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Doxorubicin (adriamycin)-induced cardiotoxicity in turkey poults: An animal model
0306-4492186 $3.00 + 0.00 (ii 1986 Pergamon Press Ltd
Camp. Biochem. Physioi. Vol. 83C, No. I, pp. 53-60, 1986 Printed in Great Britain
~OXORUBICIN ~ADRIAMYCIN)-INDUCED CARDIOTOXICITY IN TURKEY POULTS: AN ANIMAL MODEL CAROLINE M. CZARNECKI Department of Veterinary Biology, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108, USA. Telephone: (612) 376-4760 (Received 5 June 1985) Abstract-l. Doxorubicin (adriamycin, ADR)-induced cardiomyopathy was produced in turkey poults in a period of 3 weeks. 2. The cardiotoxicity is characterized by electrocardiographic changes, alterations in ultrastructural morphology, ventricular dilatation, a significantly increased number of heterophils, and significant decreases in body and heart weights, dry heart weight to body weight ratios, and number of non-granulocytic leukocytes. 3. The turkey poult is a cost-effective model system for evaluating cardiotoxic effects of anthracycline drugs.
INTRODUCTION
A serious complication
of chronic administration of ant~racyc~ine antibiotics in the treatment of solid tumors is the development of cardiac toxicity (Buja et al., 1973; Lefrak et al., 1973; Blum and Carter, 1974; Minow et al., 19’75; Lenaz and Page, 1976; Billingham et al., 1978; Bristow et al., 1978). This cardiotoxicity is characterized by electrocardiographic (ECG) abnormalities, myocardial fibrosis and necrosis, cardiomegaly and cardiac failure (Cargill et al., 1974; Olson et al., 1974; Mettler et al., 1977). Morphological features of myocardial
damage have been described in both human (Billingham et al., 1978; Ferrans, 1978) and animal hearts (Jaenke, 1974; Mettler et al., 1977; Olson and Capen, 1978; Van Vleet et al., 1978, 1979, 1980). Animals subject to the toxic effects of doxorubicin (adriamycin, ADR) include the chick embryo (Pannuti, 1972), rat (Mettler et al., 1977; Olson and Capen, 1977, 1978; Sonneveld, 1978), rabbit (Jaenke, 1974, 1976; Madanat et al., 1978), mouse (Rosenoff et al., 1975; Myers et ul., 1976, 1977; Doroshow et al., 1978; Bertazzoli et al., 1979), dog (Kehoe et al., 1978; Van Vleet et al., 1980), pig (Van Vleet et al., 1979) and monkey (Denine and Schmidt, 1975). Of these animals, the rat, mouse and rabbit have been used most extensively to demonstrate acute and chronic alterations (Cargill et a/., 1974; Olson el al., 1974; Jaenke, 1976; Mettler et al., 1977; Myers et al., 1977; Olson and Capen, 1977,1978; Lenaz ef al., 1978; Van Vleet et al., 1978; Taylor and Bulkley, 1982; Kimler et at., 19841, to study pathogenetic mechanisms (Rosenofl’ et al., 1971; Buja et al., 1973; DiMarco et ai., 1975; Young, 1975; Mimnaugh et al., 1979) and to define model systems for testing antineoplastic drugs for cardiotoxic activity (Mettler et al., 1977; Doroshow et at., 1979; Zbinden and Beilstein, 1982; Unverferth et al,, 1983: Kimler ef al., 1984). The usefulness
of
these
animal
models,
however,
is 53
limited by the time (10-20 weeks in the rat) required to induce cardiomyopathy (Mettler et al., 1977). In turkey poults, myocardial damage can be induced in a relatively short time (2-4 weeks) by fur~olidone (Czarnecki, 1980; Czarnecki and Grahn, 1980; Czarnecki et al., 1983) and alcohol (Noren et al., 1983; Czarnecki et al., 1986). These studies indicate that the turkey poult may be a more costeffective animal model for investigations of druginduced cardiomyopathy. The purpose of the present experiment was to evaluate this animal model system for testing analogs and complexes of ADR for cardiac toxicity. MATERIALS AND METHODS One-day-old Broad Breasted White turkey poults (toms) of the Nicholas strain were obtained from a single hatch from a commercial source. The poults were maintained on a normal ration for 4 weeks at which time they were screened using the electrocardiographic (ECG) technique developed by Jankus et al. (197 1) and modified by Czarnecki and Good (1980). Poults with normal ECGs were placed randomly in one of two pens: control and ADR-treated. The poults in each pen were assigned to one of three groups with six poults/group (control pen-Groups I, III and V;.ADRtreated pen-Groups II. IV and VI). ADR was reconstituted in physiological saline solution at a concentration of 2 mg/ml just prior to injection. Beginning at 4 weeks of age and continuing to 7 weeks of age, the poults in the ADR pen were treated in one of the following ways: two injections of ADR/week at a dose of I mg/kg body wt/week (Group II); two injections of ADRjweek at a dose of Zmgjkg body wt/week (Group IV); three injections of ADR/week at a dose of 1 mg/kg body wt (Group VI) during the 4th and 6th weeks only. Poults in the control pen received equivalent volumes of physiological saline and were paired with ADRtreated poults as follows: Group I (control) with Group II (ADR); Group III (control) with Group IV (ADR); Group V (controi) with Group VI (ADR). All injections were administered to the right jugular vein. Body weights and ECG recordings were obtained weekly.
54
CAROLINEM. CZARNECKI each pen. Quantitative data were analyzed using Student’s t test; P values < 0.05 were considered to be statistically significant.
Table 1. Mean body weights for turkey poults assigned to control and ADR-treated groups at 4 weeks of age Control ..._~_____ Body weight (g) 652.3 f I6.7*(18)t
ADR-treated 658.8 + 15.4(18)
*Mean k SE. tNumber of poults.
