Modulatory role of dietary Chlorella vulgaris powder against arsenic-induced immunotoxicity and oxidative stress in Nile tilapia (Oreochromis niloticus).

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YFSIM3152_proof ■ 12 October 2014 ■ 1/9

Fish & Shellfish Immunology xxx (2014) 1e9

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Modulatory role of dietary Chlorella vulgaris powder against arsenic-induced immunotoxicity and oxidative stress in Nile tilapia (Oreochromis niloticus) Q6

Eman Zahran a, *, Engy Risha b a b

Department of Internal Medicine, Infections and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt Departments of Clinical Pathology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 June 2014 Received in revised form 28 August 2014 Accepted 29 September 2014 Available online xxx

Arsenic intoxicant have long been regarded as an impending carcinogenic, genotoxic, and immunotoxic heavy metal to human and animals as well. In this respect, we evaluated biomarkers of the innate immune response and oxidative stress metabolism in gills and liver of Nile tilapia (Oreochromis niloticus) after arsenic exposure, and the protective role of Chlorella vulgaris (Ch) dietary supplementation were elucidated. Protective role of C. vulgaris (Ch), as supplementary feeds (5% and 10% of the diet) was studied in Nile tilapia (O. niloticus) against arsenic induced toxicity (NaAsO2 at 7 ppm) for 21 days exposure period. A significant down-regulation in innate immune response; including, respiratory burst, lysozyme, and bactericidal activity followed due to deliberately Asþ3 exposure. Similarly, oxidative stress response; like nitric oxide (NO), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels were significantly decreased. Combined treatment of Ch and Asþ3 significantly enhanced the innate immune response and antioxidant activity. Strikingly, Ch supplementation at 10% has been considered the optimum for Nile tilapia since it exhibited enhancement of innate immune response and antioxidant activity over the level 5%, and even better than that of control level. Thus, our results concluded that dietary Ch supplementation could protect Nile tilapia against arsenic induced immunosuppression and oxidative stresses. © 2014 Published by Elsevier Ltd.

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Keywords: Arsenic intoxications Nile tilapia Immunotoxicity Oxidative stress Micro-algae Phytoremediation

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1. Introduction Due to the expansion and advancements of human industrial activities, heavy metals found their way into the atmosphere, water, and soil that eventually lead to environmental hazards due to improper biodegradation [1], and thus wreaking adverse impacts on aquatic habitats worldwide [2]. Tilapia is one of the most important aquaculture species, in Egypt and worldwide [3], due to their faster growth rate and tolerance to varieties of the harsh environmental conditions. Culturing Tilapia in raceways, ponds or net cages in open or coastal waters provides ideal aquaculture conditions; hence water quality is an important factor in aquaculture sustainability [4]. However, the current aquaculture practices promote threats of the environmental hazards, associated not only with deterioration of water quality unsuitable for aquatic organisms, but also with a negative

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* Corresponding author. Tel.: þ20 1096535264; fax: þ20 502379952. E-mail address: [email protected] (E. Zahran).

impact on fish health and/or in the consumers [5] owing to the persistence of heavy metals, bio-accumulation, toxicity and biomagnification in the food chain [6]. In this context, heavy metal intoxications in the aquatic ecosystem has been focused as a critical environmental issue attributing subsequent public health concern [7]. Arsenic has been recognized as one of the most relevant environmental global single substance toxicants (ATSDR, 1999) contaminating groundwater [8]. Higher Asþ3 concentrations are often lethal while exposure to chronic sub-lethal concentration always results in increased risk of cancer and damage to various organs [9]. Arsenic usually exists in two oxidation states, methylated species, arsenosugars and arsenolipids, which differ in their toxicity and the combination of these two states in fish tissues results in several pathophysiological effects [10]. Fish are sensitive to the water pollutants leading to various deleterious effects upon entering their organ, due to unremitting exposure. Heavy metals contaminated water affect the gills being the highest exposed organ, directly contacted with the contaminated water [11]. Liver as the organ responsible for xenobiotic

http://dx.doi.org/10.1016/j.fsi.2014.09.035 1050-4648/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Zahran E, Risha E, Modulatory role of dietary Chlorella vulgaris powder against arsenic-induced immunotoxicity and oxidative stress in Nile tilapia (Oreochromis niloticus), Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/ j.fsi.2014.09.035

