Effect of Methimazole Treatment on Doxorubicin-induced CardiotoxicityEffect of methimazole treatment on doxorubicin-induced cardiotoxicity Abdel-Moneim M.Osman Ph.D., Marwa M.Nemnem M.Pharm.Sc.,Amany A.Emam,Ph.D.Wael T.Abou daif and Mohamed T.Khayyal Ph.D. aAuthor for correspondence: Abdel-Moneim M.Osman, Pharmacology Dept.,College of Medicine, KAAU, Jeddah,P.O.box 80202, Saudi Arabia( Fax +96626408404, e.mail. moneimosman@hotmail.com Key Words : Methimazole, Doxorubicin, cardiotoxicity, interaction Running title: Protective effect of methimazole 1. IntroductionDoxorubicin is the most widely used drugs in the treatment of a variety of human neoplasm (1,7).Unfortunately with increasing the use of doxorubicin, acute as well as chronic cumulative dose-dependent cardiomyopathy have been recognized as the major limiting factor of doxorubicin chemotherapy (21, 6). Several mechanisms for the doxorubicin-induced cardio toxicity have been proposed, including membrane lipid per oxidation, free radical formation (38,23) ,mitochondrial damage (19) and iron-dependent oxidative damage to macromolecules(27).Oxygen radicals are apparently involved in all mechanisms proposed. Methimazole is an anti-thyroid drug, containing –SH group. Although it is mainly used in the treatment of hyperthyroidism (9), it has been found to be effective in reducing the toxicity of several nephrotoxic chemotherapeutic drugs e.g. cephaloridine , (32, 3), gentamycin (12) and cisplatin (28).The effect has been attributed to its antioxidant and free radical scavenging capabilities, leading to a reduction of oxidative stress in the kidney without affecting the cytotoxic activity of the drug. Since the renal protective effect of methimazole is largely based on its free radical scavenging capability and its antioxidant effect, and since the cardio toxic effect of doxorubicin is mainly mediated through free radical generation( 34), it was considered of interest to study whether the antioxidant effect of methimazole on the kidney might the also be extend to the heart. First we determined the cardio toxicity of doxorubicin in presence and absence of methimazole. Secondly, the uptake of doxorubicin in heart and tumor tissue was evaluated in albino mice. Thirdly, the effect of methimazole treatment on the antitumor activity of doxorubicin was determined to answer the question does the protective effect of methimazole could be at the expense of abolishing the antitumor activity of doxorubicin? 2. Materials and Methods 2.1 Drugs Doxorubicin was obtained as adriablastina vials from NCI store, Cairo, Egypt (Pharmacia & Upjohn Co.S.P.A, Milan, Italy), Methimazole was purchased from Sigma chemical Co. (St.Louis, MO, USA).All other chemicals included in the current study were from Merck (Darmstadt, Germany) . 2.2 animal and tumor The investigation was performed on albino mice weighing 18-20 gm from our institute outbreed stock. The animals were housed in a conditioned atmosphere and kept on a standard diet and water ad labium .A line of Ehrlich as cites carcinoma cells (EAC) was supplied through the courtesy of Prof.Dr.A.M.Osman Via Dr.C.Benckhuisen, AVL, Amsterdam, The Netherlands. And maintains in our laboratory by weekly i.p. transplantation of 2.5x106 cells/mouse. 2.3 Measurement of the cardio toxic effect of doxorubicin Forty female Swiss albino mice were equally divided into four groups .Group I ,in which mice injected with normal saline (0.2 ml/20 gm body weight, i.p. for 11 days every other day and considered as a control group. Group II, mice were injected with methimazole 40 mg/kg ,i.p., on days 1,7, and 11 from the beginning of treatment ( depending to serve as a methimazole –positive control. Group III, mice were injected with doxorubicin in a total dose of 18 mg/kg, i.p. divided in six equal doses (3 mg/kg each) every other day over a period of two weeks to serve as a doxorubicin-positive control .Group IV, mice were injected with methimazole in a dose of 40 mg/kg, i.p., 30 min before the animals were given doxorubicin in a dose of 3 mg/kg , i.p. only on days 1, 7,and 11 from the beginning of doxorubicin treatment. After 24 hours from the end of treatment regimen, blood was collected from retro-orbital plexus of the animals and serum was separated. Heart from each group were dissected out and immersed in 10% formalin solution for histopathological investigation. LDH-Iso, CK-MB and tropnin-I activities were determined according to the methods of (35, 36 and 13,respectively). 