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The development of metabolic tolerance in the alcohol-preferring P rats: Comparison of forced and free-choice drinking of ethanol
Pharmacology Biochemistry & Behavior, Vol. 25. pp. 1013-1020, 1986. e Ankho International Inc. Printed in the U.S.A.
0091-3057/86 $3.00 + .00
The Development of Metabolic Tolerance in the Alcohol-Preferring P Rats: Comparison of Forced and Free-Choice Drinking of Ethanol' LAWRENCE
LUMENG-'
AND TING-KAI
LI
D e p a r t m e n t s of M e d i c i n e a n d B i o c h e m i s t r y I n d i a n a U n i v e r s i t y S c h o o l o f M e d i c i n e a n d The R . L. R o u d e b u s h VA M e d i c a l C e n t e r , I n d i a n a p o l i s , I N 46223 Received
15 N o v e m b e r
1985
LUMENG, L. AND T.-K. LI. 771¢, d(,vel~qmlent ~/'metabolic toleram'e in the ah'ohol-pr¢:/~,rrin~ P rats: Comparison ~/' fi~rced andJ?ee-choice drinking of ethanol. PHARMACOL BIOCHEM BEHAV 25(5) 1013-1020, 1986.--Experiments were performed to determine whether metabolic tolerance to alcohol develops in the alcohol-preferring P rats during free-choice drinking. In Experiment 1, alcohol elimination rates (AERs) in female Wistar and P rats were measured as a function of age from 26 to 180 days old. AERs calculated as mmol hr ~per kg body weight fell with age, whereas AERs expressed as mmol hr ' per rat increased to reach a constant value after 60 days of age. These data indicate that the chronic effects of ethanol on AER are most easily interpreted if experiments are performed in animals 60 days of age or older and AERs are calculated as mmol hr ~ per rat. In Experiments 2 and 3, P female rats were exposed to alcohol for 6-7 weeks either by free-choice drinking or by forced feeding with liquid diets. With free-choice drinking of alcohol, solid food containing 31 percent of the calories as protein, 10 percent ethanol (v/v) and water were made available ad lib. The liquid diets used for forced ethanol feeding were the Bio-Serv-711 diet, a protein-supplemented Bio-Serv-711 diet and the AIN diet and they contained 18, 32 and 22 percent calories as protein, respectively. When compared with pair-fed or ad lib controls, all the P rats exposed to alcohol by either free-choice or forced-feeding exhibited increased AERs (i.e., metabolic tolerance) after 6-7 weeks. However, if AERs before and after alcohol exposure in the same animals were compared, a net increase in AER was evident only in the P rats on free-choice drinking or forced-fed diets which contained at least 22 percent protein. Alcohol consumption and blood alcohol concentrations of the P rats exhibited diurnal variation during free-choice drinking or when they were forced-fed alcohol diets which contained at least 22 percent protein. The high BACs attained in the P rats given the ethanol-containing Bio-Serv-711 diet, presumably because of the lower AERs under this condition, disrupted the diurnal cycling of alcohol ingestion and blood alcohol concentrations. The studies demonstrate that the P rats on chroinic free-choice drinking of alcohol develop metabolic tolerance to much the same degree as animals forced fed ethanol contained in liquid diets. Additionally, they demonstrate that, in animals fed ethanol-containing liquid diets, a net increase in AER after alcohol exposure is evident only if dietary protein constitutes at least 22 percent of the total calories. Ethanol
Metabolic tolerance
Alcohol-preferring P rats
T H E P (alcohol-preferring) and N P ( a l c o h o l - n o n p r e f e r r i n g ) lines o f rats have b e e n raised by selective b r e e d i n g as an animal m o d e l to investigate the b i o c h e m i c a l , physiological and b e h a v i o r a l c o n c o m i t a n t s o f v o l u n t a r y alcohol c o n s u m p tion [7]. The P rats orally s e l f - a d m i n i s t e r e t h a n o l solutions (e.g., 10 p e r c e n t v/v) w h e n food and w a t e r are freely available and their b l o o d alcohol c o n c e n t r a t i o n s rise to as high as 218 mg p e r c e n t during the dark cycle [18,30]. R e c e n t studies indicate that b l o o d alcohol c o n c e n t r a t i o n s as low as 16 mg p e r c e n t are p h a r m a c o l o g i c a l l y active in the P rats since t h e y exhibit a stimulation o f s p o n t a n e o u s m o t o r activity after given d o s e s o f ethanol that p r o d u c e peak b l o o d alcohol con-
Alcohol elimination rate
Alcohol dehydrogenase
c e n t r a t i o n s in the range o f 16--70 mg p e r c e n t [29]. The P but not the N P rats also s e l f - a d m i n i s t e r e t h a n o l by the intragastric route in a f r e e - c h o i c e situation, w h e n food is available ad lib [27]. T h u s , the p o s t - i n g e s t i v e effects o f e t h a n o l is reinforcing. We h a v e also s h o w n that, with c h r o n i c f r e e - c h o i c e drinking, the P rats d e v e l o p physical dep e n d e n c e as e v i d e n c e d by signs o f w i t h d r a w a l w h e n the alcohol solution is r e m o v e d [28]. The d e v e l o p m e n t o f t o l e r a n c e to the effects o f e t h a n o l is a w e l l - d o c u m e n t e d c o n s e q u e n c e o f c h r o n i c e x p o s u r e and its acquisition with f r e e - c h o i c e drinking is a n e c e s s a r y criterion for an animal model o f a l c o h o l i s m . We have b e e n exploring
~Supported by PHS AA-03243. -'Requests for reprints should be addressed to Dr. Lawrence Lumeng, Departments of Medicine and Biochemistry, Emerson Hall Room 421. 545 Barnhill Drive, Indianapolis, IN 46223.
1013
LUMENG
1014
TABLE COMPOSITION
Protein Bio-Serv-711 (BS-711) Control Kcal/ml %Kcal Ethanol KcalYml %Kcal
A N D LI
1
OF DIETS
Fats
Carbohydrates Ethanol
Total
0.18 18 0.18 18
0.35 35 0.35 35
0.47 47 0,11 11
--0.36 36
1.00 -1.00 --
Supplemented Bio-Serv-711 (SBS-711) Control KcalYml 0.39 %Kcal 32 Ethanol Kcal/ml 0.39 %Kcal 32
0.35 29 0.35 29
0.47 39
--.36 29
1.21
Wayne-Blox solid food (WB) Kcal/g %Kcal
1.04 31
0.36 11
1.99 58
---
3.39 --
Bio-Serv-AlN-76 rAIN) Control Kcal/ml %Kcal Ethanol Kcal/ml %Kcal
0.22 22 0.22 22
0.115 11.5 0.115 11.5
0.665 66.5 0.31 31
--0.355 35.5
1.00 -1.00 --
• 11
10
--
1.21 --
METHOD
Animals
GROWTH CURVES OF P AND WISTAR FEMALE RATS M A I N T A I N E D ON COMMERCIAL SOLID RAT DIETS
F e m a l e W i s t a r and P rats were h o u s e d individually in w i r e - m e s h c a g e s a n d in a c o n t r o l l e d t e m p e r a t u r e a n d humidity e n v i r o n m e n t with fixed light-dark cycles (7 a.m. to 7 p . m . , light and 7 p.m. to 7 a . m . , dark). T h e W i s t a r rats were p u r c h a s e d from H a r l a n I n d u s t r i e s , I n d i a n a p o l i s , IN a n d rats from the P line were from the S-21 g e n e r a t i o n o f the a l c o h o l - p r e f e r r i n g rats s e l e c t i v e l y bred in o u r l a b o r a t o r y , in E x p e r i m e n t 1. the g r o w t h c u r v e s a n d alcohol e l i m i n a t i o n rates ( A E R s ) o f 2 g r o u p s o f P rats a n d a g r o u p of W i s t a r rats ( n - 6 p e r group) were m o n i t o r e d as a f u n c t i o n o f age (26 day old to 180 day old). T h e m e a n age o f the P rats at the start of E x p e r i m e n t 2 was 6 8 + 2 d a y s a n d that at the start o f Experim e n t 3 was 6 0 _+ 1 days.
300
i ~. I ~9
200
:¢
RATS .,=+ c,+u.,+
23
f-l--f']
I 50
WISTAR RATS
I 100
(n=6),
I 150
I 200
AGE,d
FIG. I. Growth curves of a group of female Wistar rats and two groups of female P rats maintained on a commercial solid rat diet (Wayne-Blox). The coefficients of variation ranged from 2 to 7 percent.
w h e t h e r the P rats will d e v e l o p m e t a b o l i c a n d n e u r o n a l t o l e r a n c e to alcohol with c h r o n i c f r e e - c h o i c e drinking. This r e p o r t d e s c r i b e s studies on the d e v e l o p m e n t o f m e t a b o l i c t o l e r a n c e as a result o f c h r o n i c e t h a n o l intake. W e i n c l u d e d as p o s i t i v e c o n t r o l s in t h e s e s t u d i e s , P rats t h a t w e r e forcedfed e t h a n o l i n c o r p o r a t e d into liquid diets. B e c a u s e liquid diets differ in protein c o n t e n t [12], we also e x a m i n e d the effect o f different diet f o r m u l a t i o n s on the d e v e l o p m e n t of m e t a b o l i c t o l e r a n c e a n d on the diurnal v a r i a t i o n of alcohol ingestion a n d b l o o d alcohol c o n c e n t r a t i o n s .
