Accepted Manuscript Title: Grafting of gallic acid onto chitosan nano particles enhances antioxidant activities in vitro and protects against ochratoxin A toxicity in catfish (Clarias gariepinus) Author: Mosaad A. Abdel-Wahhab Abdulhadi Aljawish Amany M. Kenawy Aziza A. El-Nekeety Heba S. Hamed Sekena H. Abdel-Aziem PII: DOI: Reference:
S1382-6689(15)30146-0 http://dx.doi.org/doi:10.1016/j.etap.2015.12.005 ENVTOX 2410
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
20-8-2015 13-12-2015 14-12-2015
Please cite this article as: Abdel-Wahhab, M.A., Aljawish, A., Kenawy, A.M., ElNekeety, A.A., Hamed, H.S., Abdel-Aziem, S.H.,Grafting of gallic acid onto chitosan nano particles enhances antioxidant activities in vitro and protects against ochratoxin A toxicity in catfish (Clarias gariepinus), Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.12.005 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.
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Ochratoxin-A (OTA) is a mycotoxin produced by Penicillium and Asperigillus OTA is hepatonephrotoxic, teratogenic and immunosuppressive agent Applicability of chitosan (CS) is limited due to its poor solubility CS nanoparticles (CSNPs) was modified and grafted with gallic acid or octyl gallate Modified CSNPs has higher antioxidant activity in vitro & protect against OTA in vivo
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Grafting of gallic acid onto chitosan nano particles enhances antioxidant activities in vitro and protects against ochratoxin A toxicity in catfish (Clarias gariepinus)
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Mosaad A. Abdel-Wahhaba*
[email protected] , Abdulhadi Aljawishb, Amany M. Kenawyc, Aziza A. El-Nekeetya, Heba S. Hamedd, Sekena H. Abdel-Azieme a
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Food Toxicology & Contaminants Department, National Research Center, Dokki 12622, Cairo, Egypt b Université de Lorraine, Laboratoire d’Ingénierie des Biomolécules (LIBio), 2 avenue de la Forêt de Haye, TSA40602-F-54518 Vandœuvre-lès-Nancy, France c Hydrobiology Department, National Research Center, Dokki 12622, Cairo, Egypt d Zoology department, Faculty of Women for Arts, Science & Education, Ain shams University, Cairo, Egypt e Cell Biology Dept., National Research Center, Dokki 12622, Cairo, Egypt
Abstract
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Tel: 202 2283 1943, fax: 202 3337 0931
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This study aimed to prepare and characterize enzymatic modified chitosan nanoparticles (CSNPs) with gallic acid (GA) or octyl gallate (OG) to optimize its potential in human application and to evaluate their protective role against ochrtoxin A (OTA) toxicity in catfish. The modified CSNPs have average size around 90 nm with positive charge and high scavenging activity especially GA-CSNPs. In the in vivo study, catfish were divided into 8 groups and treated for 3 weeks as follow: the control group, OTA-treated group (1 mg/ kg b.w.), the groups treated with CSNPs, GA-CSNPs or OG-CSNPs (280 mg/ kg b.w.) anole or in combination with OTA. Blood, liver and kidney samples were collected for different analyses. OTA induced a significant biochemical disturbances accompanied with oxidative stress in liver and kidney, histological changes and increase DNA fragmentation in the kidney. Co-treatment with OTA plus the different CSNPs resulted in a significant improvement in all tested parameters and histological picture of the kidney. This improvement was more pronounced in the group treated with GA-CSNPs. It could be concluded that grafting of GA or its ester improved the properties of CSNPs. Moreover, GA-CSNPs showed strong scavenging properties than OG-CSNPs due to the blocking of carboxyl groups responsible of the scavenging activity in OG. Keywords Ochratoxin A; chitosan nanoparticles; gallic acid; octyl gallate; catfish; nephrotoxicity
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1. Introduction Ochratoxin-A (OTA) is a mycotoxin produced as secondary metabolites of fungal species such as Penicillium and Asperigillus (Khalesi and Khatib, 2011; Cremer et al., 2011). OTA can be found in cereals, coffee, dried fruits, juices, nuts, grape, breast milk, human/animal blood, tissues and different animal organs (Njobeh et al., 2010, Reddy and Bhoola, 2010) and other traditional food worldwide (Ahn et al., 2016) that can cause several health problems. Human exposure to OTA is of great concern and the European Union has set a maximum limit at 5 µg/kg in raw cereals and roasted coffee and 3 µg/kg in all products derived from cereals (EC, 2005). The International Agency for Research on Cancer (IARC, 1993) has classified OTA in Group 2B which mean possibly carcinogenic agent. OTA is hepatonephrotoxic, teratogenic and immunosuppressive agent that has been implicated as an endocrine disruptor as well as a contributor to increased cancer risk (Abdel-Wahhab et al., 1999, 2005, 2008). The major target organ of OTA toxicity is the kidney and human exposure to OTA is connected to the development of Balkan endemic nephropathy and urinary bladder tumours (Miletiĉ-Medved et al., 2005; Doricakova and Vrzal, 2015). Moreover, OTA was found in the patients with chronic interstitial nephropathy in Tunisia in the range of 1.8-65 ng/ ml blood however; it was found in the range of 1.7-8.5 ng/ml blood in the healthy group (Zaied et al., 2011). OTA is capable of disturbing cellular physiology in several ways however; its principal effects are involved in phenylalanine metabolism and inhibiting protein synthesis by competing with phenylalanine in the reaction catalysed by phenylalanyl-t-RNA synthetase (Creppy et al., 1985; Abdel-Wahhab et al., 2005, 2008). Moreover, it is an inducer FPG-sensitive sites in kidney cell DNA (Laura-Ana et al., 2015). Chitosan (CS) is a natural polysaccharide corresponding to the second most naturally abundant polysaccharide, after cellulose. Despite the large applicability of CS, its utilization is limited due to its poor solubility with neutral pH, low antioxidant poor workability and its physical properties that are rigid and brittle