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E. Zahran, E. Risha / Fish & Shellfish Immunology xxx (2014) 1e9

removal is negatively affected by heavy metals contaminated water, reducing the integrity of hepatocytes and hamper metabolic processes and xenobiotic removal activities [12]. Thus induce oxidative stress (OS) through damaging mitochondrial respiration, increased reactive oxygen species (ROS) generation, Lipid peroxidation (LPO), and reduction of intracellular antioxidants within target cells [13,14]. Increased levels of ROS can modulate gene expression and induce apoptosis while decreased their levels can result in oxidative damage and cell to death [15]. Alleviation of heavy metals like Asþ3 intoxicant in aquatic environments either through neutralization or metal hydroxide precipitation using varieties of chemical compounds costs a lot. So, in managing Asþ3 toxicity, now it is essential crucial need to apply alternative eco-friendly approaches having holistic advantages. In this regard, an interest in phytoremediation like supplementation of Ch, unicellular green microalgae, in diet has gained great attention recently [16]. Besides, the proven tolerating activities of Ch, against various heavy metals, metalloids [17], detoxify water supply contaminated with arsenic in Taiwan (OU and DipION, 2010), overwhelmed our interest to investigate the protective role of Ch in combating arsenic induced toxicity. Simultaneously, we also evaluated the innate immune response and the antioxidant activities in Nile tilapia (Oreochromis niloticus) after sub-lethal Asþ3 exposure. 2. Material and methods 2.1. Arsenic exposure A three weeks exposure of Sodium arsenite (NaAsO2; 95%, manufactured by MERCK, 0082970, Art. 6287) concentration corresponding to 7 ppm was applied according to a previous toxicological assay in O. niloticus for 96 h-LC50 [18], which is equal to 1/ 10th of 96 h-LC50 (71.7 ppm). Fresh daily stock solution of NaAsO2 was prepared by dissolving the analytical grade NaAsO2 in double-distilled water, then desired concentration in part per million (ppm) was prepared from the stock solution. Unexposed fish were maintained in separate tanks without arsenic under identical conditions. 2.2. Fish Nile tilapia (O. niloticus), were obtained from private fish farm at Ad-Dakahliya province, Egypt. One hundred and twenty fish were distributed in 12 tanks; in triplicate giving 30 fish per treatment. The average weight of the fish was 90 g and 3% of their body weight was administrated daily as feed. They were maintained in aquarium tanks, which were provided with adequate aeration and under water internal power filter. About 50% of the water was exchanged daily to maintain water quality. The fish were fed twice ad lib with a commercial diet at 25 ± 2 C during the feeding period. Fish were acclimatized for two weeks and during this period no clinical signs were ever observed. 2.3. Chlorella vulgaris powder A dried powder of Chlorella vulgaris (Ch) cultures was purchased from the Institute of National Research Center, Cairo, Egypt. 2.4. Preparation of diets and experimental design Four groups were assigned under examination characterized as a control group (no Ch or Asþ3); Asþ3 group (exposed to water borne Asþ3 at 7 ppm); Ch1 (Ch at 5% þ Asþ3 at the same level of exposure); and Ch2 (Ch at 10% þ Asþ3 at the same level of exposure).

Supplementation of Ch powder was performed as described in Table 1. All ingredients were mixed with oil and then adding water until stiff dough resulted. Each diet was then extruded through a mincer. The resulting strands were shadow-dried, broken up, sieved into pellets, and stored in plastic bags at 4 C until use. Nile tilapia (O. niloticus) were distributed in the aquaria and triplicate tanks were assigned per dietary treatment (Total N ¼ 120 fish). Fish were fed ad lib twice daily in equal rations at 09.00 h and 16.00 h for 21 days. Water changed daily at about 80% during the experimental period and the water quality including, (temperature 25 ± 2 C, dissolved oxygen (mg/l) 6.62 ± 0.10, and pH 7.25 ± 0.04) was maintained during the entire experiment. The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water. The work described is in compliance with the guidelines of the Ethical committee of Mansoura University. 2.5. Sample collections Three fish from each aquarium (6fish/group) were sampled at day 7, 14 and 21. Each aquarium was sampled one at a time; the fish were sedated with a low dose of buffered tricaine (30 mg/L tricaine þ60 mg/L sodium bicarbonate), and each fish was then euthanized one at a time in a separate container having a high dose of buffered tricaine (200 mg/L tricaine þ400 mg/L sodium bicarbonate). Blood samples were taken from the caudal vein using a 23gauge needle. 2.5.1. Immunological parameters 2.5.1.1. Respiratory burst activity. Respiratory burst activity of the whole blood was quantified by the nitrobluetetrazolium (NBT) assay which measures the quantity of intracellular oxidative free radicals; according to Secombes [19], with some modification. Briefly, 100 ml of the blood suspension were added to each well of 96 well microtitre plate (Nalge-Nunc, Hereford, U.K.). The plate was incubated at 25 C, for two hr to allow attachment of cells. Unattached cells were washed off three times using fresh L-15 medium. L-15 medium was then supplemented with NBT (1 mg/ml) and phorbol 12-myristate 13-acetate (PMA, SigmaeAldrich; one mg/ml) dissolved in dimethyl sulphoxide (DMSO, Sigma), and 100 ml added to each well of a microtitre plate and incubated for 1 h at room temperature. After incubation, the supernatant removed from the plate and NBT reduction fixed with 100% methanol for 10 min. The plate was then washed with 70% methanol, and left to air dry. A mixture of 120 ml of 2 M potassium hydroxide and 140 ml DMSO was added to dissolve the resulting formazan blue crystals. The NBT reduction was measured using the micro-plate reader (Optica, Table 1 Basic components of the basal diet (air dry basis, %). Ingredient

