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PROTECTIVE EFFECT OF THYMOQUINONE AGAINST DOXORUBICIN–INDUCED CARDIOTOXICITY IN RATS: A POSSIBLE MECHANISM OF PROTECTION
Pharmacological Research, Vol. 41, No. 3, 2000 doi:10.1006rphrs.1999.0585, available online at http:rrwww.idealibrary.com on
PROTECTIVE EFFECT OF THYMOQUINONE AGAINST DOXORUBICIN – INDUCED CARDIOTOXICITY IN RATS: A POSSIBLE MECHANISM OF PROTECTION MAHMOUD N. NAGI and MAHMOUD A. MANSOURU Department of Pharmacology, College of Pharmacy, King Saud Uni¨ ersity, P.O Box 2457, Riyadh 11451, Saudi Arabia Accepted 6 August 1999
Administration of thymoquinone Ž10 mg kgy1 dayy1, p.o.. with drinking water starting 5 days before a single injection of doxorubicin Ž15 mg kgy1 i.p.. and continuing during the experimental period ameliorated the doxorubicin-induced cardiotoxicity in rats. This protection was evidenced from the significant reduction in serum enzymes: lactate dehydrogenase elevated level, 24 h and creatine phosphokinase elevated levels, 24 h and 48 h after doxorubicin administration. The cardiotoxicity of doxorubicin has been suggested to result from the generation of superoxide free-radical. The protective action of thymoquinone was examined against superoxide anion radical either generated photochemically, biochemically or derived from calcium ionophore ŽA23187. stimulated polymorphonuclear leukocytes. The results indicate that thymoquinone is a potent superoxide radical scavenger, scavenging power being as effective as superoxide dismutase against superoxide. In addition thymoquinone has an inhibitory effect on lipid peroxidation induced by Fe 3qrascorbate using rat heart homogenate. The superoxide scavenging and anti-lipid peroxidation may explain, in part, the protective effect of thymoquinone against doxorubicin-induced cardiotoxicity. Q 2000 Academic Press KEY
WORDS:
thymoquinone, lactate dehydrogenase, creatine phosphokinase, cardiotoxicity, doxorubicin.
INTRODUCTION Doxorubicin Žadriamycin. is a quinone-containing antitumour antibiotic that is used to treat several types of cancer w1x. However, its clinical use has been restricted by dose-limiting cardiotoxicity which lead to cardiomyopathy and heart failure w1]3x. At present, oxygen radical-induced injury of membrane lipids is considered to be the most important factor responsible for the development of doxorubicin-induced cardiotoxicity w4]7x. The mechanism involves one-electron reduction of doxorubicin for generation of a semiquinone radical. Doxorubicin semiquinone radical reduces oxygen to produce superoxide and to regenerate doxorubicin. The net result of this process is that doxorubicin catalyzes the reduction of oxygen by NADPH, to form a superoxide radical, which is subsequently reduced to hydrogen peroxide ŽH 2 O 2 . by the antioxidant enzyme, superoxide dismutase. In the presence of Fe 2q, the H 2 O 2 is further reduced to the extremely reactive hydroxyl radical Ž. OH., which can react with polyunsaturated
fatty acids to yield lipid hydroperoxide. This initiates a lipid radical chain reaction, which can cause oxidative damage to cell membranes w5x. Thymoquinone ŽTQ., the main constituent of the volatile oil from Nigella sati¨ a seeds has been reported to inhibit eicosanoid generation in leucocytes and non-enzymatic peroxidation in ox brain phospholipid liposome w8x. Previous studies have shown that pretreatment with TQ protected organs against oxidative damage induced by a variety of free radical generating agents, including carbon tetrachloride w9, 10x, cis-platin w11x and doxorubicin w12x. In this study, we investigated the effect of TQ on doxorubicin induced cardiotoxicity in rats, the ability of TQ to scavenge superoxide radical and the effect of TQ on the non-enzymatic lipid peroxidation of rat heart homogenate induced in ¨ itro by Fe 3qrascorbate.
MATERIALS AND METHODS
Chemicals U
Corresponding author.
