Accepted Manuscript Title: Protective role of dietary Spirulina platensis against diazinon-induced Oxidative damage in Nile tilapia; Oreochromis niloticus Authors: Nevien K.M. Abdelkhalek, Ismail A.M. Eissa, Eman Ahmed, Omnia E. Kilany, Mohamed El-Adl, Mahmoud A.O. Dawood, Ahmed M. Hassan, Mohamed M. Abdel-Daim PII: DOI: Reference:
S1382-6689(17)30185-0 http://dx.doi.org/doi:10.1016/j.etap.2017.07.002 ENVTOX 2820
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Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
12-3-2017 20-6-2017 4-7-2017
Please cite this article as: Abdelkhalek, Nevien K.M., Eissa, Ismail A.M., Ahmed, Eman, Kilany, Omnia E., El-Adl, Mohamed, Dawood, Mahmoud A.O., Hassan, Ahmed M., Abdel-Daim, Mohamed M., Protective role of dietary Spirulina platensis against diazinon-induced Oxidative damage in Nile tilapia; Oreochromis niloticus.Environmental Toxicology and Pharmacology http://dx.doi.org/10.1016/j.etap.2017.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective role of dietary Spirulina platensis against diazinon-induced Oxidative damage in Nile tilapia; Oreochromis niloticus
Nevien K. M. Abdelkhalek#1, Ismail A. M. Eissa2, Eman Ahmed3, Omnia E. Kilany4, Mohamed El-Adl5, Mahmoud A. O. Dawood6, Ahmed M. Hassan7 and Mohamed M. Abdel-Daim#* 3
Department of Internal medicine, Infectious and Fish Diseases, Faculty of Veterinary
Medicine, Mansoura University, Egypt. 2
Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Suez
Canal University, Ismailia 41522, Egypt. 3
Department of Pharmacology, Faculty of Veterinary Medicine, Suez Canal
University, Ismailia 41522, Egypt 4
Department of Clinical Pathology, Faculty of Veterinary Medicine, Suez Canal
University, Ismailia 41522, Egypt 5
Department of Biochemistry, Faculty of Veterinary Medicine, Mansoura University,
Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University,
Kafrelsheikh 33516, Egypt. 7
Department of Hygiene, Faculty of Veterinary Medicine, Suez Canal University,
Ismailia 41522, Egypt
# Both authors are equally contributed in this research work.
*Correspondence: Mohamed M. Abdel-Daim; Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt. Tel/Fax.: +20643207052 E-mail: [email protected]
, [email protected]
Diazinon induced oxidative damage in tilapia liver, kidney and gills
Spirulina has antioxidant activities against diazinon-induced damage
Diazinon induced serum biochemical alterations
Spirulina protected against diazinon-induced toxicities
Abstract The current study was performed to investigate the ameliorating effect of dietary supplementation of 0.5 and 1% Spiurolina platensis (SP) diet against the subacute toxicity of diazinon (DZN) 0.28 mg/L in Nile. At the end of experiment after 28 days, hepatic and renal damage markers (aspartate transaminase, alanine transaminase, alkaline phosphatase, urea, uric acid and creatinine), serum biochemical parameters (total proteins, albumin, cholesterol and glucose) and tissue antioxidant status (superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione and malondialdehyde) were detesrmined. The results of the current study revealed significant improvement in hepatic and renal damage markers after SP supplementation in fish exposed to DZN toxicity. Moreover, SP improved serum biochemical markers through increasing serum albumin and globulins with a significant decrease in serum glucose and cholesterol. In addition,
liver, kidneys and gills antioxidant status showed a significant improvement after SP supplemented to fish exposed to DZN where a significant increase in tissue antioxidant activity were observed with a significant decline in lipid peroxidation levels. It can be concluded that, SP supplementation attenuated the toxic effect of DZN toxicity in Nile tilapia through improving liver and kidney functions with a significant enhancement of tissue antioxidant status. Key words: diazinon; organophosphate; Spirulina; Nile tilapia;
Introduction The suitability of Nile tilapia in aquaculture system due to its higher growth
rate, marketability and price stability are considered the main factors for its worldwide distribution (Wang and Lu, 2015). The presence of those harmful pollutants stimulates imbalances between prooxidant and antioxidant defense systems due to the release of free radicals that initiates a chain reaction causing oxidative stress altering fish immune status that leads to vulnerability to disease conditions (Nabatchican et al., 2014). Organophosphorus compounds are a world-wide used insecticides that characterized by their inhibition to actylcholine esterase enzyme which are used extensively to replace the bio-accumulative hazards of chlorinated compounds such as organochlorine pesticides (Galloway and Handy, 2003). DZN is an important organophosphorus compound derivatives that maintains a potential broad spectrum effects against different insects threatens veterinary field. It is easily washed out into the surface waters affecting wide range of non-target organisms including fish (Oruc,
2011). The stability of DZN in water can persists for 6 months causing dangerous accumulative effects on aquatic organisms' tissues (Al-Ghanim, 2014). Recently, there has been an increasing concern among researchers about the undesirable effects of such chemical pollutants on the aquatic ecosystems and how to overcome such contaminants using environmentally safe drugs (Abdel-Daim et al., 2015; Abdelkhalek et al., 2015).