a direct result of the strong intra- and inter-molecular hydrogen bonding (Holmes et al., 2011). For a breakthrough in utilization, the modification of CS could be a key point to allow the introducing of desired properties and increase its potential applications through various types of side chains. Enzymatic or chemical modification of CS were carried out thanks to its reactive groups that are the free amino groups on deacetylated units at C2 and the hydroxyl groups at C3 and C6 functions. Due to safety and environmental concerns, enzymatic modifications were investigated as an attractive alternative to toxic and non-specific chemical approaches. Recently, additional groups such as phenolic compounds were enzymatically introduced onto chitosan in order to improve its antioxidant activities and the functional properties (Bozic et al., 2012a,b; Aljawish et al., 2014). Moreover, as a solution for the water-insolubility property of CS, nanoparticle formulation provides basis for enhancing oral bioavailability and therapeutic efficacy of CS and other drugs that are poorly soluble (Jia, 2005). Chitosan nanoparticles (CSNPs) exhibit more superior activities than CS and have been reported to have immune-enhancing effect, antimicrobial and anticancer activity than those of CS. Furthermore, nanoparticles have larger surface area compared to large particles which produces more dissolution pressure with a corresponding increase in saturation solubility (Müller and Böhm, 1998). The aims of the current study were to prepare and characterize enzymatic modified CNPs with Gallic acid (GA) and its ester (octyl gallate, OG) to optimize its potential in human application and to evaluate their protective role against OTA toxicity in catfish as a sensitive animal model.
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1. Material and methods 2.1. Materials, Chemical and kits Chitosan from crab shells with purity about 98.5-99 %, deacetylation degree 89% and 110-120 kDa, tyrosinase (from mushroom), gallic acid (GA) and octyl gallate (OG) with purity of 99 % were purchased from Sigma-Aldrich (France). Ochratoxin A (OTA) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Kits for total protein (TP), albumin, creatinine, uric acid creatine kinase (CK) alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) and total antioxidant capacity (TAC) were obtained from Eagle Diagnostics (Dallas, TX, USA). Other chemicals were of the highest purity commercially available.
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2.2. Preparation of modified chitosan Gallic acid (GA) and its ester octyl gallate (OG) were enzymatically grafted onto chitosan (CS) according to the method described by Sousa et al. (2009) with some modifications. One hundred mM phosphate buffer (pH 7) containing 1 g of CS fibers was mixed with tyrosinase (100 U/ mg) and the phenolic compounds (100 mM) in methanol and the oxidation reactions were carried out at 20 °C in a magnetic stirred reactor under atmospheric conditions for 24h. The reactions were performed in large open beaker under continuous stirring to prevent oxygen limitations. The enzymatic reaction was stopped after 24 h by filtering the reaction medium with Ministar-RC membranes (Sartorius, porosity 0.2 µm) under vacuum. CS was extensively washed with abundant amount of water, methanol then acetone to remove any trace of unbound phenols. This washing procedure was run until no color was detected in the washing solution and the modified CS was stocked in the desiccator until use for characterization and the in vivo study (Fig. 1).
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Preparation of chitosan nanoparticles (CSNPs) Twenty mg of each of CS or its two modifiers (GA-CS and OG-CS) were dissolved in 40 ml of acetic acid (2.0%, v/v). A 20 ml of sodium tripolyphosphate (0.75 mg/ ml) was dropped slowly with stirring. CSNPs as a suspension were stirred at 1000-2000 rpm for 10 min, the supernatant was discarded and powdered CSNPs were obtained by freeze-drying and the samples were prepared using KBr to form pellets (Tang et al., 2007). 2.4. Characterization of chitosan nanoparticles (CSNPs) FTIR spectra of CS and its nanoparticles (CSNPs, GA-CSNPs and OG-CSNPs) were determined using an infrared spectrometer (FTIR) (Thermo Fisher Scientific Inc., Nico-let iS10, USA) and the electrolyte solution of CS and its nanoparticles were obtained using a perkin-Elmer UV-Vis Lambda 35 spectrophotometer scanning 200-800 nm. The particles size and zeta potential of CS and modified CSNPs were carried out using A Zetasizer nano ZS (Malvern Instruments Ltd., Worcestershire, UK) at pH 3 and a concentration of 1 mg/ ml in aqueous acetic acid 1% (v/v). All measurements were carried out in triplicate. Grafted phenol content onto CS was determined using Folin-Ciocalteau reagent according to the method described by Singleton et al. (1999). The phenolic content was calculated by the following equation established from gallic acid as a standard: y= 0.010 x (R2 0.9997) 3 Page 3 of 21
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where x is the absorption value at 660 nm and y is the concentration of grafted phenol onto CS (mg phenol/ mg CS). The radical activity was determined according to the method described by Tang et al. (2013). The ABTS+ solution was done by the reaction between 7 mM ABTS and 2.45 mM potassium persulphate in water and the mixture was stand in the dark at room temperature for 12-16 h. The ABTS+ solution was diluted with ethanol to obtain an absorbance of 0.700 ± 0.025 at 734 nm at room temperature before use. An aliquot of each nanoparticles-containing medium (0.1 ml) was mixed with 3.9 ml of diluted ABTS+ solution. The mixture was left stirring in the dark at room temperature for 1h. The absorbance was measured at 734 nm at room temperature using a perkinElmer UV-Vis Lambda 35 spectrophotometer against ethanol as a blank without CSNPs. The scavenging ability was calculated by the following equation: ABTS Scavenging ability (%) = (1- Ab sample/Ab control) x 100 Where Abcontrol is the initial concentration of the ABTS-+ and Absample is the absorbance of the remaining concentration of ABTS+ in the presence of CSNPs. The data were recorded as the averages of triplicate experiments. 