ORIGINAL ARTICLE
Fenugreek seed extract attenuates cisplatin-induced testicular damage in Wistar rats A. A. Hamza1, H. M. Elwy2 & A. M. Badawi3 1 Hormone Evaluation Department, National Organization for Drug Control and Research (NODCAR), Giza, Egypt; 2 Physical Chemistry Department, National Organization for Drug Control and Research, Giza, Egypt; 3 Biochemistry Department, National Organization for Drug Control and Research, Giza, Egypt
Keywords Cisplatin—fenugreek—inflammation— oxidative stress—testicular damage Correspondence Alaaeldin A. Hamza, NODCAR, 6 Abu Hazem Road, Pyramids, Giza, Egypt. Tel.: 20 01201991433; Fax: +20 2 35855582; E-mail: [email protected] Accepted: March 16, 2015 doi: 10.1111/and.12435
Summary Cisplatin (CIS) provides oxidative stress and inflammations in testicular tissues. Fenugreek seed extract (FSE) is a widely used herbal medicine with potent antioxidant and anti-inflammation properties. The purpose of this study was to investigate the protective effects and the possible mechanisms of FSE against CIS-induced testicular damage in rats. Adult male Wistar rats were given vehicle, single dose of CIS alone (10 mg kg 1), single dose of FSE alone or single dose of CIS followed by FSE (50, 100 or 200 mg kg 1) every day for 5 days. On day 6, oxidative stress and apoptotic testicular toxicity were evaluated. FSE attenuated both germ cell degenerations and apoptosis in seminiferous tubules in CIS-treated rats. Furthermore, FSE counteracted CIS-induced oxidative stress in rats as assessed by the restoration of superoxide dismutase and catalase activities and reduction in the myeloperoxidase activity and malondialdehyde levels in testes. CIS increased expressions of inducible nitric oxide synthase and nuclear factor-kappa B in testicular tissues. Importantly, treatment with FSE at all doses effectively alleviated all of these inflammatory parameters in testes. Based on these results, we concluded that FSE reduces CIS-induced reproductive toxicity in rats by the suppression of testicular oxidative stress, apoptosis and inflammations.
Introduction Cisplatin (CIS) is a highly effective chemotherapeutic agent used to treat many types of solid tumours. However, its use is limited by the acute and chronic toxic side effects to multiorgan systems (Chirino & Pedraza-Chaverri, 2009). The human testis is a known target organ for injury resulting from exposure to this potent chemotherapeutic agent (Boekelheide, 2005). Owing to the relative spermiotoxicity of CIS, almost all the human patients show temporary or permanent azoospermia and testicular atrophy. Animal studies have provided evidence of CISinduced dysfunction of Leydig cells, Sertoli cells and germ cells (Cherry et al., 2004; Boekelheide, 2005). The major mechanism known to be involved in CIS-induced testicular damage includes oxidative stress (Amin & Hamza, 2006). CIS causes lipid peroxidations, atrophy in the testicular seminiferous tubules and apoptosis in spermatocytes (Seaman et al., 2003; Cherry et al., 2004; Amin et al., 2012). Based on this concept, a variety of antioxidant and natural products have been employed to counteract © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
CIS-induced testicular damage (Atessahin et al., 2006; Ilbey et al., 2009). Nevertheless, so far, there is still no single agent proved to be effective enough to prevent this adverse effect. Fenugreek (Trigonella foenum graecum Linn) is an annual herb that belongs to the family Leguminosae. The seeds of fenugreek are commonly used in the Middle East and South Asia as a spice in food preparation and used as traditional medicines in diabetes, high cholesterol, inflammations and gastrointestinal ailments (Basu & Srichamroen, 2010; BelguithHadriche et al., 2010). Recently, fenugreek seed extract (FSE) has been shown to protect against buthionine sulfoximine and cyclophosphamide urotoxicity in mice (Bhatia et al., 2006), against alcohol liver toxicity in rats (Kaviarasan & Anuradha, 2007) and against adriamycin reproductive toxicity in rats (Sakr et al., 2012). The seeds are also used as herbal medicine in many parts of the world for carminative, tonic and aphrodisiac effects (Duke et al., 2002). Fenugreek seeds are a rich source of many active phytochemicals such as alkaloids, trigonelline, steroidal saponins, salicylate, nicotinic acid and flavonoids, which are thought to account for 211
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many of its presumed therapeutic effects (Basu & Srichamroen, 2010). In view of these considerations, the functional health effect of FSE in protecting against CIS toxicity could be of current interest. Therefore, the aim of this study was to evaluate the protective effects of FSE on CIS-induced alterations in sperm characteristics and biochemical changes related to oxidative stress and inflammations in the testes of rats. Materials and methods Chemicals O-dianisidine, 2,4-dinitrophenylhydrazine, thiobarbituric acid, Folin’s reagent, pyrogallol, SOD enzyme, H2O2, bovine albumin, 2,4,6-tripyridyl triazine, gallic acid, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl triazine, 2, 2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), b-carotene, caffeic, p-coumaric, gallic and syringic acid (+)-catechin, vanillin, quercetin and rutin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit anti-rat primary antibodies of inducible nitric oxide synthase (iNOS) (Ab-1) and nuclear factor-kappa B, NF-jB-p65 (Rel A, Ab-1) were obtained from (Thermo Fisher Scientific, Lab Vision Corporation, Fremont, CA, USA). All other chemicals were obtained from local commercial suppliers. CIS was purchased from Bristol-Myers Squibb Company, USA. Preparation of plant extract Fenugreek seed extract was purchased from local herbal market, Cairo, Egypt. A sample of 10 g of ground-dried seeds was extracted with 100 ml of 70% (v/v) aqueous ethanol. The plant material was macerated for 48 h in the aqueous ethanol at 4 °C. The extracts were filtered and dried under reduced pressure in a rotary evaporator at 40 °C and kept at 20 °C. This aqueous extract was then used at doses of 50, 100 and 200 mg kg 1 body weight in a volume of 5 ml kg 1 body weight. In vitro antioxidant properties Total phenolic content was determined by the method of Singleton et al. (1999) using the Folin–Ciocalteu reagent and expressed in milligrams of gallic acid equivalent per gram dry weight of plant material. In vitro total antioxidant properties of FSE were estimated by the ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH·), 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) and b-carotene bleaching tests. The FRAP assay is based on the reducing power of antioxidants in 212
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which a potential antioxidant will reduce the oxidised ferric ions to the ferrous ions (Nenadis et al., 2007). ABTS assay is based on the reduction of blue green (ABTS·+) form by antioxidants to its original colourless ABTS form (Erel, 2004). DPPH assay is based on the reduction of DPPH· radical to a yellow-coloured compound (Nenadis et al., 2007). The ability of plant extracts to prevent bleaching of b-carotene was assessed according to a procedure based on Lim & Quah (2007) and expressed as percentage of inhibition. IC50 value (mg ml 1) is the concentration of extract that causes ABTS·+ or DPPH· radical is scavenged by 50% and the bleaching of b-carotene is prevented by 50%. Also in ABTS, FRAP and DPPH assays, the calibration curve of ascorbic acid was plotted, and the antioxidant capacity of the FSE was then expressed as lmol ascorbic acid equivalent per gram dry extract. HPLC analysis of phenolic compounds HPLC analyses were performed according to the method of Abad-Garcia et al. (2007) with an Agilent 1200 LC system (Santa Clara, CA, USA) consisting of degasser, quaternary pump (G1311A), autosampler (G1329A), column heater (G1316A) and diode array detector (DAD) (G1315C). A reversed-phase C18 (4.6 mm 9 250 mm 9 5 lm) column (Waters Corporation, Milford, MA, USA) was used. The mobile phase used in this study was a gradient of two solvents: solvent A (acetic acid–water, 0.5 : 99.5, v/v) and solvent B (methanol). The gradient profile was as follows: 0–2 min, 0% B isocratic; 2–6 min, linear gradient from 0% to 15% B; 6–12 min, 15% B isocratic; 12–17 min, linear gradient from 15% to 20% B; 17–35 min, 20% B isocratic; 35–90 min, linear gradient from 20% to 35% B; and 90– 136 min, 35% B isocratic. After each run, the system was washed and reconditioned for 10 min before analysis of the next sample. The flow rate was 0.8 ml min 1, and injection volume was 50 ll. A series of individual standard solutions of phenolic compounds (each 1.0 mg ml 1) dissolved initially in minimal amount of DMSO and diluted with aqueous methanol (50 : 50 v/v) were used to plot calibration curves. Phenolic compounds were identified and quantified in the samples using retention times and calibration curves respectively. The detection was carried out at 254, 280, 320 and 360 nm. Animals Forty-eight healthy adult male Wistar rats (3 months old, 150–200 g) were used. The animals were obtained from the Animal House, National Organization for Drug Control and Research (NODCAR). Animals were cared for in accordance with the standard guidelines (Canadian Council on Animal Care 1993). They were maintained on © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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standard pellet diet and tap water ad libitum and were kept in polycarbonate cages with wood chip bedding under a 12-h light/dark cycle and room temperature 22– 24 °C. Rats were acclimatised to the environment for 2 weeks prior to experimental use. The experimental protocol was approved by the local ethical committee of NODCAR. Experimental protocol Rats were randomly divided into six groups (eight rats each). Groups were treated as follows: control group (CON) received water orally (5 ml kg 1 body weight) for 5 days and a single intraperitoneal injection (i.p.) of saline on the first day (5 ml kg 1 body weight). The same dose volume (5 ml) was used for all other groups. FSE group received FSE (200 mg kg 