Chronic Exposure to the Opioid Antagonist Naltrexone During Pregnancy: Maternal and Offspring Effects

Physiology & Behavior, Vol. 62, No. 3, pp. 501–508, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/97 $17.00 / .00

PII S0031-9384(97)00007-3

Chronic Exposure to the Opioid Antagonist Naltrexone During Pregnancy: Maternal and Offspring Effects PATRICIA J. MCLAUGHLIN,*1 STEVEN W. TOBIAS,† C. MAX LANG† AND IAN S. ZAGON* *Departments of Neuroscience and Anatomy and †Comparative Medicine, The Milton S. Hershey Medical Center, The Pennsylvania State University, 500 University Drive, Hershey, PA 17033 Received 16 October 1996; Accepted 27 January 1997. MC LAUGHLIN, P. J., S. W. TOBIAS, C. M. LANG AND I. S. ZAGON. Chronic exposure to the opioid antagonist naltrexone during pregnancy: Maternal and offspring effects. PHYSIOL BEHAV 62(3) 501–508, 1997.—The role of endogenous opioids in pregnancy and parturition, and the influence exerted on prenatal and postnatal features of the offspring, were studied in rats. Females received daily injections of 50 mg/kg naltrexone (NTX), a dosage found to block opioids from interacting with opioid receptors for 24 h, throughout pregnancy. No effects on the length of gestation, course of pregnancy, litter size, or the viability of the mother or offspring were noted. The body weights, crown-rump lengths, and wet and dry weights of the brain, heart, kidney, liver, and skeletal muscle (triceps surae) in neonates delivered by NTX-treated rats were substantially elevated compared to newborn animals of saline-injected mothers. Offspring exposed to NTX during prenatal life were larger in body weight and length, and organ wet and dry weights on postnatal days 10 and 21. By weaning (day 21), body weights of NTX-exposed rats were 36% greater than controls, and increases were observed in the wet weights of brain (18%), heart (42%), kidney (38%), lungs (22%), liver (44%), and triceps surae (246%). These data lead us to hypothesize that native opioids are important growth-inhibiting, tonically active regulators of prenatal ontogeny, and that events occurring in prenatal life are determinants to postnatal outcome insofar as somatic development. q 1997 Elsevier Science Inc. Opioids Naltrexone Organ growth

Development

Prenatal

Postnatal

ENDOGENOUS opioids not only serve in neurotransmission and neuromodulation ( 1 ) , but these peptides function as growth factors in neural and non-neural tissues of eukaryotes, and in prokaryotes ( e.g., 2,3,13 – 17,21,24,27 – 36 ) . The pentapeptide, [ Met 5 ] -enkephalin has been identified as the native opioid involved with growth processes and to signify this physiological property has been termed the opioid growth factor ( OGF ) . OGF is a negative growth regulator in developing, renewing, healing, and neoplastic tissues, and is direct and rapidly acting, noncytotoxic, reversible in action, obedient to the intrinsic rhythms of the cell ( e.g., circadian rhythm) , and is neither species nor cell-tissue specific. This growth factor is especially targeted to events related to cell proliferation, although it appears to influence cell migration, differentiation, and tissue organization. OGF and its receptor, zeta ( z ) , are in a state of continuous and unremitting balance with respect to growth modulation. Both the peptide and receptor are associated with the cellular elements being governed. Chronic blockade of the opioids and their receptors imposed by 50 mg/kg naltrexone during postnatal life has been shown to

Maternal

Growth factors

Opioid antagonist

result in rat pups that are larger in size, and to have enhanced brain and organ weights, increased body weights, early acquisition of some physical characteristics, spontaneous motor and reflexive behaviors, and an acceleration in the proliferation and development of brain cells (including differentiation of spines and dendrites in the cerebellum and hippocampus) (8,28– 32,34,35). A few studies have begun to examine the effects of administering opioid antagonists during pregnancy ( 4,5,7 – 12,16,18 – 20,22,23,25,26 ) . Unfortunately, only one of these studies ( 7 ) designed a protocol to disrupt opioid-receptor interaction continuously from fertilization to parturition, and even Harry and Rosecrans ( 7 ) present no evidence of a continuous opioid blockade or consistent drug exposure. In fact, only a few studies ( 4,10 ) investigated whether the opioid antagonist treatment utilized was sufficient to block opioid receptors. Moreover, confounding influences such as maternal and embryo / fetal responses to the presence and then absence of opioid antagonist, ( which under certain methods may even invoke episodic blockade and rebound hyperstim-

