ORIGINAL�ARTICLE ABSTRACT Objective: To compare protective efficacy of cinnamon extract and green tea against Bisphenol A induced histological changes in rat kidney. Study Design: Randomized control trial. Place and Duration of Study: The study was conducted at animal house of national institute of health sciences, th th Islamabad from 16 September 2016 to 16 March 2017. Materials and Methods: Sixty adult male Sprague Dawley rats were placed in 4 confines having 15 rats each. Rats in control group A were given distilled water subcutaneously for 30 days. Rats in experimental group B were given Bisphenol a (BPA) s/c at dose of 30mg/kg/day for 30 days. Rats in group C were given cinnamon (200mg/kg/day) orally along with s/c injection of BPA while group D rats received green tea orally along with s/c BPA injections. Dissection was done after 30 days and right kidney of all rats were dissected out and examined grossly for weight and appearance and histologically for changes in glomerulus and Bowman's capsule. Data was entered and analyzed in SPSS version 22. Mean and standard error was calculated for the quantitative variables. Categorical variables were presented by frequency and percentage. One way analysis of variance (ANOVA) was applied for the mean comparison of quantitative variables between control and experimental groups. Post hoc tuckey's test was applied for the multiple comparisons among groups. Chi square test was applied for the comparison of qualitative variables among groups. A p value less than 0.05 was considered as a significant value. Results: Gross and histological parameters were observed in experimental and control groups. Deterioration of kidney structure was greatest in group B as compared to control group. Although both group C and D protected kidney against these changes but group D offered more nephron protection than group C. Conclusion: BPA adversely affects morphology and microstructure of kidneys by causing oxidative stress and green tea is more beneficial than cinnamon in ameliorating these nephrotoxic effects of BPA. Key Words: Bisphenol A, Cinnamomum Zeylanicum, Kidney, Oxidative Stress. polycarbonated plastics, is one of highest volume 3 environmental toxin produced worldwide. Human 4 exposure to BPA occurs mainly through food as it is used inplastic food containers, soft drink bottles, toys, inner lining of metal cans and water supply pipes. Significant BPA levels have been detected in 5 indoor and outdoor air, water , oil and canned food. It is used as color developer in electric invoices and paper towels. Hence absorbed through dermal 6 contact. BPA causes oxidative stress induced tissue 7 damage in the kidneys as it is eliminated by kidneys, therefore, increased level of BPA are observed in 8 CKD. BPA affect embryology, physiology, histomorphology 9 of various organs of animals and humans. Antioxidant therapies have nephron protective effect because they cause epithelial cell sparing and Introduction Kidneys, vital organs of human body, remove metabolic wastes of human body and conserve fluids 1 and electrolytes. The acute renal injury is induced by toxins ultimately progresses to chronic kidney disease which is now recognized as a global public 2 health problem. Bisphenol A (BPA), a polymer of Comparative Effects of Cinnamon Extract and Green Tea Against Bisphenol- A Induced Damage in Rat Kidney Huma Beenish, Rehana Rana, Shabana Ali Muhammad, Asma Ali Khan Correspondence: Dr. Huma Beenish Assistant Professor, Anatomy Islamic International Medical College Riphah International University, Islamabad E-mail: huma1186@gmail.com Department of Anatomy Islamic International Medical College Riphah International University, Islamabad Funding Source: NIL; Conflict of Interest: NIL Received: Sep 13, 2017; Revised: Jan 24, 2018 Accepted: Feb 06, 2018 Comparative Effects of Cinnamon Extract and Green TeaJIIMC 2018 Vol. 13, No.1 12 of BPA. At the end of 30 days experimental duration, rats were anesthetized, kidneys were exposed through longitudinally cutting in the abdominal 11 region. Right kidneys of all animals were dissected out and observed for gross appearance by comparing them with normal kidneys of control group. Weight and gross appearance of each kidney was noted. Transverse sections of kidney were taken and stained with hematoxylin and eosin. Slides were examined under X4, X10 and X40 power of light microscope. The presence or absence of following qualitative parameters was confirmed by observing four random fields in each slide of kidney. 