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Effects of low doses of alcohol on delta-9-tetrahydrocannabinol's effects in pregnant rats
Life Sciences, Vol. 47, pp. 1677-1682 Printed in the U.S.A.
EFFECTS
OF L O W DOSES
Pergamon Press
OF A L C O H O L ON D E L T A - 9 - T E T R A H Y D R O C A N N A B I N O L ' S E F F E C T S IN P R E G N A N T RATS
E.L.
Abel
and M.G.
Subramanian
Fetal A l c o h o l R e s e a r c h C e n t e r and D e p a r t m e n t of O b s t e t r i c s and Gynecology, W a y n e State University, C.S. M o t t C e n t e r for H u m a n G r o w t h and Development, 275 E. Hancock, Detroit, M i c h i g a n 48201. (Received in final form August 28, 1990) Summary P r e g n a n t rats w e r e i n t u b a t e d w i t h 50 m g / k g of d e l t a 9-tetrahydrocannabinol (THC) or w i t h T H C p l u s a l c o h o l to determine if a low dose of alcohol (i g/kg) would s i g n i f i c a n t l y increase b l o o d levels of THC. On the b a s i s of t h i s study, a s e c o n d study was c o n d u c t e d in w h i c h p r e g n a n t rats w e r e i n t u b a t e d w i t h THC (50, 25 or 0 mg/kg) p l u s a l c o h o l (2, 1 or 0 g/kg) from g e s t a t i o n d a y six to parturition. THC r e d u c e d b i r t h w e i g h t s b u t did not s i g n i f i c a n t l y a f f e c t litter size or p a s s i v e a v o i d a n c e learning. A l c o h o l did not h a v e a s i g n i f i c a n t e f f e c t on o f f s p r i n g b i r t h w e i g h t nor did it i n t e r a c t w i t h T H C to a f f e c t offspring. M a n y w o m e n w h o smoke m a r i j u a n a d u r i n g p r e g n a n c y a l s o u s e alcohol and o t h e r d r u g s (i-4). We h a v e p r e v i o u s l y shown that c o n c o m i t a n t treatment during pregnancy with delta-9-tetrahydrocannabinol (THC), the p s y c h o a c t i v e i n g r e d i e n t in marijuana, a n d alcohol can d r a m a t i c a l l y i n c r e a s e THC's e f f e c t s on f e t o t o x i c i t y in a n i m a l s (56). T h i s is in c o n t r a s t to p h e n o b a r b i t a l (7) and c h l o r d i a z e p o x i d e (8) w h i c h do not p o t e n t i a t e THC's fetotoxicity. In the p r e s e n t study, we j o i n t l y a d m i n i s t e r e d lower doses of T H C t h a n t h o s e we p r e v i o u s l y e v a l u a t e d for f e t o t o x i c e f f e c t s and d o s e s of a l c o h o l t h a t we h a v e p r e v i o u s l y found to have m i n i m a l e f f e c t s on o f f s p r i n g b i r t h w e i g h t (9-10), and we a l l o w e d a n i m a l s to d e l i v e r t h e i r y o u n g so t h a t we c o u l d assess the p o s s i b i l i t y t h a t alcohol p o t e n t i a t e s the e f f e c t s of THC on b i r t h weight, p o s t n a t a l g r o w t h or behavior. Our first e x p e r i m e n t d e t e r m i n e d if a low d o s e of a l c o h o l w o u l d s i g n i f i c a n t l y i n c r e a s e b l o o d THC levels and w h e n p e a k b l o o d THC levels occurred. On the basis of these data, we a d m i n i s t e r e d our a l c o h o l d o s e s so t h a t alcohol w o u l d be p r e s e n t w h e n T H C levels w e r e at t h e i r peak. Materials
and M e t h o d s
N u l l i p a r o u s L o n g - E v a n s H o o d e d rats (Charles River, Portage, MI) a p p r o x i m a t e l y t h r e e m o n t h s of age w e r e b r e d in our l a b o r a t o r y to t h r e e - m o n t h old males. P r e g n a n t female rats w e r e i n t u b a t e d w i t h 50 m g / k g T H C and 1 g / k g alcohol (N=I0) or w i t h 50 m g / k g T H C and an equal v o l u m e of d i s t i l l e d w a t e r (N=I0). T h e T H C w a s p r o v i d e d by the N a t i o n a l I n s t i t u t e on Drug A b u s e (NIDA). Tail tip b l o o d s a m p l e s (200 ul) w e r e c o l l e c t e d at one, two, four and six hours 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
