Counteracting adriamycin-induced oxidative stress by administration of N-acetyl cysteine and vitamin E

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Counteracting adriamycin-induced oxidative stress by administration of N-acetyl cysteine and vitamin E
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  Clin Chem Lab Med 2005;43(8):834–840    2005 by Walter de Gruyter  •  Berlin  •  New York. DOI 10.1515/CCLM.2005.140  Article in press - uncorrected proof Counteracting adriamycin-induced oxidative stress byadministration of N-acetyl cysteine and vitamin E Periandavan Kalaiselvi 1, *, ViswanathanPragasam 1 , Srinivasan Chinnikrishnan 1 ,Coothan Kandaswamy Veena 1 , RajaguruSundarapandiyan 2 and PalaninathanVaralakshmi 1 1 Department of Medical Biochemistry, 2 Department of Pathology,Dr. ALM. Post Graduate Institute of Basic MedicalSciences, University of Madras, Taramani Campus,India Abstract Adriamycin (ADR), a cytotoxic antineoplastic drug, isused in the treatment of various solid tumors. How-ever, its efficacy continues to be challenged by sig-nificant toxicities including nephrotoxicity. In thepresent study, the effects of N-acetyl cysteine (NAC)and vitamin E, known antioxidants, were investigatedon ADR-induced peroxidative damage in rat kidney.Adult male albino rats of Wistar strain were admin-istered ADR as a single dose (10 mg/kg body weight,i.v.). Histopathological studies indicated that ADR-treated kidney sections show focal tubular necrosisand casts. ADR-injected rats showed a significantdecline in the activities/levels of enzymic antioxidants(superoxide dismutase, catalase, glutathione peroxi-dase, glutathione reductase, glucose-6-phosphatedehydrogenase and glutathione-S-transferase) andnon-enzymic antioxidants (thiols, vitamin C and vita-min E) with high malondialdehyde levels. The extentof nephrotoxicity was evident from the increasedactivities of urinary marker enzymes (alkaline phos-phatase, lactate dehydrogenase and  g -glutamyltrans-ferase). Treatment with NAC and vitamin E (50 mg/kgb.w., i.p.) 1 day prior to ADR administration main-tained near normal activities of the enzymes, signifi-cantly reduced lipid peroxidation and prevented thenecrosis caused by ADR, thereby proving to be aneffective thiol replenishing agent and antioxidant. Keywords:  adriamycin; antioxidant; N-acetyl cysteine;nephrotoxicity; vitamin E. Introduction Adriamycin (ADR), an aminoglycosidic antibiotic, is acommonly used anticancer agent with provenefficacy *Corresponding author: Dr. P. Kalaiselvi, PhD, Lecturer,Department of Medical Biochemistry, Dr. ALM. PostGraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai – 600 113, IndiaPhone: q 91-44-2492-5548, Fax: q 91-44-2492-6709,E-mail: pkalaiselvi@yahoo.com in acute leukemias, lymphomas and a number ofsolidtumors (1). The effect of ADR toxicity is both species-and organ-dependent (2). Nephrotoxicity is the majorside effect of aminoglycoside antibiotics accountingfor 10–15% of all cases of renal failure (3). ADR-induced nephrotoxicity involves various conditionssuch as nephropathy, renal insufficiency, nephroticsyndrome, glomerulosclerosis, necrosis,hemorrhage,tubular degeneration, swelling and vacuolation ofepi-thelial cell cytoplasm with accompanying proteinuria(4). ADR nephropathy represents a model very closeto human progressive chronic renal disease (5). Themolecular mechanism by which ADR causes renaldamage is unknown. However, numerous studiessupport the hypothesis that the ADR-induced nephro-toxicity may be the consequence of oxidative stress,i.e., oxidation and cross linking of cellular thiols andmembrane lipid peroxidation (6). Several in vitro andin vivo studies have demonstrated that reactive oxy-gen species including superoxide radical, hydroxylradical and hydrogen peroxide (H 2 O 2 ) are importantmediators of tissue injury (7).Biological antioxidants contribute to the protectionof cells and tissues against the deleterious effects of reactive oxygen species and other free radicals. Glu-tathione is involved in numerous vital processeswhere the reducing potential of the thiol is used.Several disorders are believed to be characterized byan increase in oxidative burden, potentially due