Effect of Fetal Ethanol Exposure on the In Vitro Release of Growth Hormone, Somatostatin and Growth Hormone-Releasing Factor Induced by Clonidine and Growth Hormone Feedback in Male and Female Rats

This study was designed to examine the effect of fetal ethanol (ETOH) exposure on the sensitivity of the hypothalamic-growth hormone (GH) axis to clonidine (an α₂-adrenoreceptor agonist) stimulation and GH feedback. During gestation, dams were fed either a liquid diet in which 36% of the calories were derived from ETOH, or pair-fed an isocaloric control liquid diet without ETOH. A second set of controls were fed lab chow ad libitum. After birth, offspring of ETOH-fed dams were cross-fostered to a separate group of ad libitum control dams. The hypothalami and pituiiries of 10–, 20–, 30–, and 50-day-old offspring were separated by age, diet, and sex; pooled 6 to 8 per chamber; and tested in a hypothalamic-pituitary coperifusion system. Chambers were perifused with either clonidine (2 × 10^?-⁸ M) alone, which mimics the endogenous trigger for GH release, or clonidine in combination with human GH (2 × 10^?-⁹ M) to determine sensitivity of tissue to feedback regulation. Both stimuli act at the hypothalamic level and indirectly modulate GH release via effects on hypothalamic factors. Results of this study indicate that tissue from control male rats is responsive to the clonidine induced GH surge by 10 days of age and to feedback depression of GH release by 20 days of age. This sensitivity persists after puberty and is associated with corresponding changes in somatotropin-release inhibiting factor (SRIF) and OH-releasing factor (GRF) release (i.e., clonidine inhibits SRlF and stimulates GRF release, and human GH reverses this pattern). Fetal ETOH exposure depresses GH sensitivity to both stimuli in male pups (age × diet × drug: p < 0.002), and this depressed sensitivity is expressed by 30 days of age by reduced responses to α₂-adrenergic stimulation and OH feedback (drug × diet: p < 0.002 and p < 0.001 for 30 and 50 days of age, respectively). This effect of ETOH on GH release was associated with feedback insensitivity of SRlF (drug × diet: p < 0.003, at 50 days of age) and GRF [drug × diet p < 0.044 at 30 days; clonidine vs. clonidine and GH: p > 0.05 (NS) at 50 days of age for ETOH pups]. The depressed response of GH to clonidine after puberty may be attributable to a combination of the trends toward decreased sensitivity of both SRlF and GRF at this age. The female GH axis was both less sensitive to stimuli and less effected by ETOH than corresponding tissue from male rats (sex × age × drug × diet: p < 0.011). OH release from control female pituitaries was sensitive to clonidine before, but not after, puberty and insensitive to GH feedback at both developmental stages. On the other hand, there was a specific effect of ETOH on SRlF release at 10 days of age (diet × drug: p < 0.014), and SRlF release remained sensitive to clonidine in pups from all diet groups after puberty. Because GH release was not influenced by these changes in SRIF, these findings suggest that GH release is less sensitive to SRlF in females. In conclusion, this study suggests that fetal ETOH exposure interferes with the development of the sensitivity of the GH axis to α₂-adrenergic stimulation and feedback in males. Thus, the male GH axis is both more sensitive to the stimuli tested in this study and more effected by ETOH than the female axis. Furthermore, the effects of ETOH on these mechanisms do not alter GH release in males until the peripubertal period. It is likely, therefore, that the GH regulatory mechanism examined in this study does not contribute to growth retardation before puberty. If the effects of ETOH on GH release contributes to growth retardation in prepubertal males and in females, it most likely involves other regulatory mechanisms. On the other hand, because the adult pattern of GH release is programmed during development, the influence of ETOH on these developmental events may influence the male pattern of GH release and GH activity in adulthood.     

