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Dose-dependent sympathomimetic and cardioinhibitory effects of acrolein and formaldehyde in the anesthetized rat
TOXICOLOGY
AND
APPLIED
PHABMACOLOGY
28,
358-366 (1974)
Dose-Dependent Sympathomimetic and Cardioinhibitory Effects of Acrolein and Formaldehyde in the Anesthetized Rat1 JOHN L. EGLE,JR. AND PATRICIA M. HUDGINS Department of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23298 Received June 7, 1973; accepted December 14, 1973
Dose-DependentSympathomimetic and Cardioinhibitory Effects of Acrolein and Formaldehydein the AnesthetizedRat. EGLE, J. L., JR. AND HUDGINS, P. M. (1974), Toxicol. Appl. Pharmacol.
28,358-366.
Dose-effect
relationshipsfor iv formaldehyde and acrolein indicate that the cardiovascularactionsof thesealdehydesare qualitatively similarto acetaldehyde and propionaldehyde.Pressorresponses appear to result from the release of catecholaminesfrom sympatheticnerve endingsand from the adrenal medulla.Concentrationsof formaldehydeand acroleinrequiredto produce significant pressoractivity were found to be significantly lower than for acetaldehydeand propionaldehyde.The relative pressorpotency for formaldehydeand acroleinwas 10 and 100timesthat of acetaldehyde,respectively. Higher dosesof both formaldehydeand acroleinresultedin depressor responseswhich could be abolishedby vagotomy. Thus, it appearsthat thesealdehydesalsoexert a cardioinhibitory effect which is mediatedby the vagusnerve.Inhalation studieswith acroleinrevealedthat this aldehydehas significant cardiovascular activity at concentrations below those which might beencounteredin cigarettesmoke.The predominanteffect of inhaled acroleinat thesedoseswasa significantincreasein blood pressureandheart rate. It would appearthat the aldehydes,especiallyacrolein, may be more significantly involved in the cardiovascular responseto cigarette smoke than waspreviously recognized. Acrolein and formaldehyde are 2 of the aldehydes found in appreciable concentrations in the vapor phase of cigarette smoke. A 40-ml puff of cigarette smoke has been reported to contain 8.2 pg of acrolein and 4.1 pg of formaldehyde (Newsome et al., 1965). These substanceshave been widely studied in terms of irritation, ciliastasis, and
pathologic changes following respiratory tract exposure (Salem and Cullumbine, 1960; Kensler and Battista, 1963; Murphy et al., 1964; Davis et al., 1967; Puiseux and Izard, 1969; Sgibnev, 1968; Coon et al., 1970; Lyon et al., 1970; Dalhamn and Rosengren, 1971). Many aldehydes, including acetaldehyde and propionaldehyde have been shown to have indirect sympathomimetic activity when administered iv or by inhalation (Skog, 1952; Roman0 et al., 1954; Wingard et al., 1955; Eade, 1959; Akabane et al., 1964; Egle, 1972a,b; Egle et al., 1973). This finding has raised the possibility of the aldehydes 1 This study was aided by a grant from the American Medical Association Education and Research Foundation. 358 Copyright 0 1974 by Academic Press, Inc. Alt rights of reproduction Printed in Great Britain
in any form reserved.
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
359
playing a role in the development of cardiovascular disease as well as having direct effects on the respiratory tract (James et al., 1970). We have previously studied the respiratory retention by dogs of 4 aldehydes (Egle, 1972b) and the circulatory effects in the rat of iv and inhaled acetaldehyde and propionaldehyde (Egle, 1972a; Egle et al., 1973). The present study was carried out to investigate the effects of iv formaldehyde and acrolein on blood pressure and heart rate in the anesthetized rat. Various drugs and surgical procedures have been used in an attempt to establish the mechanisms of the observed responses. Inhalation experiments have been carried out in the anesthetized rat to determine whether concentrations of formaldehyde and acrolein, comparable to those encountered in smoking, have cardiovascular effects. METHODS
Male Wistar rats (250-450 g) were anesthetized with pentobarbital sodium (50 mg/kg) administered by ip injection. Cannulation of the right femoral artery allowed mean arterial blood pressure to be measured with a Statham pressure transducer. Heart rate was determined from the EKG recording by means of fine needle electrodes through the skin. A Grass model 7 polygraph was used to record all responses. The left femoral vein was cannulated to allow iv administration of drugs. The volume injected was within the range of 0.1 to 0.2 ml and was flushed with 0.2 ml of saline. Control saline injections (0.1 and 0.2 ml) were administered to all animals at the beginning of each experiment. These injections had no significant effect on resting blood pressure or heart rate (~2% of the initial resting level). In a portion of the experiments, bilateral adrenalectomy or vagotomy in the neck region was carried out 30 min prior to the first injection of aldehyde. Rats pretreated with reserpine received ip injections of 5 mg/kg at 48 hr and 1 mg/kg at 24 hr before the experiment. The cr-adrenergic antagonist, phentolamine (10 mg/kg), and anticholinergic agent, atropine (0.5 mg/kg), were used in an attempt to identify the mechanism of action of formaldehyde and acrolein. Blockade was assessed in advance of aldehyde administration by injection of the appropriate agonist. The agonists were norepinephrine (0.5 mg/kg) and acetylcholine (0.5 mg/kg), respectively. The time elapsed between antagonist administration and challenge by an agonist was 5 to 10 min. Complete selective antagonism was achieved at these doses and time periods. Vapor for inhalation was