Experimental and Toxicologic Pathology 66 (2014) 211–218

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Effect of quercetin against dichlorvos induced nephrotoxicity in rats Yurong Hou, Yan Zeng, Sifan Li, Lei Qi, Wei Xu, Hong Wang, Xiujuan Zhao ∗ , Changhao Sun ∗ Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, Heilongjiang, China

a r t i c l e

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Article history: Received 17 December 2013 Accepted 29 January 2014 Keywords: Dichlorvos Quercetin Rats Nephrotoxicity

a b s t r a c t This study was carried out to determine the effect of quercetin against renal injury induced by dichlorvos (DDVP) in rats. Rats were randomly assigned to control, DDVP-treated (7.2 mg/kg bw), three different doses of quercetin-treated (2 mg/kg bw, 10 mg/kg bw, 50 mg/kg bw) and different doses of quercetin plus DDVP-treated groups. DDVP was administered daily to rats through their drinking water, and quercetin was administered by intragastric gavage for 90 days. By the end of the 90th day in the DDVP-treated group, the following indices significantly increased compared with the control (P < 0.01): activities of catalase, glutathione peroxidase and superoxide dismutase; level of malondialdehyde in kidney tissues; serum levels of creatinine and urea nitrogen; and level of ␤2-microglobulin, level of retinol-conjugated protein, and activity of N-acetyl-␤-d-glucosaminidase in urine; by contrast, urine uric acid levels significantly decreased. However, in the quercetin (50 mg/kg bw) plus DDVP group, the aforementioned indices were significantly decreased compared with the DDVP-treated group (P < 0.05), except the urine uric acid levels were significantly increased (P < 0.05). Thus, rat exposure to DDVP caused renal injury, including renal tubular, glomerular filtration, and oxidative stress. These toxic effects were also regulated by high-dose quercetin. Histopathological examination revealed that exposure to DDVP induced extensive cell vacuolar denaturation, but milder histopathological alterations in the kidney tissues of rats co-treated with DDVP and quercetin (50 mg/kg bw) were observed. These results indicated that quercetin at 50 mg/kg bw can partly prevent the kidney injury induced by DDVP. © 2014 Elsevier GmbH. All rights reserved.

1. Introduction Dichlorvos (DDVP; 2, 2-dichlorovinyl dimethyl phosphate), is an organophosphorus (OP) insecticide widely used globally, especially in China, since its commercial introduction (Okamura et al., 2005). DDVP is commonly used as a pesticide for maintenance and growth of agricultural products, to control the internal and external parasites of farm animals, and to eliminate insects threatening the household, public health, and stored products (C¸etin et al., 2010; Ozdikicioglu et al., 2008). Its wide application inevitably increases the danger of environmental contamination and ecological imbalance, exposing human beings to DDVP. Some studies have demonstrated that DDVP endangers human health (Alavanja et al., 2004; Margni et al., 2002; Weisenburger, 1993). Therefore, finding ways to reduce the DDVP injury to human is a concern. DDVP is a nervous system poison, and its toxicity involves its ability to act as potent acetylcholinesterase inhibitor, inhibition of

∗ Corresponding authors at: Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 194 Xuefu Road, Nangang. E-mail addresses: xiujuan [email protected] (X. Zhao), [email protected] (C. Sun). 0940-2993/$ – see front matter © 2014 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2014.01.007