todepleting glutathione. Hence, administration of pre-cursors of glutathione may be effective in reducingthe oxidative stress and thereby its consequences (8).N-acetyl cysteine (NAC), a precursor of glutathione,has a broad array of biological properties underlyingits protective role in a variety of pathophysiologicalconditions. NAC is found to be effective againsta vari-ety of metabolic disorders involving oxidative stress,such as paracetamol toxicity, in vitro and in vivo (9),calcium oxalate urolithiasis (10), doxorubicin cardiactoxicity in mice (11) and radiographic-contrast agent-induced reductions in renal function (12).Vitamin E, a powerful antioxidant, quenches freeradicals and inhibits lipid peroxidation. Vitamin Ereacts with lipid peroxy and alkoxy radicals, donatesits liable hydrogen to them and scavenges the chain-propagating radicals. This process is important inmaintaining the integrity of cell membranes (13). Vita-min E was shown to reduce the progression of injuryand the development of glomerular diseases in rats(14). Supplementation of vitamin E has been shownto reduce the number and incidence of chemically-induced tumors in animals (15). The present studyaimed to find out the efficacy of a combinationtherapy of NAC and vitamin E on ADR-inducednephrotoxicity in experimental rats.  Kalaiselvi et al.: Nephrotoxicity and antioxidant 835  Article in press - uncorrected proof Materials and methods Drugs and chemicals ADR (doxorubicin hydrochloride-Adrim) was procured fromDabur Pharmaceuticals (New Delhi, India). NAC and vitaminE were obtained from Sisco Research Laboratories Pvt Ltd.(Mumbai, India). All other chemicals and solvents were of analytical grade. Animal model Adult male albino rats of Wistar strain weighing 150–200 g(12–14 weeks old) were obtained from Madras VeterinaryCollege (Chennai, India). The animals weremaintainedunderstandard conditions of humidity, temperature (25 " 2 8 C) andlight (12 h light/12 h dark). They were fed with a standard ratpelleted diet (M/s Pranav Agro Industries Ltd., India, underthe trade name Amrut rat/mice feed) and had free access towater. The animal experiments were conducted accordingtothe guidelines of the Institutional Animal Ethics Committee(IAEC). Experimental design The animals were divided into four groups of six rats each,as follows. Group I (control) received normal saline through-out the course of the experiment. Group II received a singledose intravenous injection of ADR through the tail vein(10 mg/kg body weight). Group III received intraperitonealinjections of NAC (50 mg/kg body weight) in saline and vita-min E (50 mg/kg body weight) 1 h prior to the administrationof ADR as Group II. Group IV received intraperitoneal injec-tion of NAC and vitamin E in the above mentioned concen-tration and served as drug controls.At the end of the experimental period (after 48 h) the ani-mals were housed in metabolic cages for 24-h urine collec-tion, which was collected in ice-jacketed beakers at 0 8 C. Theurine samples were centrifuged for 10 min at low speed toremove any sediment; the supernatant was dialyzed andused for enzyme assays. Animals were sacrificed by cervicaldecapitation. Kidneys were excised immediately and placedin ice-cold saline, trimmed free of connective tissues, blottedwith filter paper and weighed. Histopathological studies Immediately after sacrifice a portion of the kidney tissue wasfixed in 10% formalin. The washed tissue was dehydrated indescending grades of isopropanol and finally cleared inxylene. The tissue was then embedded in molten paraffinwax. Sections were cut at 5- m m thickness, stained withhematoxylin and eosin. The sections were then viewedunder a light microscope for histopathological changes. Biochemical analysis Antioxidant enzymes were assayed in a 10% kidney homo-genate of the control and experimental groups. Superoxidedismutase (SOD) was assayed as described (16), with oneunit of enzyme activity defined as the amount required for50% inhibition of pyrogallol auto-oxidation. Catalase (CAT)was assayed by the reduction of the dichromate in aceticacid to chromic acetate when heated in the presenceofH 2 O 2 ;the chromic acetate thus produced was measured colori-metrically