The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.4, 577-581, 2010
577 Letter
Effect of Imidacloprid on antioxidant enzymes and lipid peroxidation in female rats to derive its No Observed Effect Level (NOEL) Upasana Kapoor, Mithilesh Kumar Srivastava, Shipra Bhardwaj and Laxman Prasad Srivastava Pesticide Toxicology Division Indian Institute of Toxicology Research, (Council of Scientific and Industrial Research Govt. of India) Post Box No. 80, Mahatma Gandhi Marg Lucknow - 226 001, India (Received March 19, 2010; Accepted April 30, 2010)
ABSTRACT — Technical imidacloprid was evaluated for its effect on oxidative stress and Lipid peroxidation (LPO) in female rats for No Observed Effect Level (NOEL). Activities of Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and level of Glutathione (GSH) and LPO were estimated in liver, kidney and brain of rats after oral administration of imidacloprid (5, 10, 20 mg/kg/day) for 90 days. Imidacloprid at 5 and 10 mg/kg/day has not produced changes in SOD, CAT, GPx and level of GSH and LPO in liver, brain and kidney. However 20 mg/kg/day has produced significant changes in SOD, CAT, GPx, GSH, LPO in liver; SOD, CAT and GPx in brain and LPO in kidney. Therefore, it is concluded that imidacloprid has not generated oxidative stress at 5 and 10mg/kg/day but induced changes at 20 mg/kg/day. Hence 10 mg/kg/day may be considered as NOEL through antioxidant enzymes and LPO in female rats. Key words: Imidacloprid, NOEL, Oxidative stress, Vital organs, Female rats
INTRODUCTION Imidacloprid, 1-[(6-Chloro-3-pyridyl) methyl)-4, 5dihydroimidazol-2-yl) nitramide, a neonicotinoid has been introduced as world’s fastest growing sale insecticide in the global market (Tomizawa and Casida, 2003) and is being considered possible replacement for the widely used organophosphorus pesticide like diazinon (Jemec et al., 2007). Because of high insecticidal activity at very low volume rate (Broznil et al., 2008) it is widely used to control the pests of cereals, vegetables, tea and cotton throughout the world (Proença et al., 2005). Imidacloprid is a potential ground and surface water contaminant (PAN Pesticides database, 2006). Additionally, it may enter water bodies from spray drift or accidental spills, leading to local point-source contamination. It is an agonist to the nicotinic acetylcholine receptor (nAChR) and highly effective against many sucking insects (Tomlin, 1997). Increasing use of this insecticide and potential toxicity among humans warrants a heightened awareness about this compound (David et al., 2007).
Oxidative stress induction involves an excessive production of reactive oxygen species (ROS) resulting from impaired balance between the ROS generation and antioxidant defense capability. Induction of oxidative stress is one of the main mechanisms of the action of many pesticides. The damage of membrane lipids, protein and DNA is the endpoint biomarker of oxidative stress-inducing effects of pesticides (Tuzmen et al., 2008). Antioxidative enzyme defense system include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) that may protect the system from deleterious effects of oxygen free radicals. Although few reports on mammalian toxicity of imidacloprid are available but the information on oxidative stress of this compound after long term repeated exposure is publically unavailable (David et al., 2007; EL-Gendy et al., 2010). In our earlier study it has been observed that 10 mg/kg/day imidacloprid has not produced any significant changes in female rats as evidenced by morphological, biochemical, hematological and neuropathological parameters and this dose has been proposed to consider
Correspondence: L.P. Srivastava (E-mail:
[email protected])
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578 U. Kapoor et al.