this enzyme leads to the accumulation of acetylcholine in synapses and disruption of the nerve function, and finally death by poisoning (Dere et al., 2010; Varò et al., 2003; Yarsan et al., 1999). Some studies also have indicated that the toxic manifestations induced by OP may be associated with the enhanced production of reactive oxygen species (ROS) (Eraslan et al., 2010; Eroglu et al., 2013; Gultekin et al., 2001; Kanbur et al., 2009). Oxidative stress occurs when the production of ROS is beyond the antioxidant capability of the target cell, resulting in oxidative damage because of ROS interaction of with critical macromolecules. Oxidative stress is an imbalance of oxidation and antioxidation and results in damages to membrane lipids, protein, DNA, and tissues in the body. Tissues can either repair the damage or directly reduce the pro-oxidative state via the antioxidant system. Antioxidant systems are composed of enzymatic and non-enzymatic systems. The enzymatic system includes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) which scavenge free radicals and ROS (Padma et al., 2012; Uzun et al., 2010). The non-enzymatic mechanism involves endogenous compounds in the body, and exogenous compounds are administered into the body, such as flavonoids, vitamins E and C, urate, and melatonin. These substances may prevent ROS formation and exhibit pro-oxidative effects. Quercetin (3,5,7,3 ,4 -pentahydroxyflavanone) is a flavonoid found in onions, parsley, Brassica green vegetables, green tea, red

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grape wine, berries, citrus fruits, grains, and so on. Quercetin is a potent oxygen free radical scavenger and a metal chelator, and is capable of inhibiting lipid peroxidation in vitro and in vivo (Behling et al., 2006). In addition, it possesses a wide range of biological activities, including cardioprotection, cataract prevention, anti-cancer, anti-ulcer, anti-inflammatory, anti-allergic, antiviral and antibacterial activities (Adewole et al., 2007). Many studies have shown that the beneficial effects of quercetin may be related to its antioxidant action (Jullian et al., 2007; Kalender et al., 2012). Some papers have reported that quercetin can protect the body against oxidative stress induced by pesticides (Panemangalore and Bebe, 2009; Waheed and Mohammed, 2012). However, these studies focus on the rat liver, and information on renal protective effect of quercetin is lacking. As the urinary organ, kidney regulates the metabolism balance and has its own endocrine system. The kidney can discharge body metabolites and exogenous harmful substances. Some articles have shown that DDVP can change renal clearance and renal tubular function in albino mice (Desai and Desai, 2008). Other studies indicated that DDVP can affect the antioxidant defense mechanisms and induce lipid peroxidation in rats (Ajiboye, 2010; Celik and Suzek, 2009). Our previous study showed that DDVP induces oxidative damage in the rat kidney. Considering the antioxidant effect of quercetin, we presume that it can protect against DDVP-induced renal oxidative damage. The aim of the present study is to investigate of possible ameliorative effects of quercetin on DDVP induced nephrotoxicity.

2. Materials and methods

given to the animals via gavage. The dose of DDVP (7.2 mg/kg bw) was determined according to our previous study (Yang et al., 2011), which can cause significant toxic effect on rat’s kidney. Animals were exposed to DDVP by drinking water ad libitum. Body weight was measured once a week during the test. After 7 days acclimatization, rats were randomly divided into eight groups (n = 11/group), the weight of each group was similar to guarantee the reliability of the study: group C (Control), group Q1 (Low-dose quercetin-treated), group Q2 (Moderatedose quercetin treated), group Q3 (High-dose quercetin treated), group D (DDVP-treated), group DQ1 (low-dose quercetin plus DDVP-treated), group DQ2 (Moderate-dose quercetin plus DDVPtreated), group DQ3 (High-dose quercetin plus DDVP-treated). The quercetin-treated rats (group Q1, Q2, Q3, DQ1, DQ2, DQ3) administrated quercetin via gavage once a day according to the dose assigned to each group, whereas the animals in group C and D were given 0.5% CMC through the same administration method. For the DDVP-treated group (D, DQ1, DQ2, DQ3), animals received dichlorvos by drinking water ad libitum, whereas the rats in other groups (C, Q1, Q2, Q3) were given drinking water instead. Daily water volume given to each rat was its average amount of water consumption last week plus 5 ml drinking water. Based on the doses of the abovementioned treatment groups, dichlorvos was dissolved in total drinking water given to rats. Water consumption increased from 26 to 43 ml in the first 7 weeks after dosing and then maintained at about 40 ml until the end of the treatment. Water consumption in every week showed no significant changes between the experiment groups and the control group (P > 0.05). The rats received the treatment for a continually 90 days.