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a f t e r i n t u b a t i o n on g e s t a t i o n day 20. P l a s m a w a s s e p a r a t e d and frozen until s u b s e q u e n t a n a l y s i s of T H C and the l l - n o r - 9 - c a r b o x y l THC m e t a b o l i t e , u s i n g r a d i o i m m u n o a s s a y (RIA). The R I A kits w e r e o b t a i n e d from R e s e a r c h T r i a n g l e Institute, N.C. The a s s a y p r o c e d u r e s f o l l o w e d i n s t r u c t i o n s d e s c r i b e d for the k i t s and all samples were assayed in duplicate. Standard and experimental plasma samples were extracted with methanol. Aliquots of the supernatants were incubated with antiserum s p e c i f i c for T H C and THC-COOH. T h e a s s a y tubes w e r e i n c u b a t e d at 4°C overnight. P r e c i p i t a t i n g i m m u n o b e a d - b u f f e r s o l u t i o n was a d d e d to a s s a y t u b e s and vortexed. F o l l o w i n g an a d d i t i o n a l t h r e e h o u r i n c u b a t i o n at 4°C, the tubes w e r e c e n t r i f u g e d and s u p e r n a t a n t s w e r e discarded. The p r e c i p i t a t e in the t u b e was w a s h e d w i t h cold buffer, c e n t r i f u g e d and s u p e r n a t a n t p o u r e d off. The precipitate in the t u b e w a s c o u n t e d for r a d i o a c t i v i t y in an a u t o m a t i c gamma c o u n t e r (Model 28062, M i c r o m e d i c Systems, Inc.) and s e r u m levels of c a n n a b i n o l s w e r e calculated. C o n t r o l p l a s m a p o o l s (two from THC and t h r e e for THC-COOH) p r o v i d e d w i t h kits, w e r e a l s o c a r r i e d out in d u p l i c a t e s in each assay. T r i p l i c a t e tubes to e s t i m a t e the n o n s p e c i f i c a l l y p r e c i p i t a t e d r a d i o a c t i v i t y w e r e a l s o i n c l u d e d in e a c h assay. W i t h i n and b e t w e e n a s s a y v a r i a b i l i t y , e x p r e s s e d as c o e f f i c i e n t of variation, was less t h a n 12% for the two assays. B a s e d on the d a t a for this study, we c o n d u c t e d our s e c o n d study to e v a l u a t e p o t e n t i a l b i o l o g i c a l s y n e r g i s m b e t w e e n T H C and alcohol. On the m o r n i n g of v a g i n a l p l u g i d e n t i f i c a t i o n (gestation day 0), p r e g n a n t f e m a l e s w e r e d i v i d e d and a s s i g n e d to one of t h r e e d r u g t r e a t m e n t g r o u p s and w e r e i n d i v i d u a l l y h o u s e d in P l e x i g l a s cages. A n i m a l s in e a c h g r o u p w e r e i n t u b a t e d once p e r day w i t h T H C (50, 25 or 0 mg/kg) d i s s o l v e d in s e s a m e oil b e g i n n i n g on g e s t a t i o n day six. E a c h of t h e s e m a i n g r o u p s was s u b d i v i d e d such t h a t a n i m a l s in e a c h s u b g r o u p w e r e a l s o i n t u b a t e d w i t h e i t h e r 2, 1 or 0 g / k g of a l c o h o l (in d i s t i l l e d water). T h e s e d o s e s of a l c o h o l w e r e c h o s e n b e c a u s e t h e y h a v e no s i g n i f i c a n t e f f e c t s on b i r t h w e i g h t or p o s t n a t a l w e i g h t in rats (9-10). A n i m a l s in the two lower d o s e alcohol g r o u p s h a d t h e i r alcohol d i s s o l v e d in a m a l t o s e d e x t r i n s o l u t i o n i s o c a l o r i c to the g r o u p r e c e i v i n g the 2 g / k g alcohol dose. A n i m a l s w e r e i n t u b a t e d w i t h THC at a p p r o x i m a t e l y 9:00 a.m. and t h e n t h r e e h o u r s later w i t h alcohol. The d e l a y was b a s e d on r e s u l t s from our initial s t u d y s h o w i n g t h a t p e a k c o n c e n t r a t i o n s o c c u r r e d a r o u n d 4 hrs a f t e r intubation. B e c a u s e of t h i s slow absorption, we d e l a y e d a l c o h o l a d m i n i s t r a t i o n so t h a t w h e n it w a s administered, THC levels in the b l o o d w o u l d be at or n e a r p e a k concentrations. For p u r p o s e s of p a i r - f e e d i n g , the nine e x p e r i m e n t a l g r o u p s w e r e d i v i d e d into t r i a d s such that a n i m a l s in each of the T H C s u b g r o u p s w e r e p a i r - f e d to the s u b g r o u p r e c e i v i n g the h i g h e s t a l c o h o l dose. W i t h i n t w e l v e h o u r s a f t e r birth, p u p s in each litter w e r e counted, weighed, culled to e i g h t per litter and r e t u r n e d to t h e i r b i o l o g i c a l mothers. Animals (N=II-20 p e r sex p e r group) w e r e t e s t e d for p a s s i v e a v o i d a n c e at s e v e n t e e n days of age. No m o r e t h a n two m a l e s or females from each l i t t e r w e r e evaluated. Animals were weaned at t w e n t y - o n e d a y s of age. Due to a miscommunication, a n i m a l s w e r e not w e i g h e d at weaning. Instead, a n i m a l s w e r e w e i g h e d at t h i r t y d a y s of age.