1; p.o.) for 5 days. CIS group was given CIS in a single dose (10 mg kg 1; i.p.) on the first day of treatment, a dose that induced testicular toxicity in rats (Amin & Hamza, 2006). Rats of the protective groups (CIS + FSE groups) received FSE for 5 days, 1 h after a single dose of CIS on the first day of treatment. FSE was administrated orally at three dose levels 50, 100 and 200 mg kg 1 body weight. Doses of FSE were selected based on previously reported pharmacological properties of this plant. FSE at doses varying from 100 to 200 mg kg 1 has been reported to suppress chemically induced oxidative damage and apoptosis (Bhatia et al., 2006; Kaviarasan & Anuradha, 2007). Sample preparation On day six, the rats were weighed and sacrificed under diethyl ether by cervical decapitation. The testes and epididymides were dissected out and weighed. After the testes were weighed, one of them was immediately fixed in 10% buffered formalin for histopathological examinations. The other testis was homogenised in ice-cold Tris–HCL buffer (150 mM, pH 7.4) and stored at 20 °C for further biochemical analyses. The w/v ratio of the tissue to the homogenisation buffer was 1 : 10 w/v. Sperm motility and count Total sperm number was determined using a Neubauer haemocytometer by the method of Kenjale et al. (2008). Cauda epididymidis was dissected out, weighed, immediately minced in 5 ml of physiological saline and then incubated at 37 °C for 30 min to allow spermatozoa to leave the epididymal tubules. The percentage of motile spermatozoa was recorded using a phase contrast microscope at 4009 magnification. The total number of spermatozoa per gram of cauda was then calculated. © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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Oxidative stress in testicular tissues Determination of malondialdehyde (MDA) in testicular tissues is based on its reaction with thiobarbituric acid to form a pink-coloured complex with absorption maximum at 535 nm (Uchiyama & Mihara, 1978). Catalase (CAT) activity was determined by measuring the exponential disappearance of H2O2 at 240 nm and expressed as units per mg of protein as described by Aebi (1984). Myeloperoxidase (MPO) activity in testicular tissues was determined as described by Hillegass et al. (1990). One unit of MPO was defined as the amount of MPO present that degrades one micromolar of peroxide per minute. Superoxide dismutase (SOD) activity in testicular tissues was determined according to the method by Nandi & Chatterjee (1988). One unit of SOD is described as the amount of enzyme required to cause 50% inhibition of pyrogallol auto-oxidation. The total protein content of testis was determined according to the Lowry’s method as modified by Peterson (1977). Absorbance was recorded using a Lambda 25 UV/VIS spectrophotometer, PerkinElmer (Beacons field, UK), in all measurements. Histology For the histological examinations, small pieces of testis were fixed in 10% neutral phosphate-buffered formalin, dehydrated and then sectioned (5 lm in thickness). Sections were then stained with haematoxylin and eosin and examined under optical microscopy (Olympus CX31, London, UK). TUNEL assay and immunohistochemistry Apoptosis of testicular tissues was identified in deparaffinised sections using the terminal deoxynucleotidyl transferase-mediated triphosphate nick-end labelling (TUNEL) assay. In this assay, the manufacturer’s protocol for the Apoptag plus Peroxidase in Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA) was followed. For immunohistochemical analysis, four sequential sections per rat were analysed; antigens were unmasked by immersing the sections in 0.1 M sodium citrate buffer (pH 6) in heated water bath for 15 min. Then, endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol. Primary antibodies of iNOS and NF-jB-p65 unit (1 : 100 dilutions) were incubated overnight at 4 °C. After a standard staining protocol using EnVisionTM FLEX plus detection kit (Dako, Code K8012, Glostrup, Denmark), the sections were lightly counterstained with haematoxylin, dehydrated and mounted. Tissue images were captured by optical microscopy (Olympus DP71). The immunocytochemical staining was 213
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quantified as number of positive cells/high-power field (Table 3).
Moreover, the FSE showed high total phenol value (13.8 0.43 mg gallic acid equivalents g 1).
Statistical analysis
Amount of total phenolics and HPLC quantification of major phytochemical compounds
(version 18) statistical program (SPSS Inc., Chicago, IL, USA) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analyses of differences between the means of the treated and control groups were performed using Dunnett’s t-test. SPSS
Results In vitro antioxidant capacity and phenolic content of FSE It is clear that due to the complex nature of the different phytochemical classes present in plants, the antioxidant capacities of plant extracts cannot be evaluated using a single method. In the present work, the FRAP, ABTS, DPPH· and b-carotene methods were used to assess the antioxidant activities of FSE. The results of the total antioxidant and phenolic assays of the FSE are presented in Table 1. FRAP assay depends on the reduction of oxidised ferric ions to the ferrous ions by the antioxidant agents. In this study, each gram of dried FSE has high FRAP value, 17.16 lmol ascorbic acid equivalent. The ABTS and DPPH radical scavenging methods are widely used for evaluating the ability of plant extracts to scavenge free radical generated from ABTS and DPPH reagents. The FSE exhibited high antifree radical scavenging activity where the ascorbic acid equivalent antioxidant capacities of the FSE were 30.16 and 31.01 lmo g 1 in ABTS and DPPH assays respectively. In addition, the antioxidant activity evaluated as IC50 value (lg ml 1) revealed a similar behaviour. The ABTS and DPPH radical scavenging ability of samples (IC50) was 1.0 and 1.07 mg ml 1. b-Carotene undergoes rapid bleaching in the absence of antioxidants. The presence of antioxidants hinders the extent of bleaching by neutralising the linolic hydroperoxyl radical formed. FSE showed high ability to prevent bleaching of b-carotene (IC50 = 0.59 mg ml 1).
The total phenolic content of plant extract was determined and expressed in milligrams of gallic acid equivalent (Table 2). As shown in Table 2, the FSE showed high total phenol value (13.8 0.43 mg gallic acid equivalents g 1). The data from the qualitative–quantitative analysis of FSE made using HPLC coupled with photodiode array detection are presented in Table 2. The components of the gallic acid, ferulic acid and rutin were identified by comparison with the retention times of standards analysed under identical analytical conditions, while the quantitative data were calculated from their respective calibration curves. Rutin (3.26 mg g 1) and ferulic acid (2.07 mg g 1) were the most abundant phenolic compounds present in the plant extract. The least abundant compound present in the extract was gallic acid (0.19 mg g 1). FSE ameliorated CIS-induced depletion in testicular and epididymal weights as well as in sperm indices Treatment with CIS significantly (P < 0.001) decreased the weights of testes and epididymides as well as the caudal sperm count and motility (Fig. 1). However, treatment of CIS-intoxicated rats with FSE caused a significant improvement in the weights of testes and epididymides as well as in the caudal sperm count and motility. A dose-dependent improvement was evident in these parameters. The concomitant treatment of high dose of FSE with CIS caused the highest protective effects in these parameters. Rats receiving FSE alone did not show any significant change in the weights of both testes and epididymides as well as in the quality of sperm indices. FSE-suppressed histopathological changes and proapoptotic effect of CIS Control rats showed normal testicular architecture with an orderly arrangement of germinal cells and Sertoli cells.
Table 1 In vitro antioxidant capacity of fenugreek seed extract (FSE)
Assay
FRAP assay TAC (lmol g 1)
FSE
17.16 0.42
ABTS assay
DPPH assay
TAC (lmol g 1)
IC50 (mg ml 1)
TAC (lmol g 1)
IC50 (mg ml 1)
b-Carotene assay IC50 (mg ml 1)
30.16 0.02
1.0 0.05
31.01 0.02
1.07 0.07
0.59 0.05
Total antioxidant activity of FSE is expressed as ascorbic acid equivalents (mmol g ments.
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1
of dry extract). Values are means SEM of three experi-
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Table 2 Amount of total phenolicsa and the qualitative–quantitative analysesb of the ethanol extract from fenugreek seed extract carried out using an HPLC-DAD Compound
Retention time (min)
Amount of compounds
Total phenolic content Gallic acid Ferulic acid Rutin
– 10.13 0.01 65.03 0.01 90.08 0.01
13.80 0.19 2.07 3.26
0.43 0.01 0.01 0.06
Results are expressed as mean SEM of three experiments. Total phenolic content was expressed as mg gallic acid equivalents g 1 dried extract. b The amount of compounds was expressed as mg g 1 of dried extract. a
CIS treatment induced testicular atrophy with degeneration of germ cells in seminiferous tubules (Fig. 2A). Disorganisation and shedding of germ cells into lumina were also observed. Animals pretreated with FSE showed less degeneration of seminiferous epithelium with shedding of germ cells in few tubules. To evaluate whether FSE protected testicular tissues through inhibiting CIS-induced apoptosis, germ cell apoptosis was assessed by TUNEL assay. Brown staining indicating TUNEL-positive nuclei was visible in seminiferous tubules of control and CIStreated animals. Testicular tissues exposed to CIS contained a large number of the TUNEL-positive germ cells in contrast to those treated with FSE, indicating the
Fig. 1 Inhibitory effect of FSE on CIS-induced depletion in testicular and epididymal weights as well as on caudal sperm count and motility. Values are expressed as mean SEM (n = 8). Significance was determined by one-way analysis of variance followed by Dunnett’s t-test: ***P < 0.001,**P < 0.01, *P < 0.05 versus control group; aP < 0.001, bP < 0.01, cP < 0.05 versus CIS group.
