1Department of Psychiatry and Behavioral Sciences, Northwestern University Medical School, The Asher Center Chicago, Illinois 60611; and 2Department of Physiology, University of Adelaide, Adelaide, Australia 5005
Submitted 18 December 2002 ; accepted in final form 27 February 2003
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ABSTRACT |
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corticosterone; 11-hydroxysteroid dehydrogenase-2; fetal alcohol exposure; birth weight; placental weight; adrenalectomized
One consistent feature of numerous human epidemiological studies and animal models of low birth weight is prenatal exposure to glucocorticoids (10, 36, 66). Furthermore, even brief prenatal exposure to elevated glucocorticoids can result in permanent adverse changes in the adult offspring's cardiovascular system (13, 32). Although glucocorticoids are important in normal development, excessive exposure through administration of glucocorticoids to the mother leads to reduced birth weight (67). However, decreased levels of maternal plasma corticosterone (Cort) by maternal adrenalectomy also result in reduced birth weight, and very low levels of Cort are sufficient to normalize birth weight and increase fetal Cort levels (51). As fetal adrenals start functioning during the last week of gestation (16), maternal glucocorticoid levels are the primary regulators of fetal development during the first 2 wk of gestation. Subsequently, maternal Cort might affect fetal development indirectly through regulating fetal adrenal function during the last week of gestation. Normally, fetuses are protected from any large excursions in maternal glucocorticoids by placental 11-hydroxysteroid dehydrogenase type 2 (11
-HSD-2), which inactivates cortisol and Cort (47). The expression and activity of 11
-HSD-2, however, are developmentally regulated and also subject to external influence. Thus fetal exposure to glucocorticoids represents net steroidogenic activity of the fetus itself plus a contribution of maternal glucocorticoids, subject to modulation by placental 11
-HSD-2. Maternal alcohol ingestion is also associated with elevated glucocorticoid levels and low birth weight in the fetus (13, 19, 49). In studies with rats, we have previously shown that, in response to maternal ethanol ingestion over the last 2 wk of gestation (beginning on day 8), maternal plasma Cort levels are consistently and significantly elevated from gestational day 18 to parturition (50). In contrast, fetal Cort levels are decreased (42, 65). Thus a highly significant inverse relationship between maternal and fetal glucocorticoid levels exists during the last week of gestation (42). An inverse relationship in the opposite direction exists between fetal and maternal Cort after maternal adrenalectomy (50) such that increased fetal Cort during the last week of gestation elevates maternal Cort levels to near normal by gestational day 21. However, alcohol exposure in adrenalectomized (Adx) dams still leads to significantly decreased fetal Cort levels in both sexes, suggesting that ethanol inhibits fetal Cort production directly. Because glucocorticoids in the fetus play a key role in the regulation of growth and maturation of many organ systems, as well as the programming of the postnatal hypothalamic-pituitary-adrenal axis itself, decreased fetal Cort levels, resulting from increased maternal Cort levels and/or ethanol, have the potential to permanently alter the physiology of the offspring.
We hypothesized that, if developmental exposure to high followed by low levels of fetal glucocorticoids are involved in cardiovascular vulnerability of the fetal alcohol-exposed (FAE) offspring, then maternal adrenalectomy, and the ensuing low levels of maternal Cort, would eliminate cardiovascular changes found in adult offspring. Therefore, the aim of the present study was to systematically measure in a rat model the effects of maternal ethanol consumption and maternal adrenalectomy on fetal body weight, placental weight, placental 11-HSD-2 expression, and left ventricular weight in the adult. This information could suggest a potential mechanism of alcohol-induced fetal programming of the cardiovascular system.
