Chemopreventive effect of Quercus infectoria against chemically induced renal toxicity and carcinogenesis

In this study we have shown that Quercus infectoria attenuates FeNTA induced renal oxidative stress, hyperproliferative response and renal carcinogenesis in rats. Fe-NTA promoted DEN (N-diethyl nitrosamine) initiated renal carcinogenesis by increasing the percentage incidence of tumors and induces early tumor markers viz. ornithine decarboxylase (ODC) level and PCNA expression. FeNTA (9 mg Fe/kg body weight, intraperitoneally) enhances renal Malondialdehyde, xanthine oxidase and hydrogen peroxide generation with reduction in renal glutathione content, antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase and phase-II metabolizing enzymes such as glutathione-S-transferase and quinone reductase. It also enhances blood urea nitrogen and serum creatinine. Fe-NTA also lead to increase in levels of some inflammatory markers viz NO and MPO and some proinflammatory cytokines viz PGE-2 and TNF-α. The chemopreventive efficacy of Quercus infectoria was studied in terms of xenobiotic metabolizing enzyme activities, LPO, redox status, serum toxicity markers, inflammatory and proinflammatory markers and cell proliferation in the kidney tissue. Oral administration of Quercus infectoria at doses of 75 and 150 mg/kg b wt effectively suppressed renal oxidative stress, inflammation and tumor incidence. Chemopreventive effects of Quercus infectoria were associated with up-regulation of xenobiotic metabolizing enzyme activities and down regulation of serum toxicity markers. Present study supports Quercus infectoria as a potent chemopreventive agent and suppresses Fe-NTA-induced renal carcinogenesis and oxidative and inflammatory response in Wistar rat. *Corresponding author, Mailing address: Dr. Sarwat Sultana Molecular Carcinogenesis and Chemoprevention Division, Dept. of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India. E-mail address: [email protected] Article History:-----------------------Date of Submission: 30-04-2012 Date of Acceptance: 11-05-2012 Conflict of Interest: NIL Source of Support: NONE F U L L L e n g t h R e s e a r c h P a p e r C o v e r e d i n I n d e x C o p e r n i c u s w i t h I C V a l u e 4 . 6 8 f o r 2 0 1 0 Int. J. Drug Dev. & Res., April-June 2012, 4 (2): 336-351 Covered in Scopus & Embase, Elsevier 336 demonstrated that iron overload dramatically potentiates chemical carcinogenicity . Mechanisms whereby iron may act in carcinogenesis are induction of oxidative stress, facilitation of tumor growth and modification of the immune system. Metal ions react with superoxide anion (O ) and H2O2 to produce highly reactive species such as hydroxyl free radical (OH) and metal–oxygen complexes in biological systems, resulting in oxidative DNA damage. Since H2O2 itself is not toxic to cells, H2O2-induced oxidative DNA damage in cells has been thought to result from the formation of hydroxyl free radicals through Fenton reaction with iron . The development of RCC has been linked with innumerable risk factors including environmental exposure to different toxicants . Nitrilotriacetic acid (NTA) is an aminotricarboxylic acid with an empirical formula of C6H9NO6. Nitrilotriacetate (NTA) can make complexes with metal ions such as Fe or Cu. Fe-NTA, a complex of Feand NTA, is a strong nephrotoxic agent and a renal carcinogen. It is an established fact that an iron-chelate of nitrilotriacetate, ferric nitrilotriacetate (Fe-NTA) induces acute and sub-acute renal injury in animals . It is evidenced from previous reports that oxygen free radical was formed from redox-active iron and was detected in the serum of Fe-NTA-treated rats [7. Medicinal plants have been used by all civilization as a basis of medicines since ancient times. In the recent times natural products have been used to prevent the toxicities induced by chemicals, drugs and carcinogenic xenobiotics. Plant based products are generally considered safe and proved to be effective against various human ailments and their medicinal uses have been gradually increasing in developed countries. Epidemiological studies have also proven that consumption of vegetables and fruit have caused lower incidence of cancers . Interest in medicinal plants as a re-emerging health aid also has been encouraged by the rising costs of prescription drugs in the maintenance of personal health and wellbeing, and the bioprospecting of new plant derived drugs. Studies have shown that natural phytochemicals containing phenolic compounds derived from certain plants have the capability to prevent cancer. Quercus infectoria Olivier (Fagaceae) is a small tree or a shrub mainly present in Asia Minor, Greece, Syria and Iran. The tree yield galls that emerge on its shoots as a consequence of assault of gall wasp Cypnis gallae tincotoriae . The Quercus infectoria galls (QI) have a great medicinal value and have pharmacologically been deciphered to be astringent, antipyretic, anti-diabetic, anti-tremor, local anesthetic and anti-Parkinson . Kaur et al., 2007 reported QI is capable of protecting against oxidative damage to lipids and proteins and also chelates metal ions that catalyze the generation