Developmental toxicity of terbutaline: Critical periods for sex-selective effects on macromolecules and DNA synthesis in rat brain, heart, and liver

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1 Brain Research Bulletin, Vol. 59, No. 4, pp , 2003 Copyright 2002 Elsevier Science Inc. All rights reserved /02/$ see front matter PII: S (02) Developmental toxicity of terbutaline: Critical periods for sex-selective effects on macromolecules and DNA synthesis in rat brain, heart, and liver Melissa C. Garofolo, Frederic J. Seidler, Mandy M. Cousins, Charlotte A. Tate, Dan Qiao and Theodore A. Slotkin Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA [Received 5 August 2002; Revised 2 September 2002; Accepted 15 September 2002] ABSTRACT: β-adrenoceptors (βars) control cell replication/differentiation, and during development, signaling is not subject to desensitization. We examined the effects of terbutaline, a β 2 AR agonist used as a tocolytic, on development in rat brain regions and peripheral tissues with high βar concentrations. Prenatal terbutaline (gestational days 17 20) decreased cell numbers (DNA content) in the fetal brain and liver. Early postnatal exposure (PN2 5) reduced DNA synthesis in early-developing brain regions of females, with sensitization of the effect upon repeated terbutaline administration; after multiple terbutaline injections, DNA content was reduced in male cerebellum. The cerebellum was targeted later (PN11 14), exhibiting decreased DNA synthesis in both sexes; in contrast, cardiac DNA synthesis decreased after one injection but increased after the fourth daily injection. Our results suggest that excessive βar stimulation by terbutaline alters cell development in brain regions and peripheral tissues, with the net effect depending on sex and the timing of exposure. These effects may contribute to neuropsychiatric, cognitive, cardiovascular, and metabolic abnormalities reported in the offspring of women treated with β-agonist tocolytics Elsevier Science Inc. All rights reserved. KEY WORDS: β-adrenoceptor, Brain development, Cell development, DNA synthesis, Heart development, Liver development, Preterm labor, Terbutaline, Tocolysis. INTRODUCTION In the U.S., preterm labor occurs in up to 20% of all pregnancies, with preterm delivery, a leading cause of neonatal morbidity and mortality, in about half the cases [10]. Terbutaline and other β 2 -adrenoceptor (β 2 AR) agonists are commonly used as tocolytics, and although terbutaline is considered generally safe, it is important to note that preterm labor is not on FDA s list of approved uses. In addition to blocking uterine contractions, terbutaline penetrates the placenta to activate fetal βars [2,9,22 44,65]. In turn, βar stimulation promotes the respiratory and cardiovascular adaptations necessary for successful perinatal transition [8,32,33,35], so that terbutaline and related drugs provide a beneficial outcome in the event of preterm delivery. Nevertheless, it is increasingly evident that fetal exposure to βar tocolytics also has deleterious longer-term effects, such as alterations in postnatal glucose metabolism, tachycardia, and an elevated incidence of cardiac abnormalities [11,13,21,36,49]. More recently, neurobehavioral consequences have been identified: impaired school performance [20,25], cognitive dysfunction, and psychiatric disorders [48]. Stimulation of fetal βars is likely to be responsible both for the beneficial and harmful effects of terbutaline and related drugs. The linkage of these receptors to the production of cyclic AMP contributes to tocolytic and fetal cardiorespiratory effects [44,51,74], but is also likely to have an adverse effect on cell development in fetal tissues that have high concentrations of βars. In virtually all prokaryotic and eukaryotic cells, cyclic AMP controls cell development, generally by initiating the switch from cell replication to differentiation [12,15,24,27,66 68,78]. In addition, excessive βar stimulation can elicit both apoptosis [16,23,53,54,81] and necrosis [28,76] of cells expressing high concentrations of βars. Mature cells are protected from excessive βar stimulation by a specific adaptation, desensitization, which uncouples receptors from their ability to elicit intracellular responses [46,73]; as just one example, desensitization protects mature cardiac cells from necrosis induced by isoproterenol [28]. However, in the fetus or neonate, βars are resistant to desensitization [72,83]. In the case of terbutaline, we found that even repeated administration of high doses failed to desensitize βars in the fetal/neonatal heart, liver or brain, and instead elicited an increase in responsiveness, thus augmenting the net effect on cyclic AMP production [2,3,65]. The early appearance of βars in the fetus, well before the development of presynaptic innervation [55,61], their overexpression in many fetal tissues [61], and their linkage to cyclic AMP production through adenylyl cyclase [61], may thus render cell development vulnerable to βar stimulants such as terbutaline [30,43,44,56 58,60,64]. In the current study, we examined the effects of terbutaline administered to fetal or neonatal rats, on biomarkers of cellular development in brain regions that differ in their maturational timetables, as well as in their proportions of βar subtypes [19,38,52]; we also Abbreviations: ANOVA, analysis of variance; βar, β-adrenoceptor; GD, gestational day; PN, postnatal day Address for correspondence: Dr. T. A. Slotkin, Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710, USA. Fax: ; t.slotkin@duke.edu 319

