American Journal of Biochemistry

p-ISSN: 2163-3010    e-ISSN: 2163-3029

2016;  6(5): 113-121

doi:10.5923/j.ajb.20160605.01

 

Ameliorative Effects of Quercetin and Naringenin on Diethylnitrosamine/2-acetyl aminoflourene-Induced Nephrotoxicity in Male Wistar Rats

Adel Abdel-Moneim A. , Osama M. Ahmed , Hanaa I. Fahim , Mohamed Y. Zaky

Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt

Correspondence to: Adel Abdel-Moneim A. , Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt.

Email:

Copyright © 2016 Scientific & Academic Publishing. All Rights Reserved.

This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/

Abstract

Nephrotoxicity is one of the most widespread kidney tribulations. Quercetin and naringenin are natural flavonoids highly enriched in fruits, and vegetables which have a bioactive effect on human health. The current study was designed to evaluate the preventive effects of quercetin and naringenin on Diethylnitrosamine (DEN)/2- Acetylaminofluorene (2AAF)-induced nephrotoxicity in rats. The Wistar rats used in this study were allocated into 4 groups. The 1st group was kept as a normal group while the other 3 groups were intraperitoneally administered DEN (150 mg/kg b.w./week for two weeks) dissolved in 0.9% saline at the beginning of experiment. One week after the last injection of DEN, 2-AAF was orally administrated at dose 20 mg/kg b.w. in 1% tween 80 four days per week for three weeks. One of the three DEN/2AAF-administered groups was kept as control and the others were orally treated with quercetin and naringenin at dose 10 mg/kg b.w. every other day for 20 weeks. DEN/2AAF administration induced kidney injury evidenced by histological alterations as well as significant increases of serum creatinine, urea and uric acid concentrations. There was also a significant increase in the levels of renal lipid peroxidation and nitric oxide. Moreover, renal glutathione content and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase were significantly declined as a result DEN/2AAF administration. The treatments of DEN/2AAF-administered rats with the two flavonoids quercetin and naringenin successfully amended kidney histological perturbations and improved serum markers related to kidney function in association with alleviation of the deteriorated kidney oxidative stress and anti-oxidant defense system. In conclusion, quercetin and naringenin successfully attenuate the DEN/2AAF-induced nephrotoxicity and this effect may be mediated via suppression of the oxidative stress and stimulation of the antioxidant defense system.

Keywords: Quercetin, Naringenin, Nephrotoxicity, Diethylnitrosamine, Acetylaminofluorene, Oxidative stress

Cite this paper: Adel Abdel-Moneim A. , Osama M. Ahmed , Hanaa I. Fahim , Mohamed Y. Zaky , Ameliorative Effects of Quercetin and Naringenin on Diethylnitrosamine/2-acetyl aminoflourene-Induced Nephrotoxicity in Male Wistar Rats, American Journal of Biochemistry, Vol. 6 No. 5, 2016, pp. 113-121. doi: 10.5923/j.ajb.20160605.01.

Article Outline

1. Introduction
2. Material and Methods
    2.1. Experimental Animals
    2.2. Chemicals
    2.3. Induction of Nephrotoxicity
    2.4. Preparation of Dose of Quercetin and Naringenin
    2.5. Experimental Design
    2.6. Blood and Kidney Sampling
    2.7. Biochemical Assays
    2.8. Histopathological Study
    2.9. Statistical Analysis
3. Results
    3.1. Effect on Serum Creatinine, Urea and Uric Acid Concentrations
    3.2. Effect on Kidney Oxidative Stress and Antioxidant Defense System
        3.2.1. Effect on Kidney LPO and NO Level
        3.2.2. Effect on Kidney GSH Content and Its Metabolizing Enzymes, GPx and GST Activities
        3.2.3. Effect on Kidney SOD and CAT Activities
        3.2.4. Effect on Kidney Histological Changes
4. Discussion
ACKNOWLEDGEMENTS

1. Introduction

The kidney is the target of many xenobiotic toxicants, including environmental chemicals. An environmental chemical has the potential to adversely affect human health through the disruption of kidney functions [1]. An exposure to environmental pollutants increased risks for kidney disease [2]. Mixture of physiologic and biochemical proceedings donate to the propensity of the kidney to several distinct classes of nephrotoxicity [3]. When kidney is evaluated with other organs, the kidney is uniquely susceptible to chemical toxicity, partially because of its unduly high blood flow, or due to its intricacy both physiologically and anatomically [4]. Nephrons toxicity may be consequent to direct cytotoxic damage to kidney structures by environmental toxicants, immunologic processes, indirect toxicity due to alterations in renal hemodynamics, or the production of endogenous nephrotoxic substances [5].
Diethylnitrosamine (DEN), a well-known potent carcinogenic agent, is produced from the metabolism of some drugs and also present in tobacco, cheese and wide variety of foods [6]. DEN-induced renal damage was reported to occur through stimulation of oxidative stress and abolishment of antioxidant defense system [7]. In the same way, DEN was suggested to induce generation of free oxygen species and eventually resulting in oxidative stress and cellular injury which play an important role in the pathogenesis of kidney diseases [8]. Hence, the use of antioxidants could offer ameliorative effects against drug-induced renal damage.
Flavonoids were considered one of the most important groups of secondary metabolites and bioactive compounds in plants and good sources of natural antioxidants in human diets [9]. Flavonoids have been suggested to exhibit a powerful antioxidant activity due to their ability to reduce free radical formation and scavenge free radicals, together with the up-regulation of antioxidant defenses [9]. Several studies have estimated the anti-oxidative, tissue protective and anti-tumor effects of flavonoids [10, 11]. Quercetin, one of the most natural abundant flavonoids found in fruits and vegetables such as apples and onions, has been shown to have very potent antioxidant [12, 13], and cytoprotective effects by preventing endothelial apoptosis caused by oxidants [14]. In rats, quercetin exhibited a protective effect against cisplatin-induced oxidative damage in kidneys [15]. Naringenin, another natural flavonoid highly enriched in citrus fruit, tomatoes and cocoa, has been reported to have a bioactive effect on human health including anti-tumor [16], anti-inflammatory [17, 18], and antioxidant activities [19]. Previous studies demonstrated that naringenin significantly ameliorated oxidative and inflammatory tissue injuries in different experimental models [20, 21].
Therefore, this study was designed to assess the preventive effects of quercetin and naringenin on DEN/2AAF-induced kidney toxicity in male Wistar rats.

