Fish Physiol Biochem DOI 10.1007/s10695-013-9885-4

Effect of vitamin C dietary supplementation in reducing the alterations induced by fenitrothion in Oreochromis niloticus Eman A. Abd El-Gawad Omnia M. Abdel Hamid

•

Received: 21 May 2013 / Accepted: 28 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The objective of this study was to investigate the dietary effect of vitamin C in amelioration some of studied alterations induced by fenitrothion in Nile tilapia (Oreochromis niloticus). Nile tilapia was exposed to sub-lethal concentration of fenitrothion 0.04 mg/l (96 h LC50 value was 0.8 mg/l), and basal diet was supplemented with two different dose of vitamin C (500 and 1200 mg/kg B wt/day) for 30 days. Vitamin C supplemented groups showed significant decrease in plasma cortisol and glucose level, enzymes activity of liver and gills (catalase, glutathione-S-transferase and superoxide dismutase) and % of tail DNA damage compared to exposed group. Moreover, fish revealed significant increase in total plasma protein, albumin, globulin and A/G ratio. High dose of vitamin C dietary supplementation (1200 mg/kg B wt/day) returns these parameters to its normal levels with no significant difference compared to non exposed control group. These results indicated that incorporation of high dose of vitamin C (1200 mg) in aqua feed for 30 days could be

E. A. Abd El-Gawad (&) Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh, Kalubia 13736, Egypt e-mail: [email protected] O. M. Abdel Hamid Department of Biochemistry, Faculty of Veterinary Medicine, Benha University, Moshtohor, Toukh, Kalubia 13736, Egypt

potentially less expensive and effective in reducing the alterations induced by fenitrothion in Nile tilapia. Keywords Vitamin C
Fenitrothion
Antioxidant enzymes
Biochemical parameters
DNA damage
Nile tilapia

Introduction Pollution of the aquatic ecosystem with agricultural effluents constitute a serious threat to the aquatic organisms and human (Brinelli and Provini 2004). Organophosphorus insecticides have become the most commonly used pesticides in many countries after the great restriction on the use of organochlorine compounds (Sancho et al. 1998). Fenitrothion [O,Odimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate] is used on large scale due to its effectiveness against a wide range of pests affecting cereals grains, cotton, rice and vegetables. It is marketed as Sumithion in Egypt. Fenitrothion is considered moderately toxic to warm-water and cold-water fishes (Meister 1994). The 96 h LC50 was 0.84 mg/l for Nile Tilapia (Benli and Ozkul 2010), 1.7 mg/l for brook trout (Salvelinus fontinalis) and 2.6 mg/l for mullet (Mugil cephalus) (Worthing 1987), and 48 h LC50 was 4.1 mg/l for common carp (Cyprinus carpio) and 3.0 mg/l for rainbow trout. It is considered a

123

Fish Physiol Biochem

cholinesterase inhibitor, which led to accumulation of acetylcholine and a subsequent activation of cholinergic muscarinic and nicotinic receptors (Sarikaya et al. 2004). It is known that pesticides induce oxidative stress due to the formation of reactive oxygen species (ROS) and alterations in the antioxidant or free radicals scavenging enzymes system in the aquatic organisms (Di Giulio et al. 1989). ROS such as superoxide anion radical (O2-), hydrogen peroxide (H2O2) and highly reactive hydroxyl radical (OH-) react with susceptible biological macromolecules resulted in lipid peroxidation, DNA damage and protein oxidation (Monteiro et al. 2006). When there is an imbalance between production and elimination of ROS, oxidative stress occurs. These ROS under normal condition are counteracted by antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) (Livingston 2001), which are used as biomarker indicators to the toxic effect of pesticides (Ohe et al. 2004). Blood parameters such as cortisol and glucose are the most important reliable indicator used for assessing stress induced after exposure to pollutants (Brown 1993; Barcellos et al. 1999). In addition, total protein, albumin, globulin and albumin/globulin ratio (A/G) are strong indicators of immunosuppressive effect induced by pesticides (Nayak et al. 2004). Liver is a vital organ in the process of uptake, biotransformation and excretion of pollutant but some of metabolites produced are highly reactive, toxic and affects the integrity of DNA (Gernho¨fer et al. 2001). Comet assay is one of the most sensitive and reliable technique used to detect DNA damage (Mitchelmore and Chipman 1998). Vitamin C is one of the most natural antioxidants in living tissue, but it cannot be synthesized by many fish due to lack of enzyme L-gulonolactone oxidase (GLo, EC 1.1.3.8), which catalyzes L-gulonolactone to ascorbic acid in liver and kidney (Roy and Guma 1958); so, dietary supplementation of vitamin C in aqua feed is essential for fish growth, reproduction and counteract the stress induced by pesticides (Datta and Kaviraj 2003; Korkmaz et al. 2009). It acts by inactivating damaged free radicals produced through normal cellular activity and diverse stressors (Halver 1995) and thus protects cell membranes and others intracellular component sensitive to oxidation. Fish are being widely used as experimental models for the

123

evaluation of pollution in aquatic ecosystems, and their biological responses serve as biomarkers of environmental contaminants. Nile tilapia, Oreochromis niloticus, is one of the most common freshwater fish used as a good biological indicator for toxicological studies (Figueiredo-Fernandes et al. 2007), because it have a number of characteristics that make it an excellent model for toxicity studies such as great resistance to diseases and handling practices, easy maintenance in the laboratory and good tolerance to a wide range of environmental conditions (GarciaSantos et al. 2006). The aim of the current study was to investigate the protective effect of vitamin C dietary supplementation in reducing the alterations induced by sub-lethal concentration of fenitrothion in O. niloticus.