Diets In E x p e r i m e n t 1. the W i s t a r a n d P rats after w e a n i n g were given W a y n e - B l o x food pellets a n d w a t e r ad lib. In E x p e r i m e n t 2, the P rats w e r e divided into 6 g r o u p s ( n = 6 p e r group) a n d g i v e n different feeding r e g i m e n s for 7 w e e k s . T h e first two g r o u p s were pair-fed the c o n t r o l a n d the e t h a n o l Bio-Serv-711 (BS-711) liquid diet (Bio Serv, Inc.+ F r e n c h t o w n , NJ). T h e BS-711 liquid diet was l b r m u l a t e d a c c o r d i n g to the diet c o m p o s i t i o n p u b l i s h e d by DeCarli a n d L e i b e r in 1967 a n d is similar to the L e i b e r - D e C a r l i 1982 regular liquid diet [12]. T h e t h i r d and f o u r t h g r o u p s were also pair-fed but the c o n t r o l a n d e t h a n o l liquid diets were the " s u p p l e m e n t e d B i o - S e r v " diet (SBS-711) d e s c r i b e d by Rogers et al. [22]. T h e s u p p l e m e n t a t i o n c o n s i s t e d o f a d d i n g v i t a m i n - f r e e casein a n d zinc to the Bio-Serv-711 diet (Table I). T h e fifth g r o u p was given W a y n e - B l o x food pellets (WB) and w a t e r ad lib, a n d the sixth g r o u p was g i v e n ad lib W a y n e - B l o x food pellet a n d the f r e e - c h o i c e d r i n k i n g of 10 p e r c e n t (v/v) e t h a n o l and water. T h e a n i m a l s g i v e n the liquid
M E T A B O L I C T O L E R A N C E IN T H E P R A T S
1015
A L C O H O L E L I M I N A T I O N R A T E OF P A N D V I I S T A R F E M A L E R A T S AS A F U N C T I O N OF ,AGE
/ /
~2
I /
,
**
,
> rn
g
2.0
-10
o
R
5 2_
-i =
/
1.5
Z
E E
E
~ )
P RATS
(n=6/cJroup)
I-I--I'-I
WISTAR RATS
:i:
7
~ ©
< (n=6)
~.0
6
I
0
50
100
150
200 0
5Q
lO0
200
I50
AGE,d
FIG. 2. Change in alcohol elimination rate (AER) of the P and Wistar rats as a function of age. AERs were expressed as either mmol hr ' per rat (left panel) or mmol hr ' per kg body weight (right panel). The coefficients of variation ranged from 1 to 11 percent. Values marked with an asterisk are not different from each other and are statistically different from those without an asterisk.
TABLE 2 MEAN
DAILY
CALORIC
INTAKE
IN P RATS GIVEN
THE
DIFFERENT
DIETARY
REGIMENS
IN EXPERIMENT
2
Mean daily caloric intake per rat, Kcal/day Week 1
BS-711 (C) BS-711 (E) SBS-711 (C) SBS-711 (E) WB (C) WB (Free-choice)
Week 3-4
Week 7
Total
Pr
Ethanol
Total
Pr
Ethanol
Total
Pr
Ethanol
50.0* 51.6" 59.2 60.6 59.3 59.2
9.0* 9.3* 19.2 19.6 18.8 14.2
-18.6 -17.7 -14.4t
51.7" 51.7" 59.4 59.4 60.2 60.4
9.3* 9.3* 19.2 19.2 19.1 14.2
-18.6 -17.3 -16.4
58.6 59.2 61.3 61.2 58.5 59.4
10.6" 10.6" 20.2 19.8 17.0 14.0
-21.3 -17.9¢ -15.1"
*Significantly different from the SBS-711 (C), SBS-711 (E), WB (C) and WB (Free-choice) diets (by analysis of variance). tSignificantly different from the BS-711 (E) diets (by analysis of variance). Abbreviations: Pr, protein; C, control; E, ethanol.
diets w e r e a c c l i m a t e d to i n c r e a s i n g c o n c e n t r a t i o n s o f e t h a n o l in the diet in the m a n n e r s u g g e s t e d by the m a n u f a c t u r e r . In E x p e r i m e n t 3, the P rats w e r e s e p a r a t e d into four g r o u p s ( n = 5 per group). The first t w o g r o u p s w e r e pair-fed the c o n t r o l and the e t h a n o l - A I N liquid diet (Bio-Serv A I N - 7 6 diet) [16,21]. T h e third g r o u p was fed p o w d e r e d W a y n e - B l o x solid food and a s u c r o s e solution, while the fourth g r o u p was given the p o w d e r e d W a y n e - B l o x solid f o o d , w a t e r and 10 p e r c e n t e t h a n o l . The third and fourth g r o u p s o f rats w e r e pair-fed by providing to the c o n t r o l rats (the third group) the a m o u n t o f solid food and an a m o u n t o f the s u c r o s e solution
that is isocaloric to the a m o u n t o f I0 p e r c e n t e t h a n o l cons u m e d by the rats on f r e e - c h o i c e drinking (the fourth group).
Dcterntination o f AIchol Elimimttion Rate (AER) in Viro E t h a n o l (2 g/kg) was a d m i n i s t e r e d by the i n t r a p e r i t o n e a l injection o f a 10 p e r c e n t (v/v) solution. Tail blood s a m p l e s w e r e c o l l e c t e d as a f u n c t i o n o f time after e t h a n o l injection and the blood e t h a n o l c o n c e n t r a t i o n s w e r e m e a s u r e d by a head s p a c e m e t h o d with a gas-liquid c h r o m a t o g r a p h [13].
1016
LUMENG
A N D LI
TABLE 3 EFFECT OF DIETARY REGIMENS ON ETHANOL INTAKE IN EXPERIMENT 2 Ethanol Intake, g/kg/day Diet BS-711(E) SBS-711 (E) WB (Freechoice)
Week 1
2
3
4
10.8-+ 0.5 11.7_+0.4 8.5 ± 0.6*
13.6 ± 1.3 10.3-+0.3" 8.3 -+ 0.3*
12.5 ± 0.3 10.0+0.3" 9.3 _+ 0.7*
12.1 ± 0.4 9.8_+0.2 * 7.2 _+ 0.7*
5
6
7
12.8 + 0.4 9.8-+0.2* 7.8 ± 0.4*
12.9 ± 0.4 10.4-+0.3" 11.5 + 0.7
13.2 ± 0.2 9.5-+0.3* 8.4 ± 0.3*
*Significantly different from the BS-711 (E) group (by analysis of variance). Abbreviations: E, ethanol.
TABLE 4 EFFECT OF DIFFERENT DIETARY REGIMENS ON BODY WEIGHT AND ALCOHOL EI.IMINATION RATE IN EXPERIMENT 2 Alcohol Elimination Rate Body Weight g Diet BS-711 (C)
Before
7th Week
205 + 10
204 "+ 7
mmol/hr/rat Before -
1.90 ± .12
L BS-711 (E)
208 ±
3
7th Week
Before
1.66 -+ .051
I
2.07 ± .04
mmol/hr/kg
J
2.02 + .06
9.25 + .39
209 -+ 7
SBS-711 (E)
204 ±
6
6.95 _+ .28-I
l 9.97 ± .31
I SBS-711 (C)
7th Week
I 9.08 ± .18
J
•
262 +
,.97+.06[
1'72-+'0511
9"50+'481
6"60-+283l
~-270 + 8
1.92 + .07
2.22 + .09
9.59 + .32
8.25 _+ .26
2.03 -+ .05
2.10-+.133
9.71"+.28 I
7 "I 5 9 + ' 5 0 3
WB (C)
208 ±
6
263 "+ 4
WB (Free-Choice)
227 ± 12
274 ± 8
I
--
J
I
I
2.11,1 ± .08
2.50 + .11 9.36 "+ .17 9.14 + .27 -.J Brackets indicate that the compared values are significantly different. Body weight differences were analyzed by analysis of variance. Alcohol elimination rate differences were analyzed by two-tailed t-test. Abbreviations: C, control: E, ethanol. T h e rate o f e t h a n o l d i s a p p e a r a n c e was p s e u d o - z e r o o r d e r , a n d the A E R was c a l c u l a t e d using W i d m a r k ' s e q u a t i o n [6].
2, a n a l y s i s of v a r i a n c e a n d p o s t - h o c N e w m a n - K e u l s t e s t s w e r e used to e v a l u a t e statistical differences.
Measurement of Liver Alcohol Dehydrogenase (ADH) Activity L i v e r h o m o g e n a t e - s u p e r n a t a n t f r a c t i o n s were p r e p a r e d [13] a n d alcohol d e h y d r o g e n a s e ( A D H ) activity was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y b y m e a s u r i n g the r e d u c t i o n o f N A D + to N A D H . T h e a s s a y b u f f e r c o n t a i n e d 0.5 M Tris-HC1 (pH 7.2 a n d I - 0 . 2 ) , 2.8 m M N A D ~, a n d 5 m M e t h a n o l , a n d t h e t e m p e r a t u r e was m a i n t a i n e d at 37 ° [14]. T h e s e s u b s t r a t e c o n c e n t r a t i o n s s a t u r a t e t h e e n z y m e 88 p e r c e n t ; t h e r e f o r e , the m e a s u r e d e n z y m e a c t i v i t y w a s multiplied b y 1.14 to calculate the m a x i m u m A D H v e l o c i t y (or V m a x ) [14].
