Aqueous-ethanolic extract of morel mushroom mycelium Morchella esculenta, protects cisplatin and gentamicin induced nephrotoxicity in mice

Food and Chemical Toxicology 46 (2008) 3193–3199

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Aqueous-ethanolic extract of morel mushroom mycelium Morchella esculenta, protects cisplatin and gentamicin induced nephrotoxicity in mice B. Nitha, K.K. Janardhanan * Department of Microbiology, Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala 680 555, India

a r t i c l e

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Article history: Received 30 December 2007 Accepted 14 July 2008

Keywords: Cisplatin Gentamicin Morchella esculenta Morel mushroom mycelium Nephroprotection

a b s t r a c t Morchella esculenta (L) Pers. is an excellently edible and delicious morel mushroom found growing in the temperate forests. The mycelium of this mushroom is widely used as a flavouring agent. The current investigation was undertaken to explore the protective effect of the aqueous-ethanol extract of cultured mycelium of M. esculenta against cisplatin and gentamicin induced acute renal toxicity in Swiss albino mice. Cisplatin and gentamicin when administered induced a marked renal failure, characterized by a significant increase in serum urea and creatinine concentrations. Treatment with the extract at 250 and 500 mg/kg body weight decreased the cisplatin and gentamicin induced increase in serum creatinine and urea levels. Treatment with the extract also restored the depleted antioxidant defense system. The decreased activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) in the kidneys consequent to cisplatin and gentamicin administration was significantly elevated. The enhanced renal antioxidant defense system also prevented the tissue lipid peroxidation. The experimental results suggest that aqueous-ethanol extract of morel mushroom, M. esculenta mycelium protected cisplatin and gentamicin induced nephrotoxicity possibly by enhancing renal antioxidant system. The findings thus suggest the potential therapeutic use of morel mushroom mycelium as a novel nephroprotective agent. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Cisplatin (Cis diamine dichloroplatinum II) is a highly effective antineoplastic DNA alkylating agent used against a wide variety of cancers (Lynch et al., 2005). Although higher doses of cisplatin are more efficacious for the treatment of cancer (Cozzaglio et al., 1990; Di et al., 1990; Gandara et al., 1991) many reversible and irreversible side effects including nephrotoxicity, neurotoxicity, bone marrow toxicity, gastrointestinal toxicity and ototoxicity often limit its utility and therapeutic profile (Lynch et al., 2005). Primary targets of cisplatin in kidney are proximal straight and distal convoluted tubules where it accumulates and promotes cellular damage, by multiple mechanisms including oxidative stress, DNA damage and apoptosis (Safirstein et al., 1987; Schaaf et al., 2002; Cummings and Schnellmann, 2002; Xiao et al., 2003). Several lines of evidence suggest the role of reactive oxygen species (ROS) in the pathogenesis of nephrotoxicity (Baliga et al., 1998; KrishnaMohan et al., 2006). Cisplatin induces free radical production causing oxidative renal damage, possibly due to depletion of non-enzymatic and enzymatic antioxidant systems. Gentamicin, a typical aminoglycoside antibiotic is widely used in clinical practices for the treatment of life threatening gram-neg* Corresponding author. Tel.: +91 487 2304190; fax: +91 487 2307968. E-mail address: [email protected] (K.K. Janardhanan). 0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2008.07.007

ative infections. This antibiotic generally causes drug-induced dose-dependent nephrotoxicity in 10–20% of therapeutic courses (Karahan et al., 2005). Gentamicin induced nephrotoxicity is characterized by direct tubular necrosis, without morphological changes in glomerular structures (Cuzzocrea et al., 2002; Eisenberg et al., 1987). Gentamicin generates hydrogen peroxide in rat renal cortex mitochondria and can also enhance the generation of reactive oxygen species (ROS) (Yanagida et al., 2004; Karahan et al., 2005). Abnormal production of ROS may damage some macromolecules to induce cellular injury and necrosis via several mechanisms including peroxidation of membrane lipids, protein denaturation and DNA damage (Baliga et al., 1998; Kehrer, 1993; Parlakpinar et al., 2005). The alteration in kidney functions induced by lipid peroxidation is a proximal event in the injury cascade of gentamicin mediated nephrotoxicity (Karahan et al., 2005). Gentamicin also acts as an iron chelator and the iron–gentamicin complex is a potent catalyst of radical generation (Yanagida et al., 2004). Mushrooms are nutritional food as well as source of physiologically beneficial and non-toxic medicines. Since ancient times they have been used in folk medicine throughout the world. Mushrooms contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. A number of medicinal mushrooms have recently been reported to possess significant antioxidant activity

