Academic Sciences Asian Journal of Pharmaceutical and Clinical Research Vol 6, Issue 1, 2013 ISSN - 0974-2441

Vol. 4, Issue 3, 2011 Re search Article ISSN - 0974-2441 THE AMELIORATING EFFECTS OF GREEN TEA EXTRACT AGAINST CYROMAZINE AND CHLORPYRIFOS INDUCED LIVER TOXICITY IN MALE RATS TAREK M.HEIKAL1*; ABDEL- TAWAB H. MOSSA1; MONA A. ABDEL RASOUL2; GEHAN I. KH. MAREI2 1*Environmental Toxicology Research Unit (ETRU), Pesticide Chemistry Department, National Research Centre (NRC), Cairo, Egypt,2Department of Pest Control and Environmental Protection, Faculty of Agriculture, Damanhour University, Damanhour, Egypt, Email: [email protected] Received: 1 October 2012, Revised and Accepted: 27 October 2012 ABSTRACT In the present study, the protective effect of an aqueous extract of green tea (GT) against hepatotoxicity and oxidative damage induced by cyromazine (Cyr), chlorpyrifos (CPF) and their combination in male rats was undertaken. Eight groups containing six rats each were selected. Group I served as control. Groups II, III and IV rats were given a single daily oral doses of Cyr (169.35 mg/kg, 1/20 LD50, in corn oil), CPF (6.75 mg/kg, 1/20 LD50, in corn oil) and their combination for 28 consecutive days, respectively. Group V permitted free access to solubilised GT (1.5%) as the sole drinking fluid. Groups VI, VII and VIII rats were given the same doses as groups II, III and IV and simultaneously permitted free access to solubilised GT as the sole drinking fluid. Insecticides administration to rats resulted in significant reduction in body weight and elevation in liver weight compared to control. Insecticides administration to rats resulted in significant elevation of serum transaminases (AST & ALT), alkaline phosphatase (ALP), total protein, lipid peroxidation (LPO) expressed as malondialdehyde (MDA), lactate dehydrogenase (LDH) and decrease of serum albumin (Alb). Furthermore, significant elevation of hepatic superoxide dismutase (SOD), catalase (CAT), reduction of hepatic lactate dehydrogenase (LDH), glutathione peroxidase (GPx), depletion of hepatic glutathione reduced (GSH) and elevation of hepatic protein carbonyl (PC) content were noticed in insecticides-treated rats. Histopathological studies of liver revealed that supplementation of GT resulted in mild degeneration and necrosis of the hepatocytes. Furthermore, GT had normalized CAT, SOD, GPx, ALT, AST, ALP, serum LDH, total proteins and PC content, whereas attenuated Alb, hepatic LDH, GSH and LOP. In Conclusion, the use of green tea extract appeared to be beneficial to rats, to a great extent in attenuating and restoring the damage sustained by insecticide exposure. Keywords: Biochemical, Cyromazine, Chlorpyrifos, Green tea, Lipid peroxidation, Liver, Oxidative stress, Histopathology. INTRODUCTION Pesticides are used daily and internationally on a massive scale. In fact, herbal medicines derived from plant extracts are being They have conferred immense benefits to mankind by improving increasingly utilized as adjunct treatment options for a wide variety health and nutrition. Pesticides fall into numerous chemical classes, of clinical disease. More attention has been paid to the protective which have widely differing biological activities and thus differing effects of natural antioxidants against chemically induced toxicities potential to produce adverse effects in living organisms, including 24. The health benefits of green tea have been extensively studied in humans 1. Chlorpyrifos (CPF) is a broad-spectrum the past few decades. Nowadays, tea is considered as a source of organophosphorus insecticide (OPI) utilized extensively in dietary constituents endowed with biological and pharmacological agriculture 2, 3. CPF is thought to be primarily metabolized in the activities with potential benefits to human health. The increasing liver by multiple, specific cytochrome P450 enzymes through several interest in the health properties of tea extract and its main catechin reaction pathways 4. CPF elicits a number of additional effects, polyphenols have led to a significant rise in scientific investigation including hepatic dysfunction, haematological and immunological for prevention and therapy in several diseases 25-27. Furthermore, abnormalities, embryotoxicity, genotoxicity and neurobehavioral many previous investigators reported that green tea extract (GT) changes 5-9. displays antioxidants and free radicals scavenger properties 25, 28. Therefore the present study aimed to elucidate if Cyr has Cyromazine (Cyr), N-cyclopropyl-1,3,5-triazine-2,4,6-triamine, is an hepatotoxicity when administered orally to male rat separately or in insect growth regulator that acts by inhibiting the moulting combination with CPF as well as the hepatoprotective effect of GT processes . It is wildly used as an agricultural insecticide and a feed