2 320 GAROFOLO ET AL. compared effects in the heart, which has a majority of β 1 ARs, and the liver, in which β 2 ARs predominate [2,68]. We evaluated DNA synthesis as an index of cell replication, DNA content as a marker of cell number, DNA concentration (DNA per gram of tissue) as an assessment of cell packing density, and the protein/dna ratio as an index of relative cell size [7,63,80]. Because brain development in the newborn rat approximates the stage of neural maturation found in the third trimester human fetus [52], we also contrasted the effects of terbutaline on the fetal rat brain to those seen at two different postnatal stages. METHODS Animals and Treatments All experiments were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed-pregnant rats (Zivic Laboratories, Pittsburgh, PA) were housed in breeding cages, with a 12 h/12 h light/dark cycle and free access to food and water. For studies of prenatal treatment, dams were given daily subcutaneous injections of 10 mg/kg of terbutaline hemisulfate (Sigma Chemical Co., St. Louis, MO) or an equivalent volume (1 ml/kg) of isotonic saline vehicle on gestational days (GD) 17, 18, 19, and 20. This terbutaline regimen has been shown to elicit robust βar stimulation in the fetus, simulating the effects seen with its use as a tocolytic, including cardiac activation and enhancement of fetal lung surfactant synthesis [29,32,44]. For prenatal determinations, the individual fetus represented a single sample; to avoid bias from repeated sampling of the same litter, each dam contributed only one fetus to a given determination. The rat is altricial, so that neural development of the neonate more closely resembles that of a third trimester human fetus [52]. Accordingly, we also examined the effects of two different postnatal (PN) terbutaline treatments, one centered around the early postnatal period and the other spanning the time of eye opening. Pups from all litters were randomized on the day after birth and redistributed to the dams with litter sizes of 10 pups to ensure standardized nutrition and maternal care. Pups were then given daily injections of 10 mg/kg of terbutaline or equivalent volumes of vehicle on PN2 5 or PN11 14; treatment groups were sex-matched, containing approximately equal proportions of males and females. Each litter contributed no more than one male and one female to each determination. DNA and Protein Determinations Twenty-four hours after the last injection of terbutaline or vehicle, brains, hearts, and livers were removed and, for postnatal determinations, the brain was dissected further into three regions: blunt cuts were made through the cerebellar peduncles and the cerebellum (including flocculi) was lifted from the underlying tissue; a cut was then made rostral to the thalamus to separate the forebrain from the brainstem. Tissues were frozen in liquid nitrogen and stored at 45 C. For macromolecule determinations, tissues were thawed and homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in 19 volumes of a buffer containing 0.1 M NaCl, 1 mm EDTA, and 10 mm Tris (ph 7.4) and then sonicated (Virsonic Cell Disrupter, Virtis Company, Gardiner, NY). DNA was determined using a modified [77] fluorescent dye-binding method [34] and protein spectrophotometrically with bicinchoninic acid [69]. Utilizing the established relationship between DNA levels and cell number [80], we focused on indices of cell packing density (DNA per gram of tissue), cell number (DNA content) and cell size (protein/dna ratio) [7,63]. DNA Synthesis Incorporation of [ 3 H]thymidine into DNA was assessed 4 h after the first and last terbutaline injections, a time period corresponding to robust antimitotic effects noted for β-agonists in earlier studies [18,66,67]. Rats were given 1 µci/g b. wt. of [ 3 H]thymidine (specific activity, 2 Ci/mmol, Amersham Biosciences, Piscataway, NJ). After 30 min, tissues were dissected as already described, chilled in a minimum of 9 volumes of ice-cold water, and homogenized (Polytron). A 100-µl aliquot was digested overnight at 37 C with 1 ml of benzethonium hydroxide, acidified with 100 µl of glacial acetic acid, and counted by liquid scintillation spectrometry to determine total tissue uptake of radiolabel. In another 1-ml aliquot, DNA was precipitated with 10% trichloroacetic acid, sedimented at 1000 g for 15 min; the resultant pellet was washed twice by resuspension (Polytron) in trichloroacetic acid