Materials and methods Experimental fish A total number of 180 Nile Tilapia (O. niloticus) of average body weight 45.88 ± 5.8 g and total length 14.18 ± 0.08 cm were obtained during June 2012 from private fish farm at El-Abbassa, Sharkia, Egypt. Fish were maintained in fiber glass tank for 15 days for acclimation to the laboratory conditions. After that, fish were distributed in equal number in a glass aquarium of 108 L capacity containing 60 L of water. The water temperature, pH and dissolved oxygen were adjusted at 26 ± 2 °C, 7.4 ± 0.1 and 7.8 ± 0.4 ppm, respectively, throughout the experiment. Fish were fed daily with pelleted food (30 % crud protein) at a rate of 3 % of their body weight. Determination of lethal concentration (LC50) of fenitrothion The acute toxicity of organophosphorus insecticide fenitrothion for O. niloticus was done according to standard method (APHA 1998). Commercial grade of fenitrothion (Sumithion KZ 50 % EC)Ò was purchased from Kafr El Zayat Company for Pesticides and Chemicals, Egypt. Before starting of this experiment, feeding of fish was stopped for 48 h, and then, fish were divided into five groups (10 fish/group) in duplicate: the first four groups were exposed to different concentrations of fenitrothion 0.75, 1.0, 1.5

Fish Physiol Biochem

and 2 mg/l for 96 h, and the last fifth group was kept as control. During this experimental period (96 h), there were no water exchange and feeding. The mortality percentage was recorded at 24, 48, 72 and 96 h interval, and dead fish were removed immediately from the aquarium. The LC50 value of fenitrothion was calculated by probit analysis method (Finney 1971).

total protein (Kaplan and Szabo 1983) and albumin (Tietz 1994). The globulin level was calculated by subtracting albumin level from total protein, and A/G ratio was obtained by dividing albumin content by globulin content.

Experimental diet

After blood sampling, fish was killed for the collection of liver and gills to carry out the enzymes assay. Tissue was homogenized with chilled phosphate buffer saline (pH 7.4) using Teflon coated mechanical tissue homogenizer. The homogenate was centrifuged at 5000 rpm for 20 min. The supernatant was separated and stored at -20 °C till analysis of enzymes activity, including CAT enzyme (U/g) (Sinha 1972), SOD (U/g) (Misra and Fridovich 1972) and glutathione S transferase (U/g) (Habig et al. 1974).

Commercial product of vitamin C (C 20 %)Ò was obtained as powder from Agri-Vet Company, Egypt. Two different doses of vitamin C (500 and 1200 mg/ kg B wt/day) were used. Control diet (30 % crud protein) was used for control group, while the experimental diet was prepared by dissolving the required dose of vitamin C in appropriate amount of water then added on basal diet in a blender and mixed thoroughly. The obtained pellets were allowed to airdried at room temperature for 24 h, then packed in clean plastic jar and kept in refrigerator at 4 °C for daily use. Experimental design Total number of 80 fish was divided into four groups (10 fish/group) in duplicate. Group I were fed on control diet with no fenitrothion exposure, while group II, III and IV were exposed to sub-lethal concentration of fenitrothion (0.04 mg/l) and fed on basal diet (without vitamin C), 500 and 1200 mg/kg B wt/day vitamin C supplemented diet, respectively. Fish in all groups was fed twice daily at a rate of 3 % of their body weight for 30 days. The water of group II, III and IV was partially renewed every week with maintaining the required concentration of fenitrothion (0.04 mg/l), while water of group I was changed with normal dechlorinated tap water. At the end of the experiment, blood and tissue samples were collected from each group. Biochemical assay Blood samples were collected from the heart in 5-ml lithium heparinized tube and kept on crushed ice until centrifugation for 5 min at 3600 rpm to obtain plasma ¨ zcan Oruc 2010). The biochemical paramsamples (O eters were estimated according to the following: cortisol level (Knobil 1980), glucose (Trinder 1969),

Antioxidant enzymes assay

Evaluation of DNA damage by comet assay The alkaline single cell gel electrophoresis was done as described by Singh et al. (1988) as follows: small piece (1 g) from liver and gills was washed three times with chilled phosphate buffered saline (pH 7.4) to remove blood and then transferred to ice-cold homogenization buffer (1 ml HBSS, 20 mM EDTA, 10 % DMSO) pH 7–7.5. The tissue was then minced into fine pieces and homogenized to form single cell suspension. The suspension was centrifuged at 3000 rpm for 5 min, and then, cell pellet was resuspended in fresh mincing solution. In an eppendorf tube, 5 ll of cell suspension was mixed with 75 ll low melting point agarose (37 °C). Five ml of sample was added to an agarose precoated slide and covered gently with cover slip to make a microgel. The slides then immersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, freshly 1 % triton X-100 and 10 % DMSO), the pH adjusted to 10.0 and kept in refrigerator for at least 2 h. After that, gently remove slides from the lysing solution and laced side by side on the horizontal gel box near one end, sliding them as close together as possible. Cold electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH [ 13) was poured until completely covered the slides and left for 20 min to allow unwinding of the DNA and the expression of alkali-labile damage. Power turned on at 24 V and 300 A for 30 min. The slides then removed and washed two times by neutralizing solution (0.4 M