Statistical Analysis R e s u l t s are p r e s e n t e d as m e a n s _ + S E M . T h e two-tailed s t u d e n t ' s t-test for u n p a i r e d data was used for statistical c o m p a r i s o n s . D i f f e r e n c e s were c o n s i d e r e d to be significant w h e n the p values was less than 0.05. W h e r e results a m o n g different e x p e r i m e n t a l g r o u p s were c o m p a r e d in E x p e r i m e n t
RESULTS
Experime/lt I In p r e v i o u s studies o f m e t a b o l i c t o l e r a n c e [3, 10, 11, 15, 20, 24, 26], rats w e r e pair-fed c o n t r o l and e t h a n o l - c o n t a i n i n g diets for s e v e r a l w e e k s , and the A E R s of the t w o g r o u p s w e r e t h e n c o m p a r e d with the results e x p r e s s e d in most studies as m m o l o f e t h a n o l m e t a b o l i z e d p e r h o u r per kg body weight. In o t h e r studies, only the d i s a p p e a r a n c e rates of e t h a n o l in p l a s m a (mmol/100 ml p e r hr) w e r e c o m p a r e d . In all o f t h e s e studies, no a t t e m p t s were m a d e to d i s c e r n if e t h a n o l ingestion h a d actually i n c r e a s e d the A E R w h e n c o m p a r e d with t h a t before e t h a n o l e x p o s u r e in the same animals. T o set the stage for E x p e r i m e n t s 2 a n d 3, we d e t e r m i n e d in E x p e r i m e n t I the g r o w t h c u r v e of P f e m a l e rats and the c h a n g e in A E R as a f u n c t i o n of age. T h e p u r p o s e was to find the m o s t a p p r o p r i a t e r e f e r e n c e unit to e x p r e s s A E R s for c o m p a r i s o n of data. Since the P line of rats was originally
M E T A B O L I C T O L E R A N C E IN T H E P RATS
1017
TABLE 5 240
EFFECT OF DIFFERENT DIETARY REGIMENS ON HEPATIC ALCOHOL DEHYDROGENASE ACTIVITY IN EXPERIMENT 2
•
220
Diet
Alcohol Dehydrogenase Activity (mmol hr ~liver)
200
:"
•
180
BS-711 (C) BS-711 (E) SBS-711 (C) SBS-711 (E) WB(C) WB (Free-Choice)
2.88 2.41 2.95 3.24 3.18 2.93
+_ 0.18 +_ 0.09 +_ 0.13 +_ 0.16 _+ 0.15 _+ 0.12
z 0
140
0Z
120
o 0
derived from outbred Wistar rats [7], we also measured these parameters in a group of Wistar female rats. Figure 1 indicates that the growth curves of the P and Wistar female rats were similar. Figure 2 depicts the change in A E R as a function of age in the P and Wistar female rats, when A E R was expressed either as mmol of ethanol eliminated per hour per rat (left panel) or as mmol of ethanol eliminated per hour per kg body weight (right panel). When expressed as rate per rat+ A E R increased rapidly from 26days of age to about 60-days and it remained constant thereafter, By comparison, A E R expressed as rate per kg body weight declined sigmoidally with age and it reached a constant low only when the rats were about 120- to 180-days old. The patterns of change were similar in the P and Wistar female rats, with the curves for the Wistar rats slightly displaced to the right.
Experiment 2 In this experiment, the P rats were given different dietary regimens: groups 1 and 2 were pair-fed the ethanol and control BS-711 diet; groups 3 and 4 were pair-fed the ethanol and control SBS-711 diet: group 5 was given solid food and water ad lib and group 6 was given the solid food ad lib and the free-choice of 10 percent (v/v) ethanol and water. Table 2 summarizes the mean daily caloric intake of the P rats receiving the different dietary regimens. In the first 4 weeks of feeding, the daily caloric intake of the rats placed on the BS-711 diets was about 15 percent lower than the other groups. More importantly, owing to the low protein content of the BS-711 diets, the protein caloric intake throughout the 7-week period was only one-half of that consumed by the other groups. The daily caloric intake as ethanol in the SBS711 group tended (not statistically significant) to be lower than that in the BS-711 group. By comparison, calorie consumption as ethanol in the free-choice group was clearly lower (12 to 29 percent) than that in the BS-711 group (at least for weeks 1 and 7). Table 3 shows the effect of the different dietary regimens on ethanol intake expressed as g of ethanol kg ; per day. Partly owing to the higher daily ethanol intake per rat (cf. Table 2) and to the lesser weight gain of the rats on the BS-711 diet (Table 4), ethanol intake expressed as g kg ' per day was the highest in the BS-711 group. Table 4 shows the effect of the different dietary regimens on body weight and AER. Rats on the BS-711 diet failed to gain weight in the 7-week study. By comparison, rats in all the other groups gained at least 40 g over this period. AERs were expressed both as mmo! hr ~per rat and mmol hr ; per
160
< rr ~z ua
I 0 0 "< 0 0
IO0
..
~. ~
--.L
:
•
I °
"i
8O 60
:
"I
4O
2o
i
.*
o• .°
--T-
.%
•
o
0 2P
10 e
BS-711(E)
6A
2P
10 P
SB$-711(E)
10 P
6A
WB(FC)
FIG. 3. Blood alcohol concentrations of P rats placed on three different dietary regimens. Horizontal bar indicates the mean value. BS-711(E), SBS-711(E) and WB(FC) are abbreviations for the ethanoI-Bio-Serv-711 diet, the supplemented ethanoI-Bio-Serv-711 diet and the Wayne-Blox free-choice drinking regimen.
kg body weight. Before initiation of the feeding experiment, the mean AERs of the 6 groups of rats were similar regardless of the manner of expressing the AERs: the mean AERs varied from 1.90 to 2.11 mmol hr ; per rat and from 9.25 and 9.71 nmol hr ' per kg body weight. By the end of the 7-week experiment, the AERs of all the groups treated with alcohol were 19 to 29 percent higher than their respective controls indicating that chronic alcohol ingestion, regardless of how ethanol was consumed, produced metabolic tolerance in the P rats. However, a real increase in A ER over the 7-week experiment was evident in only 2 groups of animals, when AERs were expressed as mmol hr ' per rat, i.e., those placed on the ethanoI-SBS-711 diet and those given solid food and the free-choice drinking of 10 percent ethanol versus water. Except for the group of rats given solid food and free-choice drinking, all other groups exhibited an actual, net decline in A E R at the end of the 7-week period when A E R was calculated as mmol h r ' per kg body weight. These data, therefore, demonstrate that: (1) The P rats given solid food and the free-choice drinking of 10 percent ethanol and water develop metabolic tolerance; (2) the BS-711 diet, unless supplemented with protein, will not produce a net increase in A E R in ethanol-fed animals; and (3) interpretation of data with AERs expressed as mmol of ethanol eliminated per hr per rat is less complicated than when AERs are expressed as mmol hr ' per kg body weight. This is due to the fact that, in female Wistar and P rats, the gain in body weight and the increase in A E R as they mature from 50 to 200 days of age follow different time courses. Table 5 summarizes the effect of the different dietary regimens on A D H activity in liver. In agreement with the bulk of the literature [2, 9-11, 24, 26], chronic alcohol treatment did not lead to a discernible increase in the total ADH activity.
1018
L U M E N G A N D LI TABLE 6 MEAN DAILY CALORIC INTAKE 1N THE P RATS FORCED-FED ALCOHOL BY THE AIN DIET OR GIVEN SOLID FOOD AND THE FREE-CHOICE DRINKING OF ALCOHOL Mean daily caloric intakes per rat Week 1
Total AIN (C) AIN (E) WB (C) WB (Free-choice, water vs. 10 percent E)
Week 2
Week 4
Week 6
Pr
E
Total
Pr
E
Total
Pr
E
Total
Pr
E
53.3 54.1 59.4 61.2
14.9 15.1 14.6 15.2
19.2 -13.3'
52.2 53.9 54.5 58.9
14.6 15.1 12.7" 14.1
-19.1 -14.3"
54.2 54.1 57.8 64.0
15.2 15.1 12.7" 14.7
-19.2 -17.7"
48.1 48.3 46.7 49.1
13.5 13.5 10.4"
17.1
11.4"
14.6"
Abbreviations: C, control; E, ethanol; Pr, protein. *Significantly different from either the AIN (C) or the AIN (E) diet (by analysis of variance).
220
TABLE 7
oo
EFFECT OF ETHANOL GIVEN IN THE AIN LIQUID DIET AND BY FREE-CHOICE ON BODY WEIGHT AND ALCOHOl, ELIMINATION RATE (EXPERIMENT 3) Body Weight g Diet
Before
o3 E z 0
Alcohol Elimination Rate mmol/hr/rat
6th Week
Before
o~ Fz
6th Week
AINC
78+6 230+i]78009 7+0091
AIN (E)
176 -+ 6
1.74 + 0.08 2.09 + 0.07 .3
I WB (C)
166 -+ 5
2t-4 +
WB(Free-choice)
166 _+ 5
239 + 4
I
1.67 + 0.09 2.03 _+ 0.05-'1
I
I
/
Z © U .J
© 2£ 0
160
I
Brackets indicate that the compared values are significantly different. Abbreviations: C, control; E, ethanol.
14o 120 I00
80 6o
_J
<
40
1.68 + 0.10 2.36 + 0,08 .3
I
180
I-<
uJ
246 +
200
o o
20 0
I: 2P
• gt •
IO P
AIN(E)
Figure 3 c o m p a r e s the b l o o d alcohol c o n c e n t r a t i o n s o f the P rats r e c e i v i n g alcohol with the different dietary r e g i m e n s . Only t h o s e on the ethanol-BS-711 diet e x h i b i t e d p e r s i s t e n t l y e l e v a t e d b l o o d alcohol c o n c e n t r a t i o n s w h e n m e a s u r e d at 6 a . m . , 2 p.m. and l0 p.m. The animals given the higher protein diets, i.e., the SBS-711 or the WB diet, e x h i b i t e d m u c h l o w e r b l o o d alcohol c o n c e n t r a t i o n s and m a i n t a i n e d a diurnal cycling o f blood alcohol c o n c e n t r a t i o n s . In P rats given f r e e - c h o i c e drinking o f alcohol, b l o o d alcohol c o n c e n t r a t i o n s ranged f r o m 5 mg p e r c e n t to as high as 137 mg perc e n t during the dark cycle at the times o f m e a s u r e m e n t .