at 610 nm (17). Glutathione peroxidase (GPX) wasassayed by the method of Rotruck et al. (18), which is basedon the reaction between glutathione remaining after theaction of GPX and 5,5 9 -dithiobis-(2-nitrobenzoic acid) to givea compound that absorbs light at 412 nm. Glutathione-S-transferase (GST) was assayed as previously described (19).Glutathione reductase (GR), which utilizes NADPH to convertoxidized glutathione (GSSG) to the reduced form (GSH), wasassayed as previously described (20). Glucose-6-phosphatedehydrogenase (G6PD) was measured according to themethod of Beutler (21) in which the increase in absorbanceis measured when the reaction was started with the additionof glucose-6-phosphate. Non-enzymic antioxidants such astotal glutathione (GSH) (22), total sulfydryl groups (TSH) andnon-protein sulfydryl groups (NPSH) (23), and vitamin E (24)were estimated using standard protocols. Vitamin C (25) wasoxidized by copper to form dehydroascorbic acid and dike-toglutaric acid, and was treated with 2,4-dinitrophenylhydra-zine to form the derivative of bis-2,4-dinitrophenylhydrazine.This compound in sulfuric acid undergoes a rearrangementto form a product that was measured at 520 nm. A mildlyreducing medium with thiourea was used to prevent non-ascorbic chromogen interference. Lipid peroxidation (LPO)was assayed by the method of Devasagayam (26) in whichthe malondialdehyde (MDA) released serves as the index of LPO. Protein content was estimated by the method of Lowryet al. (27).Urinary cytotoxic marker enzymes such as alkaline phos-phatase (ALP),  g -glutamyltransferase (GGT), and lactatedehydrogenase (LDH) were assayed in the dialyzed 24-hurine sample. ALP was assayed as previously described (28)using disodium phenyl phosphate and the liberated phenolwas measured colorimetrically using Folin-Ciocalteau’s re-agent. GGT was assayed as previously described (29). LDH(30) was assayed using lithium lactate as substrate and thecolor was developed using dinitrophenyl hydrazine. Creati-nine was assayed by the method of Owens et al. (31). Statistical analysis Values are expressed as mean " SD for six animals. Theresults were computed statistically using the SPSS softwarepackage for Windows (SPSS Inc., Chicago, IL, USA). One-way analysis of variance, post-hoc testing was performedforinter-group comparisons by the least significant difference(LSD) test. A p-value - 0.001 was considered significant. Results In the present experimental study, a single high doseinjection of ADR (10 mg/kg body weight) inducedsevere biochemical changes as well as oxidativedam-age in kidneys. There was no instance of death in anyof the experimental groups during the study period.Histological examination on control andexperimen-tal rats confirmed marked foci of proximal tubularnecrosis, hyaline casts and tubular dilatations alongwith mild focal interstitial inflammatory cell infiltra-tion in ADR-administered rat kidney (Figure 1B),whereas in control (Figure 1A) and Group IV (Figure1D) a normal architecture was observed. Kidneys of rats pretreated with NAC and vitamin E along withADR administration (Group III) showed very littledamage to the tubular epithelium (Figure 1C).Table 1 shows the activities of antioxidant enzymesin the control and experimental rats. Administrationof ADR to the experimental rats (Group II) showed a  836 Kalaiselvi et al.: Nephrotoxicity and antioxidant  Article in press - uncorrected proof Figure 1  Histopathological observations in the rat kidney. (A) Control rat kidney section showing normal glomeruli andtubules; (B) ADR-administered rat kidney sections showing numerous foci of proximal tubular necrosis, hyaline casts andmild interstitial inflammatory cell infiltration; (C) ADR-administered and Vitamin E q NAC co-supplemented rat kidneysectionshowing near normal tubules with minimal epithelial damage; (D) Vitamin E- and NAC-treated rat kidney section showingnormal architecture as control rat kidney sections. Images were taken under light microscopy using hematoxylin and eosinas staining agents (100 ). = Table 1  Antioxidant enzymes in the kidney of control and experimental animals.Particulars Group I Group II Group III Group IVSOD 19.93 " 2.01 10.21 " 