adriamycininduced cardiotoxicity (cardiomyopathy …...congestive heart failure) in rats f. p....

[CANCER RESEARCH 37, 2705-2713, August 1977@

SUMMARY

The recent development of numerous analogs of the anthracycline class of oncolytic agents has resulted in anurgent need for a standardized , accurate, reproducible, andcost-effective system for candiotoxicity testing . The presentstudies were designed to determine the feasibility of usingthe matas a model for induction of the chronic type ofcandioloxicity (i.e., cardiomyopathy and congestive heartfailure). Adniamycin (ADA) was administered to rats at dosesof 1 to 2 mg/kg/week for 10 to 14 weeks. The majority ofADA-treated rats developed cardiomyopathy from 3 to 23weeks after the last injection. Forty ho70% of those rats withcardiomyopathy had gross evidence of congestive heartfailure (pleural effusions, ascites, hepahomegaly, cardiomegaly). Histological myocardial changes consisted of myocyte vacuolahion and degeneration, interstitial edema, andmild fibropiasia. In addition, damage to atnial ganglion cellswas evident in several rats. Ultrastructural alterations involved sarcoplasmic disruption with distentions of subcellulan organelles and loss of myofiiaments. Extracandiactoxic effects of ADA (nephnotoxicity, rnyelosuppression, enteropathy, arrested osleogenesis) were minor and were observed more frequently at higher cumulative doses.

The results of the present study suggest that the rat modelis an accurate, reproducible, and cost-effective system forlarge-scale cardiotoxicity testing of analogs of ADA anddaunonubicin.

INTRODUCTION

Cardiotoxicity is a well-characterized , major dose-limitingcomplication of ADA3 therapy (6, 17). Although numerousstudies have described the effects of this broad-spectrumantineopiashic agent on cellular structure and function, thepathogenesis of the candiomyopathy remains an enigma(15, 17, 43). Current investigations appear to be directed atthe identification of noncardioloxic analogs of ADA andDNA and the characterization of potential antidotal procedunes that prevent or reverse the development of card iomyopathy. More than 300 analogs of the anthracycline class ofoncolytic agents have been defined chemically. Therefore,

I NIH Fogarty International Fellow from the Veterinary Pathology Institute,

Zurich, Switzerland.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: ADA, Adriamycin: DNA, daunorubicin; CHF,congestive heart failure; ECG, electrocardiogram.

Received February 11, 1977; accepted May 16, 1977.

there is an urgent need for a standardized quantitativesystem to screen, assay, or test analogs for candioloxicactivity in comparison to the parent compounds. Of thenumber of animal models described as exhibiting ADRinduced candiotoxicity (43), the rabbit appears to be themost widely used by various laboratories. Although therabbit model provides a highly reproducible incidence ofcandiotoxicity, it is costly to use for large-scale testing ofanalogs. Therefore, the current studies were designed todetermine the feasibility of using the rat as a model forinduction of the chronic type of cardiotoxicily (i.e. , cardiomyopathy and CHF).

