Effects of the maternal consumption of alcohol on alcohol selection in rats

Alcohol. Vol. 2, pp. 323--326,1985. ©AnkhoInternationalInc. Printedin the U.S.A.

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Effects of the Maternal Consumption of Alcohol on Alcohol Selection in Rats E. REYES, K. D. G A R C I A A N D B. C. J O N E S 1 D e p a r t m e n t o f Pharmacology, School o f Medicine University o f N e w Mexico, Albuquerque, N M 87131

REYES, E., K. D. GARCIA AND B. C. JONES. Effects of the maternal consumption of alcohol on alcohol selection in rats. ALCOHOL 2(2) 323-326, 1985.--The present study was undertaken to determine the effects of the maternal administration of alcohol on alcohol selection in the adult offspring. Female Wistar rats were pair-fed liquid diets containing either alcohol (6.7% or 4% v/v) or isocaloric carbohydrates starting on day 1 of gestation. The litters were culled to 6 and the pups placed with non-alcohol treated surrogate mothers until they were weaned. At 45 days of age all rats were tested for two-choice selection of 10% (v/v) ethanol vs. water. Consumption from both drinking tubes was recorded for 30 days and selection ratios for alcohol per total fluid volume were calculated. In all conditions a significant increase in alcohol selection was observed across the 30 day test period. These data indicate that prenatal exposure to alcohol may play a part in subsequent selection of alcohol, at least initially. However, the mother's alcohol treatment did not significantly influence the offspring's selection of alcohol over the 30 day test period. Alcohol selection by an individual may be, at least in part, determined by the mother's consumption of alcohol during gestation. Alcohol

Preference

Maternal

Alcoholism

Rats

THE etiology of alcoholism is not completely understood. Several studies have been, and continue to be, conducted to determine why some individuals are at greater risk of becoming alcoholic than are others. Studies including both human and animal research, indicate that the etiology of alcoholism is complex and multifaceted. Differential alcohol selection among inbred mouse strains suggests that there is a genetic component [4] while other studies provide evidence that the maternal consumption of alcohol may play a vital role in determining an individual's drinking habits. Studies using animal models have provided evidence suggesting that the maternal consumption of alcohol may alter the offspring's taste preferences. Phillips and Stainbrook [6] showed that the in utero exposure of rats to wine increased their preference for this fluid over that of animals lacking this early exposure. Abel and York [1] have shown that prenatal exposure to " m o d e r a t e " doses of alcohol does not lead to subsequent increased alcohol intake by adults. Abel et al. [2] demonstrated that the prenatal exposure to alcohol (6 g/kg/day) can produce long-lasting tolerance to alcohol. These authors also suggest that permanent functional changes in the central nervous system result from the in utero exposure to alcohol. It is the intent of the present study to determine if an increased selection for alcohol in adults might be included as one of the effects of the maternal exposure to alcohol.

Selection

morning to establish evidence of copulation [3,9]. When spermatozoa were identified on the vaginal smear the female was placed in an individual hanging, wire bottomed cage and started on a liquid BioServ diet containing either ethanol or an isocaloric amount of carbohydrate [7,10]. A pair feeding technique, whereby the control animals received only the volume of food consumed by the alcohol treated animals, was utilized. Beginning on day I of gestation, animals were divided into three groups with 6 mothers per group. The first female to test positive for sperm was placed on a diet containing 6.7% v/v ethanol (Group I). The volume of liquid diet consumed by this animal was then used to determine the volume of food given to the control animals (Group II) or the animals receiving diets containing 4% v/v ethanol (Group III). Animals were fed the liquid diets via inverted 50 ml tubes equipped with standard metal sipper tubes. All the animals were maintained on the liquid diets throughout gestation and were weighed daily. A fourth group of rats was maintained on Lab chow ad lib and were used as surrogatefostering mothers and as an ad lib control group. On the day following parturition, the litters were culled to 6 and the pups placed on non-alcohol treated surrogate mothers until weaned. In order to estimate BAC's in Group I, blood was collected approximately every 4 days from a separate group of pregnant females treated identically to those in Group I. This was done to avoid possible confounding effects of stress associated with blood collection on the fetuses.

METHOD

Alcohol Treatment of Mothers

Blood Alcohol Concentration Determinations

Mature female Wistar rats were placed with healthy mature males overnight. Vaginal smears were performed every

At 0700 and 1700 hours at approximately 4 day intervals throughout gestation blood samples were obtained via the

1College of Santa Fe, Santa Fe, NM 87501.

323

32,1

REYES. GARCIA AND JONES

tail vein and assayed for blood alcohol levels utilizing the method described by Lunquist [5]. The increase in N A D H . which absorbs at 340 nm, resulting from the enzymatic conversion of ethanol to acetaldehyde was measured. A typical reaction mixture is as follows: 3.0 ml pyrophosphate buffer (tetrasodium pyrophosphate, 0.075 M; semicarbazide, 0.075 M and glycine, 0.022 M, pH 8.8), 0.1 ml blood supernatant fluid, 1.8 p.moles N A D and 150 units alcohol dehydrogenase. Alcohol levels are reported as mg of alcohol per 100 ml of blood.