L:vperimenl 3 The A I N liquid diet is a f o r m u l a t i o n that has a higher p r o t e i n c o n t e n t than the BS-711 diet (Table 1). Table 6 c o m p a r e s the m e a n total daily caloric intake p e r rat and the m e a n c o n t r i b u t i o n o f p r o t e i n and ethanol to the total daily caloric intake. With f o r c e d feeding o f ethanol (i.e., the A I N liquid diet with 6 p e r c e n t ethanol), the daily intake o f e t h a n o l per rat w a s initially 44 p e r c e n t ( w e e k 1) and t h e n 8 and 17 per-
6A
2p
10P
6A
WB (FC)
FIG. 4. Blood alcohol concentrations of P rats placed on the ethanol-AIN diet and the Wayne-Blox free-choice drinking regimen.
cent ( w e e k s 4 and 6) higher than that with f l e e - c h o i c e drinking o f ethanol (i.e., WB and f l e e - c h o i c e drinking o f 10 percent ethanol vs. water). W h e n the alcohol intake was exp r e s s e d as g/kg/day, f o r c e d - f e e d i n g o f ethanol as part of the A I N liquid diet also led to initially 40 p e r c e n t ( w e e k I) and then 15 p e r c e n t (week 6) higher alcohol intake than that with f l e e - c h o i c e drinking. The daily alcohol intake o f the P rats on the e t h a n o l - A I N liquid diet and t h o s e on f l e e - c h o i c e alcohol intake ranged from 10.1 to 13.8 g/kg/day and from 8.8 to 11.5 g/kg/day, r e s p e c t i v e l y . Table 7 s h o w s the a m o u n t o f weight gain and the c h a n g e in A E R s as a function of time in the P rats pair-fed the AIN liquid diets (with or without ethanol) and in t h o s e pair-fed the W a y n e - B l o x solid l a b o r a t o r y food and 10 p e r c e n t ethanol or an isocaloric a m o u n t o f a s u c r o s e solution. Weight gain o f 29 to 47 p e r c e n t was e v i d e n t in all the g r o u p s o v e r the 6-week period. A E R s , e x p r e s s e d as
M E T A B O L I C T O L E R A N C E IN T H E P RATS mmol hr ~ per rat, increased over the 6-week period in the AIN(E), WB(C) and the WB (free-choice) groups. Metabolic tolerance was demonstrated in the P rats given ethanol either by forced-feeding or by free-choice. The mean increases in AERs when the P rats were forced-fed and when they were given alcohol by free-choice were 22 and 16 percent, respectively. Figure 4 depicts the blood alcohol concentrations of the P rats when they were on the ethanol-AIN liquid diet and when they were given ethanol by free-choice. Both groups exhibited diurnal cycling of alcohol consumption, which was reflected in the diurnal variations of blood alcohol concentrations. With free-choice drinking, the blood alcohol concentrations in the P rats ranged from 17 to 127 mg percent during the dark cycle and they were similar to those of the P rats given the ethanoI-AIN diet. DISCUSSION Recent studies [7, 19, 27] of the selectively-bred Pand NP rats indicate that the P line of rats satisfies almost all of the perceived requirements for an animal model of alcoholism and that the P and NP lines are relevant models to study the biochemical, physiological and behavioral concomitants of alcohol drinking behavior. The data presented above clearly show the development of metabolic tolerance in the P rats following the free-choice drinking of ethanol for 6 to 7 weeks. In two separate experiments (Tables 4 and 7), AERs, expressed as mmol/hr/rat, increased 18-40 percent when compared with values before alcohol exposure and increased 16-20 percent when compared with values from the pair-fed and ad lib controls. In one experiment, the controls were free-fed, whereas in the other, the controls were pair-fed an amount of sucrose solution that is isocaloric to the amount of ethanol consumed by the experiment group. These findings are not unexpected because Hawkins ~'t a/. [3] have reported earlier that chronic alcohol ingestion of 8 g/kg/day can produce metabolic tolerance in vA'o in the rats. Additionally, Videla c t a / . [251 have reported that liver slices from rats exposed to alcohol in the range of 3 to 8 g/kg/day exhibited enhanced alcohol metabolic rates in vitro. Since the female P rats consumed 7-12 g of ethanol/kg/day during free-choice drinking (Tables 3 and 7), metabolic tolenmce should have developed and it did. The biochemical basis of the development metabolic tolerance in rats remains controversial. An increase in ethanol oxidation rate mediated by induction of the microsomal ethanol-oxidizing system (MEOS) has been proposed as one mechanism [8,9]. An increase in ethanol oxidation catalyzed by the ADH pathway has also been advocated as another [5. 15, 20, 23]. Although some investigators [1,17] have reported an increase in the actual amotmt of ADH in liver when rats were exposed to alcohol chronically, most [2, 9-1 I, 24, 26] have found no change. No change was observed in the P rats in this study (Table 5). At the present, an increase in ethanol oxidation mediated by the ADH pathway after chronic exposure to alcohol is best explained by enhanced turnover of N A D H . The latter can arise from increased rate of mitochondrial oxidation of N A D H and/or from an increased rate of transhydrogenation and N A D P H turnover due to induction of MEOS. In most of the previous studies of metabolic tolerance in
1019 rats, alcohol elimination was compared using simply plasma ethanol disappearance rates (mg ethanol/100 ml plasma per hr) or AERs calculated and expressed as mmol hr ' per kg body weight [3, 10, 11, 15, 20, 24, 26]. Plasma ethanol disappearance rate is not a good basis for comparison because it does not take into account changes in the volume of distribution of ethanol. Expressing the A E R as mmol of ethanol eliminated hr ~per kg is probably also less than ideal. This is because the ethanol oxidizing capacity located primarily in the liver may bear no relationship or at best, a complex relationship, with body weight. As shown by Fig. 2, when AERs expressed as mmol hr ~ per kg are plotted as a function of age, they decreased sigmoidally and became constant only at 120 to 180 days of age. By comparison, when AERs are expressed as mmol hr ~per rat, the relationship of AERs with age increases curvilinearly and becomes constant in early adulthood. Thus, as long as the rats at the beginning of the experiment are of the same age and body weight, the effect of any variable on AERs as a function of time can be more confidently interpreted if AERs are calculated as mmol hr ~ per rat. Of note, Israel e t a / . [4] have reported earlier a sigmoidal decline in AERs with growth, calculated as mmol hr ~ per kg body weight, in male Spontaneous Hypertensive and male Wistar rats. The data shown here in Fig. 2 indicates that a similar curve obtains in female Wistar and P rats. Previous studies on metabolic tolerance in rats also have not compared AERs before and after alcohol exposure. The present study examined specifically this relationship and assessed the effect of different dietary regimens. The development of metabolic tolerance to alcohol could be demonstrated with the feeding alcohol with any of the dietary regimens used by comparing the AERs of alcohol-fed rats with their concomitant controls at the end of the feeding experiment. However, a net increase through comparison of AERs before and after alcohol exposure was evident only in the P rats fed the alcohol-containing SBS-711 and AIN diets and in those given the solid food and alcohol by free-choice drinking (Tables 4 and 7). The lack of net increase in AER as a function time in the P rats fed the ethanol containing BS-711 diet is best explained by the low protein content of this diet (Table 2). The low dietary protein content is also most likely responsible for the disruption of the normal diurnal cycling of alcohol ingestion and the persistently elevated blood alcohol concentrations in the P rats given alcohol in the BS-711 diet (Fig. 3). With all the other dietary regimens wherein the protein intake was higher, food and alcohol consumption followed a diurnal rhythm. Accordingly, blood ethanol concentrations also cycled in a diurnal manner, i.e., they were low during the light cycle and high in the dark cycle. These results are in accord with those reported earlier by Rogers ¢,t a/. [22]. Since the P rats developed metabolic tolerance when they were fed alcohol with diets which did not produce persistently elevated blood alcohol concentrations, it is clear the persistent intoxication is not a necessary condition to metabolic tolerance development.
ACKNOWLEDGEMENTS The skillful technical assistance of Mary Beth Welsh is gratefully appreciated. We also thank Miss Brenda Stewart fl)r typing the manuscript.
1020
LUMENG
AND LI
REFERENCES 1. Buffs, L., G. Csabai, M. F o d o r and M. Varga. Increase of alcohol d e h y d r o g e n a s e and protein content of liver following chronic ethanol administration. F I B S Lett 183: 143-144, 1985. 2. Greenberger, N. J., R. B. C o h e n and K. J. Isselbacher. The effect of chronic ethanol administration on liver alcohol dehyd r o g e n a s e activity in the rat. Lab Invest 14: 264-271, 1965. 3. Hawkins, R. D., H. Kalant and J. M. K h a n n a . Effects of chronic intake of ethanol on rate of ethanol metabolism. ('all ,I Phvsiol P h a r m a c o l 44: 241-257, 1966. 4. Israel, Y., J. M. K h a n n a , H. Kalant, D. J. Stewart, J. A. Macdonald, G. R a c h a m i n , S. Wahid and H. Orrego. T h e spont a n e o u s l y hypertensive rat as a model for studies on metabolic tolerance to ethanol. Alcholism: ( l i n Exp Res 1: 3%42, 1977, 5. Israel, Y., L. Videla and J. Bernstein. Liver hypermetabolic state after chronic ethanol c o n s u m p t i o n : hormonal interrelations and pathogenic implications. Fed Proc 34: 2053-2059, 1975. 6. Kalant, H. Absorption, diffusion, distribution, and elimination of ethanol: Effects on biological m e m b r a n e s . In: The Biology ~1 Alcoholism, vol 1, edited by B. Kissin and H. Begleiter. N e w York: Plenum Press, 1971, pp. 1-62. 7. Li, T . - K . , L. L u m e n g , W. J. McBride and M. B. Waller. Progress toward a voluntary oral c o n s u m p t i o n model of alcoholism. l)rttg Alcohol l)ep~'ml 4: 45-60, 1979. 8. Lieber, C. S. Metabolism of ethanol. In: Metab,dic Aspect,~ ~I' Alcoholism, edited by C. S. Leiber. Baltimore: University Park Press, 1977, pp. 1-30. 9. Lieber, C. S. and L. M. DeCarli. Ethanol oxidation by hepatic m i c r o s o m e s : Adaptive increase after ethanol feeding. Sciem'~, 162: 917-918, 1968. 10. Lieber, C. S. and L. M. DeCarli. Hepatic microsomal ethanoloxidizing system: ht vitro characteristics and adaptive properties in vivo. J Biol ( ' h e m 245:2505-2512, 1970. l 1. Lieber, C. S. and L. M. DeCarli. The role of the hepatic microsomal ethanol oxidizing s y s t e m (MEOS) for ethanol metabolism iH vivo. ,I P h a r m a c o l /:\vp 7Tter 181: 279-287, 1972. 12. Lieber, C. S. and L. M. DeCarli. The feeding of alcohol in liquid diets: T w o decades of applications and 1982 update. AIcoholi,sm: C/in k,xp Res 6: 523-531, 1982. 13. L u m e n g , L., W. F. B o s r o n and T.-K. Li. Quantitative correlation of ethanol elimination rates in vivo with liver alcohol dehyd r o g e n a s e activities in fed, fasted and food-restricted rats. Biochcm P h a r m a c o l 28: 1547-1551, 1979. 14. L u m e n g , L. and D. W. Crabb. Rate-determining factors for ethanol metabolism in fasted and castrated male rats. Bi~chcm P h a r m a c o l 33: 2623-2628, 1984. 15. Mezey, E. Duration of the e n h a n c e d activity of the microsomal ethanol-oxidizing e n z y m e s y s t e m and rate o f ethanol degradation in ethanol-fed rats after withdrawal. Biochem Pharmacol 21: 137~142, 1972.