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Fig. 2 (A) Inhibitory effect of FSE on CIS-induced histopathological changes and pro-apoptotic effect in testicular tissue. (A) Representative images of haematoxylin- and eosin-stained section in the testes of all groups studied (magnifications = 1009). Testes from control (a) and FSE (b) groups exhibit typical feature of seminiferous epithelium while those from CIS (c) group displayed atrophy (*), vacuolisation and degeneration of germinal epithelium (arrow). Testes from CIS + FSE groups (d: low dose; e: medium dose; f: high dose) show nearly normal with some degeneration of seminiferous epithelium (arrow). (B) Representative images of TUNEL staining in the testicular section from all the groups studied. Magnifications = 4009. Testes from control (a) and FSE (b) groups exhibit small numbers of TUNEL-positive cells while those from CIS (c) group showed large number of TUNEL-positive cells (arrow). Testes from CIS + FSE groups (d–f) appear normal with small number of TUNEL-positive cells (arrow).
pro-apoptotic effect of CIS and anti-apoptotic effects of FSE (Fig. 2B and Table 3). FSE ameliorated CIS-induced oxidative stress in testicular tissues In CIS-intoxicated rats, testicular MDA levels and SOD and MPO activities were significantly (P < 0.001) increased and CAT activity was significantly (P < 0.001) decreased (Fig. 3), indicating the potent oxidative action of CIS on testicular tissues. Co-administration of FSE with CIS neutralised these abnormalities in MDA levels and activities of SOD, CAT and MPO, showing the effect 216
of FSE against CIS-induced oxidative stress in testes. These protective effects were dose dependent. The concomitant treatment of high dose of FSE with CIS caused the highest protective effects in these parameters. FSE ameliorated CIS-induced upregulation of iNOS and NF-jB-p65 on testicular tissues Inducible nitric oxide synthase expression is known to be regulated by the transcription factor NF-jB (Kundu & Surh, 2005). In an attempt to link the activation of induction during CIS treatment, we evaluated their expression level in all the groups. Within normal rat © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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Table 3 Effect of cisplatin (CIS) and/or fenugreek seed extract (FSE) treatment on the number of transferase-mediated triphosphate nick-end labelling (TUNEL)-, inducible nitric oxide synthase (iNOS)- and NF-jB-p65-positive cells in the testis of rat
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TUNEL a b c d e f
Control FSE CIS CIS + FSE (LD) CIS + FSE (MD) CIS + FSE (HD)
1.7 1.0 41.0 11.1 6.1 5.2
NF-jB-p65 0.33 0.58 0.59*** 1.07***,a 0.71***,a 0.42**,a
5.9 5.9 69.0 27.3 19.0 11.3
0.38 0.17 5.19*** 1.43***,a 1.58***,a 1.57**,a
iNOS 13.8 13.5 138.5 50.7 19.0 12.4
0.98 0.76 14.77*** 5.21***,a 0.58a 1.65a
The positive expression of cells in each section was calculated by counting the number of brown staining in four fields at 4009 magnifications then the number of positive cells/field. Values are expressed as mean SEM (n = 4). Significance was determined by one-way analysis of variance followed by Dunnett’s t-test: ***P < 0.001, **P < 0.01 versus control group; aP < 0.001, versus CIS group.
Fig. 3 Inhibitory effect of FSE on CIS-induced oxidative damage in rat testicular tissue. Values are expressed as mean SEM (n = 8). Significance was determined by one-way analysis of variance followed by Dunnett’s t-test: ***P < 0.001, *P < 0.05 versus control group; aP < 0.001, b P < 0.01, cP < 0.05 versus CIS group.
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testis, expression of iNOS and NF-jB was significantly low in the seminiferous tubules and intertubular tissue. There were a large number of iNOS- and NF-jB-p65positive cells in rats subjected to CIS alone (Fig. 4 and Table 3). FSE supplementation effectively inhibited the increase in the number of iNOS- and NF-jB-positive cells in the testicular tissues of CIS-treated rats. These results show that FSE suppresses CIS-induced increase in the expression of iNOS and NF-jB in testes. Discussion The human testis is a known target organ for injury resulting from exposure to CIS. Short- and long-term CIS (A)
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treatments result in germinal epithelial damage, sperm dysfunction and germ cell apoptosis in animals and humans (Cherry et al., 2004; Boekelheide, 2005). Our previous study has shown that CIS impaired rat testicular structure through inflicting oxidative stress and inducing cell apoptosis (Amin & Hamza, 2006; Amin et al., 2008). The present study further provides evidence that treatment with FSE could effectively alleviate CIS-induced testicular toxicity. In the present work, CIS treatment led to a significant decrease in testicular weight and epididymides weight with marked decrease in the caudal sperm count and motility compared with control animals. Administration of FSE restored the weights of testes and epididymides together with concomitant improvement in (B)
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Fig. 4 Inhibitory effect of FSE on CIS-induced upregulation in inducible nitric oxide synthase (iNOS) and NF-kB-p65 expression in testes (Magnifications = 1009). (A) Representative images of immunohistochemical iNOS-stained section in the testes of all groups studied. Testes from control (a) and FSE (b) groups exhibit low numbers of iNOS-positive cells, while those from CIS (c) group show large number of iNOS-positive cells (arrow). Testes from CIS + FSE groups show low number of iNOS-positive cells. (B) Representative images of immunohistochemical NF-kB-p65stained section in the testes of all groups studied. Testes from control (a) and FSE (b) groups exhibit low numbers of NF-kB-p65-positive cells, while those from CIS (c) group show large number of NF-kB-p65-positive cells. Testes from CIS + FSE groups (d: low dose; e: medium dose; f: high dose) show low number of NF-kB-p65-positive cells.
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the caudal sperm count and motility in CIS-intoxicated rats. These results were confirmed by histopathological examination which showed disorganisation, vacuolisation and degeneration of germinal epithelium in testes of rats treated with CIS alone. FSE treatment reduced these degenerative effects. Consistent with the results of histological changes, apoptosis was observed in the CIS-treated animal group. Germ cell apoptosis has been recently reported as a mechanism responsible for the damage to testes and sperm output (Boekelheide, 2005; ). CIS was reported to cause apoptosis to testicular germ cells and Sertoli cells (Seaman et al., 2003; Cherry et al., 2004). Using the TUNEL technique, apoptotic DNA fragmentation was determined in testicular tissue in the present work. Consistent with the previous studies, CIS treatment resulted in a significant increase in the testicular apoptosis, which was significantly reduced with CIS plus FSE. Accordingly, it is likely that DNA damage and apoptosis are important mechanisms for the germ cell death in the CIS-intoxicated testes and that FSE attenuates apoptotic DNA damage induced by CIS in testes. Reactive oxygen species (ROS) have been reported to play a role in CIS-induced testicular damage (Ilbey et al., 2009). They can induce lipid peroxidation and DNA fragmentation in spermatozoa and testis (Boekelheide, 2005). The negative changes observed in sperm quality in CIS-treated rats may be caused by the peroxidation of polyunsaturated fatty acids in sperm plasma membranes. Supporting this idea, an increase in lipid peroxidation product, MDA, in testes of CIS-treated rats was observed in the present work, indicating testicular oxidative damage. Additionally, our data demonstrated a significant increase in SOD activity and a significant decrease in CAT activity in testicular tissues of CIS-treated rats. This observed increased in testicular SOD activity may reflect an adaptive response to neutralise toxic superoxide radicals generated due to impairment of antioxidant status of the organ. Accordingly, an increase in the concentrations of hydrogen peroxide as end product of SOD activity in testis in response to CIS may impair the capacity of the testes to neutralise hydrogen peroxide, thereby causing excessive consumption and deactivation of CAT enzyme. These features together contribute to the sensitivity of testicular tissues to CIS-induced oxidative injury (Atessahin et al., 2006). On the other hand, the present work demonstrated that the concomitant treatment with FSE attenuated testicular MDA together with protected testicular SOD and CAT activities in rats exposed to CIS, illustrating the antioxidative actions of FSE against potent free radical-producing CIS agent. Consistent with these data, FSE treatment attenuated the testicular toxicity of the anticancer agent adriamycin in rats by decreasing lipid © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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peroxidation and increasing antioxidant levels (Sakr et al., 2012). This highlights the potential antioxidant ability of FSE as a protective