and resedimented, and the final pellet was digested to determine the radiolabel incorporated into DNA. Because the incorporation of radiolabel into DNA is dependent upon the amount of [ 3 H]thymidine taken up by the tissue, synthesis was evaluated as the ratio of trichloroacetic acid-precipitable label to total label in the tissue (fractional incorporation). Effects on incorporation of label into DNA therefore reflect changes in the synthetic rate and not differences in bioavailability or cellular uptake of radiolabel. Measurements of DNA synthesis were discontinued after PN5 for the early-developing brain regions (brainstem, forebrain) because of low activities reflecting the closure of neurogenesis [41,52,67]. Data Analysis Data are presented as means and standard errors. Differences between groups were first assessed by a global analysis of variance (ANOVA, data log-transformed because of heterogeneous variance), incorporating all factors: drug treatment, treatment period, tissue, age, and sex; however, sex differences were not assessed for fetal determinations. Because the indices of cell packing density (DNA per gram of tissue) and cell number (DNA per tissue) were based on the same measurement, these were treated as repeated measures and tested together. Depending on the treatment interactions obtained in a global test, data were then subdivided for lower order ANOVAs. When appropriate, individual differences were established post hoc using Fisher s Protected Least Significant Difference; however, where there were no interactions of treatment other variables, only the main effect was reported, without testing of individual values. Where there was a significant interaction of treatment sex, or of treatment sex other variables, separate testing was conducted for males and females; otherwise, results were combined for the two sexes. Significance for main treatment effects was assumed at p < 0.05; however, for interactions at p < 0.1, we also examined whether lower order main effects were detectable after subdivision of the interactive variables [70]. For convenience, some data are presented as the percentage change from control values; however, statistical evaluations were always carried out on the original data. RESULTS Ontogenetic Profiles of Macromolecules and DNA Synthesis The relative growth rates of the different brain regions and tissues in control rats mirrored the specific postnatal growth spurt [52]. Between PN6 and PN15, during which time the animals nearly doubled their body weights, the cerebellum increased 3.5-fold in weight, as compared to only a 60% increase in the brainstem, the earliest-developing brain region, and an 80% increase in the forebrain, which has a maturational profile intermediate between those of the brainstem and cerebellum (Fig. 1). The heart and

3 DEVELOPMENTAL TOXICITY OF TERBUTALINE 321 FIG. 1. Development of body and tissue weights in control animals. Data represent means and standard errors obtained from the number of animals shown in parentheses. ANOVA across all tissues and ages appears at the top of the panel. Values were combined for males and females because of the absence of significant sex-related differences, with the exception of body weight on PN6, where the values were: male 17.2 ± 0.3 (11), female 15.6 ± 0.3 (10), p< liver grew more slowly over the same period, by 55 and 40%, respectively. Differences in males and females were significant only for body weights on PN6. The various growth rates among tissues and brain regions were also evident in indices of cell packing density, cell number, and cell size. In keeping with the maturational timetable for neurogenesis, cell packing density increased from PN6 to PN15 in the cerebellum while decreasing in the earlier-maturing brain regions; only small increments were seen in the heart and the liver (Fig. 2A). Superimposing the changes in cell packing density on the large growth postnatal spurt of the cerebellum, the total number of cells, as assessed by the DNA content, showed a 5-fold increase by PN15, whereas the increases were far more modest for the other brain regions (Fig. 2B). The heart continued to show a substantial increase in DNA content over the same period, whereas smaller, but sizeable increments were seen in the liver. Also in keeping with these inherent differences in ontogenetic patterns, postnatal growth of the brainstem and forebrain primarily involved cell enlargement, as indicated by a rise in the protein/dna ratio, whereas in the cerebellum, the ratio remained fairly constant over the same period (Fig. 2C). The heart and liver displayed intermediate patterns, with small, but significant increases in protein/dna. [ 3 H]Thymidine incorporation into DNA, a biomarker for de novo DNA synthesis, displayed maturational profiles consistent with the postnatal peak of neurogenesis in the cerebellum. On PN2, approximately 35% of the radiolabel taken up into this brain region was incorporated into DNA within a 30-min period, declining to about 25% by