TOXICOLOGY
AND APPLIED
Induction
PHARMACOLOGY
@,269-276
(1977)
of Physical Dependence in Rats by Ethanol Inhalation without the Use of Pyrazole A. P. FERKO AND E. BOBYOCK
Hahnemann Medical College and Hospital, Philadelphia, Pennsylvania 19102 Received September 26,1976; accepted December IO, 1976
Induction of Physical Dependence in Rats by Ethanol Inhalation without the Use of Pyrazole. FERKO, A. P., AND BOBYOCK, E. (1977). Toxicol. Appl. Pharmacol. 40,269-276. Rats were exposed to increasing air ethanol concentrations (14 to 28 mg/liter) over a lo-day period without the use of pyrazole. There was a positive correlation between the rising air ethanol concentrations in the chamber and the concentrations of ethanol in the blood. On Day 10 the animals were removed from the chamber with a mean blood ethanol concentration of 3.13 mgjml and observed for signs of withdrawal over a 24-hr period. During this time all animals manifested spontaneous signs of withdrawal and convulsions on handling. There was a positive correlation between the spontaneous signs and convulsions on handling. The disappearance of ethanol from the blood followed zeroorder kinetics over the first 7 hr. In the withdrawal phase of the experiments a definite negative correlation existed between the decreasing blood ethanol concentrations and the rise in the scores for convulsions on handling. This inhalation procedure is a simple, reproducible approach for causing physical dependence on ethanol in rats without the use of pyrazole and with the maintenance of original body weight. Several investigators have produced physical dependence on ethanol in small animals (Falk et al., 1972; Deutsch and Koopmans, 1973; Freund, 1969). A review of the induction of physical dependence on alcohol in rodents was presented by Freund
(1975). One of the interesting approaches used to develop ethanol dependence in mice was an inhalation procedure introduced by Goldstein and Pal (1971) which circumvents the mice’s aversion for oral ethanol administration (Nachman et al., 1971). In order to obtain stable blood alcohol concentrations pyrazole, an inhibitor of alcohol dehydrogenase (Goldberg and Rydberg, 1969; Theorell et al., 1969) was administered; however, the use of pyrazole in this methodology is questionable (Le Blanc and Kalant, 1973; Lieber and De Carli, 1973). Rydberg and Neri (1972) state that pyrazole has an effect on the nervous system independent of its inhibition of the metabolism of ethanol. Littleton et al. (1974) report that pyrazole increased the physical dependence of mice on ethanol as shown by its potentiation of the withdrawal syndrome. The main difference observed between mice treated with pyrazole plus ethanol and ethanol alone was due to the toxic and apparently central nervous system depressant properties of pyrazole. In the absence of the other effects, they suggested that these actions, rather than the inhibition of alcohol dehydrogenase, were responsible for the potentiation of Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Britain
Great
10
269 ISSN
0041-008X
270
FERKO
AND
BOBYOCK
ethanol dependence by pyrazole. Recently Goldstein (1975) stated that the use of pyrazole introduces unwanted effects including unexplained interactions with ethanol. This present investigation attempted to study the effects of increasing air ethanol concentrations on the accumulation of ethanol in the blood of rats without the administration of pyrazole or initial ip injections of ethanol. After a period of 10 days of exposure to ethanol the animals were removed from the chamber and observed for objective criteria to see if signs of the withdrawal phenomenon were manifested.
METHODS Administration of Ethanol Vapor by Inhalation Thirty-two male Sprague-Dawley rats weighing 190 to 240 g (mean 214 g) were exposed to ethanol vapor in a chamber somewhat similar to that described by Goldstein (1972). The chamber was a cylinder 60 cm in length and 28 cm in diameter. Groups of six rats were housed in the chamber constantly, except for 30 to 45 min each day when blood samples were obtained from the rats by orbital sinus bleeding. Food and water intake and also total body weight were monitored daily. Ethanol (95 ‘A, v/v) was delivered by an infusion pump (Harvard) onto a sponge at a constant rate of 97 mg/min. Air flow (3.5 to 7 liters/min) through the flask and chamber was controlled by a twostage regulator valve and a calibrated Gilmont flow meter to give nominal ethanol vapor concentrations from 14 to 28 mg/liter of air. The ethanol vapor concentratrons measured inside the chamber were 6-7 y0 lower than the nominal vapor concentrations. The loss was due in part to ethanol being absorbed and metabolized by the rats and in part to the absorption of ethanol in bedding and urine as noted by Goldstein (1972). The chamber temperature was maintained at 26” + 1°C. The rats were exposed to the following ethanol vapor concentrations over a IO-day period: Days 1 and 2, 14 mg/liter; Day 3, 16 