2. Material and Methods

2.1. Experimental Animals

Adult male Wistar albino rats, weighing 100-120 g and aging 8-9 weeks, were used in the present study. The animals were obtained from Helwan Station for Experimental Animals, Egyptian Organization for Biological Products and Vaccines (VACSERA), Helwan, Cairo, Egypt. They were kept under observation for two weeks before the onset of the experiment to exclude any intercurrent infections. The animals were housed in good aerated polypropylene cages in the Zoology Department Animal House in Faculty of science, Beni-Suef University, Egypt at normal temperature (20-25°C) and normal daily lighting cycle (10-12 hr/day), and given enough food (balanced standard diet) and water ad libitum. All animal procedures are in accordance with the general guidelines of animal care and the recommendations of the Experimental Animal Ethics Committee of Faculty of Science, Beni-Suef University, Egypt. All efforts were done to reduce the number and suffering of animals.

2.2. Chemicals

Diethylnitrosamine and, 2- acetyl aminoflourine, quercetin and naringenin, were purchased from Sigma Chemicals Co., St. Louis, MO, USA, stored at 2-4°C. All other chemicals used for the investigation were of analytical grade.

2.3. Induction of Nephrotoxicity

Nephrotoxicity was induced by intraperitoneal injection of DEN (150 mg/kg b.w./week for two weeks) dissolved in 0.9% saline. One week after the last injection of DEN, 2-AAF (20 mg/kg b w) [22] in 1% tween 80 was administrated orally by gavage four days per week for three weeks, then rats were sacrificed after 20 weeks from the beginning of the experiment.

2.4. Preparation of Dose of Quercetin and Naringenin

Querctin and naringenin each at dose level of 10 mg/kg b. w. [23, 24] was dissolved in 5 ml of 1% carboxymethylcellulose (CMC) (1% w/v) and was administered to rats by oral gavage every other day for 20 weeks from the beginning of the experiment.

2.5. Experimental Design

The experiment was performed on forty adult male Wister rats which were randomly distributed into 4 groups, each of ten animals. The 1st group was kept as a normal group, while the other 3 groups were administered DEN, followed by 2-AAF as previously described. One of three DEN/2AAF-administered groups were kept as control while the two others were orally given quercetin and naringenin every other day for 20 weeks.

2.6. Blood and Kidney Sampling

By the end of the experiment, animals were sacrificed under mild anesthesia. Blood samples were collected from jugular veins, left to coagulate and centrifuged at 3000 rpm for 15 min to separate the serum. Kidney samples were immediately excised and perfused with ice-cold saline. Frozen kidney samples were homogenized in chilled saline (10% wt/vol) by using Telfon homogenizer (Glas-Col, Terre Haute, USA) and the homogenates were centrifuged at 3000 rpm for 10 min. The homogenate supernatants were collected and kept in deep freezer at –30°C until used for the determination of oxidative stress parameters and antioxidant defense markers.

2.7. Biochemical Assays

Serum creatinine, urea and uric acid concentrations were assayed using reagent kits purchased from Biosystems (Spain), following the methods of Fabiny and Ertingshausen [25], Tabacco et al. [26] and Fossati et al. [27], respectively.
The supernatants were used for estimation of lipid peroxidation (LPO) [28], Nitric oxide [29] using chemical reagents prepared in laboratory. Reduced glutathione (GSH) content [30], and activities of kidney antioxidant enzymes including glutathione peroxidase (GPx) [31], superoxide dismutase (SOD) [32], catalase (CAT) [33], and glutathione-S-transferase (GST) [34] were also determined using chemical reagents prepared in laboratory.

2.8. Histopathological Study

After sacrifice, decapitation and dissection, kidney from each rat was rapidly excised and then perfused in saline solution. Pieces from the kidney of rats of different groups were taken and fixed in 10% neutral buffered formalin for twenty four hours. Washing was done in tap water and serial dilutions of ethyl alcohol were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in hot air oven for twenty four hours. Paraffin wax tissue blocks were prepared, then sectioning at 4 µm thickness by slide microtome was performed. The obtained tissue sections were mounted on glass slides, deparaffinized and stained with hematoxylin and eosin (H&E) according to the method of Banchroft et al. [35].

2.9. Statistical Analysis

Results were expressed as mean ± standard error (SE). The data were analyzed using SPSS version 20 software [36] by Duncan’s method for post-hoc analysis to compare different groups at p-value, P<0.05.

3. Results

3.1. Effect on Serum Creatinine, Urea and Uric Acid Concentrations

The rats-administered DEN/2AAF exhibited a significant (P < 0.05) increase in serum creatinine, urea and uric acid concentrations as compared to the normal control rats. The treatment of DEN/2AAF-administered rats with quercetin and naringenin induced a significant (P < 0.05) improvement in the elevated serum creatinine, urea and uric acid concentrations as compared to DEN/2AAF-administered control group (Table 1).
Table 1. Effects of quercetin and naringenin on serum creatinine, urea and uric acid concentrations in DEN/2AAF-administered rats
     

3.2. Effect on Kidney Oxidative Stress and Antioxidant Defense System

3.2.1. Effect on Kidney LPO and NO Level
The administration of DEN/2AAF to normal rats produced a significant (P < 0.05) increase in the LPO and NO level. However, the treatment of DEN/2AAF- administered rats with quercetin and naringenin significantly (P < 0.05) prevented this elevation in LPO and NO level (Table 2).
Table 2. Effects of quercetin and naringenin on kidney LPO and NO level in DEN/2AAF-administered rats
     