against oxidative damage which may result from Cyr and/or CPF additive to animal breeding against fly larvae from hatching in exposure using biochemical alterations and histopathological 10,11 manure . The early investigation showed that Cyr is harmless to findings as criteria. mammalian and poultry. It was proved to cause mammary tumors in the mouse 12. Besides the amount of parent drug excreted through MATERIALS AND METHODS feces, a portion of Cyr (2–14%) is metabolized to melamine in vivo 12. The main adverse effects shown in previous studies on animals Animals were kidney related, including renal calculi, renal tubular necrosis, Healthy male Wister rats weighing 120 ±5 g were obtained from the crystalluria, haematuria and renal dysfunction 5, 13. Also, Cyr toxicity Animal Breeding House of the National Research Centre (NRC), included reduced body weight, high blood pressure, epithelial Dokki, Cairo, Egypt, and maintained in clean plastic cages in the hyperplasia of the urinary bladder among others 6, 14, 15. laboratory animal room (23 ± 2 °C). On standard pellet diet, tap Pesticides are known to increase the production of reactive oxygen water ad libitum, and daily dark/light cycle (12/12 hrs.), the rats species (ROS), which in turn generate oxidative stress in different were acclimatized for 1 week prior to the start of experiments. The tissues 6, 16-18. Many studies have implicated oxidative damage as the experimental work on rats was performed with the approval of the central mechanism of toxicity 19-21. Oxidative damage primarily Animal Care & Experimental Committee, National Research Centre, occurs through production of reactive oxygen species (ROS), Cairo, Egypt, and international guidelines for care and use of 29 including hydroxyl radicals and hydrogen peroxide that are laboratory animals . generated during the reaction and react with biological molecules, Chemicals eventually damaging membranes and other tissues 22. Many insecticides are hydrophobic molecules that bind extensively to Chlorpyrifos (97%) and Cyromazine (99%) were obtained from biological membranes, especially phospholipids bilayers 23, and they TaeGeuk Cop., South Korea. Pu-erh green tea of post-fermented tea may damage membranes by inducing lipid peroxidation (LPO) 17, 19, produced in Yunnan province, China. The kits used for biochemical 21. measurements of malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione Heikal et al. Asian J Pharm Clin Res, Vol 6, Issuel 1, 2013, 48-55 peroxidase (GPx), transaminases (ALT& AST), alkaline phosphatase Biochemical Analysis (ALP), lactate dehydrogenase (LDH), total protein and albumin were all purchased from Biodiagnostic Company, 29 Tahreer St., Dokki, The sera obtained from different treatments were subjected to Giza, Egypt. Protein carbonyl (PC) assay kit was purchased from certain biochemical analyses by using Shimadzu UV- VIS Recording Cayman Co. cat. no. 10005020 (USA). All other chemicals were of 2401 PC (Japan). reagent grades and were obtained from the local scientific Liver function tests distributors in Egypt. Serum Transaminases (ALT & AST) activities were determined by a Preparation of Green Tea Extract and Total Phenolic Content colorimetric method according to Reitman and Frankel 34. Serum Likewise, the crude aqueous extract of green tea was prepared alkaline phosphatase (ALP) activity was determined by enzymatic 35 according to Maity et al.30 and later adopted by El-Beshbishy by colorimetric method according to Young et al. . soaking 15 g of instant green tea leaves in 1 L of distilled water Oxidative stress parameter whose temperature did not exceed 90 ºC, for 5 min to obtain soluble polyphenols dissolved in the aqueous extract 31. The solution was Malondialdehyde (MDA) content as indicator of lipid peroxidation filtered to obtain the final 1.5% (w/v) green tea extract (1.2 g was determined in the serum, by a colorimetric method according to extract/ L water). This solution was substituted in the place of water Satoh et al. 36. as the sole source of drinking fluid. The reduced form of glutathione (GSH) was determined in the live Total soluble phenolics in green tea extract (GT) were determined homogenate by colorimetric method according to Beutler et al. 37. according to the method of Slinkard and Singleton with some Protein oxidation in the rat liver homogenate was measured as modifications 32. Aliquots (100 µl) of the extracts (2 mg/ml) were protein carbonyl content. The Cayman’s protein carbonyl (PC) assay transferred into test tubes and combined with 100 µl of Folin- kit utilizes the dinitrophenylhydrazine reaction to measure the PC Ciocalteu reagent; after 3 min, 100 µl of sodium carbonate solution content in homogenate according to Levine et al.38. The amount of (2% Na2CO3) was added and the volume was adjusted to a final protein-hydrazone