Abstract
Carbofuran is a highly toxic carbamate insecticide due to its low environmental persistence which can reach the aquatic system threatening aquatic organisms especially fish. The present study was designed to evaluate the half lethal concentration (LC50)/96h of carbofuran in catfish clarias gariepinus and to investigate the ability of carbofuran to induce hepatonephrotoxic side effects in catfish and its possible attenuation with lycopene (LYC). Fish were distributed into six groups, each group containing 12 fish, and received the following treatments. Fish in Group I acted as control group. Fish in Group II administered corn oil orally for 4 weeks. Fish in Groups III administered LYC (18 mg/kg b.w.) orally for 4 weeks. Fish in Group IV exposed to 1/10 LC50 carbofuran for 4 weeks. Fish in Group V administered LYC (9 mg/kg b.w.) orally and exposed to 1/10 LC50 carbofuran for 4 weeks. Fish in Group VI administered LYC (18 mg/kg b.w.) orally and exposed to 1/10 LC50 carbofuran for 4 weeks. Carbofuran treated fish revealed changes in the behavioural aspects. A significant increase in serum biochemical parameters; glucose, cortisol, AST, ALT, cholesterol, urea, creatinine, as well as hepatic and renal MDA and SOD. At the same time, serum AchE, total protein, albumin, total lipids and tissue CAT, GSH and TAC levels were reduced. LYC was able to restore all the altered serum biochemical parameters in addition to hepatorenal malondialdehyde and antioxidant biomarkers in a dose-dependent manner. Therefore, it could be concluded that LYC administration is able to alleviate the damaging effects of carbofuran.
Keywords: Carbofuran; Oxidative stress; Clarias gariepinus; Biochemistry; Lycopene .
*Corresponding author, Tel. : +20222560630 Fax: +20224157804
E-mail address: heba.salah84@yahoo.com (Heba S. Hamed)
1. Introduction
Pesticides are one of the main categories of deleterious substances used worldwide for controlling of pests in agricultural and sand control of insect vectors of human disease [1]-[2], and have many impacts that causing serious damage to aquatic life including fishes that have economic importance [3]-[5]. Among them, Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) is a widely carbamate pesticide which used to control numerous soil-borne pests and nematodes found in corn, potatoes, graps, rice, alfalfa and other crops [6]. It has very toxic effects to invertebrates, birds and relatively mammalian toxicity [7]. In fish, carbofuran also showed deleterious effects and the LC50 values in different species of fish appear to be varied due to species sensitivity to it [8]-[9]. Carbofuran 96h LC50 to different freshwater fish vary from 88 to 1990 µg/L [10]., whereas many organisms like crustaceans and insects are very sensitive with 96 h LC50 of carbofuran varying from 1.6 to 500 µg/L [11]. In fish, oxidative stress could be carbofuran, leading to the generation of free radicals with an increase of reactive oxygen species (ROS) and variation in the antioxidant profile in many species of fish [12]-[13]. Mitochondria, an aerobic organism is able to generate ROS naturally through oxidative metabolism of mitochondrial respiration [14]; thus, may be produced during detoxification process of insecticides [15]. When, there is an imbalance between the cellular antioxidant enzyme activities and ROS production, the oxidative stress occurred when ROS could not be eliminated or neutralized by the antioxidant system [16]. Almroth et al [17] ascribed that lipid peroxidation is one of the oxidative damage which involved in pesticide-induced toxicity in fish [17]. Antioxidant enzymes such as catalase (CAT) , superoxide dismutase (SOD) are the most abundant antioxidant defenses in fish, where they constitute the first line of protection against oxidative stress. SOD catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen. Hydrogen peroxide is decomposed into water and oxygen by CAT enzyme [18]. Biochemical profiles of blood are good health indicators that provide obvious evidence about the internal environment of the organism [19-21] [4], and often used when fish physiology is applied to detect and recognize the toxicological effects of harmful substances [22]. Soufy et al [23] studied the effects of chronic exposure to carbofuran on mono sex tilapia cultivated in Egyptian fish farms for 8 weeks . Their results revealed biochemical variations in AST,ALT, cholinesterase activities, creatinine and total proteins . Also, Hernández-Moreno et al [24] determined the biochemical alterations of sea bass (Dicentrarchus labrax L.) exposed to carbofuran. Lycopene (LYC), a representative of carotenoids, is a red lipophilic pigment consisting of 11 conjugated double bonds. The most plentiful sources of LYC are tomatoes and its other processed products [25]-[26]. The most known and effective antioxidants in the carotenoid family is LYC [27]-[28], where it can prevent oxidative damage, toxicity and disease by quenching singlet oxygen and other free radicals. The African catfish, Clarias gariepinus is broadly cultivated in Egypt and represent an important economic source of protein . Therefore, the catfish C. gariepinus was selected for the present study due to its culture, maintenance in the laboratory is relatively easy and its economic importance. Therefore, the present study was undertaken to
a) determine LC50 of carbofuran.
