Pharmacological Research, Vol. 39, No. 4, 1999Article No. phrs.1998.0438, available online at http:rrwww.idealibrary.com on

REVERSAL OF DOXORUBICIN-INDUCED CARDIAC METABOLICDAMAGE BY L-CARNITINE

MOHAMED M. SAYED-AHMEDa, SABRY SHAARAWY b, SAMIA A. SHOUMAN b andABDEL-MONEIM M. OSMANa,U

aPharmacology Unit, and bBiochemistry Unit, Cancer Biology Department, National CancerInstitute, Cairo Uni¨ ersity, Cairo, Egypt

Accepted 24 No¨ ember 1998

ŽBiopharmacological evaluations of the protective effects of L-carnitine a naturally occur-.ring quaternary ammonium compound against doxorubicin-induced metabolic damage

were carried out in isolated cardiac myocytes and in isolated rat heart mitochondria.Ž .Perfusion of the heart with DOX 0.5 mM caused a significant 70% inhibition of palmitate

Ž .oxidation in cardiac myocytes, while L-carnitine 5 mM perfusion caused stimulation whichaccounted for 37%. Perfusion of the heart with L-carnitine after 10-min perfusion with

Ž .DOX 0.5 mM caused 88% reversal of DOX-induced inhibition of palmitate oxidation incardiac cells. In rat heart mitochondria, DOX has no effect on either palmitate oxidation or

Žacyl-CoA synthetase activity, whereas Enoximone c-AMP-dependent phosphodiesterase. Žinhibitor , caused a significant inhibition of palmitate oxidation and acyl-CoA activity 40

.and 27%, respectively . The oxidation of palmitoyl-CoA, an index of carnitine palmitoyl-transferse reaction was significantly inhibited by DOX as a function of DOX concentration.Preincubation of mitochondria with L-carnitine caused reversal of DOX-induced inhibitionof palmitoyl-CoA oxidation depending on the concentration of L-carnitine. Moreover,L-carnitine treatment did not interfere with the cytotoxic effect of doxorubicin against thegrowth of solid Ehrlich carcinoma. The findings of this study may suggest that inhibition offatty acid oxidation in the heart is at least a part of doxorubicin cardiotoxicity and thatL-carnitine can be used to prevent the doxorubcin-induced cardiac metabolic damagewithout interfering with its antitumour activities.

Q 1999 Academic Press

KEY WORDS: doxorubicin, L-carnitine, drug interaction, fatty acid oxidation and cardiomyopathy.

INTRODUCTION

Ž .Doxorubicin DOX is an anthracycline antibioticw xwith broad spectrum antitumour activity 1 . Unfor-

tunately, its chronic administration produces a cu-mulative dose-dependent and irreversible cardiotoxi-

w xcity that limits its optimum use 2, 3 . Cardiotoxicityis the major limiting complication of DOX thataffect nearly 30]40% of the patients who receive

y2 w xmore than 500 mg m total dose 4, 5 . Althoughseveral mechanisms have been suggested to explainthis cardiotoxicity, the exact mechanism of DOXcardiotoxicity and its metabolic consequences are

w xnot clear 6, 7 . Recently, it has been suggested thatDOX may exert at least a part of its cardiotoxicity by

U Corresponding author.

1043]6618r99r040289]07r$30.00r0

inhibition of long-chain fatty acid oxidation in thew xheart 8 .

