ORIGINAL ARTICLE
Evaluation of the protective effect of quercetin against cisplatin-induced renal and testis tissue damage and sperm parameters in rats € semehmetog lu2, K. Ener1, F. Topal3, O. Evirgen3, E. Gu € rleyik4 M. Aldemir1, E. Okulu1, K. Ko 4 & A. Avcı 1 2 3 4
€rk Training and Research Hospital, Ankara, Turkey; Department of Urology, Atatu Faculty of Medicine, Department of Pathology, Hacettepe University, Ankara, Turkey; Faculty of Medicine, Department of Histology and Embryology, Ankara University, Ankara, Turkey; Faculty of Medicine, Department of Biochemistry, Ankara University, Ankara, Turkey
Keywords Cisplatin—nephrotoxicity and testicular toxicity—oxidative stress—quercetin—sperm parameters Correspondence Assoc. Prof. Mustafa Aldemir, Ankara Ataturk Training and Research Hospital Bilkent, 06800 Ankara, Turkey. Tel.: +90 533 6309102; Fax: +90 312 2912705; E-mail: [email protected]
Accepted: October 7, 2013 doi: 10.1111/and.12197
Summary The protective effect of quercetin on cisplatin-induced renal and testicular tissue damage was investigated using biochemical, histopathological and histological approaches. A total of 40 male rats were divided into 5 groups as follows: control; cisplatin alone; quercetin alone; cisplatin + quercetin; and quercetin + cisplatin. Cisplatin was administered to rats at a single dose of 7 mg kg 1 intraperitoneal. Quercetin was administered by gavage daily for 10 days at dosage 50 mg kg 1. At the end of the study serum, total antioxidant capacity (TAC) levels and total oxidant status (TOS) were determined. Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and xanthine oxidase (XO) were studied separately in serum, renal tissue and testicular tissue. Renal and testicular morphological alterations were assessed, histopathologically. Epididymal sperm concentration, motility and morphology were investigated. Testicular and renal TAC and TOS values did not alter significantly. Renal CAT levels were increased by cisplatin and cisplatin plus quercetin groups that is reversed by administration of quercetin before cisplatin. MDA, CAT, SOD ve XO levels of testicular tissue did not differ significantly. Cisplatin and cisplatin plus quercetin groups had decreased sperm motility ratio and increased abnormal spermatozoa. Quercetin partially reverses some of the cisplatin-related pathological effects on kidney and testis.
Introduction Cisplatin (cis-diamminedichloroplatinum II) is a potent anticancer agent, especially used in the treatment for solid tumours. However, clinical use of the compound is often limited by its adverse effects, including renal impairment, intestinal toxicity and myelosuppression, of which renal toxicity is the most serious dose-limiting factor (Meyer & Medias, 1994). In kidney tissue, cisplatin-induced nephrotoxicity is closely associated with increased lipid peroxidation (Choie et al., 1981). Therefore, oxygen free radicals may play a central role in cisplatin-induced renal injury. The underlying mechanism of cisplatin-induced nephrotoxicity is still not well known, but many recent in vitro and in vivo studies indicate an important role for the reactive oxygen metabolites in the pathogenesis of this effect (Matsushima et al., 1998). © 2013 Blackwell Verlag GmbH Andrologia 2013, xx, 1–9
Cisplatin-based chemotherapy may also result in impaired spermatogenesis (Cherry et al., 2004), chromosomal abnormalities in spermatozoa (Martin et al., 1999) and temporary or permanent azoospermia (Howell & Shalet, 2005). Pathogenesis of testicular damage after cisplatin exposure is generally attributed to oxidative damage (Silici et al., 2009; Salem et al., 2012). Flavonoids are phytophenolic compounds with strong antioxidant properties. Quercetin (3, 3′, 4′,5,7-pentahydroxyflavone) is one of the most abundant flavonoids in the human diet, commonly present in most edible fruits and vegetables (Heim et al., 2002). This bioflavonoid is a potent oxygen free radical scavenger and a metal chelator, capable of inhibiting lipid peroxidation in in vitro and in vivo systems (Ferry et al., 1996; Fiorani et al., 2001). The aim of this study is to investigate the protective effects of quercetin against cisplatin-induced renal and 1