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passive Avoidance. The test apparatus was a Plexiglas rectangular c h a m b e r (35 x 22 x 16 cm) d i v i d e d i n t o t w o e q u a l s e c t i o n s b y a g u i l l o t i n e door. O n e side of t h e c h a m b e r w a s c o v e r e d b y b l a c k c o l o r b o a r d , t h e o t h e r w a s clear. B o t h s i d e s h a d a g r i d f l o o r of s t a i n l e s s steel bars. A Coulbourn solid state shock generator d e l i v e r e d a 0.8 m A p u l s e of s h o c k for 0.5 seconds. T h i s t e s t and t h e age of t e s t i n g w e r e u s e d b e c a u s e p r e v i o u s r e s u l t s h a d s h o w n it s e n s i t i v e to a l c o h o l ' s in u t e r o a c t i o n s (ii). A n i m a l s w e r e r e m o v e d from t h e i r h o m e c a g e and, t h i r t y s e c o n d s later, p l a c e d in t h e c l e a r s i d e of the c h a m b e r f a c i n g a w a y from t h e d o o r w h i c h w a s a l r e a d y raised. A timer was started when a n i m a l s w e r e p l a c e d in t h e chamber. W h e n a n i m a l s s t e p p e d into the b l a c k c h a m b e r w i t h all four feet, the t i m e r w a s stopped, t h e d o o r w a s closed, a n d t h e s h o c k w a s d e l i v e r e d . Animals were removed f r o m t h e c h a m b e r a n d r e t u r n e d to t h e h o l d i n g c a g e for t h i r t y s e c o n d s a f t e r shock. This procedure was continued until criterion or e i g h t t r i a l s w e r e reached. The c r i t e r i o n w a s n o n e n t r y into the s h o c k c h a m b e r for t w o c o n s e c u t i v e 180 s e c o n d s trials. Data Analysis. T h e d a t a w e r e e x a m i n e d b y a n a l y s i s of v a r i a n c e (ANOVA). In i n s t a n c e s w h e r e all p u p s in a l i t t e r w e r e examined, e.g., b i r t h weight, t h e l i t t e r a n d not t h e i n d i v i d u a l p u p w a s u s e d as a d a t u m b e c a u s e m e a s u r e s of i n d i v i d u a l p u p s w i t h i n l i t t e r s are l i k e l y to b e c o r r e l a t e d . Results B l o o d c a n n a b i n o i d l e v e l s are s h o w n in T a b l e i. P e a k l e v e l s of THC were recorded at four hours after intubation. Alcohol s i g n i f i c a n t l y i n c r e a s e d T H C l e v e l s in b l o o d b y four a n d six h o u r s after administration (F(3,54)=5.85; p<.002) but did not s i g n i f i c a n t l y a f f e c t m e t a b o l i t e levels.
Table
1
B L O O D L E V E L S (ng/ml) OF T H C A N D ITS M E T A B O L I T E IN R A T S I N T U B A T E D W I T H T H C (50 mg/kg) PLUS A L C O H O L (i g/kg) O R W A T E R (x ± S.E.)
Time
(hr) A f t e r 1
Intubation 2
4
6
THC
THC + Alcohol
242±
37
364±52
794±160
504± 62
THC + Water
315±
36
294±52
374± 70
361± 67
COOH
THC + Alcohol
106±
18
270±35
611±
98
913±135
THC + Water
320±105
402±64
595±115
916±154
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Maternal Weiqht Gain. Treatment of pregnant females with THC significantly reduced weight gain during pregnancy (F(2,41)=49.5; p<.001). However, this was solely due to the lower weight gain of the 25 mg/kg group as indicated by a significant quadratic trend (F(I,41)=95.0; p<.001). Treatment with alcohol did not affect weight gain significantly nor was the THC x alcohol interaction significant. Litter size. Group differences were not significant for the THC or alcohol factor or their interaction. Birth Weiqht. Pups born to mothers given THC reduced birth weights (F(2,41)=3.15; p<.05). indicated that this effect was best accounted quadratic component (FI,41=5.51; p<.02). Males females (F(1,41)=43.2; p<.01). Birth weight was affected by maternal alcohol exposure nor was interaction significant. (See Table 2.)
had significantly Trend analysis for in terms of a weighed more than not significantly the THC x alcohol
Mortality. Group differences in mortality were not significant for either the THC or alcohol factors or their interactions. Weight at 30 Days. Group differences were not significant for either the THC or alcohol effects or their interaction. Males continued to weigh more than females (F(I,37)=8.54; p<.001). (See Table 2.) Passive Avoidance. Group differences were not significant for either the THC or alcohol factors or their interaction, for either first trial latencies or trials to criterion. (See Table 2.) Table 2 Effects of Combinations of THC (50, 25 or 0 mg/kg) and Alcohol (2, 1 or 0 g/kg) on Offspring (x ± S.E.) THC
(mg/kg)
50
Mat. Wt. Gain
54±3
Birth Wt.
25 (23) I
5.3±0.1
16±2
(14)
5.1±0.1
53±4
Wt. at 30 days
93±1
96±2
98±2
P.A. Latency
28±5
32±6
34.6±9
4.5~0.3
4.7~0.3
(sec.)
Trials to Crit. Alcohol
(g/kg)
Mat. Wt. Gain
~ 46~5
Birth Wt.
4.6~0.7
l (17)
5.3±0.1
39~5
(19)
5.3±0.1
45~6
95±2
94±2
96±2
P.A. Latency
28±7
27±4
39±9
Trials to Crit. I.
3.8±0.4
5.4±0.3
(14)
5.4±0.1
Wt. at 30 days (sec.)
(13)
5.6±0.1
4.6±0.6
Numbers in parentheses indicate number of litters per group.