16. Miller, S. S., M. E. Goldman, C. K. Erickson and R. L. Shorey. Induction of physical d e p e n d e n c e on and tolerance to ethanol in rats fed a new nutritionally complete and balanced liquid diet. Psychop/tarma¢'olo~,y (Berlin) 68: 55-59, 1980. 17. Mistilis, S. P and A. Garske. Induction of alcohol dehydrogenase in liver and gastro-intestinal tract. Aust AnH M c d 18: 227-231, 1969. 18. Murphy, J. M.. W. J. McBride, L. L u m e n g and T.-K. Li. Regional brain levels of m o n o a m i n e s in alcohol-preferring and -nonpreferring lines of rats. Pharmacol Biochem Behav 16: 145-149, 1982. 19. Penn, P. E., W. J. McBride, L. L u m e n g , T. M. G a f f a n d T.-K. Li. N e u r o c h e m i c a l and operant behavioral studies of a strain of alcohol-preferring rats. Pharma~'ol Bi~chem Behav 8: 475-481. 1978. 20. Rachamin, G., F. O k u n o and Y. Israel. Inhibitory effect of propylthiouracil on the d e v e l o p m e n t of metabolic tolerance to ethanol. Bioch~,m P h a r m a c , d 34: 2377-2383, 1985. 21. Report of the American Institute of Nutrition Ad Hoc Committe on Standards fur Nutritional Studies. ,l Nutr 107: 1340-1348. 1977. 22. Rogers, J., S. G. Wiener and F. E. Bloom. Long-term ethanol administration m e t h o d s for rats: A d v a n t a g e s o f inhalation over intubation or liquid diets. Behav N~,ural Biol 27: 466-486, 1979. 23. T h u r m a n , R, G., W. R. M c K e n n a and T. B. McCaffrey. Pathw a y s responsible for the adaptive increase in ethanol utilization following chronic treatment with ethanol: Inhibitor studies with the hemoglobin-free perfused rat liver. Mol t'harma('ol 12: 156-166, 1976. 24. Tobon, F. and E. Mezey. Effect of ethanol administration on hepatic ethanol and drug-metabolizing e n z y m e s and on rates o f ethanol degradation. ,I Lab ('lin M e d 77: 110-121, 1971. 25. Videla, L. and Y. Israel. Factors that modify the metabolism of ethanol in rat liver and adaptive changes produced by its chronic administration. Biochcm ,I 118: 275-281, 1970. 26. Videla, L., J. Bernstein and Y. Israel. Metabolic alterations produced in the liver by chronic ethanol administration. Biochem ,I 134: 507-514. 1973. 27. Waller, M. B., W. J. McBride. G. J. Gatto, L. L u m e n g and T.-K. Li. lntragastric self-infusion of ethanol by the P and NP (alcohol-preferring and -nonpreferring) lines of rats. Sci~,nce 225: 78-80, 1984. 28. Waller, M. B., W. J. McBride, L. L u m e n g and T.-K. Li. Induction of d e p e n d e n c e on ethanol by free-choice drinking in alcohol-preferring rats. Pharmacol Bioch~'m Behav 16: 501-507, 1982. 29. Waller, M. B., J. M. Murphy, W. J. McBride, L. L u m e n g and T.-K. Li. Effect of low dose ethanol on s p o n t a n e o u s motor acitivity in alcohol-preferring and -nonpreferring lines of rats. Pharmacol Bio¢'hem Behav. 24: 617-623, 1986. 30. Waller, M. B., J. M. Murphy, G. Gatto, W. J. McBride, L. L u m e n g and T.-K. Li. Effects of scheduling a c c e s s e s on ethanol intake by alcohol preferring P line of rats. AIcoh~d. in preparation.
0091-3057/86 $3.00 + .00
The Development of Metabolic Tolerance in the Alcohol-Preferring P Rats: Comparison of Forced and Free-Choice Drinking of Ethanol' LAWRENCE
LUMENG-'
AND TING-KAI
LI
D e p a r t m e n t s of M e d i c i n e a n d B i o c h e m i s t r y I n d i a n a U n i v e r s i t y S c h o o l o f M e d i c i n e a n d The R . L. R o u d e b u s h VA M e d i c a l C e n t e r , I n d i a n a p o l i s , I N 46223 Received
15 N o v e m b e r
1985
LUMENG, L. AND T.-K. LI. 771¢, d(,vel~qmlent ~/'metabolic toleram'e in the ah'ohol-pr¢:/~,rrin~ P rats: Comparison ~/' fi~rced andJ?ee-choice drinking of ethanol. PHARMACOL BIOCHEM BEHAV 25(5) 1013-1020, 1986.--Experiments were performed to determine whether metabolic tolerance to alcohol develops in the alcohol-preferring P rats during free-choice drinking. In Experiment 1, alcohol elimination rates (AERs) in female Wistar and P rats were measured as a function of age from 26 to 180 days old. AERs calculated as mmol hr ~per kg body weight fell with age, whereas AERs expressed as mmol hr ' per rat increased to reach a constant value after 60 days of age. These data indicate that the chronic effects of ethanol on AER are most easily interpreted if experiments are performed in animals 60 days of age or older and AERs are calculated as mmol hr ~ per rat. In Experiments 2 and 3, P female rats were exposed to alcohol for 6-7 weeks either by free-choice drinking or by forced feeding with liquid diets. With free-choice drinking of alcohol, solid food containing 31 percent of the calories as protein, 10 percent ethanol (v/v) and water were made available ad lib. The liquid diets used for forced ethanol feeding were the Bio-Serv-711 diet, a protein-supplemented Bio-Serv-711 diet and the AIN diet and they contained 18, 32 and 22 percent calories as protein, respectively. When compared with pair-fed or ad lib controls, all the P rats exposed to alcohol by either free-choice or forced-feeding exhibited increased AERs (i.e., metabolic tolerance) after 6-7 weeks. However, if AERs before and after alcohol exposure in the same animals were compared, a net increase in AER was evident only in the P rats on free-choice drinking or forced-fed diets which contained at least 22 percent protein. Alcohol consumption and blood alcohol concentrations of the P rats exhibited diurnal variation during free-choice drinking or when they were forced-fed alcohol diets which contained at least 22 percent protein. The high BACs attained in the P rats given the ethanol-containing Bio-Serv-711 diet, presumably because of the lower AERs under this condition, disrupted the diurnal cycling of alcohol ingestion and blood alcohol concentrations. The studies demonstrate that the P rats on chroinic free-choice drinking of alcohol develop metabolic tolerance to much the same degree as animals forced fed ethanol contained in liquid diets. Additionally, they demonstrate that, in animals fed ethanol-containing liquid diets, a net increase in AER after alcohol exposure is evident only if dietary protein constitutes at least 22 percent of the total calories. Ethanol
Metabolic tolerance
Alcohol-preferring P rats
T H E P (alcohol-preferring) and N P ( a l c o h o l - n o n p r e f e r r i n g ) lines o f rats have b e e n raised by selective b r e e d i n g as an animal m o d e l to investigate the b i o c h e m i c a l , physiological and b e h a v i o r a l c o n c o m i t a n t s o f v o l u n t a r y alcohol c o n s u m p tion [7]. The P rats orally s e l f - a d m i n i s t e r e t h a n o l solutions (e.g., 10 p e r c e n t v/v) w h e n food and w a t e r are freely available and their b l o o d alcohol c o n c e n t r a t i o n s rise to as high as 218 mg p e r c e n t during the dark cycle [18,30]. R e c e n t studies indicate that b l o o d alcohol c o n c e n t r a t i o n s as low as 16 mg p e r c e n t are p h a r m a c o l o g i c a l l y active in the P rats since t h e y exhibit a stimulation o f s p o n t a n e o u s m o t o r activity after given d o s e s o f ethanol that p r o d u c e peak b l o o d alcohol con-
Alcohol elimination rate
Alcohol dehydrogenase
c e n t r a t i o n s in the range o f 16--70 mg p e r c e n t [29]. The P but not the N P rats also s e l f - a d m i n i s t e r e t h a n o l by the intragastric route in a f r e e - c h o i c e situation, w h e n food is available ad lib [27]. T h u s , the p o s t - i n g e s t i v e effects o f e t h a n o l is reinforcing. We h a v e also s h o w n that, with c h r o n i c f r e e - c h o i c e drinking, the P rats d e v e l o p physical dep e n d e n c e as e v i d e n c e d by signs o f w i t h d r a w a l w h e n the alcohol solution is r e m o v e d [28]. The d e v e l o p m e n t o f t o l e r a n c e to the effects o f e t h a n o l is a w e l l - d o c u m e n t e d c o n s e q u e n c e o f c h r o n i c e x p o s u r e and its acquisition with f r e e - c h o i c e drinking is a n e c e s s a r y criterion for an animal model o f a l c o h o l i s m . We have b e e n exploring
~Supported by PHS AA-03243. -'Requests for reprints should be addressed to Dr. Lawrence Lumeng, Departments of Medicine and Biochemistry, Emerson Hall Room 421. 545 Barnhill Drive, Indianapolis, IN 46223.