RESULTS
The mean body weights of the poults assigned to the control and ADR-treated pens are shown in Table 1. These data indicate that both groups of poults were similar at the start of the experimental procedure. The mean body and heart (ventricular) weights, heart weight to body weight ratios and heart moisture content of control and ADR-treated poults at 7 weeks of age are shown in Table 2. Significant decreases in body weight (P s O.Ol), wet and dry heart weights (P I 0.001) and dry heart weight to body weight ratios (P I 0.001) were apparent in the ADR-treated poults when compared with the control poults. Although the moisture content of the hearts of ADR-treated poults was slightly greater (80.4 + 0.3) than that of control poults (79.8 f 0.2), the differences were not statistically significant. Characterizations of the ECG record and cardiac lesions in control and ADR-treated poults are presented in Table 3. Changes in ECGs were detected only in the ADR-treated poults at 6 and 7 weeks of age. The incidence and appearance of altered ECG patterns were dose-related. In the group of poults receiving the greatest dose of ADR/week (Group VI), one poult had an altered ECG pattern at 6 weeks of
When the poults were 7 weeks of age, 3 ml of blood was collected from each poult by venipuncture into heparinized syringes. Packed cell volumes were determined and blood smears were prepared and stained (Wright’s stain) for differential counts. The poults, then, were killed by cervical dislocation. For electron microscopy, thin strips of tissues were removed from the free ventricular walls, minced and fixed in preweighed vials containing 304 buffered glutaraldehyde (0.1 M phosphate buffer). Then, these tissue samples were postfixed in 1% osmium tetroxide in phosphate buffer, pH 7, dehydrated in a graded acetone series, and embedded in Epon Araldite epoxy resin. Thin sections (light gold in color) were cut on an LKB Ultrotome III, mounted on Pelco GC 300 LD copper grids, stained with uranyl acetate and lead citrate and examined and photographed with an RCA-EMU4B electron photomicroscope at 75 kV. The remainder of the heart tissue was examined for gross lesions. Dilatation of ventricles was scored as follows: 0 = none, + = slight, + + = moderate. Ventricles were removed, trimmed free of fat, weighed and then freeze-dried for 72 hr at which time moisture content was calculated. Body and heart weights were pooled for the poults in the control pen and for those in the ADR-treated pen since there were no significant differences among the poults in
Table 2. Mean body and heart (vent~calar) weights, heart weight to body weight ratios and heart moisture content in control and ADR-treated turkev ooults at 7 weeks of age
Control
ADR-treated
2062.9 f 49.6t (I 8)$ 7.26 f 0.26 (17) 3.5kO.l x 10_4(17) 1.46f0.05(17) 7.0+0.1x 10_4(17) 79.8 k 0.2 (17)
Body weigbt (g) Wet heart weight (g) Wet heart weight/body weight Dry heart weight (g) Dry heart weight/body weight Moisture content (7;)
1889.4 + 40.2 (IS)* 6.19 f 0.19 (18)** 3.3kO.l x 10-4(18) 1.21 +0.04(18)** 6.4kO.2 x 10-4(18)** 80.4 f 0.3 (18)
*Significantly different from control group: *P I 0.01; **P 5 0.001. *Mean + SE. @Jumber of poults.
Table 3. Characterizations
4
of ECG record and cardiac lesions in control and ADRtreated uoults
ECG* Age (weeks) 5 6
7
GroupS I II III IV
N(6)S N(6) N(6) N(6)
N(6) N(6) N(6) N(6)
N(6) N(6) N(6) N(6)
N(6)
V VI
N(6) N(6)
N(6) N(6)
N(6) N(5)
N(6)
W5f NV31 N(5)
Lesionst Left Right ventricle 0 + ++ 0 ~-. @ 0 0 6 I 5 0 3 6 0 0 6 I 3 2 2
ventricle f +t 0 3 0 3
0 0 0 1
0 2
0 3
41) A(l)
N(3) A(3)
6 1
0 2
0 3
6
I
*ECG: N = nxmal; A = altered. flesions: 0 = no visible gross lesions; + = slight ventricular dilatation; + + = moderate ventricular dilatation. fGroup I: two saline injections/week for 3 weeks (volume of dose equivalent to that of Group II); Group II: two ADR injections at a dose of 1mgjkg body wt/week for 3 weeks; Group III: two saline injections~week for 3 weeks (volume of dose equivalent to that of Group IV); Group IV: two ADR injections at a dose of 2 mgjkg body wt/week for 3 weeks; Group V: three saline injections/week during 4th and 6th weeks (volume of dose equivalent to that of Group VI); Group Vi: three ADR injections at a dose of I mg/kg body wt/week during 4th and 6th weeks. §Number of poults.
Doxorubicin-induced
A
6
Fig. 1. ECG pattern on lead II (aVF) for poult No. 1113 in Group VI. (A) At 5 weeks, (B) at 6 weeks (note decrease in amplitude of S wave).
age (Fig. 1). By 7 weeks of age, three out of six poults in this group had altered ECGs (Fig. 2). In Group II (poults receiving smallest dose of ADR), there were no poults exhibiting changes in ECGs, but in Group IV one poult had an altered ECG by 7 weeks of age
C
cardiotoxicity
55
(Fig. 3). Similarly, the extent of the dilatation of the ventricles appeared to be dose-related with both ventricles affected to nearly the same degree. Apparently dilatation precedes ECG changes as five poults in Group II had slight dilatation of the right ventricle while three poults in this group had slight dilatation of the left ventricle even though the ECGs for these poults were normal. Likewise, in Group IV, five poults had slight to moderate dilatation of the right ventricle and four poults showed slight to moderate dilatation of the left ventricle despite the fact that only one of these poults exhibited an altered ECG. In Group VI, five poults had dilated ventricular chambers but two of these poults had normal ECGs. Ultrastructural features of the myocardium were normal in the hearts of poults in Group II (Fig. 4). Morphological alterations were noted in the hearts of poults in Groups IV and VI (Figs 5-8) with the number and severity of changes more pronounced in the poults of Group VI. The major alteration observed was an increase in the number and size of vesicles. Some of these were formed by an outpocketing of the outer membrane of the nuclear envelope (Figs 5 and 6) while others were present among the mitochondria (Fig. 7). Other morphological changes seen included mild myolysis (Fig. 5) increase in width of the interstitial space (Fig. 6), swollen mitochondria (Figs 7 and 8) and condensed nuclei that were extensively folded (Fig. 8). The mean packed cell volume for control poults was 29.8 f 1.9 compared to 30.7 k 3.4 for ADRtreated pot&s. Although each ADR-treated group had a packed cell volume greater than its control these differences were not statistically group, significant individually nor when pooled. The differential blood count in control and ADRtreated poults at 7 weeks of age is shown in Table 4. Increased numbers of heterophils were observed in all ADR-treated poults when compared with their controls, but the number of heterophils was significantly higher (P I 0.01) only in Groups IV and VI. Decreased numbers of non-granulated leukocytes were seen in all ADR-treated poults when compared with the control poults. However, statistically significant decreases were observed only in monocytes in Groups II and VI. When the lymphocytes and monocytes were pooled for the control poults and for the ADRtreated poults, a significant decrease (P 5 0.001) in these cells was apparent in the drug-treated poults.