1043]6618r00r030283]07r$35.00r0
Thymoquinone, superoxide dismutase ŽSOD., Q 2000 Academic Press
Pharmacological Research, Vol. 41, No. 3, 2000
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Table I Effect of thymoquinone on the elevated activities of serum enzymes induced by doxorubicin. Thymoquinone (10 mg kgI1 day I1 p.o.) was given 5 days before doxorubicin (15 mg kg I1 i.p.) treatment Time after treatment
Treatment
Serum enzyme acti¨ ities Mean" SE LDH
CPK
24 h
Control 223 " 22.9 156.4" 12 TQ 216.6" 21.2 149.6" 18.6 Doxorubicin 420 " 19.9UUU 1123 " 16.5UUU TQq Dox 292 " 33.9† 425 " 14.8¶
48 h
TQ Doxorubicin TQq Dox.
220 " 29.4 340 " 52.9 229 " 23
143 " 14.6 476 " 80.5UUU 259 " 34.08‡
UUU
Significantly different from control group. †Significantly different from doxorubicin. ‡ P- 0.05, §P- 0.01, ¶P- 0.001.
glucose oxidase, NADH, phenazine methosulphate ŽPMS., nitroblue tetrazolium ŽNBT., calcium ionophore ŽA23187. were obtained from Sigma ŽSt. Louis, MO, USA.. Doxorubicin was provided as a gift from Farmitalia, Carlo Erba, Milan, Italy. All
the remaining chemicals were of the highest analytical grade. The drugs were dissolved in normal saline just before use.
Animal treatment Adult male albino rats weighing 220]250 g were obtained from the Experimental Animal Care Centre of King Saud University, Riyadh, KSA. Animals were divided into four groups of six and allowed ad libitum access to food ŽPurina chow. and tap water. One group received doxorubicin Ž15 mg kgy1 , i.p. and the other group received doxorubicin Ž15 mg kgy1 , i.p. and thymoquinone Ž10 mg kgy1 dayy1, p.o.. in the drinking water starting 5 days before doxorubicin and continuing throughout the duration of the experiment Ž48 h.. Two control groups received drinking water with or without thymoquinone.
Assessment of cardiotoxicity Blood samples were drawn from the orbital plexus, under light ether anaesthesia, into non-heparinized capillary tubes at 24 and 48 h following doxorubicin treatment. Serum was separated by centrifugation at
Fig. 1. Ža. Thymoquinone scavenging of superoxide radical as a function of its concentration measured by reduction of NBT with NADH and PMS. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
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4000 rpm for 4 min and stored at y208C until analysis. LDH and CPK levels were assayed using commercially available reagents based on the method of Buhl and Jackson w13x and Gruber w14x, respectively.
Assays for superoxide anion radical Phenazine methosulphate (PMS), NADH and NBT method [15]. The reaction mixture contained in a total volume of 1 ml, 25 mM Tris]HCl, pH 8.8, 50 m M NBT, 100 m M NADH and different concentrations of TQ or SOD. The reaction was initiated by adding 5 m M PMS and the production of formazan was followed at 560 nm at 208C for 5 min. Data were plotted as a percentage of the remaining absorbance in the presence of SOD or TQ. Glucose]glucose oxidase and NBT method [16]. The reaction mixture contained in a total volume of 1.0 ml, 10 mM glycine]NaOH, pH 9.5, 0.2 mM glucose, 50 m M NBT and different concentrations of TQ or SOD. The reaction was initiated by the addition of 10 units of glucose oxidase and the initial rate of the reaction was measured from the rate of change of absorbance at 560 nm at room temperature. Data were plotted as a percentage of the remaining activity in the presence of TQ or SOD.
285
Ribofla¨ in, methionine and NBT method [17]. The reaction mixture contained in a total volume of 2 ml, 50 mM Tris]HCl, pH 7.8, 10 mM methionine, 5 m M riboflavin, 50 m M NBT and different concentrations of TQ or SOD. Reduction of NBT after 10 min of illumination was measured at 560 nm. Data were plotted as a percentage of the remaining absorbance in the presence of SOD or TQ. Calcium ionophore (A23187) stimulated polymorphonuclear leucocytes (PMNL) and NBT [18]. The ability to reduce NBT was assayed by incubating rat PMNL 10 = 10 6 cells with 0.1% NBT Ždissolved in phosphate-buffer saline, pH 7.4. the reaction was initiated by the addition of calcium ionophore Ž10 m M. for 20 min at 378C. Termination of the assay was done by adding 0.6 ml of glacial acetic acid Žfinal assay volume 3 ml. into which the reduced NBT dye was extracted and the extract was read at 560 nm. Data were plotted as a percentage of remaining absorbance in the presence or absence of SOD or TQ.