1013
LUMENG
1014
TABLE COMPOSITION
Protein Bio-Serv-711 (BS-711) Control Kcal/ml %Kcal Ethanol KcalYml %Kcal
A N D LI
1
OF DIETS
Fats
Carbohydrates Ethanol
Total
0.18 18 0.18 18
0.35 35 0.35 35
0.47 47 0,11 11
--0.36 36
1.00 -1.00 --
Supplemented Bio-Serv-711 (SBS-711) Control KcalYml 0.39 %Kcal 32 Ethanol Kcal/ml 0.39 %Kcal 32
0.35 29 0.35 29
0.47 39
--.36 29
1.21
Wayne-Blox solid food (WB) Kcal/g %Kcal
1.04 31
0.36 11
1.99 58
---
3.39 --
Bio-Serv-AlN-76 rAIN) Control Kcal/ml %Kcal Ethanol Kcal/ml %Kcal
0.22 22 0.22 22
0.115 11.5 0.115 11.5
0.665 66.5 0.31 31
--0.355 35.5
1.00 -1.00 --
• 11
10
--
1.21 --
METHOD
Animals
GROWTH CURVES OF P AND WISTAR FEMALE RATS M A I N T A I N E D ON COMMERCIAL SOLID RAT DIETS
F e m a l e W i s t a r and P rats were h o u s e d individually in w i r e - m e s h c a g e s a n d in a c o n t r o l l e d t e m p e r a t u r e a n d humidity e n v i r o n m e n t with fixed light-dark cycles (7 a.m. to 7 p . m . , light and 7 p.m. to 7 a . m . , dark). T h e W i s t a r rats were p u r c h a s e d from H a r l a n I n d u s t r i e s , I n d i a n a p o l i s , IN a n d rats from the P line were from the S-21 g e n e r a t i o n o f the a l c o h o l - p r e f e r r i n g rats s e l e c t i v e l y bred in o u r l a b o r a t o r y , in E x p e r i m e n t 1. the g r o w t h c u r v e s a n d alcohol e l i m i n a t i o n rates ( A E R s ) o f 2 g r o u p s o f P rats a n d a g r o u p of W i s t a r rats ( n - 6 p e r group) were m o n i t o r e d as a f u n c t i o n o f age (26 day old to 180 day old). T h e m e a n age o f the P rats at the start of E x p e r i m e n t 2 was 6 8 + 2 d a y s a n d that at the start o f Experim e n t 3 was 6 0 _+ 1 days.
300
i ~. I ~9
200
:¢
RATS .,=+ c,+u.,+
23
f-l--f']
I 50
WISTAR RATS
I 100
(n=6),
I 150
I 200
AGE,d
FIG. I. Growth curves of a group of female Wistar rats and two groups of female P rats maintained on a commercial solid rat diet (Wayne-Blox). The coefficients of variation ranged from 2 to 7 percent.
w h e t h e r the P rats will d e v e l o p m e t a b o l i c a n d n e u r o n a l t o l e r a n c e to alcohol with c h r o n i c f r e e - c h o i c e drinking. This r e p o r t d e s c r i b e s studies on the d e v e l o p m e n t o f m e t a b o l i c t o l e r a n c e as a result o f c h r o n i c e t h a n o l intake. W e i n c l u d e d as p o s i t i v e c o n t r o l s in t h e s e s t u d i e s , P rats t h a t w e r e forcedfed e t h a n o l i n c o r p o r a t e d into liquid diets. B e c a u s e liquid diets differ in protein c o n t e n t [12], we also e x a m i n e d the effect o f different diet f o r m u l a t i o n s on the d e v e l o p m e n t of m e t a b o l i c t o l e r a n c e a n d on the diurnal v a r i a t i o n of alcohol ingestion a n d b l o o d alcohol c o n c e n t r a t i o n s .
Diets In E x p e r i m e n t 1. the W i s t a r a n d P rats after w e a n i n g were given W a y n e - B l o x food pellets a n d w a t e r ad lib. In E x p e r i m e n t 2, the P rats w e r e divided into 6 g r o u p s ( n = 6 p e r group) a n d g i v e n different feeding r e g i m e n s for 7 w e e k s . T h e first two g r o u p s were pair-fed the c o n t r o l a n d the e t h a n o l Bio-Serv-711 (BS-711) liquid diet (Bio Serv, Inc.+ F r e n c h t o w n , NJ). T h e BS-711 liquid diet was l b r m u l a t e d a c c o r d i n g to the diet c o m p o s i t i o n p u b l i s h e d by DeCarli a n d L e i b e r in 1967 a n d is similar to the L e i b e r - D e C a r l i 1982 regular liquid diet [12]. T h e t h i r d and f o u r t h g r o u p s were also pair-fed but the c o n t r o l a n d e t h a n o l liquid diets were the " s u p p l e m e n t e d B i o - S e r v " diet (SBS-711) d e s c r i b e d by Rogers et al. [22]. T h e s u p p l e m e n t a t i o n c o n s i s t e d o f a d d i n g v i t a m i n - f r e e casein a n d zinc to the Bio-Serv-711 diet (Table I). T h e fifth g r o u p was given W a y n e - B l o x food pellets (WB) and w a t e r ad lib, a n d the sixth g r o u p was g i v e n ad lib W a y n e - B l o x food pellet a n d the f r e e - c h o i c e d r i n k i n g of 10 p e r c e n t (v/v) e t h a n o l and water. T h e a n i m a l s g i v e n the liquid
M E T A B O L I C T O L E R A N C E IN T H E P R A T S
1015
A L C O H O L E L I M I N A T I O N R A T E OF P A N D V I I S T A R F E M A L E R A T S AS A F U N C T I O N OF ,AGE
/ /
~2
I /
,
**
,
> rn
g
2.0
-10
o
R
5 2_
-i =
/
1.5
Z
E E
E
~ )
P RATS
(n=6/cJroup)
I-I--I'-I
WISTAR RATS
:i:
7
~ ©
< (n=6)
~.0
6
I
0
50
100
150
200 0
5Q
lO0
200
I50
AGE,d
FIG. 2. Change in alcohol elimination rate (AER) of the P and Wistar rats as a function of age. AERs were expressed as either mmol hr ' per rat (left panel) or mmol hr ' per kg body weight (right panel). The coefficients of variation ranged from 1 to 11 percent. Values marked with an asterisk are not different from each other and are statistically different from those without an asterisk.
TABLE 2 MEAN
DAILY
CALORIC
INTAKE
IN P RATS GIVEN
THE
DIFFERENT
DIETARY
REGIMENS
IN EXPERIMENT
2
Mean daily caloric intake per rat, Kcal/day Week 1
BS-711 (C) BS-711 (E) SBS-711 (C) SBS-711 (E) WB (C) WB (Free-choice)
Week 3-4
Week 7
Total
Pr
Ethanol
Total
Pr
Ethanol
Total
Pr
Ethanol
50.0* 51.6" 59.2 60.6 59.3 59.2
9.0* 9.3* 19.2 19.6 18.8 14.2
-18.6 -17.7 -14.4t
51.7" 51.7" 59.4 59.4 60.2 60.4
9.3* 9.3* 19.2 19.2 19.1 14.2
-18.6 -17.3 -16.4
58.6 59.2 61.3 61.2 58.5 59.4
10.6" 10.6" 20.2 19.8 17.0 14.0
-21.3 -17.9¢ -15.1"
*Significantly different from the SBS-711 (C), SBS-711 (E), WB (C) and WB (Free-choice) diets (by analysis of variance). tSignificantly different from the BS-711 (E) diets (by analysis of variance). Abbreviations: Pr, protein; C, control; E, ethanol.
diets w e r e a c c l i m a t e d to i n c r e a s i n g c o n c e n t r a t i o n s o f e t h a n o l in the diet in the m a n n e r s u g g e s t e d by the m a n u f a c t u r e r . In E x p e r i m e n t 3, the P rats w e r e s e p a r a t e d into four g r o u p s ( n = 5 per group). The first t w o g r o u p s w e r e pair-fed the c o n t r o l and the e t h a n o l - A I N liquid diet (Bio-Serv A I N - 7 6 diet) [16,21]. T h e third g r o u p was fed p o w d e r e d W a y n e - B l o x solid food and a s u c r o s e solution, while the fourth g r o u p was given the p o w d e r e d W a y n e - B l o x solid f o o d , w a t e r and 10 p e r c e n t e t h a n o l . The third and fourth g r o u p s o f rats w e r e pair-fed by providing to the c o n t r o l rats (the third group) the a m o u n t o f solid food and an a m o u n t o f the s u c r o s e solution
that is isocaloric to the a m o u n t o f I0 p e r c e n t e t h a n o l cons u m e d by the rats on f r e e - c h o i c e drinking (the fourth group).
Dcterntination o f AIchol Elimimttion Rate (AER) in Viro E t h a n o l (2 g/kg) was a d m i n i s t e r e d by the i n t r a p e r i t o n e a l injection o f a 10 p e r c e n t (v/v) solution. Tail blood s a m p l e s w e r e c o l l e c t e d as a f u n c t i o n o f time after e t h a n o l injection and the blood e t h a n o l c o n c e n t r a t i o n s w e r e m e a s u r e d by a head s p a c e m e t h o d with a gas-liquid c h r o m a t o g r a p h [13].
1016
LUMENG
A N D LI
TABLE 3 EFFECT OF DIETARY REGIMENS ON ETHANOL INTAKE IN EXPERIMENT 2 Ethanol Intake, g/kg/day Diet BS-711(E) SBS-711 (E) WB (Freechoice)
Week 1
2
3
4
10.8-+ 0.5 11.7_+0.4 8.5 ± 0.6*
13.6 ± 1.3 10.3-+0.3" 8.3 -+ 0.3*
12.5 ± 0.3 10.0+0.3" 9.3 _+ 0.7*
12.1 ± 0.4 9.8_+0.2 * 7.2 _+ 0.7*
5
6
7
12.8 + 0.4 9.8-+0.2* 7.8 ± 0.4*
12.9 ± 0.4 10.4-+0.3" 11.5 + 0.7
13.2 ± 0.2 9.5-+0.3* 8.4 ± 0.3*
*Significantly different from the BS-711 (E) group (by analysis of variance). Abbreviations: E, ethanol.
TABLE 4 EFFECT OF DIFFERENT DIETARY REGIMENS ON BODY WEIGHT AND ALCOHOL EI.IMINATION RATE IN EXPERIMENT 2 Alcohol Elimination Rate Body Weight g Diet BS-711 (C)
Before
7th Week
205 + 10
204 "+ 7
mmol/hr/rat Before -
1.90 ± .12
L BS-711 (E)
208 ±
3
7th Week
Before
1.66 -+ .051
I
2.07 ± .04
mmol/hr/kg
J
2.02 + .06
9.25 + .39
209 -+ 7
SBS-711 (E)
204 ±
6
6.95 _+ .28-I
l 9.97 ± .31
I SBS-711 (C)
7th Week
I 9.08 ± .18
J
•
262 +
,.97+.06[
1'72-+'0511
9"50+'481
6"60-+283l
~-270 + 8
1.92 + .07
2.22 + .09
9.59 + .32
8.25 _+ .26
2.03 -+ .05
2.10-+.133
9.71"+.28 I
7 "I 5 9 + ' 5 0 3
WB (C)
208 ±
6
263 "+ 4
WB (Free-Choice)
227 ± 12
274 ± 8
I
--
J
I
I
2.11,1 ± .08
2.50 + .11 9.36 "+ .17 9.14 + .27 -.J Brackets indicate that the compared values are significantly different. Body weight differences were analyzed by analysis of variance. Alcohol elimination rate differences were analyzed by two-tailed t-test. Abbreviations: C, control: E, ethanol. T h e rate o f e t h a n o l d i s a p p e a r a n c e was p s e u d o - z e r o o r d e r , a n d the A E R was c a l c u l a t e d using W i d m a r k ' s e q u a t i o n [6].