produced was quantified spectrophotometrically volume of 2.5 mL. After 1 h of incubation in dark at room at 380 nm. temperature, the absorbance was measured at 760 nm. The amount of total phenolic compounds was calculated as mg of gallic acid Liver antioxidant enzymes equivalents (mg Eq gallic acid) from the calibration curve of gallic acid standard solution (covering the concentration range between Catalase (CAT) activity was measured according to the method 6.25 µg/ml and 100 µg/ml). The data were presented as the average described by Aebi by assaying the hydrolysis of H2O2 and the of five replicate analyses. The concentrations of phenolic compounds resulting decrease in absorbance at 240 nm over a 3 min period at were calculated according to the following equation obtained from 25 ◦C 39. The activity of CAT enzyme is expressed as mmol/mg the standard gallic acid graph. protein. Absorbance (O.D.) = 0.046 gallic acid (µg) + 0.0342. Superoxide dismutase (SOD) activity was determined according to the method described by Marklund and Marklund by assaying the Experimental Design autooxidation and illumination of pyrogallol at 440 nm for 3 min 40. One unit of SOD activity was calculated as the amount of protein that Rats were randomly divided into 8 groups each containing 6 animals caused 50% pyrogallol autooxidation inhibition. The SOD activity is (6 rats/cage). The route of administration selected for the study was expressed as U/mg protein. oral gavage for 28 consecutive days. Rats in Group I served as control and were given corn oil (0.5 ml/rat) and allowed distillate Glutathione peroxidase (GPx) activity was measured using H2O2 as water ad libitum. Rats in Group II were given chlorpyrifos (CPF) in substrate according to the method described by Paglia and Valentine 33 corn oil at a dose of 6.75 mg/kg b.wt. daily (1/20 LD50, Tomlin) . 41. The reaction was monitored indirectly as the oxidation rate of Rats in Group III were given cyromazine (Cyr) in corn oil at a dose of NADPH at 240 nm for 3 min. Enzyme activity was expressed as 33 169.35 mg/kg b.wt. daily (1/20 LD50, Tomlin) . Rats in Group IV nmol/mg protein. were given a combination of CPF (6.75 mg/kg b.wt.) and Cyr (169.35 mg/kg b. wt.). Rats in Group V were given aqueous green tea extract Total protein as the sole drinking fluid during the 28 days at a concentration of The total protein concentrations were measured as described by 1.5% (w/v) at a dose of 30 mg green tea extract/rat/day. Rats in Lowry et al. using bovine serum albumin as standard 42. Group VI, VII and VIII were given the same doses of pesticides as in groups II, III and IV, respectively and simultaneously allowed to an Indicators of liver cell necrosis aqueous green tea extract as the sole source of drinking fluid. During the experimental duration, body weights were recorded and the Liver and serum lactate dehydrogenase (LDH) activity as indicator of doses modulated according to weekly body weight gain. necrotic cell death was determined according to the method of Vassault et al. 43. After completion of treatment period, blood samples were withdrawn from the animals under light ether anesthesia by Histopathological Examination puncturing the retro-orbital venous plexus of the animals with a fine For light microscopic investigations, specimens from liver were fixed sterilized glass capillary tube. Blood samples were collected, in 10% phosphate buffer formalin, dehydrated in alcohols and subjected to serum separation and stored at -20 ⁰C for biochemical embedded in paraffin. Five micron tissue sections were stained with analysis within one week. hematoxylin and eosin stain (H&E) for general histopathological Rats were then killed by decapitation. Livers were immediately examination. One slide was prepared for each rat. Scoring of isolated, cleaned and weighed for biochemical investigation. Small histopathological changes was done as follow: (-) absent; (+) mild; pieces of liver were cut and kept in 10% of formalin solution for (++) moderate; (+++) severe, and (++++) extremely severe (Bancroft histological studies. Other portions of liver washed with saline et al.44. solution, weighed, cut in small parts, homogenized in 10% (w/v) ice- Statistical Analysis cold 100 mM phosphate buffer (pH 7.4) and used for the determination of GSH content and liver lactate dehydrogenase The results were expressed as means ± S.E.M. All data were done (LDH). Homogenates were centrifuged at 10.000xg at 4°C for 15 min, with the Statistical Package for Social Sciences (SPSS 11.0 for then the supernatants were used for the measurements of windows). The results were analyzed using one way analysis of antioxidant enzyme activities (CAT, SOD and GPx). variance (ANOVA) followed by Duncan’s test for comparison between different treatment groups. Statistical significance was set at p < 0.05.