2.1. Chemicals Dichlorvos (95% purity) was obtained from Hebei New Century Chemical Co. Ltd. (Hengshui, China). Quercetin (CAS 849061-978) was purchased from Sigma–Aldrich (Germany). Diagnostic kits for the activity or levels of SOD, GSH-Px, CAT, MDA, total protein, adenosine triphosphate (ATP), uric acid (UA), N-acetyl-␤-dglucosaminidase (NAG) were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The enzyme-linked immunosorbent assay (ELISA) kits for retinol-binding-protein (RBP), ␤2-microglobulin (␤2-MG) were obtained from Shanghai Lichen Bio Technology Co. Ltd. (Shanghai, China). The assay kits for creatinine (CR) and blood urea nitrogen (BUN) were purchased from BioSino Bio-technology and Science Inc. (Beijing, China). Carboxymethylated cellulose (CMC) and other chemicals were analytical grade.

2.3. Sample collection Urine was collected during a 24 h period in individual metabolic cages on the 90th day, then urine samples were stored at −80 ◦ C. After the collection of urine, no food was supplied, as the experimental protocol scheduled only drinking water availability on the 12 h before sacrifice. Then rats were anesthetized using pentobarbital, abdominal aorta blood samples and kidneys were collected. The blood samples were drawn and allowed to clot. Serum was analyzed by clinical chemistry methods using a Hitachi7100 automated biochemical analyzer (Hitachi Co. Japan). The collected kidneys were washed with 0.9% NaCl and immediately weighed, then one kidney was kept at −80 ◦ C for further analysis, the other was fixed in 10% formalin for histopathological examination.

2.2. Animal treatment and experimental design

2.4. Enzymatic antioxidant activities and MDA levels

A total of 88 male Wistar rats (180–200 g) were purchased from Vital Laboratory Animal Technology Co. Ltd. (Beijing, China). All animal care and experimental procedures were approved by the Institute of Zoology Animal and Medical Ethics Committee and were in accordance with the current Chinese Legislation. The rats were housed individually in wire cages at room temperature, 22 ± 2 ◦ C, with 50–60% humidity and under a 12 h/12 h light/dark cycle. They were fed with AIN-93 rodent diets and given drinking water ad libitum. Three different doses of quercetin were applied as low (2 mg/kg bw), moderate (10 mg/kg bw) and high (50 mg/kg bw) dose. The moderate dose (10 mg/kg bw) was chosen based on our previous study which investigate the quercetin daily intake of Chinese residents (Zhang et al., 2010). Quercetin was dissolved in 0.5% carboxymethyl cellulose (CMC) and according to the expected dose

The tissues were comminuted using liquid nitrogen. The activity of SOD, CAT, GSH-Px, as well as MDA levels and protein content in the kidney tissues were measured by spectrophotometer (Shimadzu UV2550, Japan) using commercial assay kits according to the manufacturer’s directions, respectively. The total protein in the tissue homogenates was determined via the Coomassie Brilliant Blue method. The SOD activity was determined via the xanthine oxides method. GSH-Px was use in the 5,5 dithiobis-2-2nitrobenzoic acid method. The CAT activity was spectrophotometrically measured and expressed as U/mg protein based on the rate of decrease of hydrogen peroxide. The concentrations of the lipid peroxidation product MDA in the kidney homogenates were determined via the thiobarbituric acid reactive substance assay base on the reaction of MDA with thiobarbituric acid to produce a complex that can be spectrophotometrically determined.

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2.5. Detection of biochemistry parameters in urine, serum and kidney Urine NAG activity and levels of CR, UA, kidney ATP activity were detected by spectrophotometer (Shimadzu UV2550, Japan) using commercial assay kits according to the manufacturer’s directions, respectively. The activity of NAG was measured at 400 nm by colorimetry. CR levels were assayed by Picric acid colorimetric method. UA levels were measured at 690 nm by phosphotungstic acid method. Urine RBP, ␤2-MG were detected by microplate reader (Molecular Devices SpectraMax M2, USA) using commercial ELISA kits according to the manufacturer’s directions, respectively. Serum CR and BUN were analyzed using the Hitachi7100 automated biochemical analyzer. The kidney ATP enzyme activity was spectrophotometrically measured and expressed as U/mgprot based on the ATP enzyme decomposed the ATP and generate the amount of inorganic phosphorus.