Quercetin and cisplatin-induced damage
testicular toxicity associated with oxidative stress in male Wistar rats. What makes this study unique is the comprehensive investigation of both biochemical and histopathological changes in kidneys, spermatids and testicles. Materials and methods Chemicals _ Cisplatin was obtained from Farmar (Istanbul, Turkey), and quercetin complex was purchased from DK Pharma_ ceuticals (Istanbul, Turkey). All surgical procedures were performed under anaesthesia by intramuscular injection _ Turkey) and of 50 mg kg 1 ketamin (Pfizer, Istanbul, 1 _ 10 mg kg xlazine (Alfasan, Istanbul, Turkey). Animals Forty Wistar Albino type male rats of 20 weeks old were used in the study. The rats weighed 220 15 g. The rats were housed in individual cages at 22–24 °C, 50–55% humidity and light:dark cycle control (12 : 12 h). The rats had free access to food and water, and water was changed daily. The protocol of this study was approved by Ankara University, School of Medicine, Animal Care and Use Committee. All experimental procedures were conducted in accordance with the Guide to the Care and Use of Laboratory Animals. Experimental design The animals were divided into 5 groups, randomly. First group was used as control (Group 1), and the second group (Group 2) was treated with a single dose of intraperitoneal cisplatin (7 mg kg 1). Third group (Group 3) was treated with 50 mg kg day 1 quercetin administered to rats by oral route for 10 days. Fourth group (Group 4) was treated with one intraperitoneal single dose 7 mg kg 1 at first day, and then, 50 mg kg day 1 oral quercetin was administered for 10 days. Fifth group (Group 5) was given 50 mg kg day 1 oral quercetin for 10 days and then single dose intraperitoneal cisplatin 7 mg kg 1 at 10th day. Biochemical analysis Renal function was assessed by estimating serum creatinine and blood urea nitrogen (BUN). Testis and kidney tissues of the rats were removed by surgical operation, washed with physiological saline solution and separated from perirenal fatty tissue. The tissues were homogenised and prepared for the assays as described previously (Kavutcu et al., 1996). Upper clear part of the tissue homogenates was used in the experiments. The protein level of 2
M. Aldemir et al.
the clear supernatants was determined by the method of Lowry et al. (1951). Malondialdehyde (MDA, nmol mg 1) levels and superoxide dismutase (SOD, U mg 1), catalase (CAT, IU mg 1) and xanthine oxidase (XO, mIU mg 1) activities were measured in the supernatant fraction (Dasgupta & Zdunek, 1992). MDA level was measured by thiobarbituric acid reactive substances (TBARS) method (Dahle et al., 1962). Superoxide dismutase activity was measured as described (Durak et al., 1996). One unit for SOD activity was expressed as the enzyme protein amount causing 50% inhibition in nitroblue tetrazolium reduction rate. CAT activity was determined by measuring decrease in hydrogen peroxide (H2O2) absorbance at 240 nm as described (Aebi, 1974). Xanthine oxidase activity was determined by measuring uric acid formation from xanthine at 293 nm as described (Hashimato, 1974). In the activity calculations, extinction coefficients of uric acid and H2O2 were used for XO and CAT and respectively. Serum total antioxidant capacity (TAC) levels and total oxidant status (TOS) were determined using a novel automated measurement method, developed by Erel (2004, 2005). Histopathological examination Kidneys and one testicle of each rat were fixed in 10% buffered formaldehyde. One half of one kidney and testicle on coronal plane were submitted to routine tissue processing, 5-lm-thick sections were stained with routine hematoxylin & eosin and examined under light microscope (Olympus BX51, Tokyo, Japan). In histopathological examination of testicles, we investigated the presence of any disruption of the normal architecture with an orderly arrangement of germinal and sertoli cells. The presence of tubular atrophy, germ cell degeneration, necrosis, shrinkage of seminiferous tubules, shredding of germ cells in lumen and interstitial oedema was noted (Fig. 1a). Severity of damage (testis damage score) was graded according to the extent of degenerative findings, as 0, no damage; 1,