agent against testicular oxidative injury induced by anticancer agents in rats. Further, the FSE was examined for its antiradical and reducing capacity in vitro and showed high antiradical and antioxidant activities. This antioxidant potential of FSE was confirmed by four different biochemical assays: scavenging activity on ABTS and DPPH radicals, reducing the ability by FRAP assay and lipid peroxidation inhibition of b-carotene-linoleate system. This antioxidant effect of FSE has been linked to certain compounds in its main constituents, flavonoids and saponins (Basu & Srichamroen, 2010), which can scavenge free radical and reduce the level of ROS. Polyphenols with their redox properties can play an important role in adsorbing and neutralising free radicals (Nichenametla et al., 2006). Our results show a good correlation between the antioxidant activity and the phenolic contents in the FSE. Rutin and ferulic acid were the main phenolic acids present in our plant extract. Previous results of HPLC suggested that the protective effect exhibited by the FSE is probably due to its phenolic contents, particularly the myricetin, chlorogenic acid, p-coumaric acid and ferulic acid in methanol extract (Jahan et al., 2013). In addition, FSE contains at least 90% steroid saponins (Petit et al., 1995). Our work showed that CIS treatment caused elevations of testicular MPO activity, a marker of neutrophil infiltration, inflammation and oxidative stress, indicating the presence of enhanced polymorphonuclear leucocyte recruitment in the inflamed tissues under the effect of oxidative stress. FSE attenuated the CIS-induced increase in testicular MPO activity, indicating the anti-inflammatory action of FSE. Additionally, the current study also showed significant increase in NF-jB and iNOS expression, as a result of CIS treatment. These results coincide with the previous finding that CIS caused a significant increase in iNOS and NF-jB-p65 expression in the testicular tissues (Ilbey et al., 2009; Amin et al., 2012). Oxidative stress is known to stimulate transcription factors, including NF-jB (Kundu & Surh, 2005). NF-jB functions as a link between oxidative damage and inflammation. Elevated levels of NF-jB and the associated inflammatory mediators such as iNOS are well-known biological markers of inflammatory responses (Chirino & PedrazaChaverri, 2009). It is well known that excessive NO production due to elevated iNOS causes cytotoxic effects and has the potential to induce germ cell apoptosis (Chirino & Pedraza-Chaverri, 2009; Jahan et al., 2013). It can be inferred that CIS-induced oxidative stress led to an increase in the expression of NF-jB, which in turn resulted in an enhanced expression of various genes such as iNOS leading to apoptosis in the testicular cells, while 219
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the administration of FSE to CIS-treated rats in this work reduced the increase in iNOS and NF-jB-p65 expression in the testicular tissues of CIS-treated rats. Thus, FSE could mediate its protective effect against testicular damage by downregulation of iNOS and NF-jB-p65. In summary, our study provides experimental evidence that concomitant treatment of FSE with CIS attenuated CIS-induced reproductive toxicity and testicular histopathological changes by antioxidants effects. We believe that the protective effects of FSE against CIS-reproductive toxicity is mediated primarily through both the inhibition of apoptosis and the abrogation of oxidative stress and iNOS and NF-jB inflammatory response associated with CIS treatment. These observations are of clinical importance as FSE could be a promising target to halt this serious testicular toxicity induced by CIS. References Abad-Garcia B, Berrueta LA, Lopez-Marquez DM, CrespoFerrer I, Gallo B, Vicente F (2007) Optimization and validation of a methodology based on solvent extraction and liquid chromatography for the simultaneous determination of several polyphenolic families in fruit juices. J Chromatogr A 1154:87–96. Aebi H (1984) Catalase. Methods Enzymol 105:121–126. Amin A, Hamza A (2006) Effects of ginger and roselle on cisplatin induced reproductive toxicity in rats. Asian J Androl 8:607–612. Amin A, Hamza A, Kambal A, Daoud S (2008) Herbal extracts counteract cisplatin-mediated cell death in rat testis. Asian J Androl 10:291–297. Amin A, Abraham C, Hamza AA, Abdalla ZA, Al-Shamsi SB, Harethi SS, Daoud S (2012) A standardized extract of Ginkgo biloba neutralizes cisplatin-mediated reproductive toxicity in rats. J Biomed Biotechnol 12:1–11. Atessahin A, Karahan I, Turk G, Giir S, Yilmaz S, Ceribasi AO (2006) Protective role of lycopene on cisplatin-induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod Toxicol 21:42–47. Basu TK, Srichamroen A (2010) Chapter 28-health benefits of fenugreek (Trigonella foenum-graecum leguminosse). In: Bioactive Food in Promoting Health Fruits and Vegetables. Basu TK, Srichamroen A (ed.). Academic Press, Harcourt Brace & Company, San Diego, pp 425–435. Belguith-Hadriche O, Bouaziz M, Jamoussi K, Et Feki A, Sayadi S, Makni-Ayedi F (2010) Lipid-lowering and antioxidant effects of an ethyl acetate extract of fenugreek seeds in high-cholesterol-fed rats. J Agric Food Chem 58:2116–2122. Bhatia K, Kaur M, Atif F, Ali M, Rehman H, Rahman S, Raisuddin S (2006) Aqueous extract of Trigonella foenumgraecum L. ameliorates additive urotoxicity of buthionine
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sulfoximine and cyclophosphamide in mice. Food Chem Toxicol 44:1744–1750. Boekelheide K (2005) Damage to fertility by cancer and its treatments. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monogr 34:6–8. Canadian Council on Animal Care (1993) Guide to the Care and Use of Experimental Animals. Olfert ED, Cross BM, Mc William AA (eds), vol. 1, CCAC, Ottawa, Canada. Cherry SM, Hunt PA, Hassold TJ (2004) Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res 564:115–128. Chirino YI, Pedraza-Chaverri J (2009) Role of oxidative and nitrosative stress in cisplatin-induced nephrotoxicity. Exp Toxicol Pathol 61:223–242. Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke PK (2002) Fenugreek (Trigonella foenum-graecum L.). In: Handbook of Medicinal Herbs. Duke JA (ed.). CRC press, Boca Raton, pp 296–297. Erel O (2004) A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 37:277–285. Hillegass LM, Griswold DE, Brickson B, Albrightson-Winslow C (1990) Assessment of myloperoxidase activity in whole rat kidney. J Pharmacol Meth 24:285–295. Ilbey YO, Ozbek E, Cekmen M, Simsek A, Otunctemur A, Somay A (2009) Protective effect of curcumin in cisplatininduced oxidative injury in rat testis: mitogeb-activated protein kinase and nuclear factor-Kappa B signaling pathways. Hum Reprod 24:1717–1725. Jahan N, Rahman K, Ali S, Asi MR (2013) Phenolic acid and flavonol contents of gemmo-modified and native extracts of some indigenous medicinal plants. Pak J Bot 45:1515–1519. Kaviarasan S, Anuradha CV (2007) Fenugreek (Trigonella foenum graecum) seed polyphenols protect liver from alcohol toxicity: a role on hepatic detoxification system and apoptosis. Pharmazie 62:299–304. Kenjale R, Shah R, Sathaye S (2008) Effect of Chlorophytum borivilianum on sexual behaviour and sperm count in male rats. Phytother Res 22:796–801. Kundu JK, Surh YJ (2005) Breaking the relay in deregulated cellular signal transduction as a rationale for chemoprevention with anti-inflammatory phytochemicals. Mutat Res 591:123–146. Lim YY, Quah EPL (2007) Antioxidant properties of different cultivars of Portulaca oleracea. Food Chem 103:734–740. Nandi A, Chatterjee IB (1988) Assay of superoxide dismutase activity in animal tissues. J Biosci 13:305–315. Nenadis N, Lazaridou O, Tsimidou MZ (2007) Use of reference compounds in antioxidant activity assessment. J Agric Food Chem 55:5452–5460.
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Nichenametla SN, Taruscio TG, Barney DL, Exon JH (2006) A review of the effects and mechanisms of polyphenolics in cancer. Crit Rev Food Sci Nutr 46:161–183. Peterson GL (1977) A simplification of the protein assay method of lowry et al. which is more generally applicable. Anal Biochem 83:346–356. Petit PR, Sauvaire YD, Hillaire-Buys DM, Leconte OM, Baissac YG, Ponsin GR, Ribes GR (1995) Steroid saponins from fenugreek seeds: extraction, purification, and pharmacological investigation on feeding behaviour and plasma cholesterol. Steroids 60:674–680. Sakr SA, El-Shenawy SM, Al-Shabka AM (2012) Aqueous fenugreek seed extract ameliorates adriamycin-induced cytotoxicity and testicular alterations in albino rats. Reprod Sci 19:70–80.
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Seaman F, Sawhney P, Giammona CJ, Richburg JH (2003) Cisplatin-induced pulse of germ cell apoptosis precedes long-term elevated apoptotic rates in C57/BL/6 mouse testis. Apoptosis 8:101–108. Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of folin–ciocalteu, reagent. In: Methods in Enzymology. Oxidants and Antioxidants, Part A., Vol 299. Packer L (ed.). Academic Press, Harcourt Brace & Company, San Diego, London, New York, Tokyo, pp 152–178. Uchiyama M, Mihara M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278.