1. Contracted glomerulus 2. Swollen glomerulus 3. Hemorrhage in glomerulus 4. Ruptured Bowman's capsule The glomeruli were labeled contracted when they appeared shrunken with increased urinary space as 17 compared to glomeruli of control group. The glomeruli with negligible urinary space were regarded as swollen. SPSS version 22 was used for data analysis. One way analysis of variance (ANOVA) was applied for the mean comparison weight between control and experimental groups. Post hoc turkey's test was applied for the multiple comparisons among groups. Chi square test was applied for the comparison of qualitative variables among groups. A p value less than 0.05 was considered as a significant value. Results Mean weight of rat kidney was significantly decreased in group B (rats were given 30mg/kg/day BPA, subcutaneously) as compared to control group A. Green tea administration in group D (rats given 200mg /kg /day green tea aqueous extract) significantly increased weight of kidney as compared to group C (rats given200mg/kg/day cinnamon aqueous extract). Appearance of kidneys was swollen in 80% experimental animals in group B while in group D only 27% kidneys were swollen. The difference of appearance of kidneys was significant between all groups (p=0.00). (Table I) (Fig. 1). All experimental animals in group B possessed contracted glomeruli while Group C and group D showed contracted glomeruli in 53.3 % and 13.3 % of experimental animals respectively. The difference of contracted appearance of glomeruli was significant 10 regeneration and cause apoptosis of my fibroblasts. Cinnamon possess ant oxidative effects which protect kidney from damage caused by diabetes and 11 various toxins. Active components of cinnamon i n c l u d e p o l y p h e n o l s , c i n n a m i c a c i d a n d cinnamaldehyde. On the other hand, green tea (Camellia sinensis) also a nephron protective 12 antioxidant derived from plants. Catechins present in green tea include epigallocatechin-3-gallate ( E G C G ) , e p i c a t e c h i n - 3 - g a l l a t e ( E C G ) , epigallocatechin (EGC) and epicatechin (EC). Beneficial effects of green tea include its ant oxidative, anti-inflammatory and ant carcinogenic 13 properties. It is also protective against renal injury. There were no histological studies seen for protection offered by green tea against BPA induced nephrotoxicity. This study has been designed to compare the modulatory effect of cinnamon n green tea consumption on daily basis to counteract the nephrotoxic changes caused by BPA. Materials and Methods This study was a randomized control trial. It was conducted in animal house of national institute of th health sciences, Islamabad from 16 September th 2016 to 16 March 2017. Research took time span of six months before results were achieved. Ethical Review Committee gave approval of synopsis of the study prior to conduction experiment. Research was carried out on sixty adult male rats of Sprague Dawley breed, weighing approximately 250 to 300 grams. Female rats and rats with any conspicuous pathology were rejected. Simple random sampling technique was used. Under supervision of animal house NIH, rats were kept in 4 confines with a number of 15 rats/confine. A standard laboratory condition indistinguishable to their class with adjusted dietary supplement was given under temperature of 27±3ºC. Rats were randomly divided by lottery method into 4 groups. (15 animals in each group). Rats in Group A serving as controls were given 1ml refined water subcutaneously. Group B rats were given 14 