as NOEL (Bhardwaj et al., 2010). Therefore the present study was designed to provide more information on development of No Observed Effect Level (NOEL) of imidacloprid through examination of antioxidant enzymes and LPO which induced oxidative damage in vital tissues of female rats. MATERIALS AND METHODS Chemicals and reagents Technical Imidacloprid (purity 96% w/w) was obtained from Bharat Rasayan Limited, New Delhi, India and all biochemicals used in experiments were obtained from Sigma Chemicals, St. Louis, MO, USA. Animals Twenty adult female rats (Rattus norvigicus Wistar strain) weight ranges from 150-155 g of Indian Institute of Toxicology Research, Lucknow breeding colony were maintained under condition of controlled temperature (22 ± 3°C) and humidity (30-70%) with 12 hr light and 12 hr dark cycle. The animals were given synthetic pellet diet (Lipton India Ltd, Mumbai, India) and water ad libitum. Rats were acclimatized for one week prior to the start of experiment. Approval from animal ethics committee of the Institute was obtained for the use of animals (ITRC/IAEC/04/2008). Experimental design Animals were divided into four groups having five animals in each. Technical imidacloprid was suspended in corn oil and administered orally through gavage in three treated groups of rats at 5, 10, 20 mg/kg/day doses corresponding to 1/90th, 1/45th and 1/22th LD50 for the period of 90 days. The control animals were given corn oil (0.4 ml/ rat/day) in a similar fashion. Tissue homogenate preparation Rats were sacrificed under ether anesthesia after 90 days treatment. Liver, brain and kidney were quickly removed, trimmed of extraneous tissue, washed with icecold physiological saline solution. Liver, brain and kidney tissues were divided into two parts and one half was homogenized with ice-cold 0.15 M KCl solution (10% w/v) for LPO, GSH, and CAT while another half were homogenized with 0.1 M phosphate buffer solution for SOD and GPx.
Biochemical measurements LPO Levels of MDA, an end product of polyunsaturated fatty acid peroxidation LPO, were measured in tissue homogenates on the basis of the reaction with thiobarbituric acid (TBA) to form a pink coloured complex, MDA produced was determined with the absorbance coefficient of the MDA-TBA complex at 532 nm on GBC Cintra 20 Spectrophotometer using 1, 1, 3, 3-tetraethoxypropane as standard (Ohkhawa et al., 1979). Determination of glutathione (GSH) GSH level was determined using 5, 5’-dithio-bis (2nitrobenzoic acid) (DTNB) for colour development and reading was taken at 412 nm on GBC Cintra 20 Spectrophotometer after 15 min. A standard curve using reduced GSH was used for calibration (Ellaman, 1959). SOD Activity of SOD was determined in the tissue homogenates by modified method of NADH-phenazinemethosulphate-nitriblue tetrazolium formazan inhibition reaction spectrophometrically measured at 560 nm on Spectrophotometer Genesys 10 UV (Kakkar et al., 1984). GPx Activity of GPx was determined and expressed in terms of μmole GSH consumed /min/mg protein measured at 420 nm on Spectrophotometer Genesys 10 UV (Flohe and Günzler, 1984) . CAT The activity of CAT was determined by the method of (Sinha, 1972) in which CAT preparation is allowed to decompose H2O2 for a fixed period of time. The reaction was then stopped by addition of dichromate- acetic acid reagent followed by heating for 15 min in boiling water bath. The remaining H2O2 was determined by measuring chromic acetate generated at 570 nm on GBC Cintra 20 Spectrophotometer. Determination of protein Protein was assayed using Bovine serum albumin (BSA) as standard and optical density read at 690 nm on GBC Cintra 20 Spectrophotometer (Lowry et al., 1951). Statistical analysis Statistical significance between control and experimental values were compared by Student‘t’ test (Fisher, 1950) and P values less than 0.05 were considered significance.
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579 No observed Effect Level (NOEL) of imidacloprid in female rats
er, brain and kidney at 5 and 10 mg/kg/day dose levels (Table 1).