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Discussion P e a k levels of THC o c c u r r e d at a r o u n d four hrs a f t e r intubation. This is s i m i l a r to o t h e r reports using the oral route of administration (12). Although blood levels of THC were s i g n i f i c a n t l y i n c r e a s e d by alcohol at four and six h o u r s after a d m i n i s t r a t i o n , we did not sample a n i m a l s long e n o u g h to k n o w if the d i f f e r e n c e p e r s i s t e d for v e r y m u c h longer. The p o i n t of this study w a s to d e t e r m i n e if c o n c o m i t a n t t r e a t m e n t w i t h a l c o h o l w o u l d i n c r e a s e b l o o d THC levels. Since we w e r e only i n t e r e s t e d in determining if THC levels w o u l d be i n c r e a s e d at all, we only s t u d i e d one d r u g c o m b i n a t i o n of THC and alcohol. The fact that a l c o h o l i n c r e a s e d THC levels even w i t h our low d o s e s u g g e s t e d that any failure to o b s e r v e b i o l o g i c a l l y s y n e r g i s t i c e f f e c t s w o u l d not be due to a b s e n c e of p h a r m a c o l o g i c a l synergism. It is possible, however, that simultaneous administration of a l c o h o l w i t h THC c o u l d h a v e i n f l u e n c e d its a b s o r p t i o n and d i s t r i b u t i o n r e s u l t i n g in even h i g h e r p e a k levels of THC. As in o t h e r s t u d i e s in this area (7, 13-14) a d m i n i s t r a t i o n of THC to p r e g n a n t rats r e s u l t e d in d e c r e a s e d b i r t h w e i g h t s in o f f s p r i n g and p o s t n a t a l catch up growth. The absence of p o s t n a t a l b e h a v i o r a l e f f e c t s in a n i m a l s p r e n a t a l l y e x p o s e d to T H C has also p r e v i o u s l y b e e n n o t e d (15) and was c o r r o b o r a t e d m o r e r e c e n t l y by Brake et. al. (16). In studies r e p o r t i n g such e f f e c t s of THC, i n t e r p r e t a t i o n of p o s i t i v e effects is e q u i v o c a l due to a p o s s i b l e c o n f o u n d i n g w i t h d e c r e a s e d food c o n s u m p t i o n (16). Fried (i) has r e p o r t e d b e h a v i o r a l a n o m a l i e s in n e o n a t e s w h o s e m o t h e r s smoke m a r i j u a n a d u r i n g pregnancy, but these e f f e c t s w e r e not p e r s i s t e n t . In a study c o n d u c t e d at t w e l v e and t w e n t y - f o u r m o n t h s of age, t h e s e c h i l d r e n did not d i f f e r from c o n t r o l s on the B a y l e y t e s t s of Infant D e v e l o p m e n t or on the H o m e O b s e r v a t i o n for M e a s u r e m e n t of the E n v i r o n m e n t scale (i), a l t h o u g h t h e y did d i f f e r at t h i r t y - s i x m o n t h s on the M c C a r t h y / R e y n e l l tests for l a n g u a g e and c o g n i t i v e a b i l i t y (18). As yet, we h a v e not b e e n able to d i s c e r n any p o s t n a t a l e f f e c t s of T H C c o m p a r a b l e to those noted in the e p i d e m i o l o g i c a l literature. One r e a s o n for t h i s d i f f e r e n c e m a y be p o o r e r p e n e t r a t i o n of THC a c r o s s the p l a c e n t a in rats c o m p a r e d to humans. For example, H u t c h i n g s et. al. (14) r e p o r t e d that in rats, m a t e r n a l THC levels w e r e a b o u t ten times h i g h e r t h a n in the fetus. By contrast, B l a c k a r d and T e n n e s (19) r e p o r t e d a 2.5:1 m a t e r n a l - f e t a l ratio in humans. W h i l e a d d i t i o n a l r i s k factors in a n i m a l s are m i n i m a l c o m p a r e d to w h a t m a y be i m p a c t i n g on p r e g n a n t women, we n e v e r t h e l e s s feel that w h i l e m a r i h u a n a is by d e f i n i t i o n a n e c e s s a r y c o n t r i b u t o r to any e f f e c t on offspring, it is not a s u f f i c i e n t c o n t r i b u t o r in and of itself. O u r study i n d i c a t e s that c o n c o m i t a n t alcohol u s e is not one of the i m p o r t a n t factors c o n t r i b u t i n g to m a r i h u a n a ' s effects. The lack of any s y n e r g i s m on b i r t h w e i g h t b e t w e e n these t w o drugs c o r r o b o r a t e D a l t e r i o et al.'s (17) o b s e r v a t i o n s . The a b s e n c e of s i g n i f i c a n t e f f e c t s of alcohol itself at the r e l a t i v e l y low doses u s e d in this study c o r r o b o r a t e our p r e v i o u s findings (9-10). A l t h o u g h we p r e v i o u s l y did o b s e r v e an i n t e r a c t i o n b e t w e e n these two d r u g s on f e t o t o x i c i t y (5-6) we u s e d doses of T H C in those s t u d i e s (i00 mg/kg) w h i c h w e r e m u c h h i g h e r t h a n in this study.
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Acknowledgement S u p p o r t e d by g r a n t A A 0 6 9 9 9 from the N a t i o n a l I n s t i t u t e on A l c o h o l A b u s e and Alcoholism. We t h a n k D. W a s e l e w s k y for t e c h n i c a l a s s i s t a n c e and Dr. R. Hawks for s u p p l y i n g the R I A kit. References i. 2. 3. 4.
5. 6. 7. 8. 9. i0. ii. 12.
13. 14. 15. 16. 17. 18. 19.