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RESULTS consistent sign in organophosphate poisoning. Except for the huddling, no other significant clinical manifestation was observed Total Phenolic Content following GT supplementation. In our study, the total polyphenolic content of GT in terms of gallic Body and Organ Weights acid equivalents (GAEs) was 28.11 ± 0.18 µg GAEs/mg (un-tabulated data). At the end of the experimental course, there was no significant difference in body and relative liver weights between GT and Clinical Signs untreated rats. However a significant loss of weekly body weight During the experiment, no death was observed in any of the gain accompanied by a significant increase in the relative liver experimental groups. Rats in the control group and in GT treated weights were recorded in rats treated with CPF, Cyr and their group did not show any sign of toxicity. However, CPF and Cyr+CPF mixture compared to the control. The administration of GT to treated rats showed varying degrees of clinical signs few minutes pesticide treated groups has an ameliorated effect either in the loss after dosing. The signs included huddling, mild tremor and diarrhea. of body weight or in the increase of relative liver weights (Table I). The observed signs were related to the cholinergic crisis; a Table 1. Effect of green tea consumption on body weight (g), liver weight (g), and relative liver weight (%) of rats treated with/without cyromazine, chlorpyrifos and their binary mixture.

Body weight Absolute liver weight (g) Relative liver weight Groups Initial (g) Final (g) % Change/week (g/100g body weight) Control 119.4±0.93ab 225.5±5.42c 22.2±0.96a 7.56±0.28ab 3.35±0.11a GT 122.1±1.16b 221.9±4.19cb 20.1±0.89ab 7.85±0.16ab 3.54±0.03ab Cyr 118.7±0.67ab 192.3±6.52ab 15.5±1.49c 7.79±0.15ab 4.07±0.16c CPF 120.7±0.47ab 184.9±4.25ab 13.3±0.99cd 8.41±0.16b 4.56±0.11d Cyr+CPF 119.6±0.93ab 175.4±7.50a 11.7±1.46d 8.27±0.30ab 4.72±0.12d Cyr+GT 120.8±0.55ab 202.9±2.55b 17.0±0.45bc 7.65±0.50ab 3.76±0.21abc CPF+GT 117.9±1.66a 195.9±7.99b 16.5±1.73bc 7.86±0.22ab 4.04±0.23bc Cyr+CPF +GT 119.9±0.77ab 189.9±6.19ab 14.6±1.41cd 7.50±0.20a 3.98±0.22bc

Each value is a mean of 6 rats ± S.E.M; a, b, c, d values are not sharing superscripts letters (a, b, c, d) differ significantly at p < 0.05; % of body weight change/week = [(final b .w t. – initial b .wt.)/ initial b .wt.]/no of weeks X 100; Cyr: Cyromazine; CPF: Chlorpyrifos; GT: Green tea. Biochemical Measurements (Fig. 1B). However, co-administration of GT to treated rats ameliorated the decrease in liver GSH levels to –17.9%, –19.8% and Serum lipid peroxidation (LPO) –24.1% for Cyr, CPF and Cyr+CPF-treated rats, respectively (Fig. 1B). Administration of Cyr, CPF and Cyr+CPF led to a significant No significant changes were observed between control and GT- increment (p < 0.05) in lipid peroxidation as evidenced by the treated groups. increase in serum MDA levels by 62.0%, 65.6%, and 85.8%, Liver protein carbonyl (PC) content respectively as compared to the control group. However, co- administration of GT to treated rats reduced the augmentation in The administration of GT to normal rats showed non-significant serum MDA levels to 13.6%, 34.3% and 42.7% for Cyr, CPF and decrease in liver protein carbonyl content (PC). However, Cyr+CPF-treated rats, respectively (Fig. 1A). administration of Cyr, CPF and Cyr+CPF significantly increased the liver PC content by 71.4%, 65.7%, and 95.7%, respectively as Liver glutathione (GSH) compared to the control group. Co-administration of GT to Glutathione, in the reduced form (GSH), acts as one of the major intoxicated rats attenuated the augmentation in liver PC content to detoxifiers in the body. A significant decrease of glutathione (GSH) 17.6%, 22.9% and 32.9% for Cyr, CPF and Cyr+CPF-treated rats, level in liver was evident in Cyr, CPF and Cyr+CPF-treated groups by respectively (Fig. 1C). –32.7%, –39.6% and –48.8%, respectively when compared to control