Assay of non-enzymatic lipid peroxidation in rat heart homogenate [8 ] The heart of a normal male rat was isolated,
Fig. 2. Ža. Thymoquinone scavenging of a superoxide radical as a function of its concentration measured by NBT reduction with riboflavin and methionine. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
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washed with saline, weighed and homogenized in ice-cold saline and 10% homogenate was made in a Branson sonifier Ž250, VWR, Scientific, Danbury, CT, USA.. The reaction mixture contained varying amounts of TQ, 0.75 ml phosphate buffer Ž50 mM, pH 7.4., 50 m l rat heart homogenate Ž10%., 0.1 ml of 1 mM ferric chloride and 0.1 ml of 1 mM ascorbic acid. The tubes were incubated at 378C for 30 min and the extent of peroxidation was measured by thiobarbituric acid ŽTBA. test. To the above test tube, the following were added 0.1 ml butylated hydroxytolune ŽBHT. Ž2% wrv. to stop further lipid peroxidation, 1.0 ml TBA Ž1% wrv in 0.05 M NaOH. and 1.0 ml trichloroacetic acid Ž2.8% wrv. and placed in a waterbath at 808C for 20 min. At the end of incubation, the tubes were cooled and centrifuged for 5 min at 3200 rpm. The chromogen was extracted with 2.0 ml n-butanol and absorbance was read at 532 mn. Tubes containing reagent blank, heart homogenate and subjected to TBA test served as control tubes. Data were plotted as a percentage of the remaining absorbance in the presence of TQ.
Statistical analysis
Data are expressed as Žmeans " SEM.. Statistical
comparison between different groups were done by using one-way analysis of variance ŽANOVA. followed by Tukey]Kramer multiple comparisons test. Significance was accepted at P- 0.05.
RESULTS
Effect of thymoquinone on the ele¨ ated serum LDH and CPK acti¨ ities induced by doxorubicin administration Serum LDH and CPK were significantly Ž P0.001. elevated 24 h after a single injection of doxorubicin reaching 420 q 19.9 and 1123 q 16.5 U ly1 , respectively, as compared with the control group. Pretreatment with TQ decreased significantly the enzyme activities by 30.5 and 62.3%, respectively ŽTable I.. After 48 h, the serum CPK level was still significantly elevated over the control values Ž P0.001.. TQ reduced significantly CPK activity ŽTable I..
Sca¨ enging of superoxide anion radical by thymoquinone Superoxide anions were generated in enzymatic
Fig. 3. Ža. Thymoquinone scavenging of superoxide radical as a function of its concentration measured by NBT reduction with calcium ionophore stimulated polymorphnuclear leukocytes. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
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Fig. 4. Ža. Thymoquinone scavenging of superoxide radical as a function of its concentration measured by NBT reduction using glucose]glucose oxidase. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
and non-enzymatic systems. In all systems used in this study, SOD inhibited the reduction of NBT in a concentration-dependent manner. Similar inhibition curves were obtained when SOD was replaced by TQ ŽFigs. 1]4.. The IC 50 values for TQ ranged from 8 to 20 m M in non-cellular system and 60 m M in the cellular system.
Effect of thymoquinone on lipid peroxidation induced by Fe 3qr ascorbate in vitro The antioxidant nature of TQ as an inhibitor of in-¨ itro non-enzymatic lipid peroxidation was examined, using rat heart homogenate. TQ inhibited lipid peroxidation in a concentration-dependent manner ŽFig. 5.. The IC 50 for TQ was 5 m M.