Requests for reprints should be addressed to Dr. Patricia J. McLaughlin, Department of Neuroscience and Anatomy, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, E-mail: [email protected]

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MC LAUGHLIN ET AL. Drug Injections On day 1 of pregnancy, animals were randomly assigned to two groups, and received daily intraperitoneal (IP) injections of either 50 mg/kg naltrexone hydrochloride (NTX; Sigma, St. Louis, MO) or an equivalent volume (0.2 ml) of saline (control). Pregnant rats were weighed daily and the dose of NTX adjusted. NTX was prepared weekly and stored at 47C. Maternal Observations Pregnant rats were weighed, and observed daily with respect to their behavior, feeding, and fluid-intake. On the first day of pregnancy some females were examined for their latency of response (e.g., licking of paws) on a hot-plate (557C; Analgesia Meter, Technilabs Instruments Inc., Pequannock, NJ). Females were evaluated for nociceptive response approximately 22 h after the last injection of NTX or saline on the day of parturition. Within 15 min of recording the baseline latency of response, females were injected with 10 mg/kg (IP) morphine sulfate. Thirty min later, the animals were placed on the hot-plate and their post-morphine latencies of response were recorded.

FIG. 1. The body weight of pregnant rats receiving daily injections of 50 mg/kg naltrexone (NTX) (n Å 11) or saline (CO) (n Å 12) throughout gestation. No differences in body weights were detected between these groups during pregnancy. Data represent means { SEM.

ulation ) , the disruption of growth processes at certain stages of development, the use of dosages that partially block opioid receptors, the lack of cross-fostering of pups at birth to eliminate further drug ( e.g., by way of the milk ) and maternal problems in adjusting to termination of the opioid antagonist administration, may have introduced difficulties in interpreting the data. This study was designed to determine if opioids function in the regulation of developmental processes during prenatal life. We chose a paradigm that used the opioid antagonist naltrexone to continuously interrupt opioid-receptor interfacing; nociceptive tests were utilized to verify that opioid receptor blockade was maintained. To eliminate the influence of residual drug accumulation and problems of maternal care because of sequelae related to drug termination, pups were fostered to control mothers at birth. The relationship of opioids to the course and outcome of pregnancy, neonatal and offspring viability, and body weights and measurements, as well as wet, dry, and relative weights of the brain, heart, kidney, liver, lungs, and skeletal muscle (i.e., triceps surae) of offspring during the preweaning period were examined. METHOD

Animals Nulliparous female (195–230 g) and male (250–300 g) Sprague–Dawley rats (Charles River Labs, Wilmington, MA) were used in this study. Animals were housed in an environment of 21 { 0.57C with a relative humidity of 50 { 10%. The room had a complete exchange of air 15–18 times per h and a 12 h light–dark cycle with no twilight; water and Harlan Teklab Purina 8604 Rodent Chow were continuously available. Animals were mated (1 male to 2 females) and the presence of sperm indicated pregnancy ( Åday 1 of gestation); all animals were maintained in stainless steel, wire-bottomed cages except where noted. Three days prior to parturition, the pregnant females were separated and placed individually into solid bottomed cages to deliver their pups.

