Beneficial Effects of Hesperidin against Cisplatin-Induced Nephrotoxicity and Oxidative Stress in Rats

Cisplatin has been frequently used for treatment of wide variety of tumors. The use of cisplatin is associated with severe cytotoxicity such as nephrotoxicity, hepatotoxicity and spermiotoxicity which radically limits its clinical use. The present study aimed to investigate the possible protective effects of multiple doses of hesperidin against cisplatin-induced nephrotoxicity induced by single i.p injection of cisplatin (7.5 mg/kg). Hesperidin was given to rats at two different doses (100 and 200 mg/kg p.o) for 7 days starting one day before cisplatin injection. Blood samples were collected for determination of serum creatinine and Blood Urea Nitrogen (BUN) levels. Kidneys were used for the determination of Malondialdehyde (MDA), Glutathione (GSH) and total nitrate and nitrite contents. Liver samples were also used for histopathological examination. Results showed that hesperidin significantly reduced cisplatin-induced elevations in serum creatinine and BUN levels. It also significantly reduced kidney MDA and NO content and elevated GSH content. In conclusion, hesperidin greatly protected kidney against cisplatin-induced toxicity in a dose-dependent manner.


INTRODUCTION
Cisplatin (cis-diammine-dichloro-platinum) has been frequently considered as the first choice chemotherapeutic agent for treatment of a wide variety of solid tumors, including ovarian, testicular, bladder, head and neck, esophageal and small cell lung cancers (Weijl et al., 1997;Previati et al., 2006;Shona et al., 2012).About 70-80% of patients respond to platinum treatment.However, such an initial effect is not robust and results in a 5-year patient survival of only 15-20%, as tumors become resistant to therapy (Siddik, 2003).The relapse of the disease and the emergence of resistance in initially responsive tumors occur with 18-24 months (Siddik, 2003;Shi et al., 2007).The dose scale necessary to overcome even a small increase in cellular resistance can lead to severe cytotoxicity in normal cells such as nephrotoxicity, hepatotoxicity and spermiotoxicity (Zicca et al., 2002;Atessahin et al., 2006;Palipoch et al., 2014) which radically limits the clinical usefulness of cisplatin-based therapy.
The mechanism of cisplatin-induced nephrotoxicity has been clearly reported through multiple mechanisms including hypoxia, the generation of free radicals, inflammation and apoptosis with an increase in the proapoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2 (Tsuruya et al., 2003).
Studies showed that the generation of Reactive Oxygen Species (ROS) such as hydroxyl radical and superoxide anion are involved in the mechanism of cisplatin toxicity (Hussein et al., 2012) which causes an elevation in Lipid Peroxidation (LPO), reduction in levels of protein bound sulfhydryl groups and glutathione (Pratibha et al., 2006;Iraz et al., 2006).
Knowing that cisplatin chemotherapy induces a fall in plasma antioxidant levels, which may reflect a failure of the antioxidant defense mechanism against the oxidative damage induced (Weijl et al.,1998), consequently the aim of the present study is that combination treatment of cisplatin and a naturally occurring antioxidant flavonoid hesperidin may be a useful strategy to protect against cisplatin-induced nephrotoxicity.
Hesperidin, flavonoid found in vegetables and fruits (Justesen et al., 1998), has many useful effects such as antiallergic, antioxidant and anti-inflammatory (Garg et al., 2001).It has also anticarcinogenic effects in different variety of tumors (Lee et al., 2010).Hesperidin was proved to have antioxidant and free radicle scavenging properties (Fraga et al., 1987).