b) evaluate the toxic effects of carbofuran on certain biochemical parameters
and oxidative stress parameters in different organs of C. gariepinus.
c) assess the protective role of lycopene against carbofuran toxicity in C. gariepinus.
2.Materials and methods
2.1. Chemicals
Carbofuran (98% purity), was purchased from Hebei Chinally International Trade Co., Hebei, China. Lycopene ((EC) No 1272/2008) was obtained from Sigma-Aldrich Chemical (St Louis, MO, USA). Biochemical kits of glucose, AchE and cortisol were purchased from Gamma Trade Co., (Cairo, Egypt). All other biochemical kits (AST, ALT, creatinine, urea , total protein, albumin, total lipids, cholesterol, MDA, SOD, CAT,GSH and TAC) were bought from Bio-Diagnostic Co., (Dokki, Cairo, Egypt).
2.2. Fish maintenance
A total number of 300 alive catfish C. gariepinus (average weight 250±50 g. and body length 33.5±2.00 cm) were brought from Abassa fish farm, El-Sharkya governorate, Egypt. Fish were kept in 100 liters well aerated fiberglass tanks and transported to the laboratory and potassium permanganate solution with (0.5% w/v) was added for 1 min to remove any dermal adherents in fish. Then, fish were adapted for 15 days in identical glass aquaria measuring (80×40×40 cm) having 80 liter of dechlorinated tap water and air pumps under laboratory circumstances (at temperature 26 ± 2 °C and natural photoperiod 12 h). The water parameters were as follows: pH 7.5±0.3, temperature 27±1 °C, dissolved oxygen 6.3±0.5 mg/l , alkalinity 120 mg/l and hardness 150 mg/l CaCO3. Fish were fed diet containing 32% protein and transferred to a fresh volume of water every 48 h to reduce impurities from metabolic wastes.
2.3. Experimental design
2.3.1. Determination 96 h LC50 of carbofuran
The half lethal concentration (LC50) of carbofuran was determined according to the method of Litchfield and Wilcoxon,1949 [29]. Briefly, five groups each of eight fish were exposed to different concentrations of carbofuran ( 0.23, 0.45, 0.90, 1.80 and 3.60 ppm.), plus the control group. The median lethal concentration (LC50) for 96 h. is the concentration which caused 50% mortality of carbofuran exposed fish.
2.3.2. Sublethal exposures
Fish were divided into six groups, each group containing 12 fish, and received the following treatments. Fish in Group I acted as control group. Fish in Group II administered corn oil orally for 4 weeks. Fish in Groups III administered LYC (18 mg/kg b.w.) orally for 4 weeks. Fish in Group IV exposed to 1/10 LC50 carbofuran for 4 weeks. Fish in Group V administered LYC (9 mg/kg b.w.) orally and exposed to 1/10 LC50 carbofuran for 4 weeks. Fish in Group VI administered LYC (18 mg/kg b.w.) orally and exposed to 1/10 LC50 carbofuran for 4 weeks. As well as three replicates of control and treated groups. To keep a constant concentration of pesticide, fish were imparted into a fresh solution of carbofuran prepared every 48 h.
2.3.3. Clinical investigation and postmortem examination
Fish were observed during the whole period of the experiment for clinical examination and postmortem injuries or deaths according to Amlacher,1970 [30].
2.3.4. Collection of blood samples
By the end of the experiment, six fish per group were collected and anesthetized with 0.02 % benzocaine solution. Blood samples were collected from the caudal vessels of fish and allowed to clot in clean dry centrifuge tubes at room temperature, then centrifuged at 3000 rpm., at 4 °C for 15 minutes and sera were detached for the determination of biochemical parameters.