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Fenugreek seed extract attenuates cisplatin-induced testicular damage in Wistar rats A. A. Hamza1, H. M. Elwy2 & A. M. Badawi3 1 Hormone Evaluation Department, National Organization for Drug Control and Research (NODCAR), Giza, Egypt; 2 Physical Chemistry Department, National Organization for Drug Control and Research, Giza, Egypt; 3 Biochemistry Department, National Organization for Drug Control and Research, Giza, Egypt
Keywords Cisplatin—fenugreek—inflammation— oxidative stress—testicular damage Correspondence Alaaeldin A. Hamza, NODCAR, 6 Abu Hazem Road, Pyramids, Giza, Egypt. Tel.: 20 01201991433; Fax: +20 2 35855582; E-mail: [email protected] Accepted: March 16, 2015 doi: 10.1111/and.12435
Summary Cisplatin (CIS) provides oxidative stress and inflammations in testicular tissues. Fenugreek seed extract (FSE) is a widely used herbal medicine with potent antioxidant and anti-inflammation properties. The purpose of this study was to investigate the protective effects and the possible mechanisms of FSE against CIS-induced testicular damage in rats. Adult male Wistar rats were given vehicle, single dose of CIS alone (10 mg kg 1), single dose of FSE alone or single dose of CIS followed by FSE (50, 100 or 200 mg kg 1) every day for 5 days. On day 6, oxidative stress and apoptotic testicular toxicity were evaluated. FSE attenuated both germ cell degenerations and apoptosis in seminiferous tubules in CIS-treated rats. Furthermore, FSE counteracted CIS-induced oxidative stress in rats as assessed by the restoration of superoxide dismutase and catalase activities and reduction in the myeloperoxidase activity and malondialdehyde levels in testes. CIS increased expressions of inducible nitric oxide synthase and nuclear factor-kappa B in testicular tissues. Importantly, treatment with FSE at all doses effectively alleviated all of these inflammatory parameters in testes. Based on these results, we concluded that FSE reduces CIS-induced reproductive toxicity in rats by the suppression of testicular oxidative stress, apoptosis and inflammations.
Introduction Cisplatin (CIS) is a highly effective chemotherapeutic agent used to treat many types of solid tumours. However, its use is limited by the acute and chronic toxic side effects to multiorgan systems (Chirino & Pedraza-Chaverri, 2009). The human testis is a known target organ for injury resulting from exposure to this potent chemotherapeutic agent (Boekelheide, 2005). Owing to the relative spermiotoxicity of CIS, almost all the human patients show temporary or permanent azoospermia and testicular atrophy. Animal studies have provided evidence of CISinduced dysfunction of Leydig cells, Sertoli cells and germ cells (Cherry et al., 2004; Boekelheide, 2005). The major mechanism known to be involved in CIS-induced testicular damage includes oxidative stress (Amin & Hamza, 2006). CIS causes lipid peroxidations, atrophy in the testicular seminiferous tubules and apoptosis in spermatocytes (Seaman et al., 2003; Cherry et al., 2004; Amin et al., 2012). Based on this concept, a variety of antioxidant and natural products have been employed to counteract © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
CIS-induced testicular damage (Atessahin et al., 2006; Ilbey et al., 2009). Nevertheless, so far, there is still no single agent proved to be effective enough to prevent this adverse effect. Fenugreek (Trigonella foenum graecum Linn) is an annual herb that belongs to the family Leguminosae. The seeds of fenugreek are commonly used in the Middle East and South Asia as a spice in food preparation and used as traditional medicines in diabetes, high cholesterol, inflammations and gastrointestinal ailments (Basu & Srichamroen, 2010; BelguithHadriche et al., 2010). Recently, fenugreek seed extract (FSE) has been shown to protect against buthionine sulfoximine and cyclophosphamide urotoxicity in mice (Bhatia et al., 2006), against alcohol liver toxicity in rats (Kaviarasan & Anuradha, 2007) and against adriamycin reproductive toxicity in rats (Sakr et al., 2012). The seeds are also used as herbal medicine in many parts of the world for carminative, tonic and aphrodisiac effects (Duke et al., 2002). Fenugreek seeds are a rich source of many active phytochemicals such as alkaloids, trigonelline, steroidal saponins, salicylate, nicotinic acid and flavonoids, which are thought to account for 211
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many of its presumed therapeutic effects (Basu & Srichamroen, 2010). In view of these considerations, the functional health effect of FSE in protecting against CIS toxicity could be of current interest. Therefore, the aim of this study was to evaluate the protective effects of FSE on CIS-induced alterations in sperm characteristics and biochemical changes related to oxidative stress and inflammations in the testes of rats. Materials and methods Chemicals O-dianisidine, 2,4-dinitrophenylhydrazine, thiobarbituric acid, Folin’s reagent, pyrogallol, SOD enzyme, H2O2, bovine albumin, 2,4,6-tripyridyl triazine, gallic acid, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl triazine, 2, 2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), b-carotene, caffeic, p-coumaric, gallic and syringic acid (+)-catechin, vanillin, quercetin and rutin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit anti-rat primary antibodies of inducible nitric oxide synthase (iNOS) (Ab-1) and nuclear factor-kappa B, NF-jB-p65 (Rel A, Ab-1) were obtained from (Thermo Fisher Scientific, Lab Vision Corporation, Fremont, CA, USA). All other chemicals were obtained from local commercial suppliers. CIS was purchased from Bristol-Myers Squibb Company, USA. Preparation of plant extract Fenugreek seed extract was purchased from local herbal market, Cairo, Egypt. A sample of 10 g of ground-dried seeds was extracted with 100 ml of 70% (v/v) aqueous ethanol. The plant material was macerated for 48 h in the aqueous ethanol at 4 °C. The extracts were filtered and dried under reduced pressure in a rotary evaporator at 40 °C and kept at 20 °C. This aqueous extract was then used at doses of 50, 100 and 200 mg kg 1 body weight in a volume of 5 ml kg 1 body weight. In vitro antioxidant properties Total phenolic content was determined by the method of Singleton et al. (1999) using the Folin–Ciocalteu reagent and expressed in milligrams of gallic acid equivalent per gram dry weight of plant material. In vitro total antioxidant properties of FSE were estimated by the ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH·), 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) and b-carotene bleaching tests. The FRAP assay is based on the reducing power of antioxidants in 212
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which a potential antioxidant will reduce the oxidised ferric ions to the ferrous ions (Nenadis et al., 2007). ABTS assay is based on the reduction of blue green (ABTS·+) form by antioxidants to its original colourless ABTS form (Erel, 2004). DPPH assay is based on the reduction of DPPH· radical to a yellow-coloured compound (Nenadis et al., 2007). The ability of plant extracts to prevent bleaching of b-carotene was assessed according to a procedure based on Lim & Quah (2007) and expressed as percentage of inhibition. IC50 value (mg ml 1) is the concentration of extract that causes ABTS·+ or DPPH· radical is scavenged by 50% and the bleaching of b-carotene is prevented by 50%. Also in ABTS, FRAP and DPPH assays, the calibration curve of ascorbic acid was plotted, and the antioxidant capacity of the FSE was then expressed as lmol ascorbic acid equivalent per gram dry extract. HPLC analysis of phenolic compounds HPLC analyses were performed according to the method of Abad-Garcia et al. (2007) with an Agilent 1200 LC system (Santa Clara, CA, USA) consisting of degasser, quaternary pump (G1311A), autosampler (G1329A), column heater (G1316A) and diode array detector (DAD) (G1315C). A reversed-phase C18 (4.6 mm 9 250 mm 9 5 lm) column (Waters Corporation, Milford, MA, USA) was used. The mobile phase used in this study was a gradient of two solvents: solvent A (acetic acid–water, 0.5 : 99.5, v/v) and solvent B (methanol). The gradient profile was as follows: 0–2 min, 0% B isocratic; 2–6 min, linear gradient from 0% to 15% B; 6–12 min, 15% B isocratic; 12–17 min, linear gradient from 15% to 20% B; 17–35 min, 20% B isocratic; 35–90 min, linear gradient from 20% to 35% B; and 90– 136 min, 35% B isocratic. After each run, the system was washed and reconditioned for 10 min before analysis of the next sample. The flow rate was 0.8 ml min 1, and injection volume was 50 ll. A series of individual standard solutions of phenolic compounds (each 1.0 mg ml 1) dissolved initially in minimal amount of DMSO and diluted with aqueous methanol (50 : 50 v/v) were used to plot calibration curves. Phenolic compounds were identified and quantified in the samples using retention times and calibration curves respectively. The detection was carried out at 254, 280, 320 and 360 nm. Animals Forty-eight healthy adult male Wistar rats (3 months old, 150–200 g) were used. The animals were obtained from the Animal House, National Organization for Drug Control and Research (NODCAR). Animals were cared for in accordance with the standard guidelines (Canadian Council on Animal Care 1993). They were maintained on © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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standard pellet diet and tap water ad libitum and were kept in polycarbonate cages with wood chip bedding under a 12-h light/dark cycle and room temperature 22– 24 °C. Rats were acclimatised to the environment for 2 weeks prior to experimental use. The experimental protocol was approved by the local ethical committee of NODCAR. Experimental protocol Rats were randomly divided into six groups (eight rats each). Groups were treated as follows: control group (CON) received water orally (5 ml kg 1 body weight) for 5 days and a single intraperitoneal injection (i.p.) of saline on the first day (5 ml kg 1 body weight). The same dose volume (5 ml) was used for all other groups. FSE group received