2.6. Histopathology For histopathological examination, kidney tissues were dissected and tissue samples were fixed in 10% formalin for at least 24 h. Then samples were dehydrated by standard procedures and embedded in paraffin, sections approximately 4 ␮m thick were cut, stained with haematoxylin and eosin, and examined by light microscope.

2.7. Statistical analysis Data were presented as the means and their standard deviation. Statistical analysis was performed on computer using SPSS version 13.0. Significant difference between the experimental groups and control group were determined using one-way ANOVA followed by the Student–Newman–Keuls test for multiple comparisons when the variance of the data was homogenous, if not the Games–Howell test was used. Statistical significance was accepted at P < 0.05.

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3.2. Kidney antioxidant enzymes and lipid peroxidation products The observed activities of antioxidant enzymes and the corresponding MDA levels are shown in Table 1. After 90 days of treatment, no statistically significant changes in SOD, GSH-Px and CAT activities, and levels of MDA were observed in the group treated with quercetin alone compared with the control group (P > 0.05). At the end of the treatment, SOD activity in DDVP-treated group was significantly increased by 12.89% compared with the control group (P < 0.01). However, when quercetin was administered in combination with DDVP (DQ groups), SOD activity significantly decreased by 4.69% in DQ2 group (P < 0.05) and by 6.73% in DQ3 group (P < 0.01) compared with the DDVP-treated group. CAT activity was significantly increased by 13.53% (P < 0.01) in the DDVP-treated group compared with the control group. However, CAT activity was statistically significant decreased by 6.39% in DQ3 group (P < 0.05) compared with the DDVP group. GSH-Px activity in the DDVP group was significantly increased by 20.02% (P < 0.01) compared with that of the control group. However, GSH-Px activity showed a significant decrease of 8.34% in DQ3 group (P < 0.05) compared with the DDVP group. The MDA content in the DDVP-treated group was significantly increased by 22.22% (P < 0.01) compared with the control group. However, MDA content significantly decreased by 9.09% in DQ3 group (P < 0.05) compared with the DDVP group. 3.3. Clinical chemistry BUN and CR concentrations were measured in the serum to monitor the toxic effect of DDVP and the protective effect of quercetin. No statistically significant changes were observed in the group treated with quercetin alone compared with the control group (P > 0.05). The BUN (Fig. 2) and CR (Fig. 3) concentrations were significantly increased by 16.33% (P < 0.01) and 21.72% (P < 0.01), respectively, in the DDVP-treated group, compared with the control group. However, the BUN and CR levels were significantly decreased by 7.02% and 9.03% in the DQ3 group, respectively, compared with the DDVP group (P < 0.05).

3. Results

3.4. Detection of biochemistry parameters in urine and kidney

3.1. The kidney-viscera coefficient

At the end of the study, no statistically significant changes in urine and kidney biochemistry parameters were found in the groups treated with quercetin alone compared with the control (P > 0.05).

The kidney-viscera coefficient was defined as the ratio of the kidney weight to the total body weight of the rats. As shown in Fig. 1, no significant changes were observed in the kidney viscera coefficients between the experimental and control groups (P > 0.05).

Fig. 1. Kidney viscera coefficients in control and experiment groups at the end of 90th day.

Fig. 2. Serum blood urea nitrogen (BUN) levels in control group and experiment groups at the end of the treatment. # Significantly different from control group at P < 0.05; ## significantly different from control group at P < 0.01; *significantly different from dichlorvos treated group at P < 0.05.

