Toxicology 201 (2004) 143–151

Protective effect of naringin, a bioflavonoid on glycerol-inducedacute renal failure in rat kidney

Devinder Singh, Vikas Chander, Kanwaljit Chopra∗

Pharmacology Division, University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh 160014, India

Received 12 March 2004; received in revised form 19 April 2004; accepted 20 April 2004

Available online 7 June 2004

Abstract

Rhabdomyolysis-induced myoglobinuric acute renal failure accounts for about 10–40% of all cases of acute renal failure(ARF). Reactive oxygen intermediates have been demonstrated to play an etiological role in myoglobinuric renal failure. Thisstudy was designed to investigate the effect of naringin, a bioflavonoid with antioxidant potential, in glycerol-induced ARF inrats. Five groups of rats were employed in this study, group I served as control, group II was given 50% glycerol (8 ml/kg,intramuscularly), group III, IV, and V were given glycerol plus naringin 100, 200, and 400 mg/kg p.o. route, respectively) 60 minprior to the glycerol injection. Renal injury was assessed by measuring plasma creatinine, blood urea nitrogen, creatinine, andurea clearance. The oxidative stress was measured by renal malondialdehyde levels, reduced glutathione levels, and by enzymaticactivity of catalase, glutathione reductase, and superoxide dismutase. Glycerol treatment resulted in a marked renal oxidative stressand significantly deranged the renal functions. Pretreatment of animals with naringin 60 min prior to glycerol injection markedlyattenuated renal dysfunction, morphological alterations, reduced elevated thiobarbituric acid reacting substances (TBARS), andrestored the depleted renal antioxidant enzymes. These results clearly demonstrate the role of oxidative stress and its relation torenal dysfunction, and suggest a protective effect of naringin in glycerol-induced renal failure in rats.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Rhabdomyolysis; Glycerol; Acute renal failure; Naringin; Oxidative stress

1. Introduction

Rhabdomyolysis is a clinical syndrome in whichinjury to the skeletal muscle results in leakage ofintracellular contents from myocytes into the cir-culation (Curry et al., 1989). The large number ofdisorders known to cause rhabdomyolysis include in-

∗ Corresponding author. Tel.:+91 0172 2534105;fax: +91 0172 2541142.

E-mail address: dr choprak@yahoo.com (K. Chopra).

trinsic muscle dysfunction (including trauma, burns,intrinsic muscle disease, excessive physical exer-tion), metabolic disorders, hypoxia, drugs, toxins,infections, temperature extremes, and idiopathic dis-orders (Salluzzo, 1992). Complications associatedwith rhabdomyolysis include disseminated intravas-cular coagulation, hyperkalemia and other metabolicimbalances, acute renal failure (ARF), and acutecardiomyopathy.

In general, about 10–40% of cases with rhabdomy-olysis develop acute renal failure and it accounts for

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2004.04.018

144 D. Singh et al. / Toxicology 201 (2004) 143–151

2–15% of all cases of ARF (Beetham, 2000). Follow-ing rhabdomyolysis, inordinate amounts of myoglobinare released into the systemic circulation and itsprompt discharge into the renal tubules sets the stagefor the initiation of renal injury process (Better andStain, 1990). Both oxidant and non-oxidant mecha-nisms are considered to be important in the complexpathophysiology of myoglobin-mediated renal injuryof rhabdomyolysis (Better and Stain, 1990; Odeh,1991; Paller, 1988; Guidet and Shah, 1989; Shah andWalker, 1988; Zager and Gamelin, 1989; Zager, 1992).Hypovolemia and metabolic acidosis facilitate tubularprecipitation of myoglobin and therefore, form thebasis for fluid-alkaline-diuresis therapy (Better andStain, 1990; Paller, 1988; Zager and Gamelin, 1989).Myoglobin, besides causing tubular obstruction, maycontribute to tubular epithelial cell injury througha probable heme-iron-mediated lipid peroxidationmechanism. This latter process further compromisesthe tubular fluid flow dynamics, and interferes withthe complete clearance off tubular heme proteins,and thus initiating the vicious cycle. Moreover, eventhe absence of heme protein precipitation, tubularinjury may still occur because of continues bathingof the tubular cells in myoglobin filtrate. The lattermay facilitate tubular cell heme-loading which alongwith the release of catalytically active iron (Zager,1992), instigate the cellular lipid peroxidation andinjury.

Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglu-coside) is a major and active flavanone glycoside ofgrapefruit and many citrus herbs as well. Naringin isreported to possess antiulcer, superoxide scavenging,and antioxidant activities (Kroyer, 1986; Chen et al.,1990). When naringin is administered orally, it ishydrolyzed by intestinal microflora to yield a majormetabolite—naringenin (4′,5,7-trihydrosyflavanone)which is the absorbable form (Ameer et al., 1996).Naringenin is shown to possess antiulcer (Parmar,1983; Motilva et al., 1994), aorta dilatation (Rojaset al., 1996), superoxide scavenging, and antioxidantactivities (Kroyer, 1986). Moreover, naringin demon-strated inhibition of breast cancer cell proliferationand delay of mammary tumerogenesis (So et al.,1996).

In the present study, we investigated the role ofnaringin in glycerol-induced myoglobinuric acute re-nal failure in rats.

2. Materials and methods

2.1. Animals

Male Wistar rats (150–200 g) bred in the central an-imal house of Punjab University (Chandigarh, India)were used. The animals were housed under standardconditions of 12 h light/dark cycles with free access tofood (Hindustan Lever Products, Kolkata, India) andwater. The experimental protocols were approved bythe institutional ethical committee of Punjab Univer-sity, Chandigarh.

2.2. Drugs

Glycerol was purchased from Ranbaxy laboratories(Mohali, India). Naringin was purchased from Sigma(St. Louis, MI, USA).

2.3. Study design

Five groups were employed in the present study,each consisting of five to seven animals. The ani-mals were allowed free access to food, but deprivedof drinking water for 24 h before glycerol injection.Group I (C) comprised of control group that receivedequivalent volume of saline for glycerol. Group II (G)animals received an intramuscular injection of 8 ml/kghypertonic glycerol as a divided dose into the hindlimbs. Groups III, IV, and V animals received naringin(100, 200, and 400 mg/kg p.o. route, respectively)60 min prior to the glycerol injection. All animals weresacrificed with a high dose of anaesthesia (60 mg/kg,thiopental sodium, i.p.) 24 h after the glycerol injectionand the blood was collected in heparinized centrifugetubes through the abdominal aorta. Freshly isolatedserum was used for the assessment of renal func-tion tests. Both the kidneys were harvested througha midline incision, the left kidney was deep frozentill further enzymatic analysis, whereas, the right kid-ney was stored in 10% formalin for the histologicalsectioning.

2.4. Assessment of renal function

Serum samples were assayed for blood urea nitro-gen (BUN) and serum creatinine by using standarddiagnostic kits (Span Diagnostics, Gujarat, India).

D. Singh et al. / Toxicology 201 (2004) 143–151 145

2.5. Post mitochondrial supernatantpreparation (PMS)

After sacrificing the animals, their kidneys werequickly removed, perfused immediately with ice coldnormal saline and homogenized in chilled potassiumchloride (1.17%) using a Potter Elvehjem homoge-nizer. The homogenate was centrifuged at 800× g for5 min at 4◦C in a refrigerated centrifuge to separatethe nuclear debris. The supernatant so obtained wascentrifuged at 10, 500× g for 20 min at 4◦C to getthe PMS which was used to assay reduced glutathione(GSH), glutathione reductase (GR), catalase (Cat), andsuperoxide dismutase (SOD) activity.

2.6. Estimation of lipid peroxidation

The Malondialdehyde (MDA) content, a measureof lipid peroxidation, was assayed in the form of thio-barbituric acid reacting substances (TBARS) (Ohkawaet al., 1979). In brief, the reaction mixture consisted of0.2 ml of 8.1% sodium lauryl sulphate, 1.5 ml of 20%acetic acid solution adjusted to pH 3.5 with sodiumhydroxide, and 1.5 ml of 0.8% aqueous solution ofthiobarbituric acid was added to 0.2 ml of 10% (w/v)of PMS. The mixture was brought up to 4.0 ml withdistilled water and heated at 95◦C for 60 min. Af-ter cooling with tap water, 1.0 ml distilled water and5.0 ml of the mixture of n-butanol and pyridine (15:1v/v) was added and centrifuged. The organic layer wastaken out and its absorbance was measured at 532 nm.TBARS were quantified using an extinction coeffi-cient of 1.56× 105 M−1 cm−1 and expressed as nmolof TBARS per milligram of protein. Tissue proteinwas estimated using Biuret method (Varley, 1988) ofprotein assay and the renal MDA content expressedas nanomoles of malondialdehyde per milligram ofprotein.