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(Jose et al., 2002; Jones and Janardhanan, 2000; Ajith et al., 2002; Lakshmi et al., 2004; Ekanem and Ubengama, 2002). Some of the most recently isolated and identified substances from mushrooms have been reported to possess significant cardiovascular, antiviral, antibacterial, antiparasitic, hepatoprotective and antidiabetic activities (Oii, 2000). Members of the genus Morchella, commonly known as morels are one of the most highly priced edible mushrooms in the world (Negi, 2006; Duncan et al., 2001). M.orchella esculenta is an excellently edible mushroom growing in temperate regions. In India, species of Morchella locally known as ‘Guchhi’ are found growing in the forests of Jammu and Kashmir and Himachal pradesh. The preparations from these mushrooms are reported to be used in health care and medicinal purposes among traditional hill societies (Prasad et al., 2002). Since commercial cultivation of morel mushrooms for the fruiting body production has not been largely successful till now, the cultured mycelium is extensively used as a flavouring agent. In this communication, we report the nephroprotective activity of the aqueous-ethanol extract of cultured mycelium of M. esculenta (L) Pers. 2. Materials and methods 2.1. Chemicals Glutathione (GSH), 5,50 dithio-dinitro bisbenzoic acid (DTNB), 1-chloro-2,4dinitrobenzene (CDNB), nitroblue tetrazolium (NBT) and thiobarbituric acid (TBA) riboflavin and sodium azide were purchased from SRL, Mumbai, hydrogen peroxide (H2O2), EDTA, n-butanol, ascorbic acid, pyridine from Merck India Ltd., Mumbai, India and 2,7-dichlorofluorescein diacetate from Sigma–Aldrich. Cisplatin (Samarath Pharma, Pvt. Ltd., Mumbai, India), Gentamicin (Biochem Pharmaceutical Industries, Mumbai, India) and vitamin E (Evion 400 of Merck India Ltd., Mumbai, India) were purchased from the Amala Hospital Pharmacy. All other chemicals and reagents used were of analytical grade.

2.5.2. Gentamicin induced nephrotoxicity Animals were divided into six groups of six animals in each group. Group I treated with (vehicle) distilled water was kept as normal. Group II injected with gentamicin (50 mg/kg body weight, i.p.) for six days was kept as control (Karahan et al., 2005). Group III and IV were treated with aqueous-ethanolic extract of M. esculenta mycelium (250 and 500 mg/kg body weight), and group V received vitamin E 250 mg/kg body weight. The extract and vitamin E were administrated orally 1 h before every gentamicin injection and continued for six days. Twenty-four hours after the last doze of gentamycin, animals were anaesthetized with ether and sacrificed. The blood was collected directly from the heart of each animal. 2.6. Biochemical analysis Creatinine was estimated using diagnostic kit (Span Diagnostics Ltd., India) and urea by diacetylmonoxime (DAM) reagent diagnostic kit (Span Diagnostics Ltd., India). Tissue lipid peroxidation (MDA) was assayed by the method of Ohkawa et al. (1979). 2.6.1. Determination of antioxidant status in the liver Kidneys were excised after sacrificing the animals and washed with ice-cold saline (0.89%) and 10% homogenate was prepared in phosphate buffer (0.05 M, pH 7) using a polytron homogenizer at 4 °C. Reduced glutathione (GSH) in the tissue was determined by the method of Moron et al. (1979), Glutathione peroxidase (GPx) activity was determined by the method of Hafemann et al. (1974) and Glutathione-S-transferase (GST) activity by the method of Habig et al., 1974. Tissue superoxide dismutase (SOD) activity was assayed according to the method of McCord and Fridovich, 1969 and tissue catalase (CAT) activity by the method of (Beer and Sizer, 1952). The protein was estimated by the method of (Bradford, 1970). 2.6.2. Isolation of kidney glomeruli Kidney glomeruli were isolated by the method of Takemoto with slight modifications (Takemoto et al., 2002).The kidneys were removed, minced into 1-mm 3 pieces, and digested in collagenase (1 mg/ml collagenase A, 100 U/ml deoxyribonuclease I in HBSS) at 37 °C for 30 min with gentle agitation. The collagenase-digested tissue was gently pressed through a 100 lm cell strainer using a flattened pestle and the cell strainer was then washed with 5 ml of HBSS. The filtered cells were passed through a new cell strainer without pressing and the cell strainer washed with 5 ml of HBSS. The cell suspension was then centrifuged at 200g for 5 min. The supernatant was discarded and the cell pellet was resuspended in 2 ml of HBSS.