Neonatal Observations Within 8 to 12 h of parturition, the number of live and dead pups, the gross appearance of offspring and the length of gestation were recorded. Pups born to females receiving NTX or saline were culled to litters of 10 and cross-fostered to untreated mothers within 8 to 12 h of birth. Inasmuch as possible, an equal number of male and female pups were included in each litter. Body Weights and Crown-Rump Lengths Pups were weighed at birth (day 0) and at three-day intervals (i.e., postnatal days 3, 6, 9, 12, 15, 18 and 21) until weaning. Crown to rump measurements were taken by measuring the distance from the base of the skull to the base of the tail using a micrometer; measurements were recorded at three-day intervals from day 0 through day 21. On postnatal day 21, weights and crown-rump measurements for males and females were recorded separately. Organ Weights On postnatal days 0, 10, and 21, at least 12 pups from each treatment group (an equal number of males and females) were randomly selected from at least 3 litters, weighed, and killed by decapitation. Separate measures for males and females were recorded on postnatal day 21. The liver, heart, lungs (right and left), triceps surae (left only), brain, and kidneys (right and left) were removed, blotted free of blood and connective tissue, and weighed. Lungs and kidneys were paired for a single weight. Organs were placed on preweighed plastic weigh boats, dried in an oven at 907C for 48 h and reweighed. Relative organ weights were calculated by dividing the wet organ weight by the corresponding body weight. Statistical Analysis Maternal body weights, hot-plate latencies, length of gestation, litter size, and number of stillborns, as well as body weights, crown to rump lengths, wet, dry, and relative organ weights of the pups, were analyzed at each time point using one-factor analysis of variance. On day 21, body weights were subjected to a two-factor analysis of variance with Sex and Treatment Group as variables. Subsequent comparisons between NTX-treated and saline-injected animals were made using Newman–Keuls tests.

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FIG. 2. Latencies (s) on the hot-plate (557C) of rats treated daily during pregnancy with 50 mg/kg naltrexone (NTX) (n Å 11) or saline (CO) (n Å 12); animals were evaluated 22 h following an injection of drug on the day of parturition. At least 10 rats in each group were tested. Data represent means { SEM. The baseline values for NTX and CO groups did not differ. Significantly different from the baseline CO latencies, as well as baseline NTX levels, at **p õ 0.01.

In addition, data for both body weights and crown to rump lengths were subjected to a repeated measure, mixed effects analysis (6) using PROC Mixed version 6.11 software (SAS Institute, Cary, NC). A series of models were fitted with main effects, quadratic terms, and interaction terms with model fits and examined by plotting residuals.

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FIG. 3. Body weights of offspring born to mothers receiving daily injections of either 50 mg/kg naltrexone (NTX) or saline (CO) throughout gestation. Pups were culled to litters of 10 each and cross-fostered at birth to untreated females. Values are means { SEM for at least 40 pups per group at each age; at 21 days, no differences between males and females were noted and sexes were combined. Significantly different from controls at *p õ 0.05 and **p õ 0.01.

On the first day of pregnancy, nociceptive tests showed no differences between the randomly assigned females; controls had a 19.8 { 1.1 s latency, whereas pregnant females in the NTX group had a mean latency of 19.3 { 1.0 s. The dosage of NTX chosen for study (i.e., 50 mg/kg) has been reported to block opioid receptors for 24 h in 21-day old rats (30). To determine whether the dosage of NTX blocked opioid receptors throughout the entire day during gestation, female rats were challenged with morphine 22 h following NTX or saline injections on the day of parturition. Control rats exhibited a baseline latency that was equivalent to that of NTX-injected animals (Fig. 2). Following

RESULTS

Maternal Observations Daily injections of 50 mg/kg NTX throughout gestation had no detrimental effects on pregnancy or maternal behavior. The body weights of NTX- and saline-treated female rats were similar on the initial day of pregnancy, and no significant differences in body weights were noted throughout gestation (Fig. 1). Following parturition, mothers of the NTX and control groups appeared to clean and nurse their young in a similar fashion. TABLE 1 EFFECTS OF DAILY INJECTIONS OF NALTREXONE OR SALINE THROUGHOUT PREGNANCY ON THE LENGTH OF GESTATION, LITTER SIZE, AND NUMBER OF STILLBORNS

Gestation Length (days) Litter Size Stillborns/Deaths

Controls

Naltrexone

22.5 { 0.2 14.7 { 0.8 0.6 { 0.2

23.1 { 0.1 12.1 { 0.9 1.1 { 0.5

Values represent means { SEM for the litters of pregnant rats injected daily with 50 mg/kg NTX (n Å 11) or saline (control) (n Å 12) throughout pregnancy. No significant differences were noted between groups in any parameter assessed.