123

Fish Physiol Biochem

significant difference in these parameters with nonexposed control group. Antioxidant enzymes activity (CAT, GST and SOD) of liver and gills was significantly (P B 0.05) increased in Nile tilapia exposed to sub-lethal concentration of fenitrothion (Fig. 1). On the contrary, dietary supplementation of vitamin C revealed significant decrease in the enzymes activity compared to fenitrothion exposed group. Moreover, high dose of vitamin C dietary supplementation showed no significant difference in the activity of antioxidant enzymes compared to nonfenitrothion exposed group (control). Significant (P B 0.05) increase in % tail DNA damage of liver and gills was recorded in fish exposed to sublethal concentration of fenitrothion (Fig. 2), while dietary supplementation of vitamin C enhanced liver and gills tissue and showed a significant (P B 0.05) decrease in % tail DNA damage compared to exposed group.

tris buffer at pH 7.5), and the slides then stained with 80 ll 19 ethidium bromide for 5 min. The slides dipped in chilled distilled water to remove excess stain and covered by cover slip to immediately analyze using 409 fluorescent microscope. One hundred of random cells were analyzed per sample, and the percent of tail DNA was calculated by comet 5 Image analysis software (Kinetic Imaging, LTd, UK). Statistical analysis All the obtained data were statistically analyzed using the SPSS (version 16.0) software. The significant of difference between groups was determined by analysis of variance (one-way ANOVA) and Duncan significant difference test. The results are presented as mean ± standard error, and the significant of difference was at (P B 0.05).

Results Discussion In the present study, the value of 96 h LC50 of fenitrothion to O. niloticus was 0.8 mg/l. Fish exposed to sub-lethal concentration of fenitrothion (0.04 mg/l) showed significant increase in plasma cortisol and glucose level compared to control (Table 1). In addition, significant decrease in total plasma protein, albumin, globulin and A/G ratio was observed, while groups fed on vitamin C incorporated diet revealed significant (P B 0.05) decrease in plasma cortisol and glucose level compared to fenitrothion exposed group. Moreover, significant increase in total plasma protein, albumin, globulin and A/G ratio was recorded in vitamin C supplemented groups. Fish received high dose of vitamin C (1200 mg/kg B wt/day) revealed no

The value of 96 h LC50 of fenitrothion for Nile tilapia was 0.8 mg/l. This result comes nearly similar to Benli and Ozkul (2010) who stated that fenitrothion is highly toxic to Nile tilapia and 96 h LC50 was 0.84 mg/l. Plasma cortisol is a primary response in fish exposed to different kinds of stressors (Wendelaar Bonga 1997). The obtained data showed a significant increase in plasma cortisol level in fenitrothion exposed fish when compared to control group. This result was concurred with Cericato et al. (2008) and Kumar et al. (2011). The increase in cortisol level might be due to activation of hypothalamo–pituitary– interrenal axis in response to pesticide stress lead to

Table 1 Effect of vitamin C supplemented diet on biochemical parameters of O. niloticus exposed to sub-lethal concentration of fenitrothion for 30 days Groups

Cortisol (lg/dl)

Glucose (mg/dl)

Total protein (g/dl)

Albumin (g/dl)

Globulin (g/dl)

A/G ratio

I

113.12 ± 0.45c

35.63 ± 0.71c

5.31 ± 0.06a

1.33 ± 0.01a

3.98 ± 0.07a

0.33 ± 0.01a

2.59 ± 0.08

c

0.47 ± 0.02

c

2.13 ± 0.06

c

0.22 ± 0.02b

3.40 ± 0.11

b

0.57 ± 0.04

b

b

5.19 ± 0.01

a

1.27 ± 0.01

a

II III IV

70.87 ± 0.67

a

117.55 ± 0.42

b

47.43 ± 0.62

b

113.87 ± 0.28

c

c

129.32 ± 0.8

a

37.57 ± 0.8

2.83 ± 0.1

3.92 ± 0.02

0.20 ± 0.01b a

0.32 ± 0.03a

Values in the same column with different superscripts were differed significantly (P B 0.05). Data expressed as mean ± SE

123

Fish Physiol Biochem

A

5

45

* 40

*

Gills

*

30 25 20

* *

15

% tail DNA

Liver

10

3.5

*

3 2.5

*

2 1.5 1 0.5 control

0 control

1.2

fenitrothion

fenitrothion + 500 mg vit C

* *

GST activity (U/g)

fenitrothion +1200mg vit C

Gills

0.8

*

*

0.6 0.4 0.2 0 control

SOD activity (U/g)

fenitrothion

20

fenitrothion +

fenitrothion

500 mg vit C

+1200mg vit C

*

18

Liver

16

Gills

14 12

*

10 8 6 4 2 0 control

fenitrothion

fenitrothion + fenitrothion 500 mg vit C +1200 mg vit C

Fig. 2 Effect of vitamin C dietary supplementation on % tail DNA damage of the liver and gills in O. niloticus exposed to sub-lethal concentration of fenitrothion for 30 days. Data are expressed as mean ± SE. *P B 0.05