Free fatty acid oxidation represents the majorsource of energy in the aerobic adult myocardiumw x9 . Therefore, inhibition of this vital pathway in theheart is usually associated with cardiotoxicity andcongestive heart failure similar to those reported by

w xDOX administration 10 . This inhibition causes sev-eral deleterious effects in cardiac tissues due todeficiency in energy supply and accumulation of

w xtoxic intermediates 11 . Although alteration of fattyacid oxidation has been associated with DOX use,the lack of understanding of fatty acid metabolism inthis cardiotoxicity and other cardiomyopathies hasled to conflicting results. In order to reduce androrcontrol DOX-related cardiotoxicity, a number of

w xdrugs has been examined 7, 12, 13 . One of theseŽdrugs is L-carnitine a naturally occurring quaternary

Q 1999 Academic Press

Pharmacological Research, Vol. 39, No. 4, 1999290

.ammonium compound which is an essential cofac- 25 mM NaHCO , 1.2 mM MgSO , 1.2 mM KH PO ,

tor for mitochondrial transport and oxidation of

w xlong-chain fatty acids. Previous studies 14 havedemonstrated that L-carnitine partially protects themyocardium against DOX-induced cardiotoxicity,but again no mechanism for this effect has beenascertained.

Therefore, this study has been initiated to de-termine the oxidation of long-chain fatty acids incardiac cells isolated from rat hearts perfused undernormal physiological condition with DOX andror

Ž .L-carnitine with the following specific aims: i de-termination of the exact site of inhibition of palmi-

Ž .tate oxidation by DOX; ii identifying the mecha-nism whereby L-carnitine reverses DOX-induced in-

Ž .hibition of palmitate oxidation; and iii to evaluatethe effect of L-carnitine on the antitumour activityof DOX.

MATERIALS AND METHODS

AnimalsMale Sprague]Dawley rats, weighing 200]250 g,

and female Swiss Albino mice, weighing 20]22 gwere obtained from the animal house of the Natio-

Ž .nal Cancer Institute NCI Cairo University. Ani-mals were allowed free access to standard diet es-sentially free from L-carnitine and water ad libitum.

Ž .A line of Ehrlich ascites carcinoma EAC was sup-plied through the courtesy of Dr C. Benckhuijsen,Amsterdam, The Netherlands, and maintained infemale mice by weekly i.p. transplantation since1982.

Materialsw 14 x w 14 x1- C Plamitate and 1- C palmitoyl-CoA were

Žpurchased from New England Nuclear Boston, MA,.USA . Sigma was the source of bovine serum al-

Ž .bumin BSA, essentially fatty acid free and palmi-toyl-CoA. Doxorubicin was a generous gift from theNCI drug store. L-Carnitine and Enoximone were

Žgenerous gifts from Dr Salah Abdel-aleem Duke.University, Medical Center, NC, USA . Collagenase,

Ž .type II was purchased from Worthington NJ, USA ,and Joklik essential medium was purchased from

Ž .Gibco laboratories NJ, USA .

Perfusion of the isolated rat heart with dox-orubicin and L-carnitine

Animals were exposed to ether in a desiccatorkept in a well-functioning hood, then killed by de-capitation. Hearts were quickly excised, placed inice-cold saline, attached through a short cannula inthe aortic root to the Langendorff perfusion appara-tus, and perfused in an open system with modifiedKrebs]Henseleit buffer as described by Pouna et al.w x15 . The perfusion buffer contained 11 mM glucose,

3 4 2 4Ž4.7 mM KCl, 118 mM NaCl, and 0.95 mM CaCl pH2

.7.4 at constant pressure equal to 60 mmHg. Theperfusion medium was continuously bubbled with amixture of 95% O r5% CO and maintained at2 2378C. After the heart was completely devoid of theblood, the perfusion medium was replaced withmedium containing 0.5 mM doxorubicin andror 5mM L-carnitine for 10 min in a closed system.

Isolation of cardiac myocytesAdult rat heart myocytes were isolated by the

w xmethod of Frangakis et al. 16 . Myocytes wereisolated with Joklik essential medium containing 5.55mM glucose, 25 mM NaHCO , 1.2 mM MgSO and3 4

Ž .0.5 mM CaCl pH 7.4 . The viability of myocytes2isolated by this procedure was 80]90% as de-termined by Trypan blue exclusion.