0.98 a *** 18.54 " 1.87 b *** 19.13 " 1.72CAT 2.53 " 0.21 1.64 " 0.13 a *** 2.15 " 0.20 a **  b *** 2.59 " 0.18GPX 18.16 " 1.63 12.81 " 1.47 a *** 17.43 " 1.65 b *** 18.39 " 2.06GST 1.32 " 0.11 0.65 " 0.06 a *** 1.24 " 0.13 b *** 1.37 " 0.12GR 9.42 " 0.83 5.32 " 0.57 a *** 8.41 " 0.73 a *  b *** 9.72 " 0.83G6PD 1.29 " 0.14 0.84 " 0.08 a *** 1.16 " 0.13 b *** 1.28 " 0.12Values are mean " SD for six rats. One unit of enzyme activity expressed as SOD-units/mg. Protein (1 U s amount of enzymerequired to bring about 50% inhibition of auto-oxidation of pyrogallol); CAT,  m mol of H 2 O 2  utilized/min/mg protein; GPX,  m gof GSH utilized/min/mg protein; GST, nmol of 1-chloro-2,4-dinitro benzene–GSH conjugateformed/min/mgprotein;GST, m molof CDNB–GSH conjugate formed/min/mg protein; GR,  m mol of NADPH oxidized/min/mg protein; G6PD,  m mol of NADPreduced/min/mg protein. Comparisons are made between:  a Group I and Groups II, III, IV;  b Group II and Group III. The symbolsrepresent statistical significance: *p - 0.05, **p - 0.01, ***p - 0.001. significant decrease (p - 0.001) in the activities of enzymic antioxidants like SOD, CAT, GPX, GR, G6PDand GST when compared to the control rats (GroupI), whereas their levels were maintained near normalin the Group III rats.Table 2 delineates the activities of non-enzymicantioxidants like GSH and thiols (protein and non-pro-tein) in control and experimental rats. In Group IIrat kidneys, thiol levels were markedly decreased(p - 0.001) when compared to the control rats, whilethey were maintained near normal in Group III rats.There was a significant decrease (p - 0.001) in theconcentrations of vitamins E and C in ADR-adminis-tered rats when compared to the control and NAC q vitamin E pretreated rats (Figure 2). In Figure 3, whichrepresents the LPO level in control and experimentalrats, a 2.02-fold increase in the LPO was observed inADR-administered rats when compared to the controlrats. Table 3 shows a significant increase (p - 0.001)in the activities of urinary cytotoxic marker enzymeslike ALP, LDH and GGT in Group II rats when com-pared to the control rats, whereas their levels weremaintained near normal in Group III rats. Discussion The nephrotoxic effect of ADR has been well docu-mented in all tumor-associated diseases. Since itsintroduction for the treatment of cancer in 1969, ADRhas been widely employed in clinical practice (1).Effective anticancer therapy with ADR and other qui-  Kalaiselvi et al.: Nephrotoxicity and antioxidant 837  Article in press - uncorrected proof Table 2  Non-enzymic antioxidant thiol in the kidneys of control and experimental animals.Particulars Group I Group II Group III Group IVGSH 2.93 " 0.24 1.36 " 0.12 a *** 2.70 " 0.24 b *** 3.06 " 0.21TSH 8.87 " 0.76 4.46 " 0.44 a *** 8.01 " 0.74 b *** 8.95 " 0.78NPSH 4.93 " 0.44 2.67 " 0.28 a *** 4.72 " 0.51 b *** 5.01 " 0.33Values are mean " SD for six rats. Units: GSH, TSH and NPSH,  m g/mg protein. Comparisons are made between:  a Group Iand Groups II, III, IV;  b Group II and Group III. The symbols represent statistical significance: ***p - 0.001. Figure 2  Levels of vitamin E and C in control and experi-mental animals. Values are mean " SD for six rats. Compar-isons are made between:  a Group I and Groups II, III, IV; b Group II and Group III. The symbols represent statisticalsig-nificance: *p - 0.05, ** p - 0.01, ***p - 0.001. Figure 3  Levels of lipid peroxidation in control and experi-mental animals. Values are mean " SD for six rats. Compar-isons are made between:  a Group I and Groups II, III, IV; b Group II and Group III. The symbols represent statisticalsig-nificance: *p - 0.05, ***p - 0.001. Table 3  Urinary marker enzymes in control and experimental animals.Particulars Group I Group II Group III Group IVALP 0.19 " 0.02 0.30 " 0.03 a *** 0.20 " 0.03 b *** 0.19 " 0.02LDH 0.39 " 0.03 0.78 " 0.06 a *** 0.42 " 0.04 b *** 0.38 " 0.04GGT 2.16 " 0.19 3.71 " 0.31 a *** 2.21 " 0.20 b *** 2.12 " 0.18Values are mean " SD for six rats. Units: ALP,  m mol of phenol liberated/h/mg creatinine; LDH,  m mol of pyruvate liberated/h/ mg creatinine; GGT,  m mol of p-nitroaniline released/h/mg creatinine. Comparisons are made between:  a Group I and