2.2. Test animals Female Swiss albino mice, six weeks old purchased from Small Animal Breeding Centre, College of Veterinary and Animal Science, Mannuthy, Kerala, India were kept for a week under environmentally controlled conditions with free access to standard food (Sai Durga Feeds and Foods, Bangalore) and water ad libitum. Mice weighing 25 ± 2 g were used for the experiments. All animal experiments were carried out according to the guidelines and approval of institutional animal ethic committee (IAEC). 2.3. Production of mushroom mycelium Culture of M. esculenta obtained from Microbial Type Culture Collection, Institute of Microbiology, Chandigarh, India, (MTCC 1795) was used for the studies. The fungus was grown in submerged culture on potato-dextrose broth (PDB) for the production of mycelial biomass. After ten days of growth in submerged culture the fungal biomass was harvested, washed thoroughly and dried at 40–50 °C (Janardhanan et al., 1970). 2.4. Preparation of extracts The dried mushroom mycelia were powdered and extracted with hot aqueousethanol (ethanol/water 50/50 v/v) for 8–10 h. The extract was concentrated and solvent completely evaporated under vacuum. The residue (6%) thus obtained was employed for the experiments.

2.6.3. Measurement of ROS in kidney glomeruli Endogenous amounts of ROS were measured by a fluorometric assay with 20 ,70 dichlorofluorescin diacetate (DCFH-DA) (Bondy and Guo, 1994). DCFH-DA is a stable, nonfluorescent molecule that is de esterified within cells to the ionized free acid, DCFH. This is trapped within cells and thus accumulated (Bass et al., 1983). DCFH is then oxidized in the presence of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) to the highly fluorescent 20 ,70 -dichlorofluorescein (DCF) (Scott et al., 1988). Cells were incubated at 37 °C for 45 min in PO4 buffer (50 mM, pH 7) containing 10 lM DCFH-DA (prepared in DMSO) in a final volume of 1 ml and fluorescence was quantified using Fluorescence spectrometer (Nanodrop fluorospectrometer, Wilmington, USA) at excitation 488 nm and emission 525 nm. 2.7. Histopathological examination Pieces of kidney from each group were fixed immediately in 10% neutral formalin for a period of at least 24 h, dehydrated in graded (50–100%) alcohol and embedded in paraffin, cut into 4–5 m thick sections and stained with hematoxylin–eosin. The sections were evaluated for the pathological symptoms of nephrotoxicity such as necrosis, fatty infiltration, fibrosis, lymphocyte infiltration, etc. 2.8. Statistical analysis All experimental data were expressed as mean ± S.D. The statistical analysis was done by one way analysis of variance (ANOVA) followed by Dunnett’s t-test using InStat3 software. p < 0.01 was considered to be significant.

2.5. Experimental design 2.5.1. Cisplatin induced nephrotoxicity Animals were divided into six groups of six animals in each group. Group I treated with (vehicle) distilled water was kept as normal. Group II injected with a single dose of cisplatin (12 mg/kg body weight, i.p.) was kept as control. Group III and IV were treated with aqueous-ethanolic extract of M. esculenta mycelium (250 and 500 mg/kg body weight), and group V received vitamin E 250 mg/kg body weight. The extract and vitamin E were administrated orally 1 h before and 24 and 48 h after cisplatin injection. Seventy two hours after cisplatin injection, animals were anaesthetized with ether and sacrificed. The blood was collected directly from the heart of each animal (Ajith et al., 2002).