Palmitate oxidation in myocytesŽ .Myocytes 2-mg cell protein suspended in 0.9 ml

of Joklik medium, containing 25 mM NaHCO , 5.553mM glucose, 1.2 mM MgSO , 0.5 mM CaCl and 104 2

Ž .mM HEPES pH 7.4 , were placed in a 25-ml Er-lenmeyer flask. To this cell suspension 0.1 ml of 0.2

w 14 x Ž 5 .mM 1- C palmitic acid 2.2=10 dpm was added.The Erlenmeyer flask was then closed with a rubberseptum containing a plastic centre well. An injectionof 0.3 ml of 1 M hyamine hydroxide was adminis-tered through the centre well septum to absorb thereleased CO , and the reaction was terminated after230 min by injecting 0.4 ml of 7% perchloric acidthrough the centre well into the incubation medium.The flasks were then shaken continuously for 2 h at378C, at which time the plastic centre well wasremoved, placed into a scintillation vial containing10 ml of Scinti Verse BD, and counted in a liquid

Ž .scintillation counter Packard Model 3385 .

Isolation of rate heart mitochondriaRat heart mitochondria were isolated by the

w xprocedure of Chappel and Hansford 17 . The isola-tion buffer contained 0.21 M mannitol, 0.07 M su-

Ž .crose, 5 mM Tris]HCl pH 7.4 , and 1 mM EGTA.

Oxidation of palmitate and palmitoyl-CoA inrat heart mitochondria

Substrate oxidation in mitochondria was measuredw xusing the method by Yang et al. 18 . The reaction

mixture, contained in a final volume of 1.0 ml, 50Ž .mM Tris]HCl pH 7.4 ; 120 mM KCl; 0.5 mM L-

Ž .carnitine and 0.5 mM EDTA-K pH 7.4 ,2 mM KP ,2 iy1 w 14 xand 0.1 mg ml BSA, 0.2 mM 1- C palmitate or 40

w 14 xmM 1- C palmitoyl-CoA, was placed in a 25-mlErlenmeyer flask. Substrate oxidation was initiated

Ž .by the addition of rat heart mitochondria 0.5]1 mgwhich was preincubated with the drug or withoutŽ .control for 10 min at 378C. The rate of oxidation of

Pharmacological Research, Vol. 39, No. 4, 1999 291

Fig. 1. Effect of DOX, L-carnitine and their combination

palmitate and palmitoyl was determined using thesame procedure described before for measuring therelease of 14 CO with myocytes.2

Assay of acyl-CoA synthetase enzymeThe activity of acyl-CoA synthetase was measured

w xaccording to the procedure described previously 19 .Briefly, the reaction mixture in a total volume of 0.5

w 14 xml, contained 0.2 mM 1- C palmitate bound toBSA, 10 mM ATP, 50 mM CoA-SH, 5 mM MgCl , 502

Ž . y1mM Tris]HCl pH 7.5 , 1 mg ml of a-cyclodextrin.The reaction was initiated by the addition of mito-

Ž .chondria 0.5 mg protein for 10 min at 378C. Reac-tions were terminated by the addition of 2.5 ml

ŽDole’s reagent isopropyl alcoholrheptaner.1NH SO , 40:10:1 by vol. . The denatured protein2 4

Ž .was removed by centrifugation. Water 0.9 ml andŽ .n-heptane 1.6 ml were added to the supernatant

fraction and mixed on a vortex mixer. The lowerphase was washed three times with 1.5-ml portionsof n-heptane. The radioactivity in the lower layerwas counted in a radioactive scintillation counter.