FIG. 4. Crown to rump lengths of rat pups born to mothers injected daily with either 50 mg/kg naltrexone (NTX) or saline (CO) throughout gestation. At birth, litters were culled to 10 pups each and cross-fostered to untreated mothers. Values are means { SEM for at least 40 pups per group. At 21 days, no differences in crown to rump lengths of male and female rats were observed and sexes were combined. Significantly different from controls at *p õ 0.05 and **p õ 0.01.

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MC LAUGHLIN ET AL. injection of 10 mg/kg morphine, the control animals had a twofold greater latency response time than their baseline levels. NTX-injected rats had a mean latency time that was comparable to baseline values. Moreover, the latency for the post-morphine level of controls differed significantly (p õ 0.03) from the postmorphine level of rats in the NTX group. Neonatal Observations The length of time for gestation, size of litters, and neonatal mortality did not differ between animals born to females in the NTX- or saline-injected groups (Table 1). The mean total pup mass of all animals (live or stillborn) from control mothers was 87.3 g, whereas the total weight of all pups born to NTX-treated females was 80.2 g. The difference in total pup mass between the two groups was not significantly different. Neonatal birth weights for NTX-treated rats were 8% greater than the birthweights of control rats; this difference was significant at p õ 0.01 (Fig. 3). Measurements of body weights on postnatal days 3, 6, 9, 12, 15, and 18 revealed that pups exposed prenatally to NTX weighed significantly more than control rats. On postnatal day 21, rats subjected to NTX during prenatal life weighed 36% more than control subjects (32.9 { 1.0 g), differing significantly (p õ 0.01) from the control animals. No differences were noted between the weights of male and females rats within either the saline- or NTX-treated groups at weaning. All repeated measures analyses indicated a substantial and significant difference in the growth patterns of the 2 groups over time. For example, at 21 days the predicted value of the NTX group was 55.9 g compared to 39.4 g of the control group. Measurements of crown to rump lengths at birth indicated that pups exposed to NTX during gestation were significantly ( p õ 0.01) longer than control rats (Fig. 4). Crown to rump lengths of rats delivered by mothers receiving NTX were significantly increased from control values on postnatal days 9, 15, 18, and 21 by 11%, 11%, 19%, and 4%, respectively (Fig. 4). Wet, dry, and relative organ weights at birth, 10 days, and 21 days for offspring of NTX and control females are presented in Figs. 5–7. No malformations could be detected for any organ in the NTX- or saline-treated pups. At birth, the wet and dry weights of the brain, heart, kidney, liver, and muscle were 14% to 65% greater for rats exposed to NTX in utero than for control animals (Fig. 5). The wet and dry weights of the lungs did not differ in neonates exposed during gestation to NTX or saline. Calculation of relative organ weights revealed that brain, kidney, and muscle of NTX-exposed rats were increased 26% to 32% above control levels, whereas relative lung weights of the NTX group were subnormal by 20%. The proportions of heart and liver wet weights to body weights were comparable in neonates delivered by mothers receiving NTX or saline. At 10 days of age, the wet and dry weights of all 6 organs examined—brain, heart, kidney, liver, lung, and muscle—were markedly increased for animals in the NTX group relative to control subjects (Fig. 6). Wet weights in the NTX group ranged from 17% (heart) to 60% (kidneys and muscle) greater than respective organ weights for control offspring. Dry weights of all

FIG. 5. Wet, dry, and relative organ weights of newborn rat pups born to mothers injected with either 50 mg/kg naltrexone (NTX) or saline (CO) throughout gestation. Brain (brn), heart (hrt), kidney (kdy), liver (liv), lung (lun), and triceps surae (mus) were weighed within 12 h of birth. Data represent means { SEM for at least 10 organs/group, with an equal number of males and females. Significantly different from controls at *p õ 0.05 and **p õ 0.01.

