Neurotoxicology and Teratology, VoL 14, pp. 313-319, 1992 0892-0362/92 $5.00 + .00 Printed in the U.S.A. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

Prenatal Cocaine Exposure in the Laboratory Mouse: Effects on Maternal Water

Consumption and Offspring Outcome

M I C H A E L W . C H U R C H .1 A N D H E L E N E C. R A U C H t

*Department o f Obstetrics~Gynecology and tDepartment o f Immunology~Microbiology, Wayne State University School o f Medicine, Detroit, Nil 48201

Received 27 Sep tember 1991; Accep ted 18 M a y 1992

CHURCH, M. W. AND H. C. RAUCH. Prenatal cocaine exposure in the laboratory mouse: Effects of maternal water consumption and offspring outcome. NEUROTOXICOL TERATOL 14(5) 313-319, 1992-Pregnant mice were given 50 mg/kg cocaine HCI (1070 solution, sc) once daily from gestation days 7 through 18 (sperm positive ffi day 0; term = day 19). Pair-fed and untreated control groups were also used. The pregnant cocaine-treated females showed normal weight gain and food consumption but had significantly increased water consumption. The cocaine-treated group had a significant increase in embryonic resorptions but no significant effects on stillbirths or postnatal mortality. The offspring of cocaine-treated females had significantly reduced birth weights and postnatal weight gains up to the age of 28 days. There was also a delay in their ca r

opening but not in other maturational milestones. Increased water consumption following cocaine treatment has been reported by other studies. We speculate that cocaine has a diuretic effect. We discuss the impfications of this effect during pregnancy.

Cocaine Dinresis Maternal water consumption Pregnancy Prenatal

Mouse Polydipsia Postnatal maturation

COCAINE consumption by women during pregnancy is asso- da ted with a variety of adverse gestational effects such as placental abruption, hypertension, preterm and precipitous labor, and poor maternal weight gain. Prenatal cocaine expo- sure also causes various morbidities in the offspring including fetal distress, postnatal mortalities, decreased birth weights, cerebral hemorrhages, delayed maturation, various birth de- fects, and neurobehavioral and sensory deficits (4).

In two previous cocaine studies that used pregnant rats, we observed an unexpected increase in maternal water consump- tion (1,8). Data published by another research group similarly suggest that cocaine-treated female rats increase their water consumption (10). This article reports that the same phenome- non is seen in mice and that the phenomenon in our mice was dependent on the state of pregnancy. We also report on other aspects of maternal and offspring outcome following cocaine treatment in the laboratory mouse.

METHOD

Female BALB/C mice were impregnated by SJL males. Mating was restricted to the hour between 8 a.m. and 9 a.m. Mating was conf'Lrmed by observing a sperm plug in the va- gina. The BALB/C females were mated with SJL males be-

cause their offspring are useful for immunologic studies. The immunologic deficiencies of our prenatally exposed mouse offspring will be described at another time.

After mating, females were housed individually in polycar- bonate cages with wood chip bedding material. The animal room was temperature (22 ° ± 1°C) and humidity (40% - 50%) controlled with a timed light cycle of 12 h per day (7:00 a.m. to 7:00 p.m.). Animals were assigned to an untreated ad lib fed control group (ADL), a pair-fed control group (PFC), or a cocaine-treated group (COC). The PFC group served two purposes. First, it helped to control for the effects of undernutrition that sometimes accompanies cocaine treatment (1-3). Second, the PFC group also received vehicle (saline) injections to assess the influence of handling stress.

The morning of mating was designated gestation day 0 (GD0). Starting on GD7 and continuing until GDI8 (term = GD19), the mated females in the COC group received subcuta- neous injections of 50 mg/kg cocaine hydrochloride dissolved in normal saline (2e/0 solution). Dose selection and route of administration were based on previous research (1-3,8,11,18). Injections were given once daily between 9:00 a.m. and 10:00 a.m. Similarly, the PFC group received isovolumetric injec- tions (SC) of saline solution.

The dams were weighed daily between 8:00 a.m. to 9:00

'Requests for reprints should be addressed to Michael W. Church, Ph.D., Fetal Alcohol Research Center, 275 East Hancock Avenue, Detroit, MI 48201.