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METHODS |
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Experiment 1a. Because low birth weight and increased placental weight are associated with prenatal exposure to increased glucocorticoids, this experiment aimed to measure plasma Cort levels of alcohol-consuming dams and fetal body weight and placental weight of their offspring. On gestational day 8, pregnant rats were randomly assigned to the following two experimental groups: FAE (n = 5) and pair fed (PF, n = 5). The FAE rats were placed on an ethanol-containing liquid diet (Lieber-DeCarli'82; BioServ) adjusted for pregnant rats containing 5% (wt/vol) ethanol (35% ethanol-derived calories), supplemented with essential minerals and vitamins. The ethanol diet was introduced in stages; 15% ethanol-derived calories was increased to 35% over 5 days, as described previously (50). The remaining rats were pair fed the same diet, with isocaloric substitution of cornstarch for the ethanol. The amount of ethanol-free diet given to rats in the PF group was based on the consumption of a corresponding dam of similar weight in the FAE group.
The liquid diets were started on gestational day 8 and were presented daily between 1600 and 1700, and daily intake was recorded. This amount of ethanol-diet consumption is not different from the alcohol intake of FAE dams reported previously, which produced blood ethanol levels of 80 mg/100 ml (39). On gestational day 21 the pregnant rats were killed by decapitation, and trunk blood was collected for Cort determination. Uterine horns were placed on ice, and fetuses were removed. The sex of each fetus was determined by anogenital distance, and then the fetus and placenta were weighed.
Experiment 1b. Low birth weight and increased placental weight (found in experiment 1a) are predisposing factors to cardiovascular vulnerability later in life. Thus we measured heart weight, specifically left ventricular weight, in adult FAE and PF offspring. The left ventricle was normalized to body weight, as it is most customary (9, 21, 23, 27, 28, 37, 60, 62, 70, 71) to control for the large sex differences in size. As in experiment 1a, pregnant rats were assigned to the same two treatment groups (PF, n = 5; FAE, n = 5). The diet administration protocol was identical, except that on gestational day 21 the diets were replaced with laboratory chow and water ad libitum, and the rats were allowed to deliver. Maternal daily alcohol consumption was similar to that in experiment 1a. Pups were weaned at 21 days of age and group housed by sex and treatment.
At 90100 days of age, adult male and female rats from both prenatal treatment groups were killed by decapitation. Animals were weighed, hearts were removed and weighed, and then ventricles were separated and individually weighed.
Experiment 2a. If the cause of decreased body weight and increased placental weight is alcohol-induced elevated plasma Cort in the dam, this elevated maternal Cort needs to have access to the fetus via decreased protection by 11-HSD-2. Thus removal of elevated maternal Cort in the alcohol-consuming dams by adrenalectomy could eliminate the increased placental weight and decreased 11
-HSD-2 expression. On gestational day 8, pregnant rats were randomly assigned to the following four experimental groups: FAE, adrenalectomized (Adx); PF, Adx; FAE, sham-adrenalectomized (Sham); and PF, Sham (n = 5).
Adrenalectomy was performed dorsally under anesthesia (n = 10, ketamine-xylazine, 87:10 mg/kg body wt), and the Sham dams (n = 10) underwent identical procedures without the removal of the glands. One-half of the Adx and Sham rats were placed on the FAE diet, which was introduced in stages, as described in experiment 1. The remaining Adx and Sham dams were placed on the PF diet. All Adx dams received their diet in 0.9% NaCl instead of water to prevent sodium depletion after adrenalectomy. To prevent resorption of the fetuses that occurs in Adx animals after surgery, a minimal replacement dose of Cort (2 µg/l; Sigma, St. Louis, MO) was included in the diet for 3 days after surgery, as described previously (50).
The liquid diets were started on gestational day 8 and were presented every day between 1600 and 1700; daily intake was recorded. Adrenalectomy did not alter alcohol metabolism, as shown in previous findings of identical blood alcohol levels in the Sham vs. Adx dams (90 ± 5.5 mg/100 ml; see Ref. 39). On gestational day 21 the pregnant rats were killed by decapitation. Fetal sex, weight, and placental weight were determined. Each placenta was frozen on dry ice and maintained at -80°C until extraction.
