Archivio Italiano di Urologia e Andrologia 2017; 89, 3192 ORIGINAL PAPER A randomized study to assess the efficacy of herbal product to prevent cisplatin-induced nephrotoxicity in a rat model Eyup Veli Kucuk 1, Ahmet Bindayi 2, Meral Mese 3, Funda Gulcu Bulmus 3, Ergun Parmaksız 3, Ali Cihangir Cetinel 4, Zerrin Bicik Bahcebasi 3, Kemal Sarica 4 1 Umraniye Training and Research Hospital Urology Clinic, Istanbul, Turkey; 2 Bahcesehir University Department of Urology, Istanbul, Turkey; 3 Dr. Lütfi Kirdar Training and Research Hospital Nephrology Clinic, Istanbul, Turkey; 4 Dr. Lütfi Kirdar Training and Research Hospital Urology Clinic, Istanbul, Turkey. Objectives: This study aimed to investigate the protective effect and antioxidant activity of an herbal product that made from multiple plants in a rat model of kidney dysfunction induced by intraperitoneal cisplatin. Materials and methods: Twenty-four rats were divided into four different groups namely: Group 1 - control healthy ani- mals without any specific medication, Group 2 - Herbal product only 5 mg/kg, Group 3 - cisplatin only and Group 4 - Herbal product 5 mg/kg + cisplatin. Results: Evaluation of our findings demonstrated a significant (p = 0.017) reduction in Catalase activities and a significant increase (p = 0.001) in renal tissue Malondialdehyde levels in cisplatin- treated rats when compared with the control group. Also, Glutathion and Glutathione peroxidase content revealed significant (p = 0.031) reduction in renal tissues of cisplatin- treated rats compared with the control group. Pre-treatment of rats with the herbal product ameliorated these cisplatin- induced changes of the antioxidant enzymes. No statistically significant changes were demonstrated in Superoxide dismutase activities in the tissue specimens of any group. Conclusions: This potent antioxidant herbal medicine was found to have potential antioxidant activity, which may in turn to be effective in the protection of kidney tissue resulting from cisplatin application. Therefore, much attention should be given to the possible role of natural dietary antioxidants for protect- ing the kidney. KEY WORDS: Animal model; Antioxidants; Cisplatin; Herbal medicine; Nephrotoxicity. Submitted 30 May 2017; Accepted 2 July 2017 Summary No conflict of interest declared. ovary (1). Of these side effects, nephrotoxicity is the most well-known, established and clinically important toxicity of this drug (2). Over the last few decades, different studies were made to investigate the pathophysiological basis of cisplatin- induced nephrotoxicity and how to protect kidney from this toxicity. These studies demonstrated number of mechanisms including oxidative stress, DNA adducts, inflammation, mitochondrial dysfunction, and direct cytotoxicity to the tubular epithelial cells (2-4). Also liter- ature revealed that Cisplatin® application might cause renal vasoconstriction that reduces blood flow, causing ischemic damage to the kidney and deteriorate the glomerular filtration rate. These alterations eventually trigger ischemia-induced oxidative stress, a well-known phenomenon that ultimately results in renal tubular cell injury and death. Cisplatin® administration in a mice model resulted in marked renal failure characterized by a significant increase in serum urea and creatinine levels due to the formation of oxidative stress as indicated by increased lipid peroxidation and decreased levels of glu- tathione (GSH), glutathione peroxidase (GSHPx), super- oxide dismutase (SOD) and catalase in renal tissues (5, 6). Being aware of the well-known side effects of Cisplatin® on the functional and morphological integrity of the kid- neys, physicians began to look for protective agents to pre- vent or at least limit the extent of the renal toxic effects of Cisplatin® based chemotherapy. Related to this issue, eval- uation of literature data demonstrated that AsAG or TMG, lutein, ascorbic acid and alpha tocopherol may reduce cis- platin-induced renal toxicity either by limiting the extent of renal functional deterioration or increasing the antioxi- dant capacity of the kidneys (3, 4, 7, 8). On