A
B Fig. 2. ECG pattern on lead II (aVF) for poult Nos 1113, 1124 and 1130 in Group VI. (A) Poult No. 1113 at 7 weeks (compare with Fig. 1 and note irregular amplitude of S wave), (B) poult No. 1124 at 6 and 7 weeks (note irregular pattern at 7 weeks), (C) poult No. 1130 at 6 and 7 weeks (note increase in amplitude of R wave at 7 weeks).
Fig. 3. ECG pattern on lead II (aVF) for poult No. 1147 in Group IV. (A) At 6 weeks, (B) at 7 weeks (note increase in amplitude of R wave).
CAROLINE M. CZARNECKI
56
Fig. 4. Longitudinal
section of myocytes in left ventricle of poult No. 1141 in Group myofibrils and nucleus (N) appear normal. x 12,060.
DISCUSSION
The etiology of the cardiotoxic effects of the anthracycline compounds is ill-defined. Considerable evidence has been compiled to suggest that acute ADR cardiotoxicity is mediated by peroxidative injury (Myers et al., 1976, 1977; Thayer, 1977; Bachur et al., 1978; Sonneveld, 1978; Doroshow et al., 1981) but alterations responsible for chronic ADR-induced cardiomyopathy are less well-defined. Animal models, reportedly used in past studies, tend to develop acute cardiotoxicity. However, it is the prolonged, repeated doses of ADR that are primarily responsible for the development of irreversible cardiomyopathy leading to congestive heart failure (Lefrak et al.,
II. Mitochondria,
1973; Minow et al., 1975; Billingham et aI., 1978; Bristow et al., 1978). Studies of chronic, repeated drug treatments are the most reliable means of evaluating adverse side effects (Peck, 1968). The results of the present study indicate that chronic, repeated small doses of ADR (1 mg/kg MWF) are more cardiotoxic than similar doses given with greater time intervals between injections (2 mg/kg MF). Cardiotoxicity, as determined by the degree of ventricular dilatation, was greater in the poults on the 1 mg/kg MWF schedule than in those on the 2 mg/kg MF regimen even though both groups of poults received similar cumulative doses of ADR during the period of this experiment. This lends credence to the hypothesis that the severity of the
Fig. 5. Oblique section of myocytes in left ventricle of poult No. II23 in Group IV. Perinuclear space is dilated with a vesicle present at arrow. Some myolysis (MY) is apparent in adjacent fiber. x 12,060.
Doxorubicin-induced
Fig. 6. Oblique (V) are present
Fig. 8. Longitudinal nuclear
membrane
57
section of myocytes in left ventricle of poult No. 1116 in Group VI. Prominent vesicles in a perinuclear position. The width of the interstitial space (IS) is increased. x 12,060.
Fig. 7. Longitudinal section of myocyte in left ventricle of poult No. 1124 in Group VI. Numerous vesicles (arrows) are present and the mitochondria appear swollen. x 12,060.
section of myocyte in left ventricle of VI. The nucleus (N) is folded and is thickened. Mitochondria are swollen. x 12,060.
poult No. 1124 in Group
cardiotoxicity
cardiotoxicity is related to the serum level of the drug as suggested by previous studies (Weiss and Manthel, 1977; Pacciarini et al., 1978; Edington et al., 1984). The selection of an animal model for testing cardiotoxic effects of drugs must take into account the responsiveness of the myocardium to the toxic agents as well as being cost-effective, accurate, reproducible and relatively simple to perform (Mettler et al., 1977). Previous studies have demonstrated that the heart of the turkey poult is responsive to toxic agents (Jankus et al., 1972; Czarnecki et al., 1974, 1986; Czarnecki, 1980; Czarnecki and Grahn, 1980; Noren et al., 1983). These studies, also, have shown that this animal model is relatively easy and inexpensive to maintain. In addition, cardiotoxic effects manifest themselves quickly and are reproducible with great accuracy. Hearse et al. (1976) reported wide species differences in the development of myocardial damage as measured by biochemical and morphological parameters For instance, hearts of the rat and mouse were found to be quite resistant while hearts of the guinea-pig and rabbit were very susceptible to cardiotoxicity. Weiss et al. (1976) observed that ADR administered at a dose of 2 mg/kg body wt/week caused dramatic cardiac damage in rabbits but not in man. In the present study, this dose of ADR produced only slight changes in the parameters measured. This indicates that the turkey poult’s heart is more similar to that of the human heart in its response to toxic drugs. Some species are prone to develop spontaneous myocardial lesions (Bajusz et al., 1969; Caulfield and Shelton, 1973; Weber et al., 1973) and should be avoided as model systems. Spontaneous cardiomyopathy is relatively rare in turkey poults, but it (or the tendency toward its development) can be detected by ECGs as early as 2 weeks of age (Czarnecki and Good, 1980). Thus, poults to be used in experimental studies can be evaluated relatively early and selection of appropriate animals can be made on the basis of normal EC&. ADR toxicity is manifested as alopecia, melanosis, body weight loss, myelosuppression, morphologic
CAROLINE M. CZARNECKI
58 Table 4. DilTerential croupt I
II III IV V VI
blood count in control and ADR-treated
Heterophils 42.8 f 53.2 f 35.0 ? 48.0 + 40.3 k 58.2 i
S.O$(6)$ 4.7 (6) 2.0 (6) 4.2 (6)‘; 4.9 (6) 4.0 (6)**
Basophils 1.5 f 3.5* 4.7 + 2.7 f 1.0 f 0.3 +
0.4(6) 1.2(6) 2.5 (6) 1.3(6) 0.5 (6) 0.2 (6)
turkev ooults at 7 weeks of see
Eosinonhils
Lvmnhocvtes
3.5 k 0.6 (6) 0.5 i 0.3 (6)*** 2.2 i_ 0.8 (6) 1.O k 0.6 (6) 1.5 k 0.8 (6) 0 f 0 (6)*
39.3 + 37.5 f 45.3 k 38.0 f 48.8 f 38.2 f
6.3 (6) 5.2 (6) 2.9 (6) 2.5 (6) 5.1 (6) 3.9 (6)
Monocvtes 13.0 F 5.3 f 12.8 f 10.3 t 8.3 f 3.3 +
2.5 (6) 1.3 (6)** 2.5 (6) 2.3 (6) 1.3 (6) 0.8 (6)***
*Significantly different from control group: *P IO.05; **P s 0.01; ***P I 0.001. tGroup I: two saline injections/week for 3 weeks (volume of dose equivalent to that of Group II); Group II: two ADR injections at a dose of 1 mg/kg body v/t/week for 3 weeks; Group III: two saline injections/week for 3 weeks (volume of dose equivalent to that of Group IV); Group IV: two ADR injections at a dose of 2 mg/kg body w/week for 3 weeks; Group V: three saline injections/week during 4th and 6th weeks (volume of dose equivalent to that of Group VI); Group VI: three ADR injections at a dose of 1 mg/kg body w/week during 4th and 6th weeks. fMean k SE. §Number of poults.