RESULTS Signs of toxicity and absolute body weight Repeated oral administration of different doses of imidacloprid (5 and 10 mg/kg/day) did not produced any signs of toxicity and mortality during 90 days exposure. However animals exposed with 20 mg/kg/day have shown significant reduction in body weight with mild diarrhea, salivation and piloerection during 90 days exposure. Effect of imidacloprid on LPO Level of LPO measured in terms of MDA was not altered in liver, brain and kidney of female rats orally administered 5 and 10 mg/kg/day of imidacloprid. A significant increase in level of MDA was found in liver and kidney at 20 mg/kg/day dose as compared to control. Level of increase was 63% in liver and 60% in kidney. MDA level formed was maximum in liver than kidney and brain (Table 1). Effect of imidacloprid on GSH A significant decrease in GSH content was observedin liver of rats exposed to imidacloprid at 20 mg/kg/day. However there was no change in GSH content in liv-
Effect of imidacloprid on SOD activity A significant decrease in SOD activity was observed in liver and brain of rats exposed to imidacloprid at 20 mg/kg/day. However no change in SOD activity was observed at 5 and 10 mg/kg/day dose levels. Percent level of decrease in SOD activity was 82% in liver and 45% in brain at 20 mg/kg/day dose (Table 1). Effect of Imidacloprid on CAT activity A significant decrease in activity of CAT was observed in liver and brain of female rats exposed to imidacloprid at 20 mg/kg/day. The level of decrease in CAT activity was 51% and 25% in liver and brain respectively. However no significant change in CAT activity was found in following tissues at 5 and 10 mg/kg/day dose levels (Table 1). Effect of imidacloprid on GPx activity A significant decrease in activity of GPx was observed in liver and brain of female rats exposed to imidacloprid at 20 mg/kg/day. The level of decrease was 14% in liver and 10% in brain. However no significant change in GPx
Table 1. Effect of Imidacloprid on LPO and antioxidant enzymes of female rats orally exposed for 90 days Tissues
Treatment (mg/kg/day)
LPO ∆
CAT●
SOD ►
GSH ■
0
16.50 ± 1.17
38.54 ± 3.98
20.67 ± 4.06
90.09 ± 7.65
260.80 ± 7.86
5
17.45 ± 0.83
32.34 ± 2.58
16.66 ± 5.07
86.72 ± 5.47
248.40 ± 7.40
10
20.87 ± 1.77
27.24 ± 2.64
9.96 ± 2.31
84.44 ± 5.13
240.20 ± 4.80
20
26.94 ± 2.73*
18.65 ± 0.59*
3.58 ± 1.30*
65.57 ± 5.16* 223.02 ± 7.69*
0
10.33 ± 0.91
13.34 ± 0.52
7.42 ± 0.58
16.32 ± 2.07
252.80 ± 5.53
5
11.44 ± 0.73
12.38 ± 0.91
6.72 ± 0.58
14.88 ± 1.14
243.20 ± 6.92
10
15.36 ± 2.30
11.93 ± 0.78
6.99 ± 0.78
14.01 ± 1.87
239.40 ± 3.27
20
15.41 ± 2.68
4.03 ± 0.74*
11.92 ± 0.65
226.80 ± 4.76*
0
18.23 ± 3.04
13.69 ± 2.05
7.77 ± 1.80
48.45 ± 3.60
287.80 ± 1.68
5
18.20 ± 0.46
12.46 ± 1.00
6.11 ± 0.93
45.17 ± 7.82
281.20 ± 4.37
10
23.14 ± 2.16
11.68 ± 1.45
5.10 ± 0.64
41.86 ± 1.60
280.40 ± 5.33
20
29.18 ± 2.26*
10.29 ± 0.73
3.73 ± 0.56
39.06 ± 1.77
270.00 ± 7.28
GPx #
Liver
Brain 9.96 ± 1.05*
Kidney
Values represent means ± S.E. (n = 5) in each group. * Significant at level of p < 0.05. MDA levels expressed as nmoles MDA/mg protein; ● CAT expressed as μmoles CAT/min/mg protein ; ►SOD expressed as Unit SOD/min/mg protein ; ■ GSH expressed as μg GSH/mg protein ; # GPx expressed as μg GSH consumed /min/mg protein. ∆
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580 U. Kapoor et al.