2.7. Estimation of antioxidant enzymes (AOE)

The AOE were estimated by the well-establishedprocedures already published elsewhere (Singh et al.,2003a). The non-protein sulfhydryl (NPSH) as amarker for reduced glutathione, was measured bythe method ofJollow et al. (1974)and the yellowcolor developed by the reduction of Ellman’s reagentby –SH groups of NPSH was read at 412 nm. The

glutathione reductase activity was measured by theNADPH oxidation method ofMohandas et al. (1984).The catalase activity was assayed by the method ofClaiborne (1985)and the rate of decomposition ofH2O2 was followed at 240 nm. The superoxide dis-mutase activity was assessed by the method ofKono(1978). The nitro blue tetrazolium (NBT) reductionby superoxide anion to blue formazon was followedat 560 nm.

2.8. Renal histology

The right kidney was isolated immediately af-ter sacrificing the animal and washed with ice-coldsaline. It was then fixed in a 10% neutral bufferedformalin solution, embedded in paraffin and used forhistopathological examination. Five micrometer (�m)thick sections were cut, deparaffinized, hydrated, andstained with hematoxylin and eosin. The renal sectionswere examined in blind fashion for hemorrhagic andhyaline casts, tubular necrosis, and apical blebbingin all treatments. A minimum of 10 fields for eachkidney slide were examined and assigned for severityof changes using scores on a scale of none (−), mild(+), moderate (++), and severe (+++) damage.

2.9. Statistical analysis

Data are presented as mean± S.E.M. Oneway analysis of variance (ANOVA) followed byStudent–Newman–Keuls test was applied to calculatethe statistical significance between various groups. Avalue ofP < 0.05 was considered to be statisticallysignificant.

3. Results

3.1. Effect of naringin on glycerol-inducedrenal dysfunction

Intramuscular injection of 8 ml/kg of hypertonicglycerol produced a marked derangement in the re-nal function and lead to a significant increase in thelevel of serum creatinine, blood urea nitrogen, and asevere fall in the clearance values of urea and creati-nine. Pretreatment of animals with all the three dosesof naringin (100, 200 and 400 mg/kg), produced a

146 D. Singh et al. / Toxicology 201 (2004) 143–151

Fig. 1. Effect of naringin (100, 200, and 400 mg/kg) on serum creatinine (a), blood urea nitrogen (BUN) (b), creatinine clearance (c), andurea clearance (d) in glycerol treated rats. The values are expressed as mean± S.E.M. ∗P < 0.05 as compared with the control group;aP < 0.05 as compared with the glycerol treated group (one-way ANOVA followed by Newman–Keuls test).

Fig. 2. Effect of naringin (100, 200, and 400 mg/kg) onglycerol-induced lipid peroxidation (MDA). The values are ex-pressed as mean±S.E.M. ∗P < 0.05 as compared with the controlgroup; aP < 0.05 as compared with the glycerol treated group(one-way ANOVA followed by Newman–Keuls test).

significant and dose dependent improvement in therenal functions (Fig. 1a–d).

3.2. Effect of naringin on glycerol-induced lipidperoxidation

Thiobarbituric acid reacting substances levels wereincreased significantly by glycerol treatment as com-pared to the control group. Pretreatment with naringin(100, 200, and 400 mg/kg) produced a significantand dose dependent reduction in TBARS in glyceroltreated rats (Fig. 2).

