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PESTICIDE
Biochemistry & Physiology

Pesticide Biochemistry and Physiology 90 (2008) 5865 www.elsevier.com/locate/ypest

Dimethoate induced biochemical perturbations in rat pancreas and its attenuation by cashew nut skin extract
Vasudeva Kamath, Apurva Kumar R. Joshi, P.S. Rajini
Received 2 April 2007; accepted 23 July 2007 Available online 1 August 2007

Food Protectants and Infestation Control Department, Central Food Technological Research Institute, Mysore 570 020, India

Abstract The signicant antiradical activity of cashew skin extract was previously described. In this investigation, the extent of protection oered by cashew nut skin extract (CSE) against the damage induced in rat pancreas by sub chronic doses dimethoate (DM), an organophosphorous pesticide was studied. Rats were supplemented with CSE at 20 mg/kg b.w./d after a daily dose of DM at 40 mg/kg/d b.w. for 2 months. Weekly random blood glucose, oral glucose tolerance test (OGTT); pancreatic damage markers like amylase and lipase; oxidative damage markers such as reactive oxygen species generated, extent of lipid peroxidation, host antioxidant defenses like reduced glutathione (GSH); GSH-dependent enzyme activities viz., glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR); free radical scavenger enzymes viz., catalase and superoxide dismutase (SOD); xenobiotic metabolizing enzymes like DT-diaphorase and NADPH-diaphorase were measured in the four dierent groups namely (1) control, (2) DM treated, (3) CSE supplemented, (4) CSE supplements following DM treatment. Random blood glucose levels increased signicantly on exposure to DM compared to that in control rats (119 5 mg/dl vs. 92 4 mg/dl), while the blood glucose levels in CSE supplemented rats were comparable to that of controls. DM treated rats exhibited impaired glucose tolerance at the end of two months as indicated by OGTT, while DM treated rats with CSE supplements showed normal glucose tolerance. Pancreatic specic marker enzymes like amylase and lipase in serum were restored to normalcy in rats supplemented with CSE following treatment with DM which otherwise was increased in the DM treated rats. Distinctly lower levels of GSH, increased levels of ROS, higher extent of lipid peroxidation, along with alterations in antioxidant enzymes and increase in xenobiotic metabolizing enzymes were evident in pancreas of DM treated rats. However, CSE supplement ameliorated the biochemical alterations in the pancreatic milieu in DM treated rats. Treatment with CSE signicantly protected rat pancreas from injury, thus ameliorating and restoring tissue antioxidant status and also conferring normal glucose tolerance. The active components present in cashew skin extract can perhaps be eective in reducing the extent of pancreatic injury and in overcoming tissue damage caused by exposure to dimethoate. 2007 Elsevier Inc. All rights reserved.
Keywords: Rat pancreas; Oral glucose tolerance; Dimethoate; Cashew nut skin extract; Amylase; Lipase; Oxidative stress

1. Introduction Organophosphorus insecticides (OPI) constitute one of the most widely used classes of pesticides being employed for both agricultural and landscape pest control. Use of OPI has increased considerably due to their low toxicity and low persistence in the mammalian system compared

Corresponding author. Fax: +91 821 2517233. E-mail address: rajini29@yahoo.com (P.S. Rajini).

to organochlorine pesticides. OPI are primarily recognized for their ability to induce toxicity in mammals through inhibition of acetylcholinesterase (AChE) and subsequent activation of cholinergic receptors [1]. The main mechanism of toxicity of OPI is due to irreversible binding to AChE as opposed to reversible binding by carbamates, thereby resulting in prolonged eect of acetylcholine [2]. Various complications have been reported in OPI intoxication cases [3], and hyperglycemia has been widely reported as one of the major adverse eects in poisoning by OPI in humans and animals [48]. Although