alterations, ECG changes, nephrotoxicity, chondroosseus toxicity and cardiomegaly (Sternberg et al., 1972; Buja et al., 1973; Cargill et al., 1974; Jaenke, 1974; Bachmann et al., 1975; Young, 1975; Mettler et al., 1977; Kehoe et al., 1978; Olson and Capen, 1978; Van Vleet et al., 1978). In the turkey poult, manifestation of ADR toxicity was limited to mild suppression of growth, slight myelosuppression of leukocytes, ECG changes and cardiomegaly. Morphological examination of myocardial tissue confirmed alterations in ultrastructural features previously described following furazolidone(Czarnecki, 1980, 1986) and alcohol-induced (Czarnecki et al., 1986) cardiomyopathy in turkey poults consonant with altered ECGs and cardiomegaly and in ADRinduced cardiotoxicity (Buja et al., 1973; Jaenke, 1974; Olson and Capen, 1977, 1978; Van Vleet et al., 1978). The data obtained in this study indicate that the turkey poult is an appropriate model system for testing the cardiotoxicity of drugs such as ADR and its analogs. Acknowledgements-The author is grateful to Carl W. Edborg III who cared for the poults, to Dr A. L. Good, Lenore Mottaz and Michael Powers who provided technical assistance and to the Dale Peterson Turkey Hatchery, Cannon Falls, MN, for the generous supply of turkey poults. This investigation was supported by a Grant-in-Aid from the American Heart Association and with funds contributed in part by the Montana Heart Association. REFERENCES Bachmann E., Weber E. and Zbinden G. (1975) Effects of seven anthracycline antibiotics on electrocardiogram and mitochondrial function of rat hearts. Agents Actions 5, 383-393. Bachur N. R., Gordon S. L. and Gee M. V. (1978) A general mechanism for microsomal activation of quinone anticancer agents to free radicals. Cancer Res. 38, 1745-1750. Bajusz E., Baker J. R., Nixon C. W. and Homburger F. (1969) Spontaneous hereditary myocardial degeneration and congestive heart failure in a strain of Syrian hamsters. Ann. N. Y. Acad. Sci. 156, 105-129. Bertazzoli C., Bellini O., Magrini U. and Tosana M. G. (1979) Quantitative experimental evaluation of adriamycin cardiotoxicity in the mouse. Cancer Treat. Rep. 63, 1877-1883. Billingham M. E., Mason J. W., Bristow M. R. and Daniels J. R. (1978) Anthracycline cardiomyopathy monitored by morphologic changes. Cancer Treat. Rep. 62, 865-872.
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cardiotoxicity
59
toxicity of adriamycin in the rat. Biochemical and ultrastructural investigations. Lab. Invest. 37, 386394. Olson H. M. and Capen C. C. (1978) Chronic cardiotoxicity of doxorubicin (adriamycin) in the rat: morphologic and biochemical investigations. Toxic. appi. Pharmac. 44, 605-616. Olson H. M., Young D. M., Prieur D. J., Leroy A. F. and Reagan R. L. (1974) Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits. Am. J. Pathol 77, 439-454. Pacciarini M. A., Barbieri B., Colombo T., Broggini M., Garattini S. and Donelh M. G. (1978) Distribution and antitumor activity of adriamycin given in a high-dose and a repeated low-dose schedule to mice. Cancer Treat. Rep. 62, 791-800. Pannuti E. (1972) Valutazione prechnica della cardiotossicita di alcune antracicline ad attivita antiblastica mediante II “test dell’uouo”. Studio microelectrocardiografio (MECG) (nota II: I’adriamicina). Rio. Patol. Clin. 27, 49-72. Peck H. M. (1968) An appraisal of drug safety evaluation in animals and the extrapolation of results to man. In Importance of Fundamenral Principles in Drug Evaluation (Edited by Tedeschi D. H. and Tedeschi R. E.), pp. 449-471. Raven Press, New York. Rosenoff S. H., Brooks E., Bostick F. and Young R. C. (1971) Alterations in DNA synthesis in cardiac tissue induced by adriamycin in uiuo-relationship to fatal toxicity. Biochem. Pharmac. 24, 1898-1901. Rosenoff S. H., Olson H. M. and Young D. M. (1975) Adriamycin-induced cardiac damage in the mouse: a small-animal model of cardiotoxicity. J. natn. Cancer Inst. 55, 191-194. Sonneveld P. (1978) Effect of alpha-tocopherol on the cardiotoxicity of adriamycin in the rat. Cancer Treat. Rep. 62, 1033-1036. Sternberg S. S., Philips F. S. and Cronin A. P. (1972) Renal tumors and other lesions in rats following a single intravenous injection of daunomycin. Cancer Res. 32, 1029-1036. Taylor A. L. and Bulkley B. H. (1982) Acute adriamycin cardiotoxicity: morphologic alterations in isolated perfused rabbit heart. Lab. Imest. 47, 459-464. Thayer W. S. (1977) Adriamycin stimulated superoxide formation in submitochondria particles. Chem. biol. Interact. 19, 265-278. Unverferth D. V., Unverferth B. J., Balcerzak S. P., Bashore T. A. and Neidhart J. A. (1983) Cardiac evaluation of Mitoxantrone. Cancer Treat. Rep. 67, 343-350. Van Vleet J. F., Greenwood L., Ferrans V. J. and Rebar A. H. (1978)Effect of selenium-vitamin E on adriamycininduced cardiomyopathy in rabbits. Am. J. cef. Res. 39, 997-1010. Van Vleet J. F., Greenwood L. A. and Ferrans V. J. (1979) Pathology of adriamycin toxicity in young pigs: I. Nonskeletal lesions. Am. J. vet. Res. 40, 1537-1552. Van Vleet J. F., Ferrans V. J. and Weirich W. E. (1980) Cardiac disease induced by chronic adriamycin: administration in dogs and an evaluation of vitamin E and selenium as cardioprotectants. Am. J. Pathol. 99, 13-42. Weber H. W., Van Der Walt J. J. and Greeff M. J. (1973) Spontaneous cardiomyopathies in Chacma baboons. In Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 2, Cardiomyopathies (Edited by Bajusz E. and Rona G.), pp. 361-375. University Park Press, Baltimore. Weiss A. J. and Manthel R. W. (1977) Experience with the use of adriamycin in combination with other anticancer agents using a weekly schedule with particular reference to lack of cardiac toxicity. Cancer 40, 20462052. Weiss A. J., Metter G. E., Fletcher W. S., Wilson W. L., Grage T. B. and Ramirez G. (1976) Studies on adriamycin using a weekly regimen demonstrating its clinical