me/liter; Day 4, 18 mg/liter; Day 5, 20 mg/liter ; Days 6 and 7, 22 mg/liter ; Day 8,24 mg/liter ; Day 9,26 mg/liter ; and Day 10, 28 mg/liter. By increasing the ethanol vapor concentration in this manner the rats were more readily able to tolerate the increasing ethanol blood concentrations over the IO-day exposure period and were able to maintain their initial body weights. A control group of rats was treated in a similar manner for 10days except that ethanol vapor was not administered. Graded Withdrawal Reactions After the lo-day exposure period the rats were taken out of the chamber and observed for signs of withdrawal. These signs were scored hourly as convulsions on handling as described by Goldstein (1972). Also the animals were observed for any spontaneous signs of withdrawal which might occur, for example: wet shakes, gnawing, tremors, and convulsions. Statistical Analysis Significance of differences for blood ethanol concentrations on various days during the exposure period was determined by Student’s t test. Correlation coefficients and level of significance (5 %) were assessed according to Bancroft (1965) for spontaneous
PHYSICAL
DEPENDENCE
ON ETHANOL
IN
271
RATS
signs of withdrawal and convulsions on handling and between decreasing blood ethanol concentrations and convulsions on handling. Assay for Ethanol in Blood and Air Ethanol concentrations in chamber vapor and blood were assayed enzymatically as described by Lundquist (1959). The ethanol chamber vapor concentration was determined daily, in duplicate, prior to the removal of the rats from the chamber. Vapor samples (0.3 ml) were obtained through a sampling port in the chamber by means of an air-tight syringe (Hamilton) and injected through serum stoppers into the head space of tubes containing 3.0 ml of reaction mixture (alcohol dehydrogenase, DPN, and buffer) and 50 ~1 of distilled water as described by Goldstein (1972). Daily blood samples (20 ~1) were obtained from half of the rats by orbital sinus bleeding. The blood samples were deproteinized with 0.2 ml of 3.4% perchloric acid solution. After centrifugation (4342 g) a 50-~1 portion of the filtrate was added to 3.0 ml of the reaction mixture. After the samples had incubated for 1 hr at room temperature, the absorbance at 340 nm was measured. On the day of withdrawal, blood samples (20 ~1) were obtained from all of the rats at 0, 1, 3, 5, and 7 hr after removal from the chamber and assayed for ethanol concentrations. RESULTS
By altering the nominal air ethanol concentrations in an increasing manner from 14 to 28 mg/liter over a IO-day period it was possible to establish increasing blood concentrations of ethanol in the rats with a mean value of 3.13 + 0.23 mg/ml on Day 10. The data are illustrated in Fig. 1. From these results it was shown that there was a
26.
-
16.
Fi z 2 c w
12
*5
a
4.0
,,,I
3.0
i
2.0
0”
, _-4.
BLOOD
/’
j
f-
I
7
6
1’
/’ I
I.0
/’
c--v I
-r'
----rd--2--2
3
4
5
6
9
IO
OAYS
FIG. 1. The increasing concentrations of ethanol in air and the subsequent accumulation of ethanol in blood of rats during 10 days of exposure using an inhalation procedure. Values plotted are the means _+ SE. Daily ethanol concentrations in air represent 12 samples and the daily blood ethanol concentrations are from 16 animals.
212
FERKO
AND
BOBYOCK
positive correlation between the rising air ethanol concentrations and the blood ethanol concentrations (r = 0.95, p c 0.05). Furthermore, the blood ethanol concentrations were significantly different between the various days once the ethanol vapor exceeded 18 mg/liter. For example, Day 5 blood ethanol values were significantly different from the blood samples obtained on Days 1 and 7 (p < 0.001). Also differences in daily blood ethanol values were seen when Day 7 was compared to Day 9 (p < 0.05), Day 8 was compared to Day 10 (p < O.OOl), and Day 9 was compared to Day 10 (p < 0.05). The first 4 or 5 days of exposure to ethanol vapor (at the end of which time the mean ethanol blood concentration was 0.5 mg/ml) were designated as an “initiation phase” which appeared to be necessary to obtain the steadily increasing ethanol blood concentrations from Day 5 to Day 10. This method of exposure “primed” the rats for exposure to the higher ethanol vapor concentrations (22 to 28 mg/liter) which they are more readily able to tolerate than if they were to receive the higher ethanol vapor concentrations initially (Days 1 to 5). TABLE 1 MODIFIED
SCORING
SYSTEM
Sign
Minimum degree
Gnawing Wet shakes Blanched ears Piloerection Lethargy Squealing on handling Tremors Limb extension Convulsions 0 Modification
FOR SPONTANEOUS
from
Sporadic l/l0 min Localized Drowsiness Occasional trembling Moderate Mild. tonic
Goldstein
SIGNS
OF ALCOHOL
Score
WITHDRAWAL
Maximal degree
0.25 Continuous 0.25 3 or more/IO min Present 0.25 Diffuse 0.50 Hypnotic Present 1.OO Continuous Shaking 1.OO Exaggerated 1.50 3 or more generalized/hour
IN RATS”
Score 0.50 0.50 0.25 0.50 1.00 1 .oo
2.00 2.00 3.00
and Pal (1971).
