Sexually dimorphic effects of maternal alcohol intake and adrenalectomy on left ventricular hypertrophy in rat offspring

Jennifer Slone Wilcoxon,1 Jeff Schwartz,2 Fraser Aird,1 and Eva E. Redei1

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


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In humans, low birth weight and increased placental weight can be associated with cardiovascular disease in adulthood. Low birth weight and increased placental size are known to occur after fetal alcohol exposure or prenatal glucocorticoid administration. Thus the effects of removing the alcohol-induced increase in maternal corticosterone by maternal adrenalectomy on predictors of cardiovascular disease in adulthood were examined in rats. Alcohol exposure of dams during the last 2 wk of gestation resulted in significantly decreased fetal weight and increased placental weight on gestational day 21. Adult female, but not male, offspring of alcohol-consuming mothers exhibited left ventricular hypertrophy. Placental 11{beta}-hydroxysteroid dehydrogenase-2 (11{beta}-HSD-2) mRNA levels, measured by Northern blot, were decreased in females but not males. Adrenalectomy of alcohol-consuming dams reversed the increase in placental weight and the decrease in female placental 11{beta}-HSD-2 expression and eliminated the left ventricular hypertrophy of adult female offspring. These data suggest that alcohol-induced changes in placental 11{beta}-HSD-2 mRNA levels and left ventricular weight are coupled in female offspring only and depend on maternal adrenal status.

corticosterone; 11{beta}-hydroxysteroid dehydrogenase-2; fetal alcohol exposure; birth weight; placental weight; adrenalectomized


HUMAN EPIDEMIOLOGICAL STUDIES have linked low birth weight with increased emergence later in life of cardiovascular and metabolic pathologies, including ischemic heart disease, hypertension, insulin resistance, and non-insulin-dependent diabetes (3, 4, 6, 7, 69). These observations have led to the hypothesis that changes in the fetal environment produce physiological adaptations by the fetus that lead to small birth weight. Meanwhile, the net effect of these changes, while beneficial before birth, may produce adverse outcomes in the long term. These adaptations are called fetal programming. Among other challenges to the fetal environment, altered maternal nutrition, such as protein or iron deficiency, modifies or programs fetal and adult morphology as well as metabolic and endocrine pathways (6, 5, 24).

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{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD-2), which inactivates cortisol and Cort (47). The expression and activity of 11{beta}-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{beta}-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{beta}-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.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. All animal procedures were approved by the Northwestern University Animal Care and Use Committee. Adult male and female Sprague-Dawley rats (viral free, 56-68 days of age; Harlan, Indianapolis, IN) were housed individually in a temperature- and humidity-controlled vivarium with regular light-dark cycles (lights on at 0700 and lights off at 1900). After 14 days acclimatization, rats were mated by placing a female in the male cage overnight. Mating was confirmed by microscopic analysis of vaginal smears for the presence of sperm the next morning. The day sperm was found was designated as gestational day 1.

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 90–100 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{beta}-HSD-2. Thus removal of elevated maternal Cort in the alcohol-consuming dams by adrenalectomy could eliminate the increased placental weight and decreased 11{beta}-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{beta}-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 90–100 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, 8–10 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 [{alpha}-32P]dCTP by random primer labeling (17) using the Random Primers DNA Labeling System kit (Life Technologies). The 11{beta}-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 936–957 and 1255–1234, respectively, of rat 11{beta}-HSD-2 cDNA (72). Plasmid containing a mouse {beta}-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{beta}-HSD-2 mRNA levels were normalized to the {beta}-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.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diet consumption, maternal weight, and litter size. There was no significant difference in the amount of diet consumed by the pregnant dams, regardless of the presence of ethanol in the diet or their adrenal status. The BioServ diets are formulated in such a way that both alcohol and PF diets contain 1 kcal/ml. Therefore, the volume of liquid diet consumed daily also provides a measure of caloric intake. The dams in the FAE groups consumed 95.2 ± 2.3 ml/day, and dams in the PF groups consumed 104.2 ± 2.7 ml/day of their respective isocaloric diets. The PF/Sham and FAE/Sham mothers consumed 106.3 ± 2.5 and 96 ± 3.1 ml/day, respectively, whereas the PF/Adx and FAE/Adx dams drank 108.6 ± 2.6 and 97.8 ± 2.8 ml/day, respectively. Although maternal weight was not was affected by adrenalectomy, a significant (P < 0.05) difference between the two diet groups was found in all of the experiments (Table 1).