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Table 1 The activity of antioxidant enzymes and MDA levels in the kidney of rats at the end of the treatment. Group

SOD (U/mgprot)

C Q1 Q2 Q3 D DQ1 DQ2 DQ3

73.7 72.9 72.2 71.4 83.2 80.6 79.3 77.6

# ## * **

± ± ± ± ± ± ± ±

4.33 3.52 2.13 2.94 3.69## 3.28## 2.10## , * 2.21# , **

CAT (U/mgprot) 51.0 50.0 49.1 49.1 57.9 56.1 55.9 54.2

± ± ± ± ± ± ± ±

GSH-Px (U/mgprot)

2.77 3.08 2.73 2.50 3.26## 2.17## 2.51## 2.65# , *

88.9 85.1 83.4 82.6 106.7 105.4 102.4 97.8

± ± ± ± ± ± ± ±

8.52 8.08 5.86 7.55 8.34## 7.92## 8.31## 3.76# , *

MDA (nmol/mgprot) 2.7 2.7 2.6 2.5 3.3 3.3 3.2 3.0

± ± ± ± ± ± ± ±

0.26 0.30 0.19 0.25 0.21## 0.22## 0.29## 0.20# , *

Significantly different from control group at P < 0.05. Significantly different from control group at P < 0.01. Significantly different from dichlorvos treated group at P < 0.05. Significantly different from dichlorvos treated group at P < 0.01.

Fig. 3. Serum creatinine (CR) levels in control group and experiment groups at the end of the treatment. # Significantly different from control group at P < 0.05; ## Significantly different from control group at P < 0.01; *significantly different from dichlorvos treated group at P < 0.05.

Fig. 4. Adenosine triphosphate (ATP) levels in control and experiment groups at the end of the treatment. # Significantly different from control group at P < 0.05.

showed no significant changes in DQ groups compared with that of the DDVP group (P > 0.05). Urine levels of RBP, ␤2-MG were significantly increased by 30.95%, 38.09% (P < 0.01) in DDVP-treated groups, respectively, compared with the control group (Table 2). Urine RBP and ␤2-MG decreased by 11.86% and 13.79% in the DQ3 group, respectively, compared with the DDVP group (P < 0.05). UA levels (Table 2) in the DDVP-treated group decreased by 15.15% (P < 0.01) compared with the control group. However, urine UA levels in DQ3 group were significantly increased by 8.46% (P < 0.05) compared with the DDVP group. NAG/CR activity was significantly increased by 45.36% (P < 0.01) in DDVP-treated group compared with the control group (Table 2). However, in DQ groups, NAG/CR was significantly decreased by 17.57% (P < 0.05) in DQ3 group compared with the DDVP group. The activity of ATP in the kidney tissue was significantly decreased by 13.69% in the DDVP-treated group (Fig. 4) compared with the control group (P < 0.05). However, the activity of ATP

3.5. Histopathological changes in the kidney Extensive cell vacuolar denaturation was observed in DDVPtreated group after 90 days of treatment. Histopathological changes in kidney were less pronounced in DQ group than DDVP-treated group after 90 days. DDVP resulted in cell vacuolar denaturation, but a slight change was observed in DQ3 group. However, no significant histopathological changes were found in kidney tissues of groups treated with quercetin alone and the control group (Fig. 5). 4. Discussion In the present study, SOD, CAT, GSH-Px and MDA were chosen as important indices to evaluate the effect of quercetin on the adverse effects of DDVP in vivo, based on lipid peroxidation and antioxidant

Table 2 Urine biochemical changes in control and experiment groups at the end of the treatment. Group

␤2-MG (mg/L)

C Q1 Q2 Q3 D DQ1 DQ2 DQ3

0.21 0.22 0.20 0.20 0.29 0.29 0.28 0.25

# ## *

± ± ± ± ± ± ± ±

0.03 0.03 0.03 0.03 0.04## 0.02## 0.02## 0.02# , *

Significantly different from control group at P < 0.05. Significantly different from control group at P < 0.01. Significantly different from dichlorvos treated group at P < 0.05.