P.A. FRIED and B. WATKINSON, Physiol. Psychol., 9 39-43 (1988). G.T. GIBSON, P.A. B A G H U R S T and D.P. COLLEY, Aust. NZ J. Obstet. Gynecol., 23 15-19 (1983). Q.H. QAZI, F. MARIANO, D.H. MILMAN, E. B E L L E R and W. C R O M B L E H O L M E , Dev. Pharmacol., 8 141-148 (1985). R. HINGSON, B. ZUCKERMAN, H. AMARO, D.A. FRANK, H. KAYNE, J.R. SORENSON, J. MITCHELL, S. PARKER, S. M O R E L O C K and R. TIMPERI, J. P u b l i c Health, 76 667-669 (1985). E.L. ABEL, Teratology, 3_!i 35-40 (1985). E.L. A B E L and B.A. DINTCHEFF, J. Stud. Alc., 47:440-443. E.L. ABEL, S.E. T A N and M. SUBRAMANIAN, Teratology, 36 193198 (1987). E.L. A B E L and S.E. TAN, Reprod. Toxicol., ! 37-40 (1987). E.L. ABEL, P s y c h o p h a r m a c o l . , 5~7 5-11 (1978). E.L. A B E L and B.A. DINTCHEFF, J. Pharmacol. Exper. Ther., 207 916-921 (1978). E.L. ABEL, Alc. Clin. Exp. Res., 6 369 (1982). S. AGURELL, J. LINDGREN, A. OHLSSON, A. LAKEMEDEL, H.K. G I L L E S P I E and L.E. HOLLISTER, A c a d e m i c Press, NY, pp. 165-183 (1984). P.A. FRIED and A.T. CHARLEBOIS, Physiol. Psychol., Z 307-310 (1979). D.E. HUTCHINGS, B. MORGAN, S.C. BRAKE, T. SHI and E. LASALLE, N e u r o t o x i c o l . Teratol., 9 39-43 (1987). E.L. ABEL, Behav. Neur. Biol., 29 137-156 (1980). S.C. BRAKE, D.E. HUTCHINGS, B. MORGAN, E. L A S A L L E and T. SHI, N e u r o t o x i c o l . Teratol., 9 45-49 (1987). S. DALTERIO, A. BARTKE, K. BLUM, and C. SWEENEY, Prog. Biochem. Pharmacol., I_88 143-154 (1981). P.A. FRIED, N e u r o t o x i c o l o g y , i0 577:184 (1989). C. B L A C K A R D and K. TENNES, N e w Eng. J. Med., 2 311 (1984).
EFFECTS
OF L O W DOSES
Pergamon Press
OF A L C O H O L ON D E L T A - 9 - T E T R A H Y D R O C A N N A B I N O L ' S E F F E C T S IN P R E G N A N T RATS
E.L.
Abel
and M.G.
Subramanian
Fetal A l c o h o l R e s e a r c h C e n t e r and D e p a r t m e n t of O b s t e t r i c s and Gynecology, W a y n e State University, C.S. M o t t C e n t e r for H u m a n G r o w t h and Development, 275 E. Hancock, Detroit, M i c h i g a n 48201. (Received in final form August 28, 1990) Summary P r e g n a n t rats w e r e i n t u b a t e d w i t h 50 m g / k g of d e l t a 9-tetrahydrocannabinol (THC) or w i t h T H C p l u s a l c o h o l to determine if a low dose of alcohol (i g/kg) would s i g n i f i c a n t l y increase b l o o d levels of THC. On the b a s i s of t h i s study, a s e c o n d study was c o n d u c t e d in w h i c h p r e g n a n t rats w e r e i n t u b a t e d w i t h THC (50, 25 or 0 mg/kg) p l u s a l c o h o l (2, 1 or 0 g/kg) from g e s t a t i o n d a y six to parturition. THC r e d u c e d b i r t h w e i g h t s b u t did not s i g n i f i c a n t l y a f f e c t litter size or p a s s i v e a v o i d a n c e learning. A l c o h o l did not h a v e a s i g n i f i c a n t e f f e c t on o f f s p r i n g b i r t h w e i g h t nor did it i n t e r a c t w i t h T H C to a f f e c t offspring. M a n y w o m e n w h o smoke m a r i j u a n a d u r i n g p r e g n a n c y a l s o u s e alcohol and o t h e r d r u g s (i-4). We h a v e p r e v i o u s l y shown that c o n c o m i t a n t treatment during pregnancy with delta-9-tetrahydrocannabinol (THC), the p s y c h o a c t i v e i n g r e d i e n t in marijuana, a n d alcohol can d r a m a t i c a l l y i n c r e a s e THC's e f f e c t s on f e t o t o x i c i t y in a n i m a l s (56). T h i s is in c o n t r a s t to p h e n o b a r b i t a l (7) and c h l o r d i a z e p o x i d e (8) w h i c h do not p o t e n t i a t e THC's fetotoxicity. In the p r e s e n t study, we j o i n t l y a d m i n i s t e r e d lower doses of T H C t h a n t h o s e we p r e v i o u s l y e v a l u a t e d for f e t o t o x i c e f f e c t s and d o s e s of a l c o h o l t h a t we h a v e p r e v i o u s l y found to have m i n i m a l e f f e c t s on o f f s p r i n g b i r t h w e i g h t (9-10), and we a l l o w e d a n i m a l s to d e l i v e r t h e i r y o u n g so t h a t we c o u l d assess the p o s s i b i l i t y t h a t alcohol p o t e n t i a t e s the e f f e c t s of THC on b i r t h weight, p o s t n a t a l g r o w t h or behavior. Our first e x p e r i m e n t d e t e r m i n e d if a low d o s e of a l c o h o l w o u l d s i g n i f i c a n t l y i n c r e a s e b l o o d THC levels and w h e n p e a k b l o o d THC levels occurred. On the basis of these data, we a d m i n i s t e r e d our a l c o h o l d o s e s so t h a t alcohol w o u l d be p r e s e n t w h e n T H C levels w e r e at t h e i r peak. Materials
and M e t h o d s
N u l l i p a r o u s L o n g - E v a n s H o o d e d rats (Charles River, Portage, MI) a p p r o x i m a t e l y t h r e e m o n t h s of age w e r e b r e d in our l a b o r a t o r y to t h r e e - m o n t h old males. P r e g n a n t female rats w e r e i n t u b a t e d w i t h 50 m g / k g T H C and 1 g / k g alcohol (N=I0) or w i t h 50 m g / k g T H C and an equal v o l u m e of d i s t i l l e d w a t e r (N=I0). T h e T H C w a s p r o v i d e d by the N a t i o n a l I n s t i t u t e on Drug A b u s e (NIDA). Tail tip b l o o d s a m p l e s (200 ul) w e r e c o l l e c t e d at one, two, four and six hours 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