the other hand, in addition to the use of these med- ical agents, recent data demonstrated that phytotherapy might prevent toxic effects of certain medications caus- ing morphologic as well as functional changes in differ- ent organ systems (3, 4, 8-10). In this study, we investi- gate the potential renoprotective effects of a herbal med- ication (Tutukon®) on cisplatin-induced oxidative stress- related nephrotoxicity in a rat model. DOI: 10.4081/aiua.2017.3.192 INTRODUCTION Despite their well-established curative effects in certain solid organ tumors, chemotherapeutic agents may exhib- it dose dependent toxic effects during the course of chemotherapy on certain organ systems, a reality that should inevitably be taken into account by all physi- cians. Cisplatin® (cis-diamminedichloroplatinum II) is one of the most widely used chemotherapeutic drugs for the treatment of various solid tumors, including those of the breast, head, neck, lung, testis, bladder and Kucuk_Stesura Seveso 28/09/17 10:17 Pagina 192 193Archivio Italiano di Urologia e Andrologia 2017; 89, 3 Prevention of cisplatin nephrotoxicity METHODS Animals This study was made with 24 male Wistar white rats, each weighing 250-300 g. Ethical committee approval (No: 94-14/2013) was obtained from the animal labora- tory of the Pendik Animal Research Laboratory (Istanbul, Turkey) and all procedures performed in studies involv- ing animals were in accordance with the ethical stan- dards of the institution or practice at which the studies were conducted. All animals were fed standard chow and kept under normal room conditions at a constant tem- perature (25°C) under a 12 hours light/dark cycle. Rats underwent a complete physical examination, biochemi- cal evaluation including blood and urine analyses and stool examination. We estimated that total of 24 subjects would be needed to detect difference among groups with α of 0.05 and a (1-β) of 80%. Rats randomly divided into four groups (each with n = 6): Group 1 (control group) received only standard rat chow and distilled drinking water without any specific medication. Group 2 (Tutukon® group) received an herbal product (Tutukon®) given via a feed- ing catheter (20 ml/kg). Group 3 (Cisplatin group) received Cisplatin® only (7.5 mg/kg once i.p.). Group 4 (Tutukon® + Cisplatin® group) received a combined med- ication (20 ml/kg of Tutukon® for seven days + 7.5 mg/kg Cisplatin® i.p. after seven days for once). Control group animals received only distilled drinking water during all study phases. After two weeks bilateral nephrectomy was performed with bilateral flank incision. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Main chemicals and drugs Cisplatin® was used to induce nephrotoxicity in rats according to the method of Prabhu et al. (11). Applied dose was 7.5 mg/kg rat body weight (intraperitoneal). Plant-based herbal medication (Tutukon®) fixed-dose combination. Dosage form: hydrolate, bottles of 600 ml, 45 ml three times/daily. Ingredients: Essential oils, flavonoids-quercetin, polysaccharides, rosemarinic acid, boldin, flavonglicozides. Composition (per 100 ml of solution): Enguisetumarvensis 570 mg, Sper gularia - rubra 330 mg, Peumusboldus 280 mg, Opuntiaficusindica 170 mg, Sideritisan gu - stifolia 170 mg, Rozmarinusofficinales 170 mg, Cyno dondaktylon 170 mg, Melissa officinalis 170 mg. Measurement of Malondialdehyde (MDA) and glutathione levels, Superoxide dismutase (SOD) and catalase (CAT) activities in renal homogenate The homogenization of tissues was car- ried out in a Teflon-glass homogenizer with a buffer containing 1.15% KCl to obtain 1/10 (w/v) whole homogenate. MDA levels were directly measured in the homogenates. The homogenates were centrifuged for 30 min at 25000 g at +4°C to determine GSH-Px and CAT activities. The obtained supernatants were centrifuged again at 25000 g, +4°C for 30 min to determine SOD activities. The MDA concentrations of tissue homogenates were measured according to a modified method from Ohkawa et al. (12) based on the reaction with TBA and the results were expressed as nmol/g protein. The GSH-Px activities were determined according to