Effect of L-carnitine andror doxorubicin on( )growth of solid Ehrlich carcinoma SEC in

miceIn this experiment, 2=106 EAC cells were trans-

planted subcutaneously in the right thigh of thelower limb of each mouse. Mice with a palpable

Ž 3.solid tumour mass 100 mm that developed within7 days after implantation were divided into fourgroups, ten animals each. Two groups were injectedi.p. with L-carnitine 600 mg kgy1 and an equivalentvolume of normal saline. The other two groups wereinjected with doxorubicin 16 mg kgy1 and a combi-nation of L-carnitine doxorubicin. The change in

Ž .tumour volume TV was measured every other dayusing a Vernier caliper and calculated by the fol-

w xlowing formula according to Osman et al. 20 .

Ž 3 . 2Tumour volume mm s0.52 AB

Where A is the minor tumour axis and B is themajor axis.

Determination of proteinProtein concentrations were determined by Bio-

Ž .Rad protein assay Bio-Rad, Richmond, VA, USAw xaccording to the method of Bradford 21 .

RESULTS

Figure 1 shows the effect of DOX, L-carnitine, andw 14 xtheir combination on 1- C palmitate oxidation in

Ž .myocytes. Perfusion of the heart with DOX 0.5 mM

for 10 min resulted in a significant inhibition ofŽ .palmitate oxidation 70% . On the other hand, L-

on palmitate oxidation in cardiac myocytes isolated fromperfused rat heart. Values are presented as mean"SD ofat least four separate experiments. UIndicates significant

Ž .change of DOX and L-carnitine ¨s control P-0.05 .aIndicates significant change of DOX plus L-carnitine ¨s

Ž .DOX P-0.05 . The control value for palmitate oxidationŽ y1 .was 3.12"0.45 nmol mg protein per 30 min .

Ž . Ž .carnitine 5 mM induced a significant increase 37%in palmitate oxidation. The addition of L-carnitine toDOX-perfused heart resulted in 88% reversal ofDOX-induced inhibition of palmitate oxidation inthe isolated myocytes although there was still 12%less than the control value.

Ž . ŽThe effects of DOX 0.5 mM and Enoximone 0.2. w 14 xmM on 1- C palmitate oxidation in rat heart mito-

chondria are shown in Fig. 2. In the contrary toheart myocytes, DOX has no effect on palmitateoxidation, while Enoximone caused 50% inhibitionof palmitate oxidation in a time-dependent manner.

ŽThe effect of a different concentration 0.05]2.mM of DOX on acyl-CoA synthetase, an enzyme

which is responsible for the activation of palmitateinto palmitoyl-CoA in rat heart mitochondria areshown in Fig. 3. Doxorubicin has no effect on this

Ž .enzyme. However, Enoximone 0.2 mM resulted asignificant 40% inhibition in acyl-CoA synthetase

Ž .activity in isolated rat heart mitochondria Fig. 4 .The dose]response curve for the effect of DOX

w 14 xon 1- C palmitoyl-CoA oxidation in isolated ratheart mitochondria in the presence of different con-centration of L-carnitine. As shown in Fig. 5 theoxidation of palmitoyl-CoA was significantly de-creased by doxorubicin in a concentration-depen-dent manner. However, preincubation of mitochon-dria with L-carnitine protected the heart from theinhibition of palmityol-CoA oxidation induced byDOX. This protection was dose-dependent for bothL-carnitine and DOX concentrations. Partial protec-tion by L-carnitine was obtained at higher concentra-

Pharmacological Research, Vol. 39, No. 4, 1999292

Ž . ŽFig. 2. Effect of DOX 0.5 mM and Enoximone 0.02Ž . ŽFig. 4. Effect of DOX 0.5 mM and Enoximone 0.02

.mM on palmitate oxidation in isolated rat heart mitochon-

Ž . Ž .dia, DOX v , Enoximone ^ . Values are presented asmean"SD of at least three separate experiments. UIndi-

Žcates significant change of enoximone ¨s control P-. Ž0.05 . The control value for palmitate oxidation was 15.08

y1 ."0.46 nmol mg protein per 30 min .