2.5. Experimental catfish Eighty apparently healthy catfish (Clarias gariepinus) with an average body weight of 200 ± 5 g irrespective to sex were purchased from El-Nobarya Fish Farm (El-Nobarya, Egypt) and transported alive in a large plastic water container supplied with battery aerators as a source of air. During transportation, catfish were treated with lidocaine, CHNO (5 mg/ L), to reduce stress. Catfish were fed twice daily on standard fish pelleted diet (32% protein ration) at rate of 3% of the fish body weight at the aquarium House, Hydrobiology Department, Veterinary Research Division, National Research Center (Dokki, Cairo, Egypt). The aquaria water was changed daily to avoid metabolite accumulations in plastic aquaria (static system). After an acclimation period of two weeks, the catfish were divided into eight experimental groups (n= 10) and each group was placed in a fully prepared aquarium containing dechlorinated tap water, the average water temperature was 20 ± 37░°C, the range of pH was 7.17-8.19 and the oxygen dissolved was 6.9 mg/ L. The experiments were carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans and EU Directive 2010/63/EU for animal experiments and the protocol was approved by National Research Center Review Committee for the use of Human or Animal Subjects. 2.6. Experimental design Catfish within different treatment groups were treated for 3 weeks according to their respective treatment as follows: group 1, untreated control; group 2, orally treated with OTA (1 mg/ kg b.w.); groups 3, 4, 5, orally treated with CSNPs, GA-CSNPs or OG-CSNPs (280 mg/ kg b.w.) respectively; groups 6, 7, 8, orally treated with OTA plus CSNPs, GA-CSNPs or OG-CSNPs respectively. After the end of the experiment period, blood samples were collected from the caudal vein of all catfish within different groups with a 5 cc syringe without anesthesia to avoid confounding effects (de Miranda Cabral Gontijo et al., 2003). Blood samples were centrifuged for 10 min at 3500 rpm and 4 °C. Sera were collected and stored at -20 °C until used for the determination of ALT, AST, CK, total protein, albumin, uric acid and createnine according to the kits instructions. Globulin was calculated by subtracting the total albumin from total protein and albumin/ globulin ratio was calculated. After the collection of blood samples, catfishes were sacrificed (by removing from water) and samples of liver and kidney from each catfish within different groups were immediately homogenized in ice-cold buffer containing 50 mM tris-HCl and 300 mM sucrose (pH 7.4) to give 10% w/v homogenate (Lin et al., 1998). The homogenate 4 Page 4 of 21
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was centrifuged at 3000 rpm at 0°C for 10 minutes and the supernatant was stored at -20 °C to the second day until analysis of malondialdehyde (MDA) and total antioxidant capacity levels. Other samples of the kidney from each catfish in different groups were fixed in 10% formal saline solution then the samples were processed by conventional method, sectioned at 5 um and stained with Heamatoxylin and Eosin (Robert, 2001). 2.7. DNA Fragmentation Analysis Kidney samples were collected and were lysed in 0.5 ml of lysis buffer containing 10 mM trisHCl (pH 8), 1 mM EDTA, 0.2 % triton X-100. The content was centrifuged at 10,000 rpm for 15 min at 4°C to separate intact chromatin in the pellet from fragmented/damaged DNA in the supernatant. The pellets were resuspended in 0.5N perchloric acid (P) and 5.5N perchloric acid was added to the supernatants (S) to obtain a final concentration of 6.0 N. Samples were incubated at 90░°C for 20 min and centrifuged at 10,000 rpm for 10 min to remove proteins. Subsequently, to each sample 160 ml of diphenylamine (DPA) solution [150 mg DPA in 10 ml glacial acetic acid, 150 ml of sulfuric acid and 50 ml acetaldehyde (16 mg/ ml)] was added and incubated at room temperature for 24 h (Gibb et al., 1997). The absorbance was measured at 600 nm using a UV doublebeam spectrophotometer (Shimdazu 160 A; Shimadzu Co., Japan). The proportion of fragmented DNA was calculated from absorbance reading at 600 nm using the formula: % Fragmented DNA= [OD(S)/ OD(S) + OD(P)] X 100 Where: OD(S) optical density of supernatant OD(P) optical density of pellet
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2.8. Agarose Gel Electrophoresis for DNA Fragmentation The analysis of DNA fragmentation was carried out by agarose gel electrophoresis according to the method described by Yokozawa and Dong (2001). DNA samples were electrophoresed on 1.4% agarose gel using Tris-borate- EDTA (TBE) buffer by the submarine gel electrophoresis machine and DNA was visualized and photographed by Ultra Vilot transilluminator. 2.9. Statistical analysis All data were statistically analyzed using the General Linear Models Procedure of the Statistical Analysis System. The significance of the differences among treatment groups was determined by Waller-Duncan k-ratio. All statements of significance were based on probability of P ≤ 0.05. 3. Results 3.1. Characterization of CS and CSNPs The FT-IR spectra of the CS and its CSNPs revealed that a new peak indicating the appearance of P=O stretching at 1256/ cm (Figs. 2a,b). Moreover, the intensity of (NH2) band at 1628/ cm found in CS was decreased dramatically and a new sorption band at 1550/ cm appeared. The GA-CNPs or OG-CNPs showed an absorption decrease at 1320 and 1380/ cm (attributed to the NH-bending of the glucosamine unit) and at 1420/ cm (the symmetric -NH3+ bending region) when compared with CSNPs. Moreover, the FT-IR spectra of GA-CNPs or OG-CNPs showed the formation of a new band at 1465/ cm. The UV-Vis spectra of CS and its NPs are presented in Fig. (2c). A significant changes in the UV/Vis spectra of CSNPs were observed at the absorbance of the range 350-400 nm and 270280 nm compared to CS. Moreover, GA-CSNPs or OG-CSNPs were absorbed in the visibleregion (colored CS) while the CS did not show any absorbance in this region (colorless CS).