3.2.2. Effect on Kidney GSH Content and Its Metabolizing Enzymes, GPx and GST Activities
DEN/2AAF-administration produced a significant (P<0.05) decrease in GSH content as well as GPx and GST activities stores as compared to the normal control group. The administration of quercetin to DEN/2AAF-administered rats significantly (P<0.05) prevented the decrease in GSH content and GPx, and GST activities. On the other hand, the administration of naringenin to DEN/2AAF-administered rats significantly (P < 0.05) improved the depleted GSH content as well as GPx activity, however, its effect on GST activity was non-significant (p > 0.05) (Table 3).
Table 3. Effect of quercetin and naringenin on kidney content of GSH and activities of Gpx and GST of DEN/ 2AAF administrated rats
     
3.2.3. Effect on Kidney SOD and CAT Activities
The kidney SOD and CAT activities were significantly (P<0.05) decreased in rats administered DEN/2AAF as compared to normal group. The treatment of DEN/2AAF-administered rats with quercetin and naringenin produced a non-significant (P < 0.05) amelioration of the decreased SOD activity, however, quercetin and naringenin administration to DEN/2AAF- rats produce a significant (P < 0.05) ameriloation in the CAT activity (Table 4).
Table 4. Effects of quercetin and naringenin on kidney SOD and CAT activities of DEN/ 2AAF administrated rats
     
3.2.4. Effect on Kidney Histological Changes
Histopatholgical examination of the kidney sections of normal rats revealed normal histological structure of renal parenchyma, glomerulus, proximal tubule and distal tubules (Figure A). On the other hand, DEN/2AAF-administration induced histpathological changes and several lesions that include karyomegaly (long arrows), sever vacuolar changes in renal tubule (Figure B). Also, DEN/2AAF-aministered group showed intraepithelial accumulation of brown pigments and perivascular oedema (Figure C), focal periglomerular mononuclear inflammatory cells aggregation and vacuolation of epithelial lining renal tubules (Figure D). The treatment of DEN/2AAF-administered rats with quercetin improved the histological architecture and integrity resulting in nearly normal renal tubules (Figure E). Also, DEN/2AAF-administered rats treated with naringenin showed congestion in the blood vessels and nearly normal renal tubules (Figure F). In comparison with DEN/2AAF-administered control rats, the kidney histological integrity and architecture as a result of treatment of DEN/2AAF-administered rats were markedly improved.
Figure 1. Photomicrographs of H&E stained kidney sections of rats in the experimental groups. Photomicrograph A showed normal histological structure of renal parenchyma, glomerulus (G), proximal tubules (PT) and distal tubules (DT) in normal rat. Photomicrographs of kidney sections of DEN/2AAF depicting several lesions that include karyomegaly (long arrows) (Photomicrograph B), severe vacuolar changes in renal tubule (V), intraepithelial accumulation of brown pigments (thin arrow) and perivascular oedema (O) (Photomicrograph C) and focal periglomerular mononuclear inflammatory cells (IF) aggregation and vacuolation (thick arrow) of epithelial lining renal tubules (Photomicrograph D). Photomicrograph of H&E stained kidney section of DEN/2AAF-administered rat treated with quercetin showed nearly normal renal tubules (Photomicrograph E). Photomicrograph of H&E stained kidney section of DEN/2AAF-administered rat treated with naringenin (Photomicrograph F) showed congestion in the blood vessels with nearly normal renal tubules (X400)

