaturated lipids are susceptible to an oxidative attack, typically by reactive oxygen species, resulting in a well-defined chain reaction with the production of end products such as malondialdehyde (MDA). Malondialdehyde was quantiﬁed according to the method of Buege and Aust (1978). To 250 µl of sample tissue extract, 250 µl TCA (20%) and 500 µl thiobarbituric acid (0.67%) was added in a centrifuge tube. The mixture boiled for 15 min. After cooling, the sample to room temperature 2 mL butanol added and the sample centrifuged at 3000×g for 15 min. Absorbance of pink supernatant measured against the reagent blank by spectrophotometer (Ex =156/mM/1cm at 532nm). Chemicals: All chemicals used in t his study were purchased from the following companies, Si gma Chemical Co. (St. Louis, MO, USA),

Fisher bioreagent (USA), MP Biom edicals, ICN (USA) and Acros Belgium and were of anal yti cal grade. Statistical Analysis: Data statistically analyzed and each readi ng represents means ± st andard deviati on. The statistical evaluations of all data were done using one-way analysi s of variance (ANOVA) followed by Dunnett’s test using a computer program (GraphPad InStat e Software, Inc). P values ≤ 0.05 regarded as st atistically si gnificant. Graphs and correlations between different param eters pl otted using GraphPad Prism Softw are, Inc. RESULTS: Physicochemical analysis of water samples: The pH, turbi dity and total dissolved soli ds val ues of wat er at different localities during the four seasons i n comparison wit h their respecti ve control were tabulated (Tabl e 1). The pH values of the sam pl es ranged from 7.2 to 8.0, turbidity val ues ranged from 4.0 t o 33.1 N TU and total dissolved solids values of the samples ranged from 112 to 430 ppm at different localities during the four seasons. The pH, turbidity and total dissolved solids values i ncreased from lower values t hrough summ er to a maximum in the aut umn i n comparison with the other seasons.

Table 1. Some physicochemical parameters in water at different areas during the four seasons Parameters

pH

Turbidity (NTU)

TDS (ppm)

Ammonia NH3 (mg/l) Nitrite NO2 (mg/l)

Nitrate NO3 (ppm)

Autumn Winter Spring Summer Autumn Winter Spring Summer Autumn winter spring Summer Autumn winter spring Summer Autumn Winter Spring Summer Autumn winter spring Summer

Standard max. permissible limit

Sampling sites

Seasons Site 1 7.5 ± 0.05 7.4 ± 0.04 7.4 ± 0.02 7.2 ± 0.02 7.2 ± 1.4 6.4 ± 1.1 6.3 ± 0.3 4.0 ± 0.1 117 ± 8.2 123 ± 10.7 121 ± 8.9 112 ± 2.3 0.06 ± 0.005± 0.062 0.002± 0.082 0.003± 0.055 0.005± 0.034 0.003± 0.032 0.002± 0.028 0.001± 0.025 0.001 8.8 ± 0.6 9.2 ± 0.7 8.3 ± 0.4 8.2 ± 0.6

Site 2 7.6 ± 0.06 7.4 ± 0.05 7.4 ± 0.03 7.3 ± 0.03 20.4 ± 1.3 18.3 ± 1.1 9.5 ± 0.5 4.2 ± 0.2 321 ± 9.8 268 ± 11.4 213 ± 15.0 137 ± 3.5 0.59 ± 0.008 0.376 ± 0.008 0.24 ± 0.006 0.11 ± 0.007 0.44 ± 0.02 0.144 ± 0.008 0.092 ± 0.005 0.038 ± 0.002 63 ± 4.1 26.1 ± 1.2 18 ± 0.9 9.6 ± 0.7

Site 3 7.7 ± 0.06 7.5 ± 0.05 7.5 ± 0.04 7.4 ± 0.04 21.6 ± 1.5 19.6 ± 1.3 11.3 ± 0.8 5.8 ± 0.3 338 ± 11.2 285 ± 13.7 225 ± 17.0 143 ± 4.0 0.62 ± 0.01 0.39 ± 0.01 0.28 ± 0.009 0.13 ± 0.005 0.51 ± 0.03 0.19 ± 0.007 0.104 ± 0.007 0.041 ± 0.003 67 ± 3.7 28.4 ± 1.9 21.8 ± 1.4 11.1 ± 0.8

Site 4 7.8 ± 0.07 7.6 ± 0.06 7.6 ± 0.05 7.5 ± 0.04 27.2 ± 1.7 22.7 ± 1.3 13.2 ± 0.6 6 ± 0.3 358 ± 13.4 291 ± 16.4 227 ± 19.0 158 ± 5.4 0.66 ± 0.02 0.423 ± 0.02 0.33 ± 0.01 0.15 ± 0.008 0.58 ± 0.03 0.272 ± 0.009 0.109 ± 0.009 0.048 ± 0.004 71.3 ± 4.3 31.5 ± 2.4 24 ± 1.5 13.4 ± 0.7

Site 5 8.0 ± 0.07 7.8 ± 0.08 7.6 ± 0.04 7.6 ± 0.04 33.1 ± 2.0 26.4 ± 1.7 15.6 ± 0.9 10 ± 0.6 430 ± 12.7 298 ± 20.4 239 ± 21.0 179 ± 6.2 0.73 ± 0.04 0.47 ± 0.02 0.36 ± 0.02 0.16 ± 0.009 0.74 ± 0.05 0.33 ± 0.008 0.122 ± 0.008 0.056 ± 0.005 75.1 ± 4.8 37.3 ± 3.3 26 ± 1.6 15.6 ± 0.9

6.5 – 7.8

< 10 NTU