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The phenolic content of the modified CSNPs using gallic acid as a standard revealed that the quantity of GA-oxidation products grafted onto CSNPs was 44.2 ± 3.0 µg/ mg which was about 2 times higher than OG-oxidation products which recorded 25.5 ± 2.5 µg/ mg. The results also revealed that the average particle size of CSNPs and GA or OG modified CSNPs were around 90 nm. CSNPs showed a positive zeta potential as CS is positively charged due to free amino groups and the recorded zeta potential was 30.6 ± 2.5 mV for CSNPs and it was decreased with the grafting of tyrosinase-catalyzed oxidation products to record 12.9 ± 1.4 mV for GA-CSNPs and 21.6 ± 3.2 mV for OG-CSNPs. The decrease in zeta potential for GA-CSNPs solution was higher than for OG-CSNPs solution when compared with the zeta potential of CSNPs. Moreover, the EC50 values of ABTS+ scavenging for CSNPs was 0.35 ± 0.30 mg/ ml, 0.07 ± 0.15 mg/ ml for GACSNPs and 0.20 ± 0.2 mg/ ml for OG-CSNPs suggesting that the scavenging activity of GACSNPs was nearly three times higher than OG-CSNPs. 3.2. In vivo results The in vivo results of the current study revealed that treatment with OTA resulted in a significant increase in serum ALT, AST, uric acid, createnine and CK accompanied with a significant decrease in TP, albumin, globulin and A/G ratio (Table 1). The results also showed that treatment with CSNPs, OG-CSNPs or GA-CSNPs alone did not induce any significant effects on these biochemical parameters except ALT in the group treated with OG-CSNPs which was found to be decreased significantly compared to the control group. However, catfish in these groups showed a significant increase in albumin, globulin and A/G ratio. Treatment with CSNPs, OGCSNPs or GA-CSNPs succeeded to normalized ALT and AST in the catfish treated with OTA however; treatment with OTA plus CSNPs succeeded to normalize TP, albumin, globulin and A/G ratio and induced a significant improvement in creatinine, uric acid and CK although these parameters were still higher than the control group. Catfish received OTA plus OG-CSNPs showed a significant improvement in TP, albumin, globulin, A/G ratio, CK and uric acid and restored createnine level compared to the control catfish (Table 1). On the other hand, treatment of catfish with OTA plus GA-CSNPs succeeded to normalize all the tested parameters except createnine which was still higher compared to the control level. The effect of different treatments on MDA as an end product of lipid peroxidation and the total antioxidant capacity (TAC) in liver and kidney of catfish is presented in Table (2). These results indicated that treatment with OTA alone resulted in a significant increase in MDA accompanied with a significant decrease in TAC in liver and kidney. Treatment with CSNPs, OG-CSNPs or GA-CSNPs alone resulted in a significant decrease in MDA in the liver but did not affect MDA in kidney tissue. However, these treatments induced a significant increase in TAC in liver and kidney (Table 2). The combined treatment with OTA plus CSNPs, OG-CSNPs or GA-CSNPs succeeded to induce a significant improvement in MDA and TAC level in the liver and kidney since MDA in the liver was in the normal range of the control in the groups treated with OTA plus CSNPs or OG-CSNPs and in the kidney in the group received OTA plus CNPs. Furthermore, MDA in the liver was significantly decreased compared to the control in the group treated with OTA plus GA-CSNPs and in the kidney of the group treated with OTA plus OGCSNPs or GA-CSNPs. It is f interest to mention that treatment with CSNPs, OG-CSNPs or GACSNPs succeeded to normalize TAC in the groups received OTA altough the levels of TAC in these groups were still higher than the control level (Table 2). The effect of OTA treatment alone or in combination with CSNPs, OG-CSNPs or GA-CSNPs on genomic DNA fragmentation in kidney tissue was determined by gel electrophoresis to access 6 Page 6 of 21
4. Discussion
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apoptotic cell death. The results presented in Table (3) and Fig. (3) revealed that treatment with OTA resulted in a significant increase in DNA fragmentation (20.93%) compared to the control group (9.39%). DNA fragmentation was insignificantly decreased in the group treated with CSNPs alone (7.67%) however it was decreased significantly in those treated with OG-CSNPs or GA-CSNPs which recorded 6.90 and 6.20% respectively. The combined treatment with OTA plus CSNPs, OG-CSNPs or GA-CSNPs succeeded to induce a significant decrease in the elevation rate of DNA damage resulted from OTA. Moreover, the improvement in DNA fragmentation was more pronounced in the group received GA-CSNPs. 