generated by passing air through acrolein or formaldehyde at room temperature. The concentrated vapor obtained was diluted in a reservoir bag to the desired concentration for inhalation experiments. Uptake of acrolein by the bags was negligible for the time period involved in the experiments. Exposure of the animals to acrolein was achieved by attaching the bag to a short metal cylinder. A latex dental dam was used to provide a seal as the cylinder was slipped over the animal’s mouth and nasal openings, Rats were exposed to various concentrations of acrolein for 1-min intervals. Periodic checks were made of the concentration of acrolein in the reservoir bag by a calorimetric assay method employing 3-methyl-2-benzothiazolone hydrazone (Sawicki et al., 1961; Hauser and Cummins, 1964). Each animal was exposed to more than one concentration of acrolein. Acrolein exposure concentrations were selected at random, and it was established that previous exposure had no effect on subsequent measurements. Blood pressure and heart rate were always permitted to return to
360
EGLE
AND
HUDGINS
control levels between exposure periods. Control inhalations consisted of 1-min exposures to air under the same conditions. Results arepresented in terms of percent change between preinjection or preinhalation values and peak response values for both blood pressure and heart rate. Significant variations were observed in initial blood pressure and heart rate readings after drug pretreatment or surgical procedures (Table 1). All results have been subjected to statistical analysis using either analysis of variance, a modified Dunnett’s test (allowing for unequal sample sizes) or Student’s t test (Steel and Torrie, 1960). TABLE INITIAL
RESTING
BLOOD
Drug or procedure Control Adrenalectomy + reserpine Phentolamine Atropine Vagotomy
PRESSURE
AND
HEART
1 RATE
IN THE ANESTHETIZED
Blood pressure Heart rate (mm Hg, mean + SE) (min-‘, mean _+SE) 107.6 + 2.8
374.2 + 7.4
78.9 + 3.7’ 98.6 + 3.5
239.4 rt 12.7” 349.7 k 17.6 372.0 + 19.3
110.8 f 10.9 104.4+ 6.4
471.1 f 13.0b
R.~T
N” 40 11 14 6 20
g Number of animals used in each experiment. b Significantly different from control (p < 0.01). c Significantly different from control (p < 0.001). RESULTS Efiects of iv Acrolein and Formaldehyde on Blood Pressure
Results of experiments in which anesthetized rats received acrolein (0.05 to 5 mg/kg), or formaldehyde (0.5 to 20 mg/kg) are summarized in Tables 2 and 3. The lower doses of both compounds produced increases in blood pressure in most instances. At intermediate dosage levels of 0.25 mg/kg with acrolein and 1 mg/kg with formaldehyde, depressor responses were observed in some instances, but an increase in blood pressure remained the predominant effect. At the highest dosage levels only depressor effects were observed in control animals following injection of either aldehyde. Responses of either type generally began within 5 set after the injection, reached a peak at 20-30 set and lasted approximately 1 min. Procedures with altered sympathetic function, such as reserpine pretreatment followed by adrenalectomy or the administration of phentolamine, resulted in an increased frequency of depressor responses at lower doses of both aldehydes. Administration of atropine attenuated the depressor response obtained at the 3 highest dosages of formaldehyde and vagotomy reversed the depressor responses to both aldehydes. After vagotomy only pressor responses were observed after acrolein administration and pressor responses after formaldehyde were more frequent at the 2 highest dosages. Effect of iv Acrolein and Formaldehyde on Heart Rate
The effects of formaldehyde (20 mg/kg) and acrolein (10 mg/kg) on heart rate are summarized in Table 4. Doses of the 2 aldehydes lower than these did not alter heart rate
f
Vagotomy
6.3 i 2.0(9/9)
6.6 & 1.9(7/7)
4.0 + 1.7 (6/6) -
8.0 L- 1.6(16/16)" -
0.05 0.25
6.1 + 2.2 (9/9)
6.4~ 2.1 (5/7) 9.5 + 2.4(2/7)
9.3 f 2.3 (3/6) 8.3 t 4.4(3/6)
12.1 + 2.6(7/10) 31.3 + 4.9 (3/10)
-_____0.5
(mg/kg)
13.5 +
2.7(8/8)
30.0 f J-0(6/6)
11.4+ 2.2 (4/10) 43.5 21 14.3 (6/10) 37.3 _+ 11.0 (6/6)
Acrolein
u Responses are calculated as mean percent change from resting blood pressure *SE. * Numbers in parentheses refer to the frequency of each response as a function of the number of observations. c Indicates that none of the animals exhibited that particular quality of response.
f 4
1
1
Direction of change
Phentolamine
Adrenaleetomy + reserpine
Control
Drug or procedure
2
(6/6) 16.9 + 5.6 (8/8)
30.5 G.1
40.2 f 6.6 (6/6)
-
11.5 + 4.8 (4/9) 66.2 f 8.5 (5/9)
1.0
5.0
34.4 f 10.8 (9/9)
16.4 & T.0 (515)
33.7 + Y.O(6/6)
50.4 &3:9(8/S)
INFLUENCE OF DRUGS AND SURGICAL PROCEDURES ON BLOOD PRESSURE RESPONSES TO INTRAVENOUS ACROLEIN”
TABLE
% * 6 g 8 i2
3 2
E 8 s 8 5 % g
c1
2
t
I
Adrenalectomy + reserpine
Phentolamine
Atropine
4.8 + 2.1(7/7)
2.8 of:2.1 (6/6) -
2.8 + 2.0 (7/7) -
4.6 + 1.4 (14/14)” 1.8 * 1.9 (7/7) -
0.5
10.0 f 5.8 (7/7)
4.7 f 0.8 (6/6) -
5.6 f 1.8 (7/7) -
5.5 f 0.7 (13/16) 17.0 + 7.0 (3/16) 3.6 212.6 (8/8) -
1.0
16.0 + 1.9 (5/10) 7.6 + 0.7 (S/10)
33.2 + 6.5 +
9.0 (6/8) 0.5 (2/8)
33.5 + 10.5 (2/6) 12.0 + 3.0 (4/6)
(7/7)
14.7 + Y.0 (7/7)
25.2 L- 5.4 (6/14) 38.6 +_ 4.6 (8/14)
18.0 + (l/6) 15.4 + 2.5 (5/6)
(12/16) (4/16) (4/7) (3/7)
1.0
21.4 + ;9
+ 3.9 I!I 8.0 + 1.1 _+ 7.0
(mg/kg)
20.7 + 5.3 (7/7)
27.6 35.8 5.8 31.3
5.0
Formaldehyde
’ Responses are calculated as mean percent change from resting blood pressure + SE. b Numbers in parentheses refer to the frequency of each response as a function of the number of observations. ’ Indicates that none of the animals exhibited that particular quality of response.