A) B) C) Serum lipid peroxidation (LPO) Liver glutathione (GSH) Liver protein carponyl content

3.50 c 0.80 b  c a b  3.00 c 100.0 bc   b a   b  0.60  2.50  d d ac cd a a  75.0 a a cd      2.00 a   bc a   0.40 a    b  50.0

1.50  (nmol/mg protein) (nmol/mg

1.00 tissue) (nmoles/g GSH 0.20

25.0 conten carbonyl Protein Malondialdehyde (nmoles/ml) Malondialdehyde 0.50

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CPF

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CPF

Control

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CPF+GT

Cyr+CPF

Control

Cyr+GT

CPF+GT

Cyr+CPF

Cyr+CPF+GT

Cyr+CPF+GT Cyr+CPF+GT

Data are expressed as mean ± S.E.M of 6 rats; Columns are not sharing above letters (a, b, c,) differ significantly at p < 0.05; Cyr: cyromazine; CPF: chlorpyrifos, GT: green tea extract; LPO: lipid peroxidation; GSH: glutathione; PC: protein carbonyl.

Figure 1: Effect of repetitive doses, for 28 consecutive days, of Cyr, CPF and their combination on serum LPO (Fig. 1A), liver glutathione (Fig. 1B) and liver protein carbonyl (Fig. 1C) levels of rat in the absence and presence of GT.

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Table 2. Effect of green tea consumption on serum levels of Enzymatic antioxidant status in liver protein, albumin and globulin of male rats treated Results in Table 4 show the influence of Cyr, CPF and their with/without cyromazine, chlorpyrifos and their binary combination on the activities of CAT, SOD and GPx. Subacute levels mixture. of the tested pesticides resulted in a state of liver injury and Total Albumin A/G extensive oxidative damage in rats as manifested by the significant Globulin(G) Treatments protein (A) ratio alteration in these enzymes. In fact, in treated rats, a significant (g/dl) (g/dl) (g/dl) increase was noted in the activities of CAT and SOD, whereas a a a a significant decrease was observed in the activity of GPx. However, Control 6.21±0.16 4.21±0.10 1.99±0.21 2.21 a a a the co-administration of GT mitigated the significant alteration in GT 6.18±0.19 4.11±0.24 2.07±0.36 2.37 c c d the activities of SOD, GPx and CAT (Table 4). Cyr 7.06±0.07 3.01±0.10 4.04±0.16 0.75 CPF 6.73±0.07bc 3.13±0.04c 3.60±0.07cd 0.87 Effects of treatments on biochemical parameters in serum and Cyr+CPF 7.16±0.13c 2.99±0.06c 4.17±0.16d 0.72 liver Cyr+GT 6.76±0.2bc 3.68±0.11b 3.09±0.22bc 1.22 CPF+GT 6.48±0.14ab 3.65±0.10b 2.82±0.17b 1.32 The oral administration of GT to normal rats produced no changes in Cyr+CPF 6.86±0.09bc 3.98±0.11ab 2.87±0.14b all the biochemical parameters tested compared to the normal +GT 1.41 control rats (Tables. 2&3). Treatments with Cyr, CPF and Cyr+CPF induced a significant increase (p < 0.05) in the total protein levels by Each value is a mean of 6 rats ± S.E.M; a, b, c, d values are not 13.6, 8.3 and 15.3%, respectively compared to the control. In sharing superscripts letters (a, b, c, d) differ significantly at p < contrast, exposure to Cyr, CPF and Cyr+CPF induced a significant 0.05; Cyr: Cyromazine; CPF: Chlorpyrifos; GT: Green tea. decrease (p < 0.05) in albumin level by -28.4, -25.7 and -28.9%, respectively, compared to the control rats. However, co- Exposure to Cyr, CPF and Cyr+CPF caused a significant elevation in administration of GT to the treated rats resulted in a partial recovery the activities of ALT, AST and ALP serum enzymes, compared to the in the above mentioned parameters (Table 2). The estimated normal control rats. However, the co-administration of GT globulin concentrations in serum of treated rat as well as the A/G attenuated the increment in serum enzymes ALT, AST and ALP levels ratio were recorded in Table 2. The A/G ratio in control rats (Table 3). Lactate dehydrogenase (LDH) activity increased in the recorded 2.21. This ratio was declined in intoxicated rats by 0.72, serum of Cyr, CPF and Cyr+CPF-treated rats by 35.0%, 32.0% and 0.87 and 72 for Cyr, CPF and the mixture Cyr+CPF, respectively, 41.5%, respectively (Fig. 2A), but decreased by –33.6%, –39.5% and however mitigation by 1.22, 1.32 and 1.41 was recorded following –45.2% in the liver of Cyr, CPF and Cyr+CPF-treated rats (Fig. 2B). administration of GT (Table 2). However, the co-administration of GT attenuated the increment in serum LDH as well as the reduction in liver LDH (Fig. 2A&B)