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Table 1. Maternal body weight, total fetal and adult litter size, and average number of males and females

 

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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Fig. 1. A: fetal weight on gestational day 21 (n = 26–34 males or females/diet). There were five litters in both the pair–fed (PF) and fetal-alcohol exposed (FAE) groups. Values are shown as means ± SE. *Significant difference from PF control, P < 0.001. B: placental weight on gestational day 21 for the animals described in A. Values are shown as means ± SE. *Significant difference from PF, P < 0.01.

 

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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Fig. 2. Ratio of left ventricular weight (LV) to body weight (BW) in adult (90–100 days of age) PF and FAE offspring (n = 24–34 male or female animals/diet). There were 5 litters/treatment group. Values are shown as means ± SE. *Significant difference from PF, P < 0.05. Left ventricular weight normalized to right ventricular weight revealed similar profiles (data not shown).

 

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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Fig. 3. A: fetal weight on gestational day 21 (n = 19–34 male or female animals/diet for each surgery). There were 5 litters/treatment group [pair fed/sham adrenalectomized (PF/Sham), fetal alcohol exposed, sham adrenalectomized (FAE/Sham), pair fed, adrenalectomized (PF/Adx), and fetal alcohol exposed, adrenalectomized (FAE/Adx)]. Values are shown as means ± SE. B: placental weight on gestational day 21 for the animals described in A. Values are shown as means ± SE. *Significant difference from PF control, P < 0.01.

 

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{beta}-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{beta}-HSD-2 mRNA compared with those of PF/Sham females. However, the placental 11{beta}-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{beta}-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{beta}-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{beta}-HSD-2 mRNA levels compared with PF females of Adx mothers.



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Fig. 4. Placental 11{beta}-hydroxysteroid dehydrogenase 2 (11{beta}-HSD-2) mRNA levels normalized to {beta}-actin (n = 4–5/group, representing 1 placenta for each sex/litter) on gestational day 21. Values are shown as means ± SE. *Significant difference from PF control, P < 0.05. +Significant difference from PF/Adx, P < 0.01.

 

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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Fig. 5. Ratio of left ventricular weight to body weight in adult (90–100 days of age) offspring (n = 18–33 animals for each sex and diet/surgery). There were 5 litters/treatment group. Values are shown as means ± SE. *Significant difference from PF, P < 0.05. Left ventricular weight normalized to right ventricular weight revealed similar profiles (data not shown).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main findings of the present study suggest that maternal adrenal hormones might contribute to the cardiovascular changes found in adult female offspring exposed to ethanol in utero. In the present study, maternal ethanol ingestion produced hypertrophy of the left ventricle in the adult FAE female offspring, which was normalized by maternal adrenalectomy. Maternal adrenalectomy also normalized the increased placental weight found in fetuses of both sexes in response to FAE.