During the lo-day period in which the animals were housed in the chamber they maintained their weight and exhibited some signs of intoxication (ataxia, abnormal sleeping posture, and some degree of lethargy). Additionally, a few animals, particularly at Days 8,9, or 10 were unconscious; however, they regained consciousness in the 30- to 45-min interval while removed from the chamber each day for bleeding, monitoring food and water intake, and changing cages. One animal died in the chamber on Day 7 and one on Day 8. This represents approximately a 6 ‘A mortality rate in the study. Following the 10 days of exposure to ethanol vapor the animals were removed from their chamber. Blood ethanol concentrations were measured over a 7-hr interval and signs of withdrawal were observed and scored hourly up to 12 hr. A final withdrawal score was recorded at 24 hr. As mentioned previously convulsions on handling as described by Goldstein (1972) were scored. Also other signs of withdrawal exhibited by the animals were graded and listed in Table 1 along with their individual scores. The results of both methods employed to grade the withdrawal phenomenon are listed in Table 2 along with a column for the total score when the two individual scores were
PHYSICAL DEPENDENCE ONETHANOL
273
IN RATS
TABLE 2 A COMPARISONOF THE SCORESFOR CONVULSIONS ON HANDLING AND SPONTANEOUS SIGNSOF WITHDRAWAL IN RATS FOLLOWING 10 DAYS OF EXPOSURE TO INCREASING ETHANOL VAPOR CONCENT-~~TI~NS
Hours 0 1
2 3 4 5 6 7 8 9 10 11
12 24
Total score’
Convulsion
0.57 +-O.Ogb 0.60 + 0.08 0.69 t- 0.05 0.64 + 0.07 1.12 It 0.20 1.22 -t 0.30 2.10 + 0.38 2.62 + 0.39 3.01 + 0.49 3.25 '1: 0.48 3.56 + 0.57 2.71 4 0.84 2.04 -L 0.93 2.262 0.51
0.00 0.00 0.00 0.00
on handling + + t +
0.31 + 0.35 + 0.58 + 0.77 5
Spontaneous
signs
0.57 + O.Ogb 0.60 f 0.08 0.69 -c 0.05 0.64 f 0.07 0.81 k 0.13 0.88 + 0.18 1.52 + 0.22 1.86 + 0.24 2.20 +_0.32 2.34? 0.31 2.38 & 0.36 2.04 + 0.52 1.38 kO.61 1.60 + 0.32
O.oob 0.00 0.00 0.00
0.12 0.15 0.19
0.20
0.81 f 0.21
0.9o?r 0.22 1.10 * 0.28
0.66& 0.24 0.66 + 0.24 0.66 + 0.23
4 Total score consists of the combined values for convulsions on handling and spontaneous signs of withdrawal. b Means + SE of 26 male rats.
combined. In comparing the convulsions on handling to the various signs which the animals exhibited a positive correlation was seen (r = 0.95, p < 0.05). Additionally, as should be expected, the total score (Table 2) indicated a positive correlation to the convulsions on handling values (r = 0.95) and to the various signs (Y = 0.99) seen during the time period in which the animals were observed.
-
5.0
1 . zz
4.0
----
BLOOD
ETHANOL
-
CONVULSIONS
ON
HANDLING
d.6 1.6
-
I
;: z 2
3.0.p
L
2.0
1.4
1
1.2 ‘b,
1.0 0.6
3-N
z
‘f
0.6
I.0
“-3
0.4 0.2
1
i.
0
I
: 2
A 3
4
5
6
7
6
3
IO
II
I2
24
HOUR s
FIG. 2. The disappearance of ethanol from the blood of rats and the increase in scores for convulsions on handling over a 24-hr period following 10 days of ethanol inhalation. The hourly values are the means 2 SE for 26 animals.
274
FERKO
AND
BOBYOCK
The change in the blood ethanol concentrations and the increase in the scores for convulsions on handling are shown in Fig. 2. As illustrated in the figure the elimination of ethanol from the blood followed zero-order kinetics. The average rate of disappearance of alcohol from the blood was 0.41 + 0.01 (SE) mg/ml/hr. The onset of the withdrawal was at 4 hr and the graded withdrawal reaction attained a maximum effect at approximately 10 hr. It appeared that as the blood ethanol values decreased the signs of the withdrawal phenomenon occurred more strongly. This was confirmed by the fact that there was a definite negative correlation between the changes in blood ethanol concentrations and the rise in the score for convulsions on handling (Y= -0.88,~ < 0.05). Several groups of control animals were placed in the chamber for 10 days. They were treated similarly to the experimental group in every respect except that ethanol was not vaporized into the chamber. On removal of these animals from the chamber following 10 days exposure they did not exhibit signs of withdrawal over a 24-hr period and behaved in a manner similar to normal Sprague-Dawley rats. DISCUSSION This investigation indicated a significant increase in blood ethanol concentrations over a period of time in rats by using an inhalation procedure. Also it was shown that upon removal of all animals from the atmosphere of ethanol a sequence of spontaneous signs of withdrawal were manifested. The effects were accomplished without the use of pyrazole and with the maintenance of initial weight of the animals. It has been stated that physical dependence develops in animals after a short period of continuous intoxication (Goldstein, 1972). In the present study ethanol blood concentrations were not held at a constant value but were steadily increased to a mean ethanol blood concentration of 3.13 + 0.23 mg/ml achieved on Day 10. The time period and the blood ethanol concentrations were selected so that significant signs of withdrawal were seen in animals which had maintained their body weight. The withdrawal appeared to reach a peak effect in 10 hr, a time which is quite similar to that of previous reports (Goldstein, 1972; Littleton et al., 1974). Based on the data obtained it appears that this approach provides a reliable method for the induction of physical dependence on ethanol. Freund (1975) listed six criteria for