the method of Beutler, (13) which records the disappearance of NADPH at 340 nm, and results were expressed as U/g protein. The CAT activities were determined by measuring the decom- position of hydrogen peroxide at 240 nm, according to the method of Aebi (14). The results were expressed as k/g protein, where k is the first-order rate constant. SOD activities were determined using the method of Sun et al. (15) and the results were expressed as U/g protein. Tissue GSH concentrations were measured by an assay using the dithionitrobenzoic acid recycling method described by Elman (16) and expressed as nmol/mg protein. Protein concentrations were meas- ured according to Lowry et al. (17). Statistical analysis For statistical analysis of the obtained data NCSS (Number Cruncher Statistical System) 2007&PASS (Power Analysis and Sample Size) 2008 Statistical Software (Utah, USA) program was used. The data was expressed in mean +/- standard deviation. Mann-Whitney U and Kruskal-Wallis tests were used to compare the parameters between two or more groups that don't fit to normal distribution. A P value of < 0.05 was considered as significant. RESULTS Evaluation of the tissue enzyme levels demonstrated the following findings (Figure 1). Figure 1. MDA, CAT, GSH-Px and kidney GSH levels according to the groups. Kucuk_Stesura Seveso 28/09/17 10:17 Pagina 193 Archivio Italiano di Urologia e Andrologia 2017; 89, 3 E. Veli Kucuk, A. Bindayi, M. Mese, F. Gulcu Bulmus, E. Parmaksız, A. Cihangir Cetinel, Z. Bicik Bahcebasi, K. Sarica 194 MDA: There were statistically significant differences among groups regarding levels of MDA (p < 0.001) (Table 1). There was a significant increase in renal tissue MDA levels in cisplatin-treated rats (Cisplatin® only or Tutukon® + Cisplatin®) compared with the control and Tutukon® groups (p < 0.05). There was no statistically sig- nificant difference with respect to MDA levels between the Tutukon® and control groups. Lastly, although not statistically significant, renal tissue MDA levels of the Tutukon + Cisplatin® group were found to be comparably lower than the Cisplatin® group (p = 0.055) (Table 2). SOD: There were no statistically significant differences among groups regarding SOD activities (p > 0.05) (Table 1 and 2). CAT: CAT activity of the cisplatin-treated rats (Cisplatin® only or Tutukon® + Cisplatin®) was lower than the other groups (Figure 1). The combined medication group showed significantly lower CAT activity than the control and Tutukon® groups (p = 0.025, p = 0.016, respective- ly). According to comparative studies, CAT activity of the Cisplatin® group was significantly lower than the control group (p = 0.037). It was also found to be lower but not statistically significant than the Tutukon® group (p = 0.055) (Table 2). GSH-Px: There was statistically significant difference of GSH-Px levels among groups (p = 0.031) (Table 1). According to dual-comparison of the groups, GSHPx levels in the Cisplatin® group showed significant reduc- tion compared with Tutukon® and combined medication groups (p = 0.037, p = 0.016, respectively) (Table 2). GSH: There were statistically significant differences among groups for GSH levels (p = 0.007) (Table 1). According to dual comparison results, GSH levels of the Cisplatin® group were significantly lower than the Tutukon® and combined medication groups (p = 0.010, p = 0.037, respectively) (Table 2). DISCUSSION Cisplatin® is a chemotherapeutic agent widely used for the treatment of various solid cancers. However, nephro- toxicity is one of the major side effects of Cisplatin® and published data have shown that an estimated 20% of patients receiving high-dose Cisplatin® suffer severe renal dysfunction and approximately one third of patients may experience kidney injury following initial treatment (18, 19). Renal tubular injury and cell death are the most prominent pathologic findings originating from reduced blood flow (ischemia) induced oxidative stress in renal tubular cells following the application of this agent. In literature, several mechanisms were documented to be important in renal tubular injury. Yonazewa et al. showed