3.3. The histological examination The results of semiquantitative scoring for histological examinations of kidney sections of catfish within different treatment groups are summarized in Table (4). The microscopic examination of the kidney sections in the control group showed normal renal structures (Fig. 4a). The kidney sections in the catfish treated with OTA showed congestion accompanied with hyperplasia in the wall of renal blood vessels (Fig. 4b). The same group showed severe necrosis, degeneration, hyalinization in the tubular epithelial and interstitial tissues and focal aggregation of melanomacrophage cells (Fig. 4c) associated with severe degenerative and necrotic changes in the tubular epithelium and interstitial tissues and focal aggregation of melanomacrophage cells (Fig. 4d). The examination of the kidney sections in the catfish in the groups treated with CNPs, OG-CSNPs or GA-CSNPs showed normal renal structure with multifocal melanomacrophage cells aggregation (Fig. 4e). However, catfish treated with OTA plus CSNPs showed vacuolization of tubular epithelium in some tubules with slight congestion and multifocal melanomacrophages infiltration (Fig. 4f). On the other hand, the kidney sections of catfish treated with OTA plus GA-CSNPs showed normal tubules with slight peritubular and peri glomerular edema (Fig. 4g). However, the kidney of catfish treated with OTA plus CSNPs, OGCSNPs or GA-CSNPs showed normal structure with apparently normal pathomorphological pictures (Fig. 4e). Furthermore, catfish treated with OTA plus GA-CSNPs showing normal renal structure (Fig. 4h).
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4.1. Characterization of chitosan nanoparticles (CSNPs) The results of FT-IR indicated by the presence of a new peak at 1256/ cm and the decreasing of intensity at 1628/ cm suggesting that anionic phosphate groups of sodium polyphosphate interacted with the free amino groups of CS (Antoniou et al., 2015; Tang et al., 2015; Qi et al., 2014). The difference between the FT-IR spectra of CSNPs and the modified GA-CSNPs or OGCSNPs was the presence of a new peak at 1420/ cm and the decreasing of band at 1320 and 1380/ cm which confirmed the presence of phenol groups onto modified CSNPs (Sousa et al., 2009; Aljawish et al., 2012). The results of UV-Vis spectra showed an increase of the absorbance in the range 350-400 nm which is probably due to the presence of sodium tripolyphosphate onto CS (Fahima et al., 2013). In addition, the absorbance at 270-280 nm is due to the presence of the tyrosinase-catalyzed oxidation products of GA and OG (Bozic et al., 2012) and the presence of sodium tripolyphosphate (Fahima et al., 2013) onto modified CS. Moreover, the absorption of GACSNPs or OG-CSNPs in the visible-region gave a strong evidence of the possibility of the presence of oxidation products onto CSNPs. The decrease of zeta potential of GA-CSNPs or OG-CSNPs reported herein is mainly due to the grafting of GA or OG products onto CS. This decrease in zeta potential for GA-CSNPs may be due to the high quantity of GA-products grafted onto CS in comparison with the OG7 Page 7 of 21
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products. These results are in accordance with the previous study indicating that the grafting of oxidation products of FA or EF onto chitosan decreased zeta potential (Aljawish et al., 2012). Moreover, the EC50 values in scavenging the ABTS+ of GA-CSNPs or OG-CSNPs were higher than that of CNPs which may be due to the presence of phenolic compounds onto CSNPs. The scavenging activity of GA-CSNPs was nearly three times higher than OG-CSNPs which may be due to the high quantity of GA-products grafted onto CS compared to the OG-products. In addition, the scavenging activity of GA-CSNPs was higher than that of OG-CSNPs suggesting the blocking of carboxyl groups responsible of the scavenging activity in OG. 4.2. The in vivo study In the in vivo study, the protective effect of CNPs and the modified OG-CNPs or GA-CNPs against OTA toxicity in catfish as a sensitive animal model was evaluated. The selected dose of OTA was literature based (Encarnação, 2006) however; the selected dose of CNPs was based on our previous work (Abdel-Aziem et al., 2011). The current results confirm and extend previous data which has demonstrated that OTA induces oxidative stress and impair liver and kidney function. The decreased level of TP, albumin and globulin may be due to the inhibition of tRNAsynthetase resulting in a reduction of protein synthesis (Creppy et al., 1995) and/or protein catabolism accompanied by kidney dysfunction (Abdel-Wahhab et al., 1999, 2005). The increased level of CK in the OTA-treated catfish is suggesting that OTA-induced myocardial infraction and cardiac or voluntary muscles injury and the presence of necrosis or acute atrophy of cardiac muscles (Apple, 1989). In the current study, the MDA concentration in liver and kidney tissues was found to be increased in OTA-treated catfish indicated that OTA induced LPO, oxidative stress and cellular damage (Vaca et al., 1998). It is well documented that OTA enhanced lipid peroxidation in experimental animals which directly results from free radicalmediated toxicity (Hohler, 1998; Abdel-Wahhab et al., 2005) and may contribute at least in part to renal toxicity and carcinogenicity in rats during long-term