a model of physical dependence on ethanol. He stated that the “ideal” method would induce physical dependence so defined: (1) reproducibly, (2) in the shortest possible period of time (days), (3) by simple procedures, (4) with introduction of the fewest variables besides the administration of ethanol, (5) with production of spontaneous major signs of withdrawal, and (6) in all treated animals. He further claimed that no presently available method could approach this ideal. However, it is quite clear from our results that all six points were satisfied. It is not necessary to examine each one of the objectives individually but a few should be reemphasized, particularly numbers 4 and 5. This method does introduce the fewest variables besides the administration of ethanol. Pyrazole was eliminated because of its toxic effects on the liver and central nervous system. Also, pyrazole can produce interactions with ethanol and other drugs which may be used in a model for drug treatment experiments. Interpretation of the results from such drug-drug interactions is difficult. Another variable that was avoided in this study was the loss of original body weight (Goldstein, 1972; Roach et al., 1973). However,
PHYSICAL
DEPENDENCE
ON ETHANOL
IN RATS
275
Branchey et al. (1971) have shown that some weight loss may not interfere with a rat model for physical dependence. Their work involved oral administration of ethanol. Since the animals are in an environment of ethanol vapor, the effects of ethanol upon lung structure will have to be examined thoroughly in future studies. In a human study Lester and Greenberg (1951) noted that 10 to 20 mg/liters of ethanol in air causes some coughing and smarting of the eyes, but these signs disappear in 5 to 10 min. Thirty milligrams per liter of ethanol in air could be tolerated. However, above 40 mg/liter the atmosphere containing ethanol was intolerable for even short periods. Point 5 of the Freund criteria indicates that the animals should demonstrate spontaneous major signs of withdrawal. Table 1 lists the signs according to the increasing order of occurrence and severity. The observable, spontaneously manifested sign’swhen compared to the incidence of convulsions on handling (Goldstein, 1972) showed a positive correlation (Table 2). In summary this study satisfied some of the earlier criticisms which had been directed at induction of ethanol dependence by previously reported inhalation procedures, i.e., weight loss and the use of pyrazole. ACKNOWLEDGMENT We thank Dr. W. S. Chernick for his assistanceand advice. REFERENCES to Biostatistics. Harper and Row, New York. G., AND KISSIN, B. (1971). Modification in the response to alcohol following the establishment of physical dependence. Psychopharmacologia 22,314-322. DE~TSCH, J. A., AND KOOPMANS, H. S. (1973). Preference enhancement for alcohol by passive exposure. Science 179, 1242-l 243. FALK, J. L., SAMSON, H. H., AND WINGER, G. (1972). Behavioral maintenance of high concentrations of blood ethanol and physical dependence in the rat. Science 177,811-813. FREUND, G. (1969). Alcohol withdrawal syndrome in mice. Arch. Neural. 21,315-320. FREUND, G. (1975). Induction of physical dependence on alcohol in rats. In Advances in Experimental Medicine and Biology (E. Majchrowicz, ed.) Vol. 56, pp, 31 l-325. Plenum, New York. GOLDBERG, L., AND RYDBERG, U. (1969). Inhibition of ethanol metabolism in vivo by administration of pyrazole. Biochem. Pharamcol. 18, 1749-1762. GOLDSTEIN, D. B. (1972). Relationship of alcohol dose to intensity of withdrawal signs in mice. J. Pharmacol. Exp. Ther. 180,203-215. GOLDSTEIN, D. B. (1975). Physical dependence on alcohol in mice. Fed. Proc. Fed. Amer. Sot. BANCROFT, BRANCHEY,
H. M.,
(1965). Introduction RAUSCHER,
Exp. Biol. 34,1953-1961.
D. B., ANDPAL, N. (1971). Alcohol dependence produced in mice.by inhalation of ethanol: Grading the withdrawal reactions. Science 172, 288-290. LE BLANC, A. E., AND KALANT, H. (1973). Central nervous system interaction of pyrazole and ethanol in the rat. Canad. J. Physiol. Pharmacol. 51, 612-615. LESTER, D., AND GREENBERG, L. A. (1951). The inhalation of ethyl alcohol by man. Quart. J. Stud. Ale. 12, 167-178. LIEBER, C. S., AND DE CARLI, L. M. (1973). Ethanol dependence and tolerance: A nutritionally controlled experimental model in the rat. Res Comm. Chem. Path. Pharm. 6,983-991. LITTLETON, J. M., GRIFFITHS, P. J., AND ORTIZ, A. (1974). The induction of ethanol dependence and the ethanol withdrawal syndrome: The effects of pyrazole. J. Pharm. Pharmacol. 26,81-91. GOLDSTEIN,
276
FERKOAND BOBYOCK
LUNDQUIST,F. (1959). The determination of ethyl alcohol in blood and tissues. Methods Biochem. Anal. 7,217-251.
NACHMAN, M., LARVE,C., AND LE MAGNEN,J. (1971). The role of olfactory and orosenory factors in the alcohol preference of inbred strain of mice. Physiol. Behau. 6, 53-59. ROACH, M. K., KHAN, M. M., COFFMAN,R., PENNINGTON,W., AND DAVIS,D. L. (1973). Brain (Na+ and K+)-activated adenosine triphosphatase activity and neurotransmitrer uptake in alcohol-dependent rats. Brain Res 63,323-329. RYDBERG,U. AND NERI, A. (1972). 4-Methylpyrazole as an inhibitor of ethanol metabolism: Differential metabolic and central nervous effects. Acfa Pharmacol. Toxicol. 31,431-432. THEORELL,H., YONETANI,T., AND SJOBERG, B. (1969). On the effect of some heterocylic compounds on the enzymic activity of liver alcohol dehydrogenase. Acta Chem. &and. 23, 255-260.