Spiurolina platensis (SP) is a filamentous cyanobacteria that is distributed in Central Africa and used extensively as human dietary supplementation. This unicellular cyanobacterium has a potent antioxidant activity, hepatoreno protective role as well as an anti-inflammatory effect. Moreover, SP can ameliorate organ toxicity induced by heavy metals (El-Desoky et al., 2013). There is growing evidence suggesting that different algal species posses proteins of high nutritive value which are suitable not only as a feed supplement among aquatic organisms (Habib et al., 2008) but also can ameliorate the toxic effect of some pyrethroid compounds such as deltamethrin (Abdelkhalek et al., 2015). However, the potential protective role of SP against other pollutants as DZN in Nile tilapia has not yet proven. Therefore, the current study was carried out to investigate the protective effect of Spirulina platensis against DZN-induced hepatorenal and gills toxicity and oxidative stress in Oreochromis niloticus. 2.
Material and methods 2.1.Chemicals: Diazinon (DZN) was purchased from Adwia pharamaecuticals (Cairo, Egypt)
containing 60% DZN as an active principle. DZN was diluted in deionized water immediately before administration. Spirulina platensis (SP) pure powder was 4
purchased from (Herbaforce, UK). All the necessary commercial kits required for the completion of analytical procedures were purchased from (Biodiagnostic, Egypt). 2.2.Experimental design and fish grouping: One hundred male mono-sex O. niloticus were obtained from a private fish farm from Dakhlia Governorate, Egypt, with an average body weight 60±6.1 g and 11±0.25 cm total length. Fish were transported alive to the laboratory of Fish Diseases and Management, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt, where the experimental and analytical procedures were followed the Research Ethical Committee. Fish were distributed equally into five groups (20 fish/group) in ten fully prepared water aquaria (10 fish/aquarium). They were kept for two weeks for acclimation. Water parameters were measured daily with SensoDirect 150 (Lovibond, Germany) for adjusting water temperature, pH and dissolved oxygen during the experimental period. The water parameters were maintained according to the requirement of O. niloticus as followed: (Temperature 22±1.2 ºC, pH 7.6-7.8, dissolved oxygen 7.05±0.5 mg/L). All studied groups of fish were fed on basal diet during the acclimation period. The first group was kept as control group and fed on basal diet. The second group (SP1%) were fed on basal diet supplemented with Spirulina platensis powder at a dose of 1% (1g/100kg basal diet) for 30 days. The third group was intoxicated with DZN with a dose of 0.28 mg/L which was considered 1/10 of DZN 96 hour LC50 in O. niloticus (2.8 mg/L) (El-Sherif et al., 2009). The fourth and fifth groups of fish were intoxicated with the previously mentioned dose of DZN but fed on Spirulina platensis powder supplemented diets with a dose of 0.5% (DZN-SP0.5%) and 1% (DZN-SP1%) , respectively. The basal diet was calculated to contain 3000 KCAL, 31.78% crude protein, 7.15% fat. The preparation of diet was done weekly and stored at 4ºC for daily use. 5
2.3.Analytical procedures: 2.3.1. Serum and tissue preparation: At the end of the experiment, fish were anesthetized with MS222 with a dose of 85 mg/kg fish body weight and buffered with a double dose of sodium bicarbonate according to the method of (Matsche, 2011). Blood samples were collected from the caudal blood vessels of every single fish. The collected blood samples were transferred into clean and sterile centrifuge tubes for serum separation procedures. After euthanasia of fish with MS222 through increase the initial dose of MS222 to 400 mg/L (Matthews and Varga, 2012), gills, kidneys and liver were collected, washed with normal saline and dried with filter paper. The collected tissues were homogenized in phosphate buffer saline, filtered and centrifuged at 3000 g for 15 minutes at 4ºC. A clear supernatant was collected and stored at -80ºC till the analytical procedures were resumed. 2.3.2. Hepatic and renal function: Freshly collected sera were used to estimate serum aspartate transaminases (AST) and alanine transaminases (ALT) according to (Reitman and Frankel, 1957) procedures. Hepato-biliary integrity was checked by the estimation of serum alkaline phosphatase (ALP) activity according to (Tietz et al., 1983). Urea, creatinine and uric acid were enzymatically measured according to the method of (Coulombe and Favreau, 1963; Larsen, 1972; Whitehead et al., 1991) respectively. 2.3.3. Serum biochemical parameters:
Serum protein profile was determined according to the methodology of (Lowry et al., 1951) and (Young et al., 2001) for estimation of serum total proteins and albumin concentration, respectively. The calculation of serum globulin and A/G ration was also performed. Serum cholesterol was enzymatically measured according to (Allain et al., 1974). The glycemic status of fish was estimated by measuring serum glucose concentration according to (Trinder, 1969). 2.3.4. Tissue antioxidant status and oxidative stress markers: Superoxide radical scavenging ability of liver, gills and kidneys of O. niloticus were expressed through the estimation of superoxide dismutase activity according to the technique of (Nishikimi et al., 1972). Moreover, the capability of tissues in scavenging hydrogen peroxide was also investigated through the determination of catalase activity according to (Aebi, 1984). In addition, glutathione peroxidase activity was also determined for further studying hydrogen peroxide scavenging capacity of studied tissues according to the method of (Paglia and Valentine, 1967). The concentration of reduced glutathione was determined according to (Beutler et al., 1963). The oxidative stress marker, malondialdehyde, was estimated according to the method of (Draper and Hadley, 1990). Finally, the evaluation of liver, gills and kidneys protein content was determined according to (Lowry et al., 1951). 3.
Results 3.1.Hepatic and renal function test: The toxic effect of DZN and preventive role of SP on liver and kidney
function of Oreochromis niloticus were represented in Table (1). DZN showed a significant elevation in liver transaminases enzymes as well as in serum ALP
activities (P ≤ 0.05). Moreover, renal damage markers (urea, uric acid and creatinine) showed a significant elevation (P ≤ 0.05) in comparison with control group. Combined treatment with DZN and SP reduced both renal and hepatic damage markers significantly (P ≤ 0.05) especially in DZN-SP1% where a significant decline in serum creatinine, urea, ALP and ALT was detected in comparison with both DZN and DZN-SP0.5%. There was no significant change (P ≤ 0.05) between SP treated and control groups in all studied parameters 3.2.Serum biochemical parameters: In Table (2), the intoxicated role of DZN and protective role of SP were investigated on serum biochemical parameters. Similarly, DZN revealed a significant decrease in serum levels of total proteins, albumin, globulins and A/G ratio (P ≤ 0.05). In addition, a significant increase in serum glucose and cholesterol was also found after DZN intoxication (P ≤ 0.05). Surprisingly, the protective role of SP was clearly observed through the significant elevation of serum total protein, albumin, globulin and A/G ratio as well as the significant decrease in serum glucose and cholesterol in treated groups (P ≤ 0.05). Moreover, DZN-SP1% restored the concentrations of all studied parameters back to normal. 3.3.Tissue antioxidant and oxidative stress markers: Tissue homogenate of gills, kidneys and liver of O.niloticus were done to investigate the antioxidant status and oxidative stress markers in DZN intoxicated group of fish and the preventive role of SP (Fig. 1-5). A significant (P ≤ 0.05) decline in branchial, renal and hepatic SOD activity in DZN group was observed which was 8
alleviated gradually by supplementation of SP 0.5 and 1%. Supplementation with SP at rate of 1% produced almost complete recovery and normalized the branchial, renal and hepatic SOD activity (Fig. 1). In Fig. 2, a significant decline of all tissue homogenates CAT activity in DZN treated group was found which began to increase gradually through the change in the concentration of SP from 0.5 to 1%. Moreover, DZN-SP1% showed a significant increase in CAT activity (P ≤ 0.05) in gills in comparison with control group. (Fig. 2). Following the same trend, a significant decrease in GSH-Px activity in intoxicated group with DZN was observed in gills, kidney and liver which also started to elevate after SP supplementation, however a non significant change was detected between SP and control group (P ≤ 0.05). Interestingly, DZN-SP1% group showed a nearly similar activity for GSH-Px enzyme in comparison with control group (P ≤ 0.05) (Fig. 3). In Fig.4, the concentration of GSH in gills, kidney and liver homogenates revealed the lowest concentration in DZN group in comparison with all studied group. A significant increase in GSH concentration (P ≤ 0.05) between in DZN-SP1% and DZN-SP0.5% in kidney and liver homogenates was observed in a concentration dependent manner. Accordingly, in DZN-SP1% group dietary supplementation of SP could restore the concentration of GSH similar to control and SP1% groups. (Fig. 4). Concerning the levels of lipid peroxidation, DZN significantly (P ≤ 0.05) increased the concentration of malondialdehyde in comparison with all studied groups in gills, kidneys and liver homogenates. In addition, DZN-SP0.5% and DZN-SP1% alleviated the levels of lipid peroxidation effectively, although it couldn't reach to the levels of control and SP1% (Fig. 5). 9