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a f t e r i n t u b a t i o n on g e s t a t i o n day 20. P l a s m a w a s s e p a r a t e d and frozen until s u b s e q u e n t a n a l y s i s of T H C and the l l - n o r - 9 - c a r b o x y l THC m e t a b o l i t e , u s i n g r a d i o i m m u n o a s s a y (RIA). The R I A kits w e r e o b t a i n e d from R e s e a r c h T r i a n g l e Institute, N.C. The a s s a y p r o c e d u r e s f o l l o w e d i n s t r u c t i o n s d e s c r i b e d for the k i t s and all samples were assayed in duplicate. Standard and experimental plasma samples were extracted with methanol. Aliquots of the supernatants were incubated with antiserum s p e c i f i c for T H C and THC-COOH. T h e a s s a y tubes w e r e i n c u b a t e d at 4°C overnight. P r e c i p i t a t i n g i m m u n o b e a d - b u f f e r s o l u t i o n was a d d e d to a s s a y t u b e s and vortexed. F o l l o w i n g an a d d i t i o n a l t h r e e h o u r i n c u b a t i o n at 4°C, the tubes w e r e c e n t r i f u g e d and s u p e r n a t a n t s w e r e discarded. The p r e c i p i t a t e in the t u b e was w a s h e d w i t h cold buffer, c e n t r i f u g e d and s u p e r n a t a n t p o u r e d off. The precipitate in the t u b e w a s c o u n t e d for r a d i o a c t i v i t y in an a u t o m a t i c gamma c o u n t e r (Model 28062, M i c r o m e d i c Systems, Inc.) and s e r u m levels of c a n n a b i n o l s w e r e calculated. C o n t r o l p l a s m a p o o l s (two from THC and t h r e e for THC-COOH) p r o v i d e d w i t h kits, w e r e a l s o c a r r i e d out in d u p l i c a t e s in each assay. T r i p l i c a t e tubes to e s t i m a t e the n o n s p e c i f i c a l l y p r e c i p i t a t e d r a d i o a c t i v i t y w e r e a l s o i n c l u d e d in e a c h assay. W i t h i n and b e t w e e n a s s a y v a r i a b i l i t y , e x p r e s s e d as c o e f f i c i e n t of variation, was less t h a n 12% for the two assays. B a s e d on the d a t a for this study, we c o n d u c t e d our s e c o n d study to e v a l u a t e p o t e n t i a l b i o l o g i c a l s y n e r g i s m b e t w e e n T H C and alcohol. On the m o r n i n g of v a g i n a l p l u g i d e n t i f i c a t i o n (gestation day 0), p r e g n a n t f e m a l e s w e r e d i v i d e d and a s s i g n e d to one of t h r e e d r u g t r e a t m e n t g r o u p s and w e r e i n d i v i d u a l l y h o u s e d in P l e x i g l a s cages. A n i m a l s in e a c h g r o u p w e r e i n t u b a t e d once p e r day w i t h T H C (50, 25 or 0 mg/kg) d i s s o l v e d in s e s a m e oil b e g i n n i n g on g e s t a t i o n day six. E a c h of t h e s e m a i n g r o u p s was s u b d i v i d e d such t h a t a n i m a l s in e a c h s u b g r o u p w e r e a l s o i n t u b a t e d w i t h e i t h e r 2, 1 or 0 g / k g of a l c o h o l (in d i s t i l l e d water). T h e s e d o s e s of a l c o h o l w e r e c h o s e n b e c a u s e t h e y h a v e no s i g n i f i c a n t e f f e c t s on b i r t h w e i g h t or p o s t n a t a l w e i g h t in rats (9-10). A n i m a l s in the two lower d o s e alcohol g r o u p s h a d t h e i r alcohol d i s s o l v e d in a m a l t o s e d e x t r i n s o l u t i o n i s o c a l o r i c to the g r o u p r e c e i v i n g the 2 g / k g alcohol dose. A n i m a l s w e r e i n t u b a t e d w i t h THC at a p p r o x i m a t e l y 9:00 a.m. and t h e n t h r e e h o u r s later w i t h alcohol. The d e l a y was b a s e d on r e s u l t s from our initial s t u d y s h o w i n g t h a t p e a k c o n c e n t r a t i o n s o c c u r r e d a r o u n d 4 hrs a f t e r intubation. B e c a u s e of t h i s slow absorption, we d e l a y e d a l c o h o l a d m i n i s t r a t i o n so t h a t w h e n it w a s administered, THC levels in the b l o o d w o u l d be at or n e a r p e a k concentrations. For p u r p o s e s of p a i r - f e e d i n g , the nine e x p e r i m e n t a l g r o u p s w e r e d i v i d e d into t r i a d s such that a n i m a l s in each of the T H C s u b g r o u p s w e r e p a i r - f e d to the s u b g r o u p r e c e i v i n g the h i g h e s t a l c o h o l dose. W i t h i n t w e l v e h o u r s a f t e r birth, p u p s in each litter w e r e counted, weighed, culled to e i g h t per litter and r e t u r n e d to t h e i r b i o l o g i c a l mothers. Animals (N=II-20 p e r sex p e r group) w e r e t e s t e d for p a s s i v e a v o i d a n c e at s e v e n t e e n days of age. No m o r e t h a n two m a l e s or females from each l i t t e r w e r e evaluated. Animals were weaned at t w e n t y - o n e d a y s of age. Due to a miscommunication, a n i m a l s w e r e not w e i g h e d at weaning. Instead, a n i m a l s w e r e w e i g h e d at t h i r t y d a y s of age.