Animals:
Adult male Wistar rats (120-150 g) obtained from the National Research Centre (Giza, Egypt) were used in the current study.They were housed 10 per cage and maintained at 25±2ºC with free access to water and food, under a 12/12 h light-dark cycle.All procedures in this study were carried out according to guidelines of Ethics Committee of Faculty of Pharmacy, Cairo University.
Experimental design: Rats were divided into 7 groups, each of 6-8 rats.It was proved that intra-peritoneal injection of cisplatin at a dose of 7.5 mg/kg causes induction of hepatptoxicity (Mansour et al., 2006): Groups 1-3 received a single i.p. saline on second day.Groups 4-7 received a single dose of cisplatin (7.5 mg/kg i.p) on second day 1 hour after vehicle or drug administration.
At the end of the experiment, rats were fasted overnight, blood was collected and serum was separated by centrifugation at 3000 r.p.m. and used for biochemical estimations including: • Serum creatinine (Cr) and Blood Urea Nitrogen (BUN) using colorimetric assay kits (Diamond Diagnostics, Egypt) For tissue biomarkers and histopathological estimations; animals were sacrificed then kidneys were dissected out, washed with cold normal saline and dried between two filter papers.
A portion of each kidney tissue was homogenized and the homogenates were centrifuged at 1000×g for 15 min and the obtained supernatants were used for the estimation of tissue biomarkers namely, reduced Glutathione (GSH) according to the method described by Beutler et al. (1963) and Arab et al. (2014) and expressed as mg%, Thiobarbituric acid Reactive Substances (TBARS) content, estimated as Malondialdehyde (MDA) according to the method described by Mihara and Uchiyama (1978) and Mehany et al. (2013) and expressed as nmol/ml and total nitrate/nitrite (NOx) content according to the method described by Miranda et al. (2001).
Histopathological examination: Another portion of each kidney tissue was preserved in formalin solution in saline for a time sufficient for hardening of tissue to be sectioned for histopathological examination.Autopsy samples were taken from the liver and kidney of rats in different groups and fixed in 10% formolsaline for twenty four hours.Washing was done in tap water then serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used for dehydration.Specimens were cleared in xylene and embedded in paraffin at 56 degree in hot air oven for twenty four hours.Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns thickness by slidgemicrotome.The obtained tissue sections were collected on glass slides, deparaffinized, stained by hematoxylin and eosin stain for routine examination then examination was done through the light electric microscope (Bancroft et al., 1996).

Statistical analysis:
The values of the measured parameters were presented as mean ± SEM.Comparisons between different treatments were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test.Differences were considered statistically significant at p<0.05.

Effect of hesperidin on cisplatin-induced elevations in serum Cr and BUN levels:
Cisplatin significantly increased serum Cr and BUN levels when compared to normal control group.Oral administration of silymarin 100 mg/kg and hesperidin 200 mg/kg significantly decreased cisplatin-induced increase in serum Cr and BUN levels when compared to cisplatin control group.Administration of hesperidin 100 mg/kg significantly reduced elevated serum Cr but did not significantly affect elevated BUN level.No significant difference was observed in serum Cr and BUN levels between normal control and silymarin or hesperidin control groups (Fig. 1 and 2).

Effect of hesperidin on renal lipid peroxides content:
Cisplatin significantly elevated renal MDA content as compared to normal control group.Silymarin 100 mg/kg p.o and hesperidin 200 mg/kg p.o significantly decreased cisplatin-induced elevation in renal MDA content when compared to cisplatin control group.Hesperidin 100 mg/kg p.o did not significantly affect elevated renal MDA content.No significant difference was observed in renal MDA content in silymarin or hesperidin control groups (Fig. 3).Effect of hesperidin on renal reduced glutathione content: Cisplatin significantly reduced renal GSH content as compared to normal control group.Oral administration of silymarin 100 mg/kg and hesperidin 100 or 200 mg/kg significantly increased cisplatininduced reduction in GSH content when compared to cisplatin control group.No significant difference was observed in renal GSH content between normal control and silymarin or hesperidin control groups (Fig. 4).