FSE (200 mg kg 1; p.o.) for 5 days. CIS group was given CIS in a single dose (10 mg kg 1; i.p.) on the first day of treatment, a dose that induced testicular toxicity in rats (Amin & Hamza, 2006). Rats of the protective groups (CIS + FSE groups) received FSE for 5 days, 1 h after a single dose of CIS on the first day of treatment. FSE was administrated orally at three dose levels 50, 100 and 200 mg kg 1 body weight. Doses of FSE were selected based on previously reported pharmacological properties of this plant. FSE at doses varying from 100 to 200 mg kg 1 has been reported to suppress chemically induced oxidative damage and apoptosis (Bhatia et al., 2006; Kaviarasan & Anuradha, 2007). Sample preparation On day six, the rats were weighed and sacrificed under diethyl ether by cervical decapitation. The testes and epididymides were dissected out and weighed. After the testes were weighed, one of them was immediately fixed in 10% buffered formalin for histopathological examinations. The other testis was homogenised in ice-cold Tris–HCL buffer (150 mM, pH 7.4) and stored at 20 °C for further biochemical analyses. The w/v ratio of the tissue to the homogenisation buffer was 1 : 10 w/v. Sperm motility and count Total sperm number was determined using a Neubauer haemocytometer by the method of Kenjale et al. (2008). Cauda epididymidis was dissected out, weighed, immediately minced in 5 ml of physiological saline and then incubated at 37 °C for 30 min to allow spermatozoa to leave the epididymal tubules. The percentage of motile spermatozoa was recorded using a phase contrast microscope at 4009 magnification. The total number of spermatozoa per gram of cauda was then calculated. © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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Oxidative stress in testicular tissues Determination of malondialdehyde (MDA) in testicular tissues is based on its reaction with thiobarbituric acid to form a pink-coloured complex with absorption maximum at 535 nm (Uchiyama & Mihara, 1978). Catalase (CAT) activity was determined by measuring the exponential disappearance of H2O2 at 240 nm and expressed as units per mg of protein as described by Aebi (1984). Myeloperoxidase (MPO) activity in testicular tissues was determined as described by Hillegass et al. (1990). One unit of MPO was defined as the amount of MPO present that degrades one micromolar of peroxide per minute. Superoxide dismutase (SOD) activity in testicular tissues was determined according to the method by Nandi & Chatterjee (1988). One unit of SOD is described as the amount of enzyme required to cause 50% inhibition of pyrogallol auto-oxidation. The total protein content of testis was determined according to the Lowry’s method as modified by Peterson (1977). Absorbance was recorded using a Lambda 25 UV/VIS spectrophotometer, PerkinElmer (Beacons field, UK), in all measurements. Histology For the histological examinations, small pieces of testis were fixed in 10% neutral phosphate-buffered formalin, dehydrated and then sectioned (5 lm in thickness). Sections were then stained with haematoxylin and eosin and examined under optical microscopy (Olympus CX31, London, UK). TUNEL assay and immunohistochemistry Apoptosis of testicular tissues was identified in deparaffinised sections using the terminal deoxynucleotidyl transferase-mediated triphosphate nick-end labelling (TUNEL) assay. In this assay, the manufacturer’s protocol for the Apoptag plus Peroxidase in Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA) was followed. For immunohistochemical analysis, four sequential sections per rat were analysed; antigens were unmasked by immersing the sections in 0.1 M sodium citrate buffer (pH 6) in heated water bath for 15 min. Then, endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol. Primary antibodies of iNOS and NF-jB-p65 unit (1 : 100 dilutions) were incubated overnight at 4 °C. After a standard staining protocol using EnVisionTM FLEX plus detection kit (Dako, Code K8012, Glostrup, Denmark), the sections were lightly counterstained with haematoxylin, dehydrated and mounted. Tissue images were captured by optical microscopy (Olympus DP71). The immunocytochemical staining was 213
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quantified as number of positive cells/high-power field (Table 3).
Moreover, the FSE showed high total phenol value (13.8 0.43 mg gallic acid equivalents g 1).
Statistical analysis
Amount of total phenolics and HPLC quantification of major phytochemical compounds
(version 18) statistical program (SPSS Inc., Chicago, IL, USA) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analyses of differences between the means of the treated and control groups were performed using Dunnett’s t-test. SPSS
Results In vitro antioxidant capacity and phenolic content of FSE It is clear that due to the complex nature of the different phytochemical classes present in plants, the antioxidant capacities of plant extracts cannot be evaluated using a single method. In the present work, the FRAP, ABTS, DPPH· and b-carotene methods were used to assess the antioxidant activities of FSE. The results of the total antioxidant and phenolic assays of the FSE are presented in Table 1. FRAP assay depends on the reduction of oxidised ferric ions to the ferrous ions by the antioxidant agents. In this study, each gram of dried FSE has high FRAP value, 17.16 lmol ascorbic acid equivalent. The ABTS and DPPH radical scavenging methods are widely used for evaluating the ability of plant extracts to scavenge free radical generated from ABTS and DPPH reagents. The FSE exhibited high antifree radical scavenging activity where the ascorbic acid equivalent antioxidant capacities of the FSE were 30.16 and 31.01 lmo g 1 in ABTS and DPPH assays respectively. In addition, the antioxidant activity evaluated as IC50 value (lg ml 1) revealed a similar behaviour. The ABTS and DPPH radical scavenging ability of samples (IC50) was 1.0 and 1.07 mg ml 1. b-Carotene undergoes rapid bleaching in the absence of antioxidants. The presence of antioxidants hinders the extent of bleaching by neutralising the linolic hydroperoxyl radical formed. FSE showed high ability to prevent bleaching of b-carotene (IC50 = 0.59 mg ml 1).
The total phenolic content of plant extract was determined and expressed in milligrams of gallic acid equivalent (Table 2). As shown in Table 2, the FSE showed high total phenol value (13.8 0.43 mg gallic acid equivalents g 1). The data from the qualitative–quantitative analysis of FSE made using HPLC coupled with photodiode array detection are presented in Table 2. The components of the gallic acid, ferulic acid and rutin were identified by comparison with the retention times of standards analysed under identical analytical conditions, while the quantitative data were calculated from their respective calibration curves. Rutin (3.26 mg g 1) and ferulic acid (2.07 mg g 1) were the most abundant phenolic compounds present in the plant extract. The least abundant compound present in the extract was gallic acid (0.19 mg g 1). FSE ameliorated CIS-induced depletion in testicular and epididymal weights as well as in sperm indices Treatment with CIS significantly (P < 0.001) decreased the weights of testes and epididymides as well as the caudal sperm count and motility (Fig. 1). However, treatment of CIS-intoxicated rats with FSE caused a significant improvement in the weights of testes and epididymides as well as in the caudal sperm count and motility. A dose-dependent improvement was evident in these parameters. The concomitant treatment of high dose of FSE with CIS caused the highest protective effects in these parameters. Rats receiving FSE alone did not show any significant change in the weights of both testes and epididymides as well as in the quality of sperm indices. FSE-suppressed histopathological changes and proapoptotic effect of CIS Control rats showed normal testicular architecture with an orderly arrangement of germinal cells and Sertoli cells.
Table 1 In vitro antioxidant capacity of fenugreek seed extract (FSE)
Assay
FRAP assay TAC (lmol g 1)
FSE
17.16 0.42
ABTS assay
DPPH assay
TAC (lmol g 1)
IC50 (mg ml 1)
TAC (lmol g 1)
IC50 (mg ml 1)
b-Carotene assay IC50 (mg ml 1)
30.16 0.02
1.0 0.05
31.01 0.02
1.07 0.07
0.59 0.05
Total antioxidant activity of FSE is expressed as ascorbic acid equivalents (mmol g ments.
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of dry extract). Values are means SEM of three experi-
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Table 2 Amount of total phenolicsa and the qualitative–quantitative analysesb of the ethanol extract from fenugreek seed extract carried out using an HPLC-DAD Compound
Retention time (min)
Amount of compounds
Total phenolic content Gallic acid Ferulic acid Rutin
– 10.13 0.01 65.03 0.01 90.08 0.01
13.80 0.19 2.07 3.26
0.43 0.01 0.01 0.06
Results are expressed as mean SEM of three experiments. Total phenolic content was expressed as mg gallic acid equivalents g 1 dried extract. b The amount of compounds was expressed as mg g 1 of dried extract. a
CIS treatment induced testicular atrophy with degeneration of germ cells in seminiferous tubules (Fig. 2A). Disorganisation and shedding of germ cells into lumina were also observed. Animals pretreated with FSE showed less degeneration of seminiferous epithelium with shedding of germ cells in few tubules. To evaluate whether FSE protected testicular tissues through inhibiting CIS-induced apoptosis, germ cell apoptosis was assessed by TUNEL assay. Brown staining indicating TUNEL-positive nuclei was visible in seminiferous tubules of control and CIStreated animals. Testicular tissues exposed to CIS contained a large number of the TUNEL-positive germ cells in contrast to those treated with FSE, indicating the
Fig. 1 Inhibitory effect of FSE on CIS-induced depletion in testicular and epididymal weights as well as on caudal sperm count and motility. Values are expressed as mean SEM (n = 8). Significance was determined by one-way analysis of variance followed by Dunnett’s t-test: ***P < 0.001,**P < 0.01, *P < 0.05 versus control group; aP < 0.001, bP < 0.01, cP < 0.05 versus CIS group.