Liver

1

C

Gills

*

0

5

B

Liver

*

4

35

CAT activity (U/g)

*

4.5

fenitrothion

fenitrothion +

fenitrothion

500 mg vit C

+1200mg vit C

Fig. 1 Effect of vitamin C supplemented diet on antioxidant enzymes activity of liver and gills: a catalase (CAT); b glutathione-S-transferase (GST) and c superoxide dismutase (SOD) in O. niloticus exposed to sub-lethal concentration of fenitrothion for 30 days. Values are expressed as mean ± SE. *P B 0.05

release of cortisol hormone in the blood stream (Firat et al. 2011). Moreover, elevation of plasma cortisol could be attributed to increase in the osmotic water influx in order to restore the hydro-mineral balance. This osmoregulatory dysfunction might be harmful

and owing to sustain high cortisol level, which may cause several deleterious changes affecting the immune competence, health status and survivability of the fish (Wendelaar bonga 1997). Dietary supplementation of vitamin C revealed significant decrease in cortisol level compared to fenitrothion exposed group, while there was no significant difference between group supplemented with high dose of vitamin C and control. The obtained results come in tune with Thangavel et al. (2005) and Cericato et al. (2008). This significant decrease might be due to the effect of vitamin C in prevention of unsaturated fatty acids to convert into cholesterol ester, which is the precursor of the hormone molecule resulted in inhibition of cortisol biosynthesis (Montero et al. 1999). On contrast, Dabrowska et al. (1991) found that high ascorbate intake did not influence the plasma cortisol level in common carp (C. carpio); so, the relationship between steroidogenesis and vitamin C is still unclear and not known in fish. Blood glucose is considered a second indicator after cortisol to pesticide stress (Sepici-Dincel et al. 2009). Fenitrothion exposed group revealed significant elevation in glucose level. This result agreed with Datta and Kaviraj (2003), Kumar et al. (2011) and AlGhanim (2012). The elevation of blood glucose level might be attributed to increase in glycogen mobilization to glucose in stressed fish (Iwama et al. 1999). Also, Pickering (1981) mentioned that stress stimuli the rapid secretion of two hormones (glucocorticoid and catecholamines) from the adrenal tissue of the fish. Both hormones are known to produce hyperglycemia due to enhanced gluconeogenesis response of

123

Fish Physiol Biochem

stressed fish in their attempt to satisfy new energy demands (Winkalar et al. 2007; Abalaka et al. 2011). The observed hyperglycemia in this study is considered an indication of a disrupt in carbohydrate metabolism possibly due to enhanced breakdown of liver glycogen and the synthesis of glucose from extrahepatic tissue protein and amino acid to produce enough energy (Gill et al. 1993). Significant decrease in plasma glucose was recorded in groups fed on vitamin C (500 and 1200 mg/kg B wt) for 30 days compared to fenitrothion exposed group. In addition, high dose of vitamin C (1200 mg/kg B wt/day) returns the glucose to its normal level compared to control. Similar results were observed in Clarias gariepinus exposed to deltamethrin and fed high dose of ascorbic acid (100 mg/100 g diet) (Datta and Kaviraj 2003) and Channa orientalis exposed to endosulfan with L-ascorbic acid (Ramdas 2013). Also, Tripathi and Shasmal (2010) mentioned that supplementation of high dose of vitamin C could antagonize the toxic effect of pesticide and hence increase their immune resistance (Hardie et al. 1991). This result indicated that dietary supplementation of high dose of vitamin C (1200 mg/kg B wt/day) in Nile tilapia for 30 days was sufficient to counteract the stress induced by fenitrothion and return the biochemical parameters to its normal level. Hypoproteinemia, hypoalbuminemia, hypoglobulinemia and decrease in A/G ratio were significantly observed in fish exposed to sub-lethal concentration of fenitrothion. These results corroborate with Kumar et al. (2011), Al-Ghanim (2012) and Amin and Hashem (2012). The decrease in plasma total protein might be due to impairment of protein synthesis in liver as result of high cortisol level, which prevents the uptake of amino acid resulted in inhibition of protein synthesis (Bradbury et al. 1987), or might be due to stimulation of protein catabolism to meet their energy demand (Korkmaz et al. 2009). Moreover, decline in total plasma protein, albumin and globulin might be resulted from high degree of hemodilution as a result of osmoregulatory imbalance of gill (Mazeoud et al. 1977). The reduction in A/G ratio indicated a shift of albumin production to globulin protein in response to stress and it is usually used as index to measure changes in composition of plasma or serum (Jacobes et al. 1990). Also, it is considered a strong indicator to immunosuppressive effect of pesticide on fish (Nayak et al. 2004). The increase in total protein, albumin,