3.3. Effect of naringin on glycerol-induced changesin the antioxidant pool

Treatment with glycerol significantly decreased theenzymatic activity of reduced glutathione, catalase,

D. Singh et al. / Toxicology 201 (2004) 143–151 147

Fig. 3. (a) Effect of naringin (100, 200, and 400 mg/kg) on reduced glutathione (GSH), glutathione reductase (GR) (b), superoxide dismutase(SOD) (c), and catalase (Cat) (d) in glycerol treated rats. The values are expressed as mean± S.E.M. ∗P < 0.05 as compared with thecontrol group;aP < 0.05 as compared with the glycerol treated group (one-way ANOVA followed by Newman–Keuls test).

glutathione reductase, and superoxide dismutase. Thisreduction was significantly and dose dependently im-proved by pretreatment with naringin (100, 200, and400 mg/kg) (Fig. 3a–d).

3.4. Effect of naringin on glycerol-induced changeson renal morphology

The histopathological changes were graded andsummarized inTable 1. The control group did notshow any morphological changes. By contrast, thekidneys of rats treated with glycerol showed markedhistological changes in the cortex and outer medulla.The renal sections showed severe epical blebbing,hyaline casts, tubular necrosis, and hemorrhagic casts.Treatment with naringin (100 mg/kg) did not show anysignificant morphological protection; however, withnaringin (200 mg/kg), the kidney section showed mild

hemorrhagic casts and apical blebbing, and naringin(400 mg/kg) preserved the normal morphology of thekidney (Fig. 4).

4. Discussion

The intramuscular administration of hypertonicglycerol induces myolysis and hemolysis and af-fords a faithful and widely utilized model of hemeprotein-induced renal injury (Zager, 1996; Dubrowand Flamenbaum, 1988). The heme protein-inducedrenal injury represents the integrated effects of threemajor pathophysiologic mechanisms: renal vaso-constriction, direct cytotoxicity, and cast formation(Zager, 1996; Dubrow and Flamenbaum, 1988). Thebiochemical findings of renal injury so induced haveproduced persuasive evidence incriminating oxidative

148 D. Singh et al. / Toxicology 201 (2004) 143–151

Table 1Effect of naringin (100, 200, and 400 mg/kg) pretreatment on morphological changes as assessed by histopathological examination ofkidney in glycerol treated rats

Groups Hyaline casts Hemorrhagic casts Tubular necrosis Epical blebbing

Control − − − −Glycerol ++ +++ +++ +++Naringin (100) ++ ++ − −Naringin (200) +/− − + −Naringin (400) − − − −None (−), mild (+), moderate (++), severe (+++).

stress and the catalytic effect of redox-active iron asimportant mechanisms for such injury (Zager, 1996;Shah and Walker, 1988; Paller, 1988; Baliga et al.,1996a). The pigments (hemoglobin and myoglobin),themselves are unlikely to induce acute renal failure,but their presence within the systemic circulation dur-ing periods of acidosis, dehydration, shock, or other

Fig. 4. Hematoxylin and eosin stained sections of rat kidneys: (a) normal kidney section, (b) kidney section of glycerol treated rat showingsevere hemorrhagic and hyaline casts, apical blebbing, (c) kidney section of naringin (200 mg/kg, p.o.)+ glycerol treated rat showing castsand blebbing, (d) kidney section of naringin (400 mg/kg, p.o.)+ glycerol treated rat showing a near normal morphology.

conditions associated with reduced renal perfusionmay lead to both direct toxic and hemodynamic ab-normalities resulting in acute renal failure (Dubrowand Flamenbaum, 1983).

Despite the above insights, the specific mediator(s)of proximal tubular necrosis during myohemoglobin-uric ARF have been difficult to define. In large part,

D. Singh et al. / Toxicology 201 (2004) 143–151 149

Table 2Effect of naringin (100, 200, and 400 mg/kg) pretreatment on body weight, water intake and urine output in glycerol (G) treated rats

Groups Body weight (g) Water intake (ml/24 h) Urine output (ml/24 h)

Control 190± 3.3 34± 5 12 ± 4Dehydrated control 181± 2.5 – 7±1a,∗Glycerol 178± 2 15 ± 2∗ 2.5 ± 2∗Naringin (100) 178± 3 22.5± 3a,∗ 7 ± 3a,∗Naringin (200) 182± 4.5 36± 1.5a 9 ± 4a

Naringin (400) 185± 3.8 37± 1.2a 10 ± 5a

The values are expressed as mean± S.E.M.a P < 0.05 as compared with the glycerol treated group (one-way ANOVA followed by Newman–Keuls test).∗ P < 0.05 as compared with the control group.