2, a n a l y s i s of v a r i a n c e a n d p o s t - h o c N e w m a n - K e u l s t e s t s w e r e used to e v a l u a t e statistical differences.
Measurement of Liver Alcohol Dehydrogenase (ADH) Activity L i v e r h o m o g e n a t e - s u p e r n a t a n t f r a c t i o n s were p r e p a r e d [13] a n d alcohol d e h y d r o g e n a s e ( A D H ) activity was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y b y m e a s u r i n g the r e d u c t i o n o f N A D + to N A D H . T h e a s s a y b u f f e r c o n t a i n e d 0.5 M Tris-HC1 (pH 7.2 a n d I - 0 . 2 ) , 2.8 m M N A D ~, a n d 5 m M e t h a n o l , a n d t h e t e m p e r a t u r e was m a i n t a i n e d at 37 ° [14]. T h e s e s u b s t r a t e c o n c e n t r a t i o n s s a t u r a t e t h e e n z y m e 88 p e r c e n t ; t h e r e f o r e , the m e a s u r e d e n z y m e a c t i v i t y w a s multiplied b y 1.14 to calculate the m a x i m u m A D H v e l o c i t y (or V m a x ) [14].
Statistical Analysis R e s u l t s are p r e s e n t e d as m e a n s _ + S E M . T h e two-tailed s t u d e n t ' s t-test for u n p a i r e d data was used for statistical c o m p a r i s o n s . D i f f e r e n c e s were c o n s i d e r e d to be significant w h e n the p values was less than 0.05. W h e r e results a m o n g different e x p e r i m e n t a l g r o u p s were c o m p a r e d in E x p e r i m e n t
RESULTS
Experime/lt I In p r e v i o u s studies o f m e t a b o l i c t o l e r a n c e [3, 10, 11, 15, 20, 24, 26], rats w e r e pair-fed c o n t r o l and e t h a n o l - c o n t a i n i n g diets for s e v e r a l w e e k s , and the A E R s of the t w o g r o u p s w e r e t h e n c o m p a r e d with the results e x p r e s s e d in most studies as m m o l o f e t h a n o l m e t a b o l i z e d p e r h o u r per kg body weight. In o t h e r studies, only the d i s a p p e a r a n c e rates of e t h a n o l in p l a s m a (mmol/100 ml p e r hr) w e r e c o m p a r e d . In all o f t h e s e studies, no a t t e m p t s were m a d e to d i s c e r n if e t h a n o l ingestion h a d actually i n c r e a s e d the A E R w h e n c o m p a r e d with t h a t before e t h a n o l e x p o s u r e in the same animals. T o set the stage for E x p e r i m e n t s 2 a n d 3, we d e t e r m i n e d in E x p e r i m e n t I the g r o w t h c u r v e of P f e m a l e rats and the c h a n g e in A E R as a f u n c t i o n of age. T h e p u r p o s e was to find the m o s t a p p r o p r i a t e r e f e r e n c e unit to e x p r e s s A E R s for c o m p a r i s o n of data. Since the P line of rats was originally
M E T A B O L I C T O L E R A N C E IN T H E P RATS
1017
TABLE 5 240
EFFECT OF DIFFERENT DIETARY REGIMENS ON HEPATIC ALCOHOL DEHYDROGENASE ACTIVITY IN EXPERIMENT 2
•
220
Diet
Alcohol Dehydrogenase Activity (mmol hr ~liver)
200
:"
•
180
BS-711 (C) BS-711 (E) SBS-711 (C) SBS-711 (E) WB(C) WB (Free-Choice)
2.88 2.41 2.95 3.24 3.18 2.93
+_ 0.18 +_ 0.09 +_ 0.13 +_ 0.16 _+ 0.15 _+ 0.12
z 0
140
0Z
120
o 0
derived from outbred Wistar rats [7], we also measured these parameters in a group of Wistar female rats. Figure 1 indicates that the growth curves of the P and Wistar female rats were similar. Figure 2 depicts the change in A E R as a function of age in the P and Wistar female rats, when A E R was expressed either as mmol of ethanol eliminated per hour per rat (left panel) or as mmol of ethanol eliminated per hour per kg body weight (right panel). When expressed as rate per rat+ A E R increased rapidly from 26days of age to about 60-days and it remained constant thereafter, By comparison, A E R expressed as rate per kg body weight declined sigmoidally with age and it reached a constant low only when the rats were about 120- to 180-days old. The patterns of change were similar in the P and Wistar female rats, with the curves for the Wistar rats slightly displaced to the right.
Experiment 2 In this experiment, the P rats were given different dietary regimens: groups 1 and 2 were pair-fed the ethanol and control BS-711 diet; groups 3 and 4 were pair-fed the ethanol and control SBS-711 diet: group 5 was given solid food and water ad lib and group 6 was given the solid food ad lib and the free-choice of 10 percent (v/v) ethanol and water. Table 2 summarizes the mean daily caloric intake of the P rats receiving the different dietary regimens. In the first 4 weeks of feeding, the daily caloric intake of the rats placed on the BS-711 diets was about 15 percent lower than the other groups. More importantly, owing to the low protein content of the BS-711 diets, the protein caloric intake throughout the 7-week period was only one-half of that consumed by the other groups. The daily caloric intake as ethanol in the SBS711 group tended (not statistically significant) to be lower than that in the BS-711 group. By comparison, calorie consumption as ethanol in the free-choice group was clearly lower (12 to 29 percent) than that in the BS-711 group (at least for weeks 1 and 7). Table 3 shows the effect of the different dietary regimens on ethanol intake expressed as g of ethanol kg ; per day. Partly owing to the higher daily ethanol intake per rat (cf. Table 2) and to the lesser weight gain of the rats on the BS-711 diet (Table 4), ethanol intake expressed as g kg ' per day was the highest in the BS-711 group. Table 4 shows the effect of the different dietary regimens on body weight and AER. Rats on the BS-711 diet failed to gain weight in the 7-week study. By comparison, rats in all the other groups gained at least 40 g over this period. AERs were expressed both as mmo! hr ~per rat and mmol hr ; per
160
< rr ~z ua
I 0 0 "< 0 0
IO0
..
~. ~
--.L
:
•
I °
"i
8O 60
:
"I
4O
2o
i
.*
o• .°
--T-
.%
•
o
0 2P
10 e
BS-711(E)
6A
2P
10 P
SB$-711(E)
10 P
6A
WB(FC)
FIG. 3. Blood alcohol concentrations of P rats placed on three different dietary regimens. Horizontal bar indicates the mean value. BS-711(E), SBS-711(E) and WB(FC) are abbreviations for the ethanoI-Bio-Serv-711 diet, the supplemented ethanoI-Bio-Serv-711 diet and the Wayne-Blox free-choice drinking regimen.
kg body weight. Before initiation of the feeding experiment, the mean AERs of the 6 groups of rats were similar regardless of the manner of expressing the AERs: the mean AERs varied from 1.90 to 2.11 mmol hr ; per rat and from 9.25 and 9.71 nmol hr ' per kg body weight. By the end of the 7-week experiment, the AERs of all the groups treated with alcohol were 19 to 29 percent higher than their respective controls indicating that chronic alcohol ingestion, regardless of how ethanol was consumed, produced metabolic tolerance in the P rats. However, a real increase in A ER over the 7-week experiment was evident in only 2 groups of animals, when AERs were expressed as mmol hr ' per rat, i.e., those placed on the ethanoI-SBS-711 diet and those given solid food and the free-choice drinking of 10 percent ethanol versus water. Except for the group of rats given solid food and free-choice drinking, all other groups exhibited an actual, net decline in A E R at the end of the 7-week period when A E R was calculated as mmol h r ' per kg body weight. These data, therefore, demonstrate that: (1) The P rats given solid food and the free-choice drinking of 10 percent ethanol and water develop metabolic tolerance; (2) the BS-711 diet, unless supplemented with protein, will not produce a net increase in A E R in ethanol-fed animals; and (3) interpretation of data with AERs expressed as mmol of ethanol eliminated per hr per rat is less complicated than when AERs are expressed as mmol hr ' per kg body weight. This is due to the fact that, in female Wistar and P rats, the gain in body weight and the increase in A E R as they mature from 50 to 200 days of age follow different time courses. Table 5 summarizes the effect of the different dietary regimens on A D H activity in liver. In agreement with the bulk of the literature [2, 9-11, 24, 26], chronic alcohol treatment did not lead to a discernible increase in the total ADH activity.
1018
L U M E N G A N D LI TABLE 6 MEAN DAILY CALORIC INTAKE 1N THE P RATS FORCED-FED ALCOHOL BY THE AIN DIET OR GIVEN SOLID FOOD AND THE FREE-CHOICE DRINKING OF ALCOHOL Mean daily caloric intakes per rat Week 1
Total AIN (C) AIN (E) WB (C) WB (Free-choice, water vs. 10 percent E)
Week 2
Week 4
Week 6
Pr
E
Total
Pr
E
Total
Pr
E
Total
Pr
E
53.3 54.1 59.4 61.2
14.9 15.1 14.6 15.2
19.2 -13.3'
52.2 53.9 54.5 58.9
14.6 15.1 12.7" 14.1
-19.1 -14.3"
54.2 54.1 57.8 64.0
15.2 15.1 12.7" 14.7
-19.2 -17.7"
48.1 48.3 46.7 49.1
13.5 13.5 10.4"
17.1
11.4"
14.6"
Abbreviations: C, control; E, ethanol; Pr, protein. *Significantly different from either the AIN (C) or the AIN (E) diet (by analysis of variance).