Ž .tion of DOX 2 mM and lower concentration ofL-carnitine. Whereas, complete reversal of the in-hibition of palmitoyl-CoA oxidation by L-carnitinewas achieved at lower concentrations of DOX andhigher concentrations of L-carnitine.

Figure 6 shows the effects of DOX, L-carnitine,and their combination on the growth of SEC. Treat-ment of the animals with 16 mg kgy1 DOX, causeda cessation of the tumour growth where the TV was

Fig. 3. Dose]response curve of the effect of DOX onacyl-CoA synthetase activity in isolated rat heart mito-

Ž .chondria. DOX v . Values are presented as mean"SDof at least three separate experiments.

.mM on acyl-CoA synthetase activity in isolated rat heartmitochondria. Values are presented as mean"SD of atleast three separate experiments. UIndicates significant

Ž .change of Enoximone ¨s control P-0.05 . The controlŽvalue for acyl-CoA synthetase activity was 3.83"0.32

y1 .mnol mg protein per min .

significantly decreased compared to the controlgroup. Administration of L-carnitine to DOX-treatedanimals, shows a similar tumour growth pattern asfor DOX-treated animals.

Fig. 5. Dose]response curve of the effect of DOX onpalmitoyl-CoA oxidation in isolated rat heart mitochon-dria in presence of varied concentrations of L-carnitine.

Ž . Ž . Ž .L-Carnitine 5 mM v , 2.5 mM ` , 0.5 mM ^ , 0.25 mMŽ .' . Values are presented as mean"SD of at least fourseparate experiments. UIndicates significant change of

Ž .DOX plus L-carnitine ¨s control P-0.05 . The controlŽvalue for palmitoyl-CoA oxidation was 9.43"0.98 nmol

y1 .mg protein per 30 min .

Pharmacological Research, Vol. 39, No. 4, 1999 293

Fig. 6. Effect of DOX, L-carnitine and their combinationŽ .on the growth of solid Ehrlich carcinoma. Control v ,

Ž . Ž . Ž .DOX ' , carnitine ` , DOXqcarnitine ^ . Data} }3 Žare presented as the TV in mm mean"SD, ns10 in

.each group . Day 1 means the day of drug administrationwhen the TV is approximately 100 mm3. UIndicates sig-nificant change of DOX and DOX plus L-carnitine ¨s

Ž .control P-0.05 . The doses of DOX and L-carnitinewere 16 mg kgy1 and 600 mg kgy1, respectively.

DISCUSSION

suggests that L-carnitine may interact with DOX forits binding site on CPT I. These results are in goodagreement with the study performed by Brady and

w xBrady 27 in isolated rat liver mitochondria. Theyhave demonstrated a concentration-dependent in-hibition of CPT I by DOX. The authors concludedthat DOX-induced cardiotoxicity may be partiallyattributed to the inhibition of CPT I. This hypothe-

w xsis was challenged by Kashfi et al. 28 who showedthat DOX-induced cardiotoxicity is unrelated to theinhibition of CPT I because many DOX-analoguesthat have lower cardiotoxicity than DOX are morepotent inhibitors of this enzyme.

The lack of inhibition of palmitate oxidation andacyl-CoA synthetase activity in isolated mitochon-

Ž .dria by DOX Figs 2 and 3 was not a matter ofinefficient separation of mitochondria, since Enoxi-mone showed inhibition of palmitate oxidation andacyl-CoA synthetase in isolated mitochondria.