0048-3575/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.07.007

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the precise mechanism(s) of OP induced hyperglycemia is not known, it is speculated to be due to inhibition of acetylcholinesterase in central and peripheral synapses that act in the endocrine regulation of glucose metabolism [9]. Acute pancreatitis is also a well-known complication of OP poisoning in both humans and animals [3,10,11]. The precise molecular mechanisms underlying OPIinduced acute pancreatitis are still undened, although it is believed to involve obstruction of pancreatic ducts and/or enhanced reactive oxygen species [12]. Involvement of oxidative stress following acute exposure to OPI has been reported recently [13] and it has been demonstrated unequivocally that lipid peroxidation is one of the molecular mechanisms involved in OPI-induced toxicity [1416]. The cellular antioxidant status determines the susceptibility to oxidative damage and is usually altered in response to oxidative stress. The cellular antioxidant action is reinforced by the presence of dietary antioxidants [17]. Accordingly, interest has recently grown in the role and usage of natural antioxidants as a strategy to prevent oxidative damage in various health disorders with oxidative stress as factor in their pathophysiology [18]. Natural antioxidants from fruits and vegetables are reported to provide substantial protection that slows down the process of oxidative damage caused by ROS [19]. Hence there has been growing interest in natural antioxidants of plant origin since they also nd use as nutracueticals due to their impact on the status of human health and disease prevention [20]. Dimethoate (O,O-dimethyl S-N-methyl carbomyl methyl phosphorodithioate) (DM) is one of the most important OPI used extensively on a large number of crops against several pests. The residue of DM and its analog were reported in a number of foods including cows milk [21]. While data on acute, subchronic and chronic toxicity of DM in laboratory animals are well documented, its potential to induce hyperglycemia and cause pancreatic damage in mammals is lately being understood. Interestingly, DM is reported to cause various toxic eects on rat pancreas following chronic exposure [5] as well as pancreatitis in humans following dermal exposure [22]. Recently we have reported [23] the potential of DM to cause alterations in glucose homeostasis and also on the toxic eects in pancreas of adult rats subjected to daily oral administration at sublethal doses for 1 month. Cashew nut (Anacardium occidentale L.) is a major cash crop in the world. Cashew nut shell liquid, a byproduct obtained during the processing of cashew nuts is reported to possess antioxidant activity [24]. The kernel of cashew nut valued in trade is covered with a thin reddish-brown skin or testa which has been reported to be a good source of hydrolysable tannins [25] with catechin and epicatechin as the major polyphenols [26]. We have recently reported the strong antioxidant activity of the ethanolic extract of cashew nut testa in dierent antioxidant assay systems and attributed the activity to the epicatechin present

[27]. The objective of this study was to determine the biochemical perturbations induced by dimethoate in rat pancreas and to assess whether this damage is amenable to modulation by an antioxidant-rich extract of cashew nut skin. 2. Materials and methods 2.1. Chemicals Thiobarbituric acid (TBA), xanthine oxidase, ethylenediamine tetra acetic acid (EDTA), hydrogen peroxide (H2O2), glutathione reductase (GR), 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), 2 0 ,7 0 -dichlorouorescin diacetate (DCFH-DA), 2 0 ,7 0 -Dichlorouorescein (DCF), epicatechin and Cytochrome c were procured from M/s Sigma Chemical Co. (St. Louis, MO, USA). Xanthine, NADPH, nicotinamide adenine dinucleotide-reduced (NADH), trichloroacetic acid (TCA), reduced glutathione (GSH), oxidized glutathione (GSSG), 1-chloro-2,4-dinitrobenzene (CDNB) and acetylthiocholine iodide were procured from M/s Sisco Research Lab, (Mumbai, India). All other chemicals used were of analytical grade. Dimethoate (technical grade, 97.4%) was gifted by M/s Hyderabad Chemical Supplies Ltd. (Hyderabad, India). 2.2. Animals and care Adult male rats (CFT-Wistar strain, 12- to 14-week-old, 280 5 g) were randomly drawn from the stock colony of our Institute Animal House facility and were housed individually in polypropylene cages under standard housing conditions (controlled atmosphere with 12:12 h light/dark cycles, 50% 5% humidity, and an ambient temperature of 25 2 C). The rats were acclimatized for 1 week prior to the start of the experiment. Rats were maintained on commercial pellet diet (Gold Mohur, supplied by M/s Lipton India Pvt., Ltd) ad libitum and had free access to water. All procedures with animals were conducted strictly in accordance with guidelines approved by the Institute Animal Ethical Committee, regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. During the experiments, maximum care was taken to minimize animal suering and in addition, the number of rats used was kept at minimum. 2.3. Preparation of cashew skin extract (CSE) Dried cashew kernel skin was procured from a cashew processing unit (M/s Mangala cashews, Mangalore, India). The skins were sun dried, pulverized in multi-mill and passed through a 0.5-mm sieve to obtain a ne powder. The skin powder (CSP) was mixed with ve parts of ethanol in a rotary shaker at 37 C for 3 h. The extract was separated by centrifugation and the resultant supernatant