123

globulin and A/G ratio in Nile tilapia fed on vitamin C supplemented diet compared to fenitrothion exposed group indicates reduction in stress induced by fenitrothion with vitamin C supplementation. Similar result was observed in Catla catla exposed to sublethal deltamethrin for 45 day and fed high dose of vitamin C 1000 mg/kg diet (Vani et al. 2011). Improvement in these parameters increased with high dose of vitamin C (1200 mg/kg B wt/day), and no significant difference compared to control was recorded. This might be due to increase in protein synthesis by vitamin C, which acts as an electron donor (co-factor) for important enzymes essential in protein biosynthesis and other physiological processes as synthesis of collagen and neurotransmitters (Eipper et al. 1993). This indicated that dietary supplementation of high dose of vitamin C can help fish to completely come out of stress induced by the insecticides and stimulate their immune system toward good health condition (Vani et al. 2011). Also, the present study revealed that vitamin C was able to reduce the enzymatic breakdown of protein for energy demand and form the first-line defense against free radicals and peroxides that are generated during cellular metabolism causing protein damage (May 2000). Antioxidant enzymes (CAT, GST and SOD) of liver and gills revealed a significant increase in their activities in O. niloticus exposed to sub-lethal concentration of fenitrothion for 30 days. These results come in harmony with Thomaz et al. (2009), Kumar et al. (2011) and Amin and Hashem (2012). The significant increase in the enzymes activity of liver and gills may be attributed to the production of free radicals, which cause oxidative damage and hence activation of antioxidant enzymes to provide protection and eliminate these free radicals (Sies 1986). Liver is the main organ in detoxification of different toxic metabolites and therefore reflects the status of antioxidant defense system (Wilhelm-Filho et al. 1993). Pesticides induced oxidative stress, which resulted in structural changes in liver leading to alteration of liver metabolism. Both CAT and SOD are the most sensitive and protective enzymes against oxidative damage induced by pesticide. SOD enzyme catalyzes the conversion of superoxide anion (O2-) to oxygen and hydrogen peroxide (H2O2), which degrade to water by CAT enzyme, while GST is known as phase II detoxifing enzymes, which promote the

Fish Physiol Biochem

conjugation of reduced glutathione with a variety of reactive electrophilic compounds resulting in the formation of less toxic substances which are easily excreted from the body (Yang et al. 2001). On contrary, Ameur et al. (2012) recorded a significant decrease in the activity of CAT and SOD of gills and liver of mullet and sea bass collected from polluted area. Also, Tripathi and Shasmal (2010) observed reduction in antioxidant enzyme (CAT) in response to chlorpyrifos treatment. This indicates that both enhancement and diminishment of antioxidant enzymes can occurs depending on time and dose of application of pesticide as well as susceptibility of exposed species (Cheung et al. 2001). The enzymes activity of CAT, GST and SOD in liver and gills was significantly decreased with vitamin C dietary supplementation compared to fenitrothion exposed group fed on basal diet. This might be due to the ability of vitamin C to react easily with free radicals in extracellular body fluids (Bendich 1990) and hence helps to reduce the effect of oxidative stress. Similar finding was recorded by (Tripathi and Shasmal 2010) who mentioned that vitamin C can repair the impairment of antioxidant enzymes induced by chlorpyrifos in Heteopneustes fossilis. Enzymes activity of liver and gills returns to its normal level in group fed on high dose of vitamin C compared to non exposed control group except (CAT) and (GST) activity of gills which was found elevated, probably to provide protection against oxidative damage. Fish exposed to sub-lethal concentration of fenitrothion showed significant increase in % tail DNA damage of liver and gills compared to control. This result showed parallelism with related investigations by Kumar et al. (2010), Dogan et al. (2011), Pandey et al. (2011) and Ameur et al. (2012). The increase in DNA damage in liver tissue might be due the action of highly reactive metabolites while in gills might be due to its close contact with water and its associated pollutants (Pandey et al. 2006). Gills are responsible for many vital physiological functions in fish such as respiration, excretion of nitrogenous wastes, acid–base balance and ion regulation; so when fish are exposed to environmental pollutants, these vital functions are deleteriously affected and the functional impairment of gills can significantly damage the health of fish (Alazemi et al. 1996). Also, it was found that gills are highly sensitive to pesticides than liver, kidneys or erythrocytes (Ali et al. 2009). Comet assay has been considered as an

strong tools and become a widespread technique for evaluating the relationship between DNA damage and exposure to genotoxins (Nogueira et al. 2009). On the other hand, significant decrease in % tail DNA damage was observed with dietary supplementation of vitamin C. Liver tissue revealed enhancement and no significant difference between group supplemented with high dose of vitamin C (1200 mg) and control. This result may be attributed to the ability of vitamin C to donate electron to unstable free radicals and scavenge ROS before initiating cellular damage. The electron oxidation products of ascorbate are relatively nontoxic and easily regenerated (Niki 1991). In conclusion, Nile tilapia exposed to fenitrothion at sub-lethal concentration revealed alterations in biochemical parameters including significant increase in plasma cortisol and glucose level and decrease in plasma total protein, albumin, globulin and A/G ratio. In addition, significant increase in the antioxidant enzymes activity of liver and gills and DNA damage was recorded. These alterations were return to great extent to normal with dietary supplementation of higher dose of vitamin C. The strength of vitamin C appears to lie in its ability to improve health status of fish, survivability and immunity against environmental pollution. Taking this into account, it is recommended in fish farms to use vitamin C as natural antioxidant by incorporating it in aqua feed of Nile tilapia at higher level (1200 mg/kg B wt/day) than normal required level to counteract pesticides stress. Acknowledgments The authors are thankful for Head of Department of Fish Diseases and Management, Faculty of Vet. Med. Benha University, Egypt Prof. Dr. Adel A. Shaheen for his continuous advice.