Vagotomy
i
Direction of change
Control
Drug
3
20.0
27.3 +
-
6.2 (6/6)
20.5 & iii.5 (4/4)
45.3 + T.9 (6/6)
30.8 *<:6 (6/6) 47.3 + 5.8 (6/7)
INFLUENCE OF DRUGS AND SURGICAL PROCEDURES ON BLOOD PRESSURE RESPONSESTO INTRAVENOUS FORMALDEHYDP
TABLE
-
CARDIOVASCULAR
EFFECTS OF ALDEHYDES
363
significantly and changes which were observed appeared to be secondary to blood pressure alterations. Both compounds produced a pronounced fall in heart rate at the doses shown. Formaldehyde causeda transient cardiac arrest. This cardioinhibitory effect of formaldehyde was significantly diminished by atropine and abolished by vagotomy. TABLE
4
mg/kg)
HEART RATE CHANGEAFTERINTRAVENOUSFORMALDEHYDE(~O OR ACROLEIN (10 mg/kg)
Drug or procedure
Formaldehyde 4100.0k 0.0 (6)*
Control Adrenalectomy + reserpine Phentolamine Atropine Vagotomy
457.7 + 13.2 (7) $70.5 + 4.1 (6)d $37.7 + 16.0 (3)
$1.2+ 2.0 (6)d
Acrolein $74.8+ 4.6 (7) $29.2 $40.8 $1.0 $1.6
IL 12.9 + 14.6 I!C 3.3 rtr 5.3
(6)’ (4) (10)d (7)d
’ Valuesare mean percent change from resting heart rate + SE. * Numbers in parentheses refer to the number of trials for each procedure. c Significantly different from control (p < 0.01). d Significantly different from control (p < 0.001). TABLE 5 EFFECTS OF ACROLEIN INHALATION ON BLOOD PRESSURE AND HEART RATE"
Acrolein (idml)
N
Exposures
93 8
150 28
0.025
8
25
0.05
9
17
0.10
11
20
0.25
10
19
0.50
11
20
1.00
10
20
2.50
8
16
5.00
6
16
None (air) 0.01
Blood pressure change JO.8 t8.5 $6.6 $7.2 $15.4 t20.4 $10.3 t21.0 $14.0 t26.5 $26.0 t31.2 422.0 y&t’
& 0.7
k 1.0 (25/28)* (3/W L- 1 .O (20/25) & 5.5 (15/25) +- 4.6 (14/17) + 1.2 (3/17) t- 2.8 (19/20)
JO.9 + 0.3 TO.1 310.8 (28/28) f0.4 f 0.7 (25/25) t7.5 + 1.3 (15/17) $4.0 (2/17) 1‘5.7 * 1.1 (20/20)
U/20) + 3.4 (18/19) u/w + 3.8 (17/20) L- 7.5 (3/20) 4.0 (19/20)
W20) f39:O #O.O t36.7 $23.5
Heart rate change
+ 3.2 + 7.5 L- 4.6 + 5.5
(13/16) (3/16) (10/16) (6/16)
t11.5 +- 2.1 (19/19) t7.2 t- 1.1 AS.0 1‘8.7 + 1.9 $32.8 + 6.5 t8.8 rf: 1.5 $57.7 It 7.5 t16.0 + 4.5 $49.0 f 6.9
(18/20) (2/20) (H/20) (5/20) ( 6/16) (10/16) (S/16) (1 l/16)
aResponses arecalculatedaspercentof changefrom restinglevels,mean+ SE. * Numbers in parentheses refer to the frequency of each response as a function of the number of observations.
364
EGLE
AND
HUDGINS
Similar results were observed in atropinized and vagotomized animals receiving acrolein in doses of 10 mg/kg. of Inhaled Acrolein on Blood Pressure and Heart Rate Inhalation of formaldehyde vapor at concentrations up to 2.0 pg/ml of air did not produce significant cardiovascular effects in the anesthetized rat. The cardiovascular effects of acrolein inhalation are summarized in Table 5. In general, as the concentration of acrolein inhaled increased, a pressor effect of increasing magnitude was observed. A statistically significant increase in blood pressure as compared to control (p < 0.01, Student’s t test) was observed after inhalation of 0.01 pg/ml of acrolein. However, at the highest concentration employed a greater number of depressor responses were observed. The lower concentrations of acrolein also produced modest increases in heart rate as the predominant response. After exposure to 0.05 pg/ml of acrolein a significant increase in heart rate was noted (p < 0.01). At the 2 highest concentrations of acrolein, a pronounced cardioinhibitory effect was observed in a majority of animals. At all concentrations, changes in blood pressure or heart rate were observed within 15 set after the onset of inhalation, reached a peak effect at 30 set and persisted at a steady level until the end of the exposure period. Within 10 set after vapor inhalation ceased, a rapid return to the control levels ensued. Efects
DISCUSSION
The aldehydes used in the present study have been widely investigated in regard to their irritant properties in both human subjects and experimental animals. Exposure to these aldehydes has been shown to be ciliatoxic (Kensler and Battista, 1963), to inhibit ciliary transport (Battista and Kensler, 1970) and to have effects on hepatic enzyme activity (Murphy et al., 1964). Egle (1972b) has shown that the respiratory tract retention of inhaled formaldehyde in the dog is essentially 100 % and 80-85 % for acrolein under normal breathing conditions. There have been several studies indicating that acetaldehyde and other compounds in the aldehyde series possess sympathomimetic activity (Skog, 1952; Roman0 et al., 1954; Eade, 1959; Akabane et al., 1964; Sgibnev, 1968; Egle et al., 1973). In the present study a dose-related rise in blood pressure was observed following iv administration of low doses of both acrolein and formaldehyde in the anesthetized rat. After rats were pretreated with reserpine for 2 days and adrenalectomy was performed, pressor effects of both aldehydes were attenuated and less frequently observed. Phentolamine administration in advance of aldehydes gave similar results. These findings indicated that catecholamines from the adrenal medulla and sympathetic nerve endings are important for the pressor effects of acrolein and formaldehyde. Intravenous administration of high doses of formaldehyde and acrolein produced a depressor effect which was associated with a severe bradycardia. After pretreatment with atropine the cardioinhibitory effect produced by formaldehyde and acrolein was significantly reduced and after vagotomy pressor effects were seen after administration of high doses of both aldehydes. There was no significant change in heart rate in vagotomized animals. These results indicate that formaldehyde and acrolein have two opposing effects on blood pressure and heart rate. With low doses, pressor effects are observed which are primarily due to release of norepinephrine from sympathetic nerve endings