Alcohol Selection Testing At 45 days of age, all animals were housed individually in cages containing two drinking tubes, both of which contained water. The volume of water consumed from each tube was measured daily for 8 days to establish baseline fluid consumption. During the following 30 days, each cage was provided with two fluid bottles, one containing tap water and the other containing a 10% (v/v) ethanol solution in tap water. Lab chow was provided ad lib. The volume of fluid consumed from each bottle was determined daily at .which time the bottles were refilled and their positions switched randomly to avoid development of side preference. In order to control for dripping, a control cage was placed on the same cage rack that housed the experimental animals. The amount of drippage was subtracted daily from the respective tubes for all animals. Data were analyzed as the proportion of volume consumed from the ethanol tube to total volume of fluid intake.

Statistics Data were statistically analyzed by analysis of variance with subsequent comparisons between means made using the Tukey HSD method. RESULTS

Alcohol related decreases in maternal weight gain, pup weights, litter sizes and pup brain weight were consistent with our earlier findings [8]. The blood alcohol concentrations (BAC) of pregnant rats receiving a liquid diet containing 6.7% (v/v) ethanol (35% of caloric intake derived from ethanol) throughout gestation are shown in Fig. 1. BAC's were found to increase over the gestational period, resulting on day 18 in a maximum BAC of 170 mg% at 0700 hours and a minimum BAC of 30 mg% at 1700 hours. The pregnant rats in Group I consumed an average of 16.85 g of ethanol per kg per day and those in Group III consumed 10.04 g. The pregnant female rats placed on the 3 different diets gained weight throughout gestation. Figure 2 shows the percent of a 10% (v/v) alcohol solution consumed versus the total fluid consumed by female offspring over a 30 day period. The data were collapsed into ten three-day blocks. Among the female offspring, Group III (n= 15) exhibited the highest selection of alcohol. At day one of testing 24.06% of their total fluid intake was made up of the 10% alcohol solution and at day thirty it was 23.55%. Group II (n= 10) and Group I (n=9) showed an increase in their selection of alcohol from 16% at day one to 20% at day thirty. The ad lib control group (n=6) showed an increase in their selection of the alcohol solution from 7.8% on day one to 12.4% on day thirty. Figure 3 shows the proportion of a 10% alcohol solution versus total fluid consumed by male offspring of the three

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FIG. I. Blood alcohol concentrations (mg per 100 ml) in pregnant rats receiving 35% of calories as ethanol. Pregnant rats were started on a liquid diet containing 6.7% {v/v) ethanol on day I of gestation and maintained on diet until parturition.

treatment groups over a 30 day test period. Male offspring (n= 16) of the mothers in Group I had the highest selection of alcohol. At day one of testing they showed an 18.47% selection of the alcohol solution and at day thirty a 26.67% selection. The control group of males (n=13) had the lowest selection at day one and at day thirty (8.76 and 23.85% respectively). The male offspring of the mothers in Group III (n=19) exhibited an intermediate selection of the alcohol solution, 13.4(1% and 25.98% at day one and day thirty, respectively. The male ad lib control (n=5) group showed an increase in their selection of the alcohol solution from 6.8% on day one to 25.0% on day thirty. Females from Group II and Group III had a higher selection of alcohol from day one to day 27 than males. At day 27 there was a cross-over and the males then manifested a higher selection of alcohol than the females. However, in Group I the situation was different. The males exhibited a higher selection of the alcohol solution throughout the test period. Table I shows that all four groups of animals increased the total volume of fluid consumed from day one to day thirty of the test period. The females in each group drank less fluid than did the males. Statistical analysis of the data from the three groups of offspring was done by analysis of variance of repeated measures. It was found that there was a significant (p<0.001) effect of blocks on alcohol selection. There was a significant (p<0.05) sex by blocks interaction in determining an animal's selection of alcohol. The mother's alcohol treatment did not significantly influence the offspring's selection of alcohol over the 30 day test period. Sex did not have a significant effect on alcohol selection in this study. There was a significant (p<0.001) influence of sex in determining the total volume of fluid consumed by an individual over the 30 day test period. There was also a significant (p<0.001) interaction between blocks and alcohol treatment of the mothers and a significant influence of blocks.

MATERNAL INFLUENCE

ON ALCOHOL SELECTION

325

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THREE DAY BLOCKS

FIG. 3. Effects of maternal administration of alcohol on two-choice selection of ethanol vs. water in adult male rats. Data are expressed in percent of ethanol consumer per total fluid intake.

FIG. 2. Effects of maternal administration of alcohol on two-choice selection of ethanol vs. water in adult female rats. Data are expressed in percent of ethanol consumed per total fluid intake.

