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The role of vitamin C as antioxidant in protection of oxidative stress induced by imidacloprid
Food and Chemical Toxicology 48 (2010) 215–221
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The role of vitamin C as antioxidant in protection of oxidative stress induced by imidacloprid Kawther S. EL-Gendy a,*, Nagat M. Aly b, Fatma H. Mahmoud b, Anter Kenawy b, Abdel Khalek H. El-Sebae a a b
Dept. of Pesticide Chemistry, Faculty of Agriculture, Alexandria, Egypt Pesticide Central Lab., Agriculture Research Center, Alexandria, Egypt
a r t i c l e
i n f o
Article history: Received 17 May 2009 Accepted 1 October 2009
Keywords: Imidacloprid Oxidative stress Lipid peroxidation Vitamin C Antioxidant enzymes
a b s t r a c t Pesticides may induce oxidative stress leading to generate free radicals and alternate antioxidant or oxygen free radical scavenging enzyme system. This study was conducted to investigate the acute toxicity of imidacloprid toward male mice and the oxidative stress of the sublethal dose (1/10 LD50) on the lipid peroxidation level (LPO), reduced glutathione content (GSH) and activities of the antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), and glutathione-S-transferase (GST). Also, the protective effect of vitamin C (200 mg/kg bw) 30 min before or after administration of imidacloprid were investigated. The results demonstrated that the median lethal dose (LD50) of imidacloprid after 24 h was 149.76 mg/kg bw. The oral administration of 14.976 mg/kg imidacloprid significantly caused elevation in LPO level and the activities of antioxidant enzymes including CAT, SOD, GPx and GST. However, G6PD activity remained unchanged, while the level of GSH content was decreased. In addition, the results showed that vitamin C might ameliorate imidacloprid-induced oxidative damage by decreasing LPO and altering antioxidant defense system in liver. The protective effect of the pre-treatment with vitamin C against imidacloprid-induced oxidative stress in liver mice is better than the post-treatment. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Imidacloprid is relatively a new pesticide which belongs to the class of the neonicotinoid compounds. It is registered to control insect pests on agricultural and nursery crops, structural pests and parasites on companion animals (USEPA, 1994). The neonicotinoids are related to nicotine in their structure and site of action at the nicotinic acetylcholine receptor (Tomizawa and Casida, 2005). Recent findings indicated that toxic manifestations induced by pesticides may be associated with the enhanced production of reactive oxygen species (ROS), which give an explanation for the multiple types of toxic responses. The production of ROS is to be caused by a mechanism in which xenobiotics, toxicants and pathological conditions may produce oxidative stress and induce various tissue damage i.e., liver, kidney and brain (Dwivedi et al., 1998; Oncu et al., 2002; Yu et al., 2008). Oxidative stress occurs when the production of ROS overrides the antioxidant capacity in the target cell, resulting in the damage of macromolecules such as nucleic acids, lipids and proteins causing alterations in the target cell function and leading to cell death (Stephan et al., 1997). * Corresponding author. Tel.: 002 03 5905029; fax: 002 03 5902766. E-mail address: [email protected] (K.S. EL-Gendy). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.10.003
Malondialdehyde (MDA) is the end product of lipid peroxidation resulting from the interaction between ROS and cellular or sub cellular membranes (Aslan et al., 1997). The cells have different mechanisms to alleviate oxidative stress and repair damaged macromolecules. The primary defense is offered by enzymatic and non-enzymatic antioxidants, which have been shown to scavenge free radicals and ROS. The antioxidant enzymes; CAT, SOD, glutathione reductase (GR), GPx, G6PD, and GST, and the non-enzymatic antioxidants including GSH, have been shown to be significantly affected by pesticides exposure (El-Gendy et al., 1990; Banerjee et al., 2001; Bebe and Panemangalore, 2005). Non-enzymatic antioxidants, can prevent the uncontrolled formation of free radicals or inhibit their reaction with biological sites, also the destruction of most free radicals rely on the oxidation of endogenous antioxidants mainly by scavenging and reducing molecules (Attia and El-Demerdash, 2002; Zama et al., 2007). Vitamin C is thought to be an important water soluble antioxidant which is reported to neutralize ROS and reduce the oxidative stress (Oncu et al., 2002; Bindhumol et al., 2003; Verma et al., 2007; Yu et al., 2008). Previous studies from our laboratory have shown that environmental contaminants can alter antioxidant system in fish (El-Gendy et al., 1990; Aly, 2005a), snails (Salama et al., 2005) and in rat (Osman et al., 2000). However, there was no reported study about
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the effect of imidacloprid on oxidative stress in animals. Therefore, the present study was planned to investigate the effect of imidacloprid on the oxidative stress biomarkers in the liver of male mice and to study the role of vitamin C in overcoming the oxidative damage induced in male mice, if administered either before or following the exposure to imidacloprid. 2. Materials and methods 2.1. Chemicals Imidacloprid [1-(6-chloro-3-pyridin-3-methyl) N-nitroimidazolidin-2-ylidenamine], 95% technical grade was obtained from Jiangzoue Agrostar Company, China. Vitamin C (L-3-ketothreohexuronic acid lactone), was purchased from El-Nassr Company for Pharmaceutical Industries, Egypt. Other chemicals used in this study were of the highest purified grades available purchased from Sigma and Merck Chemical Companies. 2.2. Animals and treatment Male Swiss albino mice (Mus musculus) were obtained from a colony raised in the animal house of the Faculty of Agriculture, Alex. Univ. Animals were housed in stainless steel cages and maintained on a 12 h. light/dark cycle, 20 ± 2 °C and 50–70% relative humidity. Food and water were provided ad libitum. Different concentrations of imidacloprid were dissolved in corn oil and were administered to mice orally with a single dose of imidacloprid. Mice serving as control had received corn oil only. After 24 h the toxicity of imidacloprid was calculated according to Weill (1952) and was expressed as LD50 value and its confidence limits. The effect of imidacloprid on the oxidative stress biomarkers in the liver of male mice and the role of vitamin C were studied by dividing the animals into five groups, each including four animals and were treated orally as follows: Group 1: Corn oil (5 ml/kg) and used as control. Group 2: Vitamin C (200 mg/kg bw). Group 3: Imidacloprid alone in a dose equivalent to 1/10 LD50. Group 4: Vitamin C (200 mg/kg bw), 30 min before administration of imidacloprid (1/10 LD50) and used as pre-treatment group. Group 5: Vitamin C (200 mg/kg bw), 30 min after administration of imidacloprid (1/10 LD50) and used as post-treatment group. 2.3. Biochemical parameters The animals were sacrificed 24 h. after imidacloprid treatment. Livers were quickly removed, and immediately rinsed in ice saline. The livers were homogenized and used for the determination of LPO level and GSH content. The liver homogenates were centrifuged at 8000g for 30 min. then the supernatants were used for the measurement of antioxidant enzyme activities (CAT, SOD, GPx, G6PD and GST).