Effect of hesperidin on renal nitrate/nitrite (NO) content:
Cisplatin significantly elevated renal NO content in cisplatin control group as compared to normal control group.Silymarin 100 mg/kg p.o and hesperidin 200 mg/kg significantly decreased cisplatininduced elevation in renal NO content when compared to cisplatin control group.No significant difference was observed in renal NO content between normal control and silymarin or hesperidin control groups (Fig. 5).
Histopathology: There was no histopathological alteration and the normal histological structure of the glomeruli and tubules at the cortex and the tubules at the corticomedullary portion were recorded in Fig. 6A.Silymarin 100 mg/kg and hesperidin 200 mg/kg on normal rats showed normal structure of the

DISCUSSION
Results of the present study states that cisplatin significantly produced nephrotoxicity in rats.Nephrotoxicity was proved by significant elevations in serum levels of creatinine and BUN as compared to normal control group.Cisplatin administration to rats showed histopathological changes in kidney including coagulation necrosis was noticed in most of the tubules associated with congestion in the glomerular tuft and cystic dilatation as well as cast formation in the tubules of both cortical and corticomedullary portions.These findings are similar to those obtained by previous investigators (Prabhu et al., 2013;Sahu et al., 2013).Abdelmeguid et al. (2010b) showed that cisplatin (5 mg/kg, i.p.), in rats caused significant deterioration of renal corpuscle structure and increased tubular necrosis after 2 weeks.Also Chirino et al. (2004) showed that cisplatin treatment induced mesangial cells contraction.
It was proved that cisplatin exerts its anticancer effect through induction of free radicles such as peroxyl radicals, superoxide radicles, hydroxy radicals and singlet oxygen, which cause kidney damage at the same time (Karadeniz et al., 2011).These free radicals attack the highly unsaturated fatty acids of the cell membrane inducing lipid peroxidation which is considered the key process in several pathological events (Weijl et al., 1997;Schinella et al., 2002).
This action of cisplatin is confirmed in the present study by significant increase in lipid peroxidation, measured as MDA level, as compared to normal control rats.Injection of cisplatin caused an increase in kidney lipid peroxides content and a reduction in the antioxidant enzymes activities which protect against tissues lipid peroxidation (Cayir et al., 2009;Goldstein and Mayor, 1983).Several cellular pathways have been suggested to contribute to induction of oxidative stress and lipid peroxidatin.Safirstein (1999) explained that the main targets of cisplatin in kidney are proximal straight and distal convoluted tubules, where it accumulates and encourages cellular damage by multiple mechanisms such as oxidative stress, DNA damage and apoptosis (Karadeniz et al., 2011).
Moreover, the decreased activities of enzymatic antioxidants caused reduction in scavenging of ROS which in turn leads to oxidative stress (Palipoch et al., 2014).This was shown in the present results by the significant reduction in tissue levels of GSH and similar results were obtained by previous studies (Mansour et al., 2006;Sahu et al., 2013).
The present work reveals that cisplatin significantly increased kidney levels of NO.Excessive production of NO causes vasodilatation and hypotension leading to organ hypoperfusion, edema and organ dysfunction.NO can interact with ROS to form peroxynitrite, which is a powerful oxidant and cytotoxic agent and may play an important role in the cellular damage associated with the overproduction of NO (Ahmad et al., 2012).Another possible explanation of cisplatin toxicity is that cisplatin induces a cascade of inflammatory reactions, that play an important pathogenic role in cisplatin-induced tissues injury (Pabla and Dong, 2008;Hussein et al., 2012).
Oral administration of silymarin to cisplatin treated rats significantly reduced the elevated serum levels of creatinine and BUN.It also improved the oxidative stress parameters by significantly decreasing the elevated levels of kidney MDA, NO and significantly increased kidney levels of GSH as compared to cisplatin control groups.
This action of silymarin is attributed to its antioxidant effect and its free radical scavenging activity, consequently protecting membrane permeability (Hakova et al., 1996;Mansour et al., 2006).