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
365
and catecholamines from the adrenal medulla. At higher doses a vagal stimulatory component becomes predominant and leads to severe bradycardia and a sharp fall in blood pressure. At intermediate doses of both aldehydes there appears to be a nearly equal chance of either pressor or depressor responses. Which of these responses is observed apparently depends upon such factors as the level of sympathetic and vagal tone in the animal prior to injection. Similar cardiovascular effects have been reported for iv acetaldehyde and propionaldehyde in the anesthetized rat (Akabane et al., 1964; Egle et al., 1973). However, there are considerable differences in the dose at which the compounds presently being studied produce these responses. Acrolein was found to produce a significant pressor effect in control animals at 0.05 mg/kg, and formaldehyde produced comparable changes at 0.5 mg/kg. Egle et al. (1973) have shown that a dose of 5 mg/kg of acetaldehyde was required for a similar pressor effect, and propionaldehyde was found to be slightly less potent than acetaldehyde. The same order of potency was observed for the cardioinhibitory action of these aldehydes at higher concentrations. Doses of 40 mg/kg of acetaldehyde or propioualdehyde were required to produce elects comparable to those seen with 10 mg/kg formaldehyde. Inhalation studies were carried out to determine the concentrations of formaldehyde and acrolein required in inspired air to produce significant effects on blood pressure. An attempt was made to test concentrations which are likely to be encountered in cigarette smoking. The concentrations of formaldehyde and acrolein in the effluent smoke stream from a burning cigarette have been reported to be 4.1 and 8.2 pg/40 ml puff, respectively (Newsome et al., 1965). However, concentration of aldehydes would undergo dilution as smoke is inhaled so that the actual concentration inspired would be considerably less than that calculated on the basis of the information above. Nevertheless, in the present study the lowest concentration of acrolein tested was 20 times less than the concentration in cigarette smoke, and these produced significant pressor effects. Although we suspect that cardiovascular changes observed after acrolein exposure are mediated by catecholamine release, the participation of upper respiratory reflexes in the responses cannot be ruled out. Formaldehyde was without effect at a concentration 20 times higher than that found in cigarette smoke. Thus, it seems unlikely that the formaldehyde concentration in cigarette smoke could produce significant cardiovascular effects. An important consideration is the fact that several aldehydes have sympathomimetic effects, and that total aldehyde content of a 40-ml puff of cigarette smoke is 112 ccg. It seems reasonable to assume that the sympathomimetic effects of these aldehydes would be additive. Additionally, there is also the possibility of additive effects with aldehydes from other sources, e.g., acetaldehyde from alcohol metabolism, and interactions with drugs acting on the cardiovascular system. While it is generally assumed that nicotine is the most important substance in cigarette smoke in regard to cardiovascular effects, it appears that the aldehydes may play a significant role in the total cardiovascular response to cigarette smoking. REFERENCES J., NAKANISHI, S., KAHOEI, H., MATSUMURA, R. AND OGATA, H. (1964). Studies on sympathomimetic action of acetaldehyde. I. Experiments with blood pressure and nictitating membrane responses. Jap. J. Pharmacol. 14, 295-307.
AKABANE,
366
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BATTISTA, S. P. AND KENSLER, C. J. (1970).Mucus production and ciliary transport activity.
In vivo studiesusing the chicken. Arch. Environ. Health 20, 326-338. COON,R. A., JONES, R. A. AND JENKINS,L. J., JR. (1970). Animal inhalation studieson ammonia, ethylene glycol, formaldehyde, demethylamineand ethanol. Toxicol. Appl. Pharmacol.16, 646-654. DALHAMN, T. AND ROSENOREN, A. (1971).Effect of different aldehydeson tracheal mucosa. Arch. Otolaryngol. 93, 496-500. DAVIS, T. R. A., BATTISTA, S. P. AND KENSLER, C. J. (1967).Mechanismof respiratory effects during exposureof guineapigs to irritants. Arch. Environ. Health 15, 412-419. EADE, N. R. (1959).Mechanismof sympathomimeticaction of aldehydes.J. Pharmacol.Exp. Ther. 127, 29-34. EGLE, J. L., JR. (1972a).Effectsof inhaledacetaldehydeandpropionaldehydeon blood pressure and heart rate. Toxicol. Appl. Pharmacol.23 131-135. EGLE,J. L., JR. (1972b).Retention of inhaledformaldehyde,propionaldehydeand acroleinin the dog. Arch. Environ. Health 25, 119-124. EGLE, J. L., JR., HUDGINS, P. M., AND LAI, F. M. (1973).Cardiovasculareffectsof intravenous acetaldehydeand propionaldehydein the anesthetizedrat. Toxicol. Appl. Pharmacol.24, 636-644. HAUSER, T. R. AND CUMMINS, R. L. (1964).Increasingsensitivityof 3-methyl-2benzothiazolone
hydrazone test for analysisof aliphatic aldehydesin air. Anal. Chem.36, 679-681. JAMES, T. N., BEAR, E. S., LAND, K. F., GREEN, E. W. AND WINTLER, H. H. (1970).Adrenergic
mechanismsin the sinusnode. Arch. Int. Med. 125, 512-547. KENSLER, C. J. AND BATTISTA S. P. (1963). Componentsof cigarette smoke with ciliary-
depressantactivity. New Engl. J. Med. 269, 1161-1166. LYON, J. P., JENKINS, L. J., JR., JONES, R. A., COON,R. A. AND SIEGEL, J. (1970).Repeatedand continuousexposureof laboratory animalsto acrolein. Toxicol. AppI. Pharmacol.17, 726732.