RBP (␮g/L) 86.9 87.4 85.5 83.2 113.8 109.8 104.1 100.3

± ± ± ± ± ± ± ±

16.52 10.20 12.39 12.75 9.59## 10.89## 9.72## 9.38# , *

NAG/CR (U/gCR) 9.6 9.6 9.6 9.6 13.9 13.3 12.57 11.49

± ± ± ± ± ± ± ±

2.03 1.91 1.70 1.86 1.60## 1.38## 1.18## 0.91# , *

UA (mg/L) 511.1 514.5 516.8 519.2 433.7 449.2 463.9 470.4

± ± ± ± ± ± ± ±

34.52 44.33 36.79 24.28 22.68## 20.63## 32.29## 14.63# , *

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Fig. 5. The photomicrographs of kidney after 90 days treatment. (A) Control group, (B) the Q1 group, (C) the Q2 group, (D) the Q3 group, (E) the DDVP group, (F) the DQ1 group, (G) the DQ2 group, (H) the DQ3 group. HE stain, magnification 200×

enzymes profiles in rat kidneys. We also measured other indices in urine and serum to provide further support to our results. The pathway of DDVP-induced renal injury and how quercetin regulates this injury are shown in Fig. 6. SOD and CAT are the most important defense mechanisms against toxic effects of ROS. SOD catalyzes the conversion of superoxide radicals to hydrogen peroxide, whereas CAT helps in the removal of the H2 O2 formed during the reaction catalyzed by SOD (Kanbur et al., 2008; Karabacak et al., 2011; Liu et al., 2010; Mansour

and Mossa, 2009). Some studies have indicated that OP pesticides can increase SOD and CAT activities in different tissues (Tuzmen et al., 2008; Uzun et al., 2010), whereas in other studies, the activities of these enzymes were found to be decreased (Ajiboye, 2012; Karaoz et al., 2002). In the present study, free radical production as an outcome of oxidative stress induced by DDVP increased the activities of SOD and CAT in the rat kidney. The increased SOD and CAT may be the adaptive response of the body to ROS attack. However, these increases were not sufficient to protect the membrane

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Fig. 6. The regulating effect of quercetin against dichlorvos induced renal injury in rats. ↑ or ↓ represent the measured indices were significantly increased or decreased in dichlorvos treated group compared with the control group. ↓ or ↑ represent high dose quercetin plus dichlorvos treated significantly decreased or increased the aforementioned indices compared with the dichlorvos group.

lipids, because the levels of MDA in DDVP-treated group were significantly increased compared with the control. Furthermore, SOD and CAT activities and MDA levels were significantly reduced when high-dose quercetin was given in combination with DDVP for a long period of time, which may be because quercetin can directly scavenge free radicals and/or modulate the biochemical markers of oxidative stress and antioxidant enzymes (Gargouri et al., 2011). The major function of GSH-Px is to reduce soluble hydrogen peroxide and alkyl peroxides in tissues, using glutathione as an essential substrate. Some studies have indicated that pesticide exposure can affect the GSH-Px activity in rats (Datta et al., 2010; Panemangalore and Bebe, 2000). In our present study, GSHPx activity in rat kidneys increased in the DDVP-treated group, and a similar increase in GSH-Px activity was reported by Hussain (Łukaszewicz-Hussain, 2009). However, compared with the DDVP-treated group, GSH-Px activity significantly decreased when high-dose quercetin combined with DDVP was administrated. Quercetin is a potent antioxidant and is known to modulate the activities of different enzymes because of its interactions with various biomolecules (de David et al., 2011), which may explain the decrease of GSH-Px activity under quercetin treatment. Serum BUN and CR are biochemical indicators of kidney damage. BUN and CR not only reflect the nitrogenous compounds metabolism in organism, but also the glomerular filtration function damage. In the present study, we found that BUN and CR were significantly increased in the DDVP-treated group, but the combined administration of high-dose quercetin plus DDVP to rats resulted in reduced levels of serum BUN and CR. Quercetin can modulate the serum BUN and CR levels to ameliorate the toxic effect of DDVP. Urine levels of RBP and ␤2-MG are sensitive markers for renal tubular injury. RBP is a low-molecular-weight plasma protein that is filtered by the glomerulus and is normally completely reabsorbed by the proximal tubules. The urinary excretion of RBP is used as a biomarker of the reabsorption capacity of proximal tubules when the glomerulus filtration rate is acting within normal limits (Iavicoli et al., 2011). ␤2-MG is a freely filterable protein under normal condition, and is almost totally reabsorbed in the proximal tubule. However, damage to a section of the nephron leads to an increase in ␤2-MG concentration in the urine (Shimoyama et al., 2006). In the present study, elevated excretion of RBP and ␤2-MG indicate that DDVP exposure can induce renal tubule dysfunction. These results are consistent with studies in which both RBP and ␤2-MG