313

314 CHURCH AND RAUCH

a.m. Daily records were also kept on maternal water and food consumption (Tekald 10% mouse pregnancy diet, Harlan Sprague-Dawley, Inc.). For dams not delivering pups, the presence or absence of pregnancy was determined by sacrific- ing the animal, removing the uteri, and staining with ammo- nium sulfate for implantation sites. For dams delivering pups, the uteri were not removed and stained until the pups were weaned on postnatal day 20. Implantation sites were still clearly visible at this time.

In past experiments with rats, we assigned newborn pups to untreated surrogate dams (1,5-8) but in a recent experi- ment, we let some cocaine-treated dams retain some of their pups and found that these pups developed as well as those assigned to surrogate dams (8). Moreover, female mice do not accept surrogate mothering as readily as female rats. Conse- quently, we did not use surrogate fostering in the present study to conserve animal resources.

The weight and sex of each pup were determined as soon as possible after delivery of the entire litter. Pups were thereafter weighed on a weekly basis and assessed for postnatal matura- tion and mortality.

Most data were first evaluated by analyses of variance (ANOVAs) or analyses of covariance (ANCOVAs). For re- peated measures analyses, the probability values were deter- mined according to the Greenhouse-Geisser method. When an ANOVA or ANCOVA indicated a significant effect, a post hoc test (Duncan's Multiple Range Test) was used to determine which groups differed significantly from each other. Although all dams were mated (i.e., they were sperm-positive), about half of these dams proved not to be pregnant as indicated by the absence of embryonic implantation sites in their uteri. This resulted in a two-way ANOVA design with the pregnancy factor having 2 levels (pregnant, nonpregnant) and the treat- ment factor having 3 levels (ADL, PFC, COC). In the ADL, PFC, and COC treatment groups, there were 6, 6, and 8 preg- nant dams and 5, 7, and 13 nonpregnant dams, respectively. For all data analyses involving the offspring, the litter average was used as the unit of measure.

RESULTS

Maternal Outcome No females died during treatment. Moreover, treatment

had no influence on gestational length. The pregnant ADL,

PFC, and COC dams had mean ( + SD) gestational lengths of 19.2 + 0.4, 19.2 + 0.4, and 19.4 + 0.5 days, respectively, F(2, 17) = 0 .50,p = 0.61.

The effects of the pregnancy and treatment conditions on maternal weight gain, food consumption, and water consump- tion during the treatment period (GD7 through GD18) are presented in Table 1 and Fig. 1. The data in Table 1 are the total weight gains, food consumption, and water consump- tion. The data in Fig. 1 show the day-to-day trends in these variables. The pregnant dams in the ADL, PFC, and COC groups gained more weight than their nonpregnant counter- parts. Cocaine treatment did not cause a reduction in weight gain among the pregnant mice. The same was true among the nonpregnant mice.

The pregnant dams in the ADL and COC groups consumed more food than their nonpregnant counterparts. Some of the pregnant PFC dams did not eat all the food allocated to them (see Table 1). Consequently, their food consumption was less than that eaten by the pregnant ADL and COC dams.

For water consumption, (a) pregnant and nonpregnant PFC dams drank similar amounts of water, (b) pregnant and nonpregnant ADL dams drank similar amounts of water, and (c) pregnant COC females drank significantly more water than their nonpregnant COC counterparts. Indeed, the pregnant COC females drank significantly more water than any of the other five groups (see Table 1).

One reason the pregnant COC females may have drank the most water was that they had greater body mass and conse- quently needed more water to sustain their fluid balance. To evaluate this possibility, the daily water consumption data were analyzed by a repeated measure ANCOVA using daily maternal weight as the covariate. The results indicated that maternal weight had little influence in that the pregnant COC females' water consumption was still significantly greater than the other pregnant and nonpregnant groups. That is, the Dose x Pregnancy interaction was significant, F(2, 38) = 9.50, p < 0.001, and all comparisons with the pregnant COC females were significant (all univariate F values = 7.25 to 15.88, df = 1,38, p values < 0.01) with the exception of the nonpregnant ADL females where the difference approached significance ( F = 2.42, df = 1,38, p = 0.065, one-tailed test).