DISCUSSION In the present study, a single dose of doxorubicin Ž15 mg kgy1 i.p.. induced cardiotoxicity manifested biochemically by a significant increase in serum LDH after 24 h and serum CPK after 24 and 48 h. These results are consistent with the previous studies reported by other investigators w2, 4, 7x that doxoru-
bicin induced cardiotoxicity in normal rats. The results of the present study clearly demonstrate that TQ, when given in the drinking water protects rats from doxorubicin-induced cardiotoxicity as evidenced from the significant reduction in serum enzymes, LDH and CPK ŽTable I.. These results confirm our previously published data on the protective action of TQ on doxorubicin-induced cardiotoxicity in mice w12x. The biochemical mechanisms involved in the development of doxorubicin cardiotoxicity have been well studied and documented. It is now believed that the formation of superoxide radical from doxorubicin recycling is a crucial factor in the pathogenesis of doxorubicin cardiotoxicity w4]7x. An important finding in this study is that TQ shows a superoxide radical scavenging activity, like superoxide dismutase, using four different systems for the generation of superoxide ŽFigs. 1]4. The IC 50 values for TQ in these assays were in the micromolar range which indicates that TQ is a good scavenger of superoxide radical. The effect of TQ on lipid peroxidation, a free radical mediated process, can provide some information to its antioxidant capability. The studies on the rat heart homogenate showed that TQ had
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Fig. 5. Effect of thymoquinone on non-enzymatic lipid peroxidation induced by Fe 3qrascorbate in ¨ itro. The assay conditions are described under Materials and Methods. Results are expressed as a percentage of control. Each point represents a mean of triplicate experiments.
an inhibitory effect, the magnitude of which was concentration-dependent ŽFig. 5.. These results are consistent with alleviation of doxorubicin mediated cardiotoxicity by other antioxidants, such as coenzyme Q 10 w19x, N-acetylcysteine w20x, alpha tocopherol w21x, vitamin A w22x, probucol w23x and butylated hydroxyanisol w24x. It has been shown that TQ pretreatment protected other organs against oxidative damage induced by a variety of free radical generating agents, including carbon tetrachloride w9, 10x and cisplatin w11x. On the basis of these findings, we suggested that TQ might play an important role as an endogenous antioxidant and could also be applicable as a cytoprotective agent against tissue damage mediated by chemotherapeutic agents. The results of this study, when combined with the knowledge that TQ has low toxicity w25x, support a potential role of TQ as an antioxidant drug in doxorubicin cardiotoxicity.
REFERENCES 1. Booser DG, Hortobagyi GN. Anthracyclin antibiotics in cancer therapy-focus on drug resistance. Drugs 1994; 47: 223]58. 2. Satliel E, McGuire W. Doxorubicin Žadriamycin. cardiomyopathy. Wist J Med 1983; 139: 332]41. 3. Young RC, Ozols RF, Myers CE. The anthracyclin antineoplastic drugs. N Engl J Med 1981; 305: 139]53 4. Myers CE, McGuire WP, Liss RH, Ifrim I, Grotzinger K, Young RC. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science 1977; 197: 165]7. 5. Mimnaugh EG, Trush MA, Bhatangar M, Gram TE. Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin. Biochem Pharmacol 1985; 34: 847]56. 6. Sarvazyan NA, Askari A, Huang WH. Effects of doxorubicin on cardiomyocytes with reduced level of superoxide dismutase. Life Sci 1995; 57: 1003]10.
7. Singal PK, Siveski-ILiskovic N, Hill M, Thomas T, Li T. Combination therapy with probucol prevents adriamycin-induced cardiomyopathy. J Mol Cell Cardiol 1995; 27: 1055]63. 8. Houghton PJ, Zarka RS, Delas Heras B, Hoult JR. Fixed oil of Nigella Sati¨ a and derived thymoquinone inhibit eicosanoids generation in leukocytes and membrane lipid peroxidation. Planta Med 1995; 61: 33]6. 9. Al-Gharably NM, Badary OA, Nagi MN, AlShabanah OA, Al-Sawaf HA, Al-Rikabi AC, Al-Bekairi AM. Protective effect of thymoquinone against carbon tetrachloride-induced hepatotoxicity in mice. Res Commun Pharmacol Toxicol 1997; 2: 41]50. 10. Nagi MN, Alam K, Badary OA, Al-Shabanah OA, Al-Sawaf HA, Al-Bekairi AM, Thymoquinone protects against carbon-tetrachloride induced hepatotoxicity via an antioxidant mechanism. Biochem Mol Biol Int 1998; 47: 153]9. 11. Badary OA, Nagi MN, Al-Shabanah OA, Al-Sawaf HA, Al-Sohaibani MO, Al-Bekairi AM. Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity. Can J Physiol Pharmacol 1997; 75: 1356]61. 12. Al-Shabanah OA, Badary OA, Nagi MN, Al-Garably N, Al-Rikabi A, Al-Bekairi AM. Thymoquinone protects against doxorubicin-induced cardiotoxicity without compromising its antitumor activity. J Exp Clin Cancer Res 1998; 17: 193]8. 13. Buhl SN, Jackson KY. Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate to pyruvate and pyruvate to lactate reactions in human serum at 25, 30 and 37 C. Clin Chem 1978; 24: 828]31. 14. Gruber W. Inhibition of creatine kinase activity by Ca2q and reversing effect of EDTA. Clin Chem 1978; 24: 177]84. 15. Nikishimi M, Rao NA, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 1972; 46: 849]54. 16. Nagi MN, Al-Bekairi AM, Al-Sawaf HA. Spectrophotometric assay for superoxide dismutase based on the nitroblue tetrazolium reduction by glucose ] glucose oxidase. Biochem Mol Biol Int 1995; 36: 633]8.
