Cardiomyopathy predisposes hamsters to trichlorofluoromethane toxicity

rOXICOLOGY

AND

APPLIED

PHARMACOLOGY

32,177-l 83 (1975)

Cardiomyopathy Predisposes Trichlorofluoromethane GEORGE J. TAYLOR’ Pharmacology

Branch, National Research Triangle Received

Hamsters Toxicity

AND ROBERTT. DREW

Institute of Environmental Health Park, North Carolina 27709

July 26, 1974;

to

accepted

October

Sciences,

28,1974

Cardiomyopathy PredisposesHamsters to Trichlorofluoromethane Toxicity. TAYLOR, G. J. AND DREW, R. T. (1975). Toxicol. Appl. Pharmacol. 32, 177-l 83. Cardiomyopathy occurs asan inherited trait in the BIO 82.62 strain of Syrian hamsters; the animals dying of frank congestiveheart failure when they are between240and 270daysold. Exposureto 7.5 or 2 % trichlorofluoromethane(F-l 1) killed four of four and four of five 240-dayold cardiomyopathic (CMP) hamsters,respectively, within 48 min. CMP hamsters,150 days old, and random bred hamstersof both agessurvived the 4-hr exposureand the 2-wk postexposureobservation period. Other groups of 240-day-old CMP hamsterssurvived 4-hr exposuresto air or 7.5 % Nz in air and lived an averageof 8 days postexposure.Pentobarbital anesthetizedrandom bred hamstersrequired exposureto 10% F-l 1 before arrhythmias (tachycardia) developed. Young (120-day-old) anesthetized CMP hamstersdeveloped arrhythmias during exposure to 2.5% F-11. Both 120- and 180-day-old anesthetizedCMP hamstersdevelopedsinus bradycardia during exposureto 5 % F-l 1. Thesestudiesshowthat F-l 1 is more toxic to CMP hamstersand that the toxicity is qualitatively different aswell. The deliberate inhalation of fluorocarbon propellant gasesfrom household aerosols has caused sudden death in humans (Bass, 1970) and has causedcardiac arrhythmias and myocardial depression in healthy laboratory animals (Harris, 1973; Taylor and Drew, 1973). We wondered whether preexisting diseasealters the responseto a commonly used fluorocarbon, trichlorofluoromethane (F-l 1). The effects of exposing normal hamsters to F-l 1 were compared to the effects of F-l 1 on hamsterswith inherited cardiomyopathy. Cardiomyopathy occurs as an inherited trait in the BIO 82.62 strain of Syrian hamsters. The biochemical and genetic character of the muscle defect is unknown, but as in the BIO 14.6 and BIO 40.54 hamsters, the diseasefollows a predictable pathological and physiological course (Gertz, 1972; Bajusz et al., 1969). Over 90 % of the hamsters die in congestive heart failure. The diseaseseparatesinto four histological and clinical phases: prenecrotic, necrotic or myolytic, hypertrophic, and terminal. During 1Presentaddress:Departmentof Medicine,The Johns Hopkins Hospital, Baltimore, Maryland 21205. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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DREW