3. Results 3.1. Effect of the mushroom extract on serum urea and creatinine concentrations Serum urea and creatinine concentrations were significantly increased in the cisplatin and gentamicin alone treated group of animals compared to the normal animals indicating the induction of severe nephrotoxicity (Figs. 1 and 2). Treatment with the extract

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and vitamin E showed marked decrease in concentrations of serum urea and creatinine compared to control group (p < 0.01).The higher dose of the extract (500 mg/kg body wt) reduced the serum urea and creatinine in the cisplatin challenged animals by 59.66% and

62.72%, respectively. The same dose of the extract in the same way efficiently decreased serum urea and creatinine in the gentamicin treated animals by 51.35% and 74.81%, respectively. 3.2. Effect of the mushroom extract on kidney antioxidant status and MDA

350 300

Urea mg/dl

250

** **

200

*

** ** **

150 100 50 0 Cisplatin

Gentamicin

Fig. 1. Effect of M. esculenta mycelium extract on serum urea concentrations in mice treated with cisplatin and gentamicin. Normal, Control, Extract 250 mg/kg, Extract 500 mg/kg, Standard. All values are mean ± S.D., (n = 6). ** * p < 0.01, p < 0.05 with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

4

Creatinine mg/dl

3.5 3

** **

2.5

**

2

** ** **

1.5 1 0.5 0

Cisplatin

Gentamicin

Fig. 2. Effect of M. esculenta mycelium extract on serum creatinine concentrations in mice treated with cisplatin and gentamicin. Normal, Control, Extract 250 mg/kg, Extract 500 mg/kg, Standard. All values are mean ± S.D., (n = 6). ** p < 0.01, with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

The activities of renal SOD, CAT and GPx in the cisplatin, gentamicin and the extract treated groups of animals are presented in Tables 1 and 2. Renal SOD activity was decreased significantly in the cisplatin or gentamicin alone treated group of animals compared to normal group. Treatment with the extract (500 mg/kg body wt) significantly elevated the SOD levels in cisplatin (79.86%) and gentamicin (52.70%) treated animals compared to untreated animals (p < 0.01). The activity of CAT in the cisplatin and gentamicin alone treated group was found to be decreased drastically. Treatment with the extract prevented the drug-induced decline of catalase activity (p < 0.01). The extract showed a restoration of catalase activity by 78.15% and 67.88% at doses of 500 mg/kg and 250 mg/kg, respectively in the animals with cisplatin induced renal damage. In gentamicin treated animals, the extract at a dose of 500 mg/kg helped to restore the catalase activity by 71.81%. Similarly, the depleted GPx activity consequent to the treatment with cisplatin and gentamicin was also restored by the higher dose of the extract to 55.36% and 54.73%, respectively (p < 0.01). The GSH and MDA levels of cisplatin, gentamicin and extract treated animals are presented in Figs. 3 and 4. The GSH level reduced consequent to renal damage was found to be boosted up by the extract. After cisplatin challenge the reduced GSH level (5.54 ± 1.73 U/mg protein) was restored to 10.23 ± 3.10 U/mg protein by treatment with 500 mg/kg body wt of mushroom extract. The same dose of the extract restored the GSH level of gentamicin treated animals by 46.87% (p < 0.01). In cisplatin induced renal damage the untreated group of animals showed a serum MDA level of 1.25 ± 0.37 nmol/ml. The two used doses (500 and 250 mg/kg) reduced serum MDA level by 34.40% and 29.60%, respectively. The raised serum MDA after gentamicin treatment was also restored by the aforesaid drug doses by 34.87% and 20.05%, respectively.