of oxidants. The main constituents found QI are tannins (50-70%) and free gallic acid and ellagic acid . No published information is available on the chemopreventive aspects of QI against renal carcinogenesis. Therefore, we examined its effects in amending the Fe-NTA nephrotoxicity and its chemopreventive efficacy against two stage renal carcinogenesis in wistar rats by studying its various potential molecular targets. We also studied the level of inflammatory markers and tumor promotion markers which are known to be dysregulated in cancer cells and which might be one of the targets of the chemopreventive action of QI. Material Methods Chemicals Oxidized and reduced glutathione, Nitrilotriacetate (NTA), N-nitrosodiethylamine (DEN), nitrilotriacetic acid, H2O2, dithionitrobenzene (DTNB), 1-chloro-2,4dinitrobenzene (CDNB), glutathione reductase, reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavine adenine dinucleotide F U L L L e n g t h R e s e a r c h P a p e r C o v e r e d i n I n d e x C o p e r n i c u s w i t h I C V a l u e 4 . 6 8 f o r 2 0 1 0 Dr. Sarwat Sultana et al: Chemopreventive effect of Quercus Infectoria against chemically induced renal toxicity and carcinogenesis Int. J. Drug Dev. & Res., April-June 2012, 4 (2): 336-351 Covered in Scopus & Embase, Elsevier 337 (FAD) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All the antibodies, chemicals and reagents used were of highest purity and standard commercially available. Rat TNF-α ELISA Ready Set Go, E-bioscience. (U.S.A), ELISA PGE2 EIA kit, Cayman chemical company, (U.S.A) Animals Young (8–10 weeks old), male wistar rats were housed in plastic (polypropylene) cages in animal house facility of Hamdard University. Experiments were conducted according to protocols approved by CPCSEA animal ethical committee, New Delhi, India, project number and date 547/CPCSEA, May 28th, 2009. The well ventilated animal rooms (room temperature set at 25C) were maintained on 12 h light–dark cycles. They were acclimatized for one week before the study and had free access to standard laboratory feed (Hindustan lever Ltd, Mumbai, India) and water ad libitum. Preparation of Fe-NTA Preparation of Fe-NTA solution was done by method given by Awai et al. (1979) [4] as modified by Athar and Iqbal (1998) . Briefly, ferric nitrate (0.16 mM) solution was mixed with a four-fold molar excess of disodium salt of NTA (0.64 nM) and the pH was adjusted to 7.4 with a sodium bicarbonate solution. The solution was prepared immediately before each protocol. Experimental Design The treatment regimen for Quercus infectoria and the proposal of verifying its chemopreventive efficacy against renal carcinogenesis was based on the preliminary dose dependent pilot study carried out in our laboratory. To study the chemo-protective effects of Quercus infectoria on biochemical and serological changes induced by toxicity of Fe-NTA in rats, 30 male Wistar rats were randomly divided into five equal groups. Quercus infectoria was dissolved in double distilled water. Rats were initiated with intraperitoneal (IP) injection of DEN at a dose of 200 mg/kg b wt and promoted with Fe-NTA at a dose of 9 mg Fe/kg b wt IP. Selection of the dose regimen is based on our own preliminary experiments and is also based on previously published data from our laboratory . Following treatment regimen was followed in our study ! Group I animals received only normal saline (0.9%) for seven consecutive days by oral gavage and thus served as untreated controls. ! Group II served as treated control and was administered a single dose of Fe-NTA on 20 day. ! Group III was pretreated with oral gavage of Quercus infectoria at a dose of 75 mg/kg b wt for 20 consecutive days followed by administration of Fe-NTA on 20 day. ! Group IV was pretreated with oral gavage of Quercus infectoria at a dose of 150 mg/kg b wt for 20 consecutive days followed by administration of Fe-NTA on 20 day. ! Group V received by oral gavage of Quercus infectoria only, at a dose of 150 mg/kg b wt for 20 consecutive days. All animals were sacrificed exactly 12 h after Fe-NTA administration. Kidney tissue was processed for biochemical estimations. Blood was collected and serum was separated out and processed for serological studies. To study the effect of pretreatment with Quercus infectoria on DEN initiated and Fe-NTA–promoted renal carcinogenesis, the rats were divided into six groups of 25 rats per group. The complete treatment regimen followed in tumor study is described below: ! Group I animals received only normal saline (0.9%) by oral gavage once daily for 16 weeks and served as controls. ! Group II also received only normal saline (0.9%) by oral gavage once daily for 16 weeks. In addition group II was given IP injection of DEN in saline on the very first day of experiment and ten days after the F U L L L e n g t h R e s e a r c h P a p e r C o v e r e d i n I n d e x C o p e r n i c u s w i t h I C V a l u e 4 . 6 8 f o r 2 0 1 0 Int. J. Drug Dev. & Res., April-June 2012, 4 (2): 336-351 Covered in Scopus & Embase, Elsevier 338 Dr. Sarwat Sultana et al: Chemopreventive effect of Quercus Infectoria against chemically induced renal toxicity and carcinogenesis