2.4 Uptake of doxorubicin in various tissues. Swiss albino mice were injected S.C. in the right hind limb with saline containing 0.25x106 Ehrlich carcinoma cells. After 8 days, the animals were divided into 3 groups. Group I was given saline and considered as a control group, group II is treated as in group III of the section 2.3,group III is treated as in group IV of section 2.3..Plasm, heart, and tumor of each group were dissected out and their content of doxorubicin was determined as mentioned by Bachur et al (2). 2.5Determination of thyroid hormones (T3 and T4) Animals were treated as mentioned in the section of 2.3 Serum was separated and thyroid hormones were determined follow the method of Yalow and Berson (37) using the principle of radioimmunoassay. 2.6 Evaluation of antitumor activity Female Swiss albino mice were inoculated s.c. in the right hind limb as mentioned in section 2.4. Only mice with palpable solid tumor mass (100 mm3) that develop within 8 days after tumor inoculation were used. They were divided at random into four groups and drug treatment was given as in section 2.4.The change in tumor volume was measured every other day using Vernier Caliper and calculated by the following formula according to Papadopoulos et al (30) 4II (A/2)2 X (B/2) Tumor volume (mm3) = --------------------------------------- 3 Where A is the minor tumor axis B is the major tumor axis 2.7 Statistical analysis Data are presented as means ± s.e.m. Individual groups were compared using the two-tailed student’s t-test as appropriate. Multiple group comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey-Kramer test for post-hoc analysis. Statistical significance was accepted at a level of p< 0.05. 3. Results 3.1 Effect of methimazole pretreatment on doxorubicin-induced cardio toxicity. Table (1) shows the effect of methimazole pretreatment on doxorubin-induced changes in cardiac enzymes in normal mice. Treatment of mice with six equal doses of doxorubicin (3 mg/kg x6 every other day) resulted in 2.6, 3 and 10.5 fold increases in the activities of CK-MB, LDH-Iso and troponin-I, respectively. Pretreatment with methimazole (40 mg/kg,i.p.) 30 min before doxorubicin, resulted in partial reversal of doxorubicin –induced increase in the cardiac enzymes. At the same time methimazole treatment alone, showed no significant difference in the serum level of the three enzymes tested compare to the control group. Histopathological investigation of the heart tissue after treatment confirmed the biochemical data, where doxorubicin treatment showed swollen cardiac muscle fibers, interstitial edema and inflammatory infiltration. Pretreatment with methimazole showed minimal interstitial edema and inflammatory exudates (fig.6). 3.2.Effect of methimazole pretreatment on the uptake of doxorubicin in various tissues of tumor-bearing animals Doxorubicin treatment showed a peak plasma concentration of 0.2 ug/ml, within half an hour from administration. the drug disappeared rapidly from the plasma, dropping by 60% in the first hour and 90% within two days (table 2). The drug was rapidly distributed to the heart (11.4 ug/gm tissue) and other tissues (liver and kidney, data not shown).Pretreatment with methimazole resulted in 3-fold increase in the plasma level of doxorubicin and a decrease in its level in cardiac tissue (fig. 1, table 2). Concerning the level in tumor tissues, Pretreatment with methimazole resulted in a small but insignificant increase in the peak concentration of doxorubicin after one hour from its administration, but it dropped by about 42% after 2 days.. 3.3. Effect of methimazole pretreatment on serum thyroid hormones of mice treated with doxorubicin Methimazole pretreatment did not significantly affect the level of T3 and T4 in the serum of doxorubicin –treated animals (table 4). 