The mRNA levels of placental 11{beta}-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{beta}-HSD-2 mRNA levels in the FAE males while in the FAE females 11{beta}-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{beta}-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{beta}-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{beta}-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{beta}-HSD-2 in rats decreases birth weight (48). Our data demonstrated that prenatal alcohol exposure affects 11{beta}-HSD-2 mRNA levels in a sexually dimorphic manner: decreasing in females and increasing in males. 11{beta}-HSD-2 mRNA levels are shown to correlate with enzyme activity (54, 55). Because testosterone can potentially downregulate 11{beta}-HSD activity, as has been found to occur in rat testis (35), placental 11{beta}-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{beta}-HSD-2 mRNA found in the male FAE placenta. This increase in 11{beta}-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{beta}-HSD-2 activity (52). The increased levels of 11{beta}-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{beta}-HSD-2 expression. Subsequently, the decreased levels of 11{beta}-HSD-2 may lead to left ventricular hypertrophy in the adult female FAE offspring, since 11{beta}-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{beta}-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{beta}-HSD-2 expression compared with those of the PF/Adx females. Placental 11{beta}-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{beta}-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{beta}-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{beta}-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{beta}-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.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. S. Wilcoxon, 303 E. Chicago Ave., Ward 9-190, Chicago, IL 60611 (E-mail: j-slone{at}northwestern.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Abel EL. Paternal and maternal alcohol consumption: effects on offspring in two strains of rats. Alcohol Clin Exp Res 13: 533–541, 1989.[ISI][Medline]
  2. Ahmed II, Shryne JE, Gorski RA, Branch BJ, and Taylor AN. Prenatal ethanol and the prepubertal sexually dimorphic nucleus of the preoptic area. Physiol Behav 49: 427–432, 1991.[ISI][Medline]
  3. Barker DJP. Deprivation in infancy and risk of ischaemic heart disease (Abstract). Lancet 20: 981, 1991.
  4. Barker DJ. Maternal nutrition and cardiovascular disease. Nutr Health 9: 99–106, 1993.[Medline]
  5. Barker DJ. Maternal and fetal origins of coronary heart disease. J R Coll Physicians Lond 28: 544–551, 1994.[ISI][Medline]
  6. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, and Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 10: 938–941, 1993.
  7. Barker DJ, Hales CN, Fall CH, Osmond C, Phipps K, and Clark PM. Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 36: 62–67, 1993.[ISI][Medline]
  8. Bellino FL and Holben L. Placental estrogen synthetase (aromatase): evidence for phosphate-dependent inactivation. Biochem Biophys Res Commun 162: 498–504, 1989.[ISI][Medline]
  9. Bernatova I, Pechanova O, and Simko F. Captopril prevents NO-deficient hypertension and left ventricular hypertrophy without affecting nitric oxide synthase activity in rats. Physiol Res 45: 311–316, 1996.[ISI][Medline]
  10. Bloom SL, Sheffield JS, McIntire DD, and Leveno KJ. Antenatal dexamethasone and decreased birth weight. Obstet Gynecol 97: 485–490, 2001.[Abstract/Free Full Text]
  11. Brown RW, Diaz R, Robson AC, Kotelevtsev YV, Mullins JJ, Kaufman MH, and Seckl JR. The ontogeny of 11{beta}-hydroxysteroid dehydrogenase type 2 and mineralocorticoid receptor gene expression reveal intricate control of glucocorticoid action in development. Endocrinology 137: 794–797, 1996.[Abstract]
  12. Clarke KA, Ward JW, Forhead AJ, Giussani DA, and Fowden AL. Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 activity in ovine placenta by fetal cortisol. J Endocrinol 172: 527–534, 2002.[Abstract/Free Full Text]