4. Discussion

The kidney is a biochemically active and essential organ in the human body and part of the urinary system carrying out many essential functions like clearance of metabolic waste products, control of fluid volume status, maintenance of electrolyte and acid-base balance, and endocrine function [37]. It removes the metabolic waste and possesses the common xenobiotic metabolizing enzymes, mainly localized in proximal tubular cells [38]. The present study showed that the administration of DEN/2AAF has induced renal injury and this was evident by the increased serum concentration of markers related to the kidney function like creatinine, urea and uric acid. It has been reported that serum creatinine concentration relates to glomerular function and its rise is an indicator of renal failure [39, 40]. Urea is a by-product of protein metabolism and is used as a marker in acute kidney injury for retention and elimination of uremic solutes [41]. Also, Serum uric acid was proposed as a potential risk factor for new onset of kidney disease [42, 43]. These findings are in agreement with other studies [7, 44-45]. Recently, it was found that DEN administration induced nephrotoxicity in rats marked by a significant elevation of the levels of serum creatinine and urea [46].
On the other hand, the DEN/2AAF-administered groups treated with quercetin and naringenin markedly decreased serum levels of creatinine, urea and uric acid suggesting nephrooprotective effects of querctin and naringenin. These finding are in accordance with the results described previously that showed quercetin modulate nephrotoxicity by reducing levels of kidney markers [47, 48]. Our study is also in agreement with Badarya et al. [49] and Vitaglione et al. [50] who showed that naringenin treatment significantly protected nephrotoxicity in rat and mice. Many investigators attributed this damage to exacerbated production of reactive oxygen species (ROS) and oxidative stress. ROS have the ability to cause oxidative damage in DNA, proteins and lipids [51]. The kidney is susceptible to injury caused by ROS because of the plenty of long chain polyunsaturated fatty acids found in the composition of renal lipids [52]. DEN has been suggested to cause the generation of ROS resulting in oxidative stress, alteration of the antioxidant defense system in tissues and cellular injury [53, 54]. In addition, some ROS interact with various tissue compounds leading to dysfunction and injury to the kidney, liver and other organs [55].
In the present study, the DEN/2AAF administration deleteriously increased the kidney MDA and NO production. MDA, an indicator of LPO, has long been used as ultimate biomarker of oxidative damage and the increase of MDA reflects the enhancement of LPO [56]. NO can be both a scavenger and a producer of free oxygen radicals since it reacts with superoxide anions to form peroxynitrite which becomes a damage-producing radical [57]. In the current study, levels of LPO and NO in kidney were elevated in response to the administration of DEN/2AAF. The kidney injury may be attributed to ROS which induce mesangial cells contraction, altering the filtration surface area and modifying the ultrafiltration coefficient factors that decrease the glomerular filtration rate [58]. It was found that DEN is degraded and contact with renal tubular epithelium and is converted to active electrophilic species following α or β hydroxylation, resulting in the formation of unstable hydroxyalkyl compounds that converted to alkyl carbonium ions bind to DNA forming adducts and generate superoxide radicals through LPO [59, 60]. Further, this may be due to an enhanced generation of superoxide radicals (O2.-) and hydrogen peroxide radicals that accelerated peroxidation of native membrane lipids [55].
On the same line, NO interacts with superoxide to generate the potent cytotoxic agent, peroxynitrite [61, 62]. The major effect of peroxynitrite is the nitration of cellular proteins leading to nitrosative stress and tissue injury [63]. Hence, the kidney injury is probably due to the deleterious effect of DEN itself and/or its metabolites which includes ethylcarbonium ions, NO and ROS [64]. Our results are in agreement with previous studies [65] that was found the level of MDA was significantly elevated in DEN administration to rats in comparison to normal control. Also, our results in accordance with Bishayee et al. [64] who stated that DEN also induced iNOS gene expression and generates NO radicals which react non enzymatically with O2- forming peroxynitrite (ONOO- a reactive nitrogen species).
In the current study, the DEN/2AAF administration caused a marked deterioration in the kidney antioxidant defense system manifested by depletion of kidney glutathione level and suppression of activities of antioxidant enzymes including GPx, GST, SOD and CAT. In this situation, it worth mentioning that for the purpose of preventing cellular damage induced by ROS, the anti-oxidant defense system may scavenge ROS that play an important role in the initiation of LPO [64]. This defense system operates through enzymatic (including SOD, GPx, GST and CAT), and non-enzymatic components (mainly GSH) [66, 67]. SOD is the primary step of defense mechanism in the antioxidant system against the oxidative stress, as it dismutates the highly toxic superoxide anions (O2-) to O2 and H2O2. Gpx and CAT can scavange H2O2 and convert it into harmless byproducts, thereby providing protection against ROS [68, 69]. GPx also has a high potency in scavenging reactive free radicals in response to oxidative stress and detoxifies peroxides and hydroperoxides that lead to the oxidation of GSH [70]. Furthermore, GST catalyzes the conjugation of the thiol functional groups of GSH to electrophilic xenobiotics, leading to elimination or conversion of xenobiotic-GSH conjugate [71]. GSH is the most important non-enzyme antioxidant in mammalian cells [72]. It is said to be involved in many cellular processes including the detoxification of endogenous and exogenous compounds and efficiently protects cells against deleterious effects of oxidative stress by scavenging free radicals, removing H2O2, and suppressing LPO [73]. GSH is a potent antioxidant which protects the cellular constituents against the damage induced by the free radicals [74] through the formation of S-conjugates with products of LPO [75]. It was reported that GSH decline leads to lowered cellular defense against free radical induced cellular injury resulting in cell death [76]. Thus, these antioxidant enzymes together with glutathione are highly effective in inhibiting various ROS-mediated injuries and could protect the kidney from DEN-induced nephrotoxicity that occurred in the present investigation.
