The aquatic and terrestrial ecosystems are continuously contaminated with chemical pollutants from industrial, agricultural and domestic activities. Insticides are a major category of toxicants, which have serious toxic impacts on aquatic life and still constitute a significant risk due to their toxicity on non-target organisms including fishes (Ghazala et al., 2014; Soloneski and Larramendy, 2012).

 

Diazinon (DNZ), [O,O-diethyl O-[6-m ethyl-2-(1-methylethyl)- 4-pyrimidinyl] phosphorothioate], an organo-phosphate insecticides is widely used in agriculture and public health (US, 2006). Few investigations reported the toxic potential effects of diazinon on certain fish (Banaee et al., 2011, 2013; Ibrahim and Banaee, 2014). But its impact on the specific antioxidant and oxidative stress biomarkers is less explored.

 

It is known that many xenobiotics like insecticides may cause oxidative stress by generating reactive oxygen species (ROS) and alterations in ROS scavenging enzymes (Ibrahim and Harabawy, 2014, Milatovic et al., 2006). Like other organisms, fish have antioxidant defense mechanisms, such as antioxidant enzymes superoxide dismutase  (SOD), catalase (CAT), and glutathione  peroxidase (GPX) and non-enzyme antioxidants (ascorbic  acid, thiols,  alpha tocopherol) to  protect  their cells from oxidative damages (Banaee et al., 2013). Superoxide dismutase converts the superoxide radical to peroxide and oxygen (Fridovich, 1978). Catalase targets hydrogen peroxide and quickly converts it to water and oxygen.

 

Non-enzymatic antioxidants such as vitamins E and Lycopene can act to overcome oxidative stress, as a part of the antioxidant system. Vitamin E, a constituent of plasma membrane, is an effective antioxidant and, as it is present at the site of free radical generation, it may neutralize the toxic effects of ROS (John et al., 2001).

 

Carotenoids are common highly efficient scavengers of singlet-oxygen and other excited oxygen species. During singlet-oxygen quenching, energy is transferred from single-oxygen to the lycopene molecule, which converts lycopene to the energy-rich triplet state. In contrast, the trapping of other ROS, such as hydroxyl, nitric oxide or peroxynitrite leads to oxidative breakdown of the lycopene molecule. Thus, lycopene may protect against the in vivo oxidation of lipids, proteins and DNA (Stahl and Sies, 2003; Wertz et al., 2004). Recently, lycopene has become a focus of interest because of its highly efficient antioxidant scavenging activity against singlet-oxygen and free radicals. Thus, lycopene may prevent oxidative damage, toxicity, and disease. Lyco­pene is one of the most effective antioxidants in the carotenoid fam­ily (Yonar, 2012; Yonar and Sakin, 2011).

 

The Nile tilapia Oreochromis niloticus is a widely distributed freshwater fish that can persist in a highly polluted habitat and can be used as a potential bio-indicator for aquatic environmental contaminants including pesticides. The activities and expression levels of antioxidant enzymes and metabolite were used as biomarkers to evaluate the influence of pollution on the biochemical pathway and enzymatic function in fish (Correia et al., 2007; Sun et al., 2006) and for monitoring unacceptable levels of environmental contamination. Therefore, this study was designed to evaluate the time and concentration dependent changes in the activity of antioxidant enzymes (SOD, CAT and Total antioxidant (TAO) activities as well as the concentration of malonaldehyde MDA as a bio-product of lipid peroxidation and DNA fragmentation and diazinon accumulation in gills, kidney and liver of Oreochromis niloticus following diazinon exposure at two sublethal concentrations for 14 and 28 days. Also, this study was designed to evaluate the ability of two antioxidants (Vitamin E and lycopene) to quench diazinon toxicity and accumulation.

Sample collection and chemicals

 

One hundred and twenty (120) healthy fish of the Nile tilapia, Oreochromis  niloticus  (157   ±  21.4 g  in   weight,  22  ±  1.72  cm  in length), were caught from the fish farm of faculty of Agriculture, Assiut University, Egypt. The fish were immediately transported to the fish laboratory in the Department of Zoology, Faculty of Science, Assiut University. The experimental fishes were reared in aerated glass tanks (160 L capacity) and acclimatized for two weeks before being used in the experimental study. The experi­mental fish fed pellets at a rate of 3% of fish body weight twice daily. Faeces and residual food were aspirated regularly. The water temperature, pH and dissolved oxygen concentrations (DO) were measured daily (22.2 ± 1.5°C, 6.9 ± 0.2 pH and 6.5 ± 1.03 mg/1 DO). Light cycle was 12 h light and 12 h dark.