Control group

Ch1 group

Ch2 group

Yellow Corn Soyabean meal Fish meal Wheat bran Corn gluten meal Sunflower Oil Vitamins & mineral premixa Salt Ch

17.65 20.50 25.0 34.0 1.0 0.85 0.50 0.5 0

17.65 20.50 25.0 29 1.0 0.85 0.50 0.5 5

17.65 20.50 25.0 24.0 1.0 0.85 0.50 0.5 10

a Trace minerals & vitamins premixes were prepared to cover the levels of the micro minerals &vitamins for tilapia fish as recommended by (NRC, 1993).Vitamins premix (IU or mg/kg diet); vit. A 5000, Vit.D3 1000, vit. E 20, vit. k3 2, vit. B1 2, vit. B2 5, vit. B6 1.5, vit. B12 0.02, Pantothenic acid 10, Folic acid 1, Biotin 0.15, Niacin 30. Mineral mixture (mg/kg diet); Fe 40, Mn 80, Cu 4, Zn 50, I 0.5, Co 0.2 & Se 0.2. Ch1 ¼ Ch at level of 5%; Ch2 ¼ Ch at level of 10%; Ch ¼ Chlorella.


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Mikura Ltd, UK) at 630 nm, and Respiratory burst activity was expressed as NBT reduction. 2.5.1.2. Serum lysozyme activity. Serum lysozyme activity was measured according to Ellis [20]; based on the lysis of Micrococcus lysodeikticus (Sigma Chemical Co), with some modifications. A 0.25 ml of serum was mixed with 0.75 mL M. lysodeikticus suspension (0.2 mg/mL in 0.05 M PBS, pH 6.2). The mixture reacted at 25 C for five min, and then the optical density (O.D.) was measured at one min intervals for five min at 540 nm (5010, Photometer, BM Co. Germany). One unit of enzyme activity was defined as the amount of enzyme causing a decrease in absorbance of, 0.001. The unit of lysozyme presents in serum (mg/mL) was obtained from a stander curve made with lyophilized hen-egg-white-lysozyme (Sigma). 2.5.1.3. Bactericidal activity. Bactericidal activity was determined according to Kampen et al. [21], with some modifications. One hundred of serum were added to 50 ml of 3 108/ml bacterial suspension of Aeromona hydrophila (A. hydrophila) in Tryptone soy broth (TSB); into duplicate wells of 96 round bottom well microtiter plate, and mixed before incubated for 2.5 h at 37 c. A blank control was also prepared by replacing the serum with sterile Hank's Balanced Salt Solution. Fifty ml of diphenyltetrazolium bromide solution (MTT; two mg/ml) were added to all wells and incubated for 20 min at room temperature to allow the formation of formazan. Plates were again centrifuged for 10 min at 2000 g. The supernatant was discarded, and the precipitate was dissolved in 200 ml dimethyl sulfoxide (DMSO). The absorbance of the dissolved formazan was read at 560 nm with a micro-titer plate reader (Optica, Mikura Ltd, UK). The bactericidal activity was calculated by subtracting the absorbance of samples from that of control and reported as absorbance units. 2.5.2. Antioxidant parameters in serum & tissue homogenates The liver and gills were perfused in ice-cold saline, and 0.2 g of each sample in cold PBS solution pH 7.4 was grounded in glass homogenizer tubes (pellet pestle motor) and the homogenate were centrifuged at 4000 rpm for 15 min at 4 C (Centrikon H-401 centrifuge), and the resultant supernatants were aliquot and stored at 80 C for later oxidative stress and antioxidant enzyme assays. The Total antioxidant capacity (TAC) in serum was measured in accordance to Koracevic et al. [22] which performed by the reaction of antioxidants in the sample with a freshly prepared exogenous H2O2, the residual H2O2 is determined calorimetrically by enzymatic reaction. GPX, MDA and NO levels in the tissue homogenate were measured spectrophotometrically (Photometer 5010, Photometer, BM Co. Germany), and expressed as U/g T and nmol/g tissue. CAT level was determined by measuring the decrease of hydrogen peroxide concentration at 240 nm according to Aebi [23]. The reduced glutathione (GSH) was determined in accordance with Beutler et al. [24], using Elmann_s reagent (DTNB). Thiobarbituric acid reacting substances (TBARS), expressed as malondialdehyde (MDA) concentration (nanomole per milliliter of extract), was determined as described by Ohkawa et al. [25]. H2O2 was determined in accordance to Aebi [23]