this is due to the multifactorial nature of the insult,which has necessitated whole animal experiments totest specific hypotheses. Nevertheless, this approachhas produced the view that heme-iron-driven hy-droxyl radical (•OH) generation is a critical mediatorof the evolving tubular damage. This conclusion issupported by the following pieces of information:(1) iron chelatoin (deferoxamine) therapy partiallymitigates the extent of tubular necrosis and filtrationfailure (Paller, 1988; Chander et al., 2003); (2) •OHscavengers (Shah and Walker, 1988; Zager, 1992)and glutathione (Abull-Ezz et al., 1991), can exertprotective effects; (3) lipid peroxidation, a biochem-ical hallmark of oxidative stress, has been reportedin the aftermath of heme protein nephrotoxicity(Paller, 1988; Shah and Walker, 1988); and (4) in-duction or suppression of heme oxygenase (HO; theenzyme which degrades heme porphyrin), has beenshown to decrease or increase the severity of myohe-moglobinuric (glycerol) ARF, respectively (Nath et al.,1992).

In the present study, all the animals were dehy-drated for 24 h. Prior dehydration allows the full ex-pression of renal injury, particularly the cast formation,compared to the non-dehydrated model (Zager, 1992).The animals lost an average of 5–8% of their bodyweight in the period of dehydration, during whichtime their food intake was one third less than thatin the non-dehydrated state. Moreover, oliguria, oranuria developed only in the rats which were dehy-drated prior to the glycerol injection. The urinary vol-ume of dehydrated control animals was significantlylower than the urinary volume in non-dehydrated ani-mals. Furthermore, micropuncture studies have shownthat the glomerular filtration rate of dehydrated con-

trols falls significantly as compared non-dehydratedcontrols (Oken et al., 1966; Table 2).

In this study, intramuscular injection of glycerollead to markedly high levels of serum creatinine andBUN and reduced the creatinine and urea clearance.This glycerol-induced renal dysfunction was asso-ciated with increased renal lipid peroxidation andseverely depleted the pools of antioxidant enzymesas evident from reduced levels of GSH, Cat, GR,and SOD enzymes. Moreover, the histological patternof glycerol-treated rats showed characteristic hem-orrhagic and hyaline cast deposits, tubular necrosis,and apical blebbing. Oxidative stress can further re-lease the free heme by destabilizing the intracellularheme containing proteins (Agarwal et al., 1995). In-terestingly, all the three tested doses of naringin aspretreatments produced significant attenuation of lipidperoxidation (as is evident from MDA levels), andprotected the severe depletion of antioxidant enzymepool (as evidenced by levels of GSH, GR, Cat, andSOD) in glycerol treated rats, however, the best resultswere obtained with the 200 and 400 mg/kg doses. Therenal functional and morphological damage was sig-nificantly improved by the 200 and 400 mg/kg dosesand moreover, naringin produced no per se hemody-namic, functional, and morphological changes in thekidney (data not shown).

Like most of the flavonoids, naringin has metalchelating, antioxidant, and free radical scaveng-ing properties (Jung et al., 1983; Chen et al.,1990) and offers some protection against mutage-nesis (Francis et al., 1989) and lipid peroxidation(Maridonneau-Parini et al., 1986). The antioxidanteffects of naringin have been shown to be similar tothat of GSH and furthermore, it is reported to inhibit

150 D. Singh et al. / Toxicology 201 (2004) 143–151

the hydrogen peroxide-induced lipid peroxidation(Kanno et al., 2003). Recently, naringin has beendemonstrated to play an important role in regulatingantioxidative capacity by increasing SOD and catalaseactivities and by up-regulating the gene expression ofSOD, catalase, and glutathione peroxidase (GSH-Px)(Jeon et al., 2001).

With the data in hand and based on our previousstudies (Chander et al., 2003a,b; Singh et al., 2003b),it can be postulated that reactive oxygen metabolitesplay a deleterious role in this glycerol-induced renalfailure model. Moreover, it could be emphasized thatnaringin possess a betted potential to counteract thisoxidative stress as compared to the deferoxamine andother antioxidant agents.

Acknowledgements

The Senior Research Fellowship of the Council ofScientific and Industrial Research (CSIR), New Delhi,is gratefully acknowledged.