GroupsII, III, IV;  b Group II and Group III. The symbols represent statistical significance: ***p - 0.001. nine anthracyclines is severely limited by its nephro-toxicity, which has been well established in a varietyof animal species (32). ADR is known to generatesuperoxide radicals, thus, the formation of free radi-cals as well as the accumulation of lipid peroxides inresponse to the treatment with ADR has been welldocumented (6). Hence, identification of compoundsthat could counteract the free radicals produced dueto ADR toxicity could undoubtedly increase the med-ical usefulness of ADR.Histopathological results confirm the nephrotoxiceffect of ADR in rat kidney. A marked proximal tubularnecrosis with casts and mild interstitial inflammatorycell infiltration are seen in ADR-administered ratswhen compared to control sections, whereas thesechanges are not observed in NAC- and vitamin E-pre-treated rats. Similar changes have been reported byMalarkodi et al. (33) in ADR-induced nephrotoxicity.In this study, ADR administration decreased theactivities of enzymic antioxidants like SOD, CAT, GPXand GST. Oxidative stress is found to play a criticalrole in nephrotoxicity. Decreases in the antioxidantenzymes have already been reported in the kidneysof doxorubicin- and other anticancer agents like cis-platin-treated rats (34, 35). SOD is responsible for thecatalytic dismutation of the potentially toxic super-oxide anion radical to H 2 O 2 . It is an effective defenseof the cells against endogenous and exogenous gen-eration of reactive oxygen species (36). CAT is presentin peroxisomes and catalyzes the decomposition of H 2 O 2  to yield O 2  and water (36). GPX is also one of the important enzymes responsible for theconversionof H 2 O 2  into water. Decline in the activities of theseantioxidant enzymes may be due to their inactivationcaused by excess ROS production (37). In the presentstudy, NAC and vitamin E pretreatment significantlyincreased the activities of these enzymes to near nor-malcy. Vitamin E has also been shown to restore theactivity of SOD and CAT in the kidneys of ADR-treatedrats (34).GST is a group of multifunctional proteins encodedby a multigene family. They perform functions rang-ing from catalyzing the detoxification of electrophiliccompounds to protecting against peroxidative dam-age. The decreased activity of GST after ADR admin-istration might be due to enhanced production of H 2 O 2  and subsequent GSH depletion. Increase in H 2 O 2  838 Kalaiselvi et al.: Nephrotoxicity and antioxidant  Article in press - uncorrected proof leads to the production of hydroxyl radicals, whichare deleterious to the cell (7). NAC and vitamin Eadministration improves the situation by increasingthe activity of GST.GSH is a non-protein endogenous thiol that detox-ifies reactive oxygen species formed during inter-mediate metabolism and drug detoxification.Depletion of cellular GSH has been reported to playan important role in tissue injury. GSH levels aremaintained by GSH generating enzyme, GR, and GSHutilizing enzymes, GST and GPX. The reduced activityof GR in ADR-treated groups might be one of the rea-sons for decreased GSH levels. ADR is also capableof reducing GSH synthesis (38). The observeddecrease in GSH levels in ADR-treated groups wasbrought to near normal values by the administrationof the NAC, which is the precursor of the GSH.GR requires reducing equivalents (NADPH) for itsactivity, which is provided by the action of G6PD. GR,the key enzyme of the GSH-redox cycle, was signifi-cantly decreased on ADR administration (39). Thismight be due to the reduced availability of NADPH.The decrease in G6PD activity and a fall in NADPHmight occur as a result of impaired flux of glucose-6-phosphate through the hexose monophosphateshunt(40). NAC pretreatment resulted in an increased avail-ability of reducing equivalents (NADPH) due toimproved G6PD activity, thereby increasing the activi-ty of GR, thus regenerating the GSH pool. Similarobservations have been made by Sandhya et al. (41),who reported an increase in the activities of GR andG6PD in the kidneys on antioxidant supplementationin gentamycin-induced nephrotoxicity. Hence, resto-ration of G6PD levels by NAC and