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Fig. 2 (A) Inhibitory effect of FSE on CIS-induced histopathological changes and pro-apoptotic effect in testicular tissue. (A) Representative images of haematoxylin- and eosin-stained section in the testes of all groups studied (magnifications = 1009). Testes from control (a) and FSE (b) groups exhibit typical feature of seminiferous epithelium while those from CIS (c) group displayed atrophy (*), vacuolisation and degeneration of germinal epithelium (arrow). Testes from CIS + FSE groups (d: low dose; e: medium dose; f: high dose) show nearly normal with some degeneration of seminiferous epithelium (arrow). (B) Representative images of TUNEL staining in the testicular section from all the groups studied. Magnifications = 4009. Testes from control (a) and FSE (b) groups exhibit small numbers of TUNEL-positive cells while those from CIS (c) group showed large number of TUNEL-positive cells (arrow). Testes from CIS + FSE groups (d–f) appear normal with small number of TUNEL-positive cells (arrow).
pro-apoptotic effect of CIS and anti-apoptotic effects of FSE (Fig. 2B and Table 3). FSE ameliorated CIS-induced oxidative stress in testicular tissues In CIS-intoxicated rats, testicular MDA levels and SOD and MPO activities were significantly (P < 0.001) increased and CAT activity was significantly (P < 0.001) decreased (Fig. 3), indicating the potent oxidative action of CIS on testicular tissues. Co-administration of FSE with CIS neutralised these abnormalities in MDA levels and activities of SOD, CAT and MPO, showing the effect 216
of FSE against CIS-induced oxidative stress in testes. These protective effects were dose dependent. The concomitant treatment of high dose of FSE with CIS caused the highest protective effects in these parameters. FSE ameliorated CIS-induced upregulation of iNOS and NF-jB-p65 on testicular tissues Inducible nitric oxide synthase expression is known to be regulated by the transcription factor NF-jB (Kundu & Surh, 2005). In an attempt to link the activation of induction during CIS treatment, we evaluated their expression level in all the groups. Within normal rat © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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Table 3 Effect of cisplatin (CIS) and/or fenugreek seed extract (FSE) treatment on the number of transferase-mediated triphosphate nick-end labelling (TUNEL)-, inducible nitric oxide synthase (iNOS)- and NF-jB-p65-positive cells in the testis of rat
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TUNEL a b c d e f
Control FSE CIS CIS + FSE (LD) CIS + FSE (MD) CIS + FSE (HD)
1.7 1.0 41.0 11.1 6.1 5.2
NF-jB-p65 0.33 0.58 0.59*** 1.07***,a 0.71***,a 0.42**,a
5.9 5.9 69.0 27.3 19.0 11.3
0.38 0.17 5.19*** 1.43***,a 1.58***,a 1.57**,a
iNOS 13.8 13.5 138.5 50.7 19.0 12.4
0.98 0.76 14.77*** 5.21***,a 0.58a 1.65a
The positive expression of cells in each section was calculated by counting the number of brown staining in four fields at 4009 magnifications then the number of positive cells/field. Values are expressed as mean SEM (n = 4). Significance was determined by one-way analysis of variance followed by Dunnett’s t-test: ***P < 0.001, **P < 0.01 versus control group; aP < 0.001, versus CIS group.
Fig. 3 Inhibitory effect of FSE on CIS-induced oxidative damage in rat testicular tissue. Values are expressed as mean SEM (n = 8). Significance was determined by one-way analysis of variance followed by Dunnett’s t-test: ***P < 0.001, *P < 0.05 versus control group; aP < 0.001, b P < 0.01, cP < 0.05 versus CIS group.
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testis, expression of iNOS and NF-jB was significantly low in the seminiferous tubules and intertubular tissue. There were a large number of iNOS- and NF-jB-p65positive cells in rats subjected to CIS alone (Fig. 4 and Table 3). FSE supplementation effectively inhibited the increase in the number of iNOS- and NF-jB-positive cells in the testicular tissues of CIS-treated rats. These results show that FSE suppresses CIS-induced increase in the expression of iNOS and NF-jB in testes. Discussion The human testis is a known target organ for injury resulting from exposure to CIS. Short- and long-term CIS (A)
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treatments result in germinal epithelial damage, sperm dysfunction and germ cell apoptosis in animals and humans (Cherry et al., 2004; Boekelheide, 2005). Our previous study has shown that CIS impaired rat testicular structure through inflicting oxidative stress and inducing cell apoptosis (Amin & Hamza, 2006; Amin et al., 2008). The present study further provides evidence that treatment with FSE could effectively alleviate CIS-induced testicular toxicity. In the present work, CIS treatment led to a significant decrease in testicular weight and epididymides weight with marked decrease in the caudal sperm count and motility compared with control animals. Administration of FSE restored the weights of testes and epididymides together with concomitant improvement in (B)
(a)
(b)
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(b)
(c)
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(c)
(d)
(e)
(f)
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Fig. 4 Inhibitory effect of FSE on CIS-induced upregulation in inducible nitric oxide synthase (iNOS) and NF-kB-p65 expression in testes (Magnifications = 1009). (A) Representative images of immunohistochemical iNOS-stained section in the testes of all groups studied. Testes from control (a) and FSE (b) groups exhibit low numbers of iNOS-positive cells, while those from CIS (c) group show large number of iNOS-positive cells (arrow). Testes from CIS + FSE groups show low number of iNOS-positive cells. (B) Representative images of immunohistochemical NF-kB-p65stained section in the testes of all groups studied. Testes from control (a) and FSE (b) groups exhibit low numbers of NF-kB-p65-positive cells, while those from CIS (c) group show large number of NF-kB-p65-positive cells. Testes from CIS + FSE groups (d: low dose; e: medium dose; f: high dose) show low number of NF-kB-p65-positive cells.
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the caudal sperm count and motility in CIS-intoxicated rats. These results were confirmed by histopathological examination which showed disorganisation, vacuolisation and degeneration of germinal epithelium in testes of rats treated with CIS alone. FSE treatment reduced these degenerative effects. Consistent with the results of histological changes, apoptosis was observed in the CIS-treated animal group. Germ cell apoptosis has been recently reported as a mechanism responsible for the damage to testes and sperm output (Boekelheide, 2005; ). CIS was reported to cause apoptosis to testicular germ cells and Sertoli cells (Seaman et al., 2003; Cherry et al., 2004). Using the TUNEL technique, apoptotic DNA fragmentation was determined in testicular tissue in the present work. Consistent with the previous studies, CIS treatment resulted in a significant increase in the testicular apoptosis, which was significantly reduced with CIS plus FSE. Accordingly, it is likely that DNA damage and apoptosis are important mechanisms for the germ cell death in the CIS-intoxicated testes and that FSE attenuates apoptotic DNA damage induced by CIS in testes. Reactive oxygen species (ROS) have been reported to play a role in CIS-induced testicular damage (Ilbey et al., 2009). They can induce lipid peroxidation and DNA fragmentation in spermatozoa and testis (Boekelheide, 2005). The negative changes observed in sperm quality in CIS-treated rats may be caused by the peroxidation of polyunsaturated fatty acids in sperm plasma membranes. Supporting this idea, an increase in lipid peroxidation product, MDA, in testes of CIS-treated rats was observed in the present work, indicating testicular oxidative damage. Additionally, our data demonstrated a significant increase in SOD activity and a significant decrease in CAT activity in testicular tissues of CIS-treated rats. This observed increased in testicular SOD activity may reflect an adaptive response to neutralise toxic superoxide radicals generated due to impairment of antioxidant status of the organ. Accordingly, an increase in the concentrations of hydrogen peroxide as end product of SOD activity in testis in response to CIS may impair the capacity of the testes to neutralise hydrogen peroxide, thereby causing excessive consumption and deactivation of CAT enzyme. These features together contribute to the sensitivity of testicular tissues to CIS-induced oxidative injury (Atessahin et al., 2006). On the other hand, the present work demonstrated that the concomitant treatment with FSE attenuated testicular MDA together with protected testicular SOD and CAT activities in rats exposed to CIS, illustrating the antioxidative actions of FSE against potent free radical-producing CIS agent. Consistent with these data, FSE treatment attenuated the testicular toxicity of the anticancer agent adriamycin in rats by decreasing lipid © 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
