Effects of disulfiram on the cardiovascular responses to ethanol in dogs and guinea pigs

TOXICOLOGY AND APPLIED PHARMACOLOGY14,439-446

Effects

of Disulfiram Ethanol

JIRO NAKANO,

JOEL

(1969)

on the Cardiovascular in Dogs and Guinea

Responses Pigs1

to

E. HOLLOWAY, AND JOHN S. SCHACKFORD

Departments of Pharmacology School of Medicine,

and Medicine, Oklahoma City,

Received

June

University Oklahoma

of Oklahoma 73104

14,1968

Effects of Disultiram on the Cardiovascular Responses to Ethanol in Dogs and Guinea Pigs. NAKANO, J., HOLLOWAY, J. E., and SCHACKFORD, J. S. (1969). Toxicol. AppLPharmacol. 14,439-446.Thecardiovascular effects of cardiac sympathetic nerve stimulation were studied in disulfiram-treated dogs. It was found that the magnitude of the increments of heart rate, systemic arterial pressure, and myocardial contractile force in disulfiramtreated dogs was significantly smaller than that in control dogs. In addition, the effects of ethanol on heart rate, systemic arterial pressure, and myocardial contractile force were studied in disulfiram-treated dogs and in isolated papillary muscle of disuhiram-treated guinea pigs. The magnitude and duration of the hypotensive and cardiac depressant effects of ethanol were found to be greater and more prolonged, respectively, in disultiram-treated than in control dogs. Likewise, the greater negative inotropic action of ethanol was observed in isolated atria of disulfiramtreated guinea pigs. The results obtained from the present study are consistent with previous observations that disulfiram, a dopamine-/3-hydroxylase inhibitor, reduces norepinephrine and epinephrine contents in the heart, blood vessels,and adrenal medullae, thereby causing the disturbance in the cardiovascular reflex mechanism. It is concluded that ethanol-disulfiram reaction may be partially due to the inadequate reflex syrnpathoadrenal stimulation following ethanol ingestion in disulfiram-treated animals or human subjects. It has been well established that a small or moderate quantity of ethanol causes marked cardiovascular manifestations, i.e., tachycardia, hypotension, syncope, and circulatory collapse in disulfiram-treated animals or human subjects (Asmussen et al., 1948; Larsen, 1948). Hald and Jacobsen (1948) and Raby (1954) ascribed this phenomenon to the accumulation of acetaldehyde in the body due to the conversion of ethanol to acetaldehyde and to the inhibition of acetaldehyde dehydrogenase by disulfiram. However, some pharmacodynamic aspects of disulfiram-ethanol reaction remain uncertain. For instance, Perman (1962) could not reproduce the cardiovascular disturbances by the administration of acetaldehyde which should simulate disulfiramethanol reaction. 1 This work was supported in part by research grants (HE 11848 and HE 8057) from the U.S. Public Health Service, and from the Oklahoma Heart Association. 439

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NAKANO,

HOLLOWAY,

AND

SCHACKFORD

Recently, Goldstein et al. (1964), Musacchio et al. (1964), and Thoenen et al. (1967) found that disulfiram inhibits significantly dopamine-P-hydroxylase, thereby causing a decrease in norepinephrine and an increase in dopamine levels in rat and cat hearts and spleens. In addition, Thoenen et al. (1965) showed that the contractile responses of the nictitating membranes and of the spleen following sympathetic stimulation decreased in disulfiram-treated cats. The present study was undertaken to examine (a) whether disulfiram could modify the hemodynamic responses to cardiac sympathetic stimulation and (b) whether the administration of ethanol could cause hemodynamic changes in disulfiram-treated animals which are different from those of control animals. METHODS