AND APPLIED
Induction
PHARMACOLOGY
@,269-276
(1977)
of Physical Dependence in Rats by Ethanol Inhalation without the Use of Pyrazole A. P. FERKO AND E. BOBYOCK
Hahnemann Medical College and Hospital, Philadelphia, Pennsylvania 19102 Received September 26,1976; accepted December IO, 1976
Induction of Physical Dependence in Rats by Ethanol Inhalation without the Use of Pyrazole. FERKO, A. P., AND BOBYOCK, E. (1977). Toxicol. Appl. Pharmacol. 40,269-276. Rats were exposed to increasing air ethanol concentrations (14 to 28 mg/liter) over a lo-day period without the use of pyrazole. There was a positive correlation between the rising air ethanol concentrations in the chamber and the concentrations of ethanol in the blood. On Day 10 the animals were removed from the chamber with a mean blood ethanol concentration of 3.13 mgjml and observed for signs of withdrawal over a 24-hr period. During this time all animals manifested spontaneous signs of withdrawal and convulsions on handling. There was a positive correlation between the spontaneous signs and convulsions on handling. The disappearance of ethanol from the blood followed zeroorder kinetics over the first 7 hr. In the withdrawal phase of the experiments a definite negative correlation existed between the decreasing blood ethanol concentrations and the rise in the scores for convulsions on handling. This inhalation procedure is a simple, reproducible approach for causing physical dependence on ethanol in rats without the use of pyrazole and with the maintenance of original body weight. Several investigators have produced physical dependence on ethanol in small animals (Falk et al., 1972; Deutsch and Koopmans, 1973; Freund, 1969). A review of the induction of physical dependence on alcohol in rodents was presented by Freund
(1975). One of the interesting approaches used to develop ethanol dependence in mice was an inhalation procedure introduced by Goldstein and Pal (1971) which circumvents the mice’s aversion for oral ethanol administration (Nachman et al., 1971). In order to obtain stable blood alcohol concentrations pyrazole, an inhibitor of alcohol dehydrogenase (Goldberg and Rydberg, 1969; Theorell et al., 1969) was administered; however, the use of pyrazole in this methodology is questionable (Le Blanc and Kalant, 1973; Lieber and De Carli, 1973). Rydberg and Neri (1972) state that pyrazole has an effect on the nervous system independent of its inhibition of the metabolism of ethanol. Littleton et al. (1974) report that pyrazole increased the physical dependence of mice on ethanol as shown by its potentiation of the withdrawal syndrome. The main difference observed between mice treated with pyrazole plus ethanol and ethanol alone was due to the toxic and apparently central nervous system depressant properties of pyrazole. In the absence of the other effects, they suggested that these actions, rather than the inhibition of alcohol dehydrogenase, were responsible for the potentiation of Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Britain
Great
10
269 ISSN
0041-008X
270
FERKO
AND
BOBYOCK
ethanol dependence by pyrazole. Recently Goldstein (1975) stated that the use of pyrazole introduces unwanted effects including unexplained interactions with ethanol. This present investigation attempted to study the effects of increasing air ethanol concentrations on the accumulation of ethanol in the blood of rats without the administration of pyrazole or initial ip injections of ethanol. After a period of 10 days of exposure to ethanol the animals were removed from the chamber and observed for objective criteria to see if signs of the withdrawal phenomenon were manifested.
METHODS Administration of Ethanol Vapor by Inhalation Thirty-two male Sprague-Dawley rats weighing 190 to 240 g (mean 214 g) were exposed to ethanol vapor in a chamber somewhat similar to that described by Goldstein (1972). The chamber was a cylinder 60 cm in length and 28 cm in diameter. Groups of six rats were housed in the chamber constantly, except for 30 to 45 min each day when blood samples were obtained from the rats by orbital sinus bleeding. Food and water intake and also total body weight were monitored daily. Ethanol (95 ‘A, v/v) was delivered by an infusion pump (Harvard) onto a sponge at a constant rate of 97 mg/min. Air flow (3.5 to 7 liters/min) through the flask and chamber was controlled by a twostage regulator valve and a calibrated Gilmont flow meter to give nominal ethanol vapor concentrations from 14 to 28 mg/liter of air. The ethanol vapor concentratrons measured inside the chamber were 6-7 y0 lower than the nominal vapor concentrations. The loss was due in part to ethanol being absorbed and metabolized by the rats and in part to the absorption of ethanol in bedding and urine as noted by Goldstein (1972). The chamber temperature was maintained at 26” + 1°C. The rats were exposed to the following ethanol vapor concentrations over a IO-day period: Days 1 and 2, 14 mg/liter; Day 3, 16 