Fig. 1. Respiratory burst activity level in Nile tilapia fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

3. Results 3.1. Immunological parameters 3.1.1. Respiratory burst activity There were no significant differences in the respiratory burst activity among any treatment groups at day 7. By day 14, respiratory burst activity was lower in Asþ3 group compared with the control, and was higher in both Ch1 and Ch2 groups compared to the Asþ3 group. Respiratory burst activity showed no significant differences between Ch1 and control groups; however, their levels were greatly increased (P < 0.05) in Ch2 group compared with the control group. At day 21, respiratory burst activity also showed a significant decrease in Asþ3 group compared to other groups and was higher (P < 0.05) levels in both Ch1 and Ch2 groups compared to the control and Asþ3 groups. There was no significant difference between Ch1 and Ch2 groups. Additionally, respiratory burst activity showed a significant higher level in Ch1 and Ch2 groups at day 14 & 21 compared to day 7 (Fig. 2). 3.1.2. Serum lysozyme activity There were no significant changes among any treatment groups at day 7. However, by day 14 & 21, the Lysozyme activity was lower in Asþ3 group (P < 0.05) compared to other treatment groups, and significantly higher (1e2-fold) increase in Ch1 &Ch2 groups, respectively compared to other treatment groups. 3.1.3. Bactericidal activity At day 7, only Ch supplement at 10% (Ch2 group) significantly increased the bactericidal activity compared to the Asþ3 group. By day 14, the bactericidal activity was significantly lowered (2- fold decrease) in Asþ3 group compared to other treatment groups. Both supplemented group Ch1 &Ch2 were able to restore the bactericidal level to the control one. Interestingly, The bactericidal activity

2.6. Statistical analysis

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All data in this experiment were subjected to one-way analysis of variance (ANOVA) using the SPSS computer software (SPSS version 17.0 for Windows). Differences between means were assessed by Duncan's multiple-range test and effects with a probability of P < 0.05 were considered significant (Fig. 1).

Fig. 2. Serum Lysozyme activity in Nile tilapia fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).


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However, there were no differences between both Ch1 and Ch2 groups, Asþ3 group and, the control one. Same trend was observed at day 21 and day 14, except that there was a significant reduction in the CAT level, in Ch2 group compared to the control one at day 14.

Fig. 3. Bactericidal activity level in Nile tilapia fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

significantly lowered in Asþ3 group at day 21 (2 fold-decrease), compared to the control one. While with Ch supplementation, bactericidal activity significantly up-regulated (3-fold increase) compared to the Asþ3 group and also showed a significant increase over the control one (Figs. 3e10). 3.2. Antioxidant parameters in serum and gills/liver homogenates 3.2.1. Antioxidant activity in serum 3.2.1.1. TAC. The TAC level showed no significant changes between Asþ3 group and control at any time points or within each group over the time. Ch supplementation in both groups, Ch1 and Ch2, showed higher level (P < 0.05) compared to other treatment groups. There were no significant changes between the two levels used of Ch over time, only at day 14 the TAOC level declined significantly (2- fold) decrease in the Ch2 group compared to other time points. 3.2.2. Antioxidant activity in gills & liver 3.2.2.1. NO. In the gills, Only NO activity was significantly higher in the Ch1 group compared to other treatment groups at day 7. By day 14 & 21, NO activity declined significantly in Asþ3 group compared to other treatment groups. NO activity was up-regulated in Ch2 group Compared to other treatment groups. In the liver, NO activity was in the same trend as in gills, except that, at day 14; both Ch1 and Ch2 groups were not significantly different from the control. 3.2.2.2. CAT activity. At day 7, only the Ch2 group showed a significant increase of the CAT level compared to other treatment groups. By day 14 & 21, the CAT level down-regulated significantly in Asþ3 group compared to the control and Ch2 group. There were no significant changes of the CAT level in any of the treatment groups over time. Similarly in liver, CAT level showed a significant reduction (2- fold) in Asþ3 group compared to the control at day 7.