Acute Prenatal Ethanol Exposure and Luteinizing Hormone-Releasing Hormone Messenger RNA Expression in the Fetal Mouse Brain

Ethanol exposure during critical periods of development results in alterations of central nervous system morphology and function. In this study, the effects of acute ethanol exposure on the number of neurons expressing luteinizing hormone-releasing hormone (LHRH) messenger RNA (mRNA) has been analyzed. Also, the expression of LHRH mRNA in the diagonal band of Broca/preoptic area (DBB/POA) was determined. Pregnant C57BL/6J mice were intubated with two doses of a 25% solution of ethanol or water (2.9 g/kg body weight) 4 hr apart on gestation day 7 (G7), G10, or G11. Animals were killed on G18, and in situ hybridization was utilized to detect neurons expressing LHRH mRNA. The number of neurons expressing LHRH mRNA was determined along their migration route from the rostrum into the forebrain. Ethanol exposure on G7 did not significantly change the number of neurons expressing LHRH mRNA on G18 compared with that in control animals. However, the number of neurons expressing LHRH mRNA in the nasal septum area in animals exposed to ethanol on G10 or G11 was significantly less than the number in control animals (p < 0.05). Prenatal ethanol exposure on any of the aforementioned treatment days did not alter the expression of LHRH mRNA at the level of the DBB/POA on G18 in ethanol-treated animals compared with control animals. Also, neuron-specific enolase mRNA expression at the level of the DBB/POA was not altered by prenatal ethanol exposure. Therefore, ethanol exposure on the aforementioned treatment days did not differentially affect LHRH mRNA expression compared with neuron-specific enolase mRNA expression at the level of the DBB/POA. These data suggest that acute ethanol exposure may have specific effects on a subset of the LHRH neuron population, depending on the day of ethanol exposure during gestation and the neuroanatomical region in which the LHRH neurons are located.     

Complex Rhythmicity of Growth Hormone Secretion in Humans

To better characterize the 24 hr GH secretion pattern in humans, we studied frequently sampled 24 hr GH profiles in 93 young (18–45 years of age) healthy, fed volunteers: men (n = 67) and women (n = 26) with BMI<26kg/m². Analysis of the composite GH series in men revealed 3 significant GH “waves” with peaks occurring at midnight (p < 0.0001), at noon (p < 0.02) and at 1800h (p < 0.0001). In women, similar pattern was seen, with three GH “waves” peaking at midnight (p < 0.0001), 1100h (p < 0.02) and at 1600h (p < 0.002). We conclude that the 24 hr rhythmicity of GH secretion is far more complex than currently appreciated. The attribution of the two daytime GH “waves” to food consumption is unlikely but cannot be excluded at the present time. The complex temporal pattern of pulsatile GH secretion may have important effects on regulation of target cell function.     

Effect of Ethanol Exposure on Circulating Levels of Insulin-Like Growth Factor I and II, and Insulin-Like Growth Factor Binding Proteins in Fetal Rats