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passive Avoidance. The test apparatus was a Plexiglas rectangular c h a m b e r (35 x 22 x 16 cm) d i v i d e d i n t o t w o e q u a l s e c t i o n s b y a g u i l l o t i n e door. O n e side of t h e c h a m b e r w a s c o v e r e d b y b l a c k c o l o r b o a r d , t h e o t h e r w a s clear. B o t h s i d e s h a d a g r i d f l o o r of s t a i n l e s s steel bars. A Coulbourn solid state shock generator d e l i v e r e d a 0.8 m A p u l s e of s h o c k for 0.5 seconds. T h i s t e s t and t h e age of t e s t i n g w e r e u s e d b e c a u s e p r e v i o u s r e s u l t s h a d s h o w n it s e n s i t i v e to a l c o h o l ' s in u t e r o a c t i o n s (ii). A n i m a l s w e r e r e m o v e d from t h e i r h o m e c a g e and, t h i r t y s e c o n d s later, p l a c e d in t h e c l e a r s i d e of the c h a m b e r f a c i n g a w a y from t h e d o o r w h i c h w a s a l r e a d y raised. A timer was started when a n i m a l s w e r e p l a c e d in t h e chamber. W h e n a n i m a l s s t e p p e d into the b l a c k c h a m b e r w i t h all four feet, the t i m e r w a s stopped, t h e d o o r w a s closed, a n d t h e s h o c k w a s d e l i v e r e d . Animals were removed f r o m t h e c h a m b e r a n d r e t u r n e d to t h e h o l d i n g c a g e for t h i r t y s e c o n d s a f t e r shock. This procedure was continued until criterion or e i g h t t r i a l s w e r e reached. The c r i t e r i o n w a s n o n e n t r y into the s h o c k c h a m b e r for t w o c o n s e c u t i v e 180 s e c o n d s trials. Data Analysis. T h e d a t a w e r e e x a m i n e d b y a n a l y s i s of v a r i a n c e (ANOVA). In i n s t a n c e s w h e r e all p u p s in a l i t t e r w e r e examined, e.g., b i r t h weight, t h e l i t t e r a n d not t h e i n d i v i d u a l p u p w a s u s e d as a d a t u m b e c a u s e m e a s u r e s of i n d i v i d u a l p u p s w i t h i n l i t t e r s are l i k e l y to b e c o r r e l a t e d . Results B l o o d c a n n a b i n o i d l e v e l s are s h o w n in T a b l e i. P e a k l e v e l s of THC were recorded at four hours after intubation. Alcohol s i g n i f i c a n t l y i n c r e a s e d T H C l e v e l s in b l o o d b y four a n d six h o u r s after administration (F(3,54)=5.85; p<.002) but did not s i g n i f i c a n t l y a f f e c t m e t a b o l i t e levels.
Table
1
B L O O D L E V E L S (ng/ml) OF T H C A N D ITS M E T A B O L I T E IN R A T S I N T U B A T E D W I T H T H C (50 mg/kg) PLUS A L C O H O L (i g/kg) O R W A T E R (x ± S.E.)
Time
(hr) A f t e r 1
Intubation 2
4
6
THC
THC + Alcohol
242±
37
364±52
794±160
504± 62
THC + Water
315±
36
294±52
374± 70
361± 67
COOH
THC + Alcohol
106±
18
270±35
611±
98
913±135
THC + Water
320±105
402±64
595±115
916±154
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Vol. 47, No. 18, 1990
Maternal Weiqht Gain. Treatment of pregnant females with THC significantly reduced weight gain during pregnancy (F(2,41)=49.5; p<.001). However, this was solely due to the lower weight gain of the 25 mg/kg group as indicated by a significant quadratic trend (F(I,41)=95.0; p<.001). Treatment with alcohol did not affect weight gain significantly nor was the THC x alcohol interaction significant. Litter size. Group differences were not significant for the THC or alcohol factor or their interaction. Birth Weiqht. Pups born to mothers given THC reduced birth weights (F(2,41)=3.15; p<.05). indicated that this effect was best accounted quadratic component (FI,41=5.51; p<.02). Males females (F(1,41)=43.2; p<.01). Birth weight was affected by maternal alcohol exposure nor was interaction significant. (See Table 2.)
had significantly Trend analysis for in terms of a weighed more than not significantly the THC x alcohol
Mortality. Group differences in mortality were not significant for either the THC or alcohol factors or their interactions. Weight at 30 Days. Group differences were not significant for either the THC or alcohol effects or their interaction. Males continued to weigh more than females (F(I,37)=8.54; p<.001). (See Table 2.) Passive Avoidance. Group differences were not significant for either the THC or alcohol factors or their interaction, for either first trial latencies or trials to criterion. (See Table 2.) Table 2 Effects of Combinations of THC (50, 25 or 0 mg/kg) and Alcohol (2, 1 or 0 g/kg) on Offspring (x ± S.E.) THC
(mg/kg)
50
Mat. Wt. Gain
54±3
Birth Wt.
25 (23) I
5.3±0.1
16±2
(14)
5.1±0.1
53±4
Wt. at 30 days
93±1
96±2
98±2
P.A. Latency
28±5
32±6
34.6±9
4.5~0.3
4.7~0.3
(sec.)
Trials to Crit. Alcohol
(g/kg)
Mat. Wt. Gain
~ 46~5
Birth Wt.
4.6~0.7
l (17)
5.3±0.1
39~5
(19)
5.3±0.1
45~6
95±2
94±2
96±2
P.A. Latency
28±7
27±4
39±9
Trials to Crit. I.
3.8±0.4
5.4±0.3
(14)
5.4±0.1
Wt. at 30 days (sec.)
(13)
5.6±0.1
4.6±0.6
Numbers in parentheses indicate number of litters per group.