CAROLINEM.
60
effectiveness and lack of cardiac toxicity. Cancer Treat. Rep. 60, 813-822. Young D. M. (1975) Pathologic effects of adriamycin (WC-123127) in experimental systems. Cancer Chemofher. Rep. 6, 159-1’75.
CZARNECKI
Zbinden G. and Beilstein A. K. (1982) Comparison of cardiotoxi~ity of two anthracenediones and doxorubicin in rats. Toxicoi. Left. 11, 289-291.
Camp. Biochem. Physioi. Vol. 83C, No. I, pp. 53-60, 1986 Printed in Great Britain
~OXORUBICIN ~ADRIAMYCIN)-INDUCED CARDIOTOXICITY IN TURKEY POULTS: AN ANIMAL MODEL CAROLINE M. CZARNECKI Department of Veterinary Biology, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108, USA. Telephone: (612) 376-4760 (Received 5 June 1985) Abstract-l. Doxorubicin (adriamycin, ADR)-induced cardiomyopathy was produced in turkey poults in a period of 3 weeks. 2. The cardiotoxicity is characterized by electrocardiographic changes, alterations in ultrastructural morphology, ventricular dilatation, a significantly increased number of heterophils, and significant decreases in body and heart weights, dry heart weight to body weight ratios, and number of non-granulocytic leukocytes. 3. The turkey poult is a cost-effective model system for evaluating cardiotoxic effects of anthracycline drugs.
INTRODUCTION
A serious complication
of chronic administration of ant~racyc~ine antibiotics in the treatment of solid tumors is the development of cardiac toxicity (Buja et al., 1973; Lefrak et al., 1973; Blum and Carter, 1974; Minow et al., 19’75; Lenaz and Page, 1976; Billingham et al., 1978; Bristow et al., 1978). This cardiotoxicity is characterized by electrocardiographic (ECG) abnormalities, myocardial fibrosis and necrosis, cardiomegaly and cardiac failure (Cargill et al., 1974; Olson et al., 1974; Mettler et al., 1977). Morphological features of myocardial
damage have been described in both human (Billingham et al., 1978; Ferrans, 1978) and animal hearts (Jaenke, 1974; Mettler et al., 1977; Olson and Capen, 1978; Van Vleet et al., 1978, 1979, 1980). Animals subject to the toxic effects of doxorubicin (adriamycin, ADR) include the chick embryo (Pannuti, 1972), rat (Mettler et al., 1977; Olson and Capen, 1977, 1978; Sonneveld, 1978), rabbit (Jaenke, 1974, 1976; Madanat et al., 1978), mouse (Rosenoff et al., 1975; Myers et ul., 1976, 1977; Doroshow et al., 1978; Bertazzoli et al., 1979), dog (Kehoe et al., 1978; Van Vleet et al., 1980), pig (Van Vleet et al., 1979) and monkey (Denine and Schmidt, 1975). Of these animals, the rat, mouse and rabbit have been used most extensively to demonstrate acute and chronic alterations (Cargill et a/., 1974; Olson el al., 1974; Jaenke, 1976; Mettler et al., 1977; Myers et al., 1977; Olson and Capen, 1977,1978; Lenaz ef al., 1978; Van Vleet et al., 1978; Taylor and Bulkley, 1982; Kimler et at., 19841, to study pathogenetic mechanisms (Rosenofl’ et al., 1971; Buja et al., 1973; DiMarco et ai., 1975; Young, 1975; Mimnaugh et al., 1979) and to define model systems for testing antineoplastic drugs for cardiotoxic activity (Mettler et al., 1977; Doroshow et at., 1979; Zbinden and Beilstein, 1982; Unverferth et al,, 1983: Kimler ef al., 1984). The usefulness
of
these
animal
models,
however,
is 53
limited by the time (10-20 weeks in the rat) required to induce cardiomyopathy (Mettler et al., 1977). In turkey poults, myocardial damage can be induced in a relatively short time (2-4 weeks) by fur~olidone (Czarnecki, 1980; Czarnecki and Grahn, 1980; Czarnecki et al., 1983) and alcohol (Noren et al., 1983; Czarnecki et al., 1986). These studies indicate that the turkey poult may be a more costeffective animal model for investigations of druginduced cardiomyopathy. The purpose of the present experiment was to evaluate this animal model system for testing analogs and complexes of ADR for cardiac toxicity. MATERIALS AND METHODS One-day-old Broad Breasted White turkey poults (toms) of the Nicholas strain were obtained from a single hatch from a commercial source. The poults were maintained on a normal ration for 4 weeks at which time they were screened using the electrocardiographic (ECG) technique developed by Jankus et al. (197 1) and modified by Czarnecki and Good (1980). Poults with normal ECGs were placed randomly in one of two pens: control and ADR-treated. The poults in each pen were assigned to one of three groups with six poults/group (control pen-Groups I, III and V;.ADRtreated pen-Groups II. IV and VI). ADR was reconstituted in physiological saline solution at a concentration of 2 mg/ml just prior to injection. Beginning at 4 weeks of age and continuing to 7 weeks of age, the poults in the ADR pen were treated in one of the following ways: two injections of ADR/week at a dose of I mg/kg body wt/week (Group II); two injections of ADRjweek at a dose of Zmgjkg body wt/week (Group IV); three injections of ADR/week at a dose of 1 mg/kg body wt (Group VI) during the 4th and 6th weeks only. Poults in the control pen received equivalent volumes of physiological saline and were paired with ADRtreated poults as follows: Group I (control) with Group II (ADR); Group III (control) with Group IV (ADR); Group V (controi) with Group VI (ADR). All injections were administered to the right jugular vein. Body weights and ECG recordings were obtained weekly.