Table 1 Effect of Morchella esculenta mycelium extract on renal, SOD, CAT and GPx activities in mice treated with cisplatin Groups

Treatment (mg/kg)

SOD (U/mg protein)

CAT (U/mg protein)

GPx (U/mg protein)

Cisplatin Normal Control (cisplatin) Extract + cisplatin Extract + cisplatin Vitamin E + cisplatin

Vehicle 12 mg/kg (i.p) 250 mg/kg (p.o) 500 mg/kg (p.o) 250 mg/kg (p.o)

13.82 ± 3.04 4.44 ± 2.60 20.62 ± 6.31** 22.05 ± 7.51** 17.25 ± 4.68**

52.37 ± 7.45 5.53 ± 3.15 17.22 ± 3.23** 25.32 ± 4.42** 25.90 ± 6.19**

26.24 ± 4.21 9.28 ± 3.48 16.21 ± 7.03ns 20.79 ± 2.51** 21.55 ± 7.41**

All values are mean ± S.D. ** p < 0.01, *p < 0.05 with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

Table 2 Effect of Morchella esculenta mycelium extract on renal, SOD, CAT and GPx activities in mice treated with gentamicin Groups

Treatment (mg/kg)

SOD (U/mg protein)

CAT (U/mg protein)

GPx (U/mg protein)

Cisplatin Normal Control (gentamicinin) Extract + gentamicin Extract + gentamicin Vitamin E + gentamicin

Vehicle 50 mg/kg (i.p) 250 mg/kg (p.o) 500 mg/kg (p.o) 250 mg/kg (p.o)

14.16 ± 3.50 7.52 ± 2.66 10.93 ± 1.94ns 15.90 ± 3.07** 16.54 ± 2.95**

43.94 ± 10.08 11.60 ± 3.38 32.18 ± 6.03** 41.16 ± 13.62** 38.82 ± 10.28**

28.43 ± 4.90 11.28 ± 1.95 19.83 ± 6.29ns 24.92 ± 10.28** 22.65 ± 6.24*

All values are mean ± S.D. ** p < 0.01, *p < 0.05 with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

B. Nitha, K.K. Janardhanan / Food and Chemical Toxicology 46 (2008) 3193–3199

18

**

GSH U/mg protein

16

**

*

**

14 12 10 8 6 4 2 0 Cisplatin

1.8 1.6 MDA (nmol/ml)

1.4 1.2

**

** **

Normal

Control

Gentamicin

Fig. 3. Effect of M. esculenta mycelium extract on kidney GSH level in mice treated with cisplatin and gentamicin. Normal, Control, Extract 250 mg/kg, Extract 500 mg/kg, Standard. All values are mean ± S.D., (n = 6). **p < 0.01, * p < 0.05 with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

1

18000 16000 14000 12000 10000 8000 6000 4000 2000 0

Relative Flourescent intensity (RFU)

3196

*

** **

0.8 0.6 0.4 0.2

Extract 500 mg/kg

Extract 250 mg/kg

Treatment Groups Fig. 6. Inhibition of gentamicin induced ROS generation in kidney glomeruli by the extract measured using DCFH-DA. **p < 0.01, with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

micin alone showed a relative fluorescence unit, representing the endogenous ROS, of 12730.23 ± 2577.31. In the extract treated groups (500 and 250 mg/kg body wt) the relative fluorescence intensity was decreased and found to be 5014.35 ± 35 (p < 0.01) and 9571.55 ± 1280.65 (p < 0.05), respectively. The extract at concentrations of 500 and 250 mg/kg body wt reduced the fluorescence intensity indicating the inhibition of endogenous ROS. The inhibition was 60.61% and 24.81%, respectively in the gentamicin treated animals as compared to untreated control group of animals.

0 Cisplatin

Gentamicin

3.4. Histopathological observations

Fig. 4. Effect of M. esculenta mycelium extract on tisuue MDA level in mice treated with cisplatin and gentamicin. Normal, Control, Extract 250 mg/kg, Extract 500 mg/kg, Standard. All values are mean ± S.D., (n = 6). ** p < 0.01,*p < 0.05 with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

3.3. Effect of the extract on endogenous ROS level in kidney glomeruli

Relative Flourescent Intensity (RFU)