The striking decease in antioxidants (SOD, CAT, GSH, GPx and GST) in DEN/2AAF-exposed rats, in the present investigation, elicits strong evidence for the involvement of oxidative damage in DEN/2AAF-induced nephrotoxicity. This may be due to ROS produced from metabolism of DEN that cause decreases the activities of renal antioxidant enzymes. Our results in agreement with Shaheen et al. [77] who reported that DEN decreased renal GSH content as well as GST and SOD activities and with Mahmoud et al. [7] and Ahmed et al. [46] who found that renal GSH content and activities of SOD, CAT, GPx and GST were significantly declined in the DEN-administered rats.
In the present study, it was found that the treatment with quercetin and naringenin reduced the kidney LPO and NO level and counteracted the formation of free radicals induced by DEN/2AAF-mediated nephrotoxicity. Moreover, quercetin and naringenin treatments successfully resulted in an improvement of the lowered GSH content, and SOD, CAT, GPx and GST activities in the kidney of DEN/2AAF-intoxicated rats. This could be due to the ability of these antioxidant substances to transfer electrons free radicals, catalayse and activate antioxidant enzymes [78] and this proves that quercetin and naringenin administration overcomes the oxidative stress by their antioxidant properties.
Quercetin significantly attenuated the LPO and NO production in the renal tissue probably because of its antioxidant capacity to scavenge oxygen free radical in the kidney tissue cells of rats. In addition to its free radicals-scavenging capability, quercetin enhanced the antioxidants enzymes SOD, CAT, GPx and GST activities and the GSH recovery in the kidney of DEN/2AAF-intoxicated rats reflecting its antioxidant activities. Many studies demonstrsted that quercetin has abroad range of pharmacological activities such as antioxidant and anti-inflammatory [79, 80]. Our results are in accordance with Almaghrabi, [81] who stated that quercetin had the ability to overcome oxidative stress through the reduction of free radical levels and elevating the antioxidant enzymes proving the renoprotective effects of qurecetin against oxidative stress induced by cisplatin. Also, naringenin ameliorated the renal damage that occurred due to DEN/2AAF-by reducing LPO and NO level and elevated SOD, CAT, GPx and GST activities and increased production of reduced form of glutathione in the kidney. Many studies reported that naringenin exerts marked antioxidant activity, scavenges ROS, suppresses LPO and maintains the antioxidant defense mechanisms [82, 83]. Our results are in agreement with the previous studies which stated that naringenin inhibits iNOS activity and therefore prevents nitrosative tissue stress [84, 85]. Recently, it was concluded that naringenin, through its antioxidant effects, may represent a therapeutic option to protect against gentamicin nephrotoxicity [86].
In addition, renal injury induced by DEN/2AAF was confirmed by the observed histpathological alternations that include severe karyomegaly, sever vacuolar changes in renal tubule, intraepithelial accumulation of brown pigments and perivascular oedema, focal periglomerular mononuclear inflammatory cells aggregation and vacuolation of epithelial lining renal tubules. Our results are in agreement with Mahmoud et al. [7] and Ahmed et al. [87] who stated that renal injury induced by DEN confirmed by the observed histological alterations, including adenoma, dysplastic renal tubules with karyomegalic nuclei, atrophy of glomerular tuft, and inflammatory cells infiltration. Recently, it was reported that DEN induced renal damage which confirmed by histological changes that include focal fibrosis, necrosis and focal inflammatory cells infiltration in between the tubule [46]. As well, DEN induced injury and lesions in other body organs like liver [88]. The treatment of DEN/2AAF-administered rats with quercetin and naringenin improved kidney architecture and integrity since the kidney tissues showed nearly normal histological structures in these animals. However, the presence of congested intetubular blood vessels in kidney section of DEN/2AAF-administered rats treated with naringenin may be attributed to the increased blood flow and/or diltation of these blood vessels. These histological results are concomitant with the biochemical results which indicated improvements of the serum markers related to kidney function and antioxidant defense system as a result of treatments of DEN/2AAF-administered rats treated with quercetin and naringenin.
In conclusion, the possible ameliorative and preventive effects of quercetin and naringenin on DEN/2AAF-induced nephrotoxicity may be attributed, at least in part, to suppression of oxidative stress and enhancement of the antioxidant defense system on the basis of oxidant-antioxidant system. Thus, these treatments may act as antioxidant preventive agents. However, further clinical studies are required to assess the safety and efficacy of these agents in human beings.