Another reason the pregnant COC dams may have drank

TABLE 1 MATERNAL WEIGHT GAIN, FOOD CONSUMPTION, AND WATER CONSUMPTION

FROM GESTATION DAYS 7 THROUGH 18 (MEAN + SD)

Nonpregnant Pregnant p values

Treat Preg Variable PFC ADL COC PFC ADL COC (T) (P) T x P

Maternal weight gain (g) 1.3 -0.1 1.0 15.1" 16.4" 14.9" ns <0.001 ns (1.6) (1.2) (2.3) (1.5) (4.8) (3.6)

Food consumption (g) 52.7 62.4 55.6 61.2 76.5* 83.6* < 0.001 < 0.001 0.013 (4.6) (9.5) (12.2) (2.6) (7.0) (10.4)

Water consumption (ml) 101.3 121.8 94.0 93.2 103.0 149.9" 0.038 ns <0.001 (15.6) (45.3) (27.0) (15.7) (7.0) (29.9)

n 7 5 13 6 6 8

*Greater than nonpregnant cohorts, p < 0.05. Abbreviations: Treat = treatment factor; Preg = pregnancy factor; ns = nonsignificant.

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GESTATIONAL DAY

FIG. 1. (Top) Maternal body weights (mean + SE) as functions of treatment group and gestational day. Pregnant (P) females showed increasing weights as a function of gestational day whereas the nonpregnant (NP) females did not. These effects were independent of treatment group (ADL = ad lib fed control, PFC = pair-fed control, COC = cocaine treated). (Middle) Maternal food consumption as a function of gestational day. The shaded region is the 95% confidence interval for the pregnant ADL females. The pregnant and nonpreg- nant COC data and pregnant PFC data (mean + SE) are shown for compari- son. Data from the other groups fell within the shaded region. (Bottom) Mater- nal water consumption as a function of gestational day. The shaded region is the 95% confidence interval for the pregnant ADL females. The pregnant COC females consumed the most water (mean + SE). The nonpregnant COC and pregnant PFC data are shown for comparison. Data from other groups fell within the shaded region, with only a few minor e~ceptions.

316 CHURCH AND RAUCH

the most water was that they ate the most food , therefore needing more fluid to balance gastrointestinal absorption. An ANCOVA using food consumption as the covariate indicated that daily food consumption did not fully account for the effect because the pregnant COC group's water consumption was still significantly greater than the other five groups. That is, the Dose x Pregnancy interaction was significant, F(2, 38) = 8.52, p < 0.001, and all comparisons with the preg- nant COC females were significant (all univariate F values = 7.83 to 12.35, df = 1,38, p values < 0.01) with the exception of the nonpregnant ADL females where the difference was significant only with a one-tailed test ( F = 3.36, p = 0.035).

Another possibility was that the pregnant COC females really were not drinking excessive water but were causing ex- cessive water spillage. If the cocaine treatment were causing the animals to spill excessive water because they were agitated, then one would expect both the pregnant and nonpregnant COC groups to show excessive water spillage. This was not the case, however, as the data in Table 1 and Fig. 1 show, the nonpregnant COC females did not exhibit excessive water consumption.

Offspring Outcome There were no significant differences in the number of em-

bryonic implantation sites, stillbirths, or postnatal mortalities. No birth defects were seen upon gross examination. There was, however, a significant increase in the number of embry- onic resorptions in the COC group (see Table 2).

The birthweights and postnatal weights of the pups in the COC group were lower than those in the PFC and ADL groups (see Table 3). Because male and female pup weights were not significantly different between postnatal days 0 to 28, their data were combined to derive the data for Table 3. In terms of achieving maturational milestones, pups in the COC group showed a significant delay in ear opening but not in other measures (see Table 4). For the data in Table 4, the male and female pup data were not different so they were combined. Also, because the PFC and ADL control groups were not different, their data were combined to increase statis- tical power.

DISCUSSION

The results with prenatal cocaine exposure in our mouse model are largely consistent with results obtained by other

animal studies (1-8,10,11,16-22,24,25). For example, prenatal cocaine exposure caused increased embryonic resorptions, de- creased birthweights, produced postnatal growth retardation, and delayed physical maturation. There were some effects re- ported by other studies which we did not observe. For exam- ple, we did not observe reduced maternal weight gain, de- creased maternal food consumption, increased stillbirths, increased postnatal mortality, or incidences of physical anom- alies. Some of these observational differences are probably attributable to strain, species, and dosing differences. Al- though numerous human studies report that cocaine-abusing women show decreased gestational length (4), our laboratory (1,8) and others (14,21,22,25) have consistently failed to ob- serve such an effect in the laboratory rat and now in the laboratory mouse. This effect when seen in humans may be due to polydrug abuse rather than cocaine per se. Alterna- tively, laboratory rats and mice may not provide adequate models of this condition.