TABLE 1 TOTAL VOLUME OF FLUID CONSUMED BY ADULT RATS PRENATALLY EXPOSED TO ALCOHOL AND CONTROLS Treatment Group Group I Blocks (3 days)

Group II

Group III

Ad Lib

F

M

F

M

F

M

F

M

I 2

24(3) 25(3)

34(5) 38(6)

24(4) 26(4)

34(4) 35(3)

26(5) 24(4)

31(5) 31(5)

78(14) 86(13)

106(10) 107(4)

3 4

26(7) 28(7)

35(6) 38(9)

27(3) 29(7)

36(4) 37(3)

25(6) 30(8)

33(6) 35(5)

94(12) 86(10)

107(6) 110(3)

5 6 7 8

26(5) 26(2) 27(4) 28(4)

35(7) 35(6) 38(8) 35(8)

29(7) 30(8) 30(8) 29(5)

36(4) 35(5) 36(4) 38(5)

28(5) 29(7) 30(8) 30(9)

34(3) 38(4) 38(6) 37(3)

84(12) 91(20) 93(27) 94( I l)

I03(II) 108(12) 115(8) 109(9)

9 10

28(6) 28(9)

35(6) 37(6)

30(4) 33(1 I)

37(6) 37(5)

31(9) 31(7)

40(7) 37(6)

89(12) 93(16)

116(11) 117(12)

The dams of groups I and III received 35 and 20% respectively of their caloric intake in the form of alcohol; those in group lI received isocaloric dextran and those in the ad lib group were untreated. Volume of fluid consumed is measured in ml with the standard deviation in parentheses. F=Female and M=male.

In this study, we found: (I) a difference b e t w e e n the sexes in ethanol selection r e s p o n s e to continued e x p o s u r e to alcohol in the t w o - c h o i c e situation. Males s h o w e d a consistent overall increase in alcohol selection across the 3-day blocks. F e m a l e s showed no such trend; (2) in u t e r o e x p o s u r e to alcohol may affect overall fluid intake and perhaps fluid balance; (3) in u t e r o e x p o s u r e to alcohol does not significantly influence the offspring's appetite for alcohol. Although not a statistically significant main or interactive effect, it is

nonetheless interesting to note that in males on 9 o f t e n 3-day blocks, animals e x p o s e d in u t e r o to alcohol exhibited higher selection than did controls. In females, a similar pattern was seen on 8 o f ten 3-day blocks. T h e s e results present preliminary e v i d e n c e that a m o t h e r ' s e x p o s u r e to alcohol during gestation may influence an offspring's initial selection o f alcohol. H o w e v e r , the continual e x p o s u r e o f the individual to alcohol seems to override the effect o f the m o t h e r ' s drinking history.

326

REYES, GARC1A AND JONES ACKNOWLEDGEMENTS The authors thank Janet Wolfe and David Sandoval for their technical assistance. This study was supported in part by NIHMBRS program grant # R R 08139 and a grant from the State of New Mexico Department of Health and Environment #73/665.4535/022.

REFERENCES 1. Abel, E. L. and J. L. York. Absence of effect of prenatal ethanol on adult emotionality and ethanol consumption in rats. J Stud Alcohol 4t1: 547-553, 1979. 2. Abel, E. L., R. Bush and B. A. Dintcheff. Exposure of rats to alcohol in utero alters drug sensitivity in adulthood. Science 212: 1531-1533, 1981. 3. Bertholet, J. Y. Mating method to produce accurate timed pregnancies. Lab Anirn Sci 31: 180, 1981. 4. Duckett, S., C. W. Schneider and R. A. Hartline. Oxidation of alcohol in free-moving mice from high and low preference strains. Pharmacol Biochem Behav 15: 495-499, 1981. 5. Lundquist, F. The determination of ethyl alcohol in blood and tissues. Methods Biochem Anal 1: 217-251, 1959.

6. Phillips, D. and G. L. Stainbrook. Effects of early alcohol exposure upon adult learning ability and taste preferences. Physiol Psychol 4: 473--475, 1976. 7. Reyes, E., J. M. Rivera, L. C. Saland and H. M. Murray. Effects of maternal administration of alcohol on fetal brain development. Neurobehav Toxicol Teratol 5: 263--267, 1983. 8. Reyes, E. The role of ~/-glutamyl transpeptidase in alcoholism. Neurobehav Toxicol Teratol 7: 171-180, 1985. 9. Szabo, K. T., S. M. Free, H. A. Birkhead and P. E. Gay. Predictability of pregnancy from various signs of mating in mice and rats. Lab Anita Care 19: 822-825, 1969. 10. Teschke, R., A. Brand and G. Strohmeyer. Induction of hepatic microsomal gamma-glutamyltransferase activity following chronic alcohol consumption. Biochem Biophys Res Comn~un 75: 718--723, 1977.