Indian Journal of Experimental Biology Vol. 40, August 2002, pp. 894-900

Protective effect of Gingko hi/aha extract against doxorubicin-induced cardiotoxicity in mice

M U R Naidu, K Vijay Kumar, I Krishna Mohan, C Sundaramt & Shashi Singht

Central Research Laboratory, Departments of Clinical Pharmacology & Therapeutics, & tpathology, Nizam's Institute of Medical Sciences, Hyderabad,

*Centre for Cellular and Molecular Biology, Hyderabad 500 082, India

Received 10 May 2001 ; revised 29 April 2002

Doxorubicin (DXR) causes dose dependent cardiotoxicity in experimental animals and in humans. In chronic doxoru­bicin cardiotoxicity model mice, the role of G. bi/oba extract (Gbe) which has an antioxidant property , was investigated. Doxorubicin treated animals showed higher mortality (68%), increased ascites, marked bradycardia, prolongation of ST and QT intervals and widening of QRS complex. Myocardial SOD and glutathione peroxidase activity were decreased and lipid peroxidation was increased. Ultrastructure of heart of DXR treated animals showed loss of myofibrils , swelling of mito­chondria, vacuolization of mitochondria. G. bi/oba extract significantly protected the mice from cardiotoxic effects of doxorubicin as evidenced by lowered mortality, ascites, myocardial lipid peroxidation, normalization of antioxidant en­zymes, reversal of ECG changes and minimal ultrastructural damage of the heart. The results indicate that administration of G. bi/oba extract protected mice from doxorubicin-induced cardiotoxicity.

Anticancer therapy is not only cytotoxic to cancer cells, but also to healthy cells. This results in a narrow therapeutic index; with adverse events frequently lim­iting the administration of optimal anticancer therapy. The anthracycline antibiotics, including doxorubicin (adriamycin), daunorubicin and epirubicin are among the most active anticancer agents used against a wide range of solid and haemopoietic malignancies I . An­thracycline-induced cardiotoxicity limits effective clinical use of these compounds. Cardiotoxicity may occur acutely with electrocardiogram changes, ar­rhythmias, or a myocarditis-pericarditis syndrome2

•

The most serious form is a chronic cumulative dose­related cardiomyopathy.

The mechanism by which anthracyclines cause cardiotoxicity and antitumor activity are thought to be different. Cardiotoxicity is thought to result from sev­eral mechanisms3

.4. The anthracycline cardiotoxicity appears to be associated with the generation of reac­tive oxygen species. Anthracyclines contain a quinone group, which undergoes electron reduction to produce a semi-quinone radical. Interaction of oxygen with this semi-quinone initiates a cascade of reactive oxy­gen species5

,6. In addition, the anthracycline-iron complex is a powerful oxidant itself and is able to initiate lipid peroxidation in the absence of oxygen free radicals7

. Anthracycline-induced injury may be multifactorial and complexB

• One mechanism com­mon to most of these is the increased oxidative stress9

.

Initial attempts at preventing anthracycline-induced cardiac damage focused on the oxygen free radical scavengers. Studies with acetyJcysteine lO and alpha­tocopherol 1 1 did not demonstrate any cardiac protec­tion in humans .. Amifostine by virtue of its potential to scavenge oxygen radicals, has been shown to re­duce doxorubicin-associated cardiotoxicity in cultured

I 12 d' . 13 M h d neonata myocytes an In mIce. ono y roxy ethylrutoside, a semisynthetic flavonoid has been shown to reduce doxorubicin cardiotoxicity in vitro and in mice '4 . In vitro studies have demonstrated that G. bi/oba extract (GBe) scavenges free radicals

,s.' 7.

GBe protected the human liver microsomes from lipid peroxidation caused by cyclosporine 'B. The extract of Gingko biloba leaves (Egb-761) is a mixture of fla­vonoid glycosides and other natural compounds. Fla­vonol glycosides and proanthocyanidine present in GBe have significant oxygen free radical scavenging activity l7. In our recent study, GBe through its anti­oxidant property protected the rats from gentamicin­induced nephrotoxicity l9. The aim of the present study was to investigate the effect of GBe against doxorubi­cin-induced cardiotoxicity in mice.