These findings are consistent with previous studieswhich have demonstrated that Enoximone inhibitedacyl-CoA synthetase enzyme in isolated rat heart

w xmitochondria 26 .In this study, the inhibition of palmitoyl-CoA oxi-

dation in isolated rat heart mitochondria by DOXpoints to the CPT I as being the inhibition target ofpalmitate oxidation. Palmitoyl-CoA is a substratethat require both L-carnitine and CPT I to betransported into the mitochondria where it is oxi-dised through a b-oxidation pathway. This finding issupported by the effect of L-carnitine in reversingthe inhibition of palmitate oxidation in myocytes andpalmitoyl-CoA oxidation in isolated mitochondria.Since the reversal of the inhibition is dependent on

Ž .both L-carnitine and DOX concentration Fig. 5 , itseems that the inactivation of palmitoyltransferasesystem is due to a competition between DOX andL-carnitine for its specific site on the CPT I. Thesefindings are consistent with previous studies thatrevealed the inhibition of CPT I and CPT II by

wDOX in isolated liver and heart mitochondria 27,x28 .The different effects of DOX on palmitate oxida-

tion in mitochondria and myocytes may be due todifferences in the two models with respect to fattyacid oxidation. In myocytes, DOX inactivates CPT Iby competing with L-carnitine for its binding site onthe enzyme, and may increase the L-carnitine leakfrom myocytes. Thus, the overall effect of DOX inmyocytes is the inhibition of palmitate oxidation.Whereas, in isolated mitochondria, L-carnitine isadded in high concentrations to the assay bufferbefore the addition of DOX to overcome the compe-tition of DOX. This hypothesis is supported by thereversal of inhibition of palmitate oxidation in my-

Ž .ocytes Fig. 1 and that of palmitoyl-CoA in mito-Ž .chondria Fig. 5 by L-carnitine which is essential for

CPT I activity.Furthermore, DOX-induced significant decrease

Myocardial fatty acid utilisation is influenced bymultiple factors including plasma substrate level,hormonal meleui and myocardial perfusion. In thisstudy, perfusion of the heart with 0.5 mM DOXunder normal physiological conditions produced asignificant decrease in palmitate oxidation in isolatedcardiac myocytes. These results are consistent with

w xprevious studies 22, 23 , which showed inhibition offatty acid oxidation in both acute and chronic DOXcardiomyopathic models.

In this study, the stimulation of palmitate oxida-tion by perfusion with L-carnitine oxidation in rat

Ž .heart Fig. 1 , may be a secondary event followingwith the increase in mitochondrial transport ofpalmitate through CPT I andror to the increase inmitochondrial CoA-SHracetyl-CoA ratio. Previ-

w x w xously, Sayed-Ahmed 24 and Abdel-aleem et al. 25demonstrated that L-carnitine increased the mito-chondrial efflux of acetyl-CoA produced from car-bohydrate oxidation in the form of acetyl carnitinethrough a reaction mediated by carnitine acetyltransferase enzyme. In addition, reducing the mito-chondrial acetyl-CoA may also activate b-oxidationof palmitate by activating 3-ketoacyl-CoA thiolasewhich control the final reaction in b-oxidation of

w xfatty acids 26 . The reversal of DOX-induced inhibi-Ž .tion of palmitate oxidation by L-carnitine Fig. 1

Pharmacological Research, Vol. 39, No. 4, 1999294

in the TV of SEC, whereas, L-carnitine has no effect aorto-coronary bypass surgery. Curr Ther Res 1986;

on the tumour growth. These results are consistent

w xwith the study of Senekoowitsch et al. 29 , whoshowed that L-carnitine has no effect on the antitu-mour activity of DOX. In conclusion, these studiessuggest that DOX causes the inhibition of long-chainfatty acid oxidation. This effect may be due to inacti-vation of the CPT I by competing with L-carnitinefor its binding site andror causes the leakage ofL-carnitine outside cardiac cells. The inhibition offatty acid oxidation by DOX may play a key role inthe pathophysiology of DOX-induced cardiotoxicity.L-Carnitine causes reversal of DOX-induced inhibi-tion of palmitate oxidation suggesting that L-carni-tine may protect the myocardium against DOX-in-duced carditoxicity without altering its antitumoureffects.

ACKNOWLEDGEMENTS

The authors are indebted to Professor M. El Merza-bani for his valuable advice and for his criticalreview of the manuscript.

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