A) B) Serum lactate dehydrogenase Liver lactate dehydrogenase (LDH) 150.0 c c a 300.0 a c     b ab  ae 250.0 ab  ab de cde   a  100.0  bcd  200.0   bc  c 

150.0  LDH (IU/L) LDH 50.0

100.0 tissue) (IU/g LDH

50.0

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GT

Cyr

Cyr

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CPF

Control

Control

Cyr+GT

Cyr+GT

CPF+GT

CPF+GT

Cyr+CPF

Cyr+CPF

Cyr+CPF+GT Cyr+CPF+GT

Data are expressed as mean ± S.E.M of 6 rats; Columns are not sharing above letters (a, b, c, d,e) differ significantly at p < 0.05; Cyr: cyromazine; CPF: chlorpyrifos, GT: green tea extract; LDH: Lactate dehydrogenase. Figure 2:Effect of repetitive doses, for 28 consecutive days, of Cyr, CPF and their combination on serum LDH (Fig. 2A) and liver LDH (Fig. 2B) of rat in the absence and presence of GT. Histopathological Examination Table 3:Effect of green tea consumption on the activity of serum liver functions enzymes of male rats treated with/without The representative pictures of histopathological examination in the cyromazine, chlorpyrifos and their binary mixture. liver tissue are shown in Figure 3 (A-F) and the semiquantitative histological scoring of liver damage is presented in Table 4. Liver AST ALT ALP Treatments sections from the control group rats showed normal hepatic (U/l) (U/l) (U/l) cytoarchitecture. They formed of hepatocytes radiating from central Control 42.4±2.74a 30.4±1.56a 107.86±5.48a vein to the periphery of the lobules (Fig. 3A). Liver lobules of Cyr or GT 44.1±3.08a 31.6±2.06a 109.63±3.89a Cyr+CPF treated rats showed degeneration and coagulative necrosis Cyr 58.2±4.59bcd 36.9±2.77a 150.26±5.14cd in hepatocytes. However, CPF-treated rat liver showing CPF 61.0±3.34cd 42.0±2.42bc 136.35±4.91cb inflammatory cells infiltration in the portal area with diffuse Kupffer Cyr+CPF 66.1±3.71d 48.2±2.06bc 159.26±7.55d cells proliferation in between the hepatocytes (Fig. 3C). Cyr+GT 49.4±2.32ab 32.6±1.94a 119.62±5.90ab Inflammatory cells infiltration in the portal area was shown in CPF CPF+GT 50.8±3.07abc 32.7±3.07a 121.64±9.40ab and Cyr+CPF treated rats (Fig. 3 C&D&F) and fatty change in Cyr+CPF +GT 54.7±3.67bc 36.2±2.08ab 124.14±5.42ab hepatocytes was shown in Cyr+GT-treated rats (Fig. 3 E). Histopathological examination of liver of green tea treated rats a, b, c, d Each value is a mean of 6 rats ± S.E.M; values are not hepatocytes was more or less similar to control group. However, the sharing superscripts letters (a, b, c, d) differ significantly at p < liver of the GT and other treated groups showed marked 0.05; Cyr: Cyromazine; CPF: Chlorpyrifos; GT: Green tea. improvement in its histological structure in comparison to the treated groups (Cyr, CPF, Cyr+CPF) alone and represented by nil to

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Heikal et al. Asian J Pharm Clin Res, Vol 6, Issuel 1, 2013, 48-55 moderate degree in degeneration of hepatocytes, fatty change in cells proliferation in portal area, diffuse kupffer cells proliferation in hepatocytes, coagulative necrosis in hepatocytes, diffuse kupffer between hepatocytes, and dilatation in central vein (Table 5). Table 4:Effect of green tea consumption on liver enzymatic antioxidant parameters of male rats treated with/without cyromazine, chlorpyrifos and their binary mixture.