that organic cation transporter 2 (OCT2) mediates the entry of Cisplatin® into the renal tubular cells and stimu- late the sensitivity of Cisplatin® in these cells. Cisplatin® reduces the activity of mitochondrial respiratory com- plexes, resulting in reactive oxygen species (ROS) gener- ation (20). The other mechanism of generation of reac- tive oxygen species is depletion of endogenous antioxi- dant, glutathione. Cisplatin® may damage the kidney by depletion of critical sulfhydryl centers, including GSH inside the cells, and may provoke damage to the cell by generating a cascade of lipid membrane peroxidation, mitochondrial dysfunction and DNA injury which reduces the internal antioxidant storages (21, 22). Likewise in our study we found that Cisplatin® treatment decreased GSH, GSHPx levels, CAT activites and increased MDA levels. Thus, all these findings clearly have shown that cisplatin- induced generation of ROS, cytokines and chemokines are directly related to its cytotoxicity. ROS produce by the xanthine-xanthine oxidase system, mitochondria, and NADPH oxidase in cells. Following treatment with Cisplatin®, ROS that produced throughout these systems, are implicated in the patho- genesis of acute renal injury (22). Related to this subject, in their original study both Maliakel and Atasayar et al. showed that administration of AsAG or TMG markedly reduced the cisplatin- induced higher plasma crea- tinine and urea levels and counteracted the deleterious effects of formed oxidative stress markers by protecting the renal tissue from the cis- platin-induced lipid peroxi- dation (3, 7). In another study, Sindhu and Kuttan applied lutein, a non-toxic carotenoid with strong antioxidant activity, to reduce cisplatin-induced renal damage in mice. As shown by the reduction of serum urea and creatinine levels, nephrotoxicity, origi- Table 1. The evaluation of MDA, SOD, CAT, GSH-Px and kidney GSH. MDA SOD CAT GSH-Px Kidney GSH (nmol/g protein) (U/g protein) (k/g protein) (U/g protein) (nmol/mg protein) Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Control 18.62 ± 2.21 50.87 ± 9.22 30.22 ± 11.3 30.21 ± 8.87 7.76 ± 1.56 Tutukon® 19.89 ± 3.73 48.55 ± 10.56 26.90 ± 4.99 32.02 ± 5.79 6.68 ± 1.49 Cisplatin® 35.47 ± 7.04 38.75 ± 16.62 14.86 ± 10.63 22.14 ± 7.95 4.19 ± 0.82 Tutukon® + Cisplatin® 27.10 ± 4.95 47.19 ± 18.59 16.30 ± 4.85 35.45 ± 6.02 5.85 ± 1.41 ap 0.001** 0.667 0.017* 0.031* 0.007** aKruskal -Wallis Test; **p < 0.01; *p < 0.05. Table 2. Dual comparison of groups. MDA SOD CAT GSH-Px Kidney GSH Group 1 vs Group 2b 0.749 0.749 0.522 0.631 0.262 Group 1 vs Group 3b 0.004** 0.200 0.037* 0.109 0.006** Group 1 vs Group 4b 0.006** 0.873 0.025* 0.262 0.055 Group 2 vs Group 3b 0.004** 0.337 0.055 0.037* 0.010* Group 2 vs Group 4b 0.037* 0.873 0.016* 0.337 0.423 Group 3 vs Group 4b 0.055 0.522 0.109 0.016* 0.037* bMann Whitney U Test; **p < 0.01; *p < 0.05. Group 1: Control; Group 2: Tutukon®; Group 3: Cisplatin®; Group 4: Tutukon® + Cisplatin® (combined medication). Kucuk_Stesura Seveso 28/09/17 10:17 Pagina 194 195Archivio Italiano di Urologia e Andrologia 2017; 89, 3 Prevention of cisplatin nephrotoxicity nating from reduced activity of the antioxidant enzymes in the kidney (SOD, as well as CAT) and increased MDA levels, was reduced by lutein treatment (4). The results of this study showed that lutein might effectively protect the kidneys of mice treated with Cisplatin®, which was also supported by the histopathologic evaluation of the kidney tissues of the treated animals. Last but not least, Ajith et al. gave 250 mg/kg or 500 mg/kg vitamins (ascor- bic acid and alpha tocopherol) to their subjects to pro- tect from the nephrotoxicty induced by Cisplatin® and they found that 500 mg/kg vitamins significantly pro- tected the kidneys. However, the protective effect of vita- mins from the cisplatin induced decline of activities of renal antioxidant enzymes such as SOD, CAT, GSHPx noted only in 500 mg/kg dosage group. Additionally, Authors have observed that both vitamins at dosages of 250 and 500 mg/kg could increase the concentration of reduced GSH and limit the extent of cisplatin-induced