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(extract) was concentrated in vacuo and reconstituted in saline to a nal concentration of 25 mg/ml. 2.4. Animal treatment and experimental protocol Adult male rats were grouped by randomized design into four groups (n = 6). Group I (control) animals received saline daily (oral) for 2 months. The second group was administered with oral dose of DM (40 mg/kg b.w./d, 1/10 of the LD50 value) in saline for two months. The dosage of DM was selected based on our earlier studies [23]. Rats of group III received CSE at 20 mg/kg b.w./d in saline for 2 months. Rats of group IV were also administered orally CSE (20 mg/kg b.w./d in addition to daily oral dose of DM at 40 mg/kg b.w. for 2 months. After 4 weeks, random blood glucose was monitored in all the four groups of rats at weekly intervals until 8 weeks. All the experimental rats were subjected to oral glucose tolerance test at the end of 2 months and were sacriced for various biochemical analyses. Immediately after euthanizing, blood was collected, serum separated, and pancreas excised, rinsed in ice-cold saline and homogenized for 5 min to obtain 10% (w/v) homogenate in phosphate buer (0.1 M, pH 7.4). The homogenate was centrifuged at 9000g at 4 C for 20 min and the supernatant was used for various biochemical analyses. 2.5. Oral glucose tolerance test Oral glucose tolerance test was conducted in rats from all the experimental groups as follows. Twenty-four hours after the last dose, blood was collected from tail vein of rats fasted overnight, and glucose was estimated using a commercial glucometer (Accu-Check from M/s Roche diagnostics, GmbH, Mannheim, Germany). Subsequently, rats were orally administered a bolus of glucose (3 g/kg b.w.) and blood was collected from tail vein of these rats at interval of 30 min up to 3 h for estimation of glucose. 2.6. Biochemical measurements 2.6.1. Assessment of pancreatic damage Lipase activity was estimated in pancreas and serum by monitoring the hydrolysis of p-nitrophenyl acetate to p-nitrophenol as described by Young et al. [28]. Enzyme activity was calculated as nmol PNP released using a p-nitrophenol standard graph and enzyme activity was expressed as nmol PNP released min1 mg1 protein. Amylase activity in pancreas and serum was measured according to the method of Street and Close [29] using a commercially available kit procured from M/s Span diagnostics, Mumbai, India. The assay is based on the degradation of starch by the enzyme amylase into reducing dextrins and small oligosaccharides. Iodine solution is reacted to give a blue coloration, which is read at 620 nm. The relative decrease in blue color is the measure of enzyme activity, which was expressed as StreetClose units.