MURPHY,S. D., DAVIS,H. V. ANDZARATZIAN,V. L. (1964).Biochemicaleffectsin rats from irritating air contaminants. Toxicol. Appl. Pharmacol.6, 520-528. NEWSOME, J. R., NORMAN, V. AND KEITH, C. H. (1965).Vapor phaseanalysisof cigarettesmoke. Tob. Sci. 9, 102-110. PUISEUX-DAO, S. ANDIZARD, M. C. (1968).The effectsof acrolein and the gaseousphaseof cigarette smokeon the cellular ultrastructure of Dunatiella bioculata. C. R. Acad. Sci. 267, 74-75.
ROMANO,C., MEYER, F. H. AND ANDERSON, H. H. (1954).Pharmacologicalrelationship betweenaldehydesand arterenol. Arch. Znt. Pharmacodyn.99, 378-390. SALEM,H. ANDCULLUMBINE, H. (1960).Inhalation toxicities of somealdehydes.Toxicol. Appl. Pharmacol.2, 183-187. SAWICKI, E., HAUSER, T. R., STANLEY, T. W. AND ELBERT, W. (1961). The 3-methyl-2-benzo thiazolone test: Sensitivenewmethodfor the detection,rapid estimationand determination of aliphatic aldehydes.Anal. Chem.33, 93-96. SGIBNEV, A. K. (1968).The action of low concentrationsof formaldehydevapor on the human organism.Gig. Tr. Prof Zabol. 12, 20-25. SKOG, E. (1952).Anestheticand haemolyticaction of lower aliphatic aldehydesand their effect on respiration and blood pressure.Acta Pharmacol.Toxicol. 8, 275-289. STEEL,R. D. G. AND TORRIE, J. H. (1960).PrinciplesandProceduresof Statistics. McGrawHill, New York. WINGARD, C., HITCHCOCK, P. AND TEAGUE, R. S. (1955).A survey of aldehydewith respectto their action on blood pressure.Arch. Int. Pharmacodyn.102, 65-84.
AND
APPLIED
PHABMACOLOGY
28,
358-366 (1974)
Dose-Dependent Sympathomimetic and Cardioinhibitory Effects of Acrolein and Formaldehyde in the Anesthetized Rat1 JOHN L. EGLE,JR. AND PATRICIA M. HUDGINS Department of Pharmacology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth University, Richmond, Virginia 23298 Received June 7, 1973; accepted December 14, 1973
Dose-DependentSympathomimetic and Cardioinhibitory Effects of Acrolein and Formaldehydein the AnesthetizedRat. EGLE, J. L., JR. AND HUDGINS, P. M. (1974), Toxicol. Appl. Pharmacol.
28,358-366.
Dose-effect
relationshipsfor iv formaldehyde and acrolein indicate that the cardiovascularactionsof thesealdehydesare qualitatively similarto acetaldehyde and propionaldehyde.Pressorresponses appear to result from the release of catecholaminesfrom sympatheticnerve endingsand from the adrenal medulla.Concentrationsof formaldehydeand acroleinrequiredto produce significant pressoractivity were found to be significantly lower than for acetaldehydeand propionaldehyde.The relative pressorpotency for formaldehydeand acroleinwas 10 and 100timesthat of acetaldehyde,respectively. Higher dosesof both formaldehydeand acroleinresultedin depressor responseswhich could be abolishedby vagotomy. Thus, it appearsthat thesealdehydesalsoexert a cardioinhibitory effect which is mediatedby the vagusnerve.Inhalation studieswith acroleinrevealedthat this aldehydehas significant cardiovascular activity at concentrations below those which might beencounteredin cigarettesmoke.The predominanteffect of inhaled acroleinat thesedoseswasa significantincreasein blood pressureandheart rate. It would appearthat the aldehydes,especiallyacrolein, may be more significantly involved in the cardiovascular responseto cigarette smoke than waspreviously recognized. Acrolein and formaldehyde are 2 of the aldehydes found in appreciable concentrations in the vapor phase of cigarette smoke. A 40-ml puff of cigarette smoke has been reported to contain 8.2 pg of acrolein and 4.1 pg of formaldehyde (Newsome et al., 1965). These substanceshave been widely studied in terms of irritation, ciliastasis, and
pathologic changes following respiratory tract exposure (Salem and Cullumbine, 1960; Kensler and Battista, 1963; Murphy et al., 1964; Davis et al., 1967; Puiseux and Izard, 1969; Sgibnev, 1968; Coon et al., 1970; Lyon et al., 1970; Dalhamn and Rosengren, 1971). Many aldehydes, including acetaldehyde and propionaldehyde have been shown to have indirect sympathomimetic activity when administered iv or by inhalation (Skog, 1952; Roman0 et al., 1954; Wingard et al., 1955; Eade, 1959; Akabane et al., 1964; Egle, 1972a,b; Egle et al., 1973). This finding has raised the possibility of the aldehydes 1 This study was aided by a grant from the American Medical Association Education and Research Foundation. 358 Copyright 0 1974 by Academic Press, Inc. Alt rights of reproduction Printed in Great Britain
in any form reserved.