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505 organs were 17% to 100% greater than control weights. The ratio of wet weight to body weight (i.e., relative organ weight) were subnormal in the brain and heart, increased in comparison to controls in the kidney and muscle, and comparable to control values in liver and lung. At weaning, the wet and dry weights for all organs examined were substantially greater (p õ 0.01) in the NTX-treated rats than in control offspring (Fig. 7). Wet weights of the organs in the NTX group ranged from 18% (brain) to 246% (muscle) greater than control levels, and dry weights of pups exposed to NTX in prenatal life were 17% (lung) to 275% (muscle) greater than their control counterparts. The ratio of wet weight to body weight for liver, heart, and muscle were significantly elevated (13%, 17%, and 114%, respectively) for 21-day old rats that were subjected by the maternal route to NTX in contrast to control levels. DISCUSSION

The present study was intended to a) understand the role of opioids in the processes of pregnancy and parturition, b) inquire as to whether opioids are involved in embryonic/fetal development, and c) address the implications of continuous opioid-receptor blockade during the entire course of pregnancy on certain aspects of preweaning ontogeny. Using a paradigm of continually interfering with the interaction of opioid peptides and opioid receptors by application of NTX, we discovered that disruption of the endogenous opioid system has no effect on the length of gestation, course of pregnancy, litter size, embryo/fetal/neonatal viability, number of stillborns, or maternal weight gain. Although not rigorously measured, maternal food and water intake appeared to be unaffected by opioid receptor blockade during gestation. Thus, one can conclude that opioids have little influence on maternal factors associated with pregnancy and parturition. Interruption of opioid-receptor interfacing during prenatal life did have a significant impact on the offspring. At birth the neonates weighed, and were longer, than their control counterparts. The increases in weight and size of the newborn animals exposed in utero to NTX appeared to be inherent to intrinsic processes of growth, and not related to the size of the litter (e.g., fewer pups/ mother) or length of gestation (e.g., longer gestation times). We also found that the entire course of postnatal development with respect to the gain in body weight and body length were markedly greater in animals subjected to opioid receptor blockade during pregnancy. Moreover, a survey of five major organs showed that at birth, postnatal day 10, and at weaning (i.e., postnatal day 21) these organs (with the exception of the lungs at birth) weighed substantially more than their control counterparts. Measures of the dry weights of these organs showed parallel increases, suggesting that total cellular content was elevated. The increase in body weight generally correlated with the increases in weight and dry weights. Only the weight of the muscle in proportion to the body was elevated at all times monitored. The organs, however, exhibited no malformations at birth or throughout the preweaning period, nor were there any differences in mortality be-

FIG. 6. Wet, dry, and relative organ weights of postnatal day 10 rat pups born to mothers injected daily with either 50 mg/kg naltrexone (NTX) or saline (CO) throughout gestation and cross-fostered to untreated lactating rats. Brain (brn), heart (hrt), kidney (kdy), liver (liv), lung (lun), and triceps surae (mus) were assessed. Data represent means { SEM for at least 10 organs/group, with an equal number of males and females. Significantly different from controls at *p õ 0.05 and **p õ 0.01.

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MC LAUGHLIN ET AL. tween the animals subjected to NTX or saline prenatally, indicating that opioid antagonist exposure in early life does not lead to biological impairments and/or inadequacies. These results are the first to document the importance of opioid-receptor interaction in the prenatal period as a determinant of offspring characteristics both in the neonate and throughout the preweaning period. The effects of NTX-induced changes in ontogeny as to the physical, mental, and emotional well-being of the neonate and infant, as well as the long-term impact on juvenile and adult animals, requires elucidation. A number of investigators have examined the effects of prenatal exposure to NTX on maternal and offspring characteristics (4,5,7,9–12,16,18–20,22,23,25,26). Despite the use of 2 different opioid antagonists—NTX and naloxone, and differences in species (rats, mice), method of exposure (e.g., oral, intraperitoneal), timing of exposure during gestation (e.g., prenatal days 2 or 3, last trimester), and the extent of exposure (days, weeks), a number of observations have come forth that allow us to understand the repercussions of opioid-receptor manipulation during prenatal life. Thus, with some exceptions (e.g., 9), the reproductive capacity of adult females used for breeding (19), incidence of pregnancy (19), maintenance of pregnancy and/or maternal weight (5,10,16,18,20,25), maternal behavior (5), implantation (18), litter size (5,10–12,16,19,20,22,25), gestation time (10,11,25), ratio of males to females (20,22,25), mortality of offspring (16,25), and body weight at birth and/or in the preweaning period (7,10,11,16,20,22,25) were not affected by NTX. This included results obtained from experimental paradigms in which nociceptive tests were administered to determine the activity of the opioid antagonist so as to insure opioid receptor blockade (4,10). The present study, which eliminated the repercussions of a restricted timetable of blockade during pregnancy, the confounding influences accompanying drug exposure and termination, and the failure in many reports to cross-foster opioid antagonist-exposed pups to normal mothers in order to reduce continuing drug exposure and/or maternal dysfunction with respect to behavior, utilized a paradigm of continuous interruption of opioid peptide-receptor blockade that extended throughout the prenatal period. The results of this investigation confirm and extend many of these earlier observations as to the lack of effects on pregnancy and parturition. However, we now can observe that opioid receptor blockade instituted in the pregnant female has extensive ramifications with respect to body and organ development in the neonate and preweaning animal. These results would infer that opioids function in the embryonic and fetal rat to tightly regulate somatic properties. It is valuable to compare and contrast the present study concerned with the effects of disruption of opioid-receptor relationship during prenatal life on postnatal development, with early data in which the influence of opioid antagonist exposure only during the preweaning period was examined (28–32). In general the effects of continuous opioid peptide-receptor blockade with an opioid antagonist during the prenatal period introduced changes in body and organ development of greater magnitude