Maternal ethanol (ETOH) exposure is associated with impaired fetal growth. Because insulin-like growth factors (IGFs) are thought to be important in the regulation of fetal somatic growth, we examined the influence of maternal ETOH exposure on fetal growth and plasma levels of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in the rat model. Control (A) dams were fed a standard rat chow ad libitum. ETOH (E) consuming dams were fed a 36% ETOH diet, and pair-fed (P) dams were fed isocaloric amounts of a control liquid diet. All animals were killed on day 20 of gestation. Plasma concentrations of IGF-I and -II were determined by radioimmunoassay after formic acid-acetone extraction and heat inactivation of IGFBPs. Levels of IGFBPs in fetal plasma were estimated by Western ligand blotting after protein separation by SDS-PAGE and electrotransfer to nitrocellulose. Membranes were probed with [¹²⁵l]IGF-l, and IGFBPs were identified by autoradiography, quantified by scanning densitometry and results expressed relative to corresponding IGFBPs in control fetal plasma. Maternal weight gain from conception to 20 days of pregnancy was reduced for E compared to P and A dams (p < 0.05 E vs. P or A). The same pattern was reflected in fetal weight that tended to be lower in P compared with A pups, and was significantly reduced in E pups compared with both groups (p < 0.0001 E vs. P or A). Thus, fetal growth was more retarded in E animals despite equal caloric and protein intake by E and P dams. Radioimmunoassay of fetal plasma revealed that there was no difference in circulating levels of IGF-II in A (65.5 ± 8.7 ng/ml) and P (65.9 ± 9.8 ng/ml) plasma. However, levels of IGF-II were higher in E (87.1 ± 6.2 ng/ml) than in corresponding P animals. At the same time, there were no differences in IGF-I levels between any of the treatment groups. Western ligand blotting demonstrated that levels of 32–34 kDa IGFBPs were elevated in plasma of P compared with A fetal plasma, consistent with an effect of reduced maternal nutrition. In contrast, levels of32–34 kDa IGFBPs were reduced in E plasma compared with P and A (p < 0.04 and p < 0.02, respectively). These data suggest that circulating levels of IGF-II (but not IGF-I) and specific IGFBPs are altered in growth-retarded fetuses exposed to ETOH. Comparison to fetal plasma obtained from pair-fed litters demonstrate that these effects on IGF and IGFBP levels are specific to ETOH, and not simply due to maternal nutrient intake. Taken together, these observations indicate that ETOH has unique effects on fetal IGF physiology compared with previously studied models of fetal growth retardation. Alterations in the expression and levels of IGFs and IGFBPs may contribute to ETOH-induced fetal growth deficiency.     

Effects of In Vitro Ethanol and Fetal Ethanol Exposure on Glutathione Stimulation of N-Methyl-D-Aspartate Receptor Function

The present studies investigated the effects of glutathione (GSH; γ-glutamylcysteinylglycine) and its oxidized form (GSSG) on neuronal N -methyl-D-aspartate (NMDA) receptor activation in both acute and chronic preparations of ethanol exposure. It was demonstrated using fura-2-loaded dissociated brain cells from newborn rat pups that both GSH and GSSG (0–4 mM) produced concentration-dependent increases in intracellular calcium similar to those produced by NMDA and other agonists of the NMDA receptor. GSH-stimulated calcium entry was not inhibited by low intoxicating concentrations of ethanol, which contrasts with ethanol's typical inhibitory effect on NMDA-stimulated receptor activation. Behavioral studies in adult rats demonstrated that ethanol-induced sleep times were significantly decreased when 10 μl of GSSG (20 mM) were administered intracere-broventricularly approximately 5 min before an intraperitoneal injection of 20% (w/v) ethanol (3 g/kg). These findings suggest that the less potent effect of ethanol on GSH-stimulated calcium entry as well as the reduction in ethanol-induced sleep times may be related to the presence of glycine in the peptide. The glycine found in GSH may activate the glycine site and block or reduce ethanol's action on this site. It appears that although GSH may play an important role in the activation of the NMDA receptor, this action does not involve a process that is sensitive to acute ethanol exposure. In contrast, when rat pups were chronically exposed to ethanol via prenatal exposure before the fura-2 preparation, increases in NMDA- and GSH-stimulated calcium entry were significantly decreased relative to those in pair-fed and ad libitum-fed controls. Thus, chronic in utero exposure to ethanol may alter the NMDA-receptor complex, such that calcium entry mediated by NMDA or GSH activation is significantly reduced.     

Prenatal Ethanol Exposure Selectively Reduces the mRNA Encoding α-1 Thyroid Hormone Receptor in Fetal Rat Brain

Some of the developmental defects characteristic of congenital or experimental hypothyroidism are also observed in children or experimental animals prenatally exposed to ethanol, suggesting that a subset of neurological defects attributable to ethanol exposure are produced by interfering with thyroid hormone action. In this article, we tested whether an ethanol treatment regimen known to produce neurological damage in rats can alter the expression of the mRNAs encoding the thyroid hormone receptor isoforms (TR α-1, TR α-2, and TR α-1) in the fetal rat brain neocortex and hippocampus. Rats were fed an ethanol-containing diet beginning on gestational day (G) 6 and continuing until sacrifice on G15, G17, or G21; controls included animals pair-fed a liquid control diet or fed lab chow. Ethanol selectively reduced the expression of TR a-1 mRNA in the neocortex and hippocampus on G21, compared with pair-fed and control fetuses. In contrast, pair-feeding selectively reduced TR a-2 mRNA in both neocortex and hippocampus on G21, and increased TR 0–1 mRNA on G17. These data support the hypothesis that ethanol may interfere with thyroid hormone action during fetal brain development In addition, these data indicate that ethanol and pair-feeding exert independent effects on thyroid hormone receptor expression in the developing brain.     