2.6.2. Measurement of oxidative stress markers Levels of ROS generated in pancreas were determined by DCFH oxidation [30]. The amount of DCF (resulting from the ROS mediated oxidation of DCFH, which is produced by hydrolytic cleavage of DCFH-DA by cellular esterases) was estimated in pancreatic homogenate and results were expressed as pmol of DCF mg1 protein min1. Lipid peroxidation was measured in pancreas by estimating the thiobarbituric acid reactive substances in pancreatic homogenate according to the method of Buege and Aust [31]. The amount of thiobarbituric acid reactive substances (TBARS) was determined using the molar extinction coecient of 1.56 105 M1 cm1 and the results were expressed as nmol MDA min1 mg1 tissue. Reduced glutathione (GSH) level in pancreatic homogenate was quantied by the method of Benke et al. [32] and glutathione concentrations were calculated from a standard graph and results were expressed as mg GSH/g tissue. 2.6.3. Activity of antioxidant enzymes Catalase (EC. 1.11.1.6) activity in pancreatic homogenate was assayed by monitoring the decomposition of H2O2 at 240 nm as described by Aebi [33] and expressed as lmol of H2O2 decomposed min1 mg1 protein. Superoxide dismutase (SOD) (EC. 1.15.1.1) was assayed employing the method of Flohe and Otting [34] and the enzyme activity was expressed as units of SOD/mg protein. One unit was dened as the amount of enzyme that decreases the initial rate of Cytochrome c reduction to 50% of its maximal value for the particular sample being analyzed. The activity of glutathione reductase (EC. 1.6.4.2) was determined by monitoring the oxidation of NADPH in the presence of GSSG and expressed as nmol NADPH oxidized min1 mg1 protein by the method of Carlberg and Manervik [35]. Activity of glutathione peroxidase (EC. 1.11.1.9) was determined using method of Paglia and Valentine [36] and was expressed as nmol of NADH oxidized min1 mg1 protein. Glutathione S-transferase activity was measured by the method of Rotruck et al. [37]. Results were expressed as lmol adduct formed min1 mg1 protein. 2.6.4. Acetylcholinesterase activity The activity of acetylcholinesterase (AChE) (EC 3.1.1.7) was assayed in pancreatic homogenates by the method of Ellman et al. [38] using acetylthiocholine iodide as the substrate and the enzyme activity was expressed as lmol of substrate hydrolyzed min1 mg1 protein. 2.6.5. Activity of phase II enzymes DT-diaphorase activity was assayed as described by Ernest et al. [39] which involved measurement the rate of reduction of 2,6-dichloropenol indophenol at 550 nm with NDAH as the electron donor. The enzyme activity was calculated using the extinction coecient 21 mM1 cm1. NADPH-diaphorase activity was assayed as described by

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2.6.6. Protein assay Protein content in tissue homogenate and in serum was measured as described by Lowry et al. [41] using bovine serum albumin (BSA) as the standard. 2.7. Statistical analysis Values are presented as mean SEM. Results were statistically analyzed by analysis of variance (ANOVA). Duncans multiple range test (DMRT) was performed to determine the signicant dierence among the groups. Statistica software from STATSOFT, USA was used to analyze the data. Results were expressed as mean SE from six rats in each group. The p values <0.05 were considered signicant. 3. Results Rats supplemented with CSE showed no signs of toxicity and gained body weight as control rats. Dimethoate (DM) did not induce any distinctive clinical signs of toxicity or mortality. However, DM treatment reduced the body weight gain in comparison to control group (7.3% vs 15.7%). DM treated rats receiving CSE supplements had normal body weight gain as in controls (13.7% vs 15.7%). Data on blood glucose levels monitored among rats of various groups is presented in Fig. 1. CSE supplements per se did not alter the blood glucose levels. The blood glucose levels among DM treated rats were signicantly elevated (119 5 mg/dl vs. 92 4 mg/dl) at all sampling weeks. However, DM treated rats receiving CSE supplements, showed blood glucose levels which were comparable to that of controls (102 6 mg/dl vs. 92 4 mg/dl). Blood glucose levels monitored for 3 h period following glucose overload in various groups are presented in Fig. 2. Blood glucose levels among all groups remained elevated
150

Blood glucose (mg/dl)

Huennckens et al. [40]. This method is based on reduction of methylene blue to leukomethylene blue during the transfer of hydrogen from NADPH to NADPH-diaphorase.

150

Control CSE DM DM+CSE

100

50 0 30 60 90 time(min) 120 150 180

Fig. 2. Eect of cashew skin extract (CSE) oral glucose tolerance test in control and dimethoate (DM) treated (40 mg/kg b.w. for 2 months) adult rats. Values are mean SEM (n = 6).

