Effect of Digoxin on Chick Embryos with Hyperthyroidism Induced by L-Thyroxin

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1 ORIGINAL ARTICLE Yoshiyama Y et al., AATEX 11(2), , Effect of Digoxin on Chick Embryos with Hyperthyroidism Induced by L-Thyroxin Yuji Yoshiyama 1, Tomoko Misaki 2 and Takashi Sugiyama 2 1 Division of Clinical Pharmacy, Kyoritsu University of Pharmacy, , Shibakoen, Minato-ku, Tokyo , Japan 2 Division of Pathophysiology, Center for Clinical Pharmacy and Clinical Sciences, School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo ,Japan Abstract The objective of this study was to demonstrate that the chick embryo with hyperthyroidism induced by treatment with L-thyroxin (TRX) was a feasible model as an alternative to animal model and to examine the pharmacological effects of digoxin using this model. When 0.5 or 1.0 µg/egg of TRX was injected into the eggs, the 16th day-chick embryos showed some characteristic changes including an increased heart rate (tachycardia), an increased relative heart weight (cardiac hypertrophy) and relative thyroid weight (thyroid hypertrophy), and an increased concentration of plasma T3 and T4. These findings suggest that TRX-treated chick embryos may be a model of the heart diseases associated with hyperthyroidism. When 50 µg/egg of digoxin was injected into the TRX-treated eggs, negative chronic action reduced. This fact suggests that digoxin decreased the sensitivity to TRX in the heart of chick embryos with hyperthyroidism as well as in dog and man. In conclusion, the TRX-treated chick embryos may prove to be an alternative to animal experiments to predict unexpected effects of cardiovascular drugs. Key words: Chick embryo; hyperthyroidism; thyroxin; digoxin; electrocardiogram Introduction Many patients with hyperthyroidism show tachycardia associated with congestive heart failure (Shirani et al, 1993). Digoxin is used as a therapeutic drug in these patients (Larsen, 1988). It has been reported that digoxin at a higher dose is usually administered to obtain therapeutic effects in patients with hyperthyroidism because of the decreased sensitivity to digoxin (Huffman et al, 1977). Accordingly, to predict the unexpected pharmacological responses to cardiotonics, such as digoxin, experimental animals with peculiar sensitivity of heart such as hyperthyroidism or hypothyroidism are required. For these purposes, a hyperthyroid animal model by administering repeated doses of L-thyroxin (TRX) to rats and dogs was reported (Sanford et al, 1978, Klein, 1988, Suga et al, 1991). The hyperthyroidism with tachycardia and cardiac hypertrophy is induced by frequent administration of TRX in rats and dogs, and many cardiovascular drugs have been evaluated using this model (Skelton, 1982, Niizoe et al, 1984, Angeras et al, 1987). With the recent concern for animal rights, experimental studies using mammals have been limited in number and methods. Thus, based on social acceptance, experimental studies using chick embryos have drawn attention. In order to develop alternative methods, we have studied the biological effects of drugs on the cardiovascular system of chick embryos using physiological techniques (Yoshiyama et al,, 2004a, 2004b, 2003, Sugiyama et al, 1996). And we have also reported that the chick embryonic model of hypothyroidism induced by treatment with thiamazole can be used to examine the pharmacological and toxicological 105