me/liter; Day 4, 18 mg/liter; Day 5, 20 mg/liter ; Days 6 and 7, 22 mg/liter ; Day 8,24 mg/liter ; Day 9,26 mg/liter ; and Day 10, 28 mg/liter. By increasing the ethanol vapor concentration in this manner the rats were more readily able to tolerate the increasing ethanol blood concentrations over the IO-day exposure period and were able to maintain their initial body weights. A control group of rats was treated in a similar manner for 10days except that ethanol vapor was not administered. Graded Withdrawal Reactions After the lo-day exposure period the rats were taken out of the chamber and observed for signs of withdrawal. These signs were scored hourly as convulsions on handling as described by Goldstein (1972). Also the animals were observed for any spontaneous signs of withdrawal which might occur, for example: wet shakes, gnawing, tremors, and convulsions. Statistical Analysis Significance of differences for blood ethanol concentrations on various days during the exposure period was determined by Student’s t test. Correlation coefficients and level of significance (5 %) were assessed according to Bancroft (1965) for spontaneous
PHYSICAL
DEPENDENCE
ON ETHANOL
IN
271
RATS
signs of withdrawal and convulsions on handling and between decreasing blood ethanol concentrations and convulsions on handling. Assay for Ethanol in Blood and Air Ethanol concentrations in chamber vapor and blood were assayed enzymatically as described by Lundquist (1959). The ethanol chamber vapor concentration was determined daily, in duplicate, prior to the removal of the rats from the chamber. Vapor samples (0.3 ml) were obtained through a sampling port in the chamber by means of an air-tight syringe (Hamilton) and injected through serum stoppers into the head space of tubes containing 3.0 ml of reaction mixture (alcohol dehydrogenase, DPN, and buffer) and 50 ~1 of distilled water as described by Goldstein (1972). Daily blood samples (20 ~1) were obtained from half of the rats by orbital sinus bleeding. The blood samples were deproteinized with 0.2 ml of 3.4% perchloric acid solution. After centrifugation (4342 g) a 50-~1 portion of the filtrate was added to 3.0 ml of the reaction mixture. After the samples had incubated for 1 hr at room temperature, the absorbance at 340 nm was measured. On the day of withdrawal, blood samples (20 ~1) were obtained from all of the rats at 0, 1, 3, 5, and 7 hr after removal from the chamber and assayed for ethanol concentrations. RESULTS
By altering the nominal air ethanol concentrations in an increasing manner from 14 to 28 mg/liter over a IO-day period it was possible to establish increasing blood concentrations of ethanol in the rats with a mean value of 3.13 + 0.23 mg/ml on Day 10. The data are illustrated in Fig. 1. From these results it was shown that there was a
26.
-
16.
Fi z 2 c w
12
*5
a
4.0
,,,I
3.0
i
2.0
0”
, _-4.
BLOOD
/’
j
f-
I
7
6
1’
/’ I
I.0
/’
c--v I
-r'
----rd--2--2
3
4
5
6
9
IO
OAYS
FIG. 1. The increasing concentrations of ethanol in air and the subsequent accumulation of ethanol in blood of rats during 10 days of exposure using an inhalation procedure. Values plotted are the means _+ SE. Daily ethanol concentrations in air represent 12 samples and the daily blood ethanol concentrations are from 16 animals.
212
FERKO
AND
BOBYOCK
positive correlation between the rising air ethanol concentrations and the blood ethanol concentrations (r = 0.95, p c 0.05). Furthermore, the blood ethanol concentrations were significantly different between the various days once the ethanol vapor exceeded 18 mg/liter. For example, Day 5 blood ethanol values were significantly different from the blood samples obtained on Days 1 and 7 (p < 0.001). Also differences in daily blood ethanol values were seen when Day 7 was compared to Day 9 (p < 0.05), Day 8 was compared to Day 10 (p < O.OOl), and Day 9 was compared to Day 10 (p < 0.05). The first 4 or 5 days of exposure to ethanol vapor (at the end of which time the mean ethanol blood concentration was 0.5 mg/ml) were designated as an “initiation phase” which appeared to be necessary to obtain the steadily increasing ethanol blood concentrations from Day 5 to Day 10. This method of exposure “primed” the rats for exposure to the higher ethanol vapor concentrations (22 to 28 mg/liter) which they are more readily able to tolerate than if they were to receive the higher ethanol vapor concentrations initially (Days 1 to 5). TABLE 1 MODIFIED
SCORING
SYSTEM
Sign
Minimum degree
Gnawing Wet shakes Blanched ears Piloerection Lethargy Squealing on handling Tremors Limb extension Convulsions 0 Modification
FOR SPONTANEOUS
from
Sporadic l/l0 min Localized Drowsiness Occasional trembling Moderate Mild. tonic
Goldstein
SIGNS
OF ALCOHOL
Score
WITHDRAWAL
Maximal degree
0.25 Continuous 0.25 3 or more/IO min Present 0.25 Diffuse 0.50 Hypnotic Present 1.OO Continuous Shaking 1.OO Exaggerated 1.50 3 or more generalized/hour
IN RATS”
Score 0.50 0.50 0.25 0.50 1.00 1 .oo
2.00 2.00 3.00
and Pal (1971).