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17. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to polyacrylamide gel. Anal Biochem 1971; 44: 276]87. 18. Das UN, Begin ME, Ells G, Huang YS, Horrobin DF. Polyunsaturated fatty acids augment free radical generation in tumor cells in ¨ itro. Biochem Biophys Res Commun 1987; 145: 15]24. 19. Choe JY, Combs AB, Folkers K. Prevention of coenzyme Q 10 of the electrocardiographic changes induced by adriamycin in rats. Res Commun Chem Pathol Pharmacol 1979; 23: 199]202. 20. Doroshow JH, Looker GY, Ifrim 1, Myers CE. Prevention of doxorubicin cardiac toxicity in the mouse by N-acetylcysteine. J Clin In¨ est 1981; 68: 1053]64. 21. Geetha A, Sankar R, Marar T, Devi CS. aTocopherol reduces doxorubicin-induced toxicity in rats histological and biochemical evidence. Ind J Physiol Pharmacol 1990; 34: 94]8.
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22. Tesoriere L, Ciaccio M, Valenza M, Bongiorno A, Maresi E, Albiero R, Liverea MA. Effect of vitamin E administration on resistance of rat heart against doxorubicin-induced cardiotoxicity and lethality. J Pharmacol Exp Therapy 1994; 269: 430]6 23. Siveski-IIiskovic N, Hill M, Chow D, Singal P. Probucol protects against adriamycin cardiomyopathy without interfering with its antitumor effect. Circulation 1995; 91: 10]5. 24. Vora J, Khaw B, Narula J, Boroujerdi M. Protective effect of butylated hydroxyanisol on adriamycininduced cardiotoxicity. J Pharm Pharmacol 1996; 48: 940]4. 25. Badary OA, Al-Shabanah OA, Nagi MN, Al-Bekairi AM, El-Mazar MM. Acute and subchronic toxicity of thymoquinone in mice. Drug De¨ elop Res 1998; 44: 56]61.
PROTECTIVE EFFECT OF THYMOQUINONE AGAINST DOXORUBICIN – INDUCED CARDIOTOXICITY IN RATS: A POSSIBLE MECHANISM OF PROTECTION MAHMOUD N. NAGI and MAHMOUD A. MANSOURU Department of Pharmacology, College of Pharmacy, King Saud Uni¨ ersity, P.O Box 2457, Riyadh 11451, Saudi Arabia Accepted 6 August 1999
Administration of thymoquinone Ž10 mg kgy1 dayy1, p.o.. with drinking water starting 5 days before a single injection of doxorubicin Ž15 mg kgy1 i.p.. and continuing during the experimental period ameliorated the doxorubicin-induced cardiotoxicity in rats. This protection was evidenced from the significant reduction in serum enzymes: lactate dehydrogenase elevated level, 24 h and creatine phosphokinase elevated levels, 24 h and 48 h after doxorubicin administration. The cardiotoxicity of doxorubicin has been suggested to result from the generation of superoxide free-radical. The protective action of thymoquinone was examined against superoxide anion radical either generated photochemically, biochemically or derived from calcium ionophore ŽA23187. stimulated polymorphonuclear leukocytes. The results indicate that thymoquinone is a potent superoxide radical scavenger, scavenging power being as effective as superoxide dismutase against superoxide. In addition thymoquinone has an inhibitory effect on lipid peroxidation induced by Fe 3qrascorbate using rat heart homogenate. The superoxide scavenging and anti-lipid peroxidation may explain, in part, the protective effect of thymoquinone against doxorubicin-induced cardiotoxicity. Q 2000 Academic Press KEY
WORDS:
thymoquinone, lactate dehydrogenase, creatine phosphokinase, cardiotoxicity, doxorubicin.