Experiment 2b. Because maternal adrenalectomy eliminated the increased placental weight and decreased 11-HSD-2 expression in the female placenta in response to alcohol, we measured heart weight in the adult offspring of Adx dams to determine if the ventricular hypertrophy found in female offspring of alcohol-consuming mothers (experiment 1b) was also abolished. As in experiment 2a, pregnant rats were assigned to the same four treatment groups (PF/Adx, n = 5; FAE/Adx, n = 5; PF/Sham, n = 5; FAE/Sham, n = 5). The diet administration protocol was identical, except that on gestational day 21 the diets were replaced with laboratory chow and water ad libitum, and the rats were allowed to deliver. Maternal diet consumption was the same as that of the dams in experiment 2a. We have previously found no differences in body weight between the Sham and Adx adult offspring, so there was no need to cross-foster.
At 90100 days of age, adult male and female rats from both prenatal treatment groups were killed by decapitation. Animals were weighed, hearts were removed and weighed, and then ventricles were separated and individually weighed.
RIA. Cort concentrations were measured as described previously (52) in unextracted plasma using 125I-labeled Cort RIA (ICN Biomedicals, Carson, CA).
RNA isolation and Northern analysis. Placental RNA was extracted using Trizol reagent, according to the manufacturer's protocol (Life Technologies, Grand Island, NY). The quality and quantity of RNA were analyzed by gel electrophoresis and spectrophotometry.
For Northern analysis, 810 µg RNA from each sample were separated by electrophoresis on a 1% agarose-formaldehyde gel, blotted on a nitrocellulose filter, and fixed by UV-cross-linking, as described previously (39). Filters were hybridized with cDNA probes overnight at 42°C in ULTRA-hyb hybridization buffer (Ambion, Austin, TX) after prehybridization according to the manufacturer's protocol. Probes were labeled with [-32P]dCTP by random primer labeling (17) using the Random Primers DNA Labeling System kit (Life Technologies). The 11
-HSD-2 probe was generated by PCR using the following primer pairs: 5'-GAC TAA TGT GAA CCT CTG GGA G and 5'-TCA GTG CTC GGG GTA GAA GGT G, corresponding to nucleotides 936957 and 12551234, respectively, of rat 11
-HSD-2 cDNA (72). Plasmid containing a mouse
-actin cDNA probe (61) was kindly provided by Dr. Michael Prystowsky, Albert Einstein University. Filters were washed two times for 15 min each in 2x saline-sodium citrate (SSC)-0.1% SDS at 42°C, two times for 30 min each in 0.1x SSC-0.1% SDS at 42°C, and exposed to Hyperfilm MP autoradiography film (Amersham Pharmacia Biotech, Piscataway, NJ) at -80°C with intensifying screens. To remove probes, filters were washed in boiling water for 30 s. Autoradiographs were scanned and analyzed using NIH Image (Wayne Rasband, NIH, Bethesda, MD). 11
-HSD-2 mRNA levels were normalized to the
-actin mRNA level of each sample
Statistics. The data were analyzed by ANOVA, either a two-factor (sex and diet in experiments 1 and 2) or a three-factor (sex, diet, and surgery in experiment 3) design. Litter was a nested factor. The Tukey honest significant difference test, with a P < 0.05, was used as a post hoc test to locate significant differences among groups.
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RESULTS |
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There were no significant differences in fetal litter size, but the litter size of the surviving FAE offspring was significantly smaller in both experiments (Table 1). This difference in litter size occurs between gestational day 21 and postnatal day 1. Because we cannot disturb the dam immediately after parturition, we were unable to determine whether the cause of this decreased litter size was stillbirth or greater death rate of newborns in FAE litters.