Dogs. Sixteen dogs weighing between 20.5 and 28.0 kg were divided into two groups. The first group of dogs was used as controls. The second group of dogs was treated with a daily intraperitoneal injection of 50 mg/kg of tetraethylthiuram disulfide (disulfiram)z for 5 days. On the day of experiment, all dogs were anesthetized with the intravenous administration of 30 mg/kg of sodium pentobarbital. The left thorax was opened under artificial respiration. The pericardium was incised and the heart suspended in a pericardial cradle. In all experiments, 2.5 mg/kg of sodium heparin was administered intravenously before cannulation of blood vessels, and this dose was repeated every half hour. Systemic arterial pressure was measured continuously with a Statham pressure transducer (P23AA) connected to a catheter placed in the left subclavian artery via the left internal mammary artery. Mean arterial pressure was obtained by electronic integration with an Electronics for Medicine (EFM) pressure amplifier. Heart rate and myocardial contractile force were measured continuously with an EFM tachometer (Model TDCl), and with a Walton-Brodie strain gauge arch (Boniface et al., 1953 ; Cotten, 1953), which was sutured directly to the left ventricular muscle. All hemodynamic parameters measured except heart rate were recorded continuously with an EFM recorder (Model DR8). The effects of stimulation of the left cardiac sympathetic nerve and the intravenous administration of a single dose (250 mg/kg) of ethanol on the different hemodynamic parameters were studied in 8 control and 8 disulfiram-treated dogs. In dogs in which the hemodynamic effects of sympathetic nerve stimulation were studied, the left cardiac sympathetic nerve was isolated and severed near the left stellate ganglion, and the peripheral nerve was stimulated with a Grass stimulator (Model S4). The voltage, duration, and frequency of direct current stimuli employed were 5 V, 5 msec, and 2 cps, respectively. Guinea pigs. Guinea pigs of either sex weighing approximately 500 g were divided into two groups. The first group of 10 guinea pigs was used as controls, and the second group of 8 guinea pigs was treated daily with the intraperitoneal injections of 50 mg/kg of disulfiram for 1 week prior to the experiment. On the day of experiment, both groups of guinea pigs were killed by cervical dislocation. The hearts were removed immediately, and the atria were excised. The atria were washed twice with, and then suspended in, a bath containing oxygenated Ringer-Locke solution (32”) through which a gas mixture of 95% O2 and 5% COZ (pH = 7.35) was bubbled. The frequency of 2 Antabuse@ disulfiram, trademark of Ayerst Laboratories.

DISULFIRAM

AND

441

CIRCULATION

atria1 contraction was kept constant at a rate of 90/min using a Grass stimulator (Model S4). The force of atria1 contraction was measured and recorded continuously using a Grass force-displacement transducer (FT-03), and a Grass polygraph (Model 5), respectively. One hour after the preparation was completed, the effect of ethanol, in final (bath) concentrations between 0.6 and 8.0 mg/ml, on the myocardial contractile force was determined. In both dog and guinea pig studies, disulfiram was mixed in 0.9 % NaCl solution or sucrose syrup to make a 20% suspension. The data in this paper were evaluated statistically employing the t test (Snedecor, 1956). RESULTS

The Cardiovascular EfSects of the Left Cardiac Sympathetic Nerve Stimulation in Dogs

The effects of the left cardiac sympathetic nerve stimulation on heart rate, mean systemic arterial pressure, and myocardial contractile force were studied in 8 control 0 EB

before after

180 beatslmln. 160 -

HR

stlmulat\on stimulation 5~. 5msec.

2cps

MO-

MSAP mmHg

1 ‘*O100 80-

MCF A%

150 100 50

CONTROL

FIG. 1. Effects of left cardiac sympathetic stimulation

DISULFIRAM

on heart rate (HR), mean systemic arterial dogs.

pressure(MSAP) and myocardial contractile force (MCF) in 8 control and 8 disulfiram-treated I-shaped bar denotes the standard errors of the means.

and 8 disulfiram-treated dogs. The results are summarized in Fig. 1. In control the stimulation of the left cardiac sympathetic nerve significantly increased the rate, mean systemic arterial pressure, and myocardial contractile force. On the hand, in dusulfiram-treated dogs, the stimulation of the left cardiac sympathetic did not cause significant changes in these three hemodynamic parameters.