were increased by OP pesticides exposure (Weiqun, 2009; Xiying and guolin, 2006). NAG is located mainly in lysosomes of proximal tubular epithelial cells (Brzoska et al., 2003). The increased activity of NAG is the sensitive marker of tubule dysfunction and damage (Bosomworth et al., 1999). In the present study, NAG/CR was significantly increased in DDVP-treated group. However, in DQ groups, the urine levels of RBP and ␤2-MG, and the activity of NAG decreased, especially under high-dose quercetin plus DDVP treatment. The antioxidant properties of quercetin may prevent renal tubular damage in rat kidneys (Morales et al., 2006). This protective effect of quercetin was confirmed when the kidney tissues were examined by light microscope, as light cell vacuolar denaturation was observed in these quercetin plus dichlorvos treated rats, compared with those that received DDVP alone. UA is the end product of purine nucleoside catabolism, and has a strong antioxidant capacity based on its ability to scavenge free radicals (Alvarez-Lario and Macarron-Vicente, 2010; Maples and Mason, 1988). In our previous studies, we found that exposure to OP pesticides, including DDVP, can decrease the urine UA levels. Decreased UA level suggested that exposure to OP pesticides induced oxidative stress and affected the antioxidant system (Feng et al., 2012). However, in the present study, UA level was significantly elevated in high-dose quercetin plus dichlorvos treated group when compared with the DDVP group. Quercetin is a potent antioxidant, as shown in Fig. 6, and regulates the UA levels and the activities of SOD, CAT, and GSH-Px to scavenge the free radicals induced by DDVP in DQ groups. UA is also considered as an important antioxidant in human plasma because it can react with free radicals directly and finally result in decreased urine UA levels (Feng et al., 2012). Some studies also indicate that the antioxidant synergy between flavonoids and urate may be attribute to the beneficial effects of the flavonoid (Filipe et al., 2001). This result suggests that quercetin helps ameliorate the oxidative stress by partly increasing UA level. ATP can provide energy and maintain renal tubular secretion and reabsorption, but in the presence of xenobiotics, ATP activity decreases and can no longer provide enough energy for the renal tubule to maintain its function. Therefore, the decrease in the activity of ATP (Fig. 6) indirectly reflects tubular damage. ATPase is a glycoprotein that catalyzes the ATP hydrolysis, provides energy for ion transportation, and has a great significance in maintaining renal tubular secretion and reabsorption. Chronic exposure to