Experiment 1a. Consistent with previous results from our laboratory (50), alcohol consumption significantly increased maternal plasma Cort levels [PF (n = 5): 97.5 ± 3.7 ng/ml; FAE (n = 5): 145.9 ± 7 ng/ml; (P < 0.01)] on gestational day 21.
Maternal ethanol consumption had significant impact on the size and physical development of the fetuses. Overall, both male and female fetuses in the FAE group were significantly [F(1,178) = 55.6; P < 0.001] smaller on gestational day 21 than those in the PF group (Fig. 1A). Furthermore, maternal ethanol ingestion also had a significant impact on the placenta (Fig. 1B). The placental weight of male and female FAE fetuses on gestational day 21 was significantly (P < 0.01) greater than that of PF fetuses.
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Experiment 1b. As previously shown in our laboratory (52), plasma Cort levels were significantly (P < 0.05) decreased in FAE female (n = 8) offspring (31.2 ± 3.5 ng/ml) compared with PF female (n = 8) offspring (60.3 ± 6.6 ng/ml), although no such difference was found in the males.
Fetal alcohol exposure led to left ventricular hypertrophy in the adult female FAE offspring. There was no effect of prenatal alcohol on body weight of adult offspring (data not shown) within the same sex. To avoid the sex difference in heart weight, the left ventricular weight was normalized to body weight. There was a significant increase [F(1,103) = 5.9; P < 0.05] in the left ventricular weight-to-body weight ratio in the adult FAE female compared with the PF female as seen in Fig. 2. Interestingly, no such difference was found in the male offspring. Left ventricular weight normalized to right ventricular weight revealed similar profiles (data not shown).
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Experiment 2a. Although maternal Adx itself had no effect on fetal weight, the absence of maternal adrenal steroids potentiated the effect of alcohol in both females and males [diet x surgery F(1,347) = 28.4; P < 0.001; Fig. 3A]. Post hoc analysis revealed that both male and female FAE/Adx fetuses weighed significantly (P < 0.001) less than their respective PF/Adx controls, although only the female FAE/Sham fetuses were significantly (P < 0.01) smaller than the PF/Sham females. The FAE/Sham male fetuses tended (P = 0.08) to weigh less than the PF/Sham male fetuses.
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There were differences between the fetal weights of Sham dams and those found in experiment 1a (Figs. 1A and 3A). These differences are likely because of the number of fetuses in each litter, since typically the larger the litter the less each individual fetus weighs. Also litter size tends to vary with the season (15), since dams tend to carry larger litters in the warmer months of the year. Experiment 1a was done in the winter, and the average litter size was smaller than in experiment 2a, which was carried out during the spring and summer. Maternal adrenalectomy eliminated the significant increase found in placental weight of both male and female FAE/Sham compared with those of PF/Sham (diet x surgery, F = 13.9; P < 0.001; Fig. 3B). Post hoc analysis showed that placental weight of male and female FAE/Sham groups was significantly greater (P < 0.01) than their respective PF/Sham controls, whereas placental weight of Adx groups did not differ.
Maternal ethanol ingestion also had a significant and sexually dimorphic impact on the expression of placental 11-HSD-2 mRNA [sex x diet F(1,37) = 4.7; P < 0.05; Fig. 4]. Post hoc analysis revealed that the placentas of female FAE/Sham fetuses had significantly (P < 0.05) decreased levels of 11
-HSD-2 mRNA compared with those of PF/Sham females. However, the placental 11
-HSD-2 mRNA levels in the FAE/Sham males were increased significantly (P < 0.05) compared with PF/Sham males. Maternal adrenalectomy eliminated the effect of alcohol on placental 11
-HSD-2 expression in male fetuses (sex x diet x surgery, F = 10.75; P < 0.01). Interestingly, the post hoc test showed that removal of maternal steroids significantly (P < 0.05) decreased placental 11
-HSD-2 mRNA levels in the female PF but not in the female FAE. Thus FAE females of Adx mothers had significantly (P < 0.05) elevated 11
-HSD-2 mRNA levels compared with PF females of Adx mothers.