the end of the second postnatal week (Fig. 2D). In contrast, incorporation values on PN2 in the brainstem and forebrain were only 15 and 10% respectively, declining to under 10% by PN5; we did not assess DNA synthesis in these regions at later time points due to extremely low values commensurate with the cessation of neurogenesis [41 67]. DNA synthesis in the heart also fell off in the second postnatal week, similar to that seen in earlier studies [50]. Global Statistical Analyses of Terbutaline Effects In order to avoid type I statistical errors in subdividing the data into the different measures, tissues, ages, and sexes, we first performed global ANOVAs on data groupings. Because we did not evaluate sex for GD20 or GD21 fetuses, one ordering examined effects across all ages, but without regard to sex. Sex was then included in separate analyses of the postnatal data points. Similarly, the fetal brain was not subdivided into its constituent regions, so that separate classification had to be considered for prenatal versus postnatal determinations. For body weights, ANOVA across all ages indicated a main treatment effect (p < 0.02). Values were not significant across the GD20 and GD21 fetuses; however, for the postnatal points (including the sex variable), we found a significant treatment effect (p <0.02) without interactions between treatment and age or sex. Considered across all ages (including GD21), peripheral tissue weights showed a significant treatment tissue interaction (p <0.03); however, there were no significant differences when evaluations were conducted for just the postnatal age points, again including the sex variable. Fetal brain weight showed no significant differences across the two age points (GD20 and GD21); however, postnatal treatments elicited a significant main effect of

4 FIG. 2. Development of cell parameters in control animals. Data represent means and standard errors obtained from the number of animals shown in parentheses. ANOVA across all tissues and ages appears at the top of each panel. Values were combined for males and females because of the absence of significant sex-related differences, with the exception of forebrain DNA concentration on PN6, where the values were: male 0.87 ± 0.01 (11), female 0.94 ± 0.02 (10), p<0.04. Despite the significant overall interaction of age sex for DNA synthesis, none of the individual values showed differences between males and females. 322 GAROFOLO ET AL.

5 DEVELOPMENTAL TOXICITY OF TERBUTALINE 323 terbutaline (p <0.04) that depended upon age, brain region, and sex (treatment age region sex, p<0.1). There were two interrelated measures of cell number (DNA content) and cell packing density (DNA concentration). We, therefore, utilized a repeated-measures design to combine the effects across the two indices. For peripheral tissues across all ages, including GD21 and, therefore, excluding the sex variable, we found significant interactions of treatment tissue age (p <0.04) and treatment tissue measure (p <0.03). For the fetal determinations, there were interactions of treatment measure (p <0.07) and treatment tissue measure (p <0.009); for postnatal determinations, including the sex variable, we found interactions of treatment age sex (p <0.07) and treatment age tissue (p <0.07). There were no significant effects for fetal whole brain across the two age points (GD20 and GD21), but there were differences in the DNA markers for postnatal ages: treatment region (p <0.1), treatment measure (p <0.05), treatment age sex region measure (p <0.1). In contrast, there were no significant differences in the protein/dna ratio for any of the data groupings, although some tissues did show alterations in the protein concentration (see below). Measurements of DNA synthesis could not be compared across all tissues at all ages because values in the forebrain and brainstem were too low for accurate assessment on PN11 and PN14. Limiting the global ANOVA to the two tissues for which all ages could be evaluated (cerebellum and heart), there were significant interactions of treatment injection number (p <0.04), treatment injection number tissue (p <0.03), treatment sex age (p <0.09), and treatment injection number age tissue (p < ). Evaluating the results on PN2 and PN5 across all the tissues, there were significant interactions of treatment injection number (p < 0.05) and treatment sex (p <0.04). For PN11 and PN14, the interactions were significant for treatment injection number (p < 0.04) and treatment tissue injection number (p <0.0005). In light of the highly interactive nature of terbutaline s effects, we separated the data along logical lines for subsequent analyses and data presentation: first, the different treatment periods, then the separate tissues, and finally, the specific measures. Treatment on GD17 20 In keeping with earlier results [65], prenatal terbutaline treatment elicited no maternal mortality and did not alter the number of fetuses. Body weights, assessed on GD20 (24 h after the third