The 4th Global Fisheries & Aquaculture Research Conference 2011, Cairo.

PHYSIOLOGICAL STUDIES ON GILL AND CORPUSCULAR OXIDATIVE STRESS IN THE FISH

OREOCHROMIS NILOTICUS EXPOSED TO NEEM OIL IN RESPONSE TO ANTIOXIDANT POWER OF LUPINE SEEDS

ASHRAF A EL-BADAWI1 AND M BASSAM AL-SALAHY2

1. Central Lab for Aquaculture Res Abbassa Abo-Hammad, Sharkia1, 2.Zool. Dept., Fac. Sci., Assiut University2, Egypt.

8/5/2011------------------------------------------------------------------------------------------------

AbstractThis study was designed to investigate the effect of Neem seed oil on the

oxidative stress in gills and erythrocytes (EC), after 1, 2 and 3 weeks (W) and to evaluate the antioxidant effect of lupine seed supplementation (LS) in the Nile tilapia Oreochromis niloticus. Two doses of Neem oil; 1/20 LC50:56 (NO1) and 1/10 LC50:112 PPM (NO2) and three periods of one, two and three weeks were used. Oxidative stress parameter, total peroxide (TP) and antioxidant enzyme activities, catalase (CAT) and superoxide dismutase (SOD) as well as glycemia were measured. NO2 markedly decreased CAT and SOD activities in gills at most periods (52 - 99% and 16 – 52 %, respectively) and in EC (22 - 62 and 48 – 63 %, respectively) in most periods, but NO1 had less effect. In turn, both doses significantly enhanced TP levels in gills and EC. Moreover, hyperglycemia was detected three weeks after control (NO) exposure. LS significantly curtailed or abolished the adverse effect of NO exposure on oxidative and antioxidant parameters in gills and EC. This may be attributed to the hypoglycemic effect of LS as well as its antioxidant efficiency. In conclusion, NO exerts oxidative damage in the fish and LS has an antioxidant potency.

Key words: Fish, oxidative stress, Lupine, neem oil, gills, erythrocytes.INTRODUCTION

Pesticides are directly applied to agricultural field to get rid of harmful pests and improve crop growth. Aqueous extract of Neem leaves, extensively used in fish-farms as alternative for the control of fish parasites and fish fry predators, for the neotropical fish Prochilodus lineatus. (Winkaler et al., 2007).

These pesticides may reach the water system where fish are found and cause harm to fish as well as aquatic fauna (Martinez, 2002; Winkaler et al., 2007; Mondal et al., 2007). The harm effects of different organophosphorus pesticides on the non-target organisms are exceeded today to large extent. So there is trend worldwide to use herbal pesticides (biopesticides) like Neem instead of the chemical ones. Neem, Azadirachta indica, provides many useful compounds that are used as pesticides and

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could be applied to protect stored seeds against insects. However, as the biopesticides Neem started to widespread, some toxic effects were showed on animal and fish organs. Neem extract is less toxic to P. lineatus than other synthetic insecticides used in fish-farming (Winkaler et al., 2007). The acute toxicity values of several Neem preparations and some for pure azadirachtin for laboratory animals and some non target species have been studied extensively (Gandhi et al., 1988; Osuala and Okwuosa, 1993; Mahboob et al., 1998; Mondal et al., 2007). Also, Fish gills showed histological alterations including hyperplasia of the epithelial cells and epithelial lifting in response to toxic exposure (Mallat, 1985; Hinton et al., 1992; Fernandes and Mazon, 2003). Non-aqueous extracts appear to be the most toxic Neem-based products and suggested that use of Neem derived pesticides as an insecticide should not be discouraged (Boeke et al., 2004). In addition, Neem oil has been reported to produce greater general toxicity in Rat (Narayanan et al., 1993).

The pollution of water with Neem may induce hypoxia for fish lesions may result in decreased oxygen- uptake capacity of the gill making the fish Prochilodus lineatus exposed to Neem less able to get adequate oxygen for its total metabolic activity (Winkaler et al., 2007). Long exposure of Tilapia zillii to low concentrations of crude extract of A.indica (Neem) delayed the growth and caused respiratory problems (Omoregie and Okpanachi, 1992). The toxic substances may cause damage to gill tissues, thereby reducing the oxygen consumption and disrupting the osmoregulatory function of aquatic organisms (Ghate and Mulherkar, 1979). Thus, gill is the primary target and uptake site for many toxicants in the water (Evans, 1987). Also, the dissolved oxygen content of water decreased with increasing doses of biopesticides (Radi et al., 1988; Mondal et al., 2007). Hypoxia enhanced the generation of TBARS induced oxidative stress in the fish organs, generating peroxides (Lushchak et al 2005; Al-Salahy, 2006). However, tilapia species have a high tolerance to hypoxic and/or anoxic conditions (Ishibashi et al., 2005). Gills are extremely important in respiration, osmoregulation, acid – base balance and excretion of nitrogenous wastes in fish and they represent the greatest area of the animal in contact with external environment (Heath, 1995). Inflammatory changes, such as swelling, lifting of lamellar epithelium and hyperplasia have also been noted in the gill lamellae of various fish species following exposure to insecticides (Nowak, 1992). Gills are considered the most vulnerable organ to external pollutants owing to their direct contact with polluted water, facilitating the lamellar epithelia penetration and ingress into blood circulation (Gernhofer et al., 2001).