ACKNOWLEDGEMENTS

We would like to thank Prof. Dr. Kawkab Abdel Aziz Ahmed, Professor of Histopathology, Faculty of Veterinary Medicine, Cairo University, Egypt and Prof. Dr. Rasha Rashad Ahmed, Professor of Molecular Cell Biology, Faculty of Science, Beni-Suef University, Egypt for their help in examinations of histological sections of detection of kidney lesions.

References

[1]  Van Vleet, T. R and Schnellmann, R. G. (2003). Toxic Nephropathy: Environmental Chemicals, Sem in Nephrol., 23 (5): 500-508.
[2]  Hendryx, M. (2009). Mortality from heart, respiratory, and kidney disease, Int. Arch. Occup. Environ. Health, 82: 243–249.
[3]  Naesens, M., Kuypers, D. R. and Sarwal, M. (2009). Calcineurin inhibitor nephrotoxicity. Clin. J. Am. Soc. Nephrol., 4(2): 481–508.
[4]  Schrier, R. W., Wang, W., Poole, B. and Mitra, A. (2004). Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. J. Clin. Invest., 114: 5–14.
[5]  Arany, I. and Safirstein, R. L. (2003). Cisplatin nephrotoxicity. Semin. Nephrol., 23: 460–464.
[6]  Verna, L., Whysner, J. and Williams, G. M. (1996). N-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation. Pharmacol. Ther., 71: 57-81.
[7]  Mahmoud, A. M., Ahmed, R. R., Soliman, H. A. and Salah, M. (2015). Ruta graveolens and its active constituent rutin protect against diethylnitrosamine-induced nephrotoxicity through modulation of oxidative stress. J. Appl. Pharm. Sci., 5: 016-021.
[8]  Bartech, H., Heathen, E. and Melville, C. (1989). Carcinogenic nitrosamines: free radical aspects of their action. Free Radic. Boil. Med., 7: 637–644.
[9]  Kim, D., Jeond, S. and Lee, C. (2003): Antioxidant capacity of phenolic phytochemicals from various cultivars of plums. Food. Chem,. 81: 321-326.
[10]  Glässer, G., Graefe, E. U., Struck, F., Veit, M. and Gebhardt, R. (2002). Comparison of antioxidative capacities and inhibitory effects on cholesterol biosynthesis of quercetin and potential metabolites. Phytomedicine. 9 (1): 33–40.
[11]  Maksimovic, Z., Malencic, D. and Kovacevic, N. (2005). Polyphenol contents and antioxidant activity of Maydis stigma extracts. Bioresour Technol., 96 (8): 873–877.
[12]  Manach, C., Scalbert, A., Morand, C., Rémésy, C. and Jiménez, L. (2004). Polyphenols: Food Sources and Bioavailability. Am. J. Clin. Nutr., 79: 727-747.
[13]  Moon, Y. J., Wang, X. and Morris, M. E. (2006). Dietary Flavonoids: Effects on Xenobiotic and Carcinogen Metabolism. Toxicol. Vitro, 20: 187-210.
[14]  Renugadevi, J., and Prabu, S. M. (2010). Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Exp. Toxicol. Pathol., 62:471-481.
[15]  Vlachodimitropoulou, E., Sharp, P.A. and Naftalin, R. J (2011). Quercetin-iron chelates are transported via glucose transporters. Free Radic. Biol. Med., 50:934–944
[16]  Sabarinathan, D., Mahalakshmi, P. A. and Vanisree, J. (2011). Naringenin, a flavanone inhibits the proliferation of cerebrally implanted C6 glioma cells in rats., Chem. Biol. Interact. 189: 26–36.
[17]  Fang, F., Tang, Y., Gao, Z. and Xu, Q. (2010). A novel regulatory mechanism of naringenin through inhibition of T lymphocyte function in contact hypersensitivity suppression, Biochem. Biophys. Res. Commun., 397: 163–169.
[18]  Soromou, L. W, Zhang, Z., Li, R., Chen, N., Guo, W., Huo, M., Guan, S., Lu, J. and Deng, X. (2012). Regulation of inflammatory cytokines in lipopolysaccharide-stimulated RAW264.7 murine macrophage by 7-O-methyl-naringenin. Molec., 17: 3574–3585.
[19]  Jagetia, G. C. and Reddy, T. K. (2005). Modulation of radiation induced alteration in the antioxidant status of mice by naringin. Life Sci., 77: 780–794.
[20]  Esmaeili, M. A. and Alilou, M. (2014). Naringenin attenuates CCl4-induced hepatic inflammation by the activation of anNrf2-mediated pathway in rats. Clin. Exp. Pharmacol. Physiol., 41: 416–422.
[21]  Ramprasath, T., Senthamizharasi, M., Vasudevan, V., Sasikumar, S., Yuvaraj, S. and Selvam, G. S. (2014). Naringenin confersprotection against oxidative stress through upregulation of Nrf2 target genes in cardiomyoblast cells. J. Physiol. Biochem., 70: 407–415.
[22]  de Luján Alvarez, M., Cerliani, J. P., Monti, J., Carnovale, C., Ronco, M.T., Pisani, G., Lugano, M. C and Carrillo, M. C. (2002). The in vivo apoptotic effect of interferon alfa-2b on rat Preneoplastic liver involves Bax protein. Hepatol., 35: 824-833.
[23]  Zargar, S., Siddiqi, N. J., Ansar, S., Alsulaimani, M. S. and El Ansary, A. K. (2016). Therapeutic role of quercetin on oxidative damage induced by acrylamide in rat brain. Pharm. Biol., 5: 1-5.
[24]  Roy, S., Ahmed, F., Banerjee, S., and Saha, U. (2016). Naringenin ameliorates streptozotocin-induced diabetic rat renal impairment by downregulation of TGF-β1 and IL-1 via modulation of oxidative stress correlates with decreased apoptotic events. Pharm. Biol., 29:1-12.
[25]  Fabiny, D. L. and Ertingshausen, G. (1971). Automated reaction-rate method for determination of serum creatinine with the CentrifiChem. Clin. Chem., 17: 696-700
[26]  Tabacco, A., Meiattini, F., Moda, E. and Tarli, P. (1979). Simplified enzymic/colorimetric serum urea nitrogen determination. Clinic. Chem., 25: 336-337.
[27]  Fossati, P., Prencipe, L. and Berti, G. (1980). Use of 3, 5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin. Chem., 26: 227–231.
[28]  Yagi, K., (1987). Lipid peroxides and human disease. Chem. Phys. Lipids., 45:337-351.
[29]  Montgomery, H. A .C and Dymock, J. F. (1961). The determination of nitrite in water. Analyst. 86:414-416.
[30]  Beutler, E. O. and Kelly, B. M. (1963). Improved method for the determination of blood glutathione. J. Lab. Clin. Med., 61:882-888.
[31]  Matkovics, B., Kotorman, M., Varga, I. S., Hai, D. Q. and Varga, C. (1998). Oxidative stress in experimental diabetes induced by streptozotocin. Acta. Physiol. Hung. 85:29-38.