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
359
playing a role in the development of cardiovascular disease as well as having direct effects on the respiratory tract (James et al., 1970). We have previously studied the respiratory retention by dogs of 4 aldehydes (Egle, 1972b) and the circulatory effects in the rat of iv and inhaled acetaldehyde and propionaldehyde (Egle, 1972a; Egle et al., 1973). The present study was carried out to investigate the effects of iv formaldehyde and acrolein on blood pressure and heart rate in the anesthetized rat. Various drugs and surgical procedures have been used in an attempt to establish the mechanisms of the observed responses. Inhalation experiments have been carried out in the anesthetized rat to determine whether concentrations of formaldehyde and acrolein, comparable to those encountered in smoking, have cardiovascular effects. METHODS
Male Wistar rats (250-450 g) were anesthetized with pentobarbital sodium (50 mg/kg) administered by ip injection. Cannulation of the right femoral artery allowed mean arterial blood pressure to be measured with a Statham pressure transducer. Heart rate was determined from the EKG recording by means of fine needle electrodes through the skin. A Grass model 7 polygraph was used to record all responses. The left femoral vein was cannulated to allow iv administration of drugs. The volume injected was within the range of 0.1 to 0.2 ml and was flushed with 0.2 ml of saline. Control saline injections (0.1 and 0.2 ml) were administered to all animals at the beginning of each experiment. These injections had no significant effect on resting blood pressure or heart rate (~2% of the initial resting level). In a portion of the experiments, bilateral adrenalectomy or vagotomy in the neck region was carried out 30 min prior to the first injection of aldehyde. Rats pretreated with reserpine received ip injections of 5 mg/kg at 48 hr and 1 mg/kg at 24 hr before the experiment. The cr-adrenergic antagonist, phentolamine (10 mg/kg), and anticholinergic agent, atropine (0.5 mg/kg), were used in an attempt to identify the mechanism of action of formaldehyde and acrolein. Blockade was assessed in advance of aldehyde administration by injection of the appropriate agonist. The agonists were norepinephrine (0.5 mg/kg) and acetylcholine (0.5 mg/kg), respectively. The time elapsed between antagonist administration and challenge by an agonist was 5 to 10 min. Complete selective antagonism was achieved at these doses and time periods. Vapor for inhalation was generated by passing air through acrolein or formaldehyde at room temperature. The concentrated vapor obtained was diluted in a reservoir bag to the desired concentration for inhalation experiments. Uptake of acrolein by the bags was negligible for the time period involved in the experiments. Exposure of the animals to acrolein was achieved by attaching the bag to a short metal cylinder. A latex dental dam was used to provide a seal as the cylinder was slipped over the animal’s mouth and nasal openings, Rats were exposed to various concentrations of acrolein for 1-min intervals. Periodic checks were made of the concentration of acrolein in the reservoir bag by a calorimetric assay method employing 3-methyl-2-benzothiazolone hydrazone (Sawicki et al., 1961; Hauser and Cummins, 1964). Each animal was exposed to more than one concentration of acrolein. Acrolein exposure concentrations were selected at random, and it was established that previous exposure had no effect on subsequent measurements. Blood pressure and heart rate were always permitted to return to
360
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control levels between exposure periods. Control inhalations consisted of 1-min exposures to air under the same conditions. Results arepresented in terms of percent change between preinjection or preinhalation values and peak response values for both blood pressure and heart rate. Significant variations were observed in initial blood pressure and heart rate readings after drug pretreatment or surgical procedures (Table 1). All results have been subjected to statistical analysis using either analysis of variance, a modified Dunnett’s test (allowing for unequal sample sizes) or Student’s t test (Steel and Torrie, 1960). TABLE INITIAL
RESTING
BLOOD
Drug or procedure Control Adrenalectomy + reserpine Phentolamine Atropine Vagotomy
PRESSURE
AND
HEART
1 RATE
IN THE ANESTHETIZED
Blood pressure Heart rate (mm Hg, mean + SE) (min-‘, mean _+SE) 107.6 + 2.8
374.2 + 7.4
78.9 + 3.7’ 98.6 + 3.5
239.4 rt 12.7” 349.7 k 17.6 372.0 + 19.3
110.8 f 10.9 104.4+ 6.4
471.1 f 13.0b
R.~T
N” 40 11 14 6 20
g Number of animals used in each experiment. b Significantly different from control (p < 0.01). c Significantly different from control (p < 0.001). RESULTS Efiects of iv Acrolein and Formaldehyde on Blood Pressure
Results of experiments in which anesthetized rats received acrolein (0.05 to 5 mg/kg), or formaldehyde (0.5 to 20 mg/kg) are summarized in Tables 2 and 3. The lower doses of both compounds produced increases in blood pressure in most instances. At intermediate dosage levels of 0.25 mg/kg with acrolein and 1 mg/kg with formaldehyde, depressor responses were observed in some instances, but an increase in blood pressure remained the predominant effect. At the highest dosage levels only depressor effects were observed in control animals following injection of either aldehyde. Responses of either type generally began within 5 set after the injection, reached a peak at 20-30 set and lasted approximately 1 min. Procedures with altered sympathetic function, such as reserpine pretreatment followed by adrenalectomy or the administration of phentolamine, resulted in an increased frequency of depressor responses at lower doses of both aldehydes. Administration of atropine attenuated the depressor response obtained at the 3 highest dosages of formaldehyde and vagotomy reversed the depressor responses to both aldehydes. After vagotomy only pressor responses were observed after acrolein administration and pressor responses after formaldehyde were more frequent at the 2 highest dosages. Effect of iv Acrolein and Formaldehyde on Heart Rate
The effects of formaldehyde (20 mg/kg) and acrolein (10 mg/kg) on heart rate are summarized in Table 4. Doses of the 2 aldehydes lower than these did not alter heart rate
f
Vagotomy
6.3 i 2.0(9/9)
6.6 & 1.9(7/7)
4.0 + 1.7 (6/6) -
8.0 L- 1.6(16/16)" -
0.05 0.25
6.1 + 2.2 (9/9)
6.4~ 2.1 (5/7) 9.5 + 2.4(2/7)
9.3 f 2.3 (3/6) 8.3 t 4.4(3/6)
12.1 + 2.6(7/10) 31.3 + 4.9 (3/10)
-_____0.5
(mg/kg)
13.5 +
2.7(8/8)
30.0 f J-0(6/6)
11.4+ 2.2 (4/10) 43.5 21 14.3 (6/10) 37.3 _+ 11.0 (6/6)
Acrolein
u Responses are calculated as mean percent change from resting blood pressure *SE. * Numbers in parentheses refer to the frequency of each response as a function of the number of observations. c Indicates that none of the animals exhibited that particular quality of response.