Red blood cells (RBCs) possess high levels of cytosolic superoxide dismutase and catalase that are likely to quench reactive oxygen species (ROS) generated by Hb autoxidation. However, it is possible that the leakage of ROS from RBC involves

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Hb binding to band 3 of the red cell membrane (Tsuneshige et al., 1987 and Walder

et al., 1984). In hypoxia, red blood cells (RBCs) produces reactive oxygen species that diffuse to endothelial cells of adjoining blood vessels (Kiefmann et al., 2008). The authors concluded that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs release H2O2 the membrane of erythrocytes is rich in polyunsaturated fatty acids, a primary target for reactions involving free radicals, and may allows the erythrocytes vulnerable to oxidative damage (Jain et al., 1993). Most studies of Neem pesticide on non target organisms such as fish were focused on the hematological indices including fish mortality, determination of LC50 estimation of number of RBCs, hematocrit and hemoglobin (Larson ,1987; Fernandez et al., 1992; Jacobson, 1995; Mondal et al., 2007). Studies in the Philippines showed that the recommended dose (5%) of Neem seed kernel extract was toxic to Nile tilapia Oreochromis niloticus (Jayaraj, 1992). The studies of Neem oil on oxidative stress is very scarce in fish (Winkaler et al., 2007) and most studies focused on mammal animals (Ricci et al., 2009). It was found that Neem oil increased level of lipid phosphorylation of cell membrane and consequent loss of function (Ricci et al., 2009). Oxidative stress arises when there is an imbalance between radical-generating and radical-scavenging activity; it may therefore cause an increase in the formation of oxidation products (Gutteridge, 1995). There is no literature concerning the effect of Neem oil on the oxidative stress on fish particularly on erythrocytes and gills in fresh water fish which usually found near the agriculture areas and polluted irrigated water.

It has been showed that most sublethal dose of pesticides induced hyperglycemia in most different species of fish such as Labeo rohita (Cypermethrin: Das and Mukherjee, 2003), Rhamdia quelen (Cypermethrin: Borges et al., 2007), Cyprinus carpio (2,4 Diamin: Oruc and Uner, 1999) and Clarias batrachus (malathion: Mukhopadhyay and Dehadrai, 1980). It was found a close relationship between persistent hyperglycemia and propagation of oxidative stress (Brownlee, 2003; Reddy et al., 2009).

It is suggested that lupine seeds may have higher antioxidant activity in lipid-soluble substances (Rubio and Seiquer; 2002 and Oomah et al., 2006). The digestion of legume protein resulted in high amount of arginine, aspartate and glycine. The amino acid arginine could ameliorate the oxidative stress in alloxan-treated rats El-Missiry et al., 2004). Also, Alpha-, gamma- and delta-tocopherols were found in the lupine oil antioxidant activity was found both in the flours and in the hulls (Lampart-Szczapa et al., 2003). It was found that aqueous suspension of lupine seeds have a prophylactic effect against prolonged hyperglycemia and its damaging effect on the kidney of the fish Clarias gariepinus (Al-Salahy and Mahmoud , 2004).

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It is well established that the most important antioxidant enzymes are superoxide dismutase (SOD), which detoxifies O2

−, catalase, which reduces H2O2, (Halliwell and Gutteridge 2000). Total peroxide (TP) measured by xylenol orange, induced in early stage of long chain of reactions which ultimately leads to form lipid peroxidation as terminal products (Lushchak et al., 2005). TP includes hydrogen peroxide and other derivatives of peroxide, produced physiologically in organisms and occur in higher concentrations in some pathological conditions (Harma et al., 2003).

Nile tilapia, Oreochromis niloticus is a native fish species of Egypt that has become popular species worldwide mainly as a valuable fish, easy to breed and grow in a variety of aquaculture systems (El-Sayed, 2007). Tilapia is an omnivorous fish (Abdelghany, 1993; Abdel-Tawwab and El-Marakby, 2004).

This study aimed to evaluate the ability of lupine seeds to counteracting NO-inducing oxidative stress in gills and RBCs in Oreochromis niloticus by monitoring of total peroxide as destructive bioindicator and activities of catalase and superoxide dismutase as antioxidants. Also, to highlight some physiological mechanism of toxicity of Neem oil and the probable recovery by lupine.