2 effects of cardiovascular drugs (Sugiyama et al, 2000). Since the thyroid grand is present in all vertebrates, a convenient abnormal cardiac model would be beneficial for examing the pharmacological actions and toxicity of cardiotonics (Turner, 1971). In this study, we examined to produce a hyperthyroidism model in chick embryos, which resulted from abnormalities in the thyroid grand by an injection of TRX, and to know whether this model can be used as an alternative to animal experiments to examine the pharmacological effects of cardiotonics. Materials and Methods Materials L-thyroxin sodium salt (TRX), urethane and -chloralose were purchased from Sigma Chemical Inc. (St. Louis, MO, U.S.A.). Digoxin was from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Amerlex-MAB free-t3 kit and free-t4 kit were from Oso Clinical Diagnostics (Tokyo, Japan). TRX was dissolved to the desired concentration with physiological saline and sterilized through a membrane filter (0.2 µm pore size, Tokyo Roshi Kaisha, Ltd., Tokyo, Japan). Digoxin was diluted to the desired concentration with sterilized saline. Urethane (450 mg/ml) and -chloralose (45 mg/ml) were dissolved in physiological saline. Eggs and incubation Fertile eggs from White Leghorn chickens were obtained from Ohmiya Poultry Science (Saitama, Japan) and incubated at 37.5 ± 0.2 C at a relative humidity of about 65.5%, and turned automatically every hour (P-1 type, Showa Incubator Laboratory, Saitama, Japan). -chloralose [4.5 mg/egg]) was performed into the air sac of eggs to record the stable ECG waves in 16-day-old embryos in which R R intervals could be clearly analyzed. ECGs were recorded 0 to 60 min after the drug injection, and heart rate was determined from R R intervals. Data represented the percentage of changes in the heart rate from each 0 time. Four small holes every 90 degrees on "the equator", one small hole on "the south pole" and one small hole on "the north pole" were made on each fertilized egg by an electric drill and sealed with Paraffin (m.p. 60 ). Specially designed needle electrodes were inserted into the appropriate holes of "the equator" and "the south pole". The two needles on "the equator" were used as a bipolar lead of the embryonic heart, and the needle on "the south pole" was used as a ground lead. These needles were connected to the memory oscilloscope (VC-11, Nihon Koden Co., Tokyo, Japan). ECGs were recorded as bipolar waves between two needles on a recorder (PowerLab System, ADInstruments Japan Co., Tokyo, Japan) (Sugiyama et al, 1996). Observations in the embryos treated with TRX After recording of ECG waves, blood samples were collected from vitelline vein using a heparinized disposal syringe (Fig.1). Next, embryos were removed from eggshells, and body, heart and thyroid were removed and weighed. The collected blood was centrifuged at 3000 rpm for 10 min and plasma was used to determine the concentrations of free-t3 and free-t4, and to measure Injection of TRX and digoxin into fertile eggs TRX (0.5 or 1.0 µg/egg) was injected into the albumen of eggs on the 9th day of incubation according to a method previously reported (Sugiyama et al, 1985). Sterilized physiological saline (0.2 ml/egg) was used as control. Digoxin (25, 50 or 75 µg/egg) was injected into the air sac of control or TRX-treated eggs on the 16th day of incubation. Electrocardiogram (ECG) recording systems from chick embryos Anesthetic injection of urethane ([45 mg/egg]+ Fig.1 Blood collection from vitelline vein of fertile egg on the 16th day of incubation 106

3 TRX-treated eggs (0.5 or 1.0 µg/egg) on the 16th day of incubation, ECG waves were recordd from 0 to 30 min after injection. Statistical analysis Statistical significance was evaluated by analysis of variance followed by Dunnett s multiple comparison when the effects of different concentrations of drugs were examined, while Student s t-test was employed when two groups were compared. P-values less than 0.05 was considered significant. Fig. 2 Heart rate of TRX-untreated and TRX-treated embryos TRX was injected into the albumen of eggs on the 9th day of incubation and the embryos were observed on the 16th day of incubation. HR was calculated from RR intervals on ECG waves. Data are the mean ± SE of 42 embryos. *: p<0.05, significantly difference to the TRX-untreated embryos. digoxin concentration. The samples were frozen (-80 ) for the determination. Observations in the embryos treated with digoxin After a single injection of digoxin alone was carried into the saline-treated eggs (control) or Results Changes of the HR and organ weights in chick embryos treated with TRX As shown in Fig. 2, the HR in chick embryos treated with 0.5 or 1.0 µg/egg of TRX significantly increased compared with that of TRX-untreated embryos. The weights of the body and the relative weights of heart and thyroid gland to body weight are shown in Fig. 3. The body weight in TRX-treated embryos decreased dependently on doses but was not significantly different from TRX-untreated embryos. The relative weights of the heart and thyroid gland to body weight at 0.5 and 1.0 µg/egg of TRX were significantly increased compared with those of TRX-untreated embryos. Fig. 3 Boday weight, ratios of heart and thyroid weights to body weight in TRX-untreated and TRX-treated embryos TRX was injected into the albumen of eggs on the 9th day of incubation and the embryos were observed on the 16th day of incubation. Data are the mean ± SE of 42 embryos. *: p<0.05, significantly different from the TRX-untreated embryos. 107

4 Fig. 4 Levels of serum f-t3 and f-t4 in TRX-untreated and TRX-treated embryos TRX was injected into the albumen of eggs on the 9th day of incubation and blood was collected from vitelline vain of the treated eggs on the 16th day of incubation. Data are the mean ± SE of 42 embryos. *: p<0.05, significantly different from the TRX-untreated embryos. Plasma free-t3 and T4 concentration in chick embryos treated with TRX Both Free-T3 and T4 concentrations in plasma in chick embryos treated with 0.5 or 1.0 µg/egg of TRX significantly increased compared with those of TRX-untreated embryos (Fig. 4). Effects of digoxin on the HR in control and TRX-treated embryos The HR in chick embryos treated with digoxin alone decreased dependently on doses (Fig. 5). And digoxin induced arrhythmias including AV block 30 min after injection (Fig.6). When 50 µg/egg of digoxin was injected into the TRX-untreated eggs, the ECG waves showe a negative chronotropic action. However, when 50 µg/egg of digoxin was injected into the TRX-treated eggs, negative chronic action reduced (Fig.7). Fig. 5 Changes of heart rate in control chick embryos treated with digoxin Digoxin was injected into the air sac of fertile eggs on the 16th day of incubation. ECG waves were recorded for 30 min after injection and the heart rate was calculated from RR intervals. Data are the mean ± SE of 6 embryos. * ; p<0.05, significantly different from the control (Digoxin 0 µg/egg). Discussion We have reported that the chick embryonic model of hypothyroidism induced by treatment with thiamazole can be used to examine the pharmacological and toxicological effects of cardiovascular drugs (Sugiyama et al, 2000). In this study, we demonstrated that the chick embryo with hyperthyroidism induced by treatment with L-thyroxin (TRX) was a feasible model as an alternative to animal model and examined the pharmacological effects of digoxn using this model. When 0.5 or 1.0 µg/egg of TRX was injected into the eggs, the 108