Materials and Methods Animals and treatment-Female Swiss albino

mice (mean weight 30-40 g) were housed under con­ditions of controlled temperature and 12 hr lighting cycle and fed standard diet. The animals were divided

NAIDU et al.: PROTECTIVE EFFECf OF GINGKO BILOBA EXTRACf AGAINST CARDlOTOXICITY IN MICE 895

into four groups of 16 animals each. The control ani­mals received normal saline intraperitoneally once weekly for 4 weeks. The second group received four equal injections (each containing 4 mg/kg BW doxorubicin) intraperitoneally, once weekly for four weeks (cumulative dose 16 mg/kg) and the third group received GBe (l00 mg/kg) orally every day for 4 weeks. The fourth group received GBe (100 mg/kg bw) orally, every day for 4 weeks along with four in­jections of doxorubicin similar to second group. All animals were observed for 3 weeks after the last injec­tion of doxorubicin pertaining to their body weight, general appearance, electrocardiograph (ECG) changes and mortality . At the end of 3rd week the animals were sacrificed and the heart tissue was evaluated for lipid peroxidation products, antioxidant enzymes and morphological appearance. This study protocol was approved by the Ethics Committee on animal experimentation of the Nizam's Institute of Medical Sciences, Hyderabad.

Electrocardiograph Before sacrificing the animals, ECG was recorded

under mild ether anesthesia. For ECG recording, nee­dle electrodes were inserted under the skin for the limb leads at position II and a Grass polygraph, USA was used. The ECG tracings were recorded at the pa­per speed at 100 mm/sec. Heart rate, ST interval and QT interval were determined from the ECG.

Lipid peroxidation products The degree of lipid peroxidation in heart tissue was

estimated by measuring TBARS as described previ­ousl/o. Portions of heart tissue were dissected and homogenized in PBS, pH 7.4. The homogenate were centrifuged and the supernatants were collected and precipitated with 20% trichloroacetic acid and centri­fuged. To the protein free supernatant, 0.33% thiobar­bituric acid was added and boiled for 1 hr at 95°C; the TBA reactive products were extracted in butanol and the intensity of the pink color was read at 520 nm. Freshly diluted tetramethoxy propane (Sigma Chemi­cal Co., USA) which yields malondialdehybe (MDA) was used as standard and data was expressed in nmo­les of MDAlg of heart tissue.

Estimation of antioxidant enzymes Superoxide dismutase (SOD) was estimated in the

heart tissue homogenate by cytochrome C reduction in a xanthine-xanthine oxidase generating system20. SOD activity was determined from a standard curve

of percentage of inhibition of cytochrome C reduction with a known SOD activity. Catalase activity was de­termined by the method as described earlier20 , with H20 2 (10 mM) and phosphate buffer (0.05 M, pH. 7.0) at 210 nm. A unit is defined as the amount of enzyme that catalyzed the dismutation of 1 )lmole of H20 2/min. The specific activity is expressed in units/mg protein. Glutathione peroxidase activity was measured by the NADPH oxidation method2o and ex­pressed as nmoles of NADPH oxidized to NADP/mg protein2o. Protein was determined by the method as described earlier20.

Total antioxidant activity The total antioxidant activity in plasma and heart

tissue was measured by decolorization assay21. The pre-formed radical monocation of 2,2' -azinobis-(3-ethylenzothiozoline-6-sulfonic acid) (ABTS*+) is gen­erated by oxidation of ABTS (Sigma Chemical Co., USA) with potassium persulfate and is reduced in the presence of hydrogen-donating antioxidants . Trolox (6-hydroxy-2,5,7 ,8-tetramethychroman-2-carbox y I ic acid; Aldrich Chemical Co., Gillingham, Dorset, UK), a water soluble vitamin-E analogue was used as a standard.

Electron microscopic studies For ultrastructural studies, 3 to 5 hearts in each

group were processed. Fixation of samples in 3% glu­taraldehyde in O.lM phosphate buffer for 3 hr at room temperature. Washed with PBS four times. Post fix in 1 % Cr04 in 0.1 M Phosphate buffer for 3 hr followed by washing in 0.1 M Phosphate buffer. Dehydrate in ascending grades of acetone, infiltrate in propylene oxide:resin 1: 1 mixture overnight. Embedded in pure resin and use at 60°C for 72 hours. 30-50 nm sections were picked up on Cu grids, stained in uranyl acetate and lead citrate and scanned in JEOL 1000 CX elec­tron microscope.