References Abalaka SE, Esievo KAN, Shoyinka SVO (2011) Evaluation of biochemical changes in Clarias gariepinus adults exposed to aqueous and ethanolic extracts of Parkia biglobosa pods. Afr J Biotechnol 10:234–240. doi:10.5897/AJB10.331 Alazemi BM, Lewis JW, Andrews EB (1996) Gill damage in the freshwater fish Gnathonemus petersii (Family: Mormyridae) exposed to selected pollutants: an ultra structural study. Environ Technol 17:225–238. doi:10.1080/ 09593331708616381 Al-Ghanim KA (2012) Acute toxicity and effects of sub-lethal malathion exposure on biochemical and haematological parameters of Oreochromis niloticus. Sci Res Essays 7(16):1674–1680. doi:10.5897/SRE12.039 Ali D, Nagpure NS, Kumar S, Kumar R, Kushwaha B, Lakra WS (2009) Assessment of gentoxic and mutogenic effects

123

Fish Physiol Biochem of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single cell gel electrophoresis. Food Chem Toxicol 47:650–656. doi:10.1016/j.fct.2008.12.021 American Public Health Association, APHA (1998) Standard method for the examination of water and wastewater. American Public Health Association, APHA, Washington Ameur WB, Lapuente JD, El Megdiche Y, Barhoumi B, Trabelsi S, Camps L, Serret J, Romos-Lopez D, GonzalezLinares J, Driss MR, Borras M (2012) Oxidative stress, genotoxicity and histopathology biomarker responses in mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) liver from Bizerte Lagoon (Tunisia). Mar Pollut Bull 64:241–251. doi:10.1016/j.marpolbul.2011.11.026 Amin KA, Hashem KS (2012) Deltamethrin induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alphatocopherol. Vet Res 45:1–8. doi:10.1186/1746-6148-8-45 Barcellos LJG, Nicolaiewsky S, De Souza SMG, Lulhier F (1999) Plasmatic levels of cortisol in the response to acute stress in Nile tilapia, Oreochromis niloticus (L.), previously exposed to chronic stress. Aquac Res 30:437–444. doi:10.1046/j.1365-2109.1999.00348.x Bendich A (1990) Antioxidant micro nutrients and immune responses. In: Bendich A, Butterworth CE (eds) Micronutrients and immune functions, vol 87. New York Academy of Science, New York, pp 168–180 Benli ACK, Ozkul A (2010) Acute toxicity and histopathological effects of sub-lethal fenitrothion on Nile tilapia, Oreochromis niloticus. Pestic Biochem Physiol 97:32–35. doi:10.1016/j.pestbp.2009.12.001 Bradbury SP, Makim JR, Coats JR (1987) Physiological responses of rainbow trout, Salmo gairdneri to acute fenvalerate intoxication. Pestic Biochem Physiol 27:275–285. doi:10.1016/0048-3575(87)90057-5 Brinelli A, Provini A (2004) Risk for human health of some POPs due to fish from Lake Iseo. Ecotoxicol Environ Saf 58(1):139–145. doi:10.1016/j.ecoenv.2003.09.014 Brown JA (1993) Endocrine responses to environmental pollutants. In: Rankin JC, Jensen FB (eds) Fish ecophysiology. Chapman & Hall, London, pp 276–296 Cericato L, Neto JG, Fagundes M, Kreutz LC, Quevedo RM, Finco J, Da Rosa JG, Koakoski G, Centenaro L, Potter E, Anziliero D, Barcellos LJ (2008) Cortisol response to acute stress in jundia´ Rhamdia quelen acutely exposed to sublethal concentration of agrichemicals. Comp Biochem Physiol 148:281–286. doi:10.1016/j.cbpc.2008.06.008 Cheung CCC, Zheng GJ, Li AMY, Richardson BJ, Lam PKS (2001) Relationships between tissue concentration of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis. Aquat Toxicol 52:189–203. doi:10.1016/S0166-445X(00)00145-4 Dabrowska H, Dabrowski K, Meyer-Burgdorff K, Hanke W, Gunther KD (1991) The effect of large doses of vitamin C and magnesium on stress responses in common carp Cyprinus carpio. Comp Biochem Physiol 99A:681–685. doi:10.1016/0300-9629(91)90150-B Datta M, Kaviraj A (2003) Ascorbic acid supplementation of diet for reduction of deltamethrin induced stress in freshwater catfish Clarias gariepinus. Chemosphere 53:883–888. doi:10.1016/S0045-6535(03)00557-5