2.3.5. Biochemical examination
Serum glucose was detected according to Trinder,1959 [31], cortisol was measured according the method described by Foster and Dunn, 1974 [32], AST and ALT were measured using a colorimetric method according to Reitman and Frankel, 1957 [33] and cholesterol was determined by the method of Allain et al. and Richmond [34]-[35]. Urea was estimated according to Coulombe and Favreau, 1963 [36] and creatinine according to Larsen, 1972 [37]. Serum AchE was measured according to Knedel and Böttger, 1967 [38], total protein was evaluated according to the method of Lowry et al., 1951 [39] and albumin according to Doumas et al., 1971 [40]. Total lipids was measured according to Frings and Dunn, 1970 [41].
2.3.6. Tissue MDA and antioxidant biomarkers:
Samples from liver and kidney tissues were homogenized in cold phosphate buffer saline (0.1M pH 7.4) using a Potter–Elvejhem glass/Teflon homogenizer.. Then, the homogenate was filtered and centrifuged for 10 min at 4 °C with velocity of 1600 rpm ; the supernatant was stored at −20 °C until analysis. MDA level was determined using the supernatant (20%) according to the method of Mihara and Uchiyama, 1978 [42]. The activity of SOD was detected by Nishikimi et al.1972 [43]. CAT enzyme was determined according to Aebi, 1984 [44]. GSH concentration was detected using the method of Beutler et al., 1963 [45] an total antioxidant capacity (TAC) was determined by Koracevic et al.,2001 [46].
2.3.6.Statistical analysis:
Data were statistically analyzed using one way "ANOVA", and Duncan's multiple range test to compare between means at P< 0.05 (SPSS, 2004), version 19.
3. Results
3.1. LC50
Results exhibited that LC50 /96 h of carbofuran in the catfish was 1.21 ppm (Table 1). To study the harmful effects of carbofuran in catfish. Fish were exposed to 1/10 LC50 (0.121 ppm) for 4 weeks.
3.2. Behavioral and clinical investigations
Fish exposed to carbofuran exhibited irregular swimming movements, swimming near the water surface, vertical and downward swimming patterns and hyper secretion of mucus. Seizures, convulsion and increase in opercular movement were also observed. Clinical signs were manifested by opening fish mouth for gasping (Fig.1a); inflammation of male genital papilla (Fig.1b) ; paleness of body surface (Fig.1c); congestion of internal organs with marked enlargement of the left lobe of liver (Fig.1d) ; severe bulge of the gall bladder, enlargement of liver and discoloration into yellowish (Fig.1e) and haemorrhage of spleen (Fig.1f) .
3.3. Biochemical Parameters
The current results revealed that fish treated with carbofuran showed marked increases in serum glucose, cortisol, liver marker enzymes (AST, ALT ) and cholesterol in comparison with the control group (Table 2). In the same way, serum renal analysis; urea and creatinine were significantly (P<0.05) elevated after carbofuran exposure, accompanied with a significant decrease in serum AchE, total protein , albumin and total lipids levels when compared with sera of the control ones (Table 2). Oral administration of LYC in the two tested doses ( 9 and 18 mg/kg b.w.) during carbofuran exposure significantly (P < 0.05) decreased the elevated hepatic and renal biochemical parameters as compared with carbofuran group (Group IV). The combined treatment with the low LYC dose ( 9 mg/kg b.w.) succeeded to normalize albumin and AchE activity to normal levels. However, the high LYC dose (18 mg/kg b.w.) succeeded to restore all the tested parameters to normal bounds (Table 2). Fish administrated LYC exhibited no significant difference in serum biomarkers compared to the control group.
3.4. Hepatic lipid peroxidation and Antioxidant enzymes
The data presented in Table (3) indicated that fish treated with carbofuran only revealed a significant (P<0.05) rise in the hepatic MDA and SOD levels and a marked (P<0.05) reduction in the hepatic antioxidant enzymes ( CAT, GSH and TAC) compared with the control group. The combined treatment of carbofuran with the two tested doses of LYC resulted in a siginificant enhancement in the hepatic oxidative stress biomarkers. This improvement was more obvious in the group exposed to carbofuran and administrated with the high LYC dose (18 mg/kg b.w.).