Blood glucose (mg/dl)

Control CSE DM DM+CSE

100

50 1 2 week 3 4

Fig. 1. Eect of cashew skin extract (CSE) on blood glucose in control and dimethoate (DM) treated (40 mg/kg b.w. for 2 months) adult rats. Values are mean SEM (n = 6).

up to 60 min. Subsequently the blood glucose in control rats decreased at further time points. No signicant dierences in the glucose levels were found among the control and rats supplemented with CSE at all time points during glucose tolerance test, while blood glucose levels of DM treated rats remained consistently higher (129 6 mg/dl) at all time points. Interestingly, the blood glucose of DM treated rats receiving CSE supplement was restored to normalcy at 90 min and beyond. Activities of marker enzymes of pancreatic damage such as lipase and amylase in the experimental groups of rats are depicted in Fig. 3. CSE supplements per se did not alter either serum or pancreatic enzyme activities. Signicantly (p < 0.05) higher activities of serum lipase (47%) and amylase (3.6-fold increase) were evident in DM treated rats. However, in pancreas the activities of lipase (2.4-fold) and amylase (12%) were signicantly lower. In DM treated rats supplemented with CSE, only the serum lipase activity (5.96 0.13 nmol PNP/min/mg protein) was restored to normalcy. Further, the amylase activity in serum and pancreas, which were signicantly elevated in DM treated rats, were normalized in DM + CSE group. In general, CSE per se had no eect on both pancreatic ROS and TBARS levels as they were comparable to those of control rats (Fig. 4). Treatment with DM caused significant increase in both ROS (1.88 0.16 pmol of DCF) as well as TBARS (38 0.75 nmol of MDA) levels in pancreas. With CSE supplementation in DM treated rats, a dramatic restoration in ROS levels in pancreas was observed (100% protection), while the TBARS levels were marginally (14%) lower. The reduced glutathione levels and activities of various antioxidant enzymes in pancreas among various groups are presented in Table 1. CSE supplement per se had no measurable eect on GSH levels and on antioxidant enzymes. In DM treated rats, the GSH levels were signicantly decreased (66%) compared to those of control rats (3.36 0.12 mg/g tissue). CSE supplements in DM treated rats signicantly oset the decrease in GSH levels (2.83 0.08 mg/g tissue) in pancreas. In DM treated rats

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12 Control CSE DM DM+CSE a 6 a

V. Kamath et al. / Pesticide Biochemistry and Physiology 90 (2008) 5865

80 Control CSE b nmol PNP/mg protein/min 60 DM DM+CSE

10 nmol PNP/mg protein/min

b 40

a 20 a

0 Serum lipase

0 Pancreatic lipase

100 c Control CSE DM DM+CSE Streat Close units

210 180 150 120 90 60 30 0

80 Streat Close units

Control CSE DM DM+CSE a a b a

60 b 40 a 20 a

0 Serum amylase

Pancreatic amylase

Fig. 3. Eect of cashew skin extract (CSE) on pancreatic marker enzymes viz., serum lipase, pancreatic lipase, serum amylase and pancreatic amylase in control and dimethoate (DM) treated (40 mg/kg b.w. for 2 months) adult rats. Values are mean SEM (n = 6). The bars with dierent letter are signicantly dierent p < 0.05 by DMRT.

2.5

Control CSE DM

40 Control CSE DM DM+CSE

c b

b 30 nmol MDA/g tissue

2 pmol DCF/mg protein/min

DM+CSE

1.5

a a

20 a a 10

0.5

0 ROS

0 TBARS

Fig. 4. Eect of cashew skin extract (CSE) on ROS generation and extent of lipid peroxidation in control and dimethoate (DM) treated (40 mg/kg b.w. for 2 months) adult rats. Values are mean SEM (n = 6). The bars with dierent letter are signicantly dierent p < 0.05 by DMRT.

V. Kamath et al. / Pesticide Biochemistry and Physiology 90 (2008) 5865 Table 1 Eect of CSE supplementation on reduced glutathione and antioxidant enzyme activity in pancreas of normal and dimethoate treated rats GSHA Control CSEG DMH DM + CSE 3.36a 0.12 3.48a 0.11 2.21c 0.14 2.83b 0.08 GRB 17.50a 1.60 16.89a 1.42 25.30b 1.30 20.56a 1.20 GPXC 27.18b 5.24 26.14b 3.73 13.85a 2.20 16.68ab 1.18 GSTD 0.029a 0.004 0.031a 0.003 0.065b 0.003 0.092c 0.004 CATE 9.07a 0.98 8.98a 0.85 5.23b 1.09 9.98a 1.09 SODF