Activity of SOD is expressed as units/mg protein. One unit of SOD activity was defined as the amount of the enzyme required for 50% inhibition of the adrenaline– adrenochrome reaction. 2.3.5. GPx activity GPx catalyzes the reduction of hydroperoxides by utilizing GSH. Determination of GPx activity is carried out according to the method of Chiu et al. (1976). The activity of this enzyme is estimated by measurement of the residual reduced glutathione remaining after the action of the enzyme with the Ellman’s reagent in the presence of cumene hydroperoxide as a secondary substrate. The specific activity of this enzyme is expressed as OD/mg protein/min. 2.3.6. G6PD activity The activity of this enzyme was determined according to the method of Glock and Mclean (1953). The principle of this method is that the reduction of NADP+ to NADPH results in the appearance of an absorption band at 340 nm. In the presence of saturating concentrations of glucose-6-phosphate and NADP+ the rate change of absorbance at this wave length is proportional to the enzyme concentration. G6PH activity was calculated as OD/mg protein/min. 2.3.7. GST activity GST activity was assayed using 4-chloro,1-3-dinitrobenzene (CDNB) as a substrate by the procedure of Vessey and Boyer (1984). The assay was conducted by monitoring the appearance of the conjugated complex CDNB and GSH at 340 nm. The specific activity of this enzyme is calculated as OD/mg protein/min. 2.3.8. Protein determination The total protein concentration of supernatant was determined according to Lowry et al. (1951). 2.4. Statistical analysis All data were expressed as mean ± standard deviation (SD). The data was analyzed using one-way analysis of variance (ANOVA) followed by the Student-Newman Keuls test to determine significance between different groups. The criterion for statistical significance was set at p < 0.05. These tests were performed using a computer software CoStat program, version 2, 1986.
3. Results 3.1. Toxicity study Data obtained from the acute toxicity study was evaluated according to the method of Weill (1952). The 24 h acute oral LD50 value of imidacloprid against male mice was 149.76 mg/ kg bw with confidence limit ± 0.145. 3.2. Biochemical findings
2.3.1. LPO level Lipid peroxidation process is determined by the thiobarbituric acid (TBA) method which estimates the malondialdehyde formation (MDA) according to Nair and Turner (1984). Briefly, a 0.33 ml of liver homogenate was mixed well with 3 ml of TBA reagent. The mixture was incubated for 20 min in a boiling water bath. After cooling, the mixture was centrifuged at 3000g for 20 min. The MDA level was measured spectrophotometrically at 532 nm. Lipid peroxidation is expressed as n moles MDA/g tissue. 2.3.2. GSH content Reduced GSH as non-enzymatic antioxidant was measured according to Owens and Belcher (1965). Determination of GSH is based on the reaction of 5,0 5-dithiobis2-nitrobenzoic acid (DTNB) with GSH which yield a yellow colored chromophore; 5-thio-nitrobenzoic acid (TNB) with a maximum absorbance at 412 nm. The amount of reduced glutathione present in the liver sample was calculated as lg/g tissue. 2.3.3. CAT activity CAT is an enzyme that scavenges hydrogen peroxide and converts it to water and oxygen molecule. The activity of this enzyme was dependent on the ultraviolet absorption of the hydrogen peroxide solution, which can be measured at 240 nm (Beers and Sizer, 1952). Activity of CAT is expressed as units/mg protein. 2.3.4. SOD activity SOD catalyzes the dismutation of superoxide radicals. Measurement of SOD activity was based on the adrenaline that transforms spontaneously to adrenochrome in the presence of air at pH 10.2 according to Misra and Fridovich (1972).
The in vivo effects of the single dose of one tenth LD50 imidacloprid (14.976 mg/kg bw) on lipid peroxidation and the antioxidant defense system in the male mice liver and also, the protective effects of vitamin C against oxidative damage induced by imidacloprid were summarized in Figs. 1–7. As shown in all experiments, no death was observed in any of the experimental groups. In addition, vitamin C treatment in the absence of imidacloprid showed no significant effect on all tested parameters when compared to the control. 3.2.1. LPO level MDA is a marker of oxidative lipid damage and it is a major oxidative product of peroxidized polyunsaturated fatty acid (Zhang et al., 2004). MDA level significantly increased in the imidacloprid-treated group compared to the control (Fig. 1). Pre supplementation with vitamin C to the mice intoxicated with imidacloprid normalized or neutralized the pesticide effect. While, post supplementation with vitamin C decreased the effect of the pesticide but could not reach the control. 3.2.2. GSH content It was significantly decreased in imidacloprid-intoxicated animals compared to the control group (p < 0.05). Either pre- or
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800 a
nmoles MDA / g tissue
700
ab b
600 500 400 300 200 100 0 Corn oil
Vit.C
Imid.
Pre treat.
Post treat.
Treatments Fig. 1. Lipid peroxidation levels in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
8000 b
b
µg GSH / g tissue
a 6000
4000
2000
0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 2. GSH levels in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
a
1.6 1.4
ab
units / mg protein
1.2 ab
1 0.8 0.6 0.4 0.2 0 Corn oil
Vit.C
Imid.
Pre treat.