123

Di Giulio RT, Washburn PC, Wenning RJ, Winston GW, Jewell CS (1989) Biochemical responses in aquatic animals: a review of oxidative stress. Environ Toxicol Chem 8:1103–1123. doi:10.1002/etc.5620081203 Dogan D, Can C, Kocyigit A, Dikilitas M, Taskin A, Bilinc H (2011) Dimethoate-induced oxidative stress and DNA damage in Oncorhynchus mykiss. Chemosphere 84:39–46. doi:10.1016/j.chemosphere.2011.02.087 Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE (1993) Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains. Protein Sci 2(4):489–497. doi:10.1002/pro. 5560020401 Figueiredo-Fernandes A, Ferreira-Cardoso JV, Gar-cia-Santos S, Monteiro SM, Carrola J, Matos P, Fontainhas-Fernandes A (2007) Histopathological changes in liver and gill epithelium of Nile tilapia, Oreochromis niloticus, exposed to waterborne copper. Pesqui Vet Bras 27(3):103–109. doi:10.1590/S0100-736X2007000300004 Finney DJ (1971) Probit analysis, 3rd edn. Cambridge University Press, Cambridge, pp 50–80 Firat O, Cogun Y, Yuzereroglu TA, Gok G, Firat O, Kargin F, Kotemen Y (2011) A comparative study on the effect of pesticide (Cypermethrin) and two metals (copper, lead) to serum biochemistry of Nile tilapia, Oreochromis niloticus. Fish Physiol Biochem 37(3):657–666. doi:10.1007/s10695011-9466-3 Garcia-Santos S, Fontainhas-Fernandes A, Wilson JM (2006) Cadmium tolerance in the Nile tilapia (Oreochromis niloticus) following acute exposure: assessment of some ionoregulatory parameters. Environ Toxicol 1:33–46. doi:10. 1002/tox.20152 Gernho¨fer M, Pawet M, Schramm M, Mu¨ller E, Triebskorn R (2001) Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated stream. J Aquat Ecosyst Stress Recover 8:241–260. doi:10.1023/A:1012958804442 Gill TS, Leitner G, Porta S, Epple H (1993) Response of plasma cortisol to environmental cadmium in the eel, Anguilla rostraralesueur. J Comp Biochem Physiol 104:489–495. doi:10.1016/0742-8413(93)90023-E Habig WH, Pabst MN, Bjacoby W (1974) Glutathion, transferase, the first enzymatic step in mercatpopunc acid formation. J Biol Chem 249:7130–7138 Halver JE (1995) Vitamin requirement study techniques. J Appl Ichthyol 11:215–224. doi:10.1111/j.1439-0426.1995. tb00021.x Hardie LJ, Fletcher TC, Secombes CJ (1991) The effect of dietary vitamin C on the immune response of the Atlantic salmon (Salmo salar L.). Aquaculture 21:39–53. doi:10. 1016/0044-8486(91)90087-N Iwama GK, Vijayan MM, Forsyth RB, Ackerman PA (1999) Heat shock proteins and physiological stress in fish. Am Zool 39:901–909. doi:10.1093/icb/39.6.901 Jacobes E, Kasten PL, Demoott WR, Wolfson WL (1990) Laboratory test handbook, 2nd edn. Williams and Wilkins, Baltimore Kaplan A, Szabo LL (1983) Clinical chemistry: interpretation and techniques, 2nd edn. Lea & Febiger, Philadelphia, p 157 Knobil K (1980) Hormonal researches. Prog Horm Res 36: 52–58

Fish Physiol Biochem Korkmaz N, Cengiz El, Unlu E, Uysal E, Yanar M (2009) Cypermethrin-induced histopathological and biochemical changes in Nile tilapia (Oreochromis niloticus) and the protective and recuperative effect of ascorbic acid. Environ Toxicol Pharmacol 28:198–205. doi:10.1016/j.etap.2009. 04.004 Kumar R, Nagpure NS, Kushwaha B, Srivastava SK, Lakra WS (2010) Investigation of the genotoxicity of malathion to freshwater teleost fish Channa punctatus (Bloch) using the micronucleus and comet assay. Arch Environ Contam Toxicol 58(1):123–130. doi:10.1007/s00244-009-9354-3 Kumar N, Antony Jesu Prabhu P, Pal AK, Remya S, Aklakur Md, Rana RS, Gupta S, Raman RP, Jadhao SB (2011) Antioxidant and immuno-hematological status of tilapia (Oreochromis mossambicus) during acute toxicity test of endosulfan. Pestic Biochem Physiol 99:45–52. doi:10. 1016/j.pestbp.2010.10.003 Livingston DR (2001) Contaminate-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar Pollut Bull 42:656–666. doi:10.1016/ S0025-326X(01)00060-1 May JM (2000) How dose ascorbic acid prevent endothelium dysfunction? Free Radic Biol Med 28:14–21 Mazeoud MM, Mazeoud F, Donaldson EM (1977) Primary and secondary effect of stress in fish, some new data with a general review. Trans Am Fish Soc 106:201–212. doi:10. 1577/1548-8659(1977)106\201 Meister RT (1994) Farm chemicals handbook. Meister publishing Co., Willoughby Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175. http://www. jbc.org/content/247/10/3170 Mitchelmore CL, Chipman JK (1998) DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutat Res 399:135–147 Monteiro DA, Almeida JA, Rantin FT, Kalinin AL (2006) Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus exposed to organophosphorus insecticide folisuper 600 (methyl parathion). Comp Biochem Physiol C 143:141–149. doi:10.1016/j.cbpc.2006.01.004 Montero D, Marrero M, Izquierdo MS, Robaina L, Vergara JM, Tort L (1999) Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture 171:269–278. doi:10.1016/S00448486(98)00387-1 Nayak AK, Das BK, Demoott WR, Wolfson WL (2004) The immunosuppressive effect of apermethrin on indian major carp, rohu (Labeo rohita). Fish Shellfish Immunol 16(1): 41–50. doi:10.1016/S1050-4648(03)00029-9 Niki E (1991) Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am J Clin Nutr 54:11195–245. http://www.ncbi.nlm.nih.gov/pubmed/1962557 Nogueira P, Pacheco M, Pereira ML, Mendo S, Rotchell S (2009) Anchoring novel molecular biomarker responses to traditional responses in fish exposed to environmental contamination. Environ Pollut 158:1783–1790. doi:10. 1016/j.envpol.2009.11.007

