ADRIAMYCIN-INPUCED CARDIOMYOPATHY. A RAT MODEL

Transcription

1 Pol. J. Pharmacol. Pharm., 1986, 8, PL ISSN ADRIAMYCIN-INPUCED CARDIOMYOPATHY. A RAT MODEL ANDRZEJ CZARNECKI, ALEKSANDER HINEK *, DOROTA SOLTYSIAK-PAWLUCZUK Department of Pharmacology, Institute of Drug Research and Control. Chelmsl<a O/J4, Warszawa, Poland. Department of Histology, Pomeranian Medical Academy, Powstanc6w Wielkopolskich 72, Szczecin, Poland Adriam11cin-induced cardiomyopathy. A rat model. A. CZARNECKI, A. HINEK, D. SOLTYSIAK-PAWLUCZUK. Pol. J. Pharm. Pharm., 1986, 8, A rat model of adriamycin-induced cardiomyopathy was elaborated. Adriamycin (ADR) in three total doses 21, 25 and 0 mg/kg ip injected in 15 equal partial doses caused the loss in body weight and hematocrite values, marked leucopenia and moderate elevation of SGOT activitiess as well as ECG changes. In histopathological examination cardiomyopathic changes were found in heart samples from rats treated with two higher ADR doses. The dose 25 mg/kg is suggested for further investigations concerning the limitation of ADR toxicity. Ad.riamycin (ADR) is a broad spectrum and potent chemiotherapeutic agent [2, ]. It clinical usefulness is hampered by late, dose-dependent cardiotoxicity leading to the development of cardiomyopathy and congestive heart failure [7]. Late toxic effects appear in man after the cumulative dose 550 mg/m2 is administered [4]. The animal models of anthracycline cardiomyopathy elaborated up to the moment by Zbinden et al. [9], Jaenke [5] and Rosenhof et al. [7] were either long-lasting, required costly animals or prepared for toxicologic screening tests. The aim of this st dy was to elaborate a model for cheap and quick evaluation of ADR toxicity to be used in further studies concerning the li!llitation of ADR-induced cardiomyopathy. MATERIALS AND METHODS Experiments were performed on male Wistar rats, of initial weight g, housed in cages receiving granulated food and water ad libitum. Groups of \0 animals were injected times a week for 5 weeks with 0.050/o solution of ADR

2 172 A. Czarnecki et al. (Adriblastina-FARMITALIA) in 0.90/o NaCl. Three total doses of the drug 21, 25 and 0 mg/kg were injected in 15 equal partial doses. The control received adequate volumes of the solvent. The evaluation of parameters of general toxicity (leucocytosis, hematocrite, SGOT, body weight) and of ECG changes lead II, paper speed 100 mm/s) was carried out before the onset of the treatment and once a week thereafter. The main evaluation o:( cardiotoxic changes consisted of histopathological and histochemical examination. Rats were sacrificed 10 and 25 days after the last ADR dose. The slices of myocardium were excised in routine manner according to the transversal line running through both ventricles in their middle length. The heart samples were fixed in buffered formalin, then frozen or embedded in paraffin. The paraffin sections were stained with hematoxylin and eosin, with methyl green and pyronin, or according to van Gieson method [6]. On the frozen sections the histochemical reactions for acid phosphatase, ATP-ase, TPP-ase, succinic dehydrogenase and Sudan black staining were performed. The pathologic grades of myocardial toxicity due to ADR administration were estimated according to the scale previously proposed by Billingham et al y. rl]. RESULTS GeneraL toxicity. Forty percent of animals rece1vmg the total ADR dose of 0 mg(kg died during the experiment. All the animals from this group and 500/o of the animals receiving 25 mg/kg of ADR dev loped ascit s. All three doses of the drug caused significant reduction of weight gain of treated animals (Fig.. 1 A). b.w.lgl 50 * ,_:=.--J* = _, ,. * *... ; Adr.do$es

3 WBC Adr doses Ht [%] so,..., ;------, tut u/ s S Adtr.doses Adtr dosts Fig. 1. Changes in general toxicity parameters in rats treated with adriamycin (ADR) ip in 15 equal partial doses times a week for 5 following weeks to total doses 21, 25 and 0 mg/kg. A - body weight; B - white blood cells (WBC) count; C - hematocrite (Ht); D - serum glutamic oxalacetic transaminase (SGOT) activities; --- saline control;- - ADR 21 mg/kg; --- ADR 25 mg/kg; --- ADR 0 mg/kg; * - p < 0.05; * * - p < 0.001

4 174 A. Czarn.ecki et al. Hematology Continuos decrease in white blood cells (WBC) count and hematocrite values was observed. It was more marked after higher ADR doses reaching the significant level in the third week of treatment (Fig. 1 B, C). Also marked i:t;1crease in SGOT activities was observed in all three experimental groups (Fig. 1 D). Electrocardiographic findings lit, ECG changes: prolongation of QRS time and reduction of QRS voltage accompanied by mild bradycardia and sporadic extrasystolia were noticed after the cumulative ADR dose of 12 mg/kg was excessed. The changes appeared ealier in the rats receiving higher ADR doses. His to pathology The hearts taken 10. days after the last ADR dose in the gro;up receiving?1 mg/kg of the drug did not show any significant histopathological and histochemical changes. The hearts sections obtained from rats treated with 25 mg/kg of ADR revealed scanty cardiocytes with early myofibrillar loss or slight vacuolisation of the cytoplasm. Such Fig. 2. Left ventricle of the rat heart 25 days after total adriamycin dose of 25 mg/kg ip, (administered in 15 equal partial doses times a week for 5 following weeks). Focal cell distension, cytoplasmic vacuolisation, some cells unchanged (hematoxylin and eosin staining, X 200)