  13. Day NL, Jasperse D, Richardson G, Robles N, Sambamoorthi U, Taylor P, Scher M, Stoffer D, and Cornelius M. Prenatal exposure to alcohol: effect on infant growth and morphologic characteristics. Pediatrics 84: 536–541, 1989.[Abstract]
  14. Dodic M, May CN, Wintour EM, and Coghlan JP. An early postnatal exposure to excess glucocorticoid leads to hypertensive offspring in sheep. Clin Sci (Lond) 94: 149–155, 1998.[ISI][Medline]
  15. Drickamer LC. Seasonal variation in fertility, fecundity and litter sex ratio in laboratory and wild stocks of house mice (Mus domesticus). Lab Anim Sci 40: 284–288, 1990.[Medline]
  16. Dubois PM and Hemming FJ. Fetal development and regulation of pituitary cell types. J Electron Microsc (Tokyo) 19: 2–20, 1991.
  17. Feinberg AP and Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13, 1983.[ISI][Medline]
  18. Gallo PV and Weinberg J. Organ growth and cellular development in ethanol-exposed rats. Alcohol 3: 261–267, 1986.[ISI][Medline]
  19. Gavin CE, Kates B, Gerken LA, and Rodier PM. Patterns of growth deficiency in rats exposed in utero to undernutrition, ethanol or the neuroteratogen methylazoxymethanol (MAM). Teratology 49: 113–121, 1994.[ISI][Medline]
  20. Gordon BH, Streeter ML, Rosso P, and Winick M. Prenatal alcohol exposure: abnormalities in placental growth and fetal amino acid uptake in the rat. Biol Neonate 47: 113–119, 1985.[ISI][Medline]
  21. Grover-McKay M, Scholz TD, Burns TL, and Skorton DJ. Myocardial collagen concentration and nuclear magnetic resonance relaxation times in the spontaneously hypertensive rat. Invest Radiol 26: 227–232, 1991.[ISI][Medline]
  22. Gunberg DL. Some effects of exogenous hydrocortisone on pregnancy in the rat. Anat Rec 129: 133–153, 1957.[ISI]
  23. Hainsey T, Csiszar A, Sun S, and Edwards JG. Cyclosporin A does not block exercise-induced cardiac hypertrophy. Med Sci Sports Exerc 34: 1249–1254, 2002.[ISI][Medline]
  24. Hales CN and Barker DJ. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 35: 595–601, 1992.[ISI][Medline]
  25. Hannigan JH. Effects of prenatal exposure to alcohol plus caffeine in rats: pregnancy outcome and early offspring development. Alcohol Clin Exp Res 19: 238–246, 1995.[ISI][Medline]
  26. Hannigan JH, Abel EL, and Kruger ML. "Population" characteristics of birthweight in an animal model of alcohol-related developmental effects. Neurotoxicol Teratol 15: 97–105, 1993.[ISI][Medline]
  27. Head GA and Minami N. Importance of cardiac, but not vascular, hypertrophy in the cardiac baroreflex deficit in spontaneously hypertensive and stroke-prone rats. Am J Med 92: 54S–59S, 1992.[Medline]
  28. Knowles JW, Esposito G, Mao L, Hagaman JR, Fox JE, Smithies O, Rockman HA, and Maede N. Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice. J Clin Invest 107: 975–984, 2001.[Abstract/Free Full Text]
  29. Lam MK, Homewood J, Taylor AJ, and Mazurski EJ. Second generation effects of maternal alcohol consumption during pregnancy in rats. Prog Neuropsychopharmacol Biol Psychiatry 24: 619–631, 2000.[ISI][Medline]
  30. Lee TM and McClintock MK. Female rats in a laboratory display seasonal variation in fecundity. J Reprod Fertil 77: 51–59, 1986.[Abstract]
  31. McTernan CL, Draper N, Nicholson H, Chalder SM, Driver P, Hewison M, Kilby MD, and Stewart PM. Reduced placental 11beta-hydroxysteroid dehydrogenase type 2 mRNA levels in human pregnancies complicated by intrauterine growth restriction: an analysis of possible mechanisms. J Clin Endocrinol Metab 86: 4979–4983, 2001.[Abstract/Free Full Text]
  32. Mosier HD Jr, Dearden LC, Jansons RA, Roberts RC, and Biggs CS. Disproportionate growth of organs, and body weight following glucocorticoid treatment of the rat fetus. Dev Pharmacol Ther 4: 89–105, 1982.[ISI][Medline]
  33. Nelson LR, Taylor AN, Lewis JW, Poland RE, Redei E, and Branch BJ. Pituitary adrenal responses to morphine and foot-shock stress- are enhanced following prenatal alcohol exposure. Alcohol Clin Exp Res 10: 243–247, 1986.