Ethanol Suppresses Growth Hormone-Mediated Cellular Responses in Liver Slices

Previous studies indicate that both acute and chronic ethanol administration inhibit protein synthesis and decrease the secretion of insulin-like growth factor-1 (IGF-1). Although IGF-1 synthesis and secretion are regulated by growth hormone secretion from the pituitary gland, we assessed whether ethanol inhibits tissue response to growth hormone. Liver slices from male Sprague-Dawley rats were prepared, placed into F-12 media, and incubated at 37°C with [³H]leucine, and either 0.25 or 1 nM rat growth hormone and 0, 37 (physiological levels), or 175 mM (toxic levels) ethanol. Tissues were removed at 0,15,30, and 60 min. Protein synthesis increased linearly during this time period, and administration of growth hormone (1 nM) significantly increased protein synthetic rate by 48% ( p < 0.01), whereas addition of 37 or 175 mM ethanol attenuated the effects of growth hormone ( p < 0.01). Analysis of IGF-1 mRNA indicated a 2-fold increase in response to growth hormone ( p < 0.01), whereas ethanol administration decreased the growth hormone-induced rise of IGF-1 mRNA. Ethanol (175 mM) inhibited the release of IGF-1 into the media ( p < 0.05). Ethanol did not alter growth hormone receptor binding, and exposure of tissue slices to ethanol did not influence the number of growth hormone receptors or the affinity of growth hormone for its receptor.
Our results demonstrate that (1) growth hormone is a potent acute regulator of IGF-1 mRNA and IGF-1 peptide release, (2) ethanol inhibits growth hormone-induced protein synthesis and induction of IGF-1 gene expression, and (3) the inhibitory effects of ethanol on growth hormone occur without changing growth hormone receptor number or binding characteristics. We conclude that ethanol suppresses growth hormone-induced signal transduction, resulting in a decrease in IGF-1 gene expression.     

Modulation of the Insulin-Like Growth Factor System by Chronic Alcohol Feeding

Insulin-like growth factor (IGF)-I is a potent anabolic agent that plays an important role in regulating muscle protein balance. Alterations in one or more of the various components of the IGF system may be in part responsible for the muscle wasting that accompanies chronic alcohol consumption. The purpose of the present study was to characterize changes in the growth hormone-IGF axis produced by chronic alcohol consumption in rats. After 8 weeks of alcohol feeding, the IGF-I concentration was decreased in plasma (31%) as well as in the liver and skeletal muscle (40–50%), compared with pair-fed control animals. In addition, alcohol consumption decreased IGF-I mRNA abundance in liver and muscle (∼50%). IGF-I content in duodenum and kidney, however, was not altered by alcohol feeding. Concomitantly, the relative concentration of IGF binding protein (IG-FBP)-1 was increased in plasma, liver, and muscle of alcohol-fed rats, compared with control values. In contrast, no changes in the plasma concentrations of IGFBP-2, -3, or -4 were detected in alcohol-fed rats at this time point. Previous studies have indicated that elevations in glucocorticoids or decreases in insulin or growth hormone might be responsible for the decrease in IGF-I and/or the increase in IGFBP-1 in other catabolic conditions. However, there was no difference in the plasma concentrations of these hormones between alcohol-fed and control animals in this study. These data indicate that chronic alcohol feeding in rats decreases IGF-I and increases IGFBP-1 in the circulation and in skeletal muscle and that these changes appear to be independent of changes in classical hormonal regulators of the IGF system. The observed alterations in the IGF system are consistent with a reduction in the anabolic actions of IGF-I induced by chronic alcohol consumption.     