[32]  Marklund, S. and Marklund, G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and convenient assay for superoxide dismutase. Eur. J. Biochem., 47: 469-474.
[33]  Cohen, G., Dembiec, D. and Marcus, J. (1970). Measurement of catalase activity in tissue. Anal.Biochem., 34:30-38.
[34]  Mannervik, B. and Gutenberg, C. (1981). Glutathione transferase (Human placenta). Meth. Enzymol., 77:231-235.
[35]  Banchroft, J. D., Stevens, A. and Turner, D. R. (1996). Theory and practice of histological techniques. Fourth Ed. Churchillivingstone, New York, London, San Francisco, Tokyo, 766 p.
[36]  IBM Crop. (2011). IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Crop.
[37]  Kakihara, T., Imai, C., Hotta, H., Ikarashi, Y., Tanaka, A. and Uchiyama, M. (2003). Impaired tubular excretory function as a late renal side effect of chemotherapy in children. J. Pediatr. Hematol. Oncol., 25: 209-214.
[38]  Lock, E. A. and Reed, C. J. (1998). Xenobiotic metabolizing enzymes of the kidney, J. Toxicol. Pathol., 26: 18–25.
[39]  Adejuwon, A. A. and Adokiye, S. B. (2008). Protective effect of the aqueous leaf and seed extract of phyllanthusamaruson gentamicin and acetaminophen-induced nephrotoxic rats. J. Ethnopharmacol., 118:318-23.
[40]  Nenad, S., Dragan, M., Slavimir, V. (2008). Glomerular basement membrane alterations induced by gentamicin administration in rats. Exp.Toxicol. Pathol, 60: 69-75.
[41]  Stevens, L. A. and Levey, A. S. (2005). Measurement of kidney function. Med. Clin. North. Am., 89: 457-473.
[42]  Obermayr, R. P., Temml, C., Gutjahr, G., Knechtelsdorfer, M., Oberbauer, R. and Klauser-Braun, R. (2008). Elevated uric acid increases the risk for kidney disease. J. Am. Soc. Nephrol., 19 (12):2407–13.
[43]  Kanda, E., Muneyuki, T., Kanno, Y., Suwa, K. and Nakajima, K. (2015). Uric acid level has a U-shaped association with loss of kidney function in healthy people: A Prospective Cohort Study. Plos One., 6; 10(2):e0118031.
[44]  Rezaie, A., Fazlara, A., Haghi-Karamolah, M., Zadeh, H. N. and Pashmforosh, M. (2013). Effects of Echinacea purpurea on hepatic and renal toxicity induced by diethylnitrosamine in rats. Jundishapur. J. Nat. Pharm. Prod., 8:60-64.
[45]  Pashmforoosh, M., Rezaie, A., Haghi-Karamallah, M., Fazlara, A., Shahriari, A. and Najafzadeh, H. (2015). Effects of caffeine on renal toxicity induced by diethylnitrosamine. Zahedan J. Res. Med. Sci., 17:7-9.
[46]  Ahmed, O. M., Mahmoud, A. M., Abou Zid, S. F. and Saber, N. Y. (2016). Silymarin and hydroethanolic extracts of Silybum marianum leaves and fruits attenuate diethylnitrosamine/phenobarbital-induced nephrotoxicity via their antioxidant and anti-inflammatory actions. Amer. J. Bio., 6(2): 21-29.
[47]  Behling, E. B., Sendão, M. C., Francescato, H. D., Antunes, L. M., Costa, R. S. and Bianchi, M. L. (2006). Comparative study of multiple dosage of quercetin against cisplatin-induced nephrotoxicity and oxidative stress in rat kidneys. Pharmacol Rep., 58: 526-532.
[48]  Sanchez-Gonzalez, P. D., Lopez-Hernandez, F. J., Perez-Barriocanal, F., Morales, A. I. and Lopez-Novoa, J. M. (2011). Quercetin reduces cisplatin nephrotoxicity in rats without compromising its anti-tumour activity. Nephrol Dial Transplant., 26:3484-3495.
[49]  Badarya, O. A., Abdel-Maksoud, S., Ahmed, W. A. and Owieda, G. H. (2005). Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sci. 76: 2125–2135.
[50]  Hermenean, A., Ardelean, A., Stan, M., Herman, H., Mihali, C. V., Costache, M., Dinischiotu, A. (2013). Protective effects of naringenin on carbon tetrachloride-induced acute nephrotoxicity in mouse kidney. Chemico-Biol. Inter. 205,138–147.
[51]  Vitaglione, P., Morisco, F., Caporaso, N. and Fogliano, V. (2004). Dietary antioxidant compounds and liver health. Crit Rev Food Sci Nutr., 44: 575–586.
[52]  Ozbek, E. (2012). Induction of oxidative stress in kidney. Int J Nephrol., 2012:465897.
[53]  Bansal, A. K., Bansal, M., Soni, G. and Bhatnagar, D. (2005). Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chemico-Biological Interactions, 156: 101-111.
[54]  Rehman, M. U., Tahir, M., Khan, A. Q., Khan, R., Lateef, A., Oday-O-Hamiza., Qamar, W., and Ali, F., Sultana, S. (2013). Chrysin suppresses renal carcinogenesis via amelioration of hyperproliferation, oxidative stress and inflammation: plausible role of NF-κB. Toxicol Lett., 216(2-3): 146-158.
[55]  Shaban, N. Z., El-Kersh, M. A., Bader-Eldin, M. M., Kato, S. A. and Hamoda, A. F. (2014). “Effect of Punica Granatum (pomegranate) juice extract on healthy liver and hepatotoxicity induced by diethylnitrosamine and phenobarbital in male rats. J. Med. Food 17 (3): 339-349.
[56]  Lykkesfeldt, J. (2007). Malondialdehyde as biomarker of oxidative damage to lipids caused by smoking. Clin. Chim. Acta. 380:50-58.
[57]  Gurel, A., Armutcu, F., Sahin, S., Sogut, S., Ozyurt, H., Gulec, M., Kutlu, N. O. and Akyol, O. (2004). Protective role of alpha-tocopherol and caffeic acid phenethyl ester on ischemia-reperfusion injury via nitric oxide and myeloperoxidase in rat kidneys. Clin Chim Acta., 339 (1–2): 33–41.
[58]  Begum, Q., Noori, S. and Mahboob, T. (2011). Antioxidant effect of sodium selenite on thioacetamide-induced renal toxicity Pak. J. Biochem. Mol. Biol., 44(1): 21-26.
[59]  Pracheta, P., Sharma, V., Singh, L., Paliwal, R., Sharma, S., Yadav, S. and Sharma, S. (2011). Chemopreventive effect of hydroethanolic extract of Euphorbia neriifolia leaves against DENA-induced renal carcinogenesis in mice. Asian Pac J Cancer Prev., 12(3): 677-683.
[60]  Marnett, L. J. (2002). Oxy Radicals, Lipid Peroxidation and DNA Damage. Toxicology 181 (2): 219-222.
[61]  Lee, I. C., Kim, S. H., Lee, S. M., Baek, H. S, Moon, C., Kim, S. H., Park, S. C., Kim, H. C. and Kim, J. C. (2012). Melatonin attenuatesgentamicin-induced nephrotoxicity and oxidative stress inrats. Arch. Toxicol. 86: 1527–1536.