3.2.2.3. GSH. GSH level in gills was down-regulated (P < 0.05) in Asþ3 group compared to Ch1 and Ch2 groups and vice versa. However, there were no significant changes between Asþ3 and control groups at any time points, but only a nominal reduction in the Asþ3 group compared to the control one. However in liver, GSH level was down-regulated (P < 0.05) in Asþ3 group compared other treatment groups at day 7 & 21. Moreover, there was a significant increase in the GSH level, in Ch2 group compared to Ch1 & control groups at day7. No significant changes were detected between both supplemented groups and the control one at day 14 & 21. 3.2.2.4. GPx. Gpx level in gills showed no significant changes at day 7. Only Ch2 group showed a higher level (P < 0.05) compared to the control and other treatment groups at day 14 & 21 respectively. None of the treatment groups showed any significant changes over time except the Ch2 group that showed a significant increase by day 21. While, in the liver, Gpx level was significantly lower in Asþ3 group compared to both Ch1 & control groups at day 7. No significant changes were detected among Asþ3, Ch1 and Ch2 groups or among both supplemented groups and control at day 14. By the day 21, the GPx level showed (>3- fold) significant decline in the Asþ3 group compared to the control one and other groups. GPx level started to increase gradually at a significant level in Ch1 group, though; its level peaked again (P < 0.05) in the Ch2 group compared to other treatment groups, notably the control one that exceed its level (>2- fold) increase. 3.2.2.5. MDA. Interestingly the MDA level in gills peaked at day 7 (4- fold) increase compared to other treatment groups and start to decline significantly over time. While Ch supplementation, especially at level of 10% (Ch2 group), restored the MDA level to the control one. Same trend was noticed at day 14 & 21. Similarly, the MDA level in liver peaked in Asþ3 group at day 7 (4- fold) increase compared to other treatment groups and start to decline significantly over time. While Ch supplementation, especially at level of 10% (Ch2 group), led to restoring the MDA level to the control one. Same trend was noticed at day 14 & 21. 3.2.2.6. H2O2. In gills, there was a significant increase in the H2O2 level, in the Asþ3 group at each time point (2-fold increase) compared to other treatment groups, while both supplemented groups Ch1 and Ch2 were able to restore (P < 0.05) the H2O2 level again to the control one. Similarly, There was a significant increase in the H2O2 level, in liver, in the Asþ3 group at each time point (2fold increase) compared to other treatment groups while both supplemented groups Ch1 &Ch2 were able to restore (P < 0.05) the H2O2 level again to the control one. 4. Discussion

Fig. 4. Total antioxidant capacity (TAC) activity in Nile tilapia fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

Fish play important roles in the trophic web, accumulate toxic substances and respond to low concentration of mutagens. Thus, using fish biomarkers as indicators in the ecotoxicological studies are of great concern that warrant early detection of the aquatic environmental problems [26]. Fish innate immune response considered the early line of defense against pathogens and pollutants can adversely affect the fish ability to protect against infection. Asþ3 is one of those heavy metals that impose higher risks to fish health.


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Fig. 5. Nitric Oxide (NO) activity in Nile tilapia (a) liver and (b) gills, fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

Fig. 6. CAT activity in Nile tilapia (a) liver and (b) gills, fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

Fig. 7. GSH activity in Nile tilapia (a) liver and (b) gills, fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

Fig. 8. GPx activity in Nile tilapia (a) liver and (b) gills, fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).


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Fig. 9. MDA activity in Nile tilapia (a) liver and (b) gills, fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

Fig. 10. H2O2 activity in Nile tilapia (a) liver and (b) gills, fed Ch diet and exposed to sodium arsenite at 7 ppm. Data is expressed as the mean of six fish ± SEM. Values with a different letter superscript are significantly different between and within groups (P < 0.05).