f 4
1
1
Direction of change
Phentolamine
Adrenaleetomy + reserpine
Control
Drug or procedure
2
(6/6) 16.9 + 5.6 (8/8)
30.5 G.1
40.2 f 6.6 (6/6)
-
11.5 + 4.8 (4/9) 66.2 f 8.5 (5/9)
1.0
5.0
34.4 f 10.8 (9/9)
16.4 & T.0 (515)
33.7 + Y.O(6/6)
50.4 &3:9(8/S)
INFLUENCE OF DRUGS AND SURGICAL PROCEDURES ON BLOOD PRESSURE RESPONSES TO INTRAVENOUS ACROLEIN”
TABLE
% * 6 g 8 i2
3 2
E 8 s 8 5 % g
c1
2
t
I
Adrenalectomy + reserpine
Phentolamine
Atropine
4.8 + 2.1(7/7)
2.8 of:2.1 (6/6) -
2.8 + 2.0 (7/7) -
4.6 + 1.4 (14/14)” 1.8 * 1.9 (7/7) -
0.5
10.0 f 5.8 (7/7)
4.7 f 0.8 (6/6) -
5.6 f 1.8 (7/7) -
5.5 f 0.7 (13/16) 17.0 + 7.0 (3/16) 3.6 212.6 (8/8) -
1.0
16.0 + 1.9 (5/10) 7.6 + 0.7 (S/10)
33.2 + 6.5 +
9.0 (6/8) 0.5 (2/8)
33.5 + 10.5 (2/6) 12.0 + 3.0 (4/6)
(7/7)
14.7 + Y.0 (7/7)
25.2 L- 5.4 (6/14) 38.6 +_ 4.6 (8/14)
18.0 + (l/6) 15.4 + 2.5 (5/6)
(12/16) (4/16) (4/7) (3/7)
1.0
21.4 + ;9
+ 3.9 I!I 8.0 + 1.1 _+ 7.0
(mg/kg)
20.7 + 5.3 (7/7)
27.6 35.8 5.8 31.3
5.0
Formaldehyde
’ Responses are calculated as mean percent change from resting blood pressure + SE. b Numbers in parentheses refer to the frequency of each response as a function of the number of observations. ’ Indicates that none of the animals exhibited that particular quality of response.
Vagotomy
i
Direction of change
Control
Drug
3
20.0
27.3 +
-
6.2 (6/6)
20.5 & iii.5 (4/4)
45.3 + T.9 (6/6)
30.8 *<:6 (6/6) 47.3 + 5.8 (6/7)
INFLUENCE OF DRUGS AND SURGICAL PROCEDURES ON BLOOD PRESSURE RESPONSESTO INTRAVENOUS FORMALDEHYDP
TABLE
-
CARDIOVASCULAR
EFFECTS OF ALDEHYDES
363
significantly and changes which were observed appeared to be secondary to blood pressure alterations. Both compounds produced a pronounced fall in heart rate at the doses shown. Formaldehyde causeda transient cardiac arrest. This cardioinhibitory effect of formaldehyde was significantly diminished by atropine and abolished by vagotomy. TABLE
4
mg/kg)
HEART RATE CHANGEAFTERINTRAVENOUSFORMALDEHYDE(~O OR ACROLEIN (10 mg/kg)
Drug or procedure
Formaldehyde 4100.0k 0.0 (6)*
Control Adrenalectomy + reserpine Phentolamine Atropine Vagotomy
457.7 + 13.2 (7) $70.5 + 4.1 (6)d $37.7 + 16.0 (3)
$1.2+ 2.0 (6)d
Acrolein $74.8+ 4.6 (7) $29.2 $40.8 $1.0 $1.6
IL 12.9 + 14.6 I!C 3.3 rtr 5.3
(6)’ (4) (10)d (7)d
’ Valuesare mean percent change from resting heart rate + SE. * Numbers in parentheses refer to the number of trials for each procedure. c Significantly different from control (p < 0.01). d Significantly different from control (p < 0.001). TABLE 5 EFFECTS OF ACROLEIN INHALATION ON BLOOD PRESSURE AND HEART RATE"
Acrolein (idml)
N
Exposures
93 8
150 28
0.025
8
25
0.05
9
17
0.10
11
20
0.25
10
19
0.50
11
20
1.00
10
20
2.50
8
16
5.00
6
16
None (air) 0.01
Blood pressure change JO.8 t8.5 $6.6 $7.2 $15.4 t20.4 $10.3 t21.0 $14.0 t26.5 $26.0 t31.2 422.0 y&t’
& 0.7
k 1.0 (25/28)* (3/W L- 1 .O (20/25) & 5.5 (15/25) +- 4.6 (14/17) + 1.2 (3/17) t- 2.8 (19/20)
JO.9 + 0.3 TO.1 310.8 (28/28) f0.4 f 0.7 (25/25) t7.5 + 1.3 (15/17) $4.0 (2/17) 1‘5.7 * 1.1 (20/20)
U/20) + 3.4 (18/19) u/w + 3.8 (17/20) L- 7.5 (3/20) 4.0 (19/20)
W20) f39:O #O.O t36.7 $23.5
Heart rate change
+ 3.2 + 7.5 L- 4.6 + 5.5
(13/16) (3/16) (10/16) (6/16)
t11.5 +- 2.1 (19/19) t7.2 t- 1.1 AS.0 1‘8.7 + 1.9 $32.8 + 6.5 t8.8 rf: 1.5 $57.7 It 7.5 t16.0 + 4.5 $49.0 f 6.9
(18/20) (2/20) (H/20) (5/20) ( 6/16) (10/16) (S/16) (1 l/16)
aResponses arecalculatedaspercentof changefrom restinglevels,mean+ SE. * Numbers in parentheses refer to the frequency of each response as a function of the number of observations.