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Experiment 2b. There were no differences in plasma Cort levels among the Adx offspring (n = 8 rats/group). However, as previously found in our laboratory (52), basal Cort levels were decreased significantly (P < 0.05) in FAE/Sham females (32.3 ± 6.75 ng/ml) compared with PF/Sham females (56.59 ± 6.4 ng/ml), whereas no differences were found in the Sham males. These findings are similar to those found in the adult offspring from experiment 1b.
Although left ventricular hypertrophy was confirmed in the adult female offspring after prenatal alcohol exposure, this effect of ethanol on the left ventricular weight-to-body weight ratio (Fig. 5) was abolished by maternal adrenalectomy [sex x diet x surgery, F(1,238) = 15.8; P < 0.001]. Post hoc analysis revealed a significant (P < 0.05) increase in the ratio of left ventricular weigh to body weight in female FAE/Sham offspring compared with PF/Sham adult females similar to those shown in Fig. 2, but no such increase was found between the female Adx groups. No differences were found in left ventricular weight-to-body weight ratio in the adult male offspring of experimental dams. As found in experiment 1b, there were no differences in body weight among the treatment groups, only the typical sex difference, and left ventricular weight normalized to right ventricular weight showed profiles similar to those of Fig. 5 (data not shown).
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DISCUSSION |
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The mRNA levels of placental 11-HSD-2 mRNA, which control the amount of Cort exposure to the fetus, were increased in the FAE male but decreased in female placentas on gestational day 21. Maternal adrenalectomy eliminated the increased 11
-HSD-2 mRNA levels in the FAE males while in the FAE females 11
-HSD-2 expression was increased significantly compared with PF females after maternal Adx. Therefore, one likely explanation for the left ventricular hypertrophy found in the adult female FAE offspring is exposure to increased maternal steroids secondary to the decreased levels of placental 11
-HSD-2 mRNA.
Increasing evidence associates events occurring early in life with permanent impact (47). For example, low birth weight and increased placental size strongly predict the subsequent occurrence of hypertension, insulin resistance, and ischemic heart disease deaths in adulthood (3, 6, 7, 46, 69). However, there is evidence for vulnerability to cardiovascular abnormalities, such as left ventricular hypertrophy, in the absence of hypertension (45). In these respects, our FAE animal model exhibits a pattern similar to these models of fetal origins of adult disease. The present study also confirmed previous findings of increased placental weight (18, 20) and low birth weight (1, 25, 26) of FAE offspring. It is of interest that this lower weight in the FAE fetus coupled with a lower body weight of the FAE dam occurred despite the similar caloric consumption of FAE and PF dams. Thus this decreased body weight of the FAE mother and fetus may be because of increased metabolic rate in the alcohol-consuming dam or the less than perfect pair-feeding paradigm used by us and many other laboratories.
Cardiac malformations exist in children with fetal alcohol syndrome (44, 53) and animal models of prenatal alcohol exposure (43, 57), and cardiac hypertrophy has been found in children with fetal alcohol syndrome (68). Prenatal ethanol exposure has been shown to cause ultrastructural abnormalities in cardiac muscle cells in mice (63) and a significant difference in left ventricular muscle width in male rats (41). The high incidence of heart defects indicates that alcoholism during pregnancy has to be considered as a serious and preventable cause of congenital heart disease.
In humans and laboratory animals, prenatal glucocorticoid administration is associated with low birth weight, increased placental weight, cardiovascular disease, and potentially permanent hypertension (11). Excess glucocorticoid exposure in utero retards fetal growth in both humans and animals (32, 34, 40, 46), and cortisol affects placental size (22). Therefore, fetal overexposure to endogenous glucocorticoids (because of prenatal stress, prenatal alcohol, or reduced activity of placental 11-HSD-2) may represent a common link between the prenatal environment, fetal growth, and adult disorders (66). However, the mechanisms by which excessive maternal glucocorticoids exert these effects is not known.