[62]  Otunctemur, A., Ozbek, E., Cekmen, M., Cakir, S. S., Dursun, M., Polat, E. C., Somay, A. and Ozbay, N. (2013). Protective effect of montelukast which is cysteinyl-leukotriene receptor antagonist on gentamicin-induced nephrotoxicity andoxidative damage in rat kidney. Ren. Fail. 35: 403–410.
[63]  Negrette-Guzmán, M., Huerta-Yepez, S., Medina-Campos, O. N., Zatarain-Barrón, Z. L., Hernández-Pando, R., Torres, I., Tapia, E. and Pedraza-Chaverri, J. (2013). Sulforaphane attenuatesgentamicin-induced nephrotoxicity: role of mitochondrialprotection. Evid. Based Complement. Alternat. Med. 2013, 135314.
[64]  Bishayee, A., Barnes, K. F., Bhatia, D., Darvesh, A. S. and Carroll, R. T. (2010). Resveratrol suppresses oxidative stress and inflammatory response in diethylnitrosamine-initiated rat hepatocarcinogenesis. Cancer Prev Res., 3: 753-763.
[65]  Zhang, C., Zeng, T., Zhao, X., Yu, L., Zhu, Z. and Xie, K. (2012). Protective Effects of Garlic Oil on Hepatocarcinoma Induced by N-Nitrosodiethylamine in Rats. Int. J. Biol. Sci., 8:363-374.
[66]  LÓpez-Lázaro, M. (2008). Anticancer and carcinogenic properties of curcumin: Considerations for its clinical development as a cancer chemo preventive and chemotherapeutic agent. Mol.Nut. Food Res., 52: S103 –S127.
[67]  Chen, B., Ning, M. and Yang, G. (2012). Effect of paeonol on antioxidant and immune regulatory activity in hepatocellular carcinoma rats. Molecules., 17: 4672-4683.
[68]  Vásquez-Garzón, V. R., Arellanes-Robledo, J., García-Román, R., Aparicio-Rautista, D.I. and Villa-Treviño, S. (2009). Inhibition of reactive oxygen species and pre-neoplastic lesions by quercetin through an antioxidant defense mechanism. Free Radic Res., 43: 128–137.
[69]  Chirino, Y. I., Hernández-Pando, R., and Pedraza-Chaverrí, J. (2004). Peroxynitrite decomposition catalyst ameliorates renal damage and protein nitration in cisplatin-induced nephrotoxicity in rats. BMC Pharmacol., 4: 20.
[70]  Usunomena, U., Ademuyiwa, A. J., Tinuade, O. O., Uduenevwo, F. E., Martin, O. and Okolie, N. P. (2012). N-nitrosodimethylamine (NDMA), liver function enzymes, renal function parameters and oxidative stress parameters: A Review. Brit. J. Pharmacol. Toxicol., 3 Suppl4: 165-176.
[71]  Rao, G. M., Rao, C. V., Pushpangadan, P. and Shirwaikar, A. (2006). Hepatoprotective effects of rubiadin, a major constituent of Rubia cordifolia Linn. J. Ethnopharmacol., 103: 484-490.
[72]  Wu, G., Fang, Y. Z., Yang, S., Lupton, J. R and Turner, N. D. (2004). Glutathione metabolism and its implications for health. J Nutr., 134:489-492.
[73]  Blair, I. A. (2006). Endogenous glutathione adducts. Current Drug Metab., 7: 853-872.
[74]  Franco, R., Schonveld, O. J., Papa, A. and Panayiotidis, M. I. (2007). The central role of glutathione in the pathophysiology of human diseases. Arch. Physiol. Biochem., 113: 234-258.
[75]  Laurent, A., Perdu-Durand, E., Alary, J., Debrauwer, L. and Cravedi, J. P. (2000). Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation in rat precision-cut liver slices. Toxicol. Lett., 114:203-214.
[76]  Srivastava, A. and Shivanandappa, T. (2010). Hepatoprotective effect of the root extract of Decalepishamiltonii against carbon tetrachloride-induced oxidative stress in rats. Food Chem., 118:411-417.
[77]  Shaheen, N. E. M. (2013). Oxidative stress of diethylnitrosamine on the functions of kidney in male rats and effective role of rutin and/or selenium. J. Appl. Sci. Res., 9: 6684-6691.
[78]  Mazen, G. M. A. (2013). The synergistic effects of rutin and urate oxidase on nephrotoxicity in rats. Arab. J. Nucl. Sci. Applic., 46(1): 205-213.
[79]  Bischoff, S. C. (2008). Quercetin: potentials in the prevention and therapy of disease. Curr Opin. Cli.n Nutr. Metab. Care, 11: 733-740.
[80]  Ciftci, O., Ozdemir, I., Vardi, N., Beytur, A., and Oguz, F. (2012). Ameliorating effects of quercetin and chrysin on 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced nephrotoxicity in rats. Toxicol. Ind. Health, 28: 947-954.
[81]  Almaghrabi, O. A. (2015). Molecular and biochemical investigations on the effect of quercetin on oxidative stress induced by cisplatin in rat kidney. Saudi J. Biol. Sci. 22: 227–231.
[82]  Al-Rejaie, S. S., Abuohashish, H. M., Al-Enazi, M. M., Al-Assaf, A. H., Parmar, M. Y. and Ahmed, M. M. (2013). Protective effect of naringenin on acetic acid-induced ulcerative colitis in rats. World J. Gastroenterol., 19: 5633–5644.
[83]  Mershiba, S. D., Dassprakash, M. V., and Saraswathy, S. D. (2013). Protective effect of naringenin on hepatic and renaldysfunction and oxidative stress in arsenic intoxicated rats. Mol. Biol. Rep., 40: 3681–3691.
[84]  Jayaraman, J., Jesudoss, V. A., Menon, V. P. and Namasivayam, N. (2012). Anti-inflammatory role of naringenin in rats with ethanolinduced liver injury. Toxicol. Mech. Methods, 22: 568–576.
[85]  Annadurai, T., Thomas, P. A., and Geraldine, P. (2013). Ameliorativeeffect of naringenin on hyperglycemia-mediatedinflammation in hepatic and pancreatic tissues of Wistar ratswith streptozotocin- nicotinamide-induced experimentaldiabetes mellitus. Free Radic. Res., 47: 793–803.
[86]  Fouada, A. A., Albualib, W. H., Zahranc, A., and Gomaad, W., (2014). Protective Effect Of Naringenin Againstgentamicin-Induced Nephrotoxicity In Rats. Enviro. Toxicol. Pharmacol., 38: 420–429.
[87]  Ahmed, R. R., Mahmoud, A. M., Ashour, M. B. and Kamel, A. M. (2015). Hesperidin protects against diethylnitrosamine-induced nephrotoxicity through modulation of oxidative stress and inflammation. Nat. J. Physiol. Pharma. Pharmacol., 5 (5): 391-397.
[88]  Ahmed, O. M., Ashour, M. B., Fahim, H. I., Mahmoud, A. M., Ahmed N. A. (2014). Preventive effect of Spirulina versicolor And Enteromorpha flexuosa ethanolic extracts against diethylnitrosamine/benzo(a)pyrene-induced hapatocarcinogencity in rats Journal Of International Academic Research For Multidisciplinary 2 (6): 634-650.