MATERIALS AND METHODS

A total of 70 male 4-week-old CDF (Fischer) mats(CharlesAiver Breeding Laboratory, Wilmington, Mass.) were usedin this study. Rats were maintained in filter-topped polycambonate cages on NIH Open Formula Rat and Mouse Ration5018 (Ralston Purina Co., St. Louis, Mo.) and waler adlibitum throughout the experiment. At 6 weeks of age, therats were divided into 5 groups for either s.c. or iv. (tailvein) injection. ADA (kindly supplied by Dr. Harry Wood,Drug Synthesis and Chemistry Branch, National CancerInstitute, NIH, Bethesda, Md.) was administered i.v. in 0.9%NaCI solution at a dose schedule of 1 mg/kg/week (Group 1)and 2 mg/kg/week (Group 2) and s.c. at 2 mg/kg/week(Group 3). Control rats received equivalent volumes of 0.9%NaC1solution i.v. (Group 4) and s.c. (Group 5) once a week.Rats were lightly anesthetized with ether prior to i.v. injeclions with 27-gauge needles. With careful technique it waspossible to avoid penivenous infiltration, sclerosis, and necrosis of the tails. In order to avoid development of skinulcerations, s.c. injections were performed at alternate siteseach week. Body weights were recorded weekly. Samplesof blood were collected from the tail veins 24 hr after injeclion on a bimonthly basis for determination of packed cellvolume (microhematocnit). Injections of ADA were discontinued after 10 weeks for rats in Group 2, between 11 and 14weeks for rats in Group 1, and at 13 weeks for matsin Group3. Rats either died of toxicity or were sacrificed in poorcondition by an overdose of ether. Terminal blood samplesfrom rats being sacrificed were collected from the abdominal aorta, and sera were analyzed for calcium (TechniconAuto Analyzer), phosphorus (Hycei phosphorus), sodium,potassium (Technicon STaT-ion), blood urea nitrogen (19,37), creatine phosphokinase, (30, 33), cholesterol (14), and

AUGUST 1977 2705

Adriamycininduced Cardiotoxicity (Cardiomyopathy andCongestive Heart Failure) in Rats

F. P. Mettler,' D. M. Young,2 and J. M. Ward

Comparative Pathology Section, Laboratory of Toxicology, Experimental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute,NIl-I, Bethesda, Maryland 20014

on July 22, 2020. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

F. P. Mettler et a!.

triglycerides (3). Necropsies were performed with collectionof the following tissues in Telly's fixative (36) for hislopathological evaluation: heart, lung, liven, kidney, spleen, intestines, diaphragm, and proximal tibia. Sections were cut at 6

@mand stained with hematoxylin and eosin and Masson'shnichnome.Samples of ventricular myocandium from 8 ADAtreated rats were minced into small cubes (0.5 mm), fixed in1.5% gluharaldehyde, poslfixed in 1c@/@osmium tetnoxide(10), dehydrated in graded ethanols, and embedded inEpon-Araldihe (22). The blocked samples were cut at 250 to500 A with a Aeichert Om U-2 ultramicrotome, stained withunanyl acetate and lead citrate (31), and examined with aJEOLCO 100B electron microscope. Samples from 3 controlhearts were processed and evaluated in a similar manner.

Blood and tissue samples from appropriately matchedcontrols were utilized in these studies, and data were analyzed statistically using Student's t test (39).

All matsmaintained similar growth rates throughout the1st 4 weeks of injections (Chart 1). Thereafter, rats in Group2 became inactive and failed to maintain weight gains asobserved in other treated and control rats. Rats given ADA,2 mg/kg s.c. , gained weight at a malesimilar to that of ratsreceiving ADA, 1 mg/kg iv. Blood collected from matsinGroup 2 revealed that the packed cell volume had decreased from a base-line range of 39 to 46%, after 10 weeksof treatment, to 15 to 25%. Microhemalocnit values from matsin Group 1 were only slightly decreased (range, 35 to 40%)compared to controls (range, 40 to 48%). One rat in Group 2died of toxicity after 9 weekly i.v. injections. The remainingADA-treated rats survived the full course of the experimentexcept for 1 rat in Group 1 and 3 matsin Group 2, which died

.0â€”â€”0

0.9% NaCI@

@0-@@

_@i 0@@ 1mg/Kg

I f I I I I

0 1 2 3 4 5 6 7 8 9 10 11 12

WEEKSAFTERFIRSTDRUGADMINISTRATION

Chart 1. Dose response to weekly iv. administration of ADA in rats. Bodyweights are means of the groups at the time of observation.

as a result of inadvertent ether overdose prior howeekly i.v.injections. Nine rats in Group 1 died from 1 ho23 weeks afterthe lash ADA injection (Table 1). The remaining 10 rats inGroup 1 were sacrificed at various times ranging from 1 to32 weeks after the final ADA injection.