INTRODUCTION Doxorubicin Žadriamycin. is a quinone-containing antitumour antibiotic that is used to treat several types of cancer w1x. However, its clinical use has been restricted by dose-limiting cardiotoxicity which lead to cardiomyopathy and heart failure w1]3x. At present, oxygen radical-induced injury of membrane lipids is considered to be the most important factor responsible for the development of doxorubicin-induced cardiotoxicity w4]7x. The mechanism involves one-electron reduction of doxorubicin for generation of a semiquinone radical. Doxorubicin semiquinone radical reduces oxygen to produce superoxide and to regenerate doxorubicin. The net result of this process is that doxorubicin catalyzes the reduction of oxygen by NADPH, to form a superoxide radical, which is subsequently reduced to hydrogen peroxide ŽH 2 O 2 . by the antioxidant enzyme, superoxide dismutase. In the presence of Fe 2q, the H 2 O 2 is further reduced to the extremely reactive hydroxyl radical Ž. OH., which can react with polyunsaturated
fatty acids to yield lipid hydroperoxide. This initiates a lipid radical chain reaction, which can cause oxidative damage to cell membranes w5x. Thymoquinone ŽTQ., the main constituent of the volatile oil from Nigella sati¨ a seeds has been reported to inhibit eicosanoid generation in leucocytes and non-enzymatic peroxidation in ox brain phospholipid liposome w8x. Previous studies have shown that pretreatment with TQ protected organs against oxidative damage induced by a variety of free radical generating agents, including carbon tetrachloride w9, 10x, cis-platin w11x and doxorubicin w12x. In this study, we investigated the effect of TQ on doxorubicin induced cardiotoxicity in rats, the ability of TQ to scavenge superoxide radical and the effect of TQ on the non-enzymatic lipid peroxidation of rat heart homogenate induced in ¨ itro by Fe 3qrascorbate.
MATERIALS AND METHODS
Chemicals U
Corresponding author.
1043]6618r00r030283]07r$35.00r0
Thymoquinone, superoxide dismutase ŽSOD., Q 2000 Academic Press
Pharmacological Research, Vol. 41, No. 3, 2000
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Table I Effect of thymoquinone on the elevated activities of serum enzymes induced by doxorubicin. Thymoquinone (10 mg kgI1 day I1 p.o.) was given 5 days before doxorubicin (15 mg kg I1 i.p.) treatment Time after treatment
Treatment
Serum enzyme acti¨ ities Mean" SE LDH
CPK
24 h
Control 223 " 22.9 156.4" 12 TQ 216.6" 21.2 149.6" 18.6 Doxorubicin 420 " 19.9UUU 1123 " 16.5UUU TQq Dox 292 " 33.9† 425 " 14.8¶
48 h
TQ Doxorubicin TQq Dox.
220 " 29.4 340 " 52.9 229 " 23
143 " 14.6 476 " 80.5UUU 259 " 34.08‡
UUU
Significantly different from control group. †Significantly different from doxorubicin. ‡ P- 0.05, §P- 0.01, ¶P- 0.001.
glucose oxidase, NADH, phenazine methosulphate ŽPMS., nitroblue tetrazolium ŽNBT., calcium ionophore ŽA23187. were obtained from Sigma ŽSt. Louis, MO, USA.. Doxorubicin was provided as a gift from Farmitalia, Carlo Erba, Milan, Italy. All
the remaining chemicals were of the highest analytical grade. The drugs were dissolved in normal saline just before use.
Animal treatment Adult male albino rats weighing 220]250 g were obtained from the Experimental Animal Care Centre of King Saud University, Riyadh, KSA. Animals were divided into four groups of six and allowed ad libitum access to food ŽPurina chow. and tap water. One group received doxorubicin Ž15 mg kgy1 , i.p. and the other group received doxorubicin Ž15 mg kgy1 , i.p. and thymoquinone Ž10 mg kgy1 dayy1, p.o.. in the drinking water starting 5 days before doxorubicin and continuing throughout the duration of the experiment Ž48 h.. Two control groups received drinking water with or without thymoquinone.