MATERIALS AND METHODSMaterials

Healthy sixty six fish Tilapia (Oreochromis niloticus) of both sexes weighing 31-37 g /fish (13 – 16 cm length) were collected from the nursery ponds of Central Laboratory for Aquaculture Research at Abbassa, Abo-Hammad, Sharkia, Egypt in month of July 2009. On arrival at the laboratory, fishes were immediately released into special five glass tanks (Aquaria) (40 x 70 x 60) containing tap water and then maintained there for 7 days for acclimatization condition. The fish groups were three. The first one was subdivided into three subgroups exposed to Neem oil (NO) for one week; control, low dose (NO1) and high dose (NO2). The second group was also subdivided into three subgroups exposed to NO for two weeks; control, NO1 and NO2. The third group was subdivided into five subgroups exposed to NO for two weeks: control, NO1, NO2, NO1+ lupine supplementation (LS) and NO2 + LS. The number of each fish subgroup was six. Air compressor was used for oxygenation of water. Dissolved oxygen concentrations ranged from 6.1 to 6.6 mg L−1. The ambient water temperature was ranged between 20–23 °C; pH was ranged between 7.1–7.4. All fish were fasted for the last 12 h of the experiment. The water medium was changed at 24 hours interval to remove the metabolic-pollutants with the same treatments according to the experimental protocol. Portable oxygen meter (DO-980)

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and Hanna Instrument HI 2210 Bench top PH meter W/Temperature Compensation meter were used.

Fish were fed on artificial feed twice daily a total of 2% of mean body weight of dry pellets of 2.5 mm. Food ratio of dry weight composed of 40 % fish meal, 18% fat, 10 % corn starch, 11% ashes

Plant preparationSeeds of lupine (Lupinus termis) were washed, kept in the incubator at 37°C to

dryness for 24 h and ground well, and then lupine powder was added by 5 % to the paste of normal ratio of fish diet before dryness. This food containing lupine was prepared to be tested as antioxidant effect as well as against oxidative stress in fish treated with bioinsecticide; Neem oil of different doses and durations.

Seed Neem oil: We purchased seed Neem oil from Trifolio-M Company. The recorded LC50 of Neem oil for the tilapia was 1124.6 PPM (Jacobson 1995). Therefore, the used Neem oil doses used in this experiment were 1/10 LC50 (112.5 PPM: high dose) and 1/20 LC50 (56.3 PPM: low dose). Before use, the Neem oil was emulsified by using emulsifier agent (SiSi-6) and the emulsifiable concentrate was 50%.

Design of the experiment. Table 1. Design of fish groups with different treatments.

For one week For two weeks For three weeks

Group1 Group2 Group3Each SG

was 8 fishSG1 SG2 SG3 SG1 SG2 SG3 SG1 SG2 SG3 SG4 SG5

Cont + + +NO1 + + +NO2 + + +NO1+LS +NO1+LS +

NO1: Neem oil of 56 PPM, NO2: Neem oil of 112 PPM, SG: subgroup, Healthy six fish were chosen from each SG. n = 6.

At the end of the experiment, blood sample was taken from the caudal peduncle by suction, and then fish were sacrificed and were dissected. Sample of gills (at right side) were excised. Ten percentage homogenates (w/v) were made in phosphate buffer (pH 7.4) using homogenizer (model IKA- WERKE, D118 BASIC, Germany). Then, homogenates were centrifuged at 5,000 rpm for 15 min to separate the homogenate. Preparation of erythrocyte hemolysate: immediately after collection, blood samples were centrifuged at 3,000 rpm for 15 min at 4 °C. The plasma and

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buffy coats were removed by aspiration. The sediment containing blood cells was washed three times by resuspending in isotonic phosphate-buffered saline, followed by re-centrifugation and removal of the supernatant fluid and the buffy coats. The crude red cells were lysed in nine volumes of ice-cold distilled water to prepare a 10% erythrocyte hemolysate. All samples were 250 L-aliquotted into Eppindorff’s tubes and stored at -40 °C till used to avoid repeated freeze-thaw cycles in different assays, except aliquots of total peroxide were assayed quickly after homogenization of tissues for more accurate estimation.

Chemicals Absolute ethanol, Ferrous sulphate, Butylated hydroxytoluene, NADPH,

EDTA, catalase, hydrogen peroxide, HCL, H2SO4, xylenol orange, epinephrine folin-ciocateau phenol, and hydrogen peroxide (Fluka, Merk and Sigma-Aldrich Companies). All other chemicals were of the highest quality available.