dogs, heart other nerve

The Cardiovascular Efects of Ethanol in Dogs

The effects of the intravenous administration of 9.5% ethanol on heart rate, mean systemic arterial pressure and myocardial contractile force were studied in 8 control dogs and in 8 disulfiram-treated dogs. The results were summarized in Fig. 2. The rapid intravenous administration of a single dose (250 mg/kg) of 95 % ethanol decreased very slightly the heart rate, mean systemic arterial pressure, and myocardial contractile force in control dogs (p > 0.05). On the other hand, the administration of 16

442

NAKANO,

HOLLOWAY,

AND

SCHACKFORD

the same dose of ethanol decreased markedly the heart rate, mean systemic arterial pressure and myocardial contractile force in disulfiram-treated dogs (p < 0.01). 0

Before

CONTROL

Ethanol

DISULFIFAM

FIG. 2. Effects of the intravenous administration of ethanol (250 mg/ml) on heart rate (HR), mean systemic arterial pressure (MSAP) and myocardial contractile force in 9 dogs with and without disulfiram. Each bar indicates the mean value of the maximum changes in each hemodynamic parameter. I-shaped bar denotes the standard errors of the means.

The Efect of Ethanol on Myocardial Contractility in IsoIated Guinea Pig Atria The results of the effect of ethanol in the isolated atria of control and disulfiramtreated guinea pigs were consistent in all experiments. Tracings of a representative experiment (control guinea pigs) are illustrated in Fig. 3, and the results of the average

MCF 1 9m 0

MCF ’ gm 0 FIG. 3. Effect of ethanol on myocardial contractile force (MY)

in guinea pig atria1 preparation.

effect of graded concentrations of ethanol are summarized in Fig. 4. The administration of ethanol in concentrations more than 0.5 mg/ml always decreased contractile force of both control and disulfiram-treated atria although the frequency of atria1 contraction was kept constant by means of electrical stimulation (Fig. 4). It was also demonstrated that the rate of the rise in contractile force (dfdt) became slower after the administration of ethanol (Fig. 3). As shown in Fig. 4, the magnitude of the negative inotropic action of ethanol was essentially proportional to the dose given. Furthermore, the decrement in myocardial contractility induced by ethanol was always much smaher in control atria than in disuIfiram-treated atria.

443

DISULFIRAM AND CIRCULATION

Control (n=lO) - oDisulfiram(n=8)

‘Y,,,

l

0.5

1.0

CONCENTRATION

2.0

‘\ 4.0

8.0

OF ETHAN@L(mg/ml)

FIG. 4. Average effect of ethanol on myocardial contractile force in atria1 preparations of 10 control and 8 disulfiram-treated guinea pigs. I-shaped bar denotes the standard errors of the means.

DISCUSSION

The deleterious cardiovascular effects of acute ingestion of ethanol in disulfiramtreated animals or in human subjects could be caused by multiple biochemical and physiological factors. As reported by Hald and Jacobsen (1948) and Raby (1954), the so-called disulfiram-ethanol reaction could be due to exaggerated accumulation of acetaldehyde in the body, since ethanol is mainly converted to acetaldehyde and disulfiram inhibits the degradation of acetaldehyde by its inhibitory action on acetaldehyde dehydrogenase activity. However, Perman (1962) found that acetaldehyde accumulation per se caused hypertension, whereas disulfiram-ethanol reaction is always associated with hypotension or cardiovascular collapse. From the present study, it is evident that ethanol in concentrations more than 1.0 mg/ml decreased myocardial contractile force of the isolated guinea pig atria essentially in proportion to the concentration. Qualitatively similar observations were made previously by Loomis (1952), Gimeno et al. (1962), and Spann et al. (1968) in isolated rabbit atria, rat atria, and cat papillary muscle, respectively. Sulzer (1924), and Wakim (1946) also showed that ethanol in concentrations more than 2.5 mg/ml depressed ventricular contraction in the dog heart-lung preparations and isolated turtle heart. The cardiovascular effects of ethanol in animals with intact circulation or in human subjects may be caused by the multiple hemodynamic mechanisms. Ethanol not only depresses directly myocardial contractility, but also decreases indirectly the function of both the heart and the remainder of the cardiovascular system in these individuals through its depressing effect on the medullary vasomotor center. In addition, the observations made by Sulzer (1924), Regan et al. (1966), and in this laboratory (Nakano, unpublished data) revealed that ethanol reduces coronary arterial blood fiow significantly thereby inducing myocardial hypoxia and depression. Haggard et al.