In the present study, it was observed that Asþ3 exposure has deleterious effects on the immunological parameters of tilapia fish. The results clearly indicated that Asþ3 exposure lowered the respiratory burst activity at each time point along the entire experiment compared to other groups. This finding is similar to Saha et al. [27] after sodium arsenite exposure to mud crab Scylla serrate led to inhibition of NO and superoxide anion generation [27]. Correspondingly, Nayak et al. [28] found significantly suppressed the ability of zebrafish embryos to release an effective respiratory burst activity response following Asþ3 exposure at 2 or 10 ppb in water. On the contrary, Guardiola et al. [29] corroborated a significant increase in respiratory burst activity by day 10 exposing 5 mM arsenic trioxide to gilthead sea bream, though that response was lowered nominally at day 30, finally. Lysozyme is one of the humoral elements of innate immunity. Lysozyme activity was significantly down-regulated in the exposed group compared to other treatment groups. Similarly, Gonzalez et al. [30] depicted down-regulation of each clones (1-11 and 1163) of lysozyme precursor in male mummichogs (Fundulus heteroclitus) exposed to arsenic at 172 ppb, but up-regulation appeared while exposed to 1720 ppb. Identical trend was observed with Mozambique tilapia exposed to zinc at high dose 5 ppm, that lysozyme activity were decreased significantly than the unexposed group [31]. Our results indicating that Asþ3 exposure led to downregulation of bactericidal activity and restored the activity again upon Ch supplementation. Similarly, silver catfish (Rhamdia quelen) exposed to 0.73 ppm of glyphosate showed a nominal increase in colony forming units after 10 days of exposure [32]. In the same context, zebrafish embryos exposed to 2 and 10 ppb arsenic and infected by Edwardsiella tarda for 4 h, resulted in at least a 17-fold increase in bacterial load in embryos [28]. Additionally, A higher increase in bacterial load along with decreased bacterial clearance potential was noticed in Clarias batrachus L. fish after long-term

exposure (150 days) to arsenic (42.42 mM) and infected with A. hydrophila [33]. In line with this, there are also several studies reporting higher bacterial load after arsenic exposure [34,35]. Together, these results suggest that the arsenic exposure compromises the host defense and decreases the ability to clear the pathogen load with the subsequent increase in the susceptibility to pathogenic infection [36,37]. Antioxidant enzymes are essential key factors in animal defense system against oxidative stress induced by xenobiotic [38]. In the present study, there were no significant changes in the TAC between exposed and unexposed groups; however, TAC value significantly increased in Ch supplemented groups compared to other treatment groups. Previous investigations done by Richetti et al. [39] found reduction of antioxidant competence on analyzing the effects of lead and mercuric chloride for 24 h on zebrafish. Our result also disagreeable with the findings of Amado et al. [40] where they observed a significant lower (P < 0.005) antioxidant capacity following 24 h microcystins intoxication to teleost fish Jenynsia multidentata. Adult male crabs (Chasmagnathus granulatus) injected with Microcystis aeruginosa aqueous extract (39.2 mg/l) at 24 h intervals for 2 days showed higher total oxyradical scavenging capacity (TOSC) values against peroxyl radicals in both anterior and posterior gills. However, no differences in TOSC values were seen when hydroxyl radicals were used [41]. This could be attributed to differences in the oxyradical generating system employed, the scavenging efficiency of non-enzymatic antioxidants measured by TOSC [42]. Asþ3 exposure lowered the NO significantly in gills and liver at day 14 & 21 in comparison to other treatment groups. These data are in line with Saha et al. [27]. Same trend was found by Chakraborty et al. [43] who reported a significant increase in NO generation in the gills of Lamellidens marginalis only after 24 h arsenite exposure, then a significant decline concomitant with higher exposure dose at 3e5 ppm for longer exposure period. Our findings