 

The insecticide Diazinon 9.0% was supplied by Bayer Company for Intermediate Chemicals, Egypt. Lycopene ((EC) No 1272/2008) was purchased from Sigma-Aldrich Chemical (St Louis, MO, USA) and DL-α-tocopherol (VE) acetate were obtained from Merck (Germany).

 

Experimental design

 

The fish were weighed, measured and classified ran­domly into 12 groups (10 fish per each tank) according to doses of DZN, lycopene, vitamin E and their combinations. The diets (maize and soy bean 34% protein, 15 g/kg fish) were pellet after addition of vitamin E and lycopene doses for the treated groups and the addition of suitable amounts of molasses and water. The diets were dried at room temperature and stored in small bags for fish feeding.

 

Stock solution (1,000 ppm) of Diazinon (DZN) (0, 0-diethyl-0-[2–isopropyl-6-methyl-4 –pyrimidinyl] phosphorothioate) was prepared and stored in clean glass bottles and diluted to concen­trations of 0.76 and 2.3 mg/l in water. Such low sublethal DZN concentrations (1/10 and 3/10 of 96 h LC₅₀) were chosen accord­ing to levels monitored by Soyingbe et al. (2012). Diazinon doses were prepared and added constantly to the aquarium for four weeks. The test water was replaced daily with the required amount of stock solution to prevent deterioration of water quality and replenish diazinon levels. Lycopene was added to the diet in concentration (10 mg/kg BW). Dose response of lycopene was described previously by Ural (2013). Also, vitamin E (a-tocopherol) was supplemented in 50 mg/kg BW. Such vitamin E concentration was chosen according to levels monitored by Ortunõ et al. (2001) (Table 1).

 

 

Fish showed abnormal behavior during the experimental period. At the start of the exposure, fish were alert, lost swimming coordination and buoyancy control with eleva-tion of opercula beat rate, which increased with time. Sometimes, they tried to avoid the toxic water by fast swimming and jumping. In tanks with DZN concen-trations, the fish swam unsteadily with jerky movements and hyperactive excitability. No fish mortality was recorded in aquaria with high dose of DZN.

 

Lipid peroxidation measurement (malonaldehyde level)

 

Lipid peroxidation (LPO) results are presented in Table 2. The LPO level was significantly increased in the liver, kidney and gills samples of the DZN groups. The main effects of DZN, LYC, VE and their interactions were significant (P>0.05) in the two periods.

 

Similarly, the time of exposure main effect was significant (P>0.05), and gills main effect was significant too (P>0.05). The level of LPO was significantly (P>0.01) decreased in liver, kidney and gills of DZN-exposed fishes fed with diets supplemented with vitamin E and/or lycopene.

 

 

 

In the present work, more or less nervous manifes­tation of DZN-treated fish in the form of jerky uncoordinated movement, fins stretching and scale loss symptoms was recorded. Changes in color and loss of appetite were also observed for some fishes.  Similar behavioral responses determined in this study have been observed with the guppy exposed to  pyrethroids (Viran et al., 2003), fresh-water catfish, Heteropneustes fossilis exposed to cypermethrin (Saha and Kaviraj, 2003) and young mirror carp, Cyprinus carpio exposed to deltamethrin (Calta and Ural, 2004). Diet supplementation with LYC and/or VE for 14 and 28 days showed improvement in behavioral changes and the fishes were noticed in better conditions. Similar results for vitamin E and lycopene were observed by Ohaida (2005), Mekkawy et al. (2011, 2013), Soltan et al. (2011) and Ural (2013).

 

Diazinon insecticides induce oxidative stress leading to generation of free radicals and alteration in antioxidant enzymes or oxygen free radicals scavenging enzyme (El-Shenawy et al., 2010). Lipid peroxidation, an auto-catalytic process caused by free radicals, is considered to be a valuable indicator of oxidative damage in cellular components (Banaee et al., 2014). The present study showed that DZN at both dosages promoted MDA increase in different tissues. This increase in MDA can most likely be ascribed to an excessive production of ROS, which could be related to antioxidant enzyme leakage. Also, the increase in intracellular levels of ROS may lead to lipid peroxidation resulting in an increased permeability of gills, kidney and liver membranes (Banaee et al., 2011). In the present investigation, due to the free radical scavenging properties, significantly increased MDA level partially returned near to the control levels by VE and/or LYC. The level of lipid peroxidation was significantly decreased in different tissues of DZN-exposed fishes fed diets supplemented with vitamin E and/or lycopene. Similar results for vitamin E were observed in different fish (Abdel-Monem et al., 2012; Soltan et al., 2011).