Statistical analysis Results are presented as mean ± SO. The signifi­

cance difference among the four groups was assessed using one way ANOV A and TUKEY'S HSD test to identify differences between the groups. Statistical significance was acceptable to a level of P<0.05.

Results Intraperitoneal administration of DXR at the total

cumulative dose of 16 mg/kg (4.0 mg/kg once per week for 4 weeks) induced clear signs of cardiac toxi­city in mice. Doxorubicin treated animals appeared

896 INDIAN J EXP BIOL, AUGUST 2002

more sick, weaker and lethargic compared to control and GBe + DXR group. Animals belonging to DXR group developed distention of the abdomen due to marked ascites, and when sacrificed, had a signifi­cantly more amount of peritoneal fluid (1.95 ±0.7ml) compared to the DXR + GBe (0.52 ± 0.3 ml) (Table 1). In addition to ascites the liver was enlarged and con­gested in all the mice who received DXR. During the 3 weeks posttreatment period, the mortality was sig­nificantly higher in the DXR group (68%) compared to GBe plus DXR group (36%) (Table 1). No deaths were observed in control or GBe group. Treatment with DXR resulted in a significant decrease in heart weight (47%) and heart to body weight ratio (37%) compared to control. In GBe + DXR group, heart to body weight ratio was similar to that of control and GBe treated group.

ECG changes Intraperitoneal administration of DXR produced

ECG changes characterised by bradycardia, prolonga­tion of ST and QT intervals (Table 2 and Fig. I). The mean heart rate in DXR-treated mice reduced to 552 ± 66 bpm than control (760 ± 82 bpm) The ST and QT intervals increased to 46 ± 26 ms and 56 ± 1.5 ms with doxorubicin respectively. GBe treatment antagonised DXR-induced bradycardia and prolongation of ST and QT intervals.

Antioxidant enzymes and TBARS products The product of lipid peroxidation measured as

TBARS in heart tissue was significantly higher in

DXR treated group (30 ± 2.5 nmoles/g of heart tissue) compared to control (19.8 ±0.6 nmoles) (Table 3). Co-administration of GBe with DXR produced a sig­nificant decrease in TBARS (22 ± 1.0 nmoles/g of heart tissue). There was no change in plasma TBARS in all the treated group indicating that DXR induced oxidative stress was limited to heart tissue. G. biloba extract, GBe plus DXR treated group showed signifi­cant increase in total antioxidant activity (Table 3). Different antioxidant enzymes were examined in heart tissue in all the groups and data are shown in Table 3. DXR produced significant decrease in SOD (24 ± 3.6 U/mg protein), glutathione peroxidase (34 ± 7.2 nmo­les/mg protein) compared to controls. In controls, the activities of SOD and glutathione peroxidase were 37 ± 7.5 U/mg protein and 55 ± 9 nmoles/mg protein respectively. The activities of SOD and glutathione peroxidase in GBe + DXR group were comparable to control.

Electron microscopic studies Morphological appearance of mitochondria, sar­

coplasmic reticulum, sarcomeres, myofibrils and in­tercalated disks from control and GBe treated mice were normal. Hearts from DXR treated animals show loss of myofibrils, swelling of mitochondria, vacuoli­zation of the cytoplasm and dilation of the sarcotubu­lar system. In addition to swelling of mitochondria, there was disarrangement and disruption of cristae (Fig. 2b). The ultrastructure of hearts from GBe + DXR group showed minimal dilation of the

Table 1-Effect of G.bi/oba extract on doxorubicin-induced changes in mortality, heart weight and ascites

[Values are mean ± SD ; n = 16 for mortality data; for other parameters, n=16 in control and GBe, n=5 in doxorubicin, n=1O in DXR+GBe group]

Parameter Control Doxorubicin

Mortality (%) Nil 68% Ascitic fluid volume (ml) Nil 1.95 ± 0.78* Heart weight (mg) 197.00 ± 14 105.00 ± 18* HeartJbody wt ratio x 102 5.40±0.1 4.20 ± 0.4*

* p < 0.05 vs control group; ** P < 0.05 vs doxorubicin group. DXR, doxorubucin ; GBe, Gingko biloba extract.