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also agreeable with those findings of Dawson & Dawson and Halliwell & Gutteridge [44,45]. This may be attributed to either suppression of cytokine-induced iNOS gene expression, or through inhibition of iNOS by phenylarsine oxide binding to the vicinal dithiol moiety of NF-kB. Also, arsenite led to inactivation of NFekB and Erk1/2 MAP kinase pathways due to its inhibitory action on lipopolysaccharideeinduced nitric oxide production in macrophage cells [46]. The increase in NO production in Ch supplemented groups might have occurred due to the enhanced macrophage activity [47], Hasegawa et al. [48] evidenced that Ch augments macrophage activity. Together, these data imply that Ch may enhance the immune responses via macrophage activation. CAT level was down-regulated after Asþ3 exposure while it upregulated again in Ch supplemented groups, to the control level. Higher level of Ch (10%) revealed a significant increase over the control late by day 21. Our results were in line with Aruljothi [11] who found nominal down-regulation in CAT level, in the brain and gill tissues of Labeo rohita exposed to arsenic (1.89 ppm) for 7 days. In the same context, CAT level showed a significant reduction in C. batrachus exposed to 1/10th LC50 (8.4 ppm) starting from day 2 to the end of the treatment period (10 days) [49]. CAT level of the hemocytes of S. serrate showed the lowest level against 3 ppm of sodium arsenite exposure [50]. On the contrary, CAT levels showed no significant changes in zebrafish exposed to different Asþ3 concentration (1, 10, and 100 mg/l) for 48 h [51]. Other studies showed a different pattern of CAT level from yellow perch fish collected from differently contaminated sites and found that CAT level were higher in head kidney tissue, in fish collected from more contaminated sites compared to moderate and less contaminated ones [52]. This may be attributed to species differences; tissue analyzed and also exposed dose. Many factors are involved in Asþ3 metabolism and detoxification that may vary in different organs [53]. GSH level decreased significantly after Asþ3 exposure compared to control and Ch supplemented group. Ch supplementation (10%) showed a significant up-regulation in GSH level compared to other treatment groups, particularly the highest level. Our results were supported by Aruljothi [11] who found a reduction of GSH & GPx levels in gill and brain tissues of L. rohita treated with sub-lethal concentration of arsenic (1.89 ppm) for 7 days. GSH level showed a significant increase in the liver of arsenic-treated Channa punctatus at day 7 then a significant decline after 14 days of exposure time. However, in kidney, GSH level was declined significantly at both times of exposure [54]. Similar results were obtained in the authors' previous work Bagnyukova et al. [55]. These differences in response may be attributed to the tissue analyzed due to their tolerance to arsenic, dose, and duration of exposure. Additionally, GSH content may show both increases and decreases in fish tissues exposed to metals due to their organ-specific responses; while, reduction of GPx activity could be attributed to the direct effects of metal ions on the active site of enzyme molecules [56]. LPO considered a biomarker for heavy metal toxicity [57,58], causing damage in cell membrane structure and function with subsequent imbalance between synthesis and degradation of enzyme protein [59,60]. Our results showed a higher level of gills and liver MDA and H2O2 in Asþ3 group compared to other treatment groups. These data are coincided with previous results showed that Asþ3 exposure at 1 ppm lead to induction of LPO in the liver, kidney, and gills after exposure periods ranging from 7 to 90 days [54]. The LPO was also elevated in gills and brain tissues of freshwater fish, L. rohita after arsenic exposure for 7 days [11]. In line with our results, Bagnyukova et al. [55] found up-regulation of LPO at day 1 and 4 after Asþ3 exposure at 200 mM sodium arsenite. In the present study, H2O2 level showed a parallel pattern to the MDA level, increasing in the exposed group compared to other treatment groups. These data confirm our results; CAT enzyme