  34. Novy MJ and Walsh SW. Dexamethasone and estradiol treatment in pregnant rhesus macaques: effects on gestational length, maternal plasma hormones and fetal growth. Am J Obstet Gynecol 145: 920–930, 1983.[ISI][Medline]
  35. Nwe KH, Morat PB, and Khalid BA. Opposite effects of sex steroids on 11 beta-hydroxysteroid dehydrogenase activity in the normal and adrenalectomized rat testis. Gen Pharmacol 28: 661–664, 1997.[Medline]
  36. Nyirenda MJ and Seckl JR. Intrauterine events and the programming of adulthood disease: the role of fetal glucocorticoid exposure. Int J Mol Med 2: 607–614, 1998.[ISI][Medline]
  37. Okoshi MP, Okoshi K, Pai VD, Pai-Silva MD, Matsubara LS, and Cicogna AC. Mechanical, biochemical, and morphological changes in the heart from chronic food-restricted rats. Can J Physiol Pharmacol 79: 754–760, 2001.[ISI][Medline]
  38. O'Regan D, Welberg LL, Holmes MC, and Seckl JR. Glucocorticoid programming of pituitary-adrenal function: mechanisms and physiological consequences. Semin Neonatol 6: 319–329, 2001.[Medline]
  39. Redei E, Halasz I, Li L, Prystowsky MB, and Aird F. Maternal adrenalectomy alters the immune and endocrine functions of fetal alcohol-exposed male offspring. Endocrinology 133: 452–460, 1993.[Abstract]
  40. Reinisch JM, Simon NG, Karwo WG, and Gandelman R. Prenatal exposure to prednisone in humans and animals retards intra-uterine growth. Science 202: 436–438, 1978.[ISI][Medline]
  41. Ren J, Wold LE, Natavio M, Ren BH, Hannigan JH, and Brown RA. Influence of prenatal alcohol exposure on myocardial contractile function in adult rat hearts: role of intracellular calcium and apoptosis. Alcohol Alcohol 37: 30–37, 2002.[Abstract/Free Full Text]
  42. Revskoy S, Halasz I, and Redei E. Corticotropin-releasing hormone and proopiomelanocortin gene expression is altered selectively in the male rat fetal thymus by maternal alcohol consumption. Endocrinology 138: 389–396, 1997.[Abstract/Free Full Text]
  43. Rifas L, Towler DA, and Avioli LV. Gestational exposure to ethanol suppresses msx2 expression in developing mouse embryos. Proc Natl Acad Sci USA 94: 7549–7554, 1997.[Abstract/Free Full Text]
  44. Sardor GG, Smith DF, and MacLeod PM. Cardiac malformations in the fetal alcohol syndrome. J Pediatr 98: 771–773, 1981.[ISI][Medline]
  45. Sebkhi A, Zhao L, Lu L, Haley CS, Nunez DJR, and Wilkins MR. Genetic determination of cardiac mass in normotensive rats: results from an F344xWKY cross. Hypertension 33: 949–953, 1999.[Abstract/Free Full Text]
  46. Seckl JR. Glucocorticoids and small babies. QJM 87: 259–262, 1994.[Medline]
  47. Seckl JR. Glucocorticoids, feto-placental 11{beta}-hydroxysteroid dehydrogenase type 2, and the early life origins of adult disease. Steroids 62: 89–94, 1997.[ISI][Medline]
  48. Seckl JR, Benediktsson R, Lindsay RS, and Brown RW. Placental 11{beta}-hydroxysteroid dehydrogenase and the programming of hypertension. J Steroid Biochem Mol Biol 55: 447–455, 1995.[ISI][Medline]
  49. Singer LT, Salvator A, Arendt R, Minnes S, Farkas K, and Kliegman R. Effects of cocaine/polydrug exposure and maternal psychological distress on infant birth outcomes. Neurotoxicol Teratol 24: 127–135, 2002.[ISI][Medline]
  50. Sinha P, Halasz I, Choi JF, McGivern RF, and Redei E. Maternal adrenalectomy eliminates a surge of plasma dehydro-epiandrosterone in the mother and attenuates the prenatal testosterone surge in the male fetus. Endocrinology 138: 4792–4797, 1997.[Abstract/Free Full Text]
  51. Slone JL and Redei E. Decreased Maternal Corticosterone Leads to Increased Activity and Decreased Learning in Young Rats. San Diego, CA: Society for Neuroscience, 2001.
  52. Slone JL and Redei EE. Maternal alcohol and adrenalectomy: asynchrony of stress response and forced swim behavior. Neurotoxicol Teratol 24: 173–178, 2002.[ISI][Medline]
  53. Steeg CN and Woolf P. Cardiovascular malformations in the fetal alcohol syndrome. Am Heart J 98: 635–637, 1979.[ISI][Medline]