Oxidative stress is a result of imbalances in the equilibrium status between prooxidants and antioxidants that would result in a significant damage to tissues and organs (Üner et al., 2006). Both mammalian and piscine metabolic systems have shown a considerable amount of similarity in toxicological responses against oxidative stress which required adequate understanding for the different mechanisms regulating oxidative stress (Tridico et al., 2010). Diazinon and other xenobiotics are capable of performing oxidative damages that can cause deteriorations in different metabolic pathways including redox enzymes and mitochondrial electron permeability (Abdel-Daim, 2016; Abdel-Daim et al., 2016). The leakage in mitochondrial permeability would result in the increase of production of reactive oxygen species. The generated reactive oxygen species would affect the permeability of hepatocyte through cellular damage resulting in outflow of liver transaminase (ALT and AST) to blood (Srivastava et al., 2004) explaining the elevated levels of ALT and AST in the present study. The increase in permeability in hepato-biliary system in Nile tilapia was also observed due to the action of DZN through the elevation in ALP activity as a result of the increase in transphosphorylation reaction in hepatocyte (Sharma, 1990) . Similarly, (Ibrahim and Banaee, 2014) suggested that sub-lethal toxicity of DZN (0.2mg/L) would achieve a significant elevation in ALP activity in Nile tilapia. In Spotted Catfish, a significant elevation in serum ALP activity was found after intoxication with monocrotophos (Agrahari et al., 2007). Diazinon extends its harmful effect to cause deleterious effects on renal function through the elevation of renal markers (urea, uric acid and creatinine). The toxic effect of DZN in kidney is attributed to its role in decreasing glomerular 10
filtration rate that is indicated by a significant increase in serum creatinine as a result of depletion in kidney glutathione (Akturk et al., 2006). In O. niloticus, a significant elevation in serum urea and creatinine after DZN intoxication with a dose of 0.76 mg/L after 4 weeks of the start of the experiment was observed (Ibrahim and Banaee, 2014). The toxic effect of DZN on renal tissue is attributed to the degeneration in renal tubular epithelium with atrophy in glomeruli. In general, DZN is incriminated in functional damage in kidney which suggested the increase in the protein catabolic products such as urea and uric acid with impairment in their excretion (Jyothi and Narayan, 1999). Spirulina platensis is characterized by its high nutritive value with a potent hepato-reno protective function; in addition it has an effective role in several toxicological studies (Mazokopakis et al., 2014). In this study, dietary supplementation with SP showed a significant improvement in liver function through decreasing the activities of both liver transaminases and alkaline phosphatase and decreased the concentration of renal damage markers especially (DZN-SP1%) group. This protective effect is attributed to its antioxidant and anti-inflammatory activities. The antioxidant chemical compound in SP such as carotene, C-phycocyanins, vitamins and minerals plays an important role in protection of renal tissues from toxicity against xenobiotics (Karadeniz et al., 2009). In O. niloticus, SP has been shown a hepato-protective effect on fish exposed to deltamethrin through decreasing the rate of enzymatic activities of liver transaminases and ALP significantly (Abdelkhalek et al., 2015). Interestingly, DZN reduced the concentration of serum protein of intoxicated fish (total proteins, albumin and globulins). The decrease in serum albumin and
globulins indicates liver dysfunction. Moreover, it is suggested that the decrease in serum protein concentration is attributed to the increase in the levels of stressors of exposed fish (Mazeaud et al., 1977). Dietary supplementation of SP (0.5 and 1%) alleviates the toxicity induced by DZN and the concentration of serum protein restored its normal level. In O. niloticus, live Spirulina significantly improve serum protein levels and A/G ratio trough restoring normal hepatic function (Abdel ‐ Tawwab and Ahmad, 2009). In O. mykiss, a significant increase in serum total proteins and albumin was indicated during supplementation with spirulina for 10 weeks (Yeganeh et al., 2015) declaring the role of albumin in sustaining fish life. In this study, serum glucose and cholesterol showed a significant increase in their concentrations in DZN group when compared with control and (DZN-SP) groups. This increase shows the extent of damage that DZN can perform in fish organs and considered an indicator of the exposure of acute stress as a result of DZN toxicity due to the action of cortisol hormone that stimulates glycogenesis and gluconeogenesis. In addition, the increase of serum cholesterol is considered an indicator of destruction of cellular structure in kidney and muscle cell membranes (Üner et al., 2006). In DZN-SP