Biochemical assays Catalase activity was assayed by the method of Beers and Sizer (1952). The reaction mixture contained 50 mM phosphate buffer (pH 7.0) and 50 mM H2O2. The reaction rate was measured at 240 nm. Sigma catalase enzyme was used as standard. Superoxide dismutase (SOD) activity was determined based on its ability to inhibit the autoxidation of epinephrine in alkaline conditions as described by Misra and Fridovich (1972). Sigma SOD enzyme was used as standard. OD was measured at 480 nm. Total peroxides (TP) was assayed based on the method of Harma et al., (2005). The reagent was prepared as follows: 9.8 mg Ammonium Ferrous sulphate was added to 10 ml of 250 mM H2SO4. This solution was added to 90 ml absolute methanol containing 79.2 mg butylated hydroxytoluene and then 7.6 mg xylenol orange was dissolved. The absorbance was measured after incubation of homogenate sample with the reagent for 30 min at 560 nm. H2O2 was used as standard. Haemoglobin (Hb) content was also, assayed by cyanomethaemoglobin method ((Jain, 1986). Blood glucose was determined by the method of Trinder (1969). Total protein in the tissue homogenates was measured using the folin reagent according the method of Lowry et al.,(1951). All assays were performed in triplicate. Spectrophotometer, UNICAM, Helios Gamma, No. UVG 060609, England was used for all biochemical assays.

Statistical analysisThe data were expressed as mean + SEM. The results were analyzed

statistically using column statistics and one way ANOVA with Newman-Keuls Multiple comparison test as a post-test. These analyses were carried out using computer statistics prism 3.0 packages (Graph pad software, Inc, San Diego. (A. USA). The minimum level of statistic significance was set at P < 0.05.

RESULTS

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Gills: It was noticeable that Catalase (CAT) activity in fish erythrocytes is five to seven-folds higher than in gills in control groups (Table 2 and Fig 1). Data obtained herein (Table 2 and Fig 1) show that the NO1 (low Neem oil dose) and NO2 (high dose) significantly diminished CAT activity in gill at the third week by -33 and -52%, vs control, respectively. At the first and second weeks, CAT activity showed significant rise in NO2 fish group, and marked drop in NO1 group.

Table 2 and Fig 1-2 shows that the activity of erythrocytic SOD was 1.5 to 1.7-fold higher than in gills in control fish group. Data shows fluctuations in the levels of SOD activities at the first and second week after exposure of fish to both NO doses were recorded; this was followed by marked decreases at the third week in response to NO1 and NO2 by -16 and -52% respectively. Regarding oxidative stress bioindicator, both NO1 and NO2 significantly enhanced total peroxide (TP) at all periods by +233, +320, +76 % and by +156, +234, +157 % ,respectively corresponding to control values, respectively. The destructive effect of NO on gills increased with increasing time and of exposure as well as the dose used (Table2 and Fig 1).

Lupine seed supplementation (LS) could curtail the perturbation of antioxidant enzyme activities as well as destructive biomarker, total peroxide (TP). LS improved CAT activity in fish exposed to NO1 and NO2 from -33 to -16.3 and from -52 to -17% vs control values, respectively. Also, LS improved SOD activity in fish exposed to NO1 and NO2 from -16 to -2 and from -52 to -10 % vs control values (Table 2). (Table 1). In addition, LS improved the TP levels in fish gills exposed to NO1 and NO2 from +76 to +18 and from +157 to +15% vs control values.

ErythrocytesTable 3 and Fig 2 shows that NO2 markedly decreased the activity of catalase

(CAT) enzyme at all periods by -22, -62.2 and -51 % respectively. Also, this dose of NO significantly decreased activity of superoxide dismutase (SOD) vs control at all periods by -49 , -63 , -48 % , respectively, while the NO1 significantly increased SOD activity at all periods by +11, 45, +27% respectively. Concerning destructive bioindicator, TP was significantly increased at all periods of NO1 by +13, +89, +87 % and NO2 by +127, +42, +240%, respectively (Table 3 and Fig 2) .

The enhancing effect of NO on TP and its lowering effect on enzymatic antioxidants; CAT and SOD activity in erythrocytes were took place (Table 3). In the fish-exposed to NO1 or NO2 and supplemented with lupine, there were good extents of recoveries in the levels of erythrocytic TP (+87 to +50 and from +240 to +9%), activities of CAT (-15 to -35 and from -51 to -20%) and SOD (-27 to -24 and from -48 to -28%, respectively) compared with corresponding control values.

GlycemiaTable 4 and Fig 3 shows that at the third week, low and high doses of NO

markedly increased blood sugar by 137.5 and 78.7 %, respectively in tilapia. LS in the fish exposed to NO, could dampens down the hyperglycemic levels to 43.8 and 25% showing hypoglycemic power.