vitamin E treatmentis a good indication for normalization and preventionof nephrotoxicity.The principal high molecular weight thiol-contain-ing compounds are proteins and their thiol groupsarein equilibrium with low molecular weight thiolspeciessuch as glutathione, and the maintenance of anappropriate concentration of these species in theirreduced state is essential for numerous cellular func-tions. GSH depletion leads to a decrease or reductionin protein thiol content and alters the membraneintegrity and increases the susceptibility to lipidperoxidative damage. NAC and vitamin E supplemen-tation is found to be effective in replenishing the pro-tein thiols and preventing damage to kidneys. Similarstudies were carried out by Tepel et al. (12), confirm-ing the prevention of chronic renal insufficiency bythe administration of NAC in radiographic-contrastagent-induced reductions in renal function.Decreases in the levels of vitamin C and vitamin Ewere observed in the kidneys of ADR-treated rats.Vitamin E, an excellent chain-breaking fat-solubleantioxidant, functions as a trap for lipid peroxy radi-cals. Reduction of vitamin E levels might be deleteri-ous to the cell membrane integrity. Vitamin E, vitaminC and GSH are interrelated with each other for therecycling process. Recycling of tocoperoxyl radical totocopherol is achieved by reaction with ascorbic acid(42). Dehydroascorbic acid formed in the above reac-tion is reduced to ascorbic acid by a non-enzymaticreaction with GSH (43). The reduced status of GSH,vitamin C and vitamin E are essential for maintainingthe reduced milieu of the cell. Hence restoration of these reducing equivalents by NAC, by the generationof GSH and vitamin E treatment is shown to be effec-tive in attenuating the nephrotoxicity induced by theoxy radicals on ADR administration.LPO not only plays an important role in the genesisof many chronic disease conditions, but also evokesadverse effects on the normal organs in which theybecome distributed. It also leads to oxidation of pro-tein thiol groups, decrease in the relative content of polyunsaturated fatty acids and changes in mem-brane receptor structure and function (7). The in-crease in lipid peroxides in the ADR-treated ratsmightresult from increased production of free radicals and/ or a decrease in antioxidant status. The ADR-inducedLPO observed in our study is in line with other reports(6, 40). Restoration of enzymic and non-enzymic anti-oxidants by NAC and vitamin E afforded protectionagainst LPO. NAC and vitamin E have been reportedto be effective in reducing the amount of hydroxylradical generated by Fenton-type reactions and alsoact as a scavenger of peroxide and superoxide radi-cals (44). The above reports corroborate well with ourfindings.ADR is reported to accumulate more in the kidneythan in any other organs (45). Renal GGT is an extrin-sic brush-border membrane protein and hence, thealteration in GGT activity might be an indicator of nephrotoxicity. Increased excretion of GGT in urinemight be due to increased tubular lesion, since thebrush-border of the proximal tubules can be a toxictarget of ADR. A similar increase in the GGT activitywas reported by Saner et al. (46). Increased levels of LDH and ALP in urine have been observed in ADR-administered rats, indicating brush-bordermembranedamage (40). NAC and vitamin E supplementationprevented the increased excretion of these enzymes,suggesting a membrane protective effect.In conclusion, ADR-induced increase in the lipidperoxides and the subsequent decrease in the anti-oxidant status of the cell, as demonstrated by thedecreased activities of SOD, CAT, GR and G6PD, areattenuated upon pretreatment with NAC and vitaminE. The role of NAC and vitamin E in the regulation of thiol status and other intracellular antioxidants maybe significant in the restoration of redox status. Thepresent study provides evidence that the peroxidativedamage brought about by this antineoplastic amino-glycoside can be counteracted by co-administrationof NAC and vitamin E. References 1. Blum RH. An overview of studies with adriamycin (NSC123127) in the United States. Cancer Chemother Rep1975;6:247–56.
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