The nephroprotective properties of silymarin are due to free radical scavenging and elevating the cellular content of glutathione that lead to inhibition of the lipid peroxidation and stabilizing plasma membrane (Karimi et al., 2011).In addition, silymarin stimulates ribosomal protein synthesis by means of stimulating RNA polymerase I and so counteracting the reduction in macromolecule synthesis in the tissues (Gaedeke et al., 1996;Saller et al., 2007;Dashti-Khavidaki et al., 2012).Moreover, silymarin maintains the effective levels of α-tocopherol, β-carotene and ascorbate, resulting in an increased cell viability and preserved functionality in several animal models and cell lines (Comelli et al., 2007).Several studies revealed that silymarin reduces the nephrotoxic effects of cisplatin without decreasing its anti-tumour activity.The renoprotective effects of silymarin were more apparent when it was administered before cisplatin (Dashti-Khavidaki et al., 2012).
In addition, silymarin greatly improved the histopathological findings observed with cisplatin control as silymarin showed nearly normal histopathological structure of the kidney.In agreement with our results, Karimi et al., (2005) and Abdelmeguid et al., (2010b) showed that silymarin post-treatment attenuated glomerular atrophy that revealed minimal erythrocytes leakage and slightly dilated urinary space.Also previous studies showed that pretreatment with silymarin 2 h before cisplatin significantly decreased the pathological changes induced by cisplatin and appeared highly protective (Abdelmeguid et al., 2010a) Administration of high dose of hesperidin 200 mg/kg significantly decreased elevated serum levels of creatinine and BUN.On the other hand administration of low dose of hesperidin 100 mg/kg only significantly decreased creatinine but failed to produce any significant decrease in BUN.It is obvious that there is dose-dependent increase in response of hesperidin as by increasing the dose the response becomes better in all measured parameters.Furthermore, hesperidin 200 mg/kg significantly decreased cisplatin-induced elevation in kidney MDA and NO while increased cisplatin-induced reduction in kidney GSH.Hesperidin 100 mg/kg failed to decrease elevated levels of kidney MDA and NO while significantly increased kidney GSH.Also these results of hesperidin indicate that there is a dose-dependent increase in response.The histopathological findings showed that hesperidin greatly protected kidneys against cisplatin-induced damage.
Studies demonstrated that hesperidin enhanced the antioxidant defense mechanisms (Garg et al., 2001;Loguercio and Federico, 2003).Hesperidin improved GSH levels in kidneys of diabetic rats and decreased DNA fragmentation, in the urine of diabetic rats (Miyake et al., 1998).Hesperidin in combination with diosmin inhibited the reactive oxygen radicals production in Zymosan-stimulated human polymorphonuclear neutrophils (Jean and Bodinier, 1994).Consequently, in-vivo and in-vitro studies have been shown that hesperidin reduced oxidative stress (Tirkey et al., 2005).
In addition studies proved that flavonoids are hydrogen-donors and free-radical scavengers and so they act as potential chainbreaking antioxidants to inhibit the low density lipoprotein oxidation (Cotelle et al., 1996;Kaur et al., 2006).Furthermore, other studies have indicated that flavonoids can chelate transition metal-ions so can inhibit free radical formation and the propagation of free-radical reactions (Morel et al., 1993;Haller and Hizoh, 2004).
In conclusion, the present study proved that oxidative stress plays an important role in cisplatininduced nephrotoxicity.
Hesperidin, decreased cisplatin-induced kidney damage, suppressed cisplatininduced generation of ROS, lipid peroxidation and oxidative stress.All these obtained results indicate that hesperidin provide protective effects on cisplatininduced nephrotoxicity and it may find use as an adjunct in the cancer chemotherapy in which cisplatin is used as first drug.

Fig. 1 :
Fig. 1: Effect of hesperidin treatment on serum creatinine level in cisplatin administered rats; N = 6-8 rats per group.Each value represents the mean ± SE of the mean; Statistics were carried out by one way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test; * : significantly different from normal control at p<0.05; @ : Significantly different from cisplatin control at p<0.05

Fig. 4 :
Fig. 4: Effect of hesperidin treatment on kidney GSH content in cisplatin administered rats; N = 6-8 rats per group.Each value represents the mean ± SE of the mean; Statistics were carried out by one way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test; * : significantly different from normal control at p<0.05; @ : Significantly different from cisplatin control at p<0.05