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catalyzes the removal of H2O2 with production of H2O and O2 [61e63], we reported in this study reduced level of CAT under Asþ3 exposure that prevents the formation of radical intermediates into water and oxygen. Thus, H2O2 level showed induction in the exposed group to free the O-2 radicals and thus producing cell damages that also confirmed earlier in mice through induced As toxicity by Yamanaka et al. and Blair et al. [64,65]. Hydrogen peroxide is an important ROS in arsenite toxicity; also activation of NADPH oxidase had been implicated in the elevation of intracellular H2O2 by arsenite [66]. Our findings are also supported by various earlier findings stated by Mena et al. and Lushchak [67e69]; in different experiments induced in rats, aquatic animals etc. It is predictable through above results, Asþ3 exposure cause alterations in the innate immune response and antioxidant defense system in fish [70]. Our results indicate that Ch was remarkably effective in ameliorating the innate immune disruption and oxidative stress responses caused by arsenic, suggesting its potential therapeutic effect in our experiment. Additionally, the effect of Ch supplementation was in a dose-dependent manner. This was in accordance with other studies concerning Ch using to detoxify heavy metals such as mercury, copper, cadmium and lead. It was also used successfully in Taiwan in cases of arsenic poisoning due to contaminated water supply [71]. In our current study, dietary Ch supplementation enhanced the innate immune responses that were adversely affected after Asþ3 exposure, that similarly predicted earlier by Suhendrayatna et al.; Mason, and Andrews et al. [17,72,73]. These results were confirmed earlier by who evedinced. The detoxification power of Ch against Asþ3 toxicity may be attributed to its contents of natural antioxidants such as chlorophyll, polyphenol, vitamins, sulfur-containing compounds that have the capacity to scavenge free radicals. Antioxidative role of Ch in phenol compounds of the methanolic extract and the antioxidative effects of hydrophilic compounds presented in the aqueous extract of Ch have been previously investigated [3,74]. Chlorophyll content of Ch may play a role in its radical-scavenging effects [75]. Ch was able to detoxify arsenite inside the cell by sequestering it into subcellular compartments [76]. Plants bioremediate heavy metals through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione [77]. Similarly, other pathways have been involved in detoxification ability of Ch, it can act as an ion exchange resin; i.e., cell wall absorbs rather large amounts of toxic metals. Specific combination of amino-acids, the Ch derived growth factor, or some other yet unknown action results in mobilization of heavy metals from within the cell [78]. In the current study, Ch intake reduced LPO in Nile tilapia after Asþ3 exposure. Similarly, fruits and vegetables rich in antioxidants were able to increase plasma antioxidant capacity in human [79]. In the same context, human intake of Ch tablets increased serum bcarotene and lutein concentrations [80]. In conclusion, our results confirmed the immunosuppressive and oxidative stress responses in gills and liver of Nile tilapia exposed to sodium arsenite; and proved that these changes are organ-specific and are related to the exposure route, the dose administered, and the chemical uptake by the organs. Therefore, it is thought that chlorella could be a helpful supplementation for improving innate immunity and antieoxidative capacity. However, the mechanism behind the antioxidant effects of chlorella needs further research to be conducted related to detoxifying system. Acknowledgments This research work was supported in by laboratory of fish diseases, department of Internal medicine, Infectious and Fish diseases


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Metacercarial infection of wild Nile tilapia (Oreochromis niloticus) from Brazil.

Physiological and oxidative stress biomarkers in the freshwater monosex Nile tilapia, Oreochromis niloticus L., exposed to pendimethalin-based herbicide.

Modulation of genotoxicity and endocrine disruptive effects of malathion by dietary honeybee pollen and propolis in Nile tilapia (Oreochromis niloticus).

Genome-wide identification, phylogeny, and gonadal expression of fox genes in Nile tilapia, Oreochromis niloticus.

Marine Collagen Peptides from the Skin of Nile Tilapia (Oreochromis niloticus): Characterization and Wound Healing Evaluation.

Draft Genome Sequences of Streptococcus agalactiae Strains Isolated from Nile Tilapia (Oreochromis niloticus) Farms in Thailand.

RING Finger Protein 38 Is a Neuronal Protein in the Brain of Nile Tilapia, Oreochromis niloticus.

Nutritional background changes the hypolipidemic effects of fenofibrate in Nile tilapia (Oreochromis niloticus).

Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus).

Physiological, biochemical and histometric responses of Nile tilapia (Oreochromis niloticus L.) by dietary organic chromium (chromium picolinate) supplementation.

Genome-wide identification, evolution and expression analysis of nuclear receptor superfamily in Nile tilapia, Oreochromis niloticus.

Effects of a homeopathic complex on the performance and cortisol levels in Nile tilapia (Oreochromis niloticus).

Figla Favors Ovarian Differentiation by Antagonizing Spermatogenesis in a Teleosts, Nile Tilapia (Oreochromis niloticus).

High temperature-induced sterility in the female Nile tilapia, Oreochromis niloticus.

Nile Tilapia Neu3 sialidases: molecular cloning, functional characterization and expression in Oreochromis niloticus.

Triploidy induction in Nile tilapia, Oreochromis niloticus L. using pressure, heat and cold shocks.

Development of a bacterial challenge test for gnotobiotic Nile tilapia Oreochromis niloticus larvae.

Feed-based vaccination regime against streptococcosis in red tilapia, Oreochromis niloticus x Oreochromis mossambicus.

High dietborne Cu and Cd induced genotoxicity of Nile tilapia (Oreochromis niloticus).

Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

Pharmacodynamic interaction of Spirulina platensis and deltamethrin in freshwater fish Nile tilapia, Oreochromis niloticus: impact on lipid peroxidation and oxidative stress.

Effects crude protein levels on female Nile tilapia (Oreochromis niloticus) reproductive performance parameters.

Analysis of differential gene expression under low-temperature stress in Nile tilapia (Oreochromis niloticus) using digital gene expression.

Antimicrobial Susceptibility of Escherichia coli Isolated from Fresh-Marketed Nile Tilapia (Oreochromis niloticus).