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peroxidation and increasing antioxidant levels (Sakr et al., 2012). This highlights the potential antioxidant ability of FSE as a protective agent against testicular oxidative injury induced by anticancer agents in rats. Further, the FSE was examined for its antiradical and reducing capacity in vitro and showed high antiradical and antioxidant activities. This antioxidant potential of FSE was confirmed by four different biochemical assays: scavenging activity on ABTS and DPPH radicals, reducing the ability by FRAP assay and lipid peroxidation inhibition of b-carotene-linoleate system. This antioxidant effect of FSE has been linked to certain compounds in its main constituents, flavonoids and saponins (Basu & Srichamroen, 2010), which can scavenge free radical and reduce the level of ROS. Polyphenols with their redox properties can play an important role in adsorbing and neutralising free radicals (Nichenametla et al., 2006). Our results show a good correlation between the antioxidant activity and the phenolic contents in the FSE. Rutin and ferulic acid were the main phenolic acids present in our plant extract. Previous results of HPLC suggested that the protective effect exhibited by the FSE is probably due to its phenolic contents, particularly the myricetin, chlorogenic acid, p-coumaric acid and ferulic acid in methanol extract (Jahan et al., 2013). In addition, FSE contains at least 90% steroid saponins (Petit et al., 1995). Our work showed that CIS treatment caused elevations of testicular MPO activity, a marker of neutrophil infiltration, inflammation and oxidative stress, indicating the presence of enhanced polymorphonuclear leucocyte recruitment in the inflamed tissues under the effect of oxidative stress. FSE attenuated the CIS-induced increase in testicular MPO activity, indicating the anti-inflammatory action of FSE. Additionally, the current study also showed significant increase in NF-jB and iNOS expression, as a result of CIS treatment. These results coincide with the previous finding that CIS caused a significant increase in iNOS and NF-jB-p65 expression in the testicular tissues (Ilbey et al., 2009; Amin et al., 2012). Oxidative stress is known to stimulate transcription factors, including NF-jB (Kundu & Surh, 2005). NF-jB functions as a link between oxidative damage and inflammation. Elevated levels of NF-jB and the associated inflammatory mediators such as iNOS are well-known biological markers of inflammatory responses (Chirino & PedrazaChaverri, 2009). It is well known that excessive NO production due to elevated iNOS causes cytotoxic effects and has the potential to induce germ cell apoptosis (Chirino & Pedraza-Chaverri, 2009; Jahan et al., 2013). It can be inferred that CIS-induced oxidative stress led to an increase in the expression of NF-jB, which in turn resulted in an enhanced expression of various genes such as iNOS leading to apoptosis in the testicular cells, while 219
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the administration of FSE to CIS-treated rats in this work reduced the increase in iNOS and NF-jB-p65 expression in the testicular tissues of CIS-treated rats. Thus, FSE could mediate its protective effect against testicular damage by downregulation of iNOS and NF-jB-p65. In summary, our study provides experimental evidence that concomitant treatment of FSE with CIS attenuated CIS-induced reproductive toxicity and testicular histopathological changes by antioxidants effects. We believe that the protective effects of FSE against CIS-reproductive toxicity is mediated primarily through both the inhibition of apoptosis and the abrogation of oxidative stress and iNOS and NF-jB inflammatory response associated with CIS treatment. These observations are of clinical importance as FSE could be a promising target to halt this serious testicular toxicity induced by CIS. References Abad-Garcia B, Berrueta LA, Lopez-Marquez DM, CrespoFerrer I, Gallo B, Vicente F (2007) Optimization and validation of a methodology based on solvent extraction and liquid chromatography for the simultaneous determination of several polyphenolic families in fruit juices. J Chromatogr A 1154:87–96. Aebi H (1984) Catalase. Methods Enzymol 105:121–126. Amin A, Hamza A (2006) Effects of ginger and roselle on cisplatin induced reproductive toxicity in rats. Asian J Androl 8:607–612. Amin A, Hamza A, Kambal A, Daoud S (2008) Herbal extracts counteract cisplatin-mediated cell death in rat testis. Asian J Androl 10:291–297. Amin A, Abraham C, Hamza AA, Abdalla ZA, Al-Shamsi SB, Harethi SS, Daoud S (2012) A standardized extract of Ginkgo biloba neutralizes cisplatin-mediated reproductive toxicity in rats. J Biomed Biotechnol 12:1–11. Atessahin A, Karahan I, Turk G, Giir S, Yilmaz S, Ceribasi AO (2006) Protective role of lycopene on cisplatin-induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod Toxicol 21:42–47. Basu TK, Srichamroen A (2010) Chapter 28-health benefits of fenugreek (Trigonella foenum-graecum leguminosse). In: Bioactive Food in Promoting Health Fruits and Vegetables. Basu TK, Srichamroen A (ed.). Academic Press, Harcourt Brace & Company, San Diego, pp 425–435. Belguith-Hadriche O, Bouaziz M, Jamoussi K, Et Feki A, Sayadi S, Makni-Ayedi F (2010) Lipid-lowering and antioxidant effects of an ethyl acetate extract of fenugreek seeds in high-cholesterol-fed rats. J Agric Food Chem 58:2116–2122. Bhatia K, Kaur M, Atif F, Ali M, Rehman H, Rahman S, Raisuddin S (2006) Aqueous extract of Trigonella foenumgraecum L. ameliorates additive urotoxicity of buthionine
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sulfoximine and cyclophosphamide in mice. Food Chem Toxicol 44:1744–1750. Boekelheide K (2005) Damage to fertility by cancer and its treatments. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monogr 34:6–8. Canadian Council on Animal Care (1993) Guide to the Care and Use of Experimental Animals. Olfert ED, Cross BM, Mc William AA (eds), vol. 1, CCAC, Ottawa, Canada. Cherry SM, Hunt PA, Hassold TJ (2004) Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation. Mutat Res 564:115–128. Chirino YI, Pedraza-Chaverri J (2009) Role of oxidative and nitrosative stress in cisplatin-induced nephrotoxicity. Exp Toxicol Pathol 61:223–242. Duke JA, Bogenschutz-Godwin MJ, duCellier J, Duke PK (2002) Fenugreek (Trigonella foenum-graecum L.). In: Handbook of Medicinal Herbs. Duke JA (ed.). CRC press, Boca Raton, pp 296–297. Erel O (2004) A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 37:277–285. Hillegass LM, Griswold DE, Brickson B, Albrightson-Winslow C (1990) Assessment of myloperoxidase activity in whole rat kidney. J Pharmacol Meth 24:285–295. Ilbey YO, Ozbek E, Cekmen M, Simsek A, Otunctemur A, Somay A (2009) Protective effect of curcumin in cisplatininduced oxidative injury in rat testis: mitogeb-activated protein kinase and nuclear factor-Kappa B signaling pathways. Hum Reprod 24:1717–1725. Jahan N, Rahman K, Ali S, Asi MR (2013) Phenolic acid and flavonol contents of gemmo-modified and native extracts of some indigenous medicinal plants. Pak J Bot 45:1515–1519. Kaviarasan S, Anuradha CV (2007) Fenugreek (Trigonella foenum graecum) seed polyphenols protect liver from alcohol toxicity: a role on hepatic detoxification system and apoptosis. Pharmazie 62:299–304. Kenjale R, Shah R, Sathaye S (2008) Effect of Chlorophytum borivilianum on sexual behaviour and sperm count in male rats. Phytother Res 22:796–801. Kundu JK, Surh YJ (2005) Breaking the relay in deregulated cellular signal transduction as a rationale for chemoprevention with anti-inflammatory phytochemicals. Mutat Res 591:123–146. Lim YY, Quah EPL (2007) Antioxidant properties of different cultivars of Portulaca oleracea. Food Chem 103:734–740. Nandi A, Chatterjee IB (1988) Assay of superoxide dismutase activity in animal tissues. J Biosci 13:305–315. Nenadis N, Lazaridou O, Tsimidou MZ (2007) Use of reference compounds in antioxidant activity assessment. J Agric Food Chem 55:5452–5460.
© 2015 Blackwell Verlag GmbH Andrologia 2016, 48, 211–221
A. A. Hamza et al.
Nichenametla SN, Taruscio TG, Barney DL, Exon JH (2006) A review of the effects and mechanisms of polyphenolics in cancer. Crit Rev Food Sci Nutr 46:161–183. Peterson GL (1977) A simplification of the protein assay method of lowry et al. which is more generally applicable. Anal Biochem 83:346–356. Petit PR, Sauvaire YD, Hillaire-Buys DM, Leconte OM, Baissac YG, Ponsin GR, Ribes GR (1995) Steroid saponins from fenugreek seeds: extraction, purification, and pharmacological investigation on feeding behaviour and plasma cholesterol. Steroids 60:674–680. Sakr SA, El-Shenawy SM, Al-Shabka AM (2012) Aqueous fenugreek seed extract ameliorates adriamycin-induced cytotoxicity and testicular alterations in albino rats. Reprod Sci 19:70–80.
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Seaman F, Sawhney P, Giammona CJ, Richburg JH (2003) Cisplatin-induced pulse of germ cell apoptosis precedes long-term elevated apoptotic rates in C57/BL/6 mouse testis. Apoptosis 8:101–108. Singleton VL, Orthofer R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of folin–ciocalteu, reagent. In: Methods in Enzymology. Oxidants and Antioxidants, Part A., Vol 299. Packer L (ed.). Academic Press, Harcourt Brace & Company, San Diego, London, New York, Tokyo, pp 152–178. Uchiyama M, Mihara M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278.
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