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Discussion P e a k levels of THC o c c u r r e d at a r o u n d four hrs a f t e r intubation. This is s i m i l a r to o t h e r reports using the oral route of administration (12). Although blood levels of THC were s i g n i f i c a n t l y i n c r e a s e d by alcohol at four and six h o u r s after a d m i n i s t r a t i o n , we did not sample a n i m a l s long e n o u g h to k n o w if the d i f f e r e n c e p e r s i s t e d for v e r y m u c h longer. The p o i n t of this study w a s to d e t e r m i n e if c o n c o m i t a n t t r e a t m e n t w i t h a l c o h o l w o u l d i n c r e a s e b l o o d THC levels. Since we w e r e only i n t e r e s t e d in determining if THC levels w o u l d be i n c r e a s e d at all, we only s t u d i e d one d r u g c o m b i n a t i o n of THC and alcohol. The fact that a l c o h o l i n c r e a s e d THC levels even w i t h our low d o s e s u g g e s t e d that any failure to o b s e r v e b i o l o g i c a l l y s y n e r g i s t i c e f f e c t s w o u l d not be due to a b s e n c e of p h a r m a c o l o g i c a l synergism. It is possible, however, that simultaneous administration of a l c o h o l w i t h THC c o u l d h a v e i n f l u e n c e d its a b s o r p t i o n and d i s t r i b u t i o n r e s u l t i n g in even h i g h e r p e a k levels of THC. As in o t h e r s t u d i e s in this area (7, 13-14) a d m i n i s t r a t i o n of THC to p r e g n a n t rats r e s u l t e d in d e c r e a s e d b i r t h w e i g h t s in o f f s p r i n g and p o s t n a t a l catch up growth. The absence of p o s t n a t a l b e h a v i o r a l e f f e c t s in a n i m a l s p r e n a t a l l y e x p o s e d to T H C has also p r e v i o u s l y b e e n n o t e d (15) and was c o r r o b o r a t e d m o r e r e c e n t l y by Brake et. al. (16). In studies r e p o r t i n g such e f f e c t s of THC, i n t e r p r e t a t i o n of p o s i t i v e effects is e q u i v o c a l due to a p o s s i b l e c o n f o u n d i n g w i t h d e c r e a s e d food c o n s u m p t i o n (16). Fried (i) has r e p o r t e d b e h a v i o r a l a n o m a l i e s in n e o n a t e s w h o s e m o t h e r s smoke m a r i j u a n a d u r i n g pregnancy, but these e f f e c t s w e r e not p e r s i s t e n t . In a study c o n d u c t e d at t w e l v e and t w e n t y - f o u r m o n t h s of age, t h e s e c h i l d r e n did not d i f f e r from c o n t r o l s on the B a y l e y t e s t s of Infant D e v e l o p m e n t or on the H o m e O b s e r v a t i o n for M e a s u r e m e n t of the E n v i r o n m e n t scale (i), a l t h o u g h t h e y did d i f f e r at t h i r t y - s i x m o n t h s on the M c C a r t h y / R e y n e l l tests for l a n g u a g e and c o g n i t i v e a b i l i t y (18). As yet, we h a v e not b e e n able to d i s c e r n any p o s t n a t a l e f f e c t s of T H C c o m p a r a b l e to those noted in the e p i d e m i o l o g i c a l literature. One r e a s o n for t h i s d i f f e r e n c e m a y be p o o r e r p e n e t r a t i o n of THC a c r o s s the p l a c e n t a in rats c o m p a r e d to humans. For example, H u t c h i n g s et. al. (14) r e p o r t e d that in rats, m a t e r n a l THC levels w e r e a b o u t ten times h i g h e r t h a n in the fetus. By contrast, B l a c k a r d and T e n n e s (19) r e p o r t e d a 2.5:1 m a t e r n a l - f e t a l ratio in humans. W h i l e a d d i t i o n a l r i s k factors in a n i m a l s are m i n i m a l c o m p a r e d to w h a t m a y be i m p a c t i n g on p r e g n a n t women, we n e v e r t h e l e s s feel that w h i l e m a r i h u a n a is by d e f i n i t i o n a n e c e s s a r y c o n t r i b u t o r to any e f f e c t on offspring, it is not a s u f f i c i e n t c o n t r i b u t o r in and of itself. O u r study i n d i c a t e s that c o n c o m i t a n t alcohol u s e is not one of the i m p o r t a n t factors c o n t r i b u t i n g to m a r i h u a n a ' s effects. The lack of any s y n e r g i s m on b i r t h w e i g h t b e t w e e n these t w o drugs c o r r o b o r a t e D a l t e r i o et al.'s (17) o b s e r v a t i o n s . The a b s e n c e of s i g n i f i c a n t e f f e c t s of alcohol itself at the r e l a t i v e l y low doses u s e d in this study c o r r o b o r a t e our p r e v i o u s findings (9-10). A l t h o u g h we p r e v i o u s l y did o b s e r v e an i n t e r a c t i o n b e t w e e n these two d r u g s on f e t o t o x i c i t y (5-6) we u s e d doses of T H C in those s t u d i e s (i00 mg/kg) w h i c h w e r e m u c h h i g h e r t h a n in this study.
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Interaction in Pregnant Rats
Vol. 47, No. 18, 1990
Acknowledgement S u p p o r t e d by g r a n t A A 0 6 9 9 9 from the N a t i o n a l I n s t i t u t e on A l c o h o l A b u s e and Alcoholism. We t h a n k D. W a s e l e w s k y for t e c h n i c a l a s s i s t a n c e and Dr. R. Hawks for s u p p l y i n g the R I A kit. References i. 2. 3. 4.
5. 6. 7. 8. 9. i0. ii. 12.
13. 14. 15. 16. 17. 18. 19.
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