During the lo-day period in which the animals were housed in the chamber they maintained their weight and exhibited some signs of intoxication (ataxia, abnormal sleeping posture, and some degree of lethargy). Additionally, a few animals, particularly at Days 8,9, or 10 were unconscious; however, they regained consciousness in the 30- to 45-min interval while removed from the chamber each day for bleeding, monitoring food and water intake, and changing cages. One animal died in the chamber on Day 7 and one on Day 8. This represents approximately a 6 ‘A mortality rate in the study. Following the 10 days of exposure to ethanol vapor the animals were removed from their chamber. Blood ethanol concentrations were measured over a 7-hr interval and signs of withdrawal were observed and scored hourly up to 12 hr. A final withdrawal score was recorded at 24 hr. As mentioned previously convulsions on handling as described by Goldstein (1972) were scored. Also other signs of withdrawal exhibited by the animals were graded and listed in Table 1 along with their individual scores. The results of both methods employed to grade the withdrawal phenomenon are listed in Table 2 along with a column for the total score when the two individual scores were
PHYSICAL DEPENDENCE ONETHANOL
273
IN RATS
TABLE 2 A COMPARISONOF THE SCORESFOR CONVULSIONS ON HANDLING AND SPONTANEOUS SIGNSOF WITHDRAWAL IN RATS FOLLOWING 10 DAYS OF EXPOSURE TO INCREASING ETHANOL VAPOR CONCENT-~~TI~NS
Hours 0 1
2 3 4 5 6 7 8 9 10 11
12 24
Total score’
Convulsion
0.57 +-O.Ogb 0.60 + 0.08 0.69 t- 0.05 0.64 + 0.07 1.12 It 0.20 1.22 -t 0.30 2.10 + 0.38 2.62 + 0.39 3.01 + 0.49 3.25 '1: 0.48 3.56 + 0.57 2.71 4 0.84 2.04 -L 0.93 2.262 0.51
0.00 0.00 0.00 0.00
on handling + + t +
0.31 + 0.35 + 0.58 + 0.77 5
Spontaneous
signs
0.57 + O.Ogb 0.60 f 0.08 0.69 -c 0.05 0.64 f 0.07 0.81 k 0.13 0.88 + 0.18 1.52 + 0.22 1.86 + 0.24 2.20 +_0.32 2.34? 0.31 2.38 & 0.36 2.04 + 0.52 1.38 kO.61 1.60 + 0.32
O.oob 0.00 0.00 0.00
0.12 0.15 0.19
0.20
0.81 f 0.21
0.9o?r 0.22 1.10 * 0.28
0.66& 0.24 0.66 + 0.24 0.66 + 0.23
4 Total score consists of the combined values for convulsions on handling and spontaneous signs of withdrawal. b Means + SE of 26 male rats.
combined. In comparing the convulsions on handling to the various signs which the animals exhibited a positive correlation was seen (r = 0.95, p < 0.05). Additionally, as should be expected, the total score (Table 2) indicated a positive correlation to the convulsions on handling values (r = 0.95) and to the various signs (Y = 0.99) seen during the time period in which the animals were observed.
-
5.0
1 . zz
4.0
----
BLOOD
ETHANOL
-
CONVULSIONS
ON
HANDLING
d.6 1.6
-
I
;: z 2
3.0.p
L
2.0
1.4
1
1.2 ‘b,
1.0 0.6
3-N
z
‘f
0.6
I.0
“-3
0.4 0.2
1
i.
0
I
: 2
A 3
4
5
6
7
6
3
IO
II
I2
24
HOUR s
FIG. 2. The disappearance of ethanol from the blood of rats and the increase in scores for convulsions on handling over a 24-hr period following 10 days of ethanol inhalation. The hourly values are the means 2 SE for 26 animals.
274
FERKO
AND
BOBYOCK
The change in the blood ethanol concentrations and the increase in the scores for convulsions on handling are shown in Fig. 2. As illustrated in the figure the elimination of ethanol from the blood followed zero-order kinetics. The average rate of disappearance of alcohol from the blood was 0.41 + 0.01 (SE) mg/ml/hr. The onset of the withdrawal was at 4 hr and the graded withdrawal reaction attained a maximum effect at approximately 10 hr. It appeared that as the blood ethanol values decreased the signs of the withdrawal phenomenon occurred more strongly. This was confirmed by the fact that there was a definite negative correlation between the changes in blood ethanol concentrations and the rise in the score for convulsions on handling (Y= -0.88,~ < 0.05). Several groups of control animals were placed in the chamber for 10 days. They were treated similarly to the experimental group in every respect except that ethanol was not vaporized into the chamber. On removal of these animals from the chamber following 10 days exposure they did not exhibit signs of withdrawal over a 24-hr period and behaved in a manner similar to normal Sprague-Dawley rats. DISCUSSION This investigation indicated a significant increase in blood ethanol concentrations over a period of time in rats by using an inhalation procedure. Also it was shown that upon removal of all animals from the atmosphere of ethanol a sequence of spontaneous signs of withdrawal were manifested. The effects were accomplished without the use of pyrazole and with the maintenance of initial weight of the animals. It has been stated that physical dependence develops in animals after a short period of continuous intoxication (Goldstein, 1972). In the present study ethanol blood concentrations were not held at a constant value but were steadily increased to a mean ethanol blood concentration of 3.13 + 0.23 mg/ml achieved on Day 10. The time period and the blood ethanol concentrations were selected so that significant signs of withdrawal were seen in animals which had maintained their body weight. The withdrawal appeared to reach a peak effect in 10 hr, a time which is quite similar to that of previous reports (Goldstein, 1972; Littleton et al., 1974). Based on the data obtained it appears that this approach provides a reliable method for the induction of physical dependence on ethanol. Freund (1975) listed six criteria for