Catalase Superoxide dismutase (SOD) Glutathione peroxidase (GPx) Treatments (CAT) (U/mg protein) (nmoles/min/mg protein) (mmoles/min/mg protein) Control 0.169±0.007a 11.93±0.59a 4.80±0.216a GT 0.173±0.006a 12.10±0.54a 4.86±0.185a Cyr 0.258±0.008c 18.16±0.86b 3.30±0.128c CPF 0.286±0.006d 19.93±0.82bc 2.95±0.102cd Cyr+CPF 0.302±0.009d 21.35±1.15c 2.77±0.114d Cyr+GT 0.183±0.005a 12.49±0.73a 4.52±0.173ab CPF+GT 0.189±0.007ab 11.87±0.65a 4.37±0.234ab Cyr+CPF+GT 0.204±0.005b 13.16±0.61a 4.19±0.192b

Each value is a mean of 6 rats ± S.E.M; a, b, c, d values are not sharing superscripts letters (a, b, c, d) differ significantly at p < 0.05; Cyr: Cyromazine; CPF: Chlorpyrifos; GT: Green tea. Table 5:Semiquantitative scoring of architectural damage on histopathological examination of the rat livers in the different treatment groups.

Cyr Cyr CPF Cyr+CPF +GT Treatments Control GT Cyr CPF +CPF +GT +GT Degeneration of hepatocytes – – +++ ++ ++++ + + + Fatty change in hepatocytes – – – – ++ + – Coagulative necrosis in hepatocytes – – ++ ++ +++ – – – Diffuse kupffer cells proliferation in portal area – – ++ ++ +++ – – + Diffuse kupffer cells proliferation in between hepatocytes – + +++ ++ +++ + + ++ Dilated central vein – – – + ++ – + –

Cyr: Cyromazine; CPF: Chlorpyrifos; GT: Green tea. (–) indicates normal, (+) indicates mild, (++) indicates moderate, (+++) indicates severe, and (++++) indicates extremely severe.

Figure 3:Histopathological examination of rat liver. (Fig. 2A) (80×) showing normal histological structure of the central vein (CV) and surrounding hepatocytes, Cyr-treated animal liver showing degeneration (d) and coagulative necrosis (c) in the hepatocytes (Fig. 2B- 80×), CPF-treated rat liver showing inflammatory cells infiltration in the portal area (m) with diffuse Kupffer cells proliferation in between the hepatocytes (Fig. 2C-80×), Cyr+CPF-treated rat liver (Fig. 2D-F) showing inflammatory cells infiltration surround the bile duct (m) with diffuse Kupffer cells proliferation in between the degenerated hepatocytes (d) (Fig. 2D -80×), Cyr+GT- treated rat liver showing alveolisation in hepatocytes indicated accumulation of lipids (arrows) (Fig. 2E -80×), showing dilatation in the central vein (cv) with diffuse Kupffer cells proliferation (arrow) in between degenerated hepatocytes (d) (Fig. 2F -80×). Specimens stained with hematoxylin and eosin.