Evaluation of the protective effect of quercetin against cisplatin-induced renal and testis tissue damage and sperm parameters in rats € semehmetog lu2, K. Ener1, F. Topal3, O. Evirgen3, E. Gu € rleyik4 M. Aldemir1, E. Okulu1, K. Ko 4 & A. Avcı 1 2 3 4
€rk Training and Research Hospital, Ankara, Turkey; Department of Urology, Atatu Faculty of Medicine, Department of Pathology, Hacettepe University, Ankara, Turkey; Faculty of Medicine, Department of Histology and Embryology, Ankara University, Ankara, Turkey; Faculty of Medicine, Department of Biochemistry, Ankara University, Ankara, Turkey
Keywords Cisplatin—nephrotoxicity and testicular toxicity—oxidative stress—quercetin—sperm parameters Correspondence Assoc. Prof. Mustafa Aldemir, Ankara Ataturk Training and Research Hospital Bilkent, 06800 Ankara, Turkey. Tel.: +90 533 6309102; Fax: +90 312 2912705; E-mail: [email protected]
Accepted: October 7, 2013 doi: 10.1111/and.12197
Summary The protective effect of quercetin on cisplatin-induced renal and testicular tissue damage was investigated using biochemical, histopathological and histological approaches. A total of 40 male rats were divided into 5 groups as follows: control; cisplatin alone; quercetin alone; cisplatin + quercetin; and quercetin + cisplatin. Cisplatin was administered to rats at a single dose of 7 mg kg 1 intraperitoneal. Quercetin was administered by gavage daily for 10 days at dosage 50 mg kg 1. At the end of the study serum, total antioxidant capacity (TAC) levels and total oxidant status (TOS) were determined. Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and xanthine oxidase (XO) were studied separately in serum, renal tissue and testicular tissue. Renal and testicular morphological alterations were assessed, histopathologically. Epididymal sperm concentration, motility and morphology were investigated. Testicular and renal TAC and TOS values did not alter significantly. Renal CAT levels were increased by cisplatin and cisplatin plus quercetin groups that is reversed by administration of quercetin before cisplatin. MDA, CAT, SOD ve XO levels of testicular tissue did not differ significantly. Cisplatin and cisplatin plus quercetin groups had decreased sperm motility ratio and increased abnormal spermatozoa. Quercetin partially reverses some of the cisplatin-related pathological effects on kidney and testis.
Introduction Cisplatin (cis-diamminedichloroplatinum II) is a potent anticancer agent, especially used in the treatment for solid tumours. However, clinical use of the compound is often limited by its adverse effects, including renal impairment, intestinal toxicity and myelosuppression, of which renal toxicity is the most serious dose-limiting factor (Meyer & Medias, 1994). In kidney tissue, cisplatin-induced nephrotoxicity is closely associated with increased lipid peroxidation (Choie et al., 1981). Therefore, oxygen free radicals may play a central role in cisplatin-induced renal injury. The underlying mechanism of cisplatin-induced nephrotoxicity is still not well known, but many recent in vitro and in vivo studies indicate an important role for the reactive oxygen metabolites in the pathogenesis of this effect (Matsushima et al., 1998). © 2013 Blackwell Verlag GmbH Andrologia 2013, xx, 1–9
Cisplatin-based chemotherapy may also result in impaired spermatogenesis (Cherry et al., 2004), chromosomal abnormalities in spermatozoa (Martin et al., 1999) and temporary or permanent azoospermia (Howell & Shalet, 2005). Pathogenesis of testicular damage after cisplatin exposure is generally attributed to oxidative damage (Silici et al., 2009; Salem et al., 2012). Flavonoids are phytophenolic compounds with strong antioxidant properties. Quercetin (3, 3′, 4′,5,7-pentahydroxyflavone) is one of the most abundant flavonoids in the human diet, commonly present in most edible fruits and vegetables (Heim et al., 2002). This bioflavonoid is a potent oxygen free radical scavenger and a metal chelator, capable of inhibiting lipid peroxidation in in vitro and in vivo systems (Ferry et al., 1996; Fiorani et al., 2001). The aim of this study is to investigate the protective effects of quercetin against cisplatin-induced renal and 1