30mg/kg/day BPA, subcutaneously. Group Crats were given200mg /kg /day cinnamon aqueous 15 extract via gavage tube 2 hr before daily subcutaneous injection of BPA. Group Drats were 16 given 200mg/kg/day green tea aqueous extract. via gavage tube 2 hr before daily subcutaneous injection 13 Comparative Effects of Cinnamon Extract and Green TeaJIIMC 2018 Vol. 13, No.1 tea against toxicity caused by BPA by measuring gross as well as microscopic parameters. It was found that nephrotoxicity caused by BPA is ameliorated by both cinnamon and green tea but green tea showed best results in protection of kidney against BPA induced damage. BPA leads to increase in kidney weight. Results showed that both cinnamon and green tea reduced kidney weight but comparison of both showed that green tea caused significantly greater reduction in kidney weight than cinnamon.(p=.021). Increase in kidney weight in group B is indicating underlying change in morphology of kidneys. The reduction in kidney weight observed in present study in group C is accordance with results of Qusti who reported a similar decrease in kidney weight caused by treatment of diabetic rat kidney with cinnamon. Author related this change with the ant oxidative 18 properties. Noori brought to light that cinnamon is unable to decrease weight of kidney in absence of toxic insult. Green tea polyphenols also act as antioxidants and 19 combat the oxidative stress induced injuries. Decrease in weight of kidney in group D is potentiated by findings of Hama Douche. Author demonstrated that increase in kidney weight caused by lead induced oxidative stress shows a relative 20 decrease after treatment with green tea. Green tea polyphenols reduced the weight of diabetic rat kidney significantly along with reduction in serum 21 creatinine and MDA levels. Improvement in renal parameters in both groups C between all the groups (p=0.00). (Table II). In control group a none of experimental animals showed contracted glomeruli, while in group B contracted glomeruli were present in 100% of experimental animals. Group C and group D showed contracted glomeruli in 53.3 % and 13.3 % of experimental animals respectively. Swollen appearance of glomeruli was neither observed in control group A nor in experimental groups B, C and D. (Table II). Hemorrhagic glomeruli were present in 86.67% experimental animals. In group B while in group C and D hemorrhage was present in 73.3 % and 26.7% of experimental animals respectively. The difference of hemorrhagic appearance of glomeruli was significant between all groups (p=0.00) (Table II). Ruptured Bowman's capsule was present in 86.7% of experimental animals in group B. In groups C 46.7% while in group D 20% experimental animals showed ruptured Bowman's capsule. The difference in presence of ruptured capsule was significant between all groups. (Table II). Table I: Distribu�on of Kidney Weight (g) and Appearance between Experimental and Control Groups N=60 Table II: Distribu�on of Contracted Glomerulus, Swollen Glomerulus, Hemorrhage in Glomerulus and Ruptured Bowman's Capsule between Experimental and Control Groups N=60 Fig 1: Distribu�on of Mean Weight between Control and Experimental Groups Discussion Present study was designed to compare the nephron protective efficacy of cinnamon extract and green 14 Comparative Effects of Cinnamon Extract and Green TeaJIIMC 2018 Vol. 13, No.1 induction of oxidative stress while epicatechin serve 20 as scavengers of free radicals. Hence all above findings strengthen our results of positive effects of green tea on glomerular histology. In present study BPA caused rupture of Bowman's capsule. Although both cinnamon and green tea improved this parameter but green tea caused significant improvement in histology of Bowman's capsule than cinnamon.