Ohe T, Watanabe T, Wakabayashi K (2004) Mutagenes in surface water: a review. Mutat Res 567:109–149. doi:10. 1016/j.mrrev.2004.08.003 ¨ zcan Oruc E (2010) Oxidative stress, steroid hormone conO centration and acetylcholinesterase activity in Oreochromis niloticus exposed to chlorpyrifos. Pestic Biochem Physiol 96:160–166. doi:10.1016/j.pestbp.2009.11.005 Pandey S, Nagpure NS, Kumar R, Sharma S, Srivastava SK, Verma MS (2006) Genotoxicity evaluation of acute doses of endosulfan to freshwater teleost Channa punctatus (Bloch) by alkaline single cell gel electrophoresis. Ecotoxicol Environ Saf 65:56–61. doi:10.1016/j.ecoenv.2005. 06.007 Pandey AK, Nagpure NS, Trivedi SP, Kumar R, Kushwaha B (2011) Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis. Mutat Res 726:209–214. doi:10.1016/ j.mrgentox.2011.09.011 Pickering AD (1981) Introduction: the concept of biological stress. In: Pickering AD (ed) Stress and fish. Academic Press, London Ramdas SB (2013) Ascorbic effect on the endosulphan induced alterations in blood glucose level of the freshwater fish, Channa orientalis (Schneider). Central Eur J Exp Biol 2:1–3 Roy RN, Guma BC (1958) Species difference in regard to the biosynthesis of ascorbic acid. Nature 182:319–320. doi:10. 1038/182319a0 Sancho E, Ferrando MD, Gamon M, Andreu-Moliner E (1998) Uptake and elimination kinetics of a pesticide in the liver of the European eel. J Environ Sci Health B 33:83–98. doi:10. 1080/03601239809373131 Sarikaya R, Selve M, Erkoc F (2004) Investigation of acute toxicity of fenitrothion on peppered corydoras (Corydoras paleatus) (Jenyns, 1842). Chemosphere 56:697–700. doi:10.1016/j.chemosphere.2004.04.008 Sepici-Dincel A, Benli ACK, Selvi M, Sarikaya R, Sahin D, Ozkull A, Erkoc F (2009) Sublethal cyfluthrin toxicity to carp (Cyprinus carpio L.) fingerlings: biochemical, haematological, histopathological alteration. Ecotoxicol Environ Saf 72:1433–1439. doi:10.1016/j.ecoenv.2009.01. 008 Sies H (1986) Biochemistry of oxidative stress. Angew Chem Int Ed 25:1058–1071 Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. doi:10.1016/ 0014-4827(88)90265-0 Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47:389–394. doi:10.1016/0003-2697(72)90132-7 Thangavel P, Sumathiral K, Karthikeyan S, Ramaswamy M (2005) Endocrine response of the freshwater teleost, Sarotherodon mossambicus (peters) to dimecron exposure. Chemosphere 61(8):1083–1092. doi:10.1016/j.chemosphere.2005.03.045 Thomaz JM, Martins ND, Monteiro DA, Rantin FT, Kalinin AL (2009) Cardio-respiratory function and oxidative stress biomarkers in Nile tilapia exposed to the organophosphate insecticides trichlorfon (NEGUVON). Ecotoxicol Environ Saf 72:1413–1424. doi:10.1016/j.ecoenv.2008.11.003

123

Fish Physiol Biochem Tietz NW (1994) Textbook of clinical chemistry, 2nd edn. Saunders WB. Company, Philadelphia Trinder P (1969) Determination of blood glucose using 4-Aminophenazone as oxygen acceptor. J Clin Pathol 22(2):246 Tripathi G, Shasmal J (2010) Reparation of chlorpyrifos-induced impairment by thyroxine and vitamin C in fish. Ecotoxicol Environ Saf 73:1397–1401. doi:10.1016/j.ecoenv.2010.07. 022 Vani T, Saharan N, Mukherjee SC, Ranjan R, Kumar R, Brahmchari RK (2011) Deltamethrin induced alteration of haematological and biochemical parameters in fingerlings of Catla catla (Ham.) and their amelioration by dietary supplement of vitamin C. Pestic Biochem Physiol 101:16–20. doi:10.1016/j.pestbp.2011.05.007 Wendelaar Bonga SE (1997) The stress response in fish. Physiol Rev 77:591–625

123

Wilhelm-Filho D, Giulivi C, Boveris A (1993) Antioxidant defences in marine fish. I. Teleosts. Comp Biochem Physiol C 106:409–413. doi:10.1016/0742-8413(93)90154-D Winkalar EU, Santosh TRM, Machdo-Neto JG, Martinez CBR (2007) Acute lethal and sublethal effects of neem leaf extracts on neotropical freshwater fish. Prochilodus lineatus. Comp Biochem Physiol C 145:236–244 Worthing CR (1987) The pesticide manual: a world compendium, 8th edn. Published by the British Crop Protection Council, Hampshire, UK Yang Y, Cheng JZ, Singhal SS, Sain M, Pandya U, Awasthu S, Awasthi YC (2001) Role of glutathione S-transverse in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation. J Biol Chem 276:19220–19230. doi:10.1074/ jbc.M100551200