activity was found in any tissues at 5 and 10 mg/kg/day dose levels. DISCUSSION A large number of xenobiotics have a capability to generate free radicals in biological system raising question whether oxidative stress is major concern for tissues damage. However antioxidant enzymes like SOD, CAT and GPx may have effect on oxidant molecules on tissues and are active in defense against oxidative cell injury by means of their being free radical scavengers. Pesticide mediated toxicity involves excessive production of ROS leading to alterations in the cellular antioxidant defense system and consequently affecting susceptibility to oxidative stress (Lopez et al., 2007). Pesticide also induces free radical generation that leads to DNA damage, protein degradation, LPO and finally culminating into damage to various vital tissues like liver, kidney and brain (Banerjee et al., 1999, Khan et al., 2005). These elevated free radicals and depressed antioxidant defense may lead to cell disruption, oxidative damage to cell membrane and hence increase susceptibility to LPO (Kapoor et al., 2009). Due to limited data on induction of free radicals scavenging enzymes in biological tissues following imidacloprid administration, hazards arising form its exposure are of great interest. Increased MDA suggests an increased production of free O2 radicals in rats (Mansour and Mossa, 2009). In present study imidacloprid significantly induced LPO and decreased other vital antioxidants in liver, brain and kidney at high dose. Susceptibility of liver, brain and kidney to this stress due to exposure of pesticides is function of overall balance between degree of oxidative stress and antioxidant capacity (Khan et al., 2005). High level of LPO in liver in the present study suggested the production of oxidative metabolites or free radicals during hepatic metabolism and this may be due to progressive nature of free radical chain reaction. Increased level of MDA in tissues of the present study supports the results of Giray et al. (2001) and Kalender et al. (2006) who have reported an increased level of MDA in hepatic and renal tissues of rats administered cypermethrin and methyl parathion. O2 free radicals and hydroperoxide collectively termed as ROS are produced by univalent reduction of dioxygen to superoxide anion (O2-) which in turn is converted into H2O2 and O2 through a reaction catalyzed by SOD (Rai and Sharma, 2007). Antioxidant enzymes (SOD and CAT) constitute first line of defense against deleterious effect of oxyradicals in cell by catalyzing dismutation of superoxide radical. Decrease in activity of SOD Vol. 35 No. 4
in liver and brain of imidacloprid intoxicated rats may be due to consumption of this enzyme in O2- to H2O2. Similar decreased activity of SOD in animals was also reported with different pesticides namely chlorpyrifos, cypermethrin, carbofuran, dimethoate and malathion, which shows decrease SOD activity in rats (Khan et al., 2005; Rai and Sharma, 2007; Mansour and Mossa, 2009). Various studies have demonstrated decrease in GPx activity due to xenobiotics which supports our findings (Kapoor et al., 2009; Mansour and Mossa, 2009). GSH plays a key role in modulating pesticide induced oxidative damage in tissues. GSH depletion is evident to intensify LPO and predispose cells to oxidant damage (Khan et al., 2005). A significant depletion of GSH in liver together with decrease in activity of GPx in liver and brain at high dose noted in present study may induce oxidant damage in tissues of animals. In addition GSH participates in detoxification of xenobiotics as substrate for enzyme Glutathion-S-transferase (GST) which is reduced by imidacloprid at high dose may be through direct utilization of GSH as an antioxidant in terminating free radical reaction. Similar reduction in GSH has been reported earlier with cypermethrin, methoxychlor, dimethoate, deltamethrin and bisphenol exposed rats (Sharma et al., 2005; Rehman et al., 2006; Rashid et al., 2009). The decrease activities of SOD, GPx, CAT and GSH content together with increase LPO may be attributed to induce free radicals in high dose imidacloprid treated rats. The toxicity of many xenobiotics is associated with the production of free radicals which are not only toxic themselves, but may also implicate in the pathophysiology of many diseases (Abdollahi et al., 2004). The results of present study have indicated that imidacloprid has not induced oxidative stress at 5 and 10 mg/kg/day doses to female rats when exposed for period for 90 days. However imidacloprid at 20 mg/kg/day dose has significantly induced oxidative stress to female rats. This may be due to dearrangement of cellular oxidative status as evidenced by increaseed level of LPO, decreased activities of CAT, SOD, and GPx and reduced GSH level in vital tissues. Thus on basis of parameters studied in the present study, a dose 10 mg/kg body weight (as proposed in our earlier study) has been confirmed as NOEL of imidacloprid in female rats. ACKNOWLEDGMENTS We are grateful to Director, Indian Institute of Toxicology Research Lucknow, India for his keen interest and encouragement. The authors are thankful to Council of Scientific and Industrial Research, New Delhi, for fund-
581 No observed Effect Level (NOEL) of imidacloprid in female rats
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