Post treat.
Treatments Fig. 3. CAT activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
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a
30 25
units / mg protein
b
b
20 15 10 5 0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 4. SOD activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
0.7
a
OD / mg protein.min
0.6
b
a
Pre treat.
Post treat.
0.5 0.4 0.3 0.2 0.1 0 Corn oil
Vit. C
Imid.
Treatments Fig. 5. Gpx activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
0.1
OD / mg protein.min
0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 6. G6PD activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
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1 0.9
a
OD / mg protein.min
0.8
a
0.7 b 0.6 0.5 0.4 0.3 0.2 0.1 0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 7. GST activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and/or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
post-treatment with vitamin C in imidacloprid-treated mice reduced the toxicity of this pesticide and maintains the levels of GSH to that of control. There were no significant changes in GSH levels between pre or post-treatment and control (Fig. 2). 3.2.3. CAT activity It was significantly increased in the imidacloprid-treated group to more than 2.3-folds when compared with the control group (p < 0.05). Pre- and post-treatments with vitamin C showed a significant decrease in the activity of CAT as compared to imidacloprid-intoxicated mice, but could not reach the control value (Fig. 3). 3.2.4. SOD activity Imidacloprid significantly increased the activity of liver SOD. Vitamin C significantly decreased the SOD activity in mice treated with the tested pesticide (Fig. 4). There were no significant differences between the two vitamin C treatments (pre or post) in the protection of the cells. 3.2.5. GPx activity The activity of GPx was significantly increased in mice intoxicated with imidacloprid and recording 1.9 times activity higher than the control group. The GPx activity was significantly decreased in pre-treatment with vitamin C comparing with the imidacloprid alone, while no significant modification was revealed in GPx activity between post-treatment with vitamin C and imidacloprid alone (Fig. 5). 3.2.6. G6PD activity No significant changes were observed in the imidacloprid or vitamin C treated groups when compared with the control (Fig. 6). 3.2.7. GST activity Fig. 7 shows that imidacloprid treatment caused a significant increase in GST activity (1.6-fold). Pre-treatment with vitamin C in imidacloprid-intoxicated mice significantly reduced the pesticide effect while, post-treatment insignificantly reduced the activity of GST compared with imidacloprid group.
4. Discussion The widespread use of pesticides in public health and agriculture programs has caused severe environmental pollution and health hazards, including cases of severe acute and chronic human poisoning (Abdollahi et al., 2004). It is well known that the oral LD50 value of any pesticide is highly dependent on the pesticide and the organism. In this study, the LD50 of imidacloprid against male mice was 149.76 mg/kg. This result is in accordance with Bomann (1989) and Kidd and James (1991) who found that the oral LD50 of imidacloprid against male mice was 130 and 131 mg/kg, respectively. Imidacloprid is a relatively new insecticide, first registered in the US in 1994 (USEPA, 1994), and its active ingredient was considered according to the World Health Organization (WHO) scheme to be moderately toxic. Nicotinoids are classified by the EPA system as toxicity class II and/or class III agents, because they block a specific neuron pathway that is more abundant in insects than warm blooded animals, so, the toxicity of these insecticides is more selective to insects than mammals (Meister, 1995; USEPA, 1994). Pesticides can induce oxidative stress by generation of free radicals that might cause lipid peroxidation, alternations in membrane fluidity, DNA damage and finally carcinogenic effects (Singh et al., 2006; Zama et al., 2007). In vertebrates, the liver is a highly metabolically active organ, with a high activity of antioxidants and associated enzymes, so that it is the main organ responsible for detoxification of xenobiotics (Hinderer and Menzer, 1976; El-Gendy et al., 1990). In the present study, imidacloprid treatment to mice indicated a marked increase in the hepatic LPO. This result coincides greatly with Gultekin et al. (2001), Oncu et al. (2002), Verma et al. (2007) and Khan and Kour (2007). LPO is the process of oxidative degradation of polyunsaturated fatty acids (PUFA) and its occurrence in biological membranes causes impaired membrane function, structural integrity, decrease in membrane fluidity and inactivation of a several membrane bound enzymes. It is plausible to speculate that imidacloprid treatment may result in peroxidation of PUFA, leading to the degradation of phospholipids and ultimately result in cellular deterioration. The present result strengthens this hypothesis and suggests that induction of oxidative stress is perhaps the central
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mechanism by which such tested pesticide exerts their cytotoxic effects (Khan and Kour, 2007). GSH is an important naturally occurring antioxidant, which prevents free radical damage and helps detoxification by conjugating with chemicals and also, it acts as an essential cofactor for antioxidant enzymes including GPx, GR and GST (Hayes et al., 2005). Under oxidative stress, GSH is consumed by GSH related enzymes to detoxify peroxides produced due to increased lipid peroxidation (Cathcart, 1985). In this study, the observed increase in lipid peroxidation and a concomitant depletion in GSH level, suggests that the increased peroxidation may be a consequence of depleted GSH stores. A marked increase in the activities of the antioxidant enzymes SOD and CAT were observed after imidacloprid treatments since they are the first lines of defense against the oxy free radicals. Such increase in the activities of these enzymes has been attributed to the defense mechanisms against oxidative stress in the process of attempted cellular repair. These results are parallel to the results of many authors; i.e., Gultekin et al. (2001), Khan and Kour (2007) and Yu et al. (2008). The biological function of GPx is to reduce lipid hydroperoxides conversion to their corresponding alcohols and to reduce free hydrogen peroxide reaction (Verma et al., 2007). In this experiment, GPx activity was significantly increased in mice treated with imidacloprid. This result is in agreement with many authors (Gultekin et al., 2001; Zama et al., 2007). G6PD is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH (required for conversion of oxidized glutathione to the reduced form) and essential for the protection of cells against oxidative damage (Marks, 1961). In the present study, G6PD remained unchanged in mice liver after imidacloprid treatment. Literature data relative to the effects of pesticides exposure on G6PD activity are different. Some studies reported that G6PD activity was decreased in animals treated with OP compounds (Singh et al., 2006; Verma et al., 2007). On the other hands, Brocardo et al. (2005) found that there was no changes in G6PD in the hippocampus of rats treated with malathion, while it was slightly increased in the cerebral cortical. Also, the treatment of the pregnant rats with glyphosate over 21 days produced an increase in G6PD in the brain of the fetuses, while no changes were observed in the pregnant rats (Daruich et al., 2001). The differences found may be due to the pesticide exposure employed regime, the tissue distribution, or the age of the animals. In addition, we also observed significant enhanced activity of GST, which is understandable in the light of depressed GSH levels. GST is a detoxifying enzyme that catalyzes the conjugation of a variety of electrophillic substrates to the thiol group of GSH, producing less toxic forms (Hayes et al., 2005). El-Gendy (1990), Aly (2005b) and Singh et al. (2006) reported that pesticides; dithiocarb, cypermethrin, dimethoate and chlorpyrifos treatment caused a significant increase in GST activity. This induction may be due to the GSH and glutathione dependent enzymes system that provide major protection against the toxic agents. The primary role of vitamin C is to neutralize free radicals, since ascorbic acid is water soluble, it can work both inside and outside the cells to combat free radical damage. The free radicals will seek out an electron to regain their stability, vitamin C is an excellent source of electrons; therefore, it can ‘‘donate electrons to free radicals such as hydroxyl and superoxide radicals and quench their reactivity” (Bendich, 1990; Bindhumol et al., 2003). The present results showed that vitamin C treatment to imidacloprid-intoxicated animals normalized the most tested antioxidant enzyme activities as well as the level of LPO and GSH content. Also, it is clear from the present results that the pre-treatment with vitamin C is better than the post-treatment.