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Pearson’s correlation (r) Pearson’s correlation (r) and regression (r2) analysis of the paired data (Table

5 and Fig 4-7) showed existence of negative correlation among a rising in TP and marked decrease in the activities of CAT and SOD in both gills and erythrocytes after three weeks of NO exposure in tilapia. This correlation was more potent in response to NO2 exposure especially in erythrocytes. In turn, there were significant positive correlations between the valuable indicator of oxidative damage; TP and the rise in glycemic level induced by NO1 and NO2 contamination in erythrocytes and in gills, respectively. This positive correlation was continued during treatment fish with LS plus NO (Table 5 and Fig 8-9).

DiscussionGills: Gills are considered the most valuable organ to external pollutants owing to their direct contact with water, facilitating the lamellar epithelia penetration and ingress into blood circulation (Gernhofer et al., 2001). Inflammatory changes, such as swelling, lifting of lamellar epithelium and hyperplasia have also been noted in the gill lamellae of various fish species following exposure to insecticides (Mallatt, 1985; Nowak, 1992). Gill epithelial cells are attractive models in aquatic toxicology since the gill is the primary target and uptake site for many toxicants in the water (Evans, 1987). Freshwater fish Prochilodus lineatus exposed to all Neem extract concentrations exhibited damaged gill and kidney tissue (Winkaler et al., 2007). Exposure to low concentrations of the crude extract of A. indica delayed the growth of this cichlid fish Tilapia zillii (Omoregie and Okpanachi, 1992). The present study showed that the high dose Neem oil (NO) (1/10 LC50 : 112.5 PPM) markedly decreased the activity of catalase (CAT) enzyme in gills after NO for two and three weeks by 62.2 and 50.8 % respectively Low dose of Neem oil (NO1: 1/20 LC50 : 56 PPM) and NO2 (NO2: 1/10 LC50 : 112 PPM) significantly diminished CAT activity in gill at the third week by -33 and -52%, vs control, respectively. At the first and second weeks, CAT activity showed significant rise in NO2 fish group, and marked drop in NO1 group. Also, Data shows fluctuations in the levels of SOD activities at the first and second week after exposure of fish to both NO doses were recorded; this was followed by marked decreases at the third week in response to NO1 and NO2 by -16 and -52.5% respectively. In turn, both doses of NO significantly enhanced the destructive oxidative biomarker; total peroxide (TP) at all periods by +233, +320, +76 % (low dose) and by +156, +234, +157 % (high dose) corresponding to control values, respectively. This result was accompanied with a severe hyperglycemia at the third week; by 137.5 and 78.7 % vs control in response to high and low doses of NO, respectively in tilapia. Moreover, this hyperglycemic effect of NO contamination may be associated with low cellular uptake of glucose. The lowering effect of NO toxicity on CAT activity may be attributed to disturbances in glucose tissue uptake, low level of Glucose-6-phosphate dehydrogenase (G6PDH), low production of NADPH or/and over production of reactive oxygen species (ROS). This concept based on G6PDH enzyme of the pentose phosphate pathway, plays a crucial role in modulating antioxidant defenses where it participate in production of NADPH necessary for CAT activity (Kirkman et al., 1999, Tian et al., 1999 and Leopold and Loscalzo, 2000). It

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has been found that decreased NADPH levels were correlated with a loss of catalase activity in human erythrocytes (Gaetani et al., 1989). Deltamethrin significantly inhibited the trout erythrocyte G6PD enzyme activity (Murat et al., 2009).

In addition, the rise in blood sugar for several hours for few days severely increased oxidative stress in body tissues due to overproduction of ROS (Cuncio et al, 1995; Ling et al., 2003; Be´meur et al., 2007). Gills and kidney which exposed to a toxic material, became oxidatively damaged facilitating copper elimination, particularly gills participate in copper excretion (Handy, 1996). In addition, copper acts as a cofactor of several metalloenzymes and other metalloproteins such as ceruloplasmin (Cp), Cu-Zn superoxide dismutase (SOD), cytochrome coxidase (Lee et al., 2002). Also, NO was found to reduce the oxygen uptake of fish fingerlings and causes mortality at a faster rate (Mondal et al., 2007). Hypoxia induced oxidative stress in fish (Lushchak et al 2005; Al-Salahy, 2006) and in mice (El-Sokkary et al., 2006). Based on these findings, inhibition of SOD activity showed in the fish-exposed to NO may resulted from: high utilization of enzyme due to marked flux of ROS, oxidative damage of gills probable facilitated Cu elimination or/ and anoxia as well as hyperglycemia- induced exceeded ROS. This concept is in harmony with the great rise in TP, which is mainly represented by H2O2 and its derivatives. This is in agreement with that reported by Halliwell and Gutteridge (2000) who reported that the tissues of xenobiotic detoxification are considered to be powerful ROS generators. In addition, fish gill is in close contact with environmental water contaminants, being the main absorption organ for those aquatic toxicants, facilitating their fast absorption and contact with phagocytes (Stagg et al., 1992).