The ability of the extract to reduce the endogenous ROS level was studied using DCFH-DA assay. The extract efficiently scavenged or reduced the production of endogenous free radicals. (Figs. 5 and 6). Treatment with cisplatin resulted in a drastic production of ROS in kidney cells. The relative fluorescence unit (RFU) in the cisplatin alone treated animals was found to be 14664 ± 1741.75 which was significantly reduced to almost the normal level (4356.66 ± 685.31) in the extract (500 mg /kg body wt) treated group of animals. The extract (500 and 250 mg/kg body wt) reduced or scavenged the endogenous ROS by 70.29% and 53.76%, respectively (p < 0.01). The group of animals which received genta18000 16000 14000 12000

**

10000

**

8000 6000 4000 2000 0 Normal

Control

Extract 500 mg/kg

Extract 250 mg/kg

Treatment Groups Fig. 5. Inhibition of cisplatin induced ROS generation in kidney glomeruli by the extract measured using DCFH-DA. **p < 0.01, with respect to control. (One way ANOVA followed by Dunnett’s t-test.)

The histological changes in kidneys and pathological manifestations are presented in Fig. 7. Treatment with both cisplatin and gentamicin induced a marked necrosis in proximal tubules. Treatment with the extract (250 and 500 mg/kg body weight) and vitamin E (250 mg/ kg body weight) ameliorated the toxic manifestations in the kidney. The histopathological observations supported this conclusion. 4. Discussion Nephrotoxicity is an undesired side effect of chemotherapy in general. Most chemotherapy drugs targets pathways that are essential to dividing cells (Hanigan and Devarajan, 2003). Several studies have now documented the importance of reactive oxygen metabolites (ROM) in cisplatin and gentamicin induced renal damage (Ueda et al., 2000). Nephrotoxicity of the drugs is usually associated with their accumulation in renal cortex, dependent upon their affinity to kidneys and on kinetics of drug trapping process (Karahan et al., 2005). A minimum dose of cisplatin (5 mg/kg body weight) was sufficient to induce nephrotoxicity in rats (Boogaard et al., 1991; Ravi et al., 1995). Cisplatin is known to accumulate in mitochondria of renal epithelial cells (Singh, 1989) and induces ROS primarily by decreasing the activity of antioxidant enzymes and by depleting intracellular concentrations of GSH (Saduzka et al., 1992; Huang et al., 2001; Haningan et al., 2003) and also causes the peroxidation of membrane lipids (Safirstein et al., 1984). Cisplatin covalently binds to DNA bases and disrupts DNA functions. The cytotoxic action of the drug is often thought to be associated with its ability to bind DNA to form cisplatin–DNA adducts (Goldstein and Mayor, 1983). Cisplatin induced oxidative stress can activate some protein kinases (MAPKs) c-Jun N-terminal kinase (JNK) and p38 which sensitize the injured cell to apoptosis (Kyriakis and Avruch, 2001). Gentamicin at a dose of 100 mg/ kg body wt intraperitoneally, for six consecutive days in rats is known to cause significant nephrotoxicity (Baliga et al., 1999; Cuzzocrea et al., 2002). Gentamicin

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Fig. 7. Nephroprotective effect of M. esculenta extract. Histopathological observations (kidney sections stained with hematoxylin-eosin, magnification-10x) (A) Normal, (B) Control (cisplatin), (C) Extract 250 mg/kg + cisplatin, (D) Extract 500 mg/kg + cisplatin, (E) Standard (Vit E, 250 mg/kg + cisplatin) (F) Control (gentamicin), (G) Extract 250 mg/ kg + gentamicin, (H) Extract 500 mg/kg + gentamicin, (I) Standard (Vit E, 250 mg/kg + gentamicin.)

usually accumulates in renal proximal tubules and enhances hydrogen peroxide generation by the mitochondria, which is mostly derived from the dismutation of superoxide (Walker and Shah, 1987). Hydrogen peroxide generated during the gentamicin induced oxidative stress in mitochondrial membranes releases iron from the mitochondria. The released iron makes a complex with gentamicin and accelerates the oxidative stress (Ueda et al., 1993). Among the main approaches used to ameliorate or protect the cisplatin and gentamicin induced nephrotoxicities, the most con-

sistent effects have been observed with the use of antioxidant agents (Mingeot-Leclerq and Tulkens, 1999; Ali, 2003). Some antioxidant agents that have been used to ameliorate cisplatin and gentamicin induced nephrotoxicity in rats include deferoxamine, methimazole, Vit E, Vit C diethyl dithiocarbamate, L-histidinol, thymoquinone (Ali, 1995; Mingeot-Leclerq and Tulkens, 1999; Somani et al., 1995; Badary et al., 1997a, b). None of these compounds have proved to be clinically efficient to provide complete protection in patients.