terbutaline injection) or GD21 (24 h after the fourth injection), showed no significant difference (Fig. 3A). Fetal brain weight was unaffected by the treatment, as was heart weight; liver weight was significantly reduced by about 10 15%. Similarly, the liver was the only tissue to show a significant change in macromolecules, FIG. 3. Effects of gestational terbutaline administration (GD17 20) on body and tissue weights and on cell parameters. Data represent means and standard errors obtained from the number of animals shown in parentheses, presented as the percent change from the corresponding control values (Figs. 1 and 2). ANOVA across all tissues appears at the top of each panel. Asterisks (*) denote the individual values showing differences between terbutaline and control groups, tested only where the ANOVA indicated a significant interaction of treatment tissue; otherwise, statistical determinations were limited to the main effects across tissues.

6 324 GAROFOLO ET AL. a decrease in the DNA content indicative of reduced numbers of cells (Fig. 3B). Although there was a small (5%) deficit in DNA concentration and content in the fetal brain on GD21, the difference fell just short of statistical significance in a two-tailed test (p <0.08). However, based on prior information about inhibition of cell replication by βar agonists in developing noradrenergic target tissues [15,30,66 68], a specific direction of change (i.e., a decrease) was expected; the deficit in DNA concentration content evoked by terbutaline in the brain on GD21 was significant in a one-tailed test of this hypothesis (p <0.04). With fetal terbutaline treatment, we did not observe any significant changes in the biomarker of cell size, the protein/dna ratio (Fig. 3C). Effects of Early Postnatal Terbutaline Treatment The fetal brain and liver overexpress β 2 ARs [19,38,42,61], so that the targeting by prenatal terbutaline administration suggested a relationship between receptor expression and adverse effects. Accordingly, we examined effects of neonatal terbutaline in brain regions and peripheral tissues possessing different proportions of βar subtypes. Terbutaline administration on PN2 5 elicited a significant, sex-dependent decrement in tissue weights: males were affected but not females, and the most notable effects were seen in the cerebellum as well as the liver (Fig. 4A). Most of the cerebellar weight deficit represented a reduction in the total number of cells, as the DNA content was reduced by a similar amount, without any commensurate change in the DNA concentration (Fig. 4B). The neonatal heart, which possesses a majority of the β 1 AR subtype [2,68], showed no changes; however, we also did not see effects on the brainstem or forebrain, brain regions which, at this stage of development, possess a relatively high proportion of the β 2 subtype [19,47]. There were no significant changes in the protein/dna ratio assessed across all the tissues (Fig. 4C); however, the forebrain showed a significant decrease (p <0.04) if evaluated separately from the other tissues, reflecting a reduction in the protein concentration (p <0.04, data not shown), rather than changes in the DNA denominator term. In light of the differential targeting of the cerebellum as compared to the other brain regions or the heart, and because of the sex FIG. 4. Effects of terbutaline administration on PN2 5. Data represent means and standard errors obtained from the number of animals shown in parentheses, presented as the percent change from the corresponding control values (Figs. 1 and 2). ANOVA across all tissues and both sexes appears at the top of each panel; when a significant treatment sex interaction was found, values were then subdivided by sex for lower order ANOVAs shown within the panels. Asterisks (*) denote the individual values showing differences between terbutaline and control groups, tested only where the ANOVA indicated a significant interaction of treatment tissue; otherwise, statistical determinations were limited to the main effects across tissues. Abbreviations: BS, brainstem; FB, forebrain; CB, cerebellum; HT, heart; LIV, liver. FIG. 5. Effects of terbutaline administration on PN2 5, on DNA synthesis measured 4 h after the first or fourth injection. Data represent means and standard errors obtained from the number of animals shown in parentheses, presented as the percent change from the corresponding control values (Figs. 1 and 2). ANOVA across all tissues, both time points and both sexes, appears at the top of the panel. Because a significant treatment sex interaction was found, values were then subdivided by sex for lower order ANOVAs shown within the panel. Asterisks (*) denote the individual values showing differences between terbutaline and control groups, tested only where the ANOVA indicated a significant interaction of treatment tissue; otherwise, statistical determinations were limited to the main effects across tissues. Abbreviations: BS, brainstem; FB, forebrain; CB, cerebellum; HT, heart.