364
EGLE
AND
HUDGINS
Similar results were observed in atropinized and vagotomized animals receiving acrolein in doses of 10 mg/kg. of Inhaled Acrolein on Blood Pressure and Heart Rate Inhalation of formaldehyde vapor at concentrations up to 2.0 pg/ml of air did not produce significant cardiovascular effects in the anesthetized rat. The cardiovascular effects of acrolein inhalation are summarized in Table 5. In general, as the concentration of acrolein inhaled increased, a pressor effect of increasing magnitude was observed. A statistically significant increase in blood pressure as compared to control (p < 0.01, Student’s t test) was observed after inhalation of 0.01 pg/ml of acrolein. However, at the highest concentration employed a greater number of depressor responses were observed. The lower concentrations of acrolein also produced modest increases in heart rate as the predominant response. After exposure to 0.05 pg/ml of acrolein a significant increase in heart rate was noted (p < 0.01). At the 2 highest concentrations of acrolein, a pronounced cardioinhibitory effect was observed in a majority of animals. At all concentrations, changes in blood pressure or heart rate were observed within 15 set after the onset of inhalation, reached a peak effect at 30 set and persisted at a steady level until the end of the exposure period. Within 10 set after vapor inhalation ceased, a rapid return to the control levels ensued. Efects
DISCUSSION
The aldehydes used in the present study have been widely investigated in regard to their irritant properties in both human subjects and experimental animals. Exposure to these aldehydes has been shown to be ciliatoxic (Kensler and Battista, 1963), to inhibit ciliary transport (Battista and Kensler, 1970) and to have effects on hepatic enzyme activity (Murphy et al., 1964). Egle (1972b) has shown that the respiratory tract retention of inhaled formaldehyde in the dog is essentially 100 % and 80-85 % for acrolein under normal breathing conditions. There have been several studies indicating that acetaldehyde and other compounds in the aldehyde series possess sympathomimetic activity (Skog, 1952; Roman0 et al., 1954; Eade, 1959; Akabane et al., 1964; Sgibnev, 1968; Egle et al., 1973). In the present study a dose-related rise in blood pressure was observed following iv administration of low doses of both acrolein and formaldehyde in the anesthetized rat. After rats were pretreated with reserpine for 2 days and adrenalectomy was performed, pressor effects of both aldehydes were attenuated and less frequently observed. Phentolamine administration in advance of aldehydes gave similar results. These findings indicated that catecholamines from the adrenal medulla and sympathetic nerve endings are important for the pressor effects of acrolein and formaldehyde. Intravenous administration of high doses of formaldehyde and acrolein produced a depressor effect which was associated with a severe bradycardia. After pretreatment with atropine the cardioinhibitory effect produced by formaldehyde and acrolein was significantly reduced and after vagotomy pressor effects were seen after administration of high doses of both aldehydes. There was no significant change in heart rate in vagotomized animals. These results indicate that formaldehyde and acrolein have two opposing effects on blood pressure and heart rate. With low doses, pressor effects are observed which are primarily due to release of norepinephrine from sympathetic nerve endings
CARDIOVASCULAR
EFFECTS
OF ALDEHYDES
365
and catecholamines from the adrenal medulla. At higher doses a vagal stimulatory component becomes predominant and leads to severe bradycardia and a sharp fall in blood pressure. At intermediate doses of both aldehydes there appears to be a nearly equal chance of either pressor or depressor responses. Which of these responses is observed apparently depends upon such factors as the level of sympathetic and vagal tone in the animal prior to injection. Similar cardiovascular effects have been reported for iv acetaldehyde and propionaldehyde in the anesthetized rat (Akabane et al., 1964; Egle et al., 1973). However, there are considerable differences in the dose at which the compounds presently being studied produce these responses. Acrolein was found to produce a significant pressor effect in control animals at 0.05 mg/kg, and formaldehyde produced comparable changes at 0.5 mg/kg. Egle et al. (1973) have shown that a dose of 5 mg/kg of acetaldehyde was required for a similar pressor effect, and propionaldehyde was found to be slightly less potent than acetaldehyde. The same order of potency was observed for the cardioinhibitory action of these aldehydes at higher concentrations. Doses of 40 mg/kg of acetaldehyde or propioualdehyde were required to produce elects comparable to those seen with 10 mg/kg formaldehyde. Inhalation studies were carried out to determine the concentrations of formaldehyde and acrolein required in inspired air to produce significant effects on blood pressure. An attempt was made to test concentrations which are likely to be encountered in cigarette smoking. The concentrations of formaldehyde and acrolein in the effluent smoke stream from a burning cigarette have been reported to be 4.1 and 8.2 pg/40 ml puff, respectively (Newsome et al., 1965). However, concentration of aldehydes would undergo dilution as smoke is inhaled so that the actual concentration inspired would be considerably less than that calculated on the basis of the information above. Nevertheless, in the present study the lowest concentration of acrolein tested was 20 times less than the concentration in cigarette smoke, and these produced significant pressor effects. Although we suspect that cardiovascular changes observed after acrolein exposure are mediated by catecholamine release, the participation of upper respiratory reflexes in the responses cannot be ruled out. Formaldehyde was without effect at a concentration 20 times higher than that found in cigarette smoke. Thus, it seems unlikely that the formaldehyde concentration in cigarette smoke could produce significant cardiovascular effects. An important consideration is the fact that several aldehydes have sympathomimetic effects, and that total aldehyde content of a 40-ml puff of cigarette smoke is 112 ccg. It seems reasonable to assume that the sympathomimetic effects of these aldehydes would be additive. Additionally, there is also the possibility of additive effects with aldehydes from other sources, e.g., acetaldehyde from alcohol metabolism, and interactions with drugs acting on the cardiovascular system. While it is generally assumed that nicotine is the most important substance in cigarette smoke in regard to cardiovascular effects, it appears that the aldehydes may play a significant role in the total cardiovascular response to cigarette smoking. REFERENCES J., NAKANISHI, S., KAHOEI, H., MATSUMURA, R. AND OGATA, H. (1964). Studies on sympathomimetic action of acetaldehyde. I. Experiments with blood pressure and nictitating membrane responses. Jap. J. Pharmacol. 14, 295-307.
AKABANE,
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