The most interesting observation was that pregnant co- caine-treated females showed increased water consumption. This effect was observed only when pregnancy and cocaine treatment were both present. Thus, the combination of co- caine treatment and pregnancy were needed to produce the effect in our mouse model. This effect on water consumption by the pregnant dams was not entirely unexpected. We ob- served this phenomenon on two prior occasions with pregnant cocaine-treated rats (1,8). Similarly, data presented by others (10) indicates that increased water consumption occurred in their nonpregnant cocaine-treated female rats.

Our speculation is that cocaine was acting as a diuretic, causing increased urinary output and thirst. The reasons for this speculation are as follows: First, drugs that increase arte- rial pressure typically have a diuretic effect because they in- crease glomerular filtration, e.g., norepinephrine (NE) and other sympathomimetics (13). Cocaine is a sympathomimetic and it interferes with NE uptake at nerve terminals. Cocaine has also been shown to enhance the contractions of intrarenal arteries (15). Second, the increased sympathetic tone caused by cocaine would increase angiotensin, aldosterone, and glu- cocorticoid levels. These hormones increase urinary output and thirst. Third, like many drugs of abuse (e.g., alcohol, narcotics, anesthetics, hypnotics), cocaine might produce a diuretic effect either by inhibiting the secretion of antidiuretic hormone (ADH) or by causing osmotic diuresis (13). We be-

TABLE 2 EFFECTS OF COCAINE ON PRE- AND POSTNATAL MORTALITIES IN THE MOUSE

Variable

Treatment Group

F PFC ADL COC (2,18) p

Number of implantations Mean 8.7 8.7 9.4 0.17 ns (SE) (0.6) (1.2) (0.9)

Number of resorptions Mean 1.8 1.6 4.0* 6.21 < 0.01 (SE) (0.4) (0.6) (0.5)

Number of stillbirths Mean 0.7 0.9 1.0 0.05 ns (SE) (0.7) (0.6) (0.9)

Number of postnatal Mean 0.2 1.6 1.4 1.07 ns mortalities (SE) (0.2) (0.7) (0.9)

*Greater than other groupsp < 0.05; ns = not significant.

COCAINE AND WATER 317

TABLE 3 EFFECTS OF COCAINE ON MOUSE OFFSPRING BIRTI-IWEIGH'I~ AND

POSTNATAL WEIGHT GAIN IN GRAMS (MALE AND FEMALE DATA COMBINED)

Treatment Group

F Age PFC ADL COC (2,18) p

Day 0 (Birth) Mean 1.65 1.57 1.46* 2.74 <0.05 (SE) (0.04) (0.03) (0.08)

Day 7 Mean 3.52 4.19 2.83t 10.11 < 0.001 (SE) (0.20) (0.10) (0.26)

Day 14 Mean 8.10 8.73 6.82:~ 4.20 <0.025 (SE) (0.40) (0.33) (0.61)

Day 21 Mean 10.92 I 1.01 9.72 2.71 < 0.05 (SE) (0.34) (0.31) (0.60)

Day 28 Mean 16.92 17.32 14.90t 3.72 <0.025 (SE) (0.50) (0.62) (0.85)

*Less than PFC group only, p < 0.05. tLess than both PFC and ADL groups, p < 0.05. ~Less than ADL group only, p < 0.05.

All p values are for directional tests.

lieve that diuresis rather than water retention occurred because there was no evidence that the mother or the fetuses were edematous.

If cocaine had a simple effect on thirst, then one would expect our nonpregnant mice to show increased water con- sumption during cocaine treatment although this was not the case. The phenomenon occurred only in our pregnant mice during cocaine treatment. Perhaps the elevated hormone levels associated with pregnancy were required. Pregnancy is associ- ated with elevated levels of estrogen, ADH, aldosterone, and angiotensin II. These hormones influence water consumption and retention which are important for increasing blood vol- ume to maintain pregnancy and support the fetus. Cocaine may have enhanced the pregnancy-related release (or response to) these hormones in our mice. One difficulty for such a theory is that Dow-Edwards et al., (10) observed a cocaine- induced water consumption in nonpregnant rats. Perhaps dos- age, route, species, genetic differences, and pregnancy status interact in complex ways to produce cocaine-induced changes in water consumption.