Pre-treatment with antioxidant vitamins decreased the generation of ROS thus prevents the pesticide induced derangement in the activities of the antioxidant enzymes (Niki et al., 1995). Our data are in agreement with Verma et al. (2007) who found that the pre-treatment with a mixture of vitamins A, E and C has protected rats from chlorpyrifos-induced oxidative stress and suggesting that this treatment alleviates the toxicity of this pesticide. Preor post-treatment with vitamin C normalized the pesticide effect and the enzyme activities reached the control levels. 5. Conclusion In view of our data, it can be concluded that vitamin C supplementation has hepato-protective effects in imidacloprid-induced liver toxicity. Vitamin C can directly and rapidly scavenge free radicals and/or inhibit their formation, additionally; it can act by upregulating endogenous antioxidant defenses. Vitamin C protects the DNA of the cells from damage caused by free radicals; it combats the effects of many toxins, including pesticides and heavy metals. It also, fights off these pollutants by stimulating enzymes in the liver that detoxify the body. Staying on top of oxidative stress is a necessity in our increasingly toxic world. Taking care to avoid those toxins as much as possible and to enrich our diets with life-giving antioxidants is a wise step to take in our endless quest for wellness. Results of the present study clearly indicate that prior feeding of antioxidant vitamin C combats oxidative stress induced by imidacloprid in mice liver. Thus, sufficient dietary intake of vitamin C by individuals who regularly come in contact with these pesticides is beneficial in combating the adverse effects of imidacloprid. Conflict of interest The authors declare that there are no conflicts of interest. References Abdollahi, M., Ranjbar, A., Shadnia, S., Nikfar, S., Rezaie, A., 2004. Pesticides and oxidative stress: a review. Med. Sci. Monit. 10, 141–147. Aly, N.M., 2005a. Biochemical effects of certain pesticides on common carp (Cyprinus carpio L). J. Adv. Agric. Res. 10 (2), 543–556. Aly, N.M., 2005b. Earthworm as a biomarker for pesticides toxicity. Alex. Sci. Exch. 26, 329–335. Aslan, R., Sekeroglu, M.R., Gultekin, F., 1997. Blood lipoperoxidation and antioxidant enzymes in healthy individuals. Relation to age, sex, habits, life style and environment. J. Environ. Sci. Health A 32, 2101–2109. Attia, A.M., El-Demerdash, F.M., 2002. Potent protective effects of melatonin on cypermethrin-induced oxidative damage in rats in vivo. J. Pest. Cont. Environ. Sci. 10 (2), 91–104. Banerjee, B.D., Seth, V., Ahmed, R.S., 2001. Pesticides induced oxidative stress: perspectives and trends. Rev. Environ. Health 16, 1–40. Bebe, F.N., Panemangalore, M., 2005. Pesticides and essential minerals modify endogenous antioxidant and cytochrome P450 in tissues of rats. Environ. Sci. Health Part (B) 40, 769–784. Beers Jr., R.F., Sizer, I.W., 1952. Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. Biol. Chem. 195, 133–140. Bendich, A., 1990. Antioxidant Micronutrients and Immune Responses, Micronutrients and Immune Functions. New York Academy of Sciences, New York. p. 175. Bindhumol, V., Chitra, K.C., Mathur, P.P., 2003. A induces reactive oxygen species generation in the liver of male rats. Toxicology 188, 117–124. Bomann, W., 1989. NTN33893-Study for acute oral toxicity to mice. Unpublished Report from Bayer AG, report No. 18593, dated 15 December 1989, GLP, unpublished. Submitted WHO by Bayer AG, Mannheim, Germany. Brocardo, P.S., Pandolfo, P., Takahashi, R.N., Rodrigues, A.L.S., Dafre, A.L., 2005. Antioxidants defenses and lipid peroxidation in the cerebral cortex and hippocampus following acute exposure to malathion and/or zinc chloride. Toxicology 207, 283–291. Cathcart, R.F., 1985. Vitamin C: the nontoxic, nonrate-limited, antioxidant free radical scavenger. Med. Hypo. 18, 61–77. Chiu, D.T.Y., Stults, F.H., Tappal, A.L., 1976. Purification and properties of rat lung soluble glutathione peroxidase. Biochem. Biophys. Acta 445, 558–566. CoStat Program 1986, Version 2, Cohort Software.