(p=0.00) Sakr noticed that same degenerative changes in renal corpuscle after c y p e r m e t h r i n t re a t m e n t a l o n g w i t h t h e i r 27 improvement with cinnamon administration. These results support our finding of improved capsular histology in group C but improvement was greater in green tea group D. In present study we found that BPA causes nephrotoxic effects in rat kidney. Effects of BPA on human kidney should be explored with reference to its accumulation and excretion by human body. Conclusion Bisphenol A causes significant changes in histomorphology of rat kidney by induction of oxidative stress. Present study shows that co administration of cinnamon and green tea along with B PA c a u s e s i m p r o v e m e n t i n g r o s s a n d microstructure of kidneys. Comparison of both shows that green tea provides better nephron protection than cinnamon against BPA toxicity. REFERENCES 1. Ross MH, Pawlina W. Histology: Lippincott Williams & Wilkins; 2006; p. 1103-33. 2. El Nahas AM, Bello AK. Chronic kidney disease: the global challenge. The Lancet. 2005; 365: 331-40. 3. Vandenberg LN, Maffini MV, Sonnenschein C, Rubin BS, Soto AM. Bisphenol A and the great divide: a review of controversies in the field of endocrine disruption. Endocrine reviews. 2009; 30: 75-95. 4. Usman A, Ahmad M. From BPA to its analogues: Is it a safe journey? Chemosphere. 2016; 158: 131-42. 5. Schecter A, Malik N, Haffner D, Smith S, Harris TR, Paepke O, et al. Bisphenol a (BPA) in US food. Environmental science & technology. 2010; 44: 9425-30. 6. Shelby M. NTP-CERHR monograph on the potential human reproductive and developmental effects of bisphenol A. Ntp Cerhr Mon. 2008; 5: 7-9. 7. Hassan ZK, Elobeid MA, Virk P, Omer SA, ElAmin M, Daghestani MH, et al. Bisphenol A induces hepatotoxicity through oxidative stress in rat model. Oxidative medicine and cellular longevity. 2012; p. 1-6. 8. Parra EG, Herrero JA, Elewa U, Bosch RJ, Arduán AO, Egido J. Bisphenol A in chronic kidney disease. International journal and D along with maximum improvement in green tea group D is also supported by a study conducted by Hasanein. Author compared the affects of green tea cinnamon and black tea on renal function tests in obese diabetic rats. The kidney dysfunction associated with diabetic rats was found to be linked with oxidative damage caused by diabetes. Both cinnamon and green tea exerted ant oxidative effects because of presence of polyphenols but RFTs improved more in green tea 22 group. Author did not focus the histological features of kidney in green tea and cinnamon treated groups. Review of literature shows most studies on BPA and antioxidants have not focused on appearance of kidneys. Study conducted by Tan support our results 23 of swollen kidneys in group B. We were unable to find effects of cinnamon and green tea on gross appearance of kidneys in literature. Hence all above literature supports our findings of maximum improvement in renal parameters with green tea as compared to cinnamon. Structure of glomeruli was observed in four different fields of vision and all the glomeruli in group B were found to be contracted. Group D showed significant improvement in this parameter than group C. Improvement in morphological appearance of glomeruli in group C in present study is supported by work of Morgan who showed glomerular congestion and hyper cellularity caused by BPA showed 15 improvement with cinnamon administration. This can be explained by presence of polyphenols in 24 cinnamon. Qusti reported that diabetes causes damage to renal corpuscle shown by shrunken glomeruli which become close to normal appearing glomeruli histologically after treatment with 18 cinnamon. But present study proved that green tea causes more improvement in above mentioned histology of glomerulus than cinnamon. Sardana noticed that degeneration in glomerular wall caused by gentamycin improved with catechins. He explained that catechins reduce NADPH oxidase 25 activity. Hence they reduce oxidative stress. Various studies support improved glomerular 26 histology with green tea in present study. Yokozawa showed that disturbed glomerular morphology in 21 diabetic kidney restored to normal with green tea. Hama douche proposed similar positive impact of green tea against nephrotoxicity by lead. Lead causes 15 Comparative Effects of Cinnamon Extract and Green TeaJIIMC 2018 Vol. 13, No.1 Based Complementary and Alternative Medicine. 