The day-to-day pattern of maternal water consumption merits discussion. Our pregnant cocaine-treated mice had ele- vated water consumption throughout the treatment period with the greatest increases occurring on the first two treatment days. Hutchings et al. (16), on the other hand, observed an initial decrease in water consumption in their cocaine-treated pregnant rats followed by a compensatory rebound. This lat- ter pattern is commonly seen when toxic substances are first given to laboratory animals. The data presented by Dow- Edwards et al. (10) show a gradual increase in daily water consumption in their cocaine-treated female rats but with no apparent decrease during the first few days of treatment. We also observed this latter pattern in an earlier study with preg- nant cocaine-treated rats. Data adapted from this latter study are shown in Fig. 2.

A cocaine-induced diuresis would have several implications for the mother and fetus. Most diuretics have the potential to cause marked natriuresis (sodium loss) and other electrolyte imbalances. Interestingly, hyponatremia has been reported in a neonate born to a cocaine-abusing woman (9). We have

TABLE 4 EFFECTS OF PRENATAL COCAINE EXPOSURE ON

THE MOUSE'S PHYSICAL MATURATION

Treatment group

F Variable Control COC (1,15)

Pinna Detachment Mean 4.5 4.2 0.34 ns (day) (SE) (0.2) (0.4)

Fur Emergence Mean 8.0 8.2 0.66 ns (day) (SE) (0.0) (0.4)

Ear Opening Mean 13.1 14.8" 7.01 0.01 (day) (SE) (0.2) (0.7)

Eye Opening Mean 14.9 15.7 2.30 ns (day) (SE) (0.2) (0.6)

*Later than control group, p < 0.01 (directional test). Control group ffi PFC + ADL groups.

318 CHURCH AND RAUCH

60 l 1 ---O ADL ~.

o+ t : 8 1 _

10

BSL 7 8 9 10 11 12 13 14 15 16 17 18 19 20

GESTATIONAL DAY

FIG. 2. Maternal water consumption as a function of gestational day in pregnant rats (mean + SE). The pregnant COC females (40 mg/kg, sc, bid, GD7-GD20) drank the most water. The pregnant PFC females, in contrast, showed lower water consumption than the ADL and COC groups. BSL = baseline derived from average water consumption during GD5 and GD6.

*Different from ADL control group; tDifferent from other two groups. Data adapted from Church et al. (1).

reported (19) that prenatal cocaine exposure in the rat can reduce bone ash content (calcium, magnesium, phosphate). Although there are several possible explanations for this ef- fect, one mechanism could involve the loss of minerals through diuresis. An imbalance in trace elements can cause growth retardation and birth defects.

Whereas several studies have now observed increased water consumption by pregnant cocaine-treated mice and rats, a few rat studies have not (2,14,16). These latter studies typically used lower cocaine doses than the former studies, suggesting a threshold must be exceeded. Additionally, the latter studies typically did not use pair-fed control groups. Such a control group offers the best comparison for cocaine's effects on wa- ter consumption. If a treatment condition results in reduced food consumption, then there should also be a reduction in water consumption. This is because eating meals produces hypovolemia. Secretion of digestive juices entails water loss. The hypovolemia leads to thirst. The less that is eaten, the less the osmotic demand and the less the thirst (12). Conse- quently, if cocaine treatment were to result in less food con-

sumption (as it frequently does) then one would expect water consumption to decrease relative to control levels. In our pre- vious rat studies, our cocaine-treated pregnant rats and their pair-fed cohorts showed decreased food consumption. But while the pair-fed group showed the expected decrease in wa- ter consumption, the cocaine-treated group showed normal to above normal water consumption. This resulted in a dramatic difference in water consumption between these two groups revealing the true effect of cocaine on maternal water con- sumption. This point is illustrated by the data in Fig. 2.

In conclusion, prenatal cocaine exposure in the mouse causes several of the same effects observed in rats and may influence water consumption in some laboratory rodents through a diuretic effect. Our mouse model was less suscepti- ble to the adverse effects of cocaine than the CF-1 strain (18).

ACKNOWLEDGEMENT

Research was supported by Grant DA05536 from the National Institute on Drug Abuse, Rockville, MD.

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