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DISCUSSION PC of Cyr, CPF, Cyr+CPF-treated rats, explains observed leakage of cellular ALT, AST and ALP to circulation due to liver injury. The liver is the largest organ in the vertebrate body, and is the major site of xenobiotic metabolism and excretion. Hepatic injury is a The cellular antioxidant status determines the susceptibility to common pathological feature which exists in many liver diseases. oxidative damage and is usually altered in response to oxidative Since liver fibrosis, cirrhosis and even liver cancer could result from stress. Hepatotoxicity could also be explained by the impaired the long existence of hepatic injury. Therefore, prevention and antioxidant enzyme activities in the liver of the rats. Indeed, the treatment of hepatic injury is a key to treat liver diseases clinically 31, antioxidant enzymes SOD, GPx and CAT limit the effects of oxidant 45. molecules in tissues and act in the defense against oxidative cell injury by means of their being free radical scavengers 73. These In toxicological studies, body, organ and relative organ weights are enzymes work together to eliminate active oxygen species. In this important criteria for evaluation of organ toxicity 17, 46. In the present respect, SOD accelerates the dismutation of H2O2, also termed as a study, oral administration of CPF and/or Cyr resulted in a significant primary defense, as it prevents further generation of free radicals reduction and elevation in body and liver weights of rats, whereas, CAT helps in the removal of H2O2 formed during the respectively. The reduction in body weight may be due to the reaction catalyzed by SOD 74. Our results indicated that CPF and/or 47-49 combined action of cholinergic and oxidative stress and/or due Cyr exposure altered SOD, CAT and GPx activities in liver of rat. to the overall increased degradation of lipids and proteins as a result These results were in line with previous studies which have shown 50 of the direct effects of CPF as an organophosphate compound . that exposure to CPF generates lipid peroxidation and alters the Moreover, the increase in liver weight could be attributed to the antioxidant status of several tissues in rats 71, 75. This alteration may relationship between liver weight increase and various toxicological be due to the decreased synthesis of enzymes or oxidative effects or to the reduction in body weight gain of experimental inactivation of enzyme protein. animals 17, 51-53. This results are consistent with many previous investigators 6, 16, 49, 54. However, co-administration of GT attenuated The histopathological alterations in the present study could be body and liver weights of intoxicated rats. summarized as follows: degeneration and coagulative necrosis in the hepatocytes, inflammatory cells infiltration, and Kupffer cells The activities of ALT, AST and ALP enzymes are the most sensitive proliferation were observed in CPF-, Cyr-, and CPF+Cyr-treated biomarkers directly implicated in the extent of hepatic damage and groups which sustained the leakage of liver enzymes. toxicity 56,57. In our findings, we demonstrated that CPF and/or Cyr administrated to rats provoked a marked elevation in serum AST, Hepatoprotective potentials of medicinal plants against pesticides ALT and ALP activities which indicating hepatocellular damage as induced hepatotoxicity remain an area that needs extensive previously reported by El-Demerdash and Kalender et al. 56, 58. This scientific research. Results of the current study revealed that GT elevation could potentially be attributed to the release of these extract reversed the elevation of lipid peroxidation. Hence, it is enzymes from the cytoplasm into the blood circulation 59, indicating possible that the mechanism of hepatoprotection of GT extract may a necrosis and inflammatory reactions 60, 61. be attributed to polyphenolic compounds (e.g. epicatechins) that scavenge a wide range of free radicals including the most active LDH can be used as an indicator for cellular damage and cytotoxicity hydroxyl radical, which initiate lipid peroxidation 25, 76. Therefore, it 62 of toxic agents . In fact, elevation in LDH activity indicates cell lysis may decrease the concentration of lipid free radicals 77. It was and death as well as the switching over of anaerobic glycolysis to reported previously that phenolic compounds chelate metal ions, 62 aerobic respiration . In the present study, LDH activity was especially iron and copper, which, in turn inhibit generation of increased in the serum of treated rats, while was decreased in the hydroxyl radicals and degradation of lipid hydroperoxides 25, 78. liver tissue. The change in LDH activity resulted from overproduction of superoxide anions and hydroxyl radicals, which The biochemical alterations accompanied by histopathological cause oxidative damage to the cell membrane 63 and increase in changes resulted from CPF and/or Cyr exposures were alleviated membrane permeability 64. following GT administration. This could be attributed to the antioxidant capacity of GT that attenuates the lipid peroxidation and Total protein and A/G ratio is done as a routine test to evaluate the liver antioxidant enzymes capacity which in turn restore the toxicological nature of various chemicals. Increases of total protein integrity of the cell membrane and improve the disturbance in and decrease of A/G ratio were observed in the present study permeability. following Cyr, CPF and CPF+Cyr treatments. In relation to decrease in the A/G ratio, the albumin level was also decreased, suggesting In conclusion, the results of the present study showed that high plasma globulin levels reflecting high protein in treated rats. Cyr and CPF induced oxidative damage and hepatotoxicity in The increase of total protein in treated groups may be due to (a) due rats. The ultimately effects was observed in their to production of enzymes lost as a result of tissue necrosis (b) to combination. In contrast, GT reduces oxidative stress by meet increased demand detoxifying the pesticide might necessitate virtue of its antioxidant properties thus improving the enhanced synthesis of enzyme proteins 65, 66. structural integrity of cell membrane and eventually alleviates the histopathological changes as well as the Oxidative damage primarily occurs through production of reactive biochemical perturbations. These beneficial effects of GT oxygen species, including hydroxyl radicals and hydrogen peroxide were able to ameliorate CPF, Cyr, Cyr+CPF-induced that are generated during the reaction and react with biological hepatotoxicity and oxidative damage. Based on our present 22 molecules, eventually damaging membranes and other tissues . observations, we propose that GT may provide a cushion for Lipid peroxidation and the oxidative protein damages, provoked by prolonged therapeutic option against toxins-induced free radicals’ attack on biological structures, have been hepatotoxicity without harmful side effects. demonstrated to play a significant role in several pathological events 67, 68. Indeed, measurement of protein carbonyl content (PC) and ACKNOWLEDGEMENT MDA have been used as a sensitive assay for oxidative damages of proteins and lipids, respectively. From the other side, GSH is an The authors are grateful of Prof. Dr. Adel Mohamed Bakeer important naturally occurring antioxidant, which prevents free Kholoussy, Professor of Pathology, Faculty of Veterinary radical damage and helps detoxification by conjugating with Medicine, Cairo University for interpreting the chemicals. 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