4. Discussion Doxorubicin is an excellent antitumor drug for treating several types of solid cancer, leukemia and lymphomas. However, acute or chronic toxicity is the major limiting complication of doxorubicin, whereas, acute cardio toxicity presents transient symptoms, such as arrhythmias, the chronic toxicity can evolve into irreversible cardiomyopathy, which affects approximately 30-40% of the patients who receive 500 mg/mm2 total dose (22). In the current study, doxorubicin was injected 6 times at 3 mg/kg, resulting a cumulative dose of 18 mg/kg nearly equivalent to 500 mg/mm2 of doxorubicin in a 50 kg human ( 20). Our experimental protocol mimics clinical situation where multiple doses of anthracycline are given to patients. Administration of doxorubicin showed increase in the activities of cardiac enzyme, LDH-Iso, CK-MB and troponin-I. The elevation of the level of different enzymes by doxorubicin treatment is probably a reflection that the drug induces cardiac toxicity, where, LDH-Iso, CK-MB and troponin-I are rather specific for myocardial damage ( 8, 18 and 10, respectively). The present biochemical data has been confirmed by the histopathological changes induced by doxorubicin, where the heart tissue showed swollen cardiac muscle fibers, interstitial edema and inflammatory infiltration (fig.5). Several hypotheses have been postulated to explain the mechanism of doxorubicin-induced cardio toxicity including generation of reactive oxygen (33). For that reason, methimazole which has sulfur nucleophile in its structure, showed a protective role against doxorubicin-induced Renal, hepatic (data not shown) and cardiotoxicty (table1 and figure 6 ). Although, methimazole has not been previously reported to reduce cardio toxicity, it was shown to control tachycardia and arrhythmia associated with elevated circulating thyroid hormone (15). Recently, it has been reported that methimazole could improve the heart in cardiac arrhythmias in sub clinical hyperthyroidism (5). These results are indirectly supported by reduction of doxorubicin concentration in the heart tissue by pretreatment with methimazole (fig.1).This in part protects the myocardium from accumulation of doxorubicin inside the cardiac cells and accordingly may prevent the accumulation of free radicals. Our results also showed that treatment with methimazole induced a nearly 3 folds increase in the plasma level of doxorubicin (table 2). This agrees with that previously reported by Gerweck et al.(17), Rancia et.al (31), and Frezard et al.(16) , where, they found a decrease the accumulation of doxorubicin in tissue and its increases in plasma which might be a function of pH gradient or P-glycoprotein over expression. Knowing that doxorubicin is a weak base, its accumulation and toxicity in tissues has been investigated as a function of extra cellular pH. Thus its level in tissues increases by increasing the extra cellular pH and consequently any drug which decrease the extra cellular pH may decrease the accumulation of doxorubicin in the tissues ( 17, 31, 16 ) On the other hand, chronic toxicity of doxorubicin was reported to be more likely related to non-reactive oxygen species ( 29, 24). This was explained due to failure of some antioxidants to show a protective effect against chronic cardiomyopathy (4, 25). Moreover, Ferreira et al ( 14)suggested an antioxidant role of doxorubicin rather than acting as prooxidant in experimental animals. This discrepancy could be due to the nature of treatment protocol of the experimental system used , the method of determining the oxidative stress in the different system. Under certain circumstances, the co-administration of compounds with antioxidative properties may reduce the antitumor activity od doxorubicin ( 11, 26).So, it was mandatory to investigate whether methimazole could interfere with the cytotoxic activity of doxorubicin?. In the present study, methimazole pretreatment did not interfere with cytotoxic activity of doxorubicin (fig.2).This was clear from the data showing that uptake of doxorubicin in the tumor cells was not affected by methimazole pretreatment ( table 3) and the percent survivor of tumor-bearing animals treated with doxorubicin was not different fron that treated with combination of methimazole and doxorubicin. 