exposure (Gautier et al., 2001). In this study, OTA was found to decrease TAC which may be explained by the conjugation of GPX with OTA or its metabolites (Abdel-Wahhab et al., 2008). Several reports suggested that OTA appears to produce several effects in the cell such as the increase of the permeability of the cell to Ca2+. The enhanced cellular concentration of Ca2+ and the presence of the prooxidant OTA uncouple oxidative phosphorylation resulting in an increased leakage of electrons from the respiratory chain which generates O2− and hence H2O2. Lack of an adequate supply of NAD(P)H and GSH to permit H2O2 consumption by the GSH dependent glutathion peroxidase and NAD(P)H dependent glutathion reductase (Hohler et al., 1997; Abdel-Wahhab et al., 2008). Moreover the increased concentration of free iron within the cell stimulates the production of OH via the Fenton like reaction due to mobilization of Fe2+ by Ca2+. This results in further cell damage and may be one of the mechanisms that OTA exerts its toxic effects (Abdel-Wahhab et al., 2005, 2008). The current study also revealed that OTA increased the percentage of DNA fragmentation indicated that OTA generate free radicals and to enhance lipid peroxidation which linked to the genotoxicity expressed by DNA adduct formation and to the disturbance of calcium homeostasis due to an impairment of the endoplasmic reticulum membrane (Pfohl-Leszkowicz et al., 1998). These results confirm that oxidative stress is an important factor in OTA genotoxicity (Gautier et al., 2001; Baldi et al., 2004). Moreover, previous reports indicated that the renal toxicity as well as the DNA damage are attributable to oxidative stress (Marin-Kuan et al., 2006). Further, DNAadducts formed through a quinone/ hydroquinone redoxcouple generated by OTA oxidation have been observed (Pfohl-Leszkowicz and Castegnaro, 2005). In this concern, Schaaf et al. (2002) 8 Page 8 of 21
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observed an elevation of ROS levels, an increase in oxidative DNA damage and depletion of GSH levels in rat proximal tubular cells and in LLC-PK1 cells and concluded that OTA-induced ROS production clearly preceded the loss of cell viability and DNA damage. Similar oxidative damage to DNA was observed using the comet assay and it was detected in target (kidney) and non-target (liver) tissues in vivo in male rats (Abdel-Wahhab et al., 2008). Moreover, OTA was suggested to alter a battery of genes in the kidney which regulated by the transcription factor Nrf2 (Nuclear factor erythroid 2-related factor 2) and involved in antioxidant defense and detoxification (Marin-Kuan et al., 2006). The histopathological results using different magnifications revealed that OTA induced significant pathological changes in the kidney tissues typical to those reported in the literature (Mansour et al., 2011). Catfish treated with OTA also showed marked degenerative and necrotic changes in the tubular epithelium and interstitial tissues and focal aggregation of melanomacrophage cells. Similar results were reported by Manning et al. (2003) in catfish fed OTA-contaminated diet (2.00 - 8.00 mg OTA/ kg diet). CS is a non-toxic and biodegradable biopolymer that exists as a cationic polyelectrolyte in acidic aqueous solution, precipitates at pH > 6. Due to the active functions of CS (amino/hydroxyl groups), it can form a complex with metals and organic contaminants from foodstuffs (Anlı et al., 2011). The current results revealed that treatment with CNPs, OG-CNPs or GA-CNP induced a significant improvement in all the tested parameters and the histological picture of the kidney. These findings are in harmony with the previous reports who suggested a protective role of CNPs against H2O2-induced RAW-264.7 cell injury through restoring the activities of endogenous antioxidants (SOD, GPx and CAT) and the enhancement of their gene expression (Wen et al., 2013). Several reports suggested that CNPs have immune-enhancing effect (Wen et al., 2011), anticancer activity (Qi et al., 2007) and antimutagenic activity (Yu et al., 2007) than those of CS. However, the other biological activities such as the antioxidant activity of CNPs are still unknown and have received less attention. Although no available report describe the protective role of CNPs against OTA-induced hepatonephrotoxicity and oxidative stress, the protective effect of CS has been documented in several reports; for instance, Jeon et al. (2003) investigated the antioxidant effect of CS on chronic CCl4- induced hepatic injury in rats and showed that CS has strong antioxidant properties, decrease the production of TBARS and increase antioxidant enzyme (catalase and SOD) activities. Subhapradha et al. (2014) reported that β-CS has a protective effect against CCl4-induced oxidative stress in rats and β-CS normalized the oxidative stress markers and plasma AST and ALT activity suggesting that β-CS may stabilize the cell membrane and prevent the leakage of intracellular enzymes into the blood stream. The overall protective