FIG. 7. Wet, dry, and relative organ weights of postnatal day 21 rat pups born to mothers injected with either 50 mg/kg naltrexone (NTX) or saline (CO) throughout gestation and cross-fostered to untreated lactating rats at birth. Brain (brn), heart (hrt), kidney (kdy), liver, (liv), lung (lun), and triceps surae (mus) were evaluated. Data represent means { SEM for at least 10 organs/group, with an equal number of males and females; no differences in males and females were noted and sexes were combined. Significantly different from controls at *p õ 0.05 and **p õ 0.01.

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than those occurring when drug exposure was restricted to the postnatal period. Thus, animals at postnatal day 21 exposed to NTX in prenatal life weighed 36% more than controls, and the wet weights of the brain, heart, kidney, liver, and skeletal muscle were increased 18%, 42%, 38%, 44%, and 247%, respectively, from control levels. On the other hand, animals exposed to NTX from birth to day 21 weighed 20% more than controls, and had increases in the brain, heart, kidney, liver, and skeletal muscle of 20%, 4%, 12%, 25%, 24%, and 60%, respectively, from control values. These data would lead us to suggest that somatic size and the weight of organs in the adult are determined both prenatally and postnatally, and that prenatal events play an important role in conferring a pattern of growth that extends (at least) into the early postnatal period. It would appear that the prenatal organism depends upon opioid peptides for ‘‘normal’’ growth. Deprivation of the signaling from opioids by the introduction of opioid receptor blockade permits an enhancement of these developmental events. Therefore, opioids must control ontogeny by exerting a repressive effect on growth, and must do so in a tonic fashion. There is sufficient evidence that at least one opioid peptide, [Met 5 ]enkephalin-termed opioid growth factor (OGF), governs cell proliferation and does so directly (e.g., 27,33–35). OGF is an

autocrine growth factor produced by both neural and non-neural cells in biological systems that are undergoing development, cellular renewal, or repair. In addition, the z opioid receptor has been identified as the mediator of OGF action with respect to growth. In the present experiments we have learned that blockade of opioids from interacting with opioid receptors has considerable meaning to the growth of the fetus and infant rat. It could be postulated that daily administration of the opioid antagonist NTX interferes with the delicate equilibrium between OGF and the z receptor—both presumably associated with developing cells of the body and organs; OGF and the z receptor have been localized to a variety of developing and renewing cells and tissues. Blockade of OGF-z receptor interaction would thereby permit cells to escape the regulating nature of OGF and accelerate replication. This would have the net effect of increasing somatic and organ development. Such a hypothesis merits further consideration in order to comprehend the importance of opioids in dictating the organization of the body and its contents. ACKNOWLEDGMENTS

We thank Dr. A. Russell Localio, Center for Biostatistics and Epidemiology, for assistance in the statistical analysis. This study was supported by NIH grants NS-20500 and HL-53557.

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