63

24.62a 0.79 25.44a 0.54 51.48b 6.23 28.69a 5.60

Values are mean SEM (n = 6); 40 mg/kg b.w./d. Mean in the same column with dierent superscript dier signicantly (p < 0.05). A mg/g tissue. B nmol/min/mg protein. C nmol/min/mg protein. D lmol/min/mg protein. E lmol/min/mg protein. F units/mg protein. G CSE, Cashew Skin Extract. H DM, Dimethoate.

there was a marked elevation in the activities of GST (2fold), GR (1.5-fold) and SOD (2-fold). While the activity of GPx (50%) and CAT (42%) were signicantly diminished. Interestingly, among DM treated rats supplemented with CSE activities of all the antioxidant enzymes were restored to normalcy. Data on the eect of CSE supplements on pancreatic AChE and Phase II xenobiotic metabolizing enzyme viz., NADPH-diaphorase and DT-diaphorase of experimental groups is presented in Table 2. CSE per se had no eect on the activities of AChE and phase II enzymes. DM treatment signicantly reduced the pancreatic AChE activity (71% inhibition). CSE supplements in DM treated rats oered signicant protection to activity of AChE since only 57% inhibition in the activity of AChE was recorded. Rats treated with DM showed increased activities of NADPHdiaphorase and DT-diaphorase enzymes up to 92% and 40% above control values, respectively. Interestingly, in DM treated rats supplemented with CSE, activities of both xenobiotic metabolizing enzymes were restored to normalcy (100% protection). 4. Discussion Organophosphorus insecticides (OPI) have been claimed to have harmful eects on the endocrinal and other bioTable 2 Eect of CSE supplementation on AChE and xenobiotic metabolizing enzyme activity in pancreas of normal and dimethoate treated rats AChEA Control CSED DME DM+CSE 4.96 0.47 5.03b 0.86 1.43a 0.21 2.11a 0.80
b

NADPH-diaohoraseB 8.78 1.27 9.12a 1.42 16.87b 0.37 11.17a 0.279

a

DT-diaphoraseC 0.62a 0.06 0.60a 0.02 0.87c 0.03 0.72b 0.03

Values are mean SEM (n = 6); 40 mg/kg b.w./d. Mean in the same column with dierent superscript dier signicantly (p < 0.05). A lmol/min/mg protein. B nmol of NADPH utilized/min/mg protein. C lmol of NADH utilized/min/mg protein. D CSE, Cashew Skin Extract. E DM, Dimethoate.

chemical functions of the pancreas [3,42]. In the present study, sub chronic administration of dimethoate for two months resulted in a signicant increase in random blood glucose levels. Further, DM treatment also impaired glucose tolerance in rats, and induced biochemical alterations in pancreatic tissue. Majority of the studies evaluating the eects of OP on glucose homeostasis have reported that exposure to OP induces hyperglycemia. Recently we reported [19] increased blood glucose and impaired glucose homeostasis in rats administered dimethoate. Increase in blood glucose has also been reported due to intoxication in rats by acephate [43], fenthion [44] malathion [16], in mice by diazinon [6] and in humans due to OP poisoning [45]. The main mechanism of action of OP occurs through the inhibition of neuronal cholinesterase activity, a key enzyme involved in neurotransmission. However, cholinergic action of OPI is also reported to result in hyperglycemia due to hyperesthesia, intermittent spasm, muscular tremors and convulsions [46]. Furthermore, it has been reported that ACh is a potent secretagogue of both insulin and glucagons [6,47]. In the present study, we observed marked inhibition in AChE activity in pancreas of DM treated rats. Probably, as proposed by Teichert-Kulizenwska et al. [48] the cholinergic eect of dimethoate may perhaps involve changes in the insulin/glucagon ratio. Exposure to OPI has been reported to evoke an imbalance in the cellular oxidative status and also lead to increased activity of enzymes associated with detoxication arsenal of experimental animals [49]. Our study indicates that subchronic administration of dimethoate causes pancreatic damage in test animals as shown by increases in serum markers of enzymes amylase and lipase (Fig. 3). This nding indicates that DM causes acute pancreatitis since increase in lipase activity by 2-fold is specic for the diagnosis of pancreatitis. Administration of another OPI, diazinon has also been shown to increase serum lipase and amylase activity in rats [50]. It has been well demonstrated that the enzymes associated with antioxidant defense mechanism are altered under the inuence of OPI and that lipid peroxidation