a model of physical dependence on ethanol. He stated that the “ideal” method would induce physical dependence so defined: (1) reproducibly, (2) in the shortest possible period of time (days), (3) by simple procedures, (4) with introduction of the fewest variables besides the administration of ethanol, (5) with production of spontaneous major signs of withdrawal, and (6) in all treated animals. He further claimed that no presently available method could approach this ideal. However, it is quite clear from our results that all six points were satisfied. It is not necessary to examine each one of the objectives individually but a few should be reemphasized, particularly numbers 4 and 5. This method does introduce the fewest variables besides the administration of ethanol. Pyrazole was eliminated because of its toxic effects on the liver and central nervous system. Also, pyrazole can produce interactions with ethanol and other drugs which may be used in a model for drug treatment experiments. Interpretation of the results from such drug-drug interactions is difficult. Another variable that was avoided in this study was the loss of original body weight (Goldstein, 1972; Roach et al., 1973). However,
PHYSICAL
DEPENDENCE
ON ETHANOL
IN RATS
275
Branchey et al. (1971) have shown that some weight loss may not interfere with a rat model for physical dependence. Their work involved oral administration of ethanol. Since the animals are in an environment of ethanol vapor, the effects of ethanol upon lung structure will have to be examined thoroughly in future studies. In a human study Lester and Greenberg (1951) noted that 10 to 20 mg/liters of ethanol in air causes some coughing and smarting of the eyes, but these signs disappear in 5 to 10 min. Thirty milligrams per liter of ethanol in air could be tolerated. However, above 40 mg/liter the atmosphere containing ethanol was intolerable for even short periods. Point 5 of the Freund criteria indicates that the animals should demonstrate spontaneous major signs of withdrawal. Table 1 lists the signs according to the increasing order of occurrence and severity. The observable, spontaneously manifested sign’swhen compared to the incidence of convulsions on handling (Goldstein, 1972) showed a positive correlation (Table 2). In summary this study satisfied some of the earlier criticisms which had been directed at induction of ethanol dependence by previously reported inhalation procedures, i.e., weight loss and the use of pyrazole. ACKNOWLEDGMENT We thank Dr. W. S. Chernick for his assistanceand advice. REFERENCES to Biostatistics. Harper and Row, New York. G., AND KISSIN, B. (1971). Modification in the response to alcohol following the establishment of physical dependence. Psychopharmacologia 22,314-322. DE~TSCH, J. A., AND KOOPMANS, H. S. (1973). Preference enhancement for alcohol by passive exposure. Science 179, 1242-l 243. FALK, J. L., SAMSON, H. H., AND WINGER, G. (1972). Behavioral maintenance of high concentrations of blood ethanol and physical dependence in the rat. Science 177,811-813. FREUND, G. (1969). Alcohol withdrawal syndrome in mice. Arch. Neural. 21,315-320. FREUND, G. (1975). Induction of physical dependence on alcohol in rats. In Advances in Experimental Medicine and Biology (E. Majchrowicz, ed.) Vol. 56, pp, 31 l-325. Plenum, New York. GOLDBERG, L., AND RYDBERG, U. (1969). Inhibition of ethanol metabolism in vivo by administration of pyrazole. Biochem. Pharamcol. 18, 1749-1762. GOLDSTEIN, D. B. (1972). Relationship of alcohol dose to intensity of withdrawal signs in mice. J. Pharmacol. Exp. Ther. 180,203-215. GOLDSTEIN, D. B. (1975). Physical dependence on alcohol in mice. Fed. Proc. Fed. Amer. Sot. BANCROFT, BRANCHEY,
H. M.,
(1965). Introduction RAUSCHER,
Exp. Biol. 34,1953-1961.
D. B., ANDPAL, N. (1971). Alcohol dependence produced in mice.by inhalation of ethanol: Grading the withdrawal reactions. Science 172, 288-290. LE BLANC, A. E., AND KALANT, H. (1973). Central nervous system interaction of pyrazole and ethanol in the rat. Canad. J. Physiol. Pharmacol. 51, 612-615. LESTER, D., AND GREENBERG, L. A. (1951). The inhalation of ethyl alcohol by man. Quart. J. Stud. Ale. 12, 167-178. LIEBER, C. S., AND DE CARLI, L. M. (1973). Ethanol dependence and tolerance: A nutritionally controlled experimental model in the rat. Res Comm. Chem. Path. Pharm. 6,983-991. LITTLETON, J. M., GRIFFITHS, P. J., AND ORTIZ, A. (1974). The induction of ethanol dependence and the ethanol withdrawal syndrome: The effects of pyrazole. J. Pharm. Pharmacol. 26,81-91. GOLDSTEIN,
276
FERKOAND BOBYOCK
LUNDQUIST,F. (1959). The determination of ethyl alcohol in blood and tissues. Methods Biochem. Anal. 7,217-251.
NACHMAN, M., LARVE,C., AND LE MAGNEN,J. (1971). The role of olfactory and orosenory factors in the alcohol preference of inbred strain of mice. Physiol. Behau. 6, 53-59. ROACH, M. K., KHAN, M. M., COFFMAN,R., PENNINGTON,W., AND DAVIS,D. L. (1973). Brain (Na+ and K+)-activated adenosine triphosphatase activity and neurotransmitrer uptake in alcohol-dependent rats. Brain Res 63,323-329. RYDBERG,U. AND NERI, A. (1972). 4-Methylpyrazole as an inhibitor of ethanol metabolism: Differential metabolic and central nervous effects. Acfa Pharmacol. Toxicol. 31,431-432. THEORELL,H., YONETANI,T., AND SJOBERG, B. (1969). On the effect of some heterocylic compounds on the enzymic activity of liver alcohol dehydrogenase. Acta Chem. &and. 23, 255-260.