 

Free radicals that are gener­ated following pesticide exposure may lead to exten­sive DNA damage (Hatjian et al., 2000). In the present study, administration of DZN resulted in DNA damage of animals exposed for 14 and 28 days. The significantly increased DNA fragmentation percentage was partially returned near to the control levels by VE and/or LYC. Diets with vitamin E and lycopene have a protective effect on DZN-induced DNA and prevent genotoxicity induced by DZN. Yassa et al. (2011) showed that vitamin E had a protective effect against DZN-induced DNA damage and protective effect of vitamin E against genotoxicity of DZN has been reported (Ibrahim and Banaee, 2014). Another possible mechanism of vitamin E might involve selec­tive removal of cells with DNA damage by apoptosis (Singh et al., 2008). Vitamin E allows free radicals to reduce a hydrogen atom from the antioxidant molecule rather than from polyunsatu­rated fatty acids thus breaking the chain of free radical reactions (Pascoe et al., 1987).

 

The first line of defense against oxidative stress consists of the antioxidant enzymes TAO, SOD and CAT, which convert superoxide radicals into hydrogen peroxide and then into water and molecular oxygen. A decrease in the activity of these enzymes changes the redox status of the cells. Thus, it is possible that an increase in the activity of these enzymes contributes to the elimination of the ROS induced by pesticide exposure from the cell (Stara et al., 2012). The present study showed a significant decrease in total antioxidant in tissues under investigation after exposure to DZN which could be related to the production of superoxide radicals or to the direct action of pesticides on enzyme synthesis (Bainy et al., 1996). However, Vitamin E and/ or lycopene adminis-tration increased the TAO tissues activity of the DZN-treated fish. The present results showed that lycopene enhanced antioxidant capacity, thus protecting tissues against the DZN-induced damages, as shown by the maintenance of the TAO activity. These results suggest that vitamin E and lycopene have a protective effect on ROS as described in other studies (Mekkawy et al., 2011, 2013).

 

SOD is a group of metallo-enzymes that plays a crucial antioxidant role and constitutes the primary defense against the toxic effects of superoxide radicals in aerobic organisms. SOD catalysis the transformation of superoxide radicals to H₂O₂ and O₂ and is the first enzyme to cope with oxygen radicals (Kohen and Nyska, 2002). The significant increase in the tissue SOD activities that was observed in this study may be meant to scavenge the overproduction of superoxide anions under the oxidative stress induced by DZN. A similar result of increased SOD activity has been reported in Oreochromis niloticus that were exposed to chlorpyrifos (CPF) (Oruç, 2010). Similarly, Yonar et al. (2012) reported that a significant increase in the SOD enzyme activity occurred in carp tissues following CPF exposure. However, the simultaneous treatments with vitamin E and/or lycopene resulted in a significant decrease in the tissue SOD activities. This decrease in SOD activity can be attributed to the inhibition of superoxide radical formation or the potential free radical scavenging activity of vitamin E and/or lycopene (Ural, 2013).

 

CAT is an enzyme that is located in the peroxisomes and facilitates the removal of hydrogen peroxide, which is metabolized to molecular oxygen and water (Van der Oost et al., 2003). The present study showed that CAT activity was significantly increased in gills, kidney and liver tissues of Oreochromis niloticus that were exposed to both DZN concentrations. This elevation due to the adaptive response to the generated free radicals, indicating the failure of the total antioxidant defense mechanism to protect the tissues from mechanical damage caused by pesticides, as evidenced by lipid peroxidation. Thus, the superoxide ion generated is dealt with by the enhanced SOD and is converted to H₂O₂ by CAT. In the present study, the administration of vitamin E and LYC were somewhat effective in restoring the activities of SOD and CAT, which might be due to its ability to scavenge the accumulated free radicals. Total antioxidant, CAT and SOD differed significantly and the effect did not normalize the values, being significance between the control and DZN plus VE and/ or LYC groups.

 

The present study showed a significant accumulation of DZN in different tissues under investigation. The accumulation pattern of DZN was gills> kidney> liver. Similar results were obtained by Yassa et al. (2011), who found that DZN residues level in gills were higher when comparing to DZN concentration among liver, kidney and muscle tissues. Administration of vitamin E and/or lycopene with DZN insignificantly reduces the residue values in the examined tissues. The increased residual levels in the kidney compared with the liver confirm that the DZN residue was much greater in kidney than that in other organs (Altuntas et al., 2004). The relative high concentration of DZN residue in kidney and gills indicates that these organs play an essential role in the excretion of DZN.