Erythrocytes RBCs possess high levels of cytosolic superoxide dismutase and catalase that

are likely to quench ROS generated by Hb autoxidation (Walder et al., 1984; Tsuneshige et al., 1987). The current study showed that NO2 contamination (high dose) significantly diminished CAT activity in erythrocytes at all periods by -22, -62.2 and -50.8% ,while the lowering effect of the low dose (NO1) on this activity was appeared after three weeks (-15%). In addition, NO2 significantly decreased activity of superoxide dismutase (SOD) vs control at all periods by -49 , -63 and -48 % , respectively, while the NO1 significantly increased SOD activity at all periods by +11, 45, +27% respectively. Also, erythrocytic destructive biomarker; TP was significantly increased at all periods of NO1 (+13, +89, +87 %) and NO2 exposure (+127, +42, +240 %, respectively). RBCs are considered prime targets for oxidative injury, mainly because of their unique biological structure which contains high concentrations of membrane polyunsaturated fatty acids (PUFAs) (Lopez-Revuelta et al., 2006). The inhibiting effect of NO on antioxidant enzyme activities of CAT and SOD accompanied with its enhancing effect on TP levels may attributed to the overproduction of ROS, following by excessive production of H2O2 in erythrocytes of tilapia. Excessive production of ROS usually results from excitation of O2 to form singlet oxygen or from the transfer of one, two or three electrons to O2 to form a superoxide radical (O2 −), hydrogen peroxide (H2O2) or a hydroxyl radical (HO•), respectively (Urban-Chmiel et al., 2009). The erythrocytic role as O2 and CO2

transporter is under constant exposure of free radicals (Harvey, 1997). It has been

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found that decreased NADPH levels were correlated with a loss of catalase activity in human erythrocytes (Gaetani et al., 1989). It was concluded that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs

release H2O2 (Kiefmann et al., 2008). The authors added that H2O2 escaped the RBC antioxidant system and diffused to the adjoining tissues. Also, it was reported that in hypoxia, red blood cells (RBCs) produces ROS that diffuse to endothelial cells of adjoining blood vessels (Rifkind et al., 2001; Kiefmann et al., 2008). In turn, pesticide including NO inducing hypoxia and tissue anoxia (Swingle 1967; Pimpão et al., 2007; Mondal et al., 2007). However, unlike the non-nucleated mammalian red blood cells, fish erythrocytes possess very large nuclei, which may occupy about 29% of the cell volume (Wilhelm-Filho et al., 1992).

In addition, hyperglycemia generate ROS through diverse pathways, leading to oxidative stress in a variety of tissues (Brownlee, 2003). Supporting these finding, the current data showed that there are significant positive correlations between oxidative damage marker; TP and the rise in glycemic level induced by both NO1 and NO2 contamination in erythrocytes and in gills. hyperglycemia is one of the important factors to increase ROS, lipid peroxidation causing the depletion of the antioxidant defense status in various tissues of rats (Reddy et al., 2009). From these findings, it could suggests that NO toxicity increase H2O2 in erythrocytes and this was facilitated in fish where gills were in close contact with water the source of contamination particularly gills are rich in blood vessels. This suggests that RBCs filled with H2O2 in fish-exposed with NO may participate in distribution and spreading of free radicals counteracting the antioxidant defense system in all tissue organs of the fish, Oreochromis niloticus.

Seeds of lupine (Lupinus termis) have a hypoglycemic action in diabetic animals (Abdel-Aal et al., 1993; Newairy et al., 2002) and in normal rats (Helmi et al., 1969; Abdel -Aal et al., 1993). Also, hypoglycemic effect of lupine was recorded in fish either treated by alloxan or loaded glucose (Mahmoud and Al-Salahy, 2005). In addition, lupine seeds have an ameliorating effect on glucose tolerance test in the fish Clarias gariepinus (Al-Salahy and Mahmoud, 2004). It had been suggested that lupine seeds might have higher antioxidant activity and contains a high amount of arginine, aspartate and glycine (Oomah et al., 2006), tocopherols (Lampart-Szczapa et al., 2003) and phenol and phospholipids (Tsaliki et al., 1999). Both arginine and tocopherol are considered as antioxidants (Kausalya and Nath, 1998; Gupta et al., 2005) and may enhance lupine seeds to be free radical scavenger and more efficient as an antioxidant. The present study showed that lupine seed supplementation (LS) reversed the adverse effect or at least induced improvement effect in both gills and erythrocytes of NO on antioxidants; CAT and SOD as well as on oxidative stress parameter, TP. This result may be partly due to the hypoglycemic role of lupine which showed three weeks after NO exposure. This may stimulate cellular uptake of glucose and probably normalizing NADPH necessary for CAT activity. Also, some constituents of lupine are considered as antioxidants and acts as free radical scavenger improving the complications of hypoxia which perhaps resulted from oxidative damage on gills. Similarly, it was found that melatonin as antioxidant could improve the oxidative damage in mice-subjected to hypoxia (El-Sokkary et al., 2006).