5 Fig. 6. Arrhythmia on chick embryos treated with digoxin Digoxin (50 mg/egg) was injected into the air sac of egg on the 16th day of incubation. AV block was observed 30 min after injection. 16th day-chick embryos showed some characteristic changes including an increased HR (tachycardia), an increased relative heart weight (cardiac hypertrophy) and relative thyroid weight (thyroid hypertrophy), and an increased concentration of plasma T3 and T4. These findings showed high similarities between rat or human and chick embryos. The results obtained suggest that TRX-treated chick embryos may be a model of the heart diseases associated with hyperthyroidism. Fig.7 Changes of the HR in TRX-untreated and TRX-treated embryos with digoxin Digoxin (50mg/egg) was injected into the air sac of control and TRX-treated Eggs on the 16th day of incubation. The HR was calculated from RR intervals 30 min after injection. Data are the mean ± SE of 5 embryos. *; p<0.05, significantly different from the control (TRX-untreated embryos). However, injection of 2 µg/egg of TRX showed a tendency to be decreased the body, heart and thyroid gland weights to the control and induced bradycardia, which are considered to be due to the over dose of TRX. We examined the pharmacological effects of digoxin using the chick embryo with hyperthyroidism induced by treatment with TRX 0.5 or 1.0 µg/egg. Growth, development and differentiation on cells in various organs of mammals and chick embryos are regulated by thyroid hormone (Freeman, 1974). Thyroid gland in chick embryos is situated near the base of neck (Romanof, 1960). Romanoff reported that the first indication of thyroid was at the end of day 2 of incubation. It is important to determine the suitable period and site for the injection of drugs into fertile eggs. We already reported that the same agent can exhibit significantly different effects in chick embryos when given at different sites or on different days of incubation (Sugiyama et al, 1982). Therefore, in the preliminary experiment, a fixed dose of TRX was injected into the albumen of eggs from 5th day to 14th day of incubation. A change of HR on the 9th day of incubation was stronger than that by other injection days (unpublished). According to these findings, in this study, we injected TRX into the albumen of fetile eggs on the 9th day of incubation. Digoxin has been widely used as a therapeutic drug in patients with congestive heart failure, atrial fibrillation or atrial flutter. However, digoxin at a higher dose is usually given to patients with hyperthyroidism than patients with hypothyroidism because of their reduced sensitivity to this drug. And we examined to know whether this fact could be proved using the chick embryo. The HR of chick embryos decreased 109