Ethanol Effects on Embryonic Craniofacial Growth and Development: Implications for Study of the Fetal Alcohol Syndrome

Fetal alcohol syndrome (FAS), which is brought about by maternal consumption of ethanol during pregnancy, is a major public health problem. To gain understanding of the etiology of this condition, a number of teratological studies have been performed in different animal systems to develop an animal model for FAS. The C57BL/6J mouse strain has been described as susceptible to the teratogenic effects of ethanol, whereas the ICR (CD-1) strain is considered relatively insensitive. We have compared the effects of ethanol on DNA and protein synthesis in cultured embryonic palate mesenchymal cells from both strains to determine if the reported differential sensitivity to ethanol is reflected in differences in ethanol's effects on cell behavior. Chronic exposure to 200 mM ethanol for 48 hr had a strong inhibitory effect on DNA synthesis in palate cells derived from both the C57BL/6J and ICR strains and a significant effect on protein synthesis in C57BL/6J palate cells. When we attempted to verify strain differences in susceptibility to ethanol teratogenesis, we were not able to observe an increased incidence of birth defects due to ethanol in either strain. High doses of ethanol (5.8 g/kg, administered by intraperitoneal injection on gestational day 8) resulted in death in both C57BL/6J and ICR mice. A lower dose (4.8 g/kg) caused decreased fetal weight and increased resorption in both strains, but did not bring about FAS-like craniofacial dysmorphology in either strain. It appears, therefore, that whereas ethanol can significantly affect the behavior of cells derived from craniofacial tissue, these effects cannot be correlated with sensitivity to ethanol teratogenesis in the mouse system. In view of these results, we suggest that the suitability of the mouse model for FAS be reviewed.     

Ethanol-Induced Alterations in the Posttranslational Processing, But Not Secretion of Luteinizing Hormone-Releasing Hormone In Vitro

The effects of ethanol (EtOH) on the male hypothalamic pituitary reproductive axis are multiple and varied. Although direct gonadal toxicity has been reported, hypothalamic-pituitary perturbations have also been noted. The difficulty of sampling the hypothalamus has made direct investigation of EtOH-induced alterations on luteinizing hormone-releasing hormone (LHRH) fraught with interpretation problems. To circumvent this, we have conducted a series of experiments exploring the effect of 200 mg% EtOH in vitro on GT1-7 cells, a newly developed LHRH secreting neural cell line. Cell lines were treated with EtOH-containing or EtOH-free media for 2,6,24, or 48 hr. EtOH caused no significant change in LHRH secretion at any time point, although there was a trend to increased secretion after 2 hr EtOH exposure when compared with control. Significantly increased total (i.e., cellular plus secreted) pro-LHRH coupled with significantly reduced cellular LHRH after 6 hr only of EtOH exposure suggested that EtOH caused a transient decrease in processing from bioinactive pro-LHRH to bioactive LHRH. However, even at this time point, LHRH secretion from these EtOH-exposed cells was no different than from control cells. Steady-state LHRH mRNA levels were not changed by EtOH at any time point. These findings are concordant with previous in vitro data using hypothalamic tissue that has similarly demonstrated no effect of EtOH on LHRH secretion. Taken together with the in vivo demonstration that EtOH reduces hypothalamic-pituitary portal blood levels of LHRH, these data indicate that EtOH exerts its effect either at an extrahypothalamic locus and/or on non-LHRH-producing cells within the hypothalamus.     