Quercetin and cisplatin-induced damage
testicular toxicity associated with oxidative stress in male Wistar rats. What makes this study unique is the comprehensive investigation of both biochemical and histopathological changes in kidneys, spermatids and testicles. Materials and methods Chemicals _ Cisplatin was obtained from Farmar (Istanbul, Turkey), and quercetin complex was purchased from DK Pharma_ ceuticals (Istanbul, Turkey). All surgical procedures were performed under anaesthesia by intramuscular injection _ Turkey) and of 50 mg kg 1 ketamin (Pfizer, Istanbul, 1 _ 10 mg kg xlazine (Alfasan, Istanbul, Turkey). Animals Forty Wistar Albino type male rats of 20 weeks old were used in the study. The rats weighed 220 15 g. The rats were housed in individual cages at 22–24 °C, 50–55% humidity and light:dark cycle control (12 : 12 h). The rats had free access to food and water, and water was changed daily. The protocol of this study was approved by Ankara University, School of Medicine, Animal Care and Use Committee. All experimental procedures were conducted in accordance with the Guide to the Care and Use of Laboratory Animals. Experimental design The animals were divided into 5 groups, randomly. First group was used as control (Group 1), and the second group (Group 2) was treated with a single dose of intraperitoneal cisplatin (7 mg kg 1). Third group (Group 3) was treated with 50 mg kg day 1 quercetin administered to rats by oral route for 10 days. Fourth group (Group 4) was treated with one intraperitoneal single dose 7 mg kg 1 at first day, and then, 50 mg kg day 1 oral quercetin was administered for 10 days. Fifth group (Group 5) was given 50 mg kg day 1 oral quercetin for 10 days and then single dose intraperitoneal cisplatin 7 mg kg 1 at 10th day. Biochemical analysis Renal function was assessed by estimating serum creatinine and blood urea nitrogen (BUN). Testis and kidney tissues of the rats were removed by surgical operation, washed with physiological saline solution and separated from perirenal fatty tissue. The tissues were homogenised and prepared for the assays as described previously (Kavutcu et al., 1996). Upper clear part of the tissue homogenates was used in the experiments. The protein level of 2
M. Aldemir et al.
the clear supernatants was determined by the method of Lowry et al. (1951). Malondialdehyde (MDA, nmol mg 1) levels and superoxide dismutase (SOD, U mg 1), catalase (CAT, IU mg 1) and xanthine oxidase (XO, mIU mg 1) activities were measured in the supernatant fraction (Dasgupta & Zdunek, 1992). MDA level was measured by thiobarbituric acid reactive substances (TBARS) method (Dahle et al., 1962). Superoxide dismutase activity was measured as described (Durak et al., 1996). One unit for SOD activity was expressed as the enzyme protein amount causing 50% inhibition in nitroblue tetrazolium reduction rate. CAT activity was determined by measuring decrease in hydrogen peroxide (H2O2) absorbance at 240 nm as described (Aebi, 1974). Xanthine oxidase activity was determined by measuring uric acid formation from xanthine at 293 nm as described (Hashimato, 1974). In the activity calculations, extinction coefficients of uric acid and H2O2 were used for XO and CAT and respectively. Serum total antioxidant capacity (TAC) levels and total oxidant status (TOS) were determined using a novel automated measurement method, developed by Erel (2004, 2005). Histopathological examination Kidneys and one testicle of each rat were fixed in 10% buffered formaldehyde. One half of one kidney and testicle on coronal plane were submitted to routine tissue processing, 5-lm-thick sections were stained with routine hematoxylin & eosin and examined under light microscope (Olympus BX51, Tokyo, Japan). In histopathological examination of testicles, we investigated the presence of any disruption of the normal architecture with an orderly arrangement of germinal and sertoli cells. The presence of tubular atrophy, germ cell degeneration, necrosis, shrinkage of seminiferous tubules, shredding of germ cells in lumen and interstitial oedema was noted (Fig. 1a). Severity of damage (testis damage score) was graded according to the extent of degenerative findings, as 0, no damage; 1,