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Attention is focused recently on the development of antioxidants from natural sources that are able to ameliorate cisplatin and gentamicin induced nephrotoxicities. These natural antioxidants may offer comparatively safer alternatives to synthetic antioxidants, which may cause serious or unacceptable adverse side effects (Ali, 2003). The results of the investigation reveal that the aqueous-ethanolic extract of M. esculenta mycelium possessed significant protective effect against both cisplatin and gentamicin induced nephrotoxicity and the effect was found to be in a dosedependent manner. The present study demonstrates that cisplatin and gentamicin induce renal injury as evident from the elevated serum urea and creatinine levels and also from the histopathological features of acute renal failure. Treatment with M. esculenta mycelium extract restored the elevated serum urea and creatinine level, indicating its significant nephroprotective effect. The decrease in SOD activity after cisplatin administration might be due to the loss of copper and zinc which are essential for the enzyme activity (Badary et al., 2005). The decreased SOD activity is insufficient to scavenge the superoxide anion produced during the normal metabolic process and could cause the initiation and propagation of lipid peroxidation in the cisplatin and gentamicin alone treated group of animals. The decrease in the activity of CAT and GPx, in turn increases H2O2 concentration and enhances the lipid peroxidation. Hence concentration of MDA, as a result of lipid peroxidation increased in the cisplatin and gentamicin treated animals (Ajith and Nivitha, 2007). Treatment with the extract prevented the lipid peroxidation by enhancing the renal CAT and GPx activities and the restoration of SOD activity can also be seen in the extract treated group of animals. One of the most important intracellular antioxidant systems is the glutathione redox cycle. Glutathione is one of the essential compounds for maintaining cell integrity (Conklin, 2000). The decreased concentration of GSH increases the sensitivity of organ to oxidative and chemical injury. The role of GSH, a non-protein thiols in the cells, in the formation of conjugates with electrophilic drug metabolites most often formed by Cytochrome P-450 linked monooxygenase is well established (Rana et al., 2002). A number of studies reveal that the metabolism of xenobiotics often produced GSH depletion (Mitchell et al., 1973; Jollow et al., 1974). Reduced renal GSH can markedly increase the toxicity of cisplatin and gentamicin. The depletion of GSH also seems to be a prime factor that permits lipid peroxidation in the cisplatin and gentamicin treated animals. Concomitant treatment with the extract and vitamin E rendered protection due to the increase in GSH concentration and could protect the renal cells from oxidative attack. Treatment with the extract enhances activity of Se-GPx (Selenium dependent GPx) compared to the cisplatin or gentamicin alone treated animals. The enhanced GPx activity could partially explains the protection of biomembrane from oxidative attack (Ajith and Nivitha, 2007). The endogenous ROS level in kidney monitored using DCFH-DA showed the efficient reduction of endogenous ROS by the extract in a dose-dependent manner. The finding reveals the ability of the extract to reduce ROS levels and to enhance the antioxidant status. Preliminary analysis of the extract revealed that the major component of the extract is a polysaccharide protein complex. TLC and HPTLC analyses indicated the presence of a number of minor components including terpenoids. The investigations carried out in our lab showed the significant antioxidant activity of the extract (unpublished data). The major components of most of the mushrooms which possess biological activity have been reported to be polysaccharides, polysaccharide–protein complexes and terpenoids. Polysaccharides and triterpenes isolated from Ganoderma lucidum, an extensively studied medicinal mushroom, showed significant antioxidant activity (Zhu et al., 1999). A polysaccharide–