Effect of Fetal Alcohol Exposure on Postnatal Pituitary Adenosine 3',5' -Cyclic Phosphate Content and Growth Hormone Release

This study examines the influence of fetal ethanol (ETOH) exposure and pair-feeding dams on postnatal, releasing factor-induced pituitary growth hormone (GH) release and adenosine 3′,5′-cyclic phosphate (CAMP) accumulation. Fetuses were exposed to ETOH in utero by feeding dams a 36% (calories derived from ETOH: 6.6% v/v) ETOH liquid diet. Postnatal body weights were measured at sacrifice to evaluate the influence of ETOH on growth. Pituitary weight and protein content were measured to determine if changes in GH secretion or cAMP are proportional to the overall effect of ETOH on the pituitary. Pituitaries from 1-, 10-, and 60-day-old pups were explanted and incubated without hormones or with either somatostatin [somatotropin-release inhibiting factor (SRIF); 10^?9 M], or GH-releasing factor (GRF; 5 × 10^?9 M). Radioimmunoassays were used to determine tissue cAMP content, after extraction, and media GH concentration. Results indicate that fetal ETOH exposure specifically reduces the weight of both male and female pups. However, by 60 days of age, this reduction is not different from that found in pups of pair-fed controls, and both groups weighed less than pups of ad libitum controls. Furthermore, both pituitary weight and protein content were proportionately reduced in ETOH-exposed pups. In regard to releasing factor sensitivity, compared with pituitaries from ad libitum controls, the capacity of GRF to simulate GH release was diminished in 10-day-old males (p < 0.00s) exposed to ETOH. On the other hand, the capacity of GRF to stimulate cAMP accumulation was generally enhanced by prenatal ETOH exposure. The capacity of SRIF to depress GH release was diminished in ETOH pups, compared with both pair-fed and ad libitum-fed controls (p < 0.0001). This difference in GH release was more apparent in pituitaries from females than males (p < 0.001). However, the depressed SRIF response was not associated with altered cAMP accumulation. These data suggest that fetal ETOH exposure has a sexually dimorphic effect on pituitary sensitivity to GH-releasing factors that may be related to altered regulation of GH release and susceptibility to growth retardation.     

Effect of Ethanol on Insulin-Like Growth Factor-II Release from Fetal Organs

This study examines the effect of ethanol (ETOH) exposure and nutrient restriction on the release of insulin-like growth factor (IGF)-II from 18- and 20-day explanted fetal organs. Fetuses were exposed to ETOH (E) in utero by feeding dams a 36% (calories derived from ETOH: 6.6% v/v) ETOH liquid diet. Control fetuses were offsprings of dams either pair-fed (P) a control liquid diet or ad libitum (A) fed a standard pelleted lab chow. Brain, heart, kidney, liver, lung, muscle, and placenta of fetuses from the same litter were pooled and explanted, and IGF-II concentration in explanted media was analyzed by radioimmunoassay. Maternal and fetal weights were determined during pregnancy and at sacrifice, respectively, to evaluate the influence of ETOH on growth. Both maternal and fetal weights were substantially reduced by ETOH on 18 and 20 days of gestation compared with both A and P controls. At 18 days of gestation, E fetuses (1.33 ± 0.03 g) weighed less than either A (1.47 ± 0.03 g) or P (1.54 ± 0.04 g) fetuses. By 20 days, A mean fetal weight (4.19 ± 0.23 g) was significantly greater than both P (3.74 ± 0.06 g) and E (3.28 ± 0.06 g) fetuses. IGF-II concentration in media from 18-day fetal explants was highest from E (brain, heart, liver, and placenta) and P tissues (kidney, lung, and muscle). IGF-II in media from A tissues (except placenta) was lower than both E and P levels. A significant difference between treatments occurred in heart. By 20 days, IGF-II levels were highest in media from all A tissues (except placenta). IGF-II in media from E tissues (except lung) was lower than those from P tissues. A significant difference between treatments occurred in the brain. With regard to the developmental pattern, IGF-II release generally increased between 18 and 20 days of gestation, with the greatest increases occurring in A tissues. Increased secretion by P tissues was greater than that by corresponding E tissues, and tended to follow the A trend. On the other hand, E brain, kidney, and placenta released only slightly more IGF-II at 20 days compared to 18 days, whereas E heart, liver, lung, and muscle released slightly less hormone. This study suggests that even moderate nutrient deprivation influences the pattern of IGF-II release from fetal organs, even though there is only a small decrease in overall body size. At the same level of nutrient deprivation, ETOH more dramatically alters both fetal weight and the pattern of IGF-II release. Because IGFs are autocrine/ paracrine factors that influence growth, differentiation, and function, the reduced availability of IGF-II may be one of the factors contributing to ETOH-induced growth retardation and impaired functional capacity of some organ systems.     