444

NAKANO, HOLLOWAY,

AND SCHACKFORD

(1941) and Loomis (1952) found that concentrations (4-10 mg/ml) of ethanol depress myocardial contraction in animals and man. Very recently, Regan et al. (1966) showed that the continuous intravenous infusion of 0.1 mg/kg/min of ethanol decreased stroke volume and the maximum rate of development of the isometric contraction (d’/dt), and decreased left ventricular end-diastolic pressure significantly. Recently, Goldstein et al. (1964), Musacchio et al. (1964) and Thoenen et al. (1967) found that disulfiram inhibits significantly dopamine-/3-hydroxylase, which is responsible for bioconversion of dopamine to norepinephrine (Fig. 5). Consequently, the administration of disulfiram resulted in an increase in dopamine levels and in a decrease in norepinephrine levels in rat and cat hearts and spleens. Furthermore, Thoenen et al. (1965) found that the magnitude of the contractile responses of the PHENYLALANINE Hydroxylase

1 TY ROSINE 4 DOPA

Ho+,“,‘2

Hydroxylase H

H

L-*romatc *$$gj$:‘2’2 I Decarboxylase mJ=j~-~ DOPAMINE

-NH2

DopaminewD’SULF’RAM 4 @-Hydroxylase HH NOREf=lNEPHRINE~~&i$-NH2 Phenylethanolamlne Transferase

4 N-Methyl EPINEPHRINE

FIG. 5. Enzymatic synthesis of catecholamines and the biochemical

site of the action of disulfiram.

nictitating membranes and of the spleens following the sympathetic stimulation in disulfiram-treated cats was significantly smaller than in control cats. The observations made by these investigators are in agreement with those made in this study on the cardiovascular effects of ethanol in disulfiram-treated animals. The present study revealed that the magnitude of the negative inotropic action of ethanol is greater in atria of the guinea pigs treated with disulfiram than in those of control guinea pigs. It is not entirely certain whether this exaggerated myocardial depression in disulfiramtreated animals is due to excessive accumulation of acetaldehyde or to the depletion of norepinephrine in the myocardium. Recently, Spann et al. (1968) showed that the magnitude of the negative inotropic action of ethanol was greater in the papillary muscle obtained from cats with heart failure than in that of control cats. It is of interest to note that the myocardial contractility of the animals with heart failure and of the animals treated with disulfiram, in which norepinephrine is depleted or reduced significantly (Chidsey et al., 1964), is more markedly depressed by the administration of ethanol. It is reasonable to assume that norepinephrine and epinephrine contents are reduced not only in the heart and spleens but also in the sympathetic nerve endings of

DISULFIRAMAND CIRCULATION

445

the entire blood vessels and in the adrenal medullae in animals treated with disulfiram. Hence, a sudden fall in systemic blood pressure as seen with the ingestion of ethanol results in markedly reduced quantities of catecholamine release through reflex sympathetic stimulation (Fig. 1). As a result, the magnitude of the hypotension was greater and its duration more prolonged after ethanol treatment of disulfiram-treated animals than in control animals. This lack of the reflex sympathetic activities in disulfiramtreated animals was evidenced by the significantly reduced increments in heart rate, systemic arterial pressure and myocardial contractile force following the cardiac sympathetic stimulation (Fig. 1). Therefore, it can be concluded that the cardiovascular manifestations of ethanol-disulfiram reaction are caused by (a) the excessive accumulation of acetaldehyde in the body, (b) the exaggeration of the negative inotropic action of the myocardium, and (c) the lack of the intact cardiovascular reflex mechanism because of catecholamine depletion in the heart and blood vessels. ACKNOWLEDGMENTS The authors are grateful to Dr. T. Robitsher of Ayerst Laboratories, New York City, New York, Dr. H. G. Schoepke of Abbott Laboratories, North Chicago, Illinois, and Dr. J. A. Strade of Organon Inc., West Orange, New Jersey, for their generous supplies of disulfiram (Antabuse), and heparin sodium (Panheparin and Liquaemin), respectively. REFERENCES E., HALD, J., and LARSEN, V., (1948). The pharmacological action of acetaldehyde on the human organism. Acta Pharmacol. Toxicol. 4, 3 1l-320. BONIFACE,K. J., BRODIE,0. J., and WALTON,R. P. (1953). Resistance strain gauge arches for direct measurement of heart contractile force in animals. Proc. Sot. Exptl. Biol. Med. 84,