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(1956) Metallon-zymes and myocardia.infarction N.England J.Med. 255:449-453 36Wuerzburg,U.,Heinrich,N.,Lang,H.,Prellwitz,W.,Neumeir,D.,Knedel,M.( 1976) Bestimmung der Aktivitfit von Kreatin-kinase MB in serum unter verwendung inhbierender anti-k6rper Klin.Wochenschz 54:357-360 37-Yalow,R. and Berson,S. Introduction and general considerations.In:Principles of competitive protein binding assay,Odell,W. and Daughaday,W.(Eds.).J.B.Lippincott,Philadelphia PP 1-19, 1971 38-Yin,X.,Wu,H.,Chen,Y,kang,YJ.( 1998) Induction of antioxidant by adriamycin in mouse heart. Biochem Pharmacol 56:87-93. Legends to figure: Figure 1 : Effect of methimazole pretreatment on the uptake of doxorubicin by heart tissue of Ehrlich carcinoma bearing mice. Each column represents the mean of 5 mice± s.e.m. *denotes significant change from doxorubicin treated group at p<0.05,Statistical analysis was carried by student’s t-test for unpaired data. Figure 2 : Effect of doxorubicin, methimazole and their combination on the growth of solid Ehrlich carcinoma –bearing mice.Each point represents the mean tumor volume (mm3) of 11 mice±s.e.m.. Statistical analysis was carried by student’s unpaired t-test. Figure 3 : Heart of control albino mouse treated with saline showing normal myocardial histology (H&E stain 200x) Figure (4) Heart of albino mouse treated with methimazole for 3 doses 40mg/kg each on day 1, 7, 11 from beginning of treatment. No myocardial lesion is apparent (H&E stain 200x) Figure(5).Heart of an albino mouse treated with doxorubicin for 6 doses, 3 mg/kg each spread over 2 weeks, showing swollen cardiac muscle fibers, interstitial edema and inflammatory infiltration (H&E stain 200x). Figure (6).Heart of albino mouse treated with methimazole 30 min before doxorubicin administration on days 1, 7, 11 from the beginning of doxorubicin treatment, showing minimal interstitial edema and inflammatory exudates (H&E stainx400) Tables (1):Cardio toxicity indices following several doses of Doxorubicin in mice pretreated with methimazolea Treatment groups Seum cardiac enzyme levels LDH-iso CK-MB (U/L) Troponin-I (ng/ml) Control 903± 71 328±25 1.25±0.08 Doxorubicin 2361+79* 3447±530* 3.6±0.60* Methimazole 1141±0.42# 868±161# 1.45±0.20# Methimazole+Dox 1869±76*,# 2041±267*,# 1.87±0.27# A-Six equal doses of doxorubicin (3 mg/kg) every other day were administered in normal mice pretreated with methimazole ( 40 mg/kg,i.p.30 min before doxorubicin) at days 1 , 7 and 11 All values are presented as means of 10 mice ± s.e.m. *Indicates significant change from respective control values at p<0.05. #Indicates significant change of methimazole treated groups (with or without doxorubicin) from doxorubicin treated group at p<0.05. Statistical analysis was carried By one way ANOVA followed by Tukey-Kramer multiple comparison test. Table (2) Effect of methimazole pretreatment on plasma doxorubicin level of Ehrlich carcinoma bearing micea Doxorubicin concentration (ug/ml plasma) Time (hrs) Doxorubicin gp. Methimazole+doxorubicin gp 0.5 0.20±0.05 0.66±.06* 1 0.08±0.01 0.14±.009* 3 0.06±0.02 0.12±.012 24 0.03±0.01 .096±.015* 48 0.02±.0.007 .062±.003* a Legends as in table (1) All values are presented as mean of 5 animals ± s.e.m. *Indicates significant change from doxorubicin treated group at p<0.05.Statistical analysis was carried by student’s t-test for unpaired data. Table (3) Effect of methimazole pretreatment on the uptake of doxorubicin in tumor of Ehrlich carcinoma bearing micea. Doxorubicin concentration (ug/gm tumor protein) Time (hrs) Doxorubicin gp. Methimazole+doxorubicin gp. 0.5 0.040±0.01 0.068±0.01 1 0.070+ 0.01 .077±0.008 3 0.060±0.017 0.58±0.001 24 0.058±.004 0.55±0.007 48 0.056±.004 0.45±0.003 alegends as in table (1) All values are presented as means of 5 mice± s.e.m. Statistical analysis was carried by student’s t-test for unpaired data. Table (4) Effect of doxorubicin and methimazole on serum thyroid hormones of Normal micea. Treatment groups Serum hormone levels T3 (ng/dl) T4 (ug/dl) Control 100±6.8 |