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The role of vitamin C as antioxidant in protection of oxidative stress induced by imidacloprid Kawther S. EL-Gendy a,*, Nagat M. Aly b, Fatma H. Mahmoud b, Anter Kenawy b, Abdel Khalek H. El-Sebae a a b
Dept. of Pesticide Chemistry, Faculty of Agriculture, Alexandria, Egypt Pesticide Central Lab., Agriculture Research Center, Alexandria, Egypt
a r t i c l e
i n f o
Article history: Received 17 May 2009 Accepted 1 October 2009
Keywords: Imidacloprid Oxidative stress Lipid peroxidation Vitamin C Antioxidant enzymes
a b s t r a c t Pesticides may induce oxidative stress leading to generate free radicals and alternate antioxidant or oxygen free radical scavenging enzyme system. This study was conducted to investigate the acute toxicity of imidacloprid toward male mice and the oxidative stress of the sublethal dose (1/10 LD50) on the lipid peroxidation level (LPO), reduced glutathione content (GSH) and activities of the antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), and glutathione-S-transferase (GST). Also, the protective effect of vitamin C (200 mg/kg bw) 30 min before or after administration of imidacloprid were investigated. The results demonstrated that the median lethal dose (LD50) of imidacloprid after 24 h was 149.76 mg/kg bw. The oral administration of 14.976 mg/kg imidacloprid significantly caused elevation in LPO level and the activities of antioxidant enzymes including CAT, SOD, GPx and GST. However, G6PD activity remained unchanged, while the level of GSH content was decreased. In addition, the results showed that vitamin C might ameliorate imidacloprid-induced oxidative damage by decreasing LPO and altering antioxidant defense system in liver. The protective effect of the pre-treatment with vitamin C against imidacloprid-induced oxidative stress in liver mice is better than the post-treatment. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Imidacloprid is relatively a new pesticide which belongs to the class of the neonicotinoid compounds. It is registered to control insect pests on agricultural and nursery crops, structural pests and parasites on companion animals (USEPA, 1994). The neonicotinoids are related to nicotine in their structure and site of action at the nicotinic acetylcholine receptor (Tomizawa and Casida, 2005). Recent findings indicated that toxic manifestations induced by pesticides may be associated with the enhanced production of reactive oxygen species (ROS), which give an explanation for the multiple types of toxic responses. The production of ROS is to be caused by a mechanism in which xenobiotics, toxicants and pathological conditions may produce oxidative stress and induce various tissue damage i.e., liver, kidney and brain (Dwivedi et al., 1998; Oncu et al., 2002; Yu et al., 2008). Oxidative stress occurs when the production of ROS overrides the antioxidant capacity in the target cell, resulting in the damage of macromolecules such as nucleic acids, lipids and proteins causing alterations in the target cell function and leading to cell death (Stephan et al., 1997). * Corresponding author. Tel.: 002 03 5905029; fax: 002 03 5902766. E-mail address: [email protected] (K.S. EL-Gendy). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.10.003
Malondialdehyde (MDA) is the end product of lipid peroxidation resulting from the interaction between ROS and cellular or sub cellular membranes (Aslan et al., 1997). The cells have different mechanisms to alleviate oxidative stress and repair damaged macromolecules. The primary defense is offered by enzymatic and non-enzymatic antioxidants, which have been shown to scavenge free radicals and ROS. The antioxidant enzymes; CAT, SOD, glutathione reductase (GR), GPx, G6PD, and GST, and the non-enzymatic antioxidants including GSH, have been shown to be significantly affected by pesticides exposure (El-Gendy et al., 1990; Banerjee et al., 2001; Bebe and Panemangalore, 2005). Non-enzymatic antioxidants, can prevent the uncontrolled formation of free radicals or inhibit their reaction with biological sites, also the destruction of most free radicals rely on the oxidation of endogenous antioxidants mainly by scavenging and reducing molecules (Attia and El-Demerdash, 2002; Zama et al., 2007). Vitamin C is thought to be an important water soluble antioxidant which is reported to neutralize ROS and reduce the oxidative stress (Oncu et al., 2002; Bindhumol et al., 2003; Verma et al., 2007; Yu et al., 2008). Previous studies from our laboratory have shown that environmental contaminants can alter antioxidant system in fish (El-Gendy et al., 1990; Aly, 2005a), snails (Salama et al., 2005) and in rat (Osman et al., 2000). However, there was no reported study about
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the effect of imidacloprid on oxidative stress in animals. Therefore, the present study was planned to investigate the effect of imidacloprid on the oxidative stress biomarkers in the liver of male mice and to study the role of vitamin C in overcoming the oxidative damage induced in male mice, if administered either before or following the exposure to imidacloprid. 2. Materials and methods 2.1. Chemicals Imidacloprid [1-(6-chloro-3-pyridin-3-methyl) N-nitroimidazolidin-2-ylidenamine], 95% technical grade was obtained from Jiangzoue Agrostar Company, China. Vitamin C (L-3-ketothreohexuronic acid lactone), was purchased from El-Nassr Company for Pharmaceutical Industries, Egypt. Other chemicals used in this study were of the highest purified grades available purchased from Sigma and Merck Chemical Companies. 2.2. Animals and treatment Male Swiss albino mice (Mus musculus) were obtained from a colony raised in the animal house of the Faculty of Agriculture, Alex. Univ. Animals were housed in stainless steel cages and maintained on a 12 h. light/dark cycle, 20 ± 2 °C and 50–70% relative humidity. Food and water were provided ad libitum. Different concentrations of imidacloprid were dissolved in corn oil and were administered to mice orally with a single dose of imidacloprid. Mice serving as control had received corn oil only. After 24 h the toxicity of imidacloprid was calculated according to Weill (1952) and was expressed as LD50 value and its confidence limits. The effect of imidacloprid on the oxidative stress biomarkers in the liver of male mice and the role of vitamin C were studied by dividing the animals into five groups, each including four animals and were treated orally as follows: Group 1: Corn oil (5 ml/kg) and used as control. Group 2: Vitamin C (200 mg/kg bw). Group 3: Imidacloprid alone in a dose equivalent to 1/10 LD50. Group 4: Vitamin C (200 mg/kg bw), 30 min before administration of imidacloprid (1/10 LD50) and used as pre-treatment group. Group 5: Vitamin C (200 mg/kg bw), 30 min after administration of imidacloprid (1/10 LD50) and used as post-treatment group. 2.3. Biochemical parameters The animals were sacrificed 24 h. after imidacloprid treatment. Livers were quickly removed, and immediately rinsed in ice saline. The livers were homogenized and used for the determination of LPO level and GSH content. The liver homogenates were centrifuged at 8000g for 30 min. then the supernatants were used for the measurement of antioxidant enzyme activities (CAT, SOD, GPx, G6PD and GST).