5 Adriamycin-induced cardiomyopathy 175 Fig.. Left ventricle of the rat heart 25 days after total adriamycin dose 0 mg/kg ip, (administered in 15 equal partial doses times a week for 5 following weeks). Loss of fibres, cytoplasmic vacuolisation (hematoxylin and eosin staining, X 200) cells were located only in the subendocardial regions of the left ventricle. Described changes suit to. the first grade of myocardial toxicity. The examined samples did not demonstrate any significant changes in histochemical reactions except for more intensive reaction for acid phosph-, atase. The hearts taken 25 - days after the last ADR dose indicated that the dose of 21 mg/kg did not evoke cardiomyopathic changes. Two higher doses, 25 and 0 mg/kg, were highly cardiotoxic and caused well developed cardiomyopathy. Suboodocardium of the left ventricle and septum were particulary involved while changes in the right ventricle were less pronounced. The involved tissue was characterized by focal cell distension, interstitial edema and perivascular fibrosis. Numerous cardiocytes iocated in the lesions demonstrated nuclear pycnosis, marked loss of myofibriles and cytoplasmic vacuolization (Fig. 2-4). The changes described above in the left ventricle and septum fit well to the grade of the Billingham scale. [1], the alterations in right ventricle may be considered as grade 1 (Tab. 1}.

6 176 A. Czarnecki et al. Fig. 4. Left ventricle of the rat heart 25 days after total adriamycin dose of 25 mg/kg ip {administered in 15 equal partial doses times a week for 5 following weeks). Marked loss of fibres (hematoxylin and eosin staining, X 200) Table 1. Pathologic grades of myocardial toxicity, evaluated ace. to Billingham et al. [I] in the rats hearts estimated 25 days after adriamycin (ADR) administration in t.hree total doses 21, 25 and 0 mgfkg ip injected in 15 equal partial doses times a week for 5 following weeks ocalisation ADR dose (mgjkg) eft ventricle 1 septum 0 right ventricle 0 DISCUSSION Cardiotoxicity is the main limitation in anticancer treatment with anthracycline antibiotics. For this reason many efforts were undertaken to diminish cardiotoxicity or to find less toxic derivatives [8]. In this study we tried to elaborate an inexpensive model for quick evaluation of ADR toxicity to be used in further experiments dealing with its limitation.

7 Adriamycin-induced cardiomyopathy 177 Obtained results show that the general ADR toxicity and electrocardiographic findings did not differ from those described previously by other investigators in rats [7], rabbits [5] and mice [6]. Histopathological examinations of the heart muscles showed fully developed cardiomyopathic changes after two higher ADR doses Morphological lesions in the myocardium were similar to those observed in various experimental studies [5, 8, 9] and in man [1]. The above mentioned models were either long lasting and required costly animals [5] or prepared only for cardiotoxicity screening [8, 9]. Zbinden et al. [9] in his study elaborated a rat model for evaluation of cardiotoxicity of new anthracycline derivatives based on electrocardiographic examination used in later studies [9]. The model proposed by us seems to be equally inexpensive but quicker. The evaluatio:n of cardiomyopathic changes is more precise. It is based on histopathological examination of hematoxylin and eosin stained heart samples in the photomicroscope with estimation of changes carried out according to Billingham et al. [1]. For further experiments dealit1g with the limitation of ADR toxicity we suggest the dose 25 mg/kg ip which caused fully developed cardiomyopathy and less expressed general toxic effects. REFERENCES 1. Billingham M. E., Mason J. W., Bristow M. R., Daniels J. R.: Anthracycline cardiomypathy monitored by morphologic changes. Cencer Treat. Rep., 1978, 62, Blum R. M.: An overview of studies with adriamycin (NSC-12127) in the United States. Cancer Chemother. Rep., 1976, 6, 247-2!>1.. Blum R. M., Carter S.: Adriamycin: a new anticancer drug with significant clinical activity. Ann. Int. Med., 1974, 80, Danysz A., Raouf Hamid M., Wierzba K.: Cytostatic and immunosuppresive drugs. In: Side Effects of Drugs Annual, Ed. Dukes M. N. G., Excerpta Medica, Amsterdam-Oxford, 1979, Jaenke R. S.: Ananthacycline antibiotic-induced cardiomyopathy in rabbits. - Lab. Invest., 1974, 0, Lilie R. D.: Histologic Technics and Practical Histochemistry. Me Grow Hill Book Co. N.Y., Toronto City, London Minow R., Benjamin R., Gottlieb J.: Adriamycin (NSC-12127) cardiomyopathy - an overview with determination of risk factors. Cancer Chemother. Rep., 1975, 6, Rosenhoff-S. H., Olson H.M., Young D. M., Young R. C.: Adriamycin-induced cardiac damage in the mouse: a small animal model of cardiotoxicity. J. Natn. Cancer Inst., 1975, 55, Zbinden G., Bachmann E., Holderegger C.: Model systems for cardiotoxic effects of anthracyclines. Antibiotics Chemother., 1978, 2, Zbinden G., Beilstein A. K.: Comparison of cardiotoxicity of two anthracenediones and doxorubicin in rats. Toxicol. Lett., 1982, 11, Received: March 4, 1985.