  54. Stewart PM, Rogerson FM, and Mason JI. Type 2 11{beta}-hydroxysteroid dehydrogenase mRNA and activity in human placenta and foetal membranes: its relationship to birth weight and putative role in foetal adrenal steroidogenesis. J Clin Endocrinol Metab 80: 885–890, 1995.[Abstract]
  55. Stewart PM, Whorwood CB, and Mason JI. Type 2 11{beta}-hydroxysteroid dehydrogenase in foetal and adult life. J Steroid Biochem Mol Biol 55: 465–471, 1995.[ISI][Medline]
  56. Sun K, Yang K, and Challis JR. Regulation of 11beta-hydroxysteroid dehydrogenase type 2 by progesterone, estrogen, and the cyclic adenosine 5'-monophoshpate pathway in cultured human placental and chorionic trophoblasts. Biol Reprod 58: 1379–1384, 1998.[Abstract]
  57. Syslak PH, Nathaniel EJ, Novak C, and Burton L. Fetal alcohol effects on the postnatal development of the rat myocardium: an ultrastructural and morphometric analysis. Exp Mol Pathol 60: 158–172, 1994.[ISI][Medline]
  58. Taylor AN, Branch BJ, Liu SH, and Kokka N. Long-term effects of fetal ethanol exposure on pituitary-adrenal response to stress. Pharmacol Biochem Behav 16: 585–589, 1982.[ISI][Medline]
  59. Taylor AN, Branch BJ, Van Zuylen JE, and Redei E. Prenatal ethanol exposure alters ACTH stress response in adult rats (Abstract). Alcohol Clin Exp Res 10: 120, 1986.
  60. Thomas CJ, Head GA, and Woods RL. ANP, and bradycardiac reflexes in hypertensive rats: influence of cardiac hypertrophy. Hypertension 32: 548–555, 1998.[Abstract/Free Full Text]
  61. Tokunaga K, Taniguchi H, Yoda K, Shimizu M, and Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal beta-actin mRNA (Abstract). Nucleic Acids Res 14: 2829, 1986.[ISI][Medline]
  62. Uggere TA, Abreu GR, Sampaio KN, Cabral AM, and Bissoli NS. The cardiopulmonary reflexes of spontaneously hypertensive rats are normalized after regression of left ventricular hypertrophy and hypertension. Braz J Med Biol Res 33: 589–594, 2000.[ISI][Medline]
  63. Uphoff C, Nyquist-Battie C, and Toth R. Cardiac muscle development in mice exposed to ethanol in utero. Teratology 30: 119–129, 1984.[ISI][Medline]
  64. Weinberg J. Differential effects of prenatal ethanol on males and females. Alcohol Clin Exp Res 12: 647–652, 1988.[ISI][Medline]
  65. Weinberg J. Prenatal ethanol exposure alters adrenocortical development of offspring. Alcohol Clin Exp Res 13: 73–83, 1989.[ISI][Medline]
  66. Welberg LA, Seckl JR, and Holmes MC. Inhibition of 11beta-hydroxysteroid dehydrogenase, the foeto-placental barrier to maternal glucocorticoids, permanently programs amygdala GR mRNA expression and anxiety-like behavior in the offspring. Eur J Neurosci 12: 1047–1054, 2000.[ISI][Medline]
  67. Welberg LA, Seckl JR, and Holmes MC. Prenatal glucocorticoid programming of brain corticosteroid receptors and corticotrophin-releasing hormone: possible implications for behaviour. Neuroscience 104: 71–79, 2001.[ISI][Medline]
  68. Wold LE, Norby FL, Hintz KK, Colligan PB, Epstein PN, and Ren J. Prenatal ethanol exposure alters ventricular myocyte contractile function in the offspring of rats: influence of maternal Mg2+ supplementation. Cardiovasc Toxicol 1: 215–224, 2001.[Medline]
  69. Yajnik CS, Fall CH, Vaidya U, Pandit AN, Bavdekar A, Bhat DS, Osmond C, Hales CN, and Barker DJ. Fetal growth and glucose and insulin metabolism in four-year-old Indian children. Diabet Med 12: 330–336, 1995.[ISI][Medline]
  70. Yang G, Meguro T, Hong C, Asai K, Takagi G, Karoor VL, Sadoshima J, Vatner DE, Bishop SP, and Vatner SF. Cyclosporine reduced left ventricular mass with chronic aortic banding in mice, which could be due to apoptosis and fibrosis. J Mol Cell Cardiol 33: 1505–1514, 2001.[ISI][Medline]
  71. Zhang Y, Carreras D, and de Bold AJ. Discoordinate re-expression of cardiac fetal genes in N(omega)-nitro-L-arginine methyl ester (L-NAME) hypertension. Cardiovasc Res 57: 158–167, 2003.[ISI][Medline]
  72. Zhou M-Y, Gomez-Sanchez EP, Cox DL, Cosby D, and Gomez-Sanchez CE. Cloning, Expression, and tissue distribution of the rat nicotinamide adenine dinucleotide-dependent 11{beta}-hydroxysteroid dehydrogenase. Endocrinology 136: 3729–3734, 1995.[Abstract]