0.5 and 1%,
serum cholesterol and glucose regained its
normal concentration indicated the anti-stress role of spirulina against xenobiotic toxicity as well as its protective effect on hepatic and renal tissues (Nachankar et al., 2005). Antioxidant status of liver, kidney and gills were studied to investigate the protective effect of spirulina against DZN toxicity. A significant decrease in SOD, CAT, GSH-Px activities as well as a significant decrease in GSH concentration was
observed in DZN in comparison with other groups. Moreover, the degree of lipid peroxidation was the highest in intoxicated group of fish with DZN when compared with other groups. The recorded damage in kidneys and liver would cause a significant depression in tissue antioxidant status due to the severe exhaustion of tissue exposed to oxidative stress (Hai et al., 1997). The inhibition of CAT was studied by the work of (Gultekin et al., 2000) and showed that chlorpyrifos-ethyl, a crystalline organo-phosphorus compound; inhibit CAT activity, which suppresses the formation of superoxide radical causing an inhibition in GSH-Px enzymatic activity. Moreover, the levels of malondialdehyde show a significant increase in DZN intoxication which is a characteristic response of its toxicity (Akturk et al., 2006). Spirulina shows its potential antioxidant activity through restoring liver and kidney functions of O. niloticus which subsequently enhances antioxidant status of fish with a reduction in lipid peroxidation in tissues. From this point, SP can effectively ameliorate the toxic effect produced by DZN in a dose dependent manner through enhancing liver and kidney function, restoring antioxidant capacity of tissues. Finally, it has an anti-stress role through controlling the increase of both glucose and cholesterol levels associated with stressors such as xenbiotics toxicity in such fish.
Conclusions Oxidative damage plays a pivotal role in DZN-induced organ toxicity in Nile tilapia. Spirulina is a potent food supplement and antioxidants which is proven to induce multi-organ protection against many chemicals and pesticides-induced toxicity. According to the current study, it is concluded that DZN exposure resulted in alteration of serum biochemical parameters and reduction of antioxidant enzyme activities as well as elevation of lipid peroxidation product. Supplementation of SP induced almost complete protection through its antioxidant and protective activities. 13
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Figure legend Fig. 1 Liver, kidney and gills superoxide dismutase activity (SOD) in control and different treated groups. Different letters indicates significantly different mean value at P ≤ 0.05 according to one way ANOVA test followed by LSD as a post Hoc test Fig. 2: Liver, kidney and gills Catalase activity (CAT) in control and different treated groups. Different letters indicates significantly different mean value at P ≤ 0.05 according to one way ANOVA test followed by LSD as a post Hoc test Fig. 3: Liver, kidney and gills enzymatic GSH-Px activity in control and different treated groups. Different letters indicates significantly different mean value at P ≤ 0.05 according to one way ANOVA test followed by LSD as a post Hoc test Fig. 4: Liver, kidney and gills reduced glutathione (GSH) concentration in control and different treated groups. Different letters indicates significantly different mean value at P ≤ 0.05 according to one way ANOVA test followed by LSD as a post Hoc test Fig. 5: Liver, kidney and gills Malondialdehyde concentration (MDA) in control and different treated groups. Different letters indicates significantly different mean value at P ≤ 0.05 according to one way ANOVA test followed by LSD as a post Hoc test\
Table (1): Hepatic and renal function tests of control and different treated groups of O. niloticus Parameters Experimental groups AST (U/L)
Uric acid (mg/dl)
Data are expressed as means ± SE (n=8). Values contain different superscripts within the same column are significantly different (P ≤ 0.05) according to one way ANOVA followed by LSD as a post Hoc test. SP; Spirulina platensis, DZN ; Diazinon, AST; Aspartate aminotransferase, ALT; alanine aminotransferase, ALP; alkaline phosphatase.
Table (2): Serum biochemical parameters in control and different treated groups of O. niloticus
Total protein (g/dl)
Data are expressed as means ± SE (n=8). Values contain different superscripts within the same column are significantly different (P ≤ 0.05) according to one way ANOVA followed by LSD as a post Hoc test. A/G; Albumin/globulin ratio. SP; Spirulina platensis, DZN; Diazinon