7 DEVELOPMENTAL TOXICITY OF TERBUTALINE 325 differences, we next evaluated the effects of terbutaline on DNA synthesis (Fig. 5). With the first injection of the β 2 AR agonist, we did not observe any significant decrement in DNA synthesis in either males or females, and if anything, values tended to be elevated in males. With the fourth injection, we found significant impairment of DNA synthesis, but surprisingly, the effects did not match those seen for tissue weight or DNA content. Reductions were significant in females, not males, and no changes were seen for the cerebellum, whereas other brain regions and the heart were affected. Effects of Late Postnatal Terbutaline Treatment In contrast to the effects of terbutaline administered on PN2 5, administration on PN11 14 failed to cause a decrease in tissue weight in the cerebellum or liver; instead, heart weight was significantly elevated (Fig. 6A). Again, we found sex-dependent alterations in DNA concentration and content (Fig. 6B). The sex differences primarily reflected tendencies toward reduced values in the brainstem of males but not females, and increases in cardiac and hepatic DNA content in females but not males; however, none of these effects achieved statistical significance individually. As with the earlier terbutaline treatment, there was no effect on the protein/dna ratio; the liver protein concentration was significantly elevated (p <0.05, data not shown), albeit not sufficiently to alter the protein/dna ratio. With terbutaline treatment on PN11 14, measurements of DNA synthesis were limited to the cerebellum and heart, the two tissues that maintain high rates of [ 3 H]thymidine incorporation into the second postnatal week (Fig. 7). The first injection of terbutaline elicited a significant decrement in cardiac DNA synthesis; by the fourth injection, however, the effect was reversed, with the terbutaline group showing an equally large increase in incorporation. The cerebellum showed a different pattern: across the first and fourth injection values, DNA synthesis was decreased significantly; rather than showing reversal by the fourth injection, the adverse effect of terbutaline was intensified. FIG. 6. Effects of terbutaline administration on PN Data represent means and standard errors obtained from the number of animals shown in parentheses, presented as the percent change from the corresponding control values (Figs. 1 and 2). ANOVA across all tissues and both sexes appears at the top of each panel; when a significant treatment sex interaction was found, values were then subdivided by sex for lower order ANOVAs shown within the panels. Asterisks (*) denote the individual values showing differences between terbutaline and control groups, tested only where the ANOVA indicated a significant interaction of treatment tissue; otherwise, statistical determinations were limited to the main effects across tissues. Abbreviations: BS, brainstem; FB, forebrain; CB, cerebellum; HT, heart; LIV, liver. DISCUSSION Unlike the adult, βars in the fetus and neonate are resistant to agonist-induced desensitization and, after prolonged exposure to receptor stimulants, actually increase their ability to generate cyclic AMP, primarily by inducing adenylyl cyclase activity [2,3,65,72,83]. In turn, the close involvement of cyclic AMP in the switch from cell replication to differentiation [12,15,24,27,66 68,78], suggested to us that tocolytic drugs, such as terbutaline, might have an adverse effect on cell development. As shown in the current study, neonatal rats given a single injection of terbutaline on PN2 did not show any significant reduction in DNA synthesis; however, decreases were readily demonstrable by the fourth injection on PN5, the period over which agonist-induced sensitization of adenylyl cyclase develops [2,3,65,83]. Similarly, with terbutaline administration commencing on PN11, there was only a small reduction in cerebellar DNA synthesis, but by the fourth injection, a larger response was obtained, rather than the desensitization that is typically seen for cyclic AMP-mediated responses in the adult [46,73]. If that were the only effect of terbutaline on cell development, then it would be expected that the total number of cells (DNA content) and cell packing density (DNA concentration) would both decline after repeated treatment, and we found a decrease in the fetal brain after terbutaline administration from GD For the PN2 5 regimen, we also saw a deficit in DNA content in the cerebellum on PN6, but this region actually showed little or no reduction of DNA synthesis. Equally surprising, terbutaline treatment on PN11 14, which did decrease DNA synthesis in the cerebellum, had little or no effect