Assessment of cardiotoxicity Blood samples were drawn from the orbital plexus, under light ether anaesthesia, into non-heparinized capillary tubes at 24 and 48 h following doxorubicin treatment. Serum was separated by centrifugation at
Fig. 1. Ža. Thymoquinone scavenging of superoxide radical as a function of its concentration measured by reduction of NBT with NADH and PMS. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
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4000 rpm for 4 min and stored at y208C until analysis. LDH and CPK levels were assayed using commercially available reagents based on the method of Buhl and Jackson w13x and Gruber w14x, respectively.
Assays for superoxide anion radical Phenazine methosulphate (PMS), NADH and NBT method [15]. The reaction mixture contained in a total volume of 1 ml, 25 mM Tris]HCl, pH 8.8, 50 m M NBT, 100 m M NADH and different concentrations of TQ or SOD. The reaction was initiated by adding 5 m M PMS and the production of formazan was followed at 560 nm at 208C for 5 min. Data were plotted as a percentage of the remaining absorbance in the presence of SOD or TQ. Glucose]glucose oxidase and NBT method [16]. The reaction mixture contained in a total volume of 1.0 ml, 10 mM glycine]NaOH, pH 9.5, 0.2 mM glucose, 50 m M NBT and different concentrations of TQ or SOD. The reaction was initiated by the addition of 10 units of glucose oxidase and the initial rate of the reaction was measured from the rate of change of absorbance at 560 nm at room temperature. Data were plotted as a percentage of the remaining activity in the presence of TQ or SOD.
285
Ribofla¨ in, methionine and NBT method [17]. The reaction mixture contained in a total volume of 2 ml, 50 mM Tris]HCl, pH 7.8, 10 mM methionine, 5 m M riboflavin, 50 m M NBT and different concentrations of TQ or SOD. Reduction of NBT after 10 min of illumination was measured at 560 nm. Data were plotted as a percentage of the remaining absorbance in the presence of SOD or TQ. Calcium ionophore (A23187) stimulated polymorphonuclear leucocytes (PMNL) and NBT [18]. The ability to reduce NBT was assayed by incubating rat PMNL 10 = 10 6 cells with 0.1% NBT Ždissolved in phosphate-buffer saline, pH 7.4. the reaction was initiated by the addition of calcium ionophore Ž10 m M. for 20 min at 378C. Termination of the assay was done by adding 0.6 ml of glacial acetic acid Žfinal assay volume 3 ml. into which the reduced NBT dye was extracted and the extract was read at 560 nm. Data were plotted as a percentage of remaining absorbance in the presence or absence of SOD or TQ.
Assay of non-enzymatic lipid peroxidation in rat heart homogenate [8 ] The heart of a normal male rat was isolated,
Fig. 2. Ža. Thymoquinone scavenging of a superoxide radical as a function of its concentration measured by NBT reduction with riboflavin and methionine. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
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washed with saline, weighed and homogenized in ice-cold saline and 10% homogenate was made in a Branson sonifier Ž250, VWR, Scientific, Danbury, CT, USA.. The reaction mixture contained varying amounts of TQ, 0.75 ml phosphate buffer Ž50 mM, pH 7.4., 50 m l rat heart homogenate Ž10%., 0.1 ml of 1 mM ferric chloride and 0.1 ml of 1 mM ascorbic acid. The tubes were incubated at 378C for 30 min and the extent of peroxidation was measured by thiobarbituric acid ŽTBA. test. To the above test tube, the following were added 0.1 ml butylated hydroxytolune ŽBHT. Ž2% wrv. to stop further lipid peroxidation, 1.0 ml TBA Ž1% wrv in 0.05 M NaOH. and 1.0 ml trichloroacetic acid Ž2.8% wrv. and placed in a waterbath at 808C for 20 min. At the end of incubation, the tubes were cooled and centrifuged for 5 min at 3200 rpm. The chromogen was extracted with 2.0 ml n-butanol and absorbance was read at 532 mn. Tubes containing reagent blank, heart homogenate and subjected to TBA test served as control tubes. Data were plotted as a percentage of the remaining absorbance in the presence of TQ.
Statistical analysis
Data are expressed as Žmeans " SEM.. Statistical
comparison between different groups were done by using one-way analysis of variance ŽANOVA. followed by Tukey]Kramer multiple comparisons test. Significance was accepted at P- 0.05.