GBe DXR+GBe

Nil 36% Nil 0.52 ± 0.3**

195.00 ± 8.40 150.00 ± 22** 5.40 ± 0.21 5.30 ± 1.0**

Table 2-Effect of G.biloba extract and doxorubucin on ECG changes in mice

[Values are mean ± SD; n = 6 in control, GBe and DXR+GBe group; n=5 in doxorubicinl

Parameter Control

Heart rate (bpm) 760.0 ± 82 ST interval (msec) 13.2 ± 0.5 QT interval (msec) 23.6 ± 0.8

*p < 0.05 vs control; **p < 0.05 vs doxorubicin DXR, doxorubicin ; GBe, Gingko biloba extract

Doxorubicin

552.0 ± 66* 46.0 ± 2.6* 56.0 ± 1.5*

GBe DXR+GBe

756.0 ± 82 736.0 ± 76** 14.0 ± 0.6 18.6 ± 1.0** 24.1 ±0.8 26.0 ± 9.0**

NAIDU et al.: PROTECTIVE EFFECT OF GINGKO BILOBA EXTRACT AGAINST CARDIOTOXICITY IN MICE 897

~ig. l-Electr?cardiograph .(ECG) of heart from (a) control mice; (b) mice treated with doxorubicin showing prolongation of ST and QT mtervals; (c) mice treated with doxorubicin + G. biloba extract prevented the prolongation of ST and QT intervals.

sarcotubular system and occasional vacuolization of the cytoplasm (Fig. 2c).

Discussion Cancer chemotherapy with anthracyclines of which

DXR is the main representative is limited by its car­diomyopathy, developing in animals and patients after cumulative dosing. Intraperitoneal administration of DXR produced cardiotoxicity in mice, which is char­acterised by increased mortality, decreased body weight : heart weight ratio, and accumulation of as­citic fluid. In rats, DXR produced increased mortality and ascites as compared to controt22

-24

. Administration of GBe lowered DXR induced mortality in mice. Similarly, lipid lowering drug and antioxidant, probu­col and potent antioxidant melatonin has been shown to reduce doxorubicin-induced deaths in rats23

•24

. Sev­eral authors have described the changes in ECG of laboratory animals with anthracyline and have dem­onstrated that, the severity of ECG changes paralled with known doxorubicin-cardiotoxicity in man25

•26

. In our experiment, DXR produced marked bradycardia, prolongation of ST and QT interval. Doxorubicin has profound influence on the shape of the ECG. The QT and ST intervals were increased, this is in accordance with other reports in animals I4

.27

. ECG changes noted in our experiment were similar to those reported in

. 28 GB mice. e was able to prevent the development of ECG changes induced by doxorubicin. Parachini and coworkers have demonstrated the protective effect of several spin traps by measuring changes in ST and QT interval in rats29

. Changes in ST interval have been

found to correlate with degree of cardiotoxicity in 30 d . 31 I . rat an nuce . n a study, Venuruton, which con-

tains 10% of monoHER, was able to completely pre­vent the cardiotoxicity in mouse. Both ICRF-187 a iron chelating agent and monoHER an antioxidant was able to protect against DXR-induced ECG changes 13

.

Myocardial damage is specific to all anthracycline antibiotics, including myofibrillar degeneration, mito­chondrial dilatation, cellular vacuolization and finally

d 32.33 . myocyte ropout . As seen In the present study, DXR treatment caused significant ultrastructural changes including marked myofibrillar loss, vacuoli­zation and mitochondrial damage. In mice treated with GBe, these DXR-induced ultrastructure changes were minimal suggesting protection of cellular dam­age by GBe. In rats DXR exhibited myocardial le­sions mainly cytoplasmic vacuolization, myofibrillar loss and swelling of mitochondria, disarrangement and mitochondrial degeneration, formation of ly­sosomal bodies, dilation of sarcotubular sytem25

.26.

Doxorubicin-induced ultrastructural changes in rats treated with melatonin and probucol were almost in­distinguishable from the control, with regular myofi­bril arrangement and weakly preserved mitochondria, only in few specimens, peri mitochondrial edema was observed24

• Reduction in scores of myocardial le­sions-induced by DXR was reported with Venoruton and ICRF-18734

. In mice, we also found similar in scores of myocardial lesion with GBe.

In mice and rats, DXR significantly increased MDA levels in myocardial tissue24

•25

. In our study,

898 INDIAN J EXP BIOL, AUGUST 2002

a

Fig. 2-Ultrastructurc of heart from (a) control mice treated only with normal saline; (b) myocardi al ti ssuc trcated with doxorubicin showing loss of myofibrils (MF), swelling of mitochondria (M), vacuolization of cytoplasm (V) and di lat ion of sarcotubular systcm (S): (c) heart tissue treatcd with doxorubicin + G. bi/aba extract showing minimal dilation of the sarcotubular system and occasional vacuoli­zat ion of the cytoplasm.