220
TABLE 7
oo
EFFECT OF ETHANOL GIVEN IN THE AIN LIQUID DIET AND BY FREE-CHOICE ON BODY WEIGHT AND ALCOHOl, ELIMINATION RATE (EXPERIMENT 3) Body Weight g Diet
Before
o3 E z 0
Alcohol Elimination Rate mmol/hr/rat
6th Week
Before
o~ Fz
6th Week
AINC
78+6 230+i]78009 7+0091
AIN (E)
176 -+ 6
1.74 + 0.08 2.09 + 0.07 .3
I WB (C)
166 -+ 5
2t-4 +
WB(Free-choice)
166 _+ 5
239 + 4
I
1.67 + 0.09 2.03 _+ 0.05-'1
I
I
/
Z © U .J
© 2£ 0
160
I
Brackets indicate that the compared values are significantly different. Abbreviations: C, control; E, ethanol.
14o 120 I00
80 6o
_J
<
40
1.68 + 0.10 2.36 + 0,08 .3
I
180
I-<
uJ
246 +
200
o o
20 0
I: 2P
• gt •
IO P
AIN(E)
Figure 3 c o m p a r e s the b l o o d alcohol c o n c e n t r a t i o n s o f the P rats r e c e i v i n g alcohol with the different dietary r e g i m e n s . Only t h o s e on the ethanol-BS-711 diet e x h i b i t e d p e r s i s t e n t l y e l e v a t e d b l o o d alcohol c o n c e n t r a t i o n s w h e n m e a s u r e d at 6 a . m . , 2 p.m. and l0 p.m. The animals given the higher protein diets, i.e., the SBS-711 or the WB diet, e x h i b i t e d m u c h l o w e r b l o o d alcohol c o n c e n t r a t i o n s and m a i n t a i n e d a diurnal cycling o f blood alcohol c o n c e n t r a t i o n s . In P rats given f r e e - c h o i c e drinking o f alcohol, b l o o d alcohol c o n c e n t r a t i o n s ranged f r o m 5 mg p e r c e n t to as high as 137 mg perc e n t during the dark cycle at the times o f m e a s u r e m e n t .
L:vperimenl 3 The A I N liquid diet is a f o r m u l a t i o n that has a higher p r o t e i n c o n t e n t than the BS-711 diet (Table 1). Table 6 c o m p a r e s the m e a n total daily caloric intake p e r rat and the m e a n c o n t r i b u t i o n o f p r o t e i n and ethanol to the total daily caloric intake. With f o r c e d feeding o f ethanol (i.e., the A I N liquid diet with 6 p e r c e n t ethanol), the daily intake o f e t h a n o l per rat w a s initially 44 p e r c e n t ( w e e k 1) and t h e n 8 and 17 per-
6A
2p
10P
6A
WB (FC)
FIG. 4. Blood alcohol concentrations of P rats placed on the ethanol-AIN diet and the Wayne-Blox free-choice drinking regimen.
cent ( w e e k s 4 and 6) higher than that with f l e e - c h o i c e drinking o f ethanol (i.e., WB and f l e e - c h o i c e drinking o f 10 percent ethanol vs. water). W h e n the alcohol intake was exp r e s s e d as g/kg/day, f o r c e d - f e e d i n g o f ethanol as part of the A I N liquid diet also led to initially 40 p e r c e n t ( w e e k I) and then 15 p e r c e n t (week 6) higher alcohol intake than that with f l e e - c h o i c e drinking. The daily alcohol intake o f the P rats on the e t h a n o l - A I N liquid diet and t h o s e on f l e e - c h o i c e alcohol intake ranged from 10.1 to 13.8 g/kg/day and from 8.8 to 11.5 g/kg/day, r e s p e c t i v e l y . Table 7 s h o w s the a m o u n t o f weight gain and the c h a n g e in A E R s as a function of time in the P rats pair-fed the AIN liquid diets (with or without ethanol) and in t h o s e pair-fed the W a y n e - B l o x solid l a b o r a t o r y food and 10 p e r c e n t ethanol or an isocaloric a m o u n t o f a s u c r o s e solution. Weight gain o f 29 to 47 p e r c e n t was e v i d e n t in all the g r o u p s o v e r the 6-week period. A E R s , e x p r e s s e d as
M E T A B O L I C T O L E R A N C E IN T H E P RATS mmol hr ~ per rat, increased over the 6-week period in the AIN(E), WB(C) and the WB (free-choice) groups. Metabolic tolerance was demonstrated in the P rats given ethanol either by forced-feeding or by free-choice. The mean increases in AERs when the P rats were forced-fed and when they were given alcohol by free-choice were 22 and 16 percent, respectively. Figure 4 depicts the blood alcohol concentrations of the P rats when they were on the ethanol-AIN liquid diet and when they were given ethanol by free-choice. Both groups exhibited diurnal cycling of alcohol consumption, which was reflected in the diurnal variations of blood alcohol concentrations. With free-choice drinking, the blood alcohol concentrations in the P rats ranged from 17 to 127 mg percent during the dark cycle and they were similar to those of the P rats given the ethanoI-AIN diet. DISCUSSION Recent studies [7, 19, 27] of the selectively-bred Pand NP rats indicate that the P line of rats satisfies almost all of the perceived requirements for an animal model of alcoholism and that the P and NP lines are relevant models to study the biochemical, physiological and behavioral concomitants of alcohol drinking behavior. The data presented above clearly show the development of metabolic tolerance in the P rats following the free-choice drinking of ethanol for 6 to 7 weeks. In two separate experiments (Tables 4 and 7), AERs, expressed as mmol/hr/rat, increased 18-40 percent when compared with values before alcohol exposure and increased 16-20 percent when compared with values from the pair-fed and ad lib controls. In one experiment, the controls were free-fed, whereas in the other, the controls were pair-fed an amount of sucrose solution that is isocaloric to the amount of ethanol consumed by the experiment group. These findings are not unexpected because Hawkins ~'t a/. [3] have reported earlier that chronic alcohol ingestion of 8 g/kg/day can produce metabolic tolerance in vA'o in the rats. Additionally, Videla c t a / . [251 have reported that liver slices from rats exposed to alcohol in the range of 3 to 8 g/kg/day exhibited enhanced alcohol metabolic rates in vitro. Since the female P rats consumed 7-12 g of ethanol/kg/day during free-choice drinking (Tables 3 and 7), metabolic tolenmce should have developed and it did. The biochemical basis of the development metabolic tolerance in rats remains controversial. An increase in ethanol oxidation rate mediated by induction of the microsomal ethanol-oxidizing system (MEOS) has been proposed as one mechanism [8,9]. An increase in ethanol oxidation catalyzed by the ADH pathway has also been advocated as another [5. 15, 20, 23]. Although some investigators [1,17] have reported an increase in the actual amotmt of ADH in liver when rats were exposed to alcohol chronically, most [2, 9-1 I, 24, 26] have found no change. No change was observed in the P rats in this study (Table 5). At the present, an increase in ethanol oxidation mediated by the ADH pathway after chronic exposure to alcohol is best explained by enhanced turnover of N A D H . The latter can arise from increased rate of mitochondrial oxidation of N A D H and/or from an increased rate of transhydrogenation and N A D P H turnover due to induction of MEOS. In most of the previous studies of metabolic tolerance in
1019 rats, alcohol elimination was compared using simply plasma ethanol disappearance rates (mg ethanol/100 ml plasma per hr) or AERs calculated and expressed as mmol hr ' per kg body weight [3, 10, 11, 15, 20, 24, 26]. Plasma ethanol disappearance rate is not a good basis for comparison because it does not take into account changes in the volume of distribution of ethanol. Expressing the A E R as mmol of ethanol eliminated hr ~per kg is probably also less than ideal. This is because the ethanol oxidizing capacity located primarily in the liver may bear no relationship or at best, a complex relationship, with body weight. As shown by Fig. 2, when AERs expressed as mmol hr ~ per kg are plotted as a function of age, they decreased sigmoidally and became constant only at 120 to 180 days of age. By comparison, when AERs are expressed as mmol hr ~per rat, the relationship of AERs with age increases curvilinearly and becomes constant in early adulthood. Thus, as long as the rats at the beginning of the experiment are of the same age and body weight, the effect of any variable on AERs as a function of time can be more confidently interpreted if AERs are calculated as mmol hr ~ per rat. Of note, Israel e t a / . [4] have reported earlier a sigmoidal decline in AERs with growth, calculated as mmol hr ~ per kg body weight, in male Spontaneous Hypertensive and male Wistar rats. The data shown here in Fig. 2 indicates that a similar curve obtains in female Wistar and P rats. Previous studies on metabolic tolerance in rats also have not compared AERs before and after alcohol exposure. The present study examined specifically this relationship and assessed the effect of different dietary regimens. The development of metabolic tolerance to alcohol could be demonstrated with the feeding alcohol with any of the dietary regimens used by comparing the AERs of alcohol-fed rats with their concomitant controls at the end of the feeding experiment. However, a net increase through comparison of AERs before and after alcohol exposure was evident only in the P rats fed the alcohol-containing SBS-711 and AIN diets and in those given the solid food and alcohol by free-choice drinking (Tables 4 and 7). The lack of net increase in AER as a function time in the P rats fed the ethanol containing BS-711 diet is best explained by the low protein content of this diet (Table 2). The low dietary protein content is also most likely responsible for the disruption of the normal diurnal cycling of alcohol ingestion and the persistently elevated blood alcohol concentrations in the P rats given alcohol in the BS-711 diet (Fig. 3). With all the other dietary regimens wherein the protein intake was higher, food and alcohol consumption followed a diurnal rhythm. Accordingly, blood ethanol concentrations also cycled in a diurnal manner, i.e., they were low during the light cycle and high in the dark cycle. These results are in accord with those reported earlier by Rogers ¢,t a/. [22]. Since the P rats developed metabolic tolerance when they were fed alcohol with diets which did not produce persistently elevated blood alcohol concentrations, it is clear the persistent intoxication is not a necessary condition to metabolic tolerance development.
ACKNOWLEDGEMENTS The skillful technical assistance of Mary Beth Welsh is gratefully appreciated. We also thank Miss Brenda Stewart fl)r typing the manuscript.
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