Placental 11-HSD-2 serves as the barrier to protect the fetus from excess maternal glucocorticoids. Its activity correlates with birth weight (47), and inhibition of placental 11
-HSD-2 in rats decreases birth weight (48). Our data demonstrated that prenatal alcohol exposure affects 11
-HSD-2 mRNA levels in a sexually dimorphic manner: decreasing in females and increasing in males. 11
-HSD-2 mRNA levels are shown to correlate with enzyme activity (54, 55). Because testosterone can potentially downregulate 11
-HSD activity, as has been found to occur in rat testis (35), placental 11
-HSD-2 can be higher in females than in males. However, testosterone levels are decreased in the male FAE fetus (2, 50) in correspondence with the increased levels of 11
-HSD-2 mRNA found in the male FAE placenta. This increase in 11
-HSD-2 mRNA could additionally be attributable to the lower levels of estradiol via the aromatization of decreased testosterone levels in the FAE male fetus. Placental estrogen is the product of the aromatase cytochrome P-450 enzyme that uses androgens as substrates (8), and estrogens inhibit placental 11
-HSD-2 activity (52). The increased levels of 11
-HSD-2 mRNA found in the FAE male placenta might protect the fetus, and subsequently the adult offspring, from left ventricular hypertrophy later in life. In contrast, estradiol is increased in the female FAE fetus compared with PF control (2), and these increased levels of estrogen could lead to increased estrogen-induced inhibition of 11
-HSD-2 expression. Subsequently, the decreased levels of 11
-HSD-2 may lead to left ventricular hypertrophy in the adult female FAE offspring, since 11
-HSD-2 has an important role in regulating fetal growth and the subsequent development of cardiovascular disease in adulthood (31).
Previous studies have indicated that adult females are more susceptible than males to some of the effects of prenatal alcohol (29, 33, 58, 59, 64). Our study appears to follow the same sexually dimorphic pattern. Adult female FAE rats demonstrated left ventricular hypertrophy in this study, and this hypertrophy was abolished by maternal adrenalectomy. Although placental 11-HSD-2 mRNA levels were decreased in the females on gestational day 21 in response to maternal ethanol ingestion, the combination of maternal adrenalectomy and prenatal alcohol increased placental 11
-HSD-2 expression compared with those of the PF/Adx females. Placental 11
-HSD-2 activity is regulated by fetal cortisol levels in an inhibitory fashion in sheep (12). Our laboratory has previously shown that maternal Adx resulted in compensatory increases in fetal Cort levels that were attenuated in fetuses of Adx dams on alcohol (50). Accordingly, the lower placental 11
-HSD-2 mRNA levels in the PF/Adx females, compared with those of PF/Sham, and the higher levels in the female FAE/Adx placenta may reflect the effect of these differences in fetal Cort levels on this placental enzyme. In Adx dams, alcohol cannot elevate maternal Cort levels, but plasma Cort levels of fetal origin still rise in FAE and PF Adx dams equally by gestational day 21 (50). However, the levels of maternal Cort in the Adx dams are still significantly lower than in Sham dams. Thus the lower maternal Cort in the Adx dams together with the higher placental 11
-HSD-2 expression in the female FAE placenta appear to protect the female FAE fetus and may indeed be the cause of the elimination of left ventricular hypertrophy in the adult FAE female offspring of Adx dams consuming alcohol.
The present findings suggest that the FAE-induced changes in placental 11-HSD-2 mRNA levels and left ventricular heart weight are coupled in the female offspring and depend on maternal adrenal status. In contrast, increased placental 11
-HSD-2 levels in FAE males may protect the male fetus from subsequent ventricular hypertrophy. These experiments support the hypothesis that adaptations to the fetal environment, which result in low birth weight, also "program" physiological changes in the adult.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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