Activity of SOD is expressed as units/mg protein. One unit of SOD activity was defined as the amount of the enzyme required for 50% inhibition of the adrenaline– adrenochrome reaction. 2.3.5. GPx activity GPx catalyzes the reduction of hydroperoxides by utilizing GSH. Determination of GPx activity is carried out according to the method of Chiu et al. (1976). The activity of this enzyme is estimated by measurement of the residual reduced glutathione remaining after the action of the enzyme with the Ellman’s reagent in the presence of cumene hydroperoxide as a secondary substrate. The specific activity of this enzyme is expressed as OD/mg protein/min. 2.3.6. G6PD activity The activity of this enzyme was determined according to the method of Glock and Mclean (1953). The principle of this method is that the reduction of NADP+ to NADPH results in the appearance of an absorption band at 340 nm. In the presence of saturating concentrations of glucose-6-phosphate and NADP+ the rate change of absorbance at this wave length is proportional to the enzyme concentration. G6PH activity was calculated as OD/mg protein/min. 2.3.7. GST activity GST activity was assayed using 4-chloro,1-3-dinitrobenzene (CDNB) as a substrate by the procedure of Vessey and Boyer (1984). The assay was conducted by monitoring the appearance of the conjugated complex CDNB and GSH at 340 nm. The specific activity of this enzyme is calculated as OD/mg protein/min. 2.3.8. Protein determination The total protein concentration of supernatant was determined according to Lowry et al. (1951). 2.4. Statistical analysis All data were expressed as mean ± standard deviation (SD). The data was analyzed using one-way analysis of variance (ANOVA) followed by the Student-Newman Keuls test to determine significance between different groups. The criterion for statistical significance was set at p < 0.05. These tests were performed using a computer software CoStat program, version 2, 1986.
3. Results 3.1. Toxicity study Data obtained from the acute toxicity study was evaluated according to the method of Weill (1952). The 24 h acute oral LD50 value of imidacloprid against male mice was 149.76 mg/ kg bw with confidence limit ± 0.145. 3.2. Biochemical findings
2.3.1. LPO level Lipid peroxidation process is determined by the thiobarbituric acid (TBA) method which estimates the malondialdehyde formation (MDA) according to Nair and Turner (1984). Briefly, a 0.33 ml of liver homogenate was mixed well with 3 ml of TBA reagent. The mixture was incubated for 20 min in a boiling water bath. After cooling, the mixture was centrifuged at 3000g for 20 min. The MDA level was measured spectrophotometrically at 532 nm. Lipid peroxidation is expressed as n moles MDA/g tissue. 2.3.2. GSH content Reduced GSH as non-enzymatic antioxidant was measured according to Owens and Belcher (1965). Determination of GSH is based on the reaction of 5,0 5-dithiobis2-nitrobenzoic acid (DTNB) with GSH which yield a yellow colored chromophore; 5-thio-nitrobenzoic acid (TNB) with a maximum absorbance at 412 nm. The amount of reduced glutathione present in the liver sample was calculated as lg/g tissue. 2.3.3. CAT activity CAT is an enzyme that scavenges hydrogen peroxide and converts it to water and oxygen molecule. The activity of this enzyme was dependent on the ultraviolet absorption of the hydrogen peroxide solution, which can be measured at 240 nm (Beers and Sizer, 1952). Activity of CAT is expressed as units/mg protein. 2.3.4. SOD activity SOD catalyzes the dismutation of superoxide radicals. Measurement of SOD activity was based on the adrenaline that transforms spontaneously to adrenochrome in the presence of air at pH 10.2 according to Misra and Fridovich (1972).
The in vivo effects of the single dose of one tenth LD50 imidacloprid (14.976 mg/kg bw) on lipid peroxidation and the antioxidant defense system in the male mice liver and also, the protective effects of vitamin C against oxidative damage induced by imidacloprid were summarized in Figs. 1–7. As shown in all experiments, no death was observed in any of the experimental groups. In addition, vitamin C treatment in the absence of imidacloprid showed no significant effect on all tested parameters when compared to the control. 3.2.1. LPO level MDA is a marker of oxidative lipid damage and it is a major oxidative product of peroxidized polyunsaturated fatty acid (Zhang et al., 2004). MDA level significantly increased in the imidacloprid-treated group compared to the control (Fig. 1). Pre supplementation with vitamin C to the mice intoxicated with imidacloprid normalized or neutralized the pesticide effect. While, post supplementation with vitamin C decreased the effect of the pesticide but could not reach the control. 3.2.2. GSH content It was significantly decreased in imidacloprid-intoxicated animals compared to the control group (p < 0.05). Either pre- or
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800 a
nmoles MDA / g tissue
700
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600 500 400 300 200 100 0 Corn oil
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Imid.
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Treatments Fig. 1. Lipid peroxidation levels in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
8000 b
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a 6000
4000
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Treatments Fig. 2. GSH levels in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
a
1.6 1.4
ab
units / mg protein
1.2 ab
1 0.8 0.6 0.4 0.2 0 Corn oil
Vit.C
Imid.
Pre treat.
Post treat.
Treatments Fig. 3. CAT activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
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a
30 25
units / mg protein
b
b
20 15 10 5 0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 4. SOD activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
0.7
a
OD / mg protein.min
0.6
b
a
Pre treat.
Post treat.