the first stage no clinical, gross, or histological pathological changes are notable. The second phase is only noticeable on histological examination which at the maximal response consists of focal myolosis and necrosis. Healing begins at about 60 days and is usually complete by 90 days, but there is still a low incidence of both lesions. In the third or hypertrophic phase, clinical signs are still absent but animals killed at this time have secondary calcification of the myocardial fibers and the heart begins to dilate. During the fourth phase, the clinical signs of heart failure appear. The animals appear cyanotic, accumulate fluid, and die several days later. The particular strain of hamster used in this study develops cardiac hypertrophy and at the age of 240 days the hamsters are in frank congestive heart failure. The 240-dayold hamsters weighed an average of 24 g more than 180-day-old hamsters, appeared edematous, and on necropsy had dilated hearts and hepatic changes characteristic of chronic passive congestion. Groups of age- and sex-matched, random-bred Syrian hamsters were studied with each group of cardiomyopathic (CMP) hamsters. We are reporting the effects of exposing CMP hamsters to F-l 1, both in terms of mortality and alterations in cardiac rhythm. METHODS Four-hour exposures. Three groups of 16 hamsters, with each group divided into four subgroups of four animals each (150- and 240-day-old CMP hamsters and 150- and 240day-old random-bred hamsters) were exposed for 4 hr to either room air, 7.5 % nitrogenroom air, or 7.5 % F-l l-room air mixtures on the same day (Table 1). Cardiomyopathic and random-bred hamsters of a given age were stratified by weight and then assigned to one of the three exposure groups at random. A fourth group of 16 hamsters was exposed to 2 % F-l 1 in room air on a separate day, and another group of four 240-day-old random bred hamsters was exposed to lOok F-11-room air. The exposures were carried out in a 30-liter chamber with the gas mixture flow rate at 7 liters/min. Ten-milliliter samples of gas were drawn from the chamber for F-l 1 analysis at 1,2, 3, and 4 hr of exposure, and immediately injected into a flask containing 10 ml benzene. A l-p1 aliquot of this benzene solution was then injected into a Varian Aerograph 1400 gas chromatograph equipped with a flame ionization detector. The 6-ft by & in. stainless steel column of the chromatograph was packed with 3 % SE 30 on 60/80 GCP. Column temperature was 50°C detector temperature 225°C and the flow rate of the nitrogen carrier gas was 15 ml/min. The amount of F-l 1 in the gas mixture was determined by comparing the peak height to that of benzene solutions of known F-11 concentration. A standard curve was determined to be linear over the F-l 1 concentrations studied. During these exposures, the hamsters were observed and an animal was presumed dead when there were no visible respirations over a 2-3 min period. Exposure was continued for 4 hr regardless of the presence of dead animals in the exposure chamber. Cardiac arrhythmias. Hamsters were anesthetized with 50 mg/kg sodium pentobarbital and secured in the prone position. Needle electrodes were inserted subcutaneously. Leads I, II, and III of the electrocardiogram were monitored simultaneously using a photographic recorder (Electronics for Medicine DR-8) at a paper speed of 100 mm/set with 0.04 set time lines. F-l 1 or nitrogen was mixed with air and delivered at 3 liters/

CARDIOMYOPATHY

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FLUOROCARBON-l

179

1

min through a cone whose maximum diameter was 2 cm. The hamster’s snout was loosely placed in the cone, and exposure was begun by metering F-l 1 or nitrogen into the air flow. During exposure, 10 ml of gas was sampled at the base of the cone for F-l 1 analysis. Exposures were continued for 5 min or until the appearance of a cardiac arrhythmia. Groups of random-bred hamsters either 120 or 180 days old were exposed to either a mixture of 7.5 $2:N2 in air or up to 10% F-l 1 in air (Table 2). Cardiomyopathic hamsters also 120 or 180 days old were exposed to either 7.5 “; N, in air or 2.5 or 5 PI; F-l Iair mixtures. RESULTS

Four-hour exposures.All random-bred hamsters survived 4-hr exposures to room air, 7.5 ‘?/, nitrogen-room air or 2 or 7.5 % F-l l-room air mixtures (Table 1). Mortality among random-bred hamsters occurred only during exposures to 10% F-11-air mixtures. Survivors of these exposures were alive 3 wk later. Young (150-day-old) CMP hamsters survived all exposures up to and including 7.5 % F-l 1 mixtures, the highest concentration of F-l 1 tested, and all were alive 3 wk after the exposure. Old (240-day-old) CMP hamsters in congestive heart failure survived exposures to room air or 7.5% nitrogen-room air mixtures, and lived an average of 8.1 days (range 3-l 5 days) and 9.5 days (range 4-16 days) respectively, after the exposure. The four 240day-old CMP hamsters exposed on the same day to 7.5 “/, F-l l-room air mixtures all died during the first 30 min of exposure. F-l 1 concentrations in the chamber were 7.2, 7.6, 7.8, and 7.4% measured at 1, 2, 3, and 4 hr of this exposure. Examination of the dead CMP hamsters at the end of the exposure showed grossly dilated hearts. A subsequent 4-hr exposure of a group of 17 hamsters to 2 % F-l l-room air mixtures resulted in the death of four of five 240 day old CMP hamsters within 48 min of exposure (Table 1). The survivor died 2 days later. Younger myopathic, and all random-bred hamsters tolerated this 4-hr exposure and were alive 3 wk later. TABLE

1

MORTALITY DURING A 4-HR EXPOSURE TO Rook AIR, NITR~CEN (PLACEBO), OR RCIOM AIR CONTAINING TRICHLQROFLUOROMETHANE (F-l 1)

Age (days) Random bred

150 240

Myopathic

150 240

Number dead/number exposed Room air

7.5 % Nitrogen

2 % F-l 1

7.5:/, F-11

10% F-11

O/4 O/4

O/4 O/4

O/4 o,l4

O/4 O/4

214

O/4 O/4”

O/4 0/4b

O/4 415=

o/4 4/4d

-

a Died 3-15 days (av. 8.1 days) after the exposure. b Died 4-16 days (av. 9.5 days) after the exposure. c Four died within 48 min of exposure; the survivor died 2 days later. d All died within 30 min of exposure.