References

Abull-Ezz, S.R., Walker, P.D., Shah, S.V., 1991. Role of glutathionein an animal model of myoglobinuric acute renal failure. Proc.Natl. Acad. Sci. U.S.A. 88, 9833–9837.

Agarwal, A., Balla, J., Alam, J., Croatt, A.J., Nath, K.A., 1995.Induction of heme oxygenase in toxic renal injury: A protectiverole in cisplatin nephropathy in the rat. Kidney Int. 48, 1298–1307.

Ameer, B., Weintraub, R.A., Johnson, J.V., Yost, R.A., Rouseff,R.L., 1996. Flavanone absorption after naringin, hesperidin,and citrus administration. Clin. Pharmacol. Ther. 60, 34–40.

Baliga, R., Ueda, N., Walker, P.D., Shah, S.V., 1996a. Oxidantmechanisms in toxic acute renal failure. Am. J. Kidney Dis.29, 465–477.

Beetham, R., 2000. Biochemical investigation of suspectedrhabdomyolysis. Ann. Clin. Biochem. 37, 581–587.

Better, O.S., Stain, J.H., 1990. Early management of shock andprophylaxis of acute renal failure in traumatic rhabdomyolysis.N. Engl. J. Med. 322, 825–827.

Chander, V., Singh, D., Chopra, K., 2003a. Attenuation ofglycerol-induced acute renal failure in rats by trimetazidineand deferoxamine. Pharmacology 67, 41–48.

Chander, V., Singh, D., Chopra, K., 2003b. Catechin, anatural antioxidant protects against rhabdomyolysis-inducedmyoglobinuric acute renal failure. Pharmacol. Res. 48, 503–509.

Chen, Y.T., Zheng, R.L., Jia, Z.J., Ju, Y., 1990. Flavonoids assuperoxide scavengers and antioxidants. Free Radic. Biol. Med.9, 19–21.

Claiborne, A., 1985. In: Greenwald, R.A. (Ed.), CRC Handbookof Methods for Oxygen Radical Reaserch, CRC Press, BocaRaton, FL, pp. 283–284.

Curry, S.C., Chang, D., Connor, D., 1989. Drug- and toxin-inducedrhabdomyolysis. Ann. Emer. Med. 18, 1068–1084.

Dubrow, A., Flamenbaum, W., 1983. In: Soles, K., Whelton, A.(Eds.), Acute Renal Failure, New York, Marcel Dekker, pp.279–293.

Dubrow, A., Flamenbaum, W., 1988. In: Brenner, B.M., Lazarus,J.M. (Eds.), Acute Renal Failure, New York, ChurchillLivingstone, pp. 279–293.

Francis, A.R., Shetty, T.K., Bhattacharya, R.K., 1989. Modu-lating effect of plant flavonoids on the mutagenicityof N-methyl-N′-nitro-N-nitrosoguanidine. Carcinogenesis 10,1953–1955.

Guidet, B., Shah, S.V., 1989. Enhanced in vivo hydrogen peroxidegeneration by rat kidney in glycerol-induced acute renal failure.Am. J. Physiol. 257, F440–F445.

Jeon, S.M., Bok, S.H., Jang, M.K., Lee, M.K., Nam, K.T., Park,Y.B., Rhee, S.J., Choi, M.S., 2001. Antioxidative activity ofnaringin and lovastatin in high cholesterol-fed rabbits. Life Sci.69, 2855–2866.

Jollow, D.J., Mitchell, L.R., Zampaglione, N., Gillete, J.R.,1974. Bromobenze induced liver necrosis: protective role ofglutathione and evidence for 3,4-bromobenzeneoxide as thehepatotoxic intermediate. Pharmacol. 11, 151–169.

Jung, G., Hennings, G., Pfeifer, M., Bessler, W.G., 1983. Inte-raction of metal-complexing compounds with lymphocytes andlymphoid cell lines. Mol. Pharmacol. 23, 698–702.

Kanno, S., Shouji, A., Asou, K., Ishikawa, M., 2003. Effectsof naringin on hydrogen peroxide-induced cytotoxicity andapoptosis in p388 cells. J. Pharmacol. Sci. 92, 166–170.