8 326 GAROFOLO ET AL. FIG. 7. Effects of terbutaline administration on PN11 14, on DNA synthesis measured 4 h after the first or fourth injection. Data represent means and standard errors obtained from the number of animals shown in parentheses, presented as the percent change from the corresponding control values (Figs. 1 and 2). ANOVA across both tissues, both time points and both sexes, appears at the top of the panel. Because a significant treatment sex interaction was not found, values were not subdivided by sex. Asterisks (*) denote the individual values showing differences between terbutaline and control groups, tested only where the ANOVA indicated a significant interaction of treatment injection #; otherwise, statistical determinations were limited to the main effects across injections. on cerebellar DNA content or concentration; furthermore, in the other brain regions, there were robust effects of the early postnatal treatment regimen on DNA synthesis but there was no corresponding decrease in indices of cell number or packing density. The sex-dependence also was different for effects on DNA synthesis versus DNA content/concentration; the reason for sex differences will be discussed below. The discrepancies between terbutaline s effects on DNA synthesis and on DNA content/concentration suggests that additional mechanisms contribute to the outcome. Prolonged βar stimulation is likely to result in apoptosis [16,23,53,54,81] or necrosis [28,76]. Whereas receptor uncoupling protects mature cells from these effects [28], the fact that stimulation is unrestrained by desensitization in the immature organism may render the cells especially vulnerable and cell death is indeed seen in neonatal cardiac tissue after tocolytic therapy [11,21]. For these effects, differential expression of βar subtypes may contribute to the pattern of vulnerability. Brain regions that, in the adult, express a predominance of the β 1 subtype, instead express higher proportions of β 2 ARs in the fetus or neonate [19,38,47]. Whereas β 1 AR stimulation elicits apoptosis, β 2 AR effects appear to be protective [1,14,16,17,82,84,85]; the higher proportion of β 1 ARs in the heart may thus be the reason why cell death occurs in response to fetal terbutaline exposure. Future studies will thus need to address whether terbutaline similarly elicits apoptosis in the developing brain, and whether the window of vulnerability and pattern of effects mirror the ontogenetic patterns of receptor subtype expression. Receptor subtype differences certainly cannot explain the differential susceptibility of brain regions to inhibition of DNA synthesis in the early neonatal period (PN2 5). The cerebellum, the region with the highest proportion of β 2 ARs [19,38,47], showed the lowest reactivity to terbutaline in terms of inhibition of DNA synthesis in the first postnatal week. Then, at later stages, after the closure of neurogenesis in the other regions, the cerebellum showed an increased ability of terbutaline to repress DNA synthesis. Accordingly, regional timetables of cell maturation must play a critical role. βars are coupled to inhibition of DNA synthesis for only a brief period, commencing just before the normal ontogenetic decline in cell replication [15,18,66,67]. Prior to that time, low levels of tonic stimulation provide a trophic signal that sustains cell replication [18,31,50,59,62]. In the current study, we saw some evidence of this in the tendency of terbutaline to enhance DNA synthesis on PN2. The much later timetable for cerebellar neurogenesis, centering around the second postnatal week [52], is likely to contribute to the shift in terbutaline s targeting of DNA synthesis from the brainstem and forebrain with early treatment, to the cerebellum with later treatment. The net effect on DNA content/concentration thus represents the summation of stimulatory and inhibitory effects on replication, likely superimposed on apoptosis, in populations of cells whose maturational vulnerabilities change with development. Consequently, even for those treatments and regions where cell numbers remain apparently unchanged, it is possible that there are alterations in the types of cells that are present; studies are underway to examine specific markers for different cell types as well as morphological indices to