In Utero Ethanol Exposure Elicits Oxidative Stress in the Rat Fetus

Prior studies in our laboratory have shown that exposure of cultured fetal rat hepatocytes to ethanol (E) blocks epidermal growth factor-dependent replication and that this is paralleled by cell membrane damage, mitochondrial dysfunction, membrane lipid peroxidation (LP), and enhanced generation of reactive oxygen species. These measures of E-mediated oxidative stress (OS) were mitigated by treatment with antioxidants, and cell replication could be normalized by maintaining cell glutathione (GSH) pools. We have now extended these studies to an in vivo model. Rats were administered E (4 g/kg, po) at 12-hr intervals on days 17 and 18 of gestation and killed on day 19,1 hr following a final dose of E (a total of 5 doses). Fetal and maternal brain and liver were assayed for signs of OS. The 2-day in utero E exposure increased membrane LP in fetal brain as evidenced by increased malondialdehyde (MDA) levels from 1.76 ± 0.12 se (nMol/mg protein) to 2.00 ± 0.08 (p < 0.05) and conjugated dienes from 0.230 ± 0.006 se (OD₂₃₃/mg lipid) to 0.282 ± 0.006 (p < 0.05). In fetal liver, MDA levels increased from 2.39 ± 0.08 se (nMol/mg protein) to 2.87 ± 0.08 (p < 0.05), whereas dienes differed significantly only between ad libitum controls and the E and pair-fed control groups (p < 0.05). E decreased GSH levels in fetal brain by 19%, from 19.88 ± 0.72 to 16.13 ± 1.06 (nMol/mg protein) (p < 0.05). A10% decrease in GSH was seen in fetal liver (p < 0.05). GSH in maternal brain was decreased by 44% from 47.29 ± 3.38 to 26.60 ± 2.29 (p < 0.05). Other E-related increases in these OS measures were not observed in maternal organs. E did not decrease α-tocopherol levels in fetal and maternal brain or in fetal liver (p < 0.05), whereas maternal liver α-tocopherol content was reduced by 31% (p < 0.05) by E treatment. It is concluded that maternal E consumption can induce an OS in fetal tissues that may contribute to the fetotoxic effects of E.     

Ethanol Promotes Cell Death by Inhibition of the Insulin-Like Growth Factor I Receptor

The mechanism by which chronic alcohol abuse induces widespread cell and tissue damage is unknown. Insulin-like growth factor I (IGF-I) is an important inhibitor of apoptosis in many cell types, in addition to its ability to stimulate proliferation. We have demonstrated previously (J, Biol. Chem . 268:21777–21782,1993; Lab. Invest . 71657–662, 1994) that ethanol in low concentrations inhibits the tyrosine auto-phosphorylation of the IGF-I receptor (IGF-IR) and IGF-I-mediated cell proliferation. We now demonstrate that ethanol reverses the antiapoptotic action of the IGF-IR in a tumor necrosis factor-a (TNF-α) model of apoptosis. In serum-depleted medium, IGF-I markedly protected BALB/c3T3 cells from TNF-α-induced apoptosis. Ethanol reversed the protective action of IGF-I, but did not enhance TNF-α killing in the absence of IGF-I. Hatf-maximal effective concentrations of ethanol were 5 to 10 mM. In the presence of 5 to 10% fetal bovine serum, TNF-α was cytotoxic for 3T3 cells only in the presence of ethanol. Mouse embryo fibroblasts with targeted knockout of the IGF-IR were completely insensitive to ethanol, in contrast with the ethanol-induced potentiation of apoptosis in wild-type cells. These results indicate that ethanol directly interacts with cellular factors that inhibit apoptosis and could provide a novel mechanism for ethanol-induced cytotoxicity in general.     