0.5 0.4 0.3 0.2 0.1 0 Corn oil
Vit. C
Imid.
Treatments Fig. 5. Gpx activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
0.1
OD / mg protein.min
0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 6. G6PD activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and /or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
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1 0.9
a
OD / mg protein.min
0.8
a
0.7 b 0.6 0.5 0.4 0.3 0.2 0.1 0 Corn oil
Vit. C
Imid.
Pre treat.
Post treat.
Treatments Fig. 7. GST activities in hepatic tissues of male mice orally treated with imidacloprid (1/10 LD50) and/or vitamin C (200 mg/kg bw). Values are expressed as mean ± SD for four animals per group. aP < 0.05 compared to control group. bP < 0.05 compared to imidacloprid group.
post-treatment with vitamin C in imidacloprid-treated mice reduced the toxicity of this pesticide and maintains the levels of GSH to that of control. There were no significant changes in GSH levels between pre or post-treatment and control (Fig. 2). 3.2.3. CAT activity It was significantly increased in the imidacloprid-treated group to more than 2.3-folds when compared with the control group (p < 0.05). Pre- and post-treatments with vitamin C showed a significant decrease in the activity of CAT as compared to imidacloprid-intoxicated mice, but could not reach the control value (Fig. 3). 3.2.4. SOD activity Imidacloprid significantly increased the activity of liver SOD. Vitamin C significantly decreased the SOD activity in mice treated with the tested pesticide (Fig. 4). There were no significant differences between the two vitamin C treatments (pre or post) in the protection of the cells. 3.2.5. GPx activity The activity of GPx was significantly increased in mice intoxicated with imidacloprid and recording 1.9 times activity higher than the control group. The GPx activity was significantly decreased in pre-treatment with vitamin C comparing with the imidacloprid alone, while no significant modification was revealed in GPx activity between post-treatment with vitamin C and imidacloprid alone (Fig. 5). 3.2.6. G6PD activity No significant changes were observed in the imidacloprid or vitamin C treated groups when compared with the control (Fig. 6). 3.2.7. GST activity Fig. 7 shows that imidacloprid treatment caused a significant increase in GST activity (1.6-fold). Pre-treatment with vitamin C in imidacloprid-intoxicated mice significantly reduced the pesticide effect while, post-treatment insignificantly reduced the activity of GST compared with imidacloprid group.
4. Discussion The widespread use of pesticides in public health and agriculture programs has caused severe environmental pollution and health hazards, including cases of severe acute and chronic human poisoning (Abdollahi et al., 2004). It is well known that the oral LD50 value of any pesticide is highly dependent on the pesticide and the organism. In this study, the LD50 of imidacloprid against male mice was 149.76 mg/kg. This result is in accordance with Bomann (1989) and Kidd and James (1991) who found that the oral LD50 of imidacloprid against male mice was 130 and 131 mg/kg, respectively. Imidacloprid is a relatively new insecticide, first registered in the US in 1994 (USEPA, 1994), and its active ingredient was considered according to the World Health Organization (WHO) scheme to be moderately toxic. Nicotinoids are classified by the EPA system as toxicity class II and/or class III agents, because they block a specific neuron pathway that is more abundant in insects than warm blooded animals, so, the toxicity of these insecticides is more selective to insects than mammals (Meister, 1995; USEPA, 1994). Pesticides can induce oxidative stress by generation of free radicals that might cause lipid peroxidation, alternations in membrane fluidity, DNA damage and finally carcinogenic effects (Singh et al., 2006; Zama et al., 2007). In vertebrates, the liver is a highly metabolically active organ, with a high activity of antioxidants and associated enzymes, so that it is the main organ responsible for detoxification of xenobiotics (Hinderer and Menzer, 1976; El-Gendy et al., 1990). In the present study, imidacloprid treatment to mice indicated a marked increase in the hepatic LPO. This result coincides greatly with Gultekin et al. (2001), Oncu et al. (2002), Verma et al. (2007) and Khan and Kour (2007). LPO is the process of oxidative degradation of polyunsaturated fatty acids (PUFA) and its occurrence in biological membranes causes impaired membrane function, structural integrity, decrease in membrane fluidity and inactivation of a several membrane bound enzymes. It is plausible to speculate that imidacloprid treatment may result in peroxidation of PUFA, leading to the degradation of phospholipids and ultimately result in cellular deterioration. The present result strengthens this hypothesis and suggests that induction of oxidative stress is perhaps the central
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mechanism by which such tested pesticide exerts their cytotoxic effects (Khan and Kour, 2007). GSH is an important naturally occurring antioxidant, which prevents free radical damage and helps detoxification by conjugating with chemicals and also, it acts as an essential cofactor for antioxidant enzymes including GPx, GR and GST (Hayes et al., 2005). Under oxidative stress, GSH is consumed by GSH related enzymes to detoxify peroxides produced due to increased lipid peroxidation (Cathcart, 1985). In this study, the observed increase in lipid peroxidation and a concomitant depletion in GSH level, suggests that the increased peroxidation may be a consequence of depleted GSH stores. A marked increase in the activities of the antioxidant enzymes SOD and CAT were observed after imidacloprid treatments since they are the first lines of defense against the oxy free radicals. Such increase in the activities of these enzymes has been attributed to the defense mechanisms against oxidative stress in the process of attempted cellular repair. These results are parallel to the results of many authors; i.e., Gultekin et al. (2001), Khan and Kour (2007) and Yu et al. (2008). The biological function of GPx is to reduce lipid hydroperoxides conversion to their corresponding alcohols and to reduce free hydrogen peroxide reaction (Verma et al., 2007). In this experiment, GPx activity was significantly increased in mice treated with imidacloprid. This result is in agreement with many authors (Gultekin et al., 2001; Zama et al., 2007). G6PD is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH (required for conversion of oxidized glutathione to the reduced form) and essential for the protection of cells against oxidative damage (Marks, 1961). In the present study, G6PD remained unchanged in mice liver after imidacloprid treatment. Literature data relative to the effects of pesticides exposure on G6PD activity are different. Some studies reported that G6PD activity was decreased in animals treated with OP compounds (Singh et al., 2006; Verma et al., 2007). On the other hands, Brocardo et al. (2005) found that there was no changes in G6PD in the hippocampus of rats treated with malathion, while it was slightly increased in the cerebral cortical. Also, the treatment of the pregnant rats with glyphosate over 21 days produced an increase in G6PD in the brain of the fetuses, while no changes were observed in the pregnant rats (Daruich et al., 2001). The differences found may be due to the pesticide exposure employed regime, the tissue distribution, or the age of the animals. In addition, we also observed significant enhanced activity of GST, which is understandable in the light of depressed GSH levels. GST is a detoxifying enzyme that catalyzes the conjugation of a variety of electrophillic substrates to the thiol group of GSH, producing less toxic forms (Hayes et al., 2005). El-Gendy (1990), Aly (2005b) and Singh et al. (2006) reported that pesticides; dithiocarb, cypermethrin, dimethoate and chlorpyrifos treatment caused a significant increase in GST activity. This induction may be due to the GSH and glutathione dependent enzymes system that provide major protection against the toxic agents. The primary role of vitamin C is to neutralize free radicals, since ascorbic acid is water soluble, it can work both inside and outside the cells to combat free radical damage. The free radicals will seek out an electron to regain their stability, vitamin C is an excellent source of electrons; therefore, it can ‘‘donate electrons to free radicals such as hydroxyl and superoxide radicals and quench their reactivity” (Bendich, 1990; Bindhumol et al., 2003). The present results showed that vitamin C treatment to imidacloprid-intoxicated animals normalized the most tested antioxidant enzyme activities as well as the level of LPO and GSH content. Also, it is clear from the present results that the pre-treatment with vitamin C is better than the post-treatment.