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C. A., HAISER,G. A., and SONNENBLICK, E. (1964). Cardiac norepinephrine stores in experimental heart failure in the dog. J. Clin. Invest. 43,2386-2393. COTTEN, M. DEV. (1953). Circulatory changes affecting measurement of heart force in situ with strain gauge arches. Am. J. Physiol. 174, 365-370. GIMENO, A. L., GIMENO, M. F., and WEBB, J. L. (1962). Effects of ethanol on cellular membrane potentials and contractility of isolated rat atrium, Am. J. Physiol. 203, 194-196. GOLDSTEIN, M., ANAGNOSTE, B., LAUBER,E., and MCKEREGHAN, M. R. (1964). Inhibition of dopamine-/%hydroxylase by disulfrram. Life Sci. 3, 763-767. HAGGARD,H. W., GREENBERG, L. A., COHEN,L. H., and RAKIETEN,N. (1941). Studies on the absorption, distribution and elimination of alcohol. IX. The concentration of alcohol in the blood causing primary cardiac failure. J. Pharmacol. Exptl. Therap. 71, 358-361. HALD, J., and JACOBSEN, E. (1948). The formation of acetaldehyde in the organism after ingestion of antabuse (tetraethylthiuramdisulphide) and alcohol. Acta Pharmacol. 4, 305310. LARSEN, V. (1948). The effect upon experimental animals of antabuse (tetraethylthiuramdisulphide) in combination with alcohol. Acta Pharmacol. 4, 321-332. LOOMIS, T. A. (1952). The effect of alcohol on myocardial and respiratory function. Qunrt. J. CHIDSEY,

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J., Kopm, I. J., and SNYDER, S. (1964). Effects of disulfiram on tissue norepinephrine content and subcellular distribution of dopamine, tyramine and their /3-hydroxylated metabolites. Life Sci. 3, 769-775. PERMAN, E. S. (1962). Studies on the Antibuse-alcohol reaction in rabbits. Acta Pkysiol. Scand. 55, Suppl. 190, l-46. MUSACCHIO,

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K. (1954). Relation of blood acetaldehyde level to clinical symptoms in the disulfiramalcohol reaction. Quart. J. Studies Ale. 15, 21-32. REGAN, T. J., KOROXENDIS, G., MOSCHOS, C. B., ALDEWURTEL, H. A., LEHAN, P. H., and HELLEMS, H. K. (1966). The acute metabolic and hemodynamic responses of the left ventricle to ethanol. J. Clin. Invest. 45, 270-280. SNEDECOR, G. W. (1956). Statistical Methods. Iowa State Univ. Press, Ames. SPANN, J. E., JR., MASON, D. T., BEISER, G. D., and GOLD, H. K. (1968). Actions of ethanol on the contractile state of the normal and failing cat papillary muscle. Clin. Res. 16, 249. SULZER, R. (1924). The influence of alcohol on the isolated mammalian heart. Heart 11, 141150. THOENEN, H., HAEFELY, W., GEY, K. F., and HIJERLIMANN, A. (1965). Diminished effects of sympathetic nerve stimulation in cats pretreated with disulfiram, liberation of dopamine as sympathetic transmitter. Life Sci. 4, 2033-2038. THOENEN, H., HAEFELY, W., GEY, K. F., and HUERLIMANN, A. (1967). Quantitative aspects of the replacement of norepinephrine by dopamine as a sympathetic transmitter after inhibition of dopamine-p-hydroxylase by disulfiram. J. Pharmacol. Exptl. Tfierap. 156, 246-251. WAKIM, K. G. (1346). The effects of ethanol on the isolated heart. Federation Proc. 5, 109. RABY,