Influence of Fetal Alcohol Exposure on the GABAergic Regulation of Growth Hormone Release in Postnatal Rats

BACKGROUND: Disruption of the growth hormone (GH) axis by maternal ethanol (ETOH) consumption may contribute to abnormalities in offspring. Interestingly, gamma-aminobutyric acid (GABA) neurotransmission, which is vulnerable to fetal ETOH exposure, also regulates the hypothalamic-pituitary GH axis. This study examines whether GABAergic control of this axis is disrupted by prenatal ETOH exposure. METHODS: Pregnant dams were fed either rat chow ad libitum or a 36% ETOH diet (by calories), or were pair-fed an isocaloric control diet. Hypothalami and pituitaries from offspring were coperfused, in vitro, with muscimol, a GABA(A) agonist, either alone or in combination with bicuculline, a GABA(A) antagonist. Perfusates were analyzed by radioimmunoassay for GH, somatostatin (SRIF), and GH-releasing factor (GRF). RESULTS: Normal development of GABA regulation was evaluated first in control offspring. Sensitivity to muscimol (measured by percent increase in GH above basal levels) occurred at all ages and generally was greater in male compared to female tissue. Furthermore, the efficacy of bicuculline in depressing muscimol-induced GH secretion increased with age in both sexes. In males, this response correlated with increased SRIF release. In females, releasing factor data were highly variable relative to the percent change and are not presented. Maternal ETOH consumption altered the development of GABAergic regulation of the GH axis in offspring. flowever, because ETOH induced changes in the response of releasing factors to muscimol appear to offset each other, a disruption in GH release was not evident. More apparent was the reduced capacity of bicuculline to reverse muscimol-induced GH release from male tissue. This ETOH effect was evident at 35-days of age and was associated with reduced SRIF release. In female tissue, a reduced bicuculline response was also suggested at 35 days of age. After puberty no response was elicited by muscimol in either tissue from pair-fed or ETOH-exposed female offspring. CONCLUSION: In summary, fetal ETOH exposure influences the development of GABAergic regulation of the hypothalamic-pituitary GH axis in an age and gender specific manner. Vulnerability of the male axis is expressed by the reduced capacity of bicuculline to depress GH release and altered releasing factor sensitivity to GABA(A)-receptor stimulation or inhibition. There is also some suggestion that the female axis is less sensitive to bicuculline during early puberty, and, unlike the male, is insensitive to both muscimol and bicuculline after puberty. The latter, however, may be attributable to stress or nutritional deprivation, rather than to the direct effect of prenatal ETOH.     

Cultured Postnatal Rat Medial Septal Neurons Respond to Acute Ethanol Treatment and Nerve Growth Factor By Changing Intracellular Calcium Levels

Ethanol neurotoxicity results in the loss of neurons during the development of the nervous system. Nerve growth factor (NGF) can ameliorate the neurotoxic effects of ethanol (EtOH) in rat medial septal (MS) neurons. These experiments study the effects of EtOH and NGF on neuronal calcium (Ca²⁺) homeostasis in cultured postnatal day of birth (PO) rat MS neurons. Previously, we observed that EtOH and NGF modulate intracellular Ca²⁺ levels ([Ca²⁺]i) in unstimulated and high potassium stimulated (30 mM KCI) cultured rat embryonic day 21 (E21) MS neurons (Webb et al., Brain Res 701:61-74, 1995). The purpose of the present study was to explore whether the effects of EtOH and NGF on Ca²⁺ homeostasis were altered by developmental stage. The hypotheses tested were the following: treatment with EtOH affects Ca²⁺ homeostasis in postnatal day of birth (PO) rat MS neurons by causing transient and persistent changes in [Ca²⁺]i; NGF modulates Ca²⁺ homeostasis in MS neurons by regulating [Ca²⁺]i; the action of NGF changes the response of MS neurons to EtOH, thus altering Ca²