In Group 2, 8 of the 17 rats died from 1 week before to 5weeks after the last injection of ADA (Table 1). The remaining 9 rats were sacrificed from 1 ho 12 weeks after the lastADA injection. Analysis of terminal serum samples from ratsin Group 2 revealed (mean Â±SE.) hypocalcemia (8.9 Â±1.8mg/100 ml), hyperkalemia (6.9 Â±2.3 mEq/liter), hypemphosphahemia (9.3 Â±2.8 mg/100 ml), elevated blood urea nitrogen levels (73 Â±23 mg/100 ml), and hypencholesterolemia(396 Â±128 mg/100 ml). These values were significantlydifferent (p < 0.05) from corresponding control means ofcalcium (10.6 Â±0.2 mg/100 ml), potassium (4.8 Â±0.3 mEq/liter), phosphorus (5.4 Â±1.5 mg/100 ml), blood urea nitrogen (22 Â±1 mg/100 ml), and cholesterol (110 Â±24 mg/100ml). Changes in serum parameters of terminal samplesfrom other ADA-treated groups did not differ significantlyfrom control values.

CHF was evident grossly at necropsy (pleural effusions,ascites, candiomegaly) in 14 of the ADA-treated rats (Table1). One rat in Group 1 died 23 weeks after the last of 14weekly ADA injections (84 mg/sq m total dose) with fulminating CHF.

Due to severe postmortem aulolysis, heart samples werenot collected for light microscopy from 8 rats in Group 1and 4 matsin Group 2. Histopathological evaluation of heartsfrom the majority of the remaining ADR rats revealed varying degrees of myocandial alterations (Table 1). The mostconsistent and predominant finding was marked vacuolization of myocytes, especially in subepicardial and subendocandial regions in addition to penivasculan locations (Figs. 1,3, and 4). Although vacuolan lesions were observed frequently in ventricles and sepIa, several rats had severevacuolahion of atnial myocytes (Fig. 2). In addition, vacuolardegeneration of ganglion cells was observed in 2 of 3 ratswhere atnial sections were found to include clusters ofcardiac neurons (Fig. 5). Three rats in Group 3 had focalatmial thromboses evident histopathologically. Myocyholysis, interstitial edema, and mild fibrosis were evident inoccasional areas of more severely affected rat hearts. Theincidence and severity of the cardiomyopathy appeared tobe dose dependent (Table 1).

Ultrastructural evaluation of samples of myocandium fromADR-treated rats revealed varying degrees of sancoplasmicvacuolation (Fig. 6). The vacuoles occurred predominantlyin a peninuclear position and consisted of distentions of thesarcoplasmic reticulum, the T-tubule system, and Golgi yesides. In myocyles where vacuolation was severe, there wasa concomitant reduction in the quantity of myofibens. A fewscattered myocytes were found ho contain fragmented sancomeres with disruption of the characteristic parallel arraysof myofilaments. In general, mitochondnia did not appear hobe altered markedly, although several were swollen withdisruption of cnistae. Formation of myelin figures was not apredominant feature of mitochondrial alterations.

Histopathological evaluation of other tissues from ADAmatsrevealedlesionsin kidneys,liven,small intestine,

RESULTS

350@

250@

150-

100

50'

2706 CANCER RESEARCH VOL. 37



ADA-induced cardiotoxicity in rats receiving weeklyinjectionsMeantotalADACardiotoxicityTreat

RouteofdoseDied/mentNo.ofadminis (mg/sqMean wk sun sacni Cardiomy

groupratstrationTreatment schedulem)vivalficedCHFopathya119iv.ADA,1 mg/kg/wk for11-14

wk7531(12@44)â€•9/102 4(36)'21'iv.ADA,

2 mg/kg/wk for 10wk12013 (9-22)8/98 12(92)310s.c.ADA,2 mg/kg/wk for 13 wk15616 (14-20)5/54 10(100)410iv.0.9%NaCI solution, 1 ml/

kg/wkfor14wk00/1000510s.c.0.9%NaClsolution,lmI/kg/

wk for13 wk00/1000

Various reported systemsto test for anthracycline-inducedcardiotoxicityParameters

and end-point evaluaSpeciesIn vivo/invitrolionsRefs.Chick

embryo+ECG evaluation, microscopic pathological evaluation26,

27Mouse

Rat+

+

+

++Survival

time, accumulative dose,microscopic and ultrastructuralpathological evalution

Survival time, heart wt, myocardialmalondialdehydecontent

Myocardial cell cu'lture, beatingfrequency, automaticity, andrhythmic contractile activity

Altered myocardial DNAsynthesisECG evaluation, accumulativedose18,

35

21, 23

9, 24

341,5,46Rabbit+

++

+Survival

time; accumulative dose;gross, microscopic, and ultrastructural pathological evalualion

Myocardial calcium accumullionInhibition of sodium-potassium

ATPase(heart, kidney)Inhibition of mitochondrial respira

tory enzymes15,

16, 43, 45

2541

12Monkey+Survival

time, accumulative dose,gross and microscopic pathological evaluation11â€•

ADR-induced Cardiotoxicity in Rats

Table1

(I Determined by histopathological evaluation.

b Numbers in parentheses, range.

â€˜.Numbers in parentheses, percentage of incidence based on number of hearts evaluated histologically.

Table 2

I, E. J. Gralla, A. W. Fleischman, Y. K. Luthra, and S. W. Standnicki. Toxicology Studies

in Beagle Dogs and Rhesus Monkeys Given Adriamycin (NSC 123,127) Intravenously,submitted for publication to Cancer TreatmentReports.

cecum, and bone. Renal changes were characterized bydilated tubules, vacuolaled glomenuli, and thickening ofBowman's capsule (Fig. 7). One rat in Group 1 died withhistopathological evidence of chronic renal failure and secondary hyperparathynoidism 17 weeks after the last ADAinjection. Rats with CHF had various degrees of centrolobulamhepahic necrosis. Midsagittal sections of proximal tibiaefrom the majority of ADA rats contained lesions in theepiphyseal-metaphyseal area consisting of a deficiency oftnabeculae, osleoblasts, and osteoclasts (Fig. 8). Thesechanges were interpreted as consistent with an arrest ofchondroosheogenesis. Lesions observed in the small intestine and cecum consisted of varying degrees of epithelial

regeneration. One rat in Group 1 had a squamous cellcarcinoma of Zymbal's gland at 22 weeks after the last ADAinjection. No other neoplastic processes were observedgrossly on histopathologically in either ADA-treated on controlrats.

DISCUSSION

The need for a model system to test analogs of ADA andDNA for candiotoxicity has resulted in numerous investigalions and several proposed experimental systems (Table 2).These systems vary considerably between species with me

AUGUST 1977 2707




sped to suggested markers or indices of cardiotoxicity.None of the recommended procedures has been testedrigorously on a large-scale basis to determine the accuracyand reproducibility of its predicliveness. Any biological syshem used to assay for anthracycline-induced cardiotoxicityshould be: (a) cost-effective with respect ho lange-scalescreening of ADA-DNA analogs; (b) accurate and reproducible for predicting the development of candiomyopathy andCHF; and (C) relatively simple to develop and perform inorder to be used readily in various laboratories withoutspecial facilities, equipment, or highly trained personnel.

The results of the present study indicate that the ratmodel may be the closest to an â€œideal animal modelâ€•forcamdiotoxicity testing (43). Large numbers of animals can beplaced on lest and maintained at a reasonable cost. Theinjection procedures and evaluation of end-point paramelens are simple and routinely used in a number of labomalonies. The induction of the candiomyopathy occurs with ahigh reproducible incidence and appears to be time anddose related. The myocardial lesion has histological charactenisitics similar to those reported in humans and rabbits(i.e., myocyte vacuolation and degeneration, edema, mildfibrosis) (4, 15-17, 43, 45). In addition, the cardiotoxicily isdelayed, progressive, and frequently manifested by development of CHF with measurable quantities of fluid retainedin body cavities. The rat model could be utilized in dualexperimentation to determine oncolytic and cardioloxic achivity of analogs. ADA and DNA have been found ho bemoderately active against s.c-implanted Walker 256 cancinosarcoma in rats (J. Vendilti, personal communication).One limitation of the rat model as utilized in our studies isthe time required for induction of the candiomyopathy (10 to20 weeks). Studies are being designed to determinewhether a characteristic subchronic ECG change correlatesconsistently and reproducibly with eventual candiomyopathy and CHF in the rat model. Zbinden et a!. (1, 5, 46)have proposed the use of EGG evaluation in rats as anexperimental system to detect anlhracycline-ind uced card iotoxicity. They administered analogs and parent agents i.p.on a daily basis for 5 to 25 injections. ECG's were recordedin unanesthelized mats1 to 4 limes weekly, and the rats werekept alive for 8.5 weeks. The cumulative dose of ADA (120mg/sq m) administered is comparable to that for rats in ourstudy (Table 1). Widening of the QRS complex and meanQAS duration plus S-wave trough were used as end pointsin evaluation of EGG's. In this respect, Zbinden et a!. (46)calculated the â€œminimal cumulative cardiotoxic doseâ€•forADA as 68 mg/sq m. No significant hishopathologicalchanges were evident in myocardia of test rats, and minoralterations (â€œslightfocal fiber degeneration or atrophy withmodest proliferation of interstitial connective tissueâ€•)werenot correlated with EGG findings (46). Perhaps lest ratswere sacrificed too early for manifestations of the chanaclenislic candiomyopalhy and fulminating CHF. The cumulalive cardiohoxic doses for ADA in our studies ranged from 75to 156 mg/sq m, which is slightly higher than Zbinden'sminimal cumulative candiotoxic dose. The major difference between these 2 rat studies is the time of evaluation ofend-point parameters (i.e. , 8.5 versus 9 to 44 weeks). Funthen studies are needed hodetermine if there is a significant

correlation between Zbindens acute-subchnonic nat modeland our proposed chronic rat model. A comprehensivecomparative study may lead to the identification of a subchronic parameter alteration which could serve as a preciseindex or marker of ADA-induced cardiotoxicity in rats.

Chalcrofletal. (7) did not observe myocardial changes bylight microscopy in rats receiving single iv. doses of DNA,25 mg/kg. Myocardial tissues were collected from test rats 1to 5 days after injection and prepared for light and electronmicroscopic evaluation. Ultrastructumal changes in myocytes consisted of mihochondnial alterations with swelling,loss of cnislae, degeneration, and formation of myelin figunes. Milochondnial changes were not predominant featunes of myocardial lesions in rats from our study. Thisdiscrepancy is most probably related to the differences inexperimental design (i.e. , acute, single high dose versuschronic, multiple low dose). Studies by Philips et a!. (29)revealed the absence of hislopathological changes in myocandia 1 to 15 weeks after single high iv. dosing of ADR(10 and 20 mg/kg) in rats. They also reported the effects ofsupralethal iv. doses (10 and 20 mg/kg) in rabbits. Although myocardial lesions were evident 5 to 24 hr after ADAadministration , the histopathological characteristics weredissimilar from those reported after chronic, multiple, lowdose treatment (15, 43). These results appear to underscoreagain the lack of similarity of findings between acute andchronic studies of anthracycline-induced cardiohoxicily. It iswell known in toxicology investigations that acute studiesare the least reliable, whereas chronic, repeated dose studies are the most reliable in evaluating the side effects ofdrugs (28).

The observation of lesions in atnial ganglia of matsfromour study was a serendipitous finding. The predominanthistopathological alteration was cyloplasmic vacuolation.Occasional ganglia cells appeared swollen,' with sparsegranular cytoplasm . The adverse effects of anhhracyclineson nerve cells and ganglia have been a subject of considerable debate. Smith (38) reported on damage to intrinsiccardiac neurons induced by DNA in patients with cancer.The alterations were evident at the light microscopic leveland consisted of unusual linclonial properties, degeneralion and death of neurons, proliferation of amphicyhes, neuronophagia, and swollen and misshapen processes. Studies by other investigators evaluating animal and humanmyocardia after treatment with DNA on ADA have failed toconfirm the finding of neuronal lesions (4, 15, 43). Hishofluonimelnic studies by Mazzanti et a!. (20) suggest that DNAis rapidly taken up by nerve cell cytoplasm in the Auerbachplexuses of rats and guinea pigs receiving i.p. injections.Other animal studies indicate that histopathological degeneralion of spinal root ganglia occurs regularly after ADAadministration (E-S. Cho, personal communication). Furthen studies are required to characterize and elucidate thepathogenesis of anthracycline-induced changes in nervecells.

The extracardiac toxic effects of ADA in the matmodel(i.e. , nephnohoxicily, hematopoietic suppression, arrestedosheogenesis) are minor and occur more frequently withhigher doses. The nephrotoxic effects of ADA and DNAhave been well characterized in rats (29, 40) and rabbits





(43). The chondroosseous toxic effects of ADA in growingrabbits have been reported in detail previously (44). Therefore, the observed extracardiac toxic effects of ADA in ratswere not an unexpected finding.

The selection of an animal model for candiotoxicity lesting should take into account that the response of the myocardium to toxic agents appears ho vary considerablyamong species. Hearse et a!. (13) found major species diffenences in development of myocardial damage as measured by biochemical and ultrastructural evaluation. Ratand mouse hearts were resistant whereas guinea pig andrabbit hearts were susceptible ho early onset of damage.These findings correlate well with the observation that nabbit myocardium is uniquely sensitive to the candiomyopathiceffects of ADA (15, 16, 43, 45). Nutritional and physiologicalpeculanities between species should also be examined.Rabbits have a rather unusual calcium homeostahic mechanism and an unusual, yet undefined, nutritional interactionof selenium and vitamin E (8). Perhaps the normally highserum calcium levels play a role in the sensitivity of thisspecies to ADA. These species differences should be considered in attempts to identify markers on indices of ADAmduced cardiotoxicily. Biochemical or ultrastruclu nalallenahions of injured myocardia may not be readily extrapolatedacross species lines. In addition, some species may be moreprone to develop spontaneous myocandial lesions (2, 42).

The results of this study suggest that the rat model is anefficient, reprod ucible , cost-effective system for large-scaletesting of ADA and DNA analogs. Rats develop candiomyopathy at considerably lower total doses of ADA comparedto rabbits (15, 16, 43, 45). For convenience and ease ofstandardization, it is recommended that rats receive analogs s.c. weekly for 13 consecutive weeks. Initial test dosesshould be one-tenth to one-fifth of the 50% lethal doseadministered s .c . Rats should be observed and weighedregularly. Six weeks after the last dose all rats should besacrificed. Necropsies should be performed on all rats withspecial notation for evidence of CHF. The heart should beremoved, trimmed, and weighed. Tissues should be collecled for histopathological evaluation, with the heart (including atnia) sectioned transversely at 3 ho 5 equidistantlevels. The following qualitative changes should be mecorded: vacuolation of myocytes, interstitial edema, myocyte degeneration, and fibroplasia. The lesions can beassigned a semiquantitative score (16) or a more refinedquanlilalion using morphometnic analysis (32). Measurement of the aforementioned parameters would allow for anaccurate, reproducible, and precise evaluation of the card iotoxic activity of ADR and DNR analogs.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to Myra Grabin and BernardTaylor for animal care and administration of injections, Reginald Reagan forelectron microscopy services, and Jo F. Pelham and Cindy Bunnell fortechnical and editorial assistance.

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39. Steel, A. G. D., and Torrie, J. H. Principles and Procedures of Statistics,pp. 427-471 . New York: McGraw-Hill Book Co., Inc., 1960.

40. Sternberg, S. S., Philips, F. S., and Cronin, A. P. Renal Tumors andOther Lesions in Rats Following a Single Intravenous Injection of Daunomycin. Cancer Res., 32: 1029-1036, 1972.

41. Van Rossum, G. D. V.. and Gosalvez, M. Inhibition of Ion-Transport bythe Antitumor Antibiotic Adriamycin. Federation Proc. , 35: 787, 1976.

42. Weber, H. W. and Van der Walt, J. J. Cardiomyopathy in CrowdedRabbits: A Preliminary Report. S. African Med. J., 47: 1591-1595, 1973.

43. Young. D. M. Pathologic Eftects of Adriamycin (NSC-123127) in Experimental Systems. Cancer Chemotherapy Rept., 6: 159-175, 1975.

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46. Zbinden, G., and Brandle, E. Toxicologic Screening of Daunorubicin(NSC-82151), Adriamycin (NSC-123127), and Their Derivatives in Rats.Cancer Chemotherapy Rept. , 59: 707-715, 1975.

Fig. 1. 5ection of ventricular myocardium from a rat receiving ADA, 2 mg/kg/week iv., for 10weeks (120 mg/sq m). The rat died with CHF 5 weeks after thelast dose. Note the vacuolar destruction of a myofiber (center) and interstitial edema (e). H & E, x 330.

Fig. 2. Section of atrial wall from ADA rat (Fig. 1). Note the diffuse vacuolation of atrial myocytes. H & E, x 130.Fig. 3. Section of myocardium from a rat receiving ADA, 2 mg/kg/week iv., for 10 weeks (120 mg/sq m). The rat was sacrificed in moribund condition 4

weeks after the last injection with fulminating CHF. Note vacuolated myocytes adjacent to arteriole (arrows). H & E, x 220.Fig. 4. Section of myocardium from a rat receiving ADA 2 mg/kg/week s.c. for 13 weeks (156 mg/sq m). The rat was sacrificed 3 weeks after the last

injection with evidence of CHF. Note the varying degrees of vacuolar destruction of myocytes and interstitial edema (e). H & E, x 350.Fig. 5. Section of a cluster of atrial neurons from ADA rat (Fig. 4). Note the vacuolar destruction of neurons (arrows). H & E, x 220.Fig. 6. Ventricular myocyte from a rat receiving ADA, 2 mg/kg/week iv., for 10 weeks (120 mg/sq m). The rat was sacrificed 1 week after the last injection

with CHF. Note the perinuclear vacuolation of the sarcoplasmic reticulum (V), swelling of mitochondria with disruption of cristae, and fragmentation anddisorientation of myofilaments. Loss of myofilaments and spreading of Z-band material are also evident (arrows). Uranyl acetate and lead citrate, x 7500.

Fig. 7. Section of kidney from a rat sacrificed 2 weeks after the last ADA injection (120 mg/sq m). Note the glomerular vacuoles (V) and thickening ofBowman's capsule. H & E, x 220.

Fig. 8. Midsagittal section of proximal tibia from an ADA rat (Fig. 3). Note the absense of primary and secondary trabeculae in the metaphyseal area. E,epiphyseal plate: M, metaphyseal marrow. H & E, x 180.

2710 CANCERRESEARCHVOL. 37





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1977;37:2705-2713. Cancer Res F. P. Mettler, D. M. Young and J. M. Ward Congestive Heart Failure) in RatsAdriamycin-induced Cardiotoxicity (Cardiomyopathy and

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