6 dependently on doses of digoxin. When 50 µg/egg of digoxin was injected into the saline-treated eggs, the ECG waves showed a negative chronotropic action as in mammals. However, when 50 µg/egg of digoxin was injected into the TRX-treated eggs, negative chronic action reduced. It is suggested that digoxin decreased the sensitivity to TRX in the heart of chick embryos with hyperthyroidism as well as in dog and man. Marrow et al. reported that the myocardial responses of ouabain, cardiotonic alkaloid derivatives were influenced in dog with hyperthyroidism (Marrow et al, 1963). Thyroid hormones increase protein syntheses of Na +,K + -ATPase and atrial natriuretic peptide (ANP), and the activity of these proteins is higher in hyperthyroidism than in the normal conditions (Larsen, 1988). However, the cardiac glycosides, digoxin and ouabain are drugs frequency used in a cardiac insufficiency treatment. From previously reported findings, the main action mechanism of digoxin in hyperthyroidism is the suppression of the Na+, K+ATPase activity (Forfar and Caldwell, 1985). Although further investigations are necessary to clarify the mechanism underlying the cardiac function induced by digoxin in hyperthyroidism of chick embryos treated with T-4, this model may offer in alternative research possibilities to predict unexpected effects of cardiovascular drugs on patients with hyperthyroidism. Acknowledgements The authors express their gratitude to Ms. Michiko Shoiri and Ms. Tomomi Ikeda for their assistance in this study. This study was supported in part by the Japanese Society of Alternatives to Animal Experiments (JSAAE). References Angeras U, Jagenburg R, Lindstedt G, Hasselgren PO., (1987) Effects of beta-blocking agents on urinary excretion of 3-methylhistidine during experimental hyperthyroidism in rats, Eur. Surg. Res., 19, Forfar, J.C., and Caldwell, G.C., (1985) Hyperthyroid heart disease, Clin Endocrinol Metab., 14, Freeman, B.M., (1960) Hormones in Development of the Avian Embryo, pp , John Wily & Sons, Inc., New York. Huffman, D.H., Klaassen, C.D., and Hartman, C.R., (1977) Digoxin in hyperthyroidism, Clin. Phar- Pharmacol. Ther., 22, Klein. I., (1988) Thyroxine-induced cardiac hypertrophy: time course of development and inhibition by propranolol, Endocrinology, 123, Larsen, P.R., (1988) Cecil Textbook of Medicine, pp , W.B. Saunders Co., Philadelphia. Marrow, D.H., Gafeney, T.E., Graunwal, D.E., (1963) Studies ondigitals.. Influence of hyper- and hypothyrodism on the myocardial responses to oubain, J. Pharmacol. Exp. Ther., 140, Niizoe, K., Ogawa, K., and Satake, T., (1984) Long-term effects of triiodothyronine and thiouracil on myocardial beta-adrenergic receptor numbers and cyclic AMP concentration in rats, Jpn. Circ. J., 48, Romanof A.L., (1960) The Avian Embryo, pp , The Macmillan Co., New York. Romanoff A.F., (1972) Pathogenesis of the Avian Embryo, John Wily & Sons, Inc., New York. Sanford, C.F., Griffin, E.E., and Wildenthal, K., (1978) Synthesis and degradation of myocardial protein during the development and regression of thyroxine-induced cardiac hypertrophy in rats, Circ. Res., 43, Shirani, J. Barron, M.M., Pierre-Louis, M.L., and Roberts, W.C., (1993) Congestive heart failure, dilated cardiac ventricles, and sudden death in hyperthyroidism, Am. J. Cardiol., 72, Skelton, C.L., (1982) The heart and hyperthyroidism, N. Engl. J. Med., 307, Suga, H., Tanaka, N., Ohgoshi, Y., Saeki, Y., Nakanishi, T., Futaki, S., Yaku, H., Goto, Y., (1991) Hyperthyroid dog left ventricle has the same oxygen consumption versus pressure-volume area (PVA) relation as euthyroid dog, Heart Vessels, 6, Sugiyama, T., Suguro. N., and Hayashida. A., (1982) Effects of the sites of injection of aminoguanidine sulfate on developing eggs, J. Pharmacobiodyn., 5, 1-12 Sugiyama T, Miyamoto K, Katagiri S., (1985) Histological studies on developing chick embryos treated with aminoguanidine sulfate, J. Toxicol. Sci., 10, Sugiyama, T., Miyazaki, H., Saito, K., Shimada, H. and Miyamoto, K. (1996) Chick embryos as an alternative experimental animal for cardiovascular investigations: stable recording of electro- 110

7 cardiogram of chick embryos in ovo on the 16 th day of incubation, Toxicol. Appl. Pharmacol., 138, Sugiyama, T., Saito, K., Shimada, H., Tsuchimoto, K. and Yoshiyama, Y. (2000) Effect of digoxicin on chick embryos with hypothyroidism induced by thiamazole, Altern. Animal Test. Experiment., 6, Turner, C.D., and Bagnara, J.T., (1971) General Rndocrinology, W.B. Saunders Co., Philadelphia. Yoshiyama Y., Sugiyama T. and Kanke M. (2003) Toxic effects of trastuzumab (Herceptin) combined with doxorubicin in chick embryos, Altern. Animal Test. Experiment., 9, Yoshiyama, Y., Sugiyama T., Tsuchimoto K. and Kanke M. (2004a) Drug-drug interactions between antiarrhythmic drugs in chick embryos, Biol. Pharm. Bull., 27, Yoshiyama Y., Sugiyama T. and Kanke M. (2004b) Toxic interactions between amitriptyline and disopyramide in chick embryos., Altern. Animal Test. Experiment., 10, Yoshiyama, Y. and Kanke M. () Toxic interaction between fluconazole and disopyramide in chick embryos, Biol. Pharm. Bull., 28, (Received: December 13, 2004 / Accepted: August 4, ) Corresponding author: Yuji Yoshiyama, Ph.D. Division of Clinical Pharmacy, Kyoritsu University of Pharmacy, , Shibakoen, Minato-ku, Tokyo , Japan Tel Fax yoshiyama-yj@kyoritsu-ph.ac.jp 111