RESULTS
Effect of thymoquinone on the ele¨ ated serum LDH and CPK acti¨ ities induced by doxorubicin administration Serum LDH and CPK were significantly Ž P0.001. elevated 24 h after a single injection of doxorubicin reaching 420 q 19.9 and 1123 q 16.5 U ly1 , respectively, as compared with the control group. Pretreatment with TQ decreased significantly the enzyme activities by 30.5 and 62.3%, respectively ŽTable I.. After 48 h, the serum CPK level was still significantly elevated over the control values Ž P0.001.. TQ reduced significantly CPK activity ŽTable I..
Sca¨ enging of superoxide anion radical by thymoquinone Superoxide anions were generated in enzymatic
Fig. 3. Ža. Thymoquinone scavenging of superoxide radical as a function of its concentration measured by NBT reduction with calcium ionophore stimulated polymorphnuclear leukocytes. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
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Fig. 4. Ža. Thymoquinone scavenging of superoxide radical as a function of its concentration measured by NBT reduction using glucose]glucose oxidase. Each point represents the mean of three different experiments. Žb. Same as in Ža. except thymoquinone was replaced by different concentrations of superoxide dismutase.
and non-enzymatic systems. In all systems used in this study, SOD inhibited the reduction of NBT in a concentration-dependent manner. Similar inhibition curves were obtained when SOD was replaced by TQ ŽFigs. 1]4.. The IC 50 values for TQ ranged from 8 to 20 m M in non-cellular system and 60 m M in the cellular system.
Effect of thymoquinone on lipid peroxidation induced by Fe 3qr ascorbate in vitro The antioxidant nature of TQ as an inhibitor of in-¨ itro non-enzymatic lipid peroxidation was examined, using rat heart homogenate. TQ inhibited lipid peroxidation in a concentration-dependent manner ŽFig. 5.. The IC 50 for TQ was 5 m M.
DISCUSSION In the present study, a single dose of doxorubicin Ž15 mg kgy1 i.p.. induced cardiotoxicity manifested biochemically by a significant increase in serum LDH after 24 h and serum CPK after 24 and 48 h. These results are consistent with the previous studies reported by other investigators w2, 4, 7x that doxoru-
bicin induced cardiotoxicity in normal rats. The results of the present study clearly demonstrate that TQ, when given in the drinking water protects rats from doxorubicin-induced cardiotoxicity as evidenced from the significant reduction in serum enzymes, LDH and CPK ŽTable I.. These results confirm our previously published data on the protective action of TQ on doxorubicin-induced cardiotoxicity in mice w12x. The biochemical mechanisms involved in the development of doxorubicin cardiotoxicity have been well studied and documented. It is now believed that the formation of superoxide radical from doxorubicin recycling is a crucial factor in the pathogenesis of doxorubicin cardiotoxicity w4]7x. An important finding in this study is that TQ shows a superoxide radical scavenging activity, like superoxide dismutase, using four different systems for the generation of superoxide ŽFigs. 1]4. The IC 50 values for TQ in these assays were in the micromolar range which indicates that TQ is a good scavenger of superoxide radical. The effect of TQ on lipid peroxidation, a free radical mediated process, can provide some information to its antioxidant capability. The studies on the rat heart homogenate showed that TQ had
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Fig. 5. Effect of thymoquinone on non-enzymatic lipid peroxidation induced by Fe 3qrascorbate in ¨ itro. The assay conditions are described under Materials and Methods. Results are expressed as a percentage of control. Each point represents a mean of triplicate experiments.
an inhibitory effect, the magnitude of which was concentration-dependent ŽFig. 5.. These results are consistent with alleviation of doxorubicin mediated cardiotoxicity by other antioxidants, such as coenzyme Q 10 w19x, N-acetylcysteine w20x, alpha tocopherol w21x, vitamin A w22x, probucol w23x and butylated hydroxyanisol w24x. It has been shown that TQ pretreatment protected other organs against oxidative damage induced by a variety of free radical generating agents, including carbon tetrachloride w9, 10x and cisplatin w11x. On the basis of these findings, we suggested that TQ might play an important role as an endogenous antioxidant and could also be applicable as a cytoprotective agent against tissue damage mediated by chemotherapeutic agents. The results of this study, when combined with the knowledge that TQ has low toxicity w25x, support a potential role of TQ as an antioxidant drug in doxorubicin cardiotoxicity.
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