Pre-treatment with antioxidant vitamins decreased the generation of ROS thus prevents the pesticide induced derangement in the activities of the antioxidant enzymes (Niki et al., 1995). Our data are in agreement with Verma et al. (2007) who found that the pre-treatment with a mixture of vitamins A, E and C has protected rats from chlorpyrifos-induced oxidative stress and suggesting that this treatment alleviates the toxicity of this pesticide. Preor post-treatment with vitamin C normalized the pesticide effect and the enzyme activities reached the control levels. 5. Conclusion In view of our data, it can be concluded that vitamin C supplementation has hepato-protective effects in imidacloprid-induced liver toxicity. Vitamin C can directly and rapidly scavenge free radicals and/or inhibit their formation, additionally; it can act by upregulating endogenous antioxidant defenses. Vitamin C protects the DNA of the cells from damage caused by free radicals; it combats the effects of many toxins, including pesticides and heavy metals. It also, fights off these pollutants by stimulating enzymes in the liver that detoxify the body. Staying on top of oxidative stress is a necessity in our increasingly toxic world. Taking care to avoid those toxins as much as possible and to enrich our diets with life-giving antioxidants is a wise step to take in our endless quest for wellness. Results of the present study clearly indicate that prior feeding of antioxidant vitamin C combats oxidative stress induced by imidacloprid in mice liver. Thus, sufficient dietary intake of vitamin C by individuals who regularly come in contact with these pesticides is beneficial in combating the adverse effects of imidacloprid. Conflict of interest The authors declare that there are no conflicts of interest. References Abdollahi, M., Ranjbar, A., Shadnia, S., Nikfar, S., Rezaie, A., 2004. Pesticides and oxidative stress: a review. Med. Sci. Monit. 10, 141–147. Aly, N.M., 2005a. Biochemical effects of certain pesticides on common carp (Cyprinus carpio L). J. Adv. Agric. Res. 10 (2), 543–556. Aly, N.M., 2005b. Earthworm as a biomarker for pesticides toxicity. Alex. Sci. Exch. 26, 329–335. Aslan, R., Sekeroglu, M.R., Gultekin, F., 1997. Blood lipoperoxidation and antioxidant enzymes in healthy individuals. Relation to age, sex, habits, life style and environment. J. Environ. Sci. Health A 32, 2101–2109. Attia, A.M., El-Demerdash, F.M., 2002. Potent protective effects of melatonin on cypermethrin-induced oxidative damage in rats in vivo. J. Pest. Cont. Environ. Sci. 10 (2), 91–104. Banerjee, B.D., Seth, V., Ahmed, R.S., 2001. Pesticides induced oxidative stress: perspectives and trends. Rev. Environ. Health 16, 1–40. Bebe, F.N., Panemangalore, M., 2005. Pesticides and essential minerals modify endogenous antioxidant and cytochrome P450 in tissues of rats. Environ. Sci. Health Part (B) 40, 769–784. Beers Jr., R.F., Sizer, I.W., 1952. Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. Biol. Chem. 195, 133–140. Bendich, A., 1990. Antioxidant Micronutrients and Immune Responses, Micronutrients and Immune Functions. New York Academy of Sciences, New York. p. 175. Bindhumol, V., Chitra, K.C., Mathur, P.P., 2003. A induces reactive oxygen species generation in the liver of male rats. Toxicology 188, 117–124. Bomann, W., 1989. NTN33893-Study for acute oral toxicity to mice. Unpublished Report from Bayer AG, report No. 18593, dated 15 December 1989, GLP, unpublished. Submitted WHO by Bayer AG, Mannheim, Germany. Brocardo, P.S., Pandolfo, P., Takahashi, R.N., Rodrigues, A.L.S., Dafre, A.L., 2005. Antioxidants defenses and lipid peroxidation in the cerebral cortex and hippocampus following acute exposure to malathion and/or zinc chloride. Toxicology 207, 283–291. Cathcart, R.F., 1985. Vitamin C: the nontoxic, nonrate-limited, antioxidant free radical scavenger. Med. Hypo. 18, 61–77. Chiu, D.T.Y., Stults, F.H., Tappal, A.L., 1976. Purification and properties of rat lung soluble glutathione peroxidase. Biochem. Biophys. Acta 445, 558–566. CoStat Program 1986, Version 2, Cohort Software.
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