54
CAROLINEM. CZARNECKI each pen. Quantitative data were analyzed using Student’s t test; P values < 0.05 were considered to be statistically significant.
Table 1. Mean body weights for turkey poults assigned to control and ADR-treated groups at 4 weeks of age Control ..._~_____ Body weight (g) 652.3 f I6.7*(18)t
ADR-treated 658.8 + 15.4(18)
*Mean k SE. tNumber of poults.
RESULTS
The mean body weights of the poults assigned to the control and ADR-treated pens are shown in Table 1. These data indicate that both groups of poults were similar at the start of the experimental procedure. The mean body and heart (ventricular) weights, heart weight to body weight ratios and heart moisture content of control and ADR-treated poults at 7 weeks of age are shown in Table 2. Significant decreases in body weight (P s O.Ol), wet and dry heart weights (P I 0.001) and dry heart weight to body weight ratios (P I 0.001) were apparent in the ADR-treated poults when compared with the control poults. Although the moisture content of the hearts of ADR-treated poults was slightly greater (80.4 + 0.3) than that of control poults (79.8 f 0.2), the differences were not statistically significant. Characterizations of the ECG record and cardiac lesions in control and ADR-treated poults are presented in Table 3. Changes in ECGs were detected only in the ADR-treated poults at 6 and 7 weeks of age. The incidence and appearance of altered ECG patterns were dose-related. In the group of poults receiving the greatest dose of ADR/week (Group VI), one poult had an altered ECG pattern at 6 weeks of
When the poults were 7 weeks of age, 3 ml of blood was collected from each poult by venipuncture into heparinized syringes. Packed cell volumes were determined and blood smears were prepared and stained (Wright’s stain) for differential counts. The poults, then, were killed by cervical dislocation. For electron microscopy, thin strips of tissues were removed from the free ventricular walls, minced and fixed in preweighed vials containing 304 buffered glutaraldehyde (0.1 M phosphate buffer). Then, these tissue samples were postfixed in 1% osmium tetroxide in phosphate buffer, pH 7, dehydrated in a graded acetone series, and embedded in Epon Araldite epoxy resin. Thin sections (light gold in color) were cut on an LKB Ultrotome III, mounted on Pelco GC 300 LD copper grids, stained with uranyl acetate and lead citrate and examined and photographed with an RCA-EMU4B electron photomicroscope at 75 kV. The remainder of the heart tissue was examined for gross lesions. Dilatation of ventricles was scored as follows: 0 = none, + = slight, + + = moderate. Ventricles were removed, trimmed free of fat, weighed and then freeze-dried for 72 hr at which time moisture content was calculated. Body and heart weights were pooled for the poults in the control pen and for those in the ADR-treated pen since there were no significant differences among the poults in
Table 2. Mean body and heart (vent~calar) weights, heart weight to body weight ratios and heart moisture content in control and ADR-treated turkev ooults at 7 weeks of age
Control
ADR-treated
2062.9 f 49.6t (I 8)$ 7.26 f 0.26 (17) 3.5kO.l x 10_4(17) 1.46f0.05(17) 7.0+0.1x 10_4(17) 79.8 k 0.2 (17)
Body weigbt (g) Wet heart weight (g) Wet heart weight/body weight Dry heart weight (g) Dry heart weight/body weight Moisture content (7;)
1889.4 + 40.2 (IS)* 6.19 f 0.19 (18)** 3.3kO.l x 10-4(18) 1.21 +0.04(18)** 6.4kO.2 x 10-4(18)** 80.4 f 0.3 (18)
*Significantly different from control group: *P I 0.01; **P 5 0.001. *Mean + SE. @Jumber of poults.
Table 3. Characterizations
4
of ECG record and cardiac lesions in control and ADRtreated uoults
ECG* Age (weeks) 5 6
7
GroupS I II III IV
N(6)S N(6) N(6) N(6)
N(6) N(6) N(6) N(6)
N(6) N(6) N(6) N(6)
N(6)
V VI
N(6) N(6)
N(6) N(6)
N(6) N(5)
N(6)
W5f NV31 N(5)
Lesionst Left Right ventricle 0 + ++ 0 ~-. @ 0 0 6 I 5 0 3 6 0 0 6 I 3 2 2
ventricle f +t 0 3 0 3
0 0 0 1
0 2
0 3
41) A(l)
N(3) A(3)
6 1
0 2
0 3
6
I
*ECG: N = nxmal; A = altered. flesions: 0 = no visible gross lesions; + = slight ventricular dilatation; + + = moderate ventricular dilatation. fGroup I: two saline injections/week for 3 weeks (volume of dose equivalent to that of Group II); Group II: two ADR injections at a dose of 1mgjkg body wt/week for 3 weeks; Group III: two saline injections~week for 3 weeks (volume of dose equivalent to that of Group IV); Group IV: two ADR injections at a dose of 2 mgjkg body wt/week for 3 weeks; Group V: three saline injections/week during 4th and 6th weeks (volume of dose equivalent to that of Group VI); Group Vi: three ADR injections at a dose of I mg/kg body wt/week during 4th and 6th weeks. §Number of poults.