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LS able to improve the TP levels in gills exposed to NO1 and NO2 from +76 to +18 and from +157 to +15% , respectively vs control values and in erythrocyte from +87 to +50 and from +240 to +9%, respectively. This result suggests a lowering effect of LS on tissue containing excess of H2O2 and its derivatives confirming its antioxidant role.

In conclusion, NO is very harmful on non-target organism such as fresh water fish particularly these fish usually found near the agricultural areas. In turn, addition of lupine seeds in fish diet can protect against some pests-induced oxidative stress.

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دراسات فسيولوجية لمضادات االكسدة للترمس على التوتر التأكسدى فى الخياشيم وكرات الدم الحمراء فى اسماك

البلطى النيلى المعاملة بزيت النيم² د. محمد بسام الصالحى، ¹د. اشرف احمد البدوى محمد

- المعمل المركزى لبحوث الثروة السمكية بالعباسة – الشرقية1- قسم علم الحيوان - كلية العلوم – جامعة اسيوط2

تم تصميم هذة الدراسة للكشف عن تAAأثير زيت بAAذور نبAAات الAAنيم على تAAوتر االكسAAدة اسابيع لتقييم التأثير المضاد1،2،3الذى يحدث فى الخياشيم وكرات الدم الحمراء بعد

للتأكسد لمسحوق بذور الترمس المضافة لعليقة غذاء اسAماك البلطى الAنيلى. وقAد تم LC₅₀ (56 من 1/20اسAAتخدام جرعAAتين من زيت بAAذور الAAنيم كمبيAAد حشAAرى وهى

ppm) من 1/10 و LC₅₀ ( 112 ppm)وتم قياس فوق االكسيد الكلى . التوتر التأكسدى( ونشاط االنزيمAAات المضAAادة لالكسAAدة ) الكتAAاليز والسAAوبر( مؤشر

اكسيد ديسميوتيز( وتقدير مستوى سكر الدم. وقد وجد انخفAAاض هAAام فى مسAAتوى نشAAاط الكتAAاليز والسAAوبر اكسAAيد ديسAAميوتيز فى

% على الAAترتيب(52 الى 16% و 99 الى 52الخياشAAيم فى معظم الفAAترات )بنسAAبة % على الAAترتيب( فى63 الى 48% و 62 الى 22وفى كAAرات الAAدم الحمAAراء ) بنسAAبة

معظم فAAترات التجربAAة عنAAد اسAAتخدام جرعAAة المبيAAد المرتفعAAة. ولكن عنAAد اسAAتخدام الجرعة االصغر كان التأثير اقل. فى المقابAAل وجAAد ان كال من جرعAAتى المبيAAد قAAد ادت الى ارتفاع معنوى فى مستوى فوق اكسيد الكلى فى كAAل من الخياشAيم وكAAرات الAدم الحمراء. فضال عن ذلك وجد ان كل من جرعتى المبيد قد ادت الى ارتفAAاع معنAAوى فى سكر الدم. وعنAAد اضAAافة مسAAحوق الAAترمس الى عليقAAة االسAAماك المعرضAAة لكAAل من جرعتى المبيد وجد تحسن او اختفاء للتأثيرات السلبية للمبيد على القياسAAات المقاسAAة

فى التجربة فى كل من الخياشيم وكرات الدم الحمراء. وقد اقترحت الدراسة ان هذا التأثير للترمس قد يرجع الى دورها المخفض لسكر الAAدم وكفاءتها كمضاد للتأكسد. ويمكن استنتاج ان مبيد زيت بذور نبات النيم قد احدثت تلف تأكسديا فى كل من الخياشيم وكرات الدم الحمراء السماك البلطى النيلى وان اضAAافة مسحوق بذور الترمس للعليقة الغذائية لالسماك يمكن ان تقAAاوم التAAأثير السAAلبى لهAAذا

المبيد.

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Table 2. Effect of lupine supplementation (LS) on catalase (CAT), superoxide dismutase (SOD) activities and total peroxide in gills of the Nile fish Oreochromis niloticus contaminated with neem oil (doses:NO1= 56 and NO2 =112 ppm ).

Data are presented as means SEM. Columns in each period with different letters differ significantly (P < 0.05), while those with the same letters do not differ significantly. NO: neem oil, L: Lupine, n= 6.