3.5.:Renal lipid peroxidation and Antioxidant enzymes
C. gariepinus exposed to 1/10 LC50 of carbofuran showed a significant (P< 0.05) increase in the renal MDA and SOD levels and a marked (P<0.05) decline in the renal antioxidant enzymes ( CAT, GSH and TAC) compared with the control fish. The combined treatment of carbofuran with the two tested doses of LYC resulted in a siginificant improvement in the renal oxidative stress biomarkers. This improvement was more pronounced in the group received the high LYC dose (18 mg/kg b.w.) plus carbofuran (Table 4).
4. Discussion
Carbamates are extremely toxic to insects and are being used widely since last few years in cultivation. Carbofuran 96 h LC50 in freshwater fishes range from 0.08 mg/L in bluegill sunfish, Lepomis macrochirus to 1.99 mg/L in fathead minnow, Pimephales promelas [10]. In this study, the LC50 of carbofuran for 96h was 1.21 ppm. Pessoa et al. and Campos-Garcia et al. [47]-[48] found that the values of carbofuran 96 h LC50 in tilapia fish, O. niloticus were 0.21 mg/L and 2.46 mg/L, respectively. This difference may be due to the variance of the tested fish species or hardiness of water quality parameters. Behavioral changes are considered as sensitive indicators of toxicant effect. Abnormal swimming performance and clinical signs observed during carbofuran exposure of catfish agree with observations reported by other authors reporting on toxicity of pesticides [49],[24],[4],[50]-[51]. Abnormal swimming and loss of equilibrium was caused by the deficiency in nervous and muscular coordination which may be due accretion of acetylcholine in synaptic and neuromuscular junctions observed by Rao et al. and Prasanth et al. [52]-[53]. Oral administration with LYC for 4 weeks showed enhancement in behavioral abnormalities and the fish were noticed in better conditions. Similar results for LYC were observed by Mekkawy et al. [4] , [50] , Ibrahim [51] , Ural [54]. Carbofuran induced hyperglycemia in C. gariepinus to satisfy its critical needs of energy by enhancing the process of gluconeogenesis in stressed fish [55]. Similar results were recorded by Bakhshwan et al., Mekkawy et al. [49]-[50], Hamed [56]- [57] , Harabawy and Ibrahim [58] , after exposure to different pesticides. Interestingly, supplementation of LYC at both used doses tends to normalize the level of glucose, particularly, the sixth group which received the higher dose of LYC. This finding is consistent with Mekkawy et al. [4], [50]. Cortisol is usually used as an indicator of stress in fish by increasing the energy through glycogenolysis, lipolysis and gluconeogenesis [19]. In the current study, cortisol level increased (P<0.05) significantly in carbofuran intoxicated fish. The results are in accordance with the previous investigators [56] – [57] , [59] – [60]. The increment in cortisol could be explained by the increase in osmotic water- influx to restore the hydromineral balance. This osmoregulatory dysfunction might be harmful perse; and owing to sustained high cortisol level which may cause serious physiological changes, affecting the immuno-competence, health status and survival of fish [61]. On the other hand, LYC along with carbofuran in the fifth and sixth groups resulted in statistically significant depletion in serum cortisol level (Table 2), which was more evident in the sixth group received (18 mg LYC/kg b.w.). Carbofuran intoxication increased serum liver markers, AST, ALT and cholesterol. Also, it increased serum kidney levels; urea and creatinine which could be attributed to hepatorenal dysfunction under the effect oxidative damage and generation of ROS. Increased activities of AST, ALT, cholesterol, urea and creatinine were also reported by El- Sayed et al., Abdelkhalek et al. and Hamed [62]-[64] in different fishes exposed to various pesticides. Consequently, administration of LYC with carbofuran exposure decreased the serum liver biomarkers and serum kidney products in the sixth group received the higher dose of LYC (Table 2). The data are corroborated by previous reports of Mekkawy et al. [4] , [50] who recorded a marked decrease in serum ALT, AST levels after cadmium and atrazine -induced stress in C. gariepinus and O. niloticus, respectively. AChE is considered to be a sensitive biomarker for monitoring the toxicity of pesticides such as carbofuran. Results of this study revealed a marked inhibition of AchE activity in carbofuran exposed fish. Decreased AchE levels were reported in sea bass, Dicentrarchus labrax L. (D. labrax L.) [24] and carps, Cyprinus carpio [65] exposed to carbofuran. The impairment of AchE enzyme activity could be due to the oxidative stress generated by pesticide exposure (Salbego et al., 2010). On the other hand, Glusczak et al., [67] proposed the inhibition of AChE activity to the effect of the surfactant rather than to an effect of pesticide itself. Oral supplementation with the both doses of LYC up regulate the level