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DDVP can decrease the ATPase activity in liver (Binukumar et al., 2010). Other studies have indicated that the ATPase is susceptible to free radical-induced damage and quercetin applied at high dose (>100 mg/kg bw) can reverse the xenobiotics-induced reduction in ATPase activity (Lees et al., 1990; Lv et al., 2012). In the present study, DDVP-treated group showed significantly decreased activity of ATPase in kidney tissue, and quercetin treatment failed to reverse this change, which may be because the dose of quercetin was too low to restore the damage. In conclusion, rats exposure to DDVP caused renal injury and quercetin can partly regulate this injury. The regulation way is shown in Fig. 6. Compared with the control group, biochemical indices mentioned above significantly changed in the DDVP group, which suggests that exposure to DDVP can induce renal injury. As shown in Fig. 6, the toxic effect of DDVP was observed in the renal tubular, glomerular filtration, and antioxidant defense system. These toxic effects were regulated by quercetin. However, even though quercetin plus DDVP treatment ameliorate this change, significant differences between high-dose quercetin plus DDVP-treated group and the control group were still observed. These results indicate that quercetin can partly prevent DDVPinduced kidney injury. Histopathological changes in the kidney give a further support of this conclusion (Fig. 5). In relation to the public health, we should consume food rich in quercetin if there is a possibility of pesticide contamination in our diet. Acknowledgment Financial support from the National Natural Science Foundation of China (81172672) is gratefully acknowledged. References Adewole SO, Caxton-Martins EA, Ojewole JA. Protective effect of quercetin on the morphology of pancreatic ␤-cells of streptozotocin-treated diabetic rats. Afr J Tradit Complement Altern Med 2007;4:64–74. Ajiboye TO. Oxidative insults of 2,2-dichlorovinyl-dimethyl phosphate (DDVP), anorganophosphate insecticide in rats brain. Fountain J Nat Appl Sci 2012;1:1–8. Ajiboye TO. Redox status of the liver and kidney of 2,2-dichlorovinyl dimethyl phosphate (DDVP) treated rats. Chem Biol Interact 2010;185:202–7. Alavanja MC, Hoppin JA, Kamel F. Health effects of chronic pesticide exposure: cancer and neurotoxicity. Annu Rev Public Health 2004;25:155–97. Alvarez-Lario B, Macarron-Vicente J. Uric acid and evolution. Rheumatology (Oxf) 2010;49:2010–5. Behling EB, Sendão MC, Francescato HD, Antunes LM, Costa RS, Bianchi Mide L. Comparative study of multiple dosage of quercetin against cisplatininduced nephrotoxicity and oxidative stress in rat kidneys. Pharmacol Rep 2006;58:526–32. Binukumar BK, Bal A, Kandimalla R, Sunkaria A, Gill KD. Mitochondrial energy metabolism impairment and liver dysfunction following chronic exposure to dichlorvos. Toxicology 2010;270:77–84. Bosomworth MP, Aparicio SR, Hay AW. Urine N-acetyl-␤-d-glucosaminidase – a marker of tubular damage? Nephrol Dial Transplant 1999;14:620–6. Brzoska MM, Kaminski M, Supernak-Bobko D, Zwierz K, Moniuszko-Jakoniuk J. Changes in the structure and function of the kidney of rats chronically exposed to cadmium. I. Biochemical and histopathological studies. Arch Toxicol 2003;77:344–52. Celik I, Suzek H. Effects of subacute exposure of dichlorvos at sublethal dosages on erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats. Ecotoxicol Environ Saf 2009;72:905–8. C¸etin E, Kanbur M, Silici S, Eraslan G. Propetamphos-induced changes in haematological and biochemical parameters of female rats: protective role of propolis. Food Chem Toxicol 2010;48:1806–10. Datta S, Dhar P, Mukherjee A, Ghosh S. Influence of polyphenolic extracts from Enydra fluctuans on oxidative stress induced by acephate in rats. Food Chem Toxicol 2010;48:2766–71. de David C, Rodrigues G, Bona S, Meurer L, Gonzalez-Gallego J, Tunon MJ, et al. Role of quercetin in preventing thioacetamide-induced liver injury in rats. Toxicol Pathol 2011;39:949–57. Dere E, Ari F, Ugur S. The effect of dichlorvos on acetylcholinesterase activity in some tissues in rats. Acta Vet (Beogr) 2010;60:123–31. Desai SN, Desai PV. Changes in renal clearance and renal tubular function in albino mice under the influence of Dichlorvos. Pestic Biochem Physiol 2008;91:160–9. Eraslan G, Kanbur M, Silici S, Karabacak M. Beneficial effect of pine honey on trichlorfon induced some biochemical alterations in mice. Ecotoxicol Environ Saf 2010;73:1084–91.

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Effect of quercetin against dichlorvos induced nephrotoxicity in rats.

This study was carried out to determine the effect of quercetin against renal injury induced by dichlorvos (DDVP) in rats. Rats were randomly assigned...
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