NAIDU et al.: PROTECTIVE EFFECT OF GINGKO BlLOBA EXTRACT AGAINST CARDIOTOXICITY IN MICE 899

Table 3-Effect of doxorubicin and G.biloba extract on myocardial lipid peroxidation, total antioxi­dant activity and antioxidant enzymes

[Values are mean ± SD; n = 6 in control, GBe and DXR+GBe, n=5 in Doxorubicin group]

Parameter Control DXR GBe DXR+GBe

MDA (nm/g of heart) 19.80 ± 0.6 30.00 ± 2.5* 19.00 ± 0.8 22.00 ± 1.0** Total antioxidant activity:

i. Plasma (nm/L) 1.89 ± 0.07 1.85 ± 0.07 2.25 ± 0.40t 2.28 ± 0.14t ii. Heart tissue (nm/mg protein) 1.70 ± 0.Q7 1.44 ± 0.11 * 2.37 ± O.4t 2 .20 ± 0.36t

Superoxide dismutase 37.00 ± 7 .5 24.00 ± 3.6* 39.00 ± 8.0 38.00 ± 10** Catalase 6 .20 ± 1.0 5.80 ± 1.4 6.28 ± 1.2 6.00 ± 1.4 Glutathione peroxidase 55.00 ± 9.0 34.00 ± 7.2* 56.00 ± 8.6 56.00 ± 8.8**

* P < 0.05 vs control; ** P < 0.05 vs doxorubicin; t P < 0.05 vs control and doxorubicin . DXR, doxorubicin; GBe, Gingko bi/oba extract. SOD is expressed as U/mg of protein; Catalase, U/mg protein; Glutathione peroxidase, nm/mg protein)

DXR treated mice showed increase in heart tissue MDA levels with decrease in levels of SOD and glu­tathione peroxidase. GBe prevented the DXR-induced changes in MDA and enzyme levels. Increase in total antioxidant activity in plasma and heart tissue in GBe and GBe plus DXR treated groups may be due to the flavonoid content in GBe. Probucol, a cholesterol lowering agent and an antioxidant prevented DXR­induced changes in glutathione peroxidase and MDA levels in rat heart23

. In another study in rats, increase in heart tissue TBARS level induced by DXR was prevented by melatonin, a potent hydroxyl radical scavenger24. In vitro studies have demonstrated that GBe scavenges free radicals l8 . It reduces lipid peroxi­dation in rat microsomes and human liver micro­somes. Flavonoids have long been recognized as ex­cellent scavengers of superoxide, hydroxyl and per­oxyl radical and as potent inhibitors of lipid peroxida­tion35-37. The Folium Gingko contain a wide variety of phytochemicals, the major constituents are flavonoids of mono, di and tri glycosides and coumaric acid es­ters that are based on flavonols, kaempferol and quercetin38

In the present study the cardioprotective effect of GBe may be due to its flavonoid content. Monohy­droxy ethylrutoside a semisynthetic flavonoid protects against DXR-induced cardiotoxicity, without influ­encing the antitumor activity of that drug in vitro and in viv039 . Similarly , amifostine by virtue of its poten­tial to scavenge oxygen radical has been shown to reduce DXR-toxicity in rat heart myocytes l2 and mice l3. The protection from DXR-induced cardiotox­icity by GBe may also have some other mechanisms. The gingkolides B is known antagonists of platelet activating factor (PAF/o. PAF is a potent inducer of oxygen free radicals and gingkolides B specifically inhibited PAF-induced degranulation, superoxide

generation and chemotaxis of neutrophils41 . In isolated rat hearts, calcineurin inhibitor FK 506, the PAF antagonist plus G. biloba extract (EGb 761) synergis­tically produce cardioprotective effects42 .

In conclusion, we observed good cardioprotective effect of GBe against chronic doxorubicin-induced cardiotoxicity in mice thus GBe merits further investi­gation as a possible cardioprotective agent against doxorubicin-induced chronic cardiotoxicity in patients.

Acknowledgement The authors thank MS.Reshma Shetty for technical

assistance and Mis Ranbaxy Research Laboratories, New Delhi, India for providing powder of Gingko biloba extract

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