elucidate this issue. In agreement with this interpretation, earlier work identified specific interference with the development of cerebellar noradrenergic neurons after fetal terbutaline exposure [43,56,57,64]. Interestingly, abnormalities of cerebellar structure and catecholaminergic function are implicated in the etiology of autism [5,6,40]. It would be worthwhile to examine a potential relationship to this disorder, parallel to the other neurodevelopmental problems that have already been identified for β-agonist tocolytics [20,25,48]. Our finding of distinct sex-selectivity for the developmental toxicity of terbutaline is of further interest. In the adult, there are significant sex differences in βar responses [79] and during development, both the concentration of βars and their linkage to adenylyl cyclase display disparities between males and females

9 DEVELOPMENTAL TOXICITY OF TERBUTALINE 327 [4,39], including differences in effects on cell replication [39]. Furthermore, estrogen receptors are highly expressed in a variety of brain regions and influence the rate of cell turnover [45,75]. The impact of terbutaline on brain development is thus likely to reflect, in part, the hormonal factors that prime the cellular responses to βar input, as well as the underlying sex differences in the patterns of cell replication and differentiation. At this time, however, we do not have specific information to explain all the sex differences seen here for the effects of terbutaline on cell replication and cell number. Not surprisingly, we found that the adverse effects of terbutaline on cell development extended to peripheral tissues (liver and heart) that highly express βars during fetal and neonatal development [55,61]. Hepatic βars are highest during the fetal period and decline to low levels in adulthood [26,42,61]. The fact that nearly all the hepatic receptors are of the β 2 subtype [2,61] renders cell signaling in this tissue especially vulnerable to terbutaline [2 4], and accordingly we found sizeable reduction in fetal liver weight and in the number of cells. As hepatic cells maintain their ability to divide into adulthood, it is unlikely that this deficiency plays a long-term role in the developmental toxicity of terbutaline; however, it may contribute to abnormalities of glucose metabolism in the immediate postnatal period [13]. The changes seen in the heart may be of greater importance, given that myocardial cells, like neurons, exit the cell cycle during the perinatal period [15,37,50,66]. βars become linked to the inhibition of DNA synthesis by the end of the first postnatal week, shortly before the onset of peripheral sympathetic function [15,50,55,66]. Before that time, receptor stimulation is promotional for cell replication [31,62] and prolonged overstimulation elicits myocardial apoptosis and necrosis [11 49]. As was true for the brain, then, the net outcome for cardiac cell development will depend on the balance of the effects occurring during the specified period of terbutaline exposure: cell replication, cell differentiation, and cell death. With terbutaline administered on PN2 5, we found inhibition of DNA synthesis, but only after repeated administration had sensitized βar signaling [3,4]. With administration on PN11 14, we saw a response even to the first dose of terbutaline, in keeping with the fact that, during this period, βars are highly coupled to the ability to arrest cardiac cell replication [50,66];nevertheless, the treatments did not change the cardiac DNA content. However, unlike the situation in the brain, cardiac cells become binucleated during the postnatal period [71], so that changes (or absence of changes) in DNA may be a misleading index of the impact on cell development. Again, morphological examinations will be needed to resolve this issue. In any case, we obtained evidence of adaptive effects in the heart that were not present in the brain: after the fourth injection of terbutaline on PN15, cardiac DNA synthesis was markedly stimulated instead of showing inhibition, an effect accompanied by significant cardiac hypertrophy. This reversal may thus serve to replace damaged cardiac cells, an effect that assumes greater importance in light of the fact that terbutaline elicits myocardial apoptosis and/or necrosis [11,21,36,49]. In conclusion, terbutaline exposure altered cellular development in the developing brain and in peripheral tissues that express high levels of βars. 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