protein complex isolated from Pleurotus ostreatus was reported to be used as a nephroprotective agent (Noda-Shokukin, 1998). In conclusion, the results of the present studies indicate that the aqueous-ethanolic extract of M. esculenta mycelium possesses profound nephroprotective activity. The experimental results also reveal that the nephroprotective activity of the extract is comparable to that of vitamin E. The activity elicited by the extract might be due to its ability to activate antioxidant enzymes. The findings suggest the potential use of the aqueous-ethanolic extract of cultured mycelia of morel mushroom, M. esculenta as a novel therapeutically useful nephroprotective agent. Conflict of interest statement Morchella esculenta is an excellently edible, delicious mushroom. The present study reveals the significant nephroprotective activity of this mushroom. As the search for drugs from natural sources without side effects is increasing day by day, this mushroom can serve as a nephroprotective agent without any side effects. Funding source: Amala Cancer Research Centre. Acknowledgements The valuable help of Dr. Ramanathan V., Associate Professor, Department of Physiology, College of Veterinary and Animal Sciences Mannuthy, Thrissur, Kerala, India during the spectrofluorimeter analysis and the help of Dr. Ajith. T.A, Associate Professor, Department of Biochemistry, Amala Institute of Medical Sciences, Thrissur, Kerala, India during the experimental work and in the preparation of the manuscript are gratefully acknowledged. References Ajith, T.A., Jose, N., Janardhanan, K.K., 2002. Amelioration of cisplatin induced nephrotoxicity in mice by ethyl acetate extract of a polypore fungus, Phellinus rimosus. J. Exp. Clin. Cancer. Res. 21, 213–217. Ajith, T.A., Usha, S., Nivitha, V., 2007. Ascorbic acid and a-tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice: a comparative study. Clin. Chim. Acta 375, 82–86. Ali, B.H., 1995. Gentamicin nephrotoxicity in humans and animals some recent research. Gen. Pharmocol. 26, 1477–1487. Ali, B.H., 2003. Agents ameliorating or augmenting experimental gentamicin nephrotoxicity: some recent research. Food. Chem. Toxicol. 41, 1447–1452. Badary, O.A., Nagi, M.N., Al-Shabanah, O.A., Al-Sawaf, H.A., Al-Sohaibani, M.O., AlBeikairi, A.M., 1997a. Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity. Can. J. Physiol. Pharmacol. 75, 1356–1361. Badary, O.A., Nagi, M.N., Al-Sawaf, H.A., Al-Harbi, M., Al-Beikairi, A.M., 1997b. Effect of L-histidinol on cisplatin nephrotoxicity in rat. Nephron 77, 435–439. Badary, O.A., Abdel-Maksoud, S., Ahmed, W.A., Owieda, G.H., 2005. Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sci. 76, 2125–2135. Baliga, R., Zhang, Z., Baliga, M., Ueda, N., Shah, S.V., 1998. In vitro and in vivo evidence suggesting a role for iron in cisplatin induced nephrotoxicity. Kidney Int. 53, 394–401. Baliga, R., Ueda, N., Walker, P.D., Shah, S.V., 1999. Oxidant mechanisms in toxic acute renal failure. Rev. Drug. Metabol. 31, 971–997. Bass, D.A., Parce, J.W., Dechatelet, L.R., Sjeda, P., Seeds, M.C., Thomas, M., 1983. Flow cytometric studies of oxidative product formation by neutrophils: a graded dose response to membrane stimulation. J. Immunol. 130, 1910. Beer, R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 1, 133–140. Bondy, S.C., Guo, S.X., 1994. Effect of ethanol treatment on indices of cumulative oxidative stress. Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. Sect. 270, 349– 355. Boogaard, P.J., Lempers, E.L., Mulder, G.J., Meerman, J.H.N., 1991. 4Methylthiobenzoic acid reduces cisplatin nephrotoxicity in rats without compromising anti-tumor activity. Biochem. Pharmacol. 41, 1997–2003. Bradford, M.A., 1970. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 240–254. Conklin, K.A., 2000. Dietary antioxidants during cancer chemotherapy: impact on chemotherapeutic effectiveness and development of side effects. Nutr. Cancer. 37, 1–18. Cozzaglio, L.R., Doci, G., Colla, G., Zumino, F., Casciarri, G., Gennari, L., 1990. A feasibility study of high-dose cisplatin and 5-flurouracil with glutathione protection in the treatment of advanced colorectal cancer. Tumori. 76, 590–594.

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