of AchE enzyme compared to carbofuran intoxicated group. Proteins are the most important and copious macromolecules in living organisms, which play an essential role in construction and physiology of the cell and in cellular metabolism [68]. Results revealed hypoproteinaemia and hypoalbuminaemia in carbofuran exposed fish as compared with the control group suggesting strong indicators of stress effect of the carbofuran. Similar results were verified in O. niloticus up on exposure to malathion [56] and C. gariepinus up on exposure to carbofuran [58]. The decline of total protein may be due to destruction of protein-synthesizing subcellular structures and inhibition of hepatic synthesis of blood protein [69]. Reduction of protein from damaged kidney could contribute further to the observed hypoproteinemia [70]. Administration of LYC restores total protein and albumin to normal levels (Table.2), The findings are in agreement with the previous investigators [4], [50]. Lipids are considered as the source of stored chemical energy in the body and characterized by their fast metabolic transformation, therefore they represent the transient body material [71]. In the present investigation, the decline of total lipids level were recorded in catfish after carbofuran exposure. The result is confirmed with Mekkawy et al., [4] who recorded a significant decrease in serum total lipids of catfish exposed to 1.7 and 3.4 mg/L atrazine for 15 and 30 days. This could be attributed to increase the secretion of catecholamines and corticosteroids with enhanced metabolic rate and in turn reduced metabolic reserves [72]. However, the simultaneous treatments with LYC resulted in a significant increase in total lipids level which was more pronounced in the sixth group received the high dose of LYC. This finding is corroborated by the previous report of Mekkawy et al., [4]. Many studies showed that carbofuran induces oxidative stress leading to the generation of free radicals with an increase of (ROS), since alteration in the antioxidant status in different species of fish [12]-[13]. In the current study, the antioxidant profile of carbofuran exposed catfish changed through the increase of hepatic and renal MDA and SOD and the decrease of CAT, GSH and TAC levels. Ibrahim and Harabawy [60] recorded that lipid peroxidation level inecreased in liver and kidney of catfish after exposure to carbofuran. Hamed [56] ascribed the increase in MDA level of catfish tissues exposed to pesticide to the excess production of ROS, which could be related to antioxidant enzyme leakage. Interestingly, administration of LYC restores the hepatorenal MDA level to nearly normal level, particularly the sixth group which received the high dose of LYC. This view was in a good agreement with Mekkawy et al. and Ibrahim [50]–[51]. The key antioxidant enzymes (SOD, CAT and TAC) in all organisms are essential for the conversion of ROS into harmless metabolites, and they may be increased or decreased under chemical stress [65]. Similarly to our results, increased SOD activity and decreased TAC level have already been reported in hepatic and renal tissues of O. niloticus [51]. The increase in the tissue SOD level may be attributed to the elimination of ROS from the cell induced by pesticide exposure [73]. Meanwhile, Sun et al [74] ascribed the decrease in CAT level to the changes in the redox status of the cells; where, ROS are generated in excess, free radical chain reactions are stimulated and interactions with protein, lipids and nucleic acids cause cellular injury and even systemic disease in carbofuran exposed fish. On the other hand, Bainy et al. [75] proposed that the reduction in TAC level of catfish tissues after exposure to carbofuran could be due to the production of superoxide radicals or to the direct action of pesticides on enzyme production [75]. Carbofuran was found to reduce GSH level of C. gariepinus tissues [60]. The decline in GSH activity in hepatorenal tissues may reflect its utilization in opposing the prevailing oxidative damage under the influence of ROS generated from carbofuran oxidative stress. Therefore, GSH provide the foremost defense against ROS-induced cellular damage [76]. While, oral administration with LYC corrected markedly the hepatic and renal antioxidant enzymes and TAC level compared to the control and carbofuran-treated groups of C. gariepinus. These findings are verified with the previous report of Ibrahim [51] who recorded a significant decrease in SOD level and an increase in TAC level of O. niloticus tissues exposed to diazinon .
5. Conclusion
From the current study, it could be concluded that carbofuran has deleterious effects in the aquatic biota especially fish even through the sublethal concentrations which revealed in alteration in behaviuor, biochemical parameters and oxidative stress. LYC succeeded to alleviate the harmful effects of carbofuran and tend to restored all studied parameters.