Doxorubicin-induced
A
6
Fig. 1. ECG pattern on lead II (aVF) for poult No. 1113 in Group VI. (A) At 5 weeks, (B) at 6 weeks (note decrease in amplitude of S wave).
age (Fig. 1). By 7 weeks of age, three out of six poults in this group had altered ECGs (Fig. 2). In Group II (poults receiving smallest dose of ADR), there were no poults exhibiting changes in ECGs, but in Group IV one poult had an altered ECG by 7 weeks of age
C
cardiotoxicity
55
(Fig. 3). Similarly, the extent of the dilatation of the ventricles appeared to be dose-related with both ventricles affected to nearly the same degree. Apparently dilatation precedes ECG changes as five poults in Group II had slight dilatation of the right ventricle while three poults in this group had slight dilatation of the left ventricle even though the ECGs for these poults were normal. Likewise, in Group IV, five poults had slight to moderate dilatation of the right ventricle and four poults showed slight to moderate dilatation of the left ventricle despite the fact that only one of these poults exhibited an altered ECG. In Group VI, five poults had dilated ventricular chambers but two of these poults had normal ECGs. Ultrastructural features of the myocardium were normal in the hearts of poults in Group II (Fig. 4). Morphological alterations were noted in the hearts of poults in Groups IV and VI (Figs 5-8) with the number and severity of changes more pronounced in the poults of Group VI. The major alteration observed was an increase in the number and size of vesicles. Some of these were formed by an outpocketing of the outer membrane of the nuclear envelope (Figs 5 and 6) while others were present among the mitochondria (Fig. 7). Other morphological changes seen included mild myolysis (Fig. 5) increase in width of the interstitial space (Fig. 6), swollen mitochondria (Figs 7 and 8) and condensed nuclei that were extensively folded (Fig. 8). The mean packed cell volume for control poults was 29.8 f 1.9 compared to 30.7 k 3.4 for ADRtreated pot&s. Although each ADR-treated group had a packed cell volume greater than its control these differences were not statistically group, significant individually nor when pooled. The differential blood count in control and ADRtreated poults at 7 weeks of age is shown in Table 4. Increased numbers of heterophils were observed in all ADR-treated poults when compared with their controls, but the number of heterophils was significantly higher (P I 0.01) only in Groups IV and VI. Decreased numbers of non-granulated leukocytes were seen in all ADR-treated poults when compared with the control poults. However, statistically significant decreases were observed only in monocytes in Groups II and VI. When the lymphocytes and monocytes were pooled for the control poults and for the ADRtreated poults, a significant decrease (P 5 0.001) in these cells was apparent in the drug-treated poults.
A
B Fig. 2. ECG pattern on lead II (aVF) for poult Nos 1113, 1124 and 1130 in Group VI. (A) Poult No. 1113 at 7 weeks (compare with Fig. 1 and note irregular amplitude of S wave), (B) poult No. 1124 at 6 and 7 weeks (note irregular pattern at 7 weeks), (C) poult No. 1130 at 6 and 7 weeks (note increase in amplitude of R wave at 7 weeks).
Fig. 3. ECG pattern on lead II (aVF) for poult No. 1147 in Group IV. (A) At 6 weeks, (B) at 7 weeks (note increase in amplitude of R wave).
CAROLINE M. CZARNECKI
56
Fig. 4. Longitudinal
section of myocytes in left ventricle of poult No. 1141 in Group myofibrils and nucleus (N) appear normal. x 12,060.
DISCUSSION
The etiology of the cardiotoxic effects of the anthracycline compounds is ill-defined. Considerable evidence has been compiled to suggest that acute ADR cardiotoxicity is mediated by peroxidative injury (Myers et al., 1976, 1977; Thayer, 1977; Bachur et al., 1978; Sonneveld, 1978; Doroshow et al., 1981) but alterations responsible for chronic ADR-induced cardiomyopathy are less well-defined. Animal models, reportedly used in past studies, tend to develop acute cardiotoxicity. However, it is the prolonged, repeated doses of ADR that are primarily responsible for the development of irreversible cardiomyopathy leading to congestive heart failure (Lefrak et al.,
II. Mitochondria,
1973; Minow et al., 1975; Billingham et aI., 1978; Bristow et al., 1978). Studies of chronic, repeated drug treatments are the most reliable means of evaluating adverse side effects (Peck, 1968). The results of the present study indicate that chronic, repeated small doses of ADR (1 mg/kg MWF) are more cardiotoxic than similar doses given with greater time intervals between injections (2 mg/kg MF). Cardiotoxicity, as determined by the degree of ventricular dilatation, was greater in the poults on the 1 mg/kg MWF schedule than in those on the 2 mg/kg MF regimen even though both groups of poults received similar cumulative doses of ADR during the period of this experiment. This lends credence to the hypothesis that the severity of the
Fig. 5. Oblique section of myocytes in left ventricle of poult No. II23 in Group IV. Perinuclear space is dilated with a vesicle present at arrow. Some myolysis (MY) is apparent in adjacent fiber. x 12,060.
Doxorubicin-induced
Fig. 6. Oblique (V) are present
Fig. 8. Longitudinal nuclear
membrane
57
section of myocytes in left ventricle of poult No. 1116 in Group VI. Prominent vesicles in a perinuclear position. The width of the interstitial space (IS) is increased. x 12,060.