effect of CS is probably due to a counteraction of free radicals by its antioxidant nature and/or to its ability to inhibit lipid accumulation by its antilipidemic property (Ramasamy and Shanmugam, 2014). In the same concern, Santhosh et al. (2006) reported that treatment with CS may prevent antitubercular drugs-induced hepatotoxicity in rats. Furthermore, CS was effective against TCDD-induced hepatotoxicity (El-Fattah, 2013) and was proved to protect liver against oxidative damage induced by radiotherapy (Mohamed, 2011). The current results revealed that enzymatic modification of CNPs enhanced its antioxidant activity; GA-CNPs showed a potential antioxidant activity followed by OG-CNPs then CNPs. These results supported the in vitro results of the current study which showed that the scavenging of ABTS+ for GA-CNPs is three time higher compared to OG-CNPs. Taken together, the in vitro and the in vivo results suggested that the modification of CS and the preparation of CNPs 9 Page 9 of 21
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enhance its antioxidant activity and scavenging properties which may be due to the high quantity of GA-products grafted onto CNPs compared to OG as well as the blocking of carboxyl groups responsible of the scavenging activity in OG. According to Ji et al. (2006), GA is attractive for conjugating onto CS for a novel green antioxidant because of (i) the high reducing potential and low O-H bond dissociation enthalpy of the tri-hydroxyl groups on the benzene ring; (ii) the possibility that the bulky group of the benzene ring of GA obstructs the inter- and intramolecular hydrogen bond network of CS; (iii) the multi-functional hydrophilicity based on the hydroxyl and carboxyl groups; (iv) the carboxylic acid group for conjugation with CS; and (v) it being a natural product. Indeed, CS is soluble in acid conditions which render this polymer inapplicable in the neutral aqueous or in the non derivatized form. Consequently, it can be expected that the CS-conjugated GA may show antioxidant activity and water-solubility. In general, incorporating GA onto CS performs multi-functional and has unique properties; i.e. (i) offers preventive antioxidation activity due to the metal ion deactivator of the CS, (ii) is a chain breaking antioxidant due to the H-atom donating ability of the trihydroxyl group of GA, (iii) has watersolubility due to the bulky hydrophilic group of GA, and (iv) has a synergistic effect in the HOscavenging activity under the Fenton reaction (Pasanphan and Chirachanchai, 2008). Moreover, it was suggested that CS form a complex with OTA leading to the block of active site of this molecule and the CS adsorbent was used as alternative fining agents to reduce OTA in red wine (Quintela et al., 2012) or to detect of OTA (Kaushik et al., 2008). Consequently, the enzymatic grafting of GA or OG improved the capacity of CSNPs for the block of OTA and prevents its toxic effect. These results are in agreement with Xie et al. (2014) who suggested that the antioxidant capacity of GA-CS was much higher than that of the plain CS as examined by several assays (DPPH, superoxide, ABTS radicals scavenging activities, reducing power, chelating power, inhibition of lipid peroxidation, ferric reducing antioxidant potential and βcarotene-linoleic). Particularly, GA-g-CS showed higher antioxidant activity than GA in βcarotene-linoleic acid assay. Conclusion The grafting of GA onto CSNPs improved its solubility, enhanced its radical scavenging properties and antioxidant activity. OTA induced oxidative stress on liver and kidney as indicated by the disturbances in the biochemical parameters tested and the histological changes in the kidney. OTA also has cytogenetic effects as indicated by the increased percentage of DNA fragmentation in the kidney. These effects are mainly due to its oxidative stress and the generation of free radicals. CSNPs or its enzymatic modified derivatives OG-CSNPs and GACSNPs succeeded to counteract these toxic effects. Moreover, GA-CSNPs was found to be the superior due to its high content of phenolic compounds. Taken together, the in vitro and in vivo results of the current study, the grafting of GA onto CSNPs is promise alternative to improve the problems associated with chitosan solubility and increase its antioxidant activity. 1. Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by the National Research Center, Dokki, Cairo, Egypt, Project # 10070112.