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V. Kamath et al. / Pesticide Biochemistry and Physiology 90 (2008) 5865

is one of the molecular mechanisms involved in OPinduced tissue damage [15,16,49]. However, OPI-induced oxidative stress in pancreas and its involvement in the ensuing hyperglycemia have not been reported. In the present study, we observed signicant increase in the ROS production in pancreas of rats treated with dimethoate. Concomitantly, there was marked reduction in GSH and signicant alterations in the activities of various antioxidant enzymes in pancreas. Activities of the GSHdependent phase II detoxifying enzymes in pancreas GPx, GR and GST and GSH-independent phase II detoxifying enzymes like DT-diaphorase and NADPHdiaphorase were also signicantly altered in the pesticide-treated rats along with free radical scavenging enzymes, SOD and CAT. Oxidative damage primarily occurs through production of ROS, including hydroxyl radicals and subsequently reacts with biological molecules, causing damage to membranes and other tissues [51]. The decrease in GSH shows that there is a net suppression in the total antioxidant capacity in the pancreatic tissue. The increase in ROS generation and subsequent increase in levels of lipid peroxidation in the pancreatic tissue upon pesticide exposure points to the underlying membrane damage stemming from a substantial loss of membrane integrity in the pancreatic tissue. The increase in serum amylase and lipase and decrease in the pancreatic tissue amylase and lipase is probably an outcome of this phenomenon. Our other question was that if oxidative stress has a role in DM-induced pancreatic damage and the ensuing altered glucose homeostasis, whether an antioxidant rich natural extract can ameliorate the eect. In the present study, we employed an ethanolic extract of cashew nut skin (CSE) to study its ameliorative potential. Our earlier studies [27] had clearly demonstrated the strong radical quenching potential of CSE. DM administration followed by CSE supplements signicantly resulted in decreased oxidative stress in the pancreas. CSE supplemented rats showed signicantly lower ROS generation and lipid peroxidation in pancreas. Further CSE treatment normalized the levels of pancreatic enzymes as well as the xenobiotic metabolizing enzymes, and also decreased the levels of serum lipase and amylase in DM treated rats. DM-induced hyperglycemia and also impaired glucose tolerance were reversed by CSE supplementation. However, CSE supplementation did not reverse AChE inhibition in pancreas induced by DM. Cashew nut skin extract contains polyphenols such as catechin and epicatechin. These polyphenols are also reportedly present in black tea and green tea extract [20]. Black tea and green tea have been shown to selectively induce phase I and Phase II metabolic enzymes which increase the formation and excretion of detoxied metabolites resulting from xenobiotic metabolism [52]. Black tea has shown to have protective eect on pesticide induced toxicity in mice liver [20]. Our results are in accordance with the earlier studies and further strengthen the impor-

tance of active compounds present in natural foodstus to ameliorate pesticide toxicity. Our ndings indicate that the hyperglycemia and impaired glucose tolerance evoked by DM in rats probably occurs as a result of oxidative damage in pancreas. Our results rule out the cholinergic involvement in the DM-induced pancreatic damage/dysfunction. Further our nding also establishes the protective role of an antioxidant-rich extract from a waste by product, cashew nut skin, against OPI induced toxicity with special reference to pancreatic damage and altered glucose homeostasis. Acknowledgments We thank the Director, CFTRI, Mysore for his support in this study. Financial assistance to the rst author (V.K.) from Indian Council of Medical Research (ICMR), New Delhi, India, in the form of Senior Research Fellowship (SRF) is gratefully acknowledged. The authors also acknowledge with thanks Dr. Muralidhara, Scientist, Biochemistry and Nutrition Department for critically going through the manuscript. References
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