Fig. 7. Longitudinal section of myocyte in left ventricle of poult No. 1124 in Group VI. Numerous vesicles (arrows) are present and the mitochondria appear swollen. x 12,060.
section of myocyte in left ventricle of VI. The nucleus (N) is folded and is thickened. Mitochondria are swollen. x 12,060.
poult No. 1124 in Group
cardiotoxicity
cardiotoxicity is related to the serum level of the drug as suggested by previous studies (Weiss and Manthel, 1977; Pacciarini et al., 1978; Edington et al., 1984). The selection of an animal model for testing cardiotoxic effects of drugs must take into account the responsiveness of the myocardium to the toxic agents as well as being cost-effective, accurate, reproducible and relatively simple to perform (Mettler et al., 1977). Previous studies have demonstrated that the heart of the turkey poult is responsive to toxic agents (Jankus et al., 1972; Czarnecki et al., 1974, 1986; Czarnecki, 1980; Czarnecki and Grahn, 1980; Noren et al., 1983). These studies, also, have shown that this animal model is relatively easy and inexpensive to maintain. In addition, cardiotoxic effects manifest themselves quickly and are reproducible with great accuracy. Hearse et al. (1976) reported wide species differences in the development of myocardial damage as measured by biochemical and morphological parameters For instance, hearts of the rat and mouse were found to be quite resistant while hearts of the guinea-pig and rabbit were very susceptible to cardiotoxicity. Weiss et al. (1976) observed that ADR administered at a dose of 2 mg/kg body wt/week caused dramatic cardiac damage in rabbits but not in man. In the present study, this dose of ADR produced only slight changes in the parameters measured. This indicates that the turkey poult’s heart is more similar to that of the human heart in its response to toxic drugs. Some species are prone to develop spontaneous myocardial lesions (Bajusz et al., 1969; Caulfield and Shelton, 1973; Weber et al., 1973) and should be avoided as model systems. Spontaneous cardiomyopathy is relatively rare in turkey poults, but it (or the tendency toward its development) can be detected by ECGs as early as 2 weeks of age (Czarnecki and Good, 1980). Thus, poults to be used in experimental studies can be evaluated relatively early and selection of appropriate animals can be made on the basis of normal EC&. ADR toxicity is manifested as alopecia, melanosis, body weight loss, myelosuppression, morphologic
CAROLINE M. CZARNECKI
58 Table 4. DilTerential croupt I
II III IV V VI
blood count in control and ADR-treated
Heterophils 42.8 f 53.2 f 35.0 ? 48.0 + 40.3 k 58.2 i
S.O$(6)$ 4.7 (6) 2.0 (6) 4.2 (6)‘; 4.9 (6) 4.0 (6)**
Basophils 1.5 f 3.5* 4.7 + 2.7 f 1.0 f 0.3 +
0.4(6) 1.2(6) 2.5 (6) 1.3(6) 0.5 (6) 0.2 (6)
turkev ooults at 7 weeks of see
Eosinonhils
Lvmnhocvtes
3.5 k 0.6 (6) 0.5 i 0.3 (6)*** 2.2 i_ 0.8 (6) 1.O k 0.6 (6) 1.5 k 0.8 (6) 0 f 0 (6)*
39.3 + 37.5 f 45.3 k 38.0 f 48.8 f 38.2 f
6.3 (6) 5.2 (6) 2.9 (6) 2.5 (6) 5.1 (6) 3.9 (6)
Monocvtes 13.0 F 5.3 f 12.8 f 10.3 t 8.3 f 3.3 +
2.5 (6) 1.3 (6)** 2.5 (6) 2.3 (6) 1.3 (6) 0.8 (6)***
*Significantly different from control group: *P IO.05; **P s 0.01; ***P I 0.001. tGroup I: two saline injections/week for 3 weeks (volume of dose equivalent to that of Group II); Group II: two ADR injections at a dose of 1 mg/kg body v/t/week for 3 weeks; Group III: two saline injections/week for 3 weeks (volume of dose equivalent to that of Group IV); Group IV: two ADR injections at a dose of 2 mg/kg body w/week for 3 weeks; Group V: three saline injections/week during 4th and 6th weeks (volume of dose equivalent to that of Group VI); Group VI: three ADR injections at a dose of 1 mg/kg body w/week during 4th and 6th weeks. fMean k SE. §Number of poults.
alterations, ECG changes, nephrotoxicity, chondroosseus toxicity and cardiomegaly (Sternberg et al., 1972; Buja et al., 1973; Cargill et al., 1974; Jaenke, 1974; Bachmann et al., 1975; Young, 1975; Mettler et al., 1977; Kehoe et al., 1978; Olson and Capen, 1978; Van Vleet et al., 1978). In the turkey poult, manifestation of ADR toxicity was limited to mild suppression of growth, slight myelosuppression of leukocytes, ECG changes and cardiomegaly. Morphological examination of myocardial tissue confirmed alterations in ultrastructural features previously described following furazolidone(Czarnecki, 1980, 1986) and alcohol-induced (Czarnecki et al., 1986) cardiomyopathy in turkey poults consonant with altered ECGs and cardiomegaly and in ADRinduced cardiotoxicity (Buja et al., 1973; Jaenke, 1974; Olson and Capen, 1977, 1978; Van Vleet et al., 1978). The data obtained in this study indicate that the turkey poult is an appropriate model system for testing the cardiotoxicity of drugs such as ADR and its analogs. Acknowledgements-The author is grateful to Carl W. Edborg III who cared for the poults, to Dr A. L. Good, Lenore Mottaz and Michael Powers who provided technical assistance and to the Dale Peterson Turkey Hatchery, Cannon Falls, MN, for the generous supply of turkey poults. This investigation was supported by a Grant-in-Aid from the American Heart Association and with funds contributed in part by the Montana Heart Association. REFERENCES Bachmann E., Weber E. and Zbinden G. (1975) Effects of seven anthracycline antibiotics on electrocardiogram and mitochondrial function of rat hearts. Agents Actions 5, 383-393. Bachur N. R., Gordon S. L. and Gee M. V. (1978) A general mechanism for microsomal activation of quinone anticancer agents to free radicals. Cancer Res. 38, 1745-1750. Bajusz E., Baker J. R., Nixon C. W. and Homburger F. (1969) Spontaneous hereditary myocardial degeneration and congestive heart failure in a strain of Syrian hamsters. Ann. N. Y. Acad. Sci. 156, 105-129. Bertazzoli C., Bellini O., Magrini U. and Tosana M. G. (1979) Quantitative experimental evaluation of adriamycin cardiotoxicity in the mouse. Cancer Treat. Rep. 63, 1877-1883. Billingham M. E., Mason J. W., Bristow M. R. and Daniels J. R. (1978) Anthracycline cardiomyopathy monitored by morphologic changes. Cancer Treat. Rep. 62, 865-872.
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