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Fig. 1 Schematic diagram illustrate the experimental design and the outcome. Fig. 2 (A) FT-IR spectra of CS; (B) FT-IR spectra of CS (a), OG-CSNPs (b) and GA-CSNPs (c) and (C) UV-Vis spectra of CS (x) and its CSNPs: CSNPs (▪), OG-CSNPs (▲) and GA-CSNPs (●). Fig. 3 DNA fragmentation analysis: 1.5% of agarose gel electrophoresis of DNA samples extracted from kidney tissues of different groups of catfish induced by OTA and prevention of CSNPs. Lane M: 100-bp ladder marker; Lanes 1, 2: Control; Lane 3, 4: CSNPs; Lane 5,6: OGCSNPs; Lane 7, 8: GA-CSNPs; Lane 9, 10: OTA; Lanes 11, 12: OTA + CSNPs; Lane 13, 14: OTA + OG-CSNPs and Lane 15, 16: OTA + GA-CSNPs. Fig. 4 Photomicrograph of kidney section of: (a) control group showing normal renal structure, (b) catfish treated with OTA showing congestion accompanied with hyperplasia in the wall of renal blood vessels and Focal aggregation of melanomacrophages, (c) catfish of the same group (OTA) showing degeneration, necrosis, hyalinization in the tubular epithelial, congestion, hyperplasia in the wall of renal blood vessels, multifocal melanomacrophage cells aggregation, (d) OTA-treated group also showing severe degenerative and necrotic changes in the tubular epithelium and interstitial tissues and focal aggregation of melanomacrophage cells, (e) catfish treated with CSNPs, OG-CSNPs or GA-CSNPs showing normal renal structure with multifocal melanomacrophage cells aggregation, (f) catfish treated with OTA plus CSNPs showing vaculaization of tubular epithelium in some tubules with slight congestion and multifocal melanomacrophages infiltration, (g) catfish treated with OTA plus OG-CSNPs showing normal tubules with slight peritubular and peri glomerular edema and (h) catfish treated with OTA plus GA-CSNPs showing normal renal tissues
Table 1 Effect of CSNPs and its derivatives on the biochemical parameters in catfish treated with OTA Control OTA CSNPs OGGAOTA + OTA + Groups CSNPs CSNPs CSNPs OGParameters CSNPs
OTA + GACSNPs
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48.24 ± 1.57a 31.67 ± 2.34a 4.89 ± 0.23a 1.50 ± 0.17a 2.97 ± 0.42a 0.53 ± 0.12a 1.62 ± 0.3a 2.78 ± 0.19c 0.28 ± 0.02a
M
an
us
cr
ip t
ALT 49.06 ± 78.58 ± 49.9 ± 43.78 ± 51.2 ± 51.42 ± 47.3 ± a b a c a a (U/L) 2.0 1.06 2.41 3.37 2.24 2.48 1.39a AST (U/L) 31.68 ± 44.33 ± 34.7 ± 31.86 ± 31.37 ± 33.94 ± 30.85 ± a b a a a a 1.96 1.69 1.90 2.21 2.26 1.92 2.15a TP (g/dl) 4.32 ± 2.42 ± 4.72 ± 4.82 ± 4.62 ± 4.46 ± 3.14 ± a b a a a a 0.12 0.34 0.28 0.75 0.2 0.51c 0.17 Alb (g/dl) 1.56 ± 0.62 ± 2.08 ± 1.74 ± 1.70 ± 1.51 ± 0.95 ± 0.16a 0.16b 0.23c 0.28d 0.11d 0.21a 0.04d Glob 2.99 ± 1.55 ± 3.04 ± 3.8 ± 3.94 ± 2.32 ± 2.19 ± (g/dl) 0.34a 0.07b 0.26c 0.17d 0.58d 0.50a 0.22c A/G ratio 0.60 ± 0.38 ± 1.14 ± 0.72 ± 1.03 ± 0.61 ± 0.43 ± (%) 0.11a 0.11b 0.51c 0.25a 0.51d 0.24a 0.06e Uric acid 1.64 ± 2.27 ± 1.66 ± 1.78 ± 1.75 ± 1.98 ± 2.11 ± (mg/dl) 0.10a 0.10b 0.07a 0.07a 0.04a 0.18c 0.22d Creatinine 2.18 ± 4.18 ± 2.06 ± 2.26 ± 2.5 ± 3.08 ± 2.16 ± 0.20b 0.16a 0.20a 0.06a 0.19d 0.14a (mg/dl) 0.10a 0.56 ± 0.29 ± 0.30 ± 0.30 ± 0.48 ± 0.42 ± CK (U/L) 0.26 ± 0.01b 0.01a 0.02a 0.02a 0.02c 0.02c 0.01a Within each row, means superscript with different letters are significantly different (P ≤ 0.05)
Ac ce p
te
d
Table 2 The effect of different treatments on MDA and TAC in liver and kidney of catfish Parameters Groups MDA (nmol/g tissue) TAC (mM/L) Liver Kidney Liver Kidney a a Control 290.26 ± 63.96 ± 15.71 1.44 ± 0.07 1.26 ± 0.29a a 10.64 OTA 418.21 ± 107.43 ± 9.12b 0.94 ± 0.03b 0.59 ± 0.06b b 44.85 66.54 ± 9.04a CSNPs 277.18 ± 1.81 ± 0.10c 1.44 ± 0.01c c 11.18 OG-CSNPs 269.23 ± 9.39c 70.64 ± 3.45a 1.91 ± 0.27c 1.60 ± 0.12d GA-CSNPs 262.82 ± 4.44d 61.41 ± 8.01a 2.0 ± 0.07d 1.68 ± 0.02c a a d OTA + CSNPs 281.92 ± 6.61 66.15 ± 7.62 2.06 ± 0.02 1.53 ± 0.15d OTA +OG-CSNPs 283.33 ± 57.69 ± 7.77d 2.06 ± 0.03d 1.65 ± 0.05c a 12.25 OTA + GA-CNPs 275.64 ± 8.04c 56.03 ± 3.64d 2.17 ± 0.02d 1.77 ± 0.05c Within each row, means superscript with different letters are significantly different (P ≤ 0.05)
Table 3 Effect of CSNPs and its derivatives on the rate of DNA fragmentation in the kidney tissue of catfish treated with OTA
Treatment Control
% DNA fragmentation in kidney tissues 9.39 ± 0.3
% Change --
17 Page 17 of 21
20.93 ±0.3 7.67 ± 0.5 6.90 ± 0.9 6.20 ±0.6 14.79±0.8 12.40 ±0.9 11.3 ± 0.8
+ 11.54 - 1.72 - 2.49 - 3.19 +7.4 + 5.01 + 3.91
ip t
OTA CSNPs OG-CSNPs GA-CSNPs OTA + CSNPs OTA + OG-CSNPs OTA + GA-CSNPs
Table 4 Semiquantitative scoring for histopathology of the kidney OTA
-
+++ +++ +++ +++ +++ +++ +++
an
-Degeneration, necrosis, in the tubular epithelial -Contraction of glomeruli Disruption of haematopoietic tissues Peritubular and periglomerular edema Interstitial mononuclear infiltration Congestion Stromal fibrous connective tissue proliferation
Control
CSNPs
groups OG-SNPs
GA-CSNPs
-
-
-
us
lesions
cr
Within each row, means superscript with different letters are significantly different (P ≤ 0.05)
OTA + CSNPs ++ ++ + ++ ++ ++ +
Ac ce p
te
d
M
-none: +mild occurrence; ++moderate occurrence; +++severe occurrence
Fig. 1
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B
cr
ip t
A
M
an
us
C
Ac ce p
te
d
Fig. 2.
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Ac ce p
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d
M
an
Fig. 3.
us
cr
ip t
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
20 Page 20 of 21
ip t cr
b
a
(H & E X 200)
an
us
(H & E X 1000)
d
M
c
(H & E X 200)
Ac ce p
te
d
(H & E X 200)
f
e
(H & E X 200)
(H & E X 400)
h
g (H & E X 200)
(H & E X 1000)
Fig. 4.
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