2016; 2016: 5614564. 19. Carloni P, Tiano L, Padella L, Bacchetti T, Customu C, Kay A, et al. Antioxidant activity of white, green and black tea obtained from the same tea cultivar. Food research international. 2013; 53: 900-8. 20. Hamadouche N, Hadi A. The protective effect of green tea extract on lead induced oxidative and damage on rat kidney. International Journal of Pharmacy and Biological Sciences. 2015; 6: 97-07. 21. Yokozawa T, Nakagawa T, Oya T, Okubo T, Juneja LR. Green tea polyphenols and dietary fibre protect against kidney damage in rats with diabetic nephropathy. Journal of pharmacy and pharmacology. 2005; 57: 773-80. 22. Hasanein MA, Gawad H, El-Megeid AAA. Effect of water extract prepared from green tea, black tea and cinnamon on obese rats suffering from diabetes. World Applied Sciences Journal. 2012; 20: 976-87. 23. Tan BL, Kassim NM, Mohd MA. Assessment of pubertal development in juvenile male rats after sub-acute exposure to bisphenol A and nonylphenol. Toxicology letters. 2003; 143: 261-70. 24. Azab KS, Mostafa AHA, Ali EM, Aziz MAA. Cinnamon extract ameliorates ionizing radiation-induced cellular injury in rats. Ecotoxicology and environmental safety. 2011; 74: 2324-9. 25. S a r d a n a A , K a l r a S , K h a n n a D, B a l a k u m a r P. Nephroprotective effect of catechin on gentamicin-induced experimental nephrotoxicity. Clinical and experimental nephrology. 2015; 19: 178-84. 26. Veljkovic M, Ilic S, Stojiljkovic N, Velickovic L, Pavlovic D, Radenkovic M, et al. Beneficial Effects of Green Tea Extract in Gentamicin-Induced Acute Renal Failure in Rats/Povoljni efekti ekstrakta zelenog caja na akutnu bubrežnu insuficijenciju uzrokovanu gentamicinom kod pacova. Acta Facultatis Medicae Naissensis. 2015; 32: 51-8. 27. Sakr SA, Albarakai AY. Effect of cinnamon on cypermethrin- induced nephrotoxicity in albino rats. Int J Adv Res. 2014; 2: 578-86. of nephrology. 2013; 2013: 437857. 9. Michalowicz J. Bisphenol A–sources, toxicity and biotransformation. Environmental toxicology and pharmacology. 2014; 37: 738-58. 10. Wojcikowski K, Wohlmuth H, Johnson DW, Rolfe M, Gobe G. An in vitro investigation of herbs traditionally used for kidney and urinary system disorders: potential therapeutic and toxic effects. Nephrology. 2009; 14: 70-9. 11. Mhammad HA, Jubrail AMS, Najeeb MK. Impact of Cinnamon Extract on Liver, Kidneys and Spleen of Diabetic Rats. Inter J of Chemical and Biomolecular Science. 2015; pp. 24854. 12. Gad SB, Zaghloul DM. Beneficial effects of green tea extract on liver and kidney functions, ultrastructure, lipid profile and hematological parameters in aged male rats. Global Vet. 2013; 11: 191-205. 13. Wang Y, Wang B, Du F, Su X, Sun G, Zhou G, et al. Epigallocatechin-3-gallate attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis in mice. Journal of Histochemistry & Cytochemistry. 2015; 63: 270- 9. 14. Badawi MM, Soliman MG, Kawi NAA, Abozaid NM. Physiological and Histopathological studies on Bisphenol-A compound as xenoestrogen in male albino rats. The Egyptian Journal of Hospital Medicine. 2013; 50: 127-36. 15. Morgan AM, El-Ballal SS, El-Bialy BE, EL-Borai NB. Studies on the potential protective effect of cinnamon against bisphenol A-and octylphenol-induced oxidative stress in male albino rats. Toxicology Reports. 2014; 1: 92-101. 16. Isbrucker R, Edwards J, Wolz E, Davidovich A, Bausch J. Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and short-term toxicity studies. Food and chemical toxicology. 2006; 44: 636-50. 17. Ahmed W, Moselhy W, Nabil T. Bisphenol A toxicity in adult m a l e r a t s : h e m a t o l o g i c a l , b i o c h e m i c a l a n d histopathological approach. Glob Veternaria. 2015; 14: 228-38. 18. Qusti S, El Rabey HA, Balashram SA. The Hypoglycemic and Antioxidant Activity of Cress Seed and Cinnamon on Streptozotocin Induced Diabetes in Male Rats. 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