Kono, Y., 1978. Generation of superoxide radical during auto-xidation of hydroxylamine and an assay for superoxidedismutase. Arch. Biochem. Biophys. 186, 189–195.

Kroyer, G., 1986. The antioxidant activity of citrus fruit peels. Z.Ernahrungswiss 25, 63–69.

Maridonneau-Parini, I., Braquet, P., Garay, R.P., 1986. Hetero-geneous effect of flavonoids on K+-loss and lipid peroxidationinduced by oxygen free radicals in human red cells. Pharmacol.Res. Commun. 18, 61–72.

Mohandas, J., Marshall, J.J., Duggin, G.G., Horvath, J.S., Tiller,D., 1984. Low activities of glutathione-related enzymes asfactors in the genesis of urinary bladder cancer. Cancer Res.44, 5086–5091.

Motilva, V., Alarcon de la, L.C., Martin, M.J., 1994. Ulcer-protecting effects of naringenin on gastric lesions induced byethanol in rat: role of endogenous prostaglandins. J. Pharm.Pharmacol. 46, 91–94.

Nath, K.A., Balla, G., Vercellotti, G.M., Balla, J., Jacob, H.S.,Levitt, M.D., Rosenberg, M.E., 1992. Induction of hemeoxygenase is a rapid protective response in rhabdomyolysis inthe rat. J. Clin. Invest. 90, 267–270.

D. Singh et al. / Toxicology 201 (2004) 143–151 151

Odeh, M., 1991. The role of reperfusion-induced injury in thepathogenesis of crush syndrome. N. Engl. J. Med. 324, 1417–1422.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipidperoxides in animal tissues by thiobarbituric acid reaction.Anal. Biochem. 95, 351–358.

Oken, D.E., Arce, M.L., Wilson, D.R., 1966. Glycerol-inducedhemoglobinuric acute renal failure in the rat. I. Micropuncturestudy of the development of oliguria. J. Clin. Invest. 45, 724–735.

Paller, M.S., 1988. Hemoglobin- and myoglobin-induced acuterenal failure in rats: role of iron in nephrotoxicity. Am. J.Physiol. 255, 539–544.

Parmar, N.S., 1983. The gastric anti-ulcer activity of naringenin, aspecific histidine decarboxylase inhibitor. Int. J. Tissue React.5, 415–420.

Rojas, D., Sanchez, V.R., Somoza, B., Ortega, T., Villar, A., 1996.Vasodilatory effect of naringenin in rat aorta. Phytother. Res.10, S123–S125.

Salluzzo, R.F., 1992. Rhabdomyolysis. In: Rosen et al. (Ed.),Emergency Medicine Concepts and Clinical Practice, St. Louis,Mosby.

Shah, S.V., Walker, P.D., 1988. Evidence suggesting a role forhydroxyl radical in glycerol-induced acute renal failure. Am.J. Physiol. 255, F438–F443.

Singh, D., Chander, V., Chopra, K., 2003a. Carvediloland trimetazidine attenuates ferric nitrilotriacetate-inducedoxidative renal injury in rats. Toxicology 191, 143–151.

Singh, D., Chander, V., Chopra, K., 2003b. Carvedilol, anantihypertensive drug with antioxidant properties, protectsagainst glycerol-induced acute renal failure. Am. J. Nephrol.23, 415–421.

So, F.V., Guthrie, N., Chambers, A.F., Moussa, M., Carroll, K.K.,1996. Inhibition of human breast cancer cell proliferation anddelay of mammary tumorigenesis by flavonoids and citrusjuices. Nutr. Cancer 26, 167–181.

Varley, H., 1988. In: Harold, Varley (Ed.), Practical ClinicalBiochemistry, CBS, Delhi.

Zager, R.A., 1996. Rhabdomyolysis and myohemoglobinuric acuterenal failure. Kidney Int. 49, 314–326.

Zager, R.A., 1992. Combined mannitol and deferoxamine therapyfor myohemoglobinuric renal injury and oxidant tubular stress.Mechanisms and therapeutic implications. J. Clin. Invest. 90,711.

Zager, R.A., Gamelin, L.M., 1989. Pathogenic mechanisms inexperimental hemoglobinuric acute renal failure. Am. J.Physiol. 256, F446–F455.