lipid peroxidation (8). Recent data showed that herbal medicine might prevent some toxic effects of certain medications (3, 4, 8-10). Phytotherapy on this aspect may be applied in a comple- mentary fashion to ameliorate these effects. Majority of these phytotherapeutic agents have diuretic, anti-inflam- matory, antioxidant and vasodilating effects. The active ingredients like essential oils, flavonoids, saponins, xan- thine derivatives and glycosides were found responsible of these protective effects (23-25). In a study, administration of plant extract mixtures pro- duced improvements in biochemical, histopathological and cytogenetic parameters (26). Other similar studies focused on the possible protective effects of plant extracts with certain ingredients demon- strating the protective effects on both the kidney as well as their specific protective effects on other pathologies in different organ systems (27, 28). Antioxidant, diuretic and antidiabetic effects of such agents also might be effec- tive in limiting the toxic effects induced by Cisplatin® administration. Taking into account the established effects of antioxi- dants as well as anti-inflammatory agents in the preven- tion of ischemia-induced injury in renal tubular epitheli- um, we aimed to evaluate the possible limitation of the extent of oxidative stress resulting from cisplatin- induced toxicity by using a potential antioxidant and anti-inflammatory agent (Tutukon®) in a rat model. Tutukon® (Grand Medical, Spain) is a medication com- posed of different herbal ingredients demonstrating cer- tain biological effects in tissues. Among these effects, the antioxidant potential is one of the most important char- acteristics of this drug due to its active ingredients. Among the eight different ingredients of Tutukon®, alka- loids rosmarinic acid, flavonoids, apigenin, luteolin, ekvizetonin saponin, and essential oils are well known for their antioxidant as well as anti-inflammatory effects shown in different studies (23, 25, 29). Regarding the mechanism of action, studies showed that these active ingredients may reduce the permeability of kidney capil- laries, dilate kidney blood vessels and ureters, restore the tubular epithelial function, induce an osmotic effect, inhibit synthesis and activation of inflammatory media- tors and thus prevent inflammatory alterations (24, 30). Our current findings clearly showed that pre-treatment with this herbal product also might significant reduction in MDA levels and significant elevation of CAT activities and GSH levels of the renal tissues in cisplatin-treated rats. Evaluation of the antioxidant enzyme (Catalase and GSHPx) content revealed significant reduction in renal tissues of cisplatin-treated rats compared with the con- trol group as well as in the groups receiving Tutukon®. These findings may provide evidence of the antioxidant effect of Tutukon® against cisplatin-induced oxidative stress and lipid peroxidation. Parallel to the data report- ed in the literature so far, we found that reduction in the activity of antioxidant enzyme (CAT), increased lipid peroxidation (MDA) and depletion of GSH in renal tis- sues were implicated in the pathogenesis of Cisplatin® nephrotoxicity. 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Antispasmodic effects of an alkaloid extracted from Phyllanthus sellowianus: a comparative study with papaverine. Braz J Med Biol Res. 1984; 17:313-21. Correspondence Eyup Veli Kucuk, MD eyupveli@gmail.com Umraniye Training and Research Hospital Urology Clinic, Istanbul, Turkey Ahmet Bindayi, MD, FEBU ahmetbindayi@gmail.com Bahcesehir University Department of Urology, Istanbul, Turkey Meral Mese, MD mesemeral@gmail.com Funda Gulcu Bulmus, MD fundagulcu@yahoo.com.tr Ergun Parmaksız, MD drergnprmksz@hotmail.com Zerrin Bicik Bahcebasi, MD zerrinbicik@yahoo.com Dr. Lütfi Kirdar Training and Research Hospital Nephrology Clinic, Istanbul, Turkey Ali Cihangir Cetinel, MD cihangircetinel@gmail.com Kemal Sarica, MD saricakemal@gmail.com Dr. Lütfi Kirdar Training and Research Hospital Urology Clinic, Istanbul, Turkey Kucuk_Stesura Seveso 28/09/17 10:17 Pagina 196