After one week After two weeks After three weeks

Gills Cont NO1 NO2 Cont NO1 NO2 Cont. NO1 NO2 NO1+L NO2+L

CAT activityU/min/mg protein

1.68a±0.16

1.28a±0.08

3.26b±0.30 2.02a±

0.05

1.74a±0.22

4.03b±0.32

2.08a±0.18

1.40b±0.16

1.00c±0.08

1.77ab ±0.17

1.72ab±0.22

SOD activityU/min/mg protein

3.01a±0.52

3.88b±0.28

2.64a±0.47 3.80a±

0.28

3.29a±0.30

5.92b±0.31

3.47a±0.33

2.91a±0.28

1.67b±0.20

3.40a±0.40

3.13a±0.33

total Peroxide nMol/mg protein

9.99a±1.08

33.26b±3.28

25.64c±1.99

12.19a±1.06

51.28b±2.38

40.68c±2.22

14.59a±1.22

25.64b±1.99

37.44c±2.40

17.18ad±1.63

12.46ad±1.49

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Table 3. Effect of lupine supplementation (LS) on catalase (CAT), superoxide dismutase (SOD) activities and total peroxide in blood erythrocytes of the Nile fish Oreochromis niloticus contaminated with neem oil (doses:NO1= 56 and NO2 =112 ppm ).

After one week After two weeks After three weeks

Erythrocytes Cont NO1 NO2 Cont NO1 NO2 Cont. NO1 NO2 NO1+L NO2+L

CAT activityU/min/mg Hb

12.20ab ±1.77

16.60a ±1.53

9.72b ±0.90 10.90a ±

1.44

13.23a±1.68

4.11c ±0.45

10.74ab±1.05

9.12a±0.93

5.28d±0.60

6.93cd±0.63

8.61bc±0.75

SOD activityU/min/mg Hb 4.32

a±

0.72

5.50a±0.26

3.14b±0.41

6.56a±0.42

9.52b±0.78

2.24c±0.22

6.08a±0.54

7.74b±0.30

3.18cd±0.32

4.64d±0.50

4.36d±0.46

total Peroxide nMol/mg Hb

3.99a±0.47

4.51a±0.55

9.05b±0.58

5.19a ±0.55

9.80b ±0.60

7.39c±0.46

4.30a ±0.44

8.04b±0.26

14.63c±0.423

6.46d±0.76

4.70a±0.51

Data are presented as means SEM. Columns in each period with different letters differ significantly (P < 0.05), while those with the same letters do not differ significantly. NO: neem oil, L: Lupine, n= 6.

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Control NO1 NO2 NO1+ LS NO2+ LS

Blood sugar mg/dL

16.00 ±1.378 38.00*± 2.550

28.60*± 1.030

23.00± 1.673

20.60± 1.435

Table 4. Effect of lupine supplementation (LS) on blood sugar (glycemia) in the fish Oreochromis niloticus contaminated with neem oil (doses: NO1= 56 and NO2 =112 ppm).

Data are presented as means SEM. *: Significant at P<0.5.

Table 5. Linear regression (r2) analysis of paired data of individual in Oreochromis niloticus exposed to NO and NO plus LS of TP in gills and erythrocytes with either CAT or SOD activities in these organs or glycemic level.

Linear regression (r2) of: NO1 NO2 NO1+LS NO2+LS

Gill TP with CAT activity

r2 = 0,8598**P=0,0077

NC

r2=0,6718*P=0,0459

NC

r2=0,8506**P=0,0088

NC

r2=0,6650*P=0,0469

NCGill TP with SOD activity

r2=0,8125*P=0,0141

r2=0,6622*P=0,0488

r2=0,7962*P=0,0168

r2=0,8075*P=0,0149

Erythrocyte TP with CAT activity

r2=0,1153P=0,5102Non Sign

r2= 0,0493P=0,6721Non Sign

r2=0,8145*P=0,0138

NC

r2=0,8094*P=0,0146

NCErythrocyte TP with SOD activity

r2=0,7488*P=0.0260

NC

r2=0,1895P=0,3884

NC

r=0,8696**P=0,0067

NC

r2=0,6808*P=0,0432

NCGill TP with glycemia

r2=0,1421P=0,4614Non Sign

r2=0,7892*P=0,0180

PC

r2=0,6892*P=0,0408

PC

r2=0,7023*P=0,0372

PCErythrocyte TP with glycemia

r2=0,8781*P=0,0211

PC

r2=0,7611*P=0,0234

PC

r2=0,8236*P=0,0170

PC

r2=0,8305*P=0,0114

PC

Data are presented as means SEM. *: Significant at P<0.5.*: Significant at P<0.5, **: Significant at P< 0.01 and ***: Significant at P< 0.001LS: Lupine supplementation, NO: neem oil, NC: Negative correlation, PC: Positive correlation. , n=6.

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