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TAYLOR

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DREW

Cardiac arrhythmias. Random-bred hamsters tolerated 5-min exposures to 2.5, 5, and 7.5% F-11-air mixtures without developing arrhythmias (Table 2). Similarly, CMP and random-bred hamsters tolerated 5-min exposure to 7.5 % nitrogen-room air mixtures without developing arrhythmias. Four random bred, 180-day-old hamsters breathing 10% F-11-air mixtures developed tachycardia with widened QRS complexes (Fig. 1). There were no visible P waves suggesting that this tachyarrhythmia could originate in the ventricles or atrioventricular node (Bellet, 1971). The arrhythmia appeared at 60-240 set (av 159 set) of exposure, and disappeared within 30 set after stopping the exposure. TABLE

2

CARDIAC ARRHYTHMIAS DURING A SMIN EXPOSURE TO NITR~CEN (PLACEBO~AIR MIXTURES CONTAINING TRICHLOROFLUOROMETHANE (F-l 1)

OR AIR

Number with arrhythmias/number exposed Hamster Random bred Myopathic

Age

7.5 % Nitrogen

2.5 % F-l 1 5 % F-l 1 7.5 % F-l 1 10 % F-l 1

120

014

180

014

O/4 O/4

o/4 O/4

o/4

4/4”

120

O/4 O/4

414” l/4”

414” 516’

-

-

180

* Tachyarrhythtnia with widened QRS (Fig. 1). b Shift in QRS axis and variable P-R interval (Fig. 2). c Bradycardia (Fig. 3).

Cardiomyopathic hamsters developed arrhythmias breathing lower concentrations of F-11-air mixtures than controls, but had no arrhythmia during 7.5% nitrogenroom air exposures. After an exposure of 200-245 set (av 215 set), four 120-day-old CMP hamsters breathing 2.5% F-l l-air mixtures had a shift in the direction of the QRS axis in the frontal plane, and a variable P-R interval without any change in the P-P interval or in P wave configuration (Fig. 2). This pattern suggests altered atrioventricular conduction (Bellet, 1971). Another group of 120-day-old CMP hamsters The effect of 10% fluorocarbon bred hamsters(ECG IeadII)

II on random I.4 0.08sec.

Con!rol

lO%fluoro-, carbon11 80 sec.

Imv

FIG. 1. Electrocardiogram (lead II) of a random-bred hamster. (Top) Control, breathing room air. (Bottom) Tachycardia, QRS widening without visible P waves during the 80th second of inhalation of 10 % F-l l-air mixture.

CARDIOMYOPATHY

AND

FLUOROCARBON-

The effect of 25%flrrorocarbon cordiomyopathic hamster

11

181

II on a 120dayold w

0.08

sec.

lead I

IeadII

I mv I

leadIll

25%fluomcarbon I I 205 sec.

FIG. 2. Electrocardiogram (leads I, II, and III) of a 120-day-old, BIO 82.62 hamster. (Top) Control, breathing room air. (Bottom Changing QRS axis with variable P-R interval during the 205th second of inhalation of 2.5 % F-l l-air mixture.

breathing 5 % F-l l-air mixtures developed sinus bradycardia within 45-185 set (av 95 set) of exposure, with heart rate falling from 310 f 24 beats/min to 162 -I 28 beats/ min (mean + SE). In contrast to the 120-day-old hamsters, only one of four 180-day-old cardiomyopathic hamsters developed an arrhythmia (sinus bradycardia) during exposure to 2.5 % F-l l-air mixtures. Six 180-day-old cardiomyopathic hamsters were exposed to 5 % F-l l-air mixtures, and five of these animals developed sinus bradycardia at 45-210 set (av 82 set) of exposure (Fig. 3); none of the 180-day-old hamsters had a change in QRS configuration. All of the arrhythmias observed in both controls and cardiomyopathic hamsters were rapidly reversed with cessation of exposure, and all animals were alive 3 wk later. The effect of 5%fluorocarbcnllona cardiomyapathic hamster (ECG

Control

5%fluoracarbon

BOdayold IeadIU

t40.08sec Imv I

II

70sec’llll~iiIl!//II/lllllliIl1lllIIII~/IIiIlII/II FIG. 3. Electrocardiogram (lead II) of a HO-day-old, BIO 82.62 hamster. (Top), Control, breathing room air. (Bottom) Sinoatrial slowing during the 70th second of inhalation of 5 ‘A F-l l-air mixture.

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DISCUSSION

These data show that F-l 1 is more toxic to cardiomyopathic hamsters than to random-bred hamsters. Cardiac arrhythmias develop in cardiomyopathic hamsters at lower inhaled concentrations of F-l 1 than in random-bred controls. Cardiomyopathic hamsters tolerated 7.5 % nitrogen-room air exposures without cardiac arrhythmias. The character of cardiac arrhythmias observed in CMP hamsters exposed to F-11 varied with age. Young hamsters in the necrotic phase of the cardiomyopathy developed conduction changes while breathing 2.5 % F-l l-air mixtures, while 180-day-old hamsters did not. At higher concentrations of F-l 1 both 120- and I SO-day-old CMP hamsters developed sinus bradycardia, suggesting an effect on the sinoatrial node. This bradycardia occurred earlier during exposure than the arrhythmias seen during 2.5 % F-l 1 in air exposures. It is possible that the conduction abnormalities observed in young hamsters may have resolved with progression of the cardiomyopathy. The use of pentobarbital anesthesia adds a variable which may alter the effect of inhaled F-l 1 directly, by depressing respiration, or through its known myocardial depressant effect. Since control and 120- and 180-day-old CMP hamsters that were not in congestive heart failure all received pentobarbital, one must postulate a greater effect of anesthesia on the cardiomyopathic group to explain observed differences on that basis. Conscious CMP hamsters in congestive heart failure died during exposures to concentrations of F-l 1 that were tolerated by all other groups studied. Young nonanesthetized CMP hamsters survived a 4-hr exposure to 7.5 % F-l 1 in air, but anesthetized young CMP hamsters developed sinus bradycardia during 5 min exposures to 5 % F-l lair mixtures. It is possible that the bradyarrhythmias resulted from the anesthesia combined with F-l 1. It is also possible that during a prolonged exposure the young CMP hamsters developed the arrhythmia but tolerated the lower heart rate. Older hamsters in congestive heart failure, unable to maintain a viable cardiac output at a lower heart rate, may have died from bradycardia caused by F-l 1 during prolonged exposure. Another possible mechanism of death in the hamsters in heart failure is depression of cardiac contractility and stroke volume by F-l 1 (Harris, 1973; Taylor and Drew, 1973; Abelmann et al., 1972). There are known differences between the conduction system of hamsters and that of other species. There is no known correlation between human heart disease and the genetic cardiomyopathy of the BIO 82.62 hamster. It is therefore inappropriate to draw direct conclusions from these data about increased risk in patients with heart disease and congestive heart failure during aerosol use. On the other hand, these results raise the possibility of increased toxicity of aerosol propellant gases in patients with decreased cardiac reserves, especially during the abuse (Bass, 1970) or misuse (Inman and Adelstein, 1969) of aerosols which may lead to inspired concentrations of propellant gases as high as those in the present study. These data do indicate that hamsters whose physiological reserves have been depleted by congestive heart failure are at greater risk when exposed to trichlorofluoromethane, a common constituent of aerosol containers. The present study also indicates that the cardiac arrhythmias seen in normal animals during fluoroalkane exposure may not accurately predict those seen in diseased animals. The abnormalities seen in

CARDIOMYOPATHY AND FLUOROCARBON- 11

normal animals and normal people during toxicological seen in patients or animals with disease.

183

testing may not predict those

REFERENCES

ABELMANN,W. H., JEFFERY, F. E. ANDWAGNER,R. (1972). Circulatory dynamicsin heart failure of Syrian hamsters.Progr. Exp. Tumor Res. 16, 261-273. BAJUSZ,E., BAKER,J. R., ND(ON,C. W. ANDHOMBURGER, F. (1969).Spontaneous,hereditary myocardial degenerationand congestiveheart failure in a strain of Syrian hamsters.Ann. N. Y. Acad. Sci. 156,105128.

BASS,M. (1970).Suddensniffing death. J. Amer. Med. Ass. 212,2075-2079. BELLET,S. (1971). Clinical Disorders of’ the Heart Beat. 3rd ed. Chaps.5, 11,and 15. Lea and Febiger, Philadelphia. Gertz, E. W. (1972).Cardiomyopathic Syrian hamster: A possiblemodel of humandisease. Progr. Exp. Tumor Res. 16,242-260.

HARRIS,W. S. (1973).Toxic effectsof aerosolpropellantson the heart. Arch. ht. Med. 131, 162-l 66.

INMAN,W. H. W. ANDADELSTEIN, A. M. (1969). Riseand fall of asthmamortality in England and Walesin relation to useof pressurizedaerosols.Lancet 2,279-285. TAYLOR,G. J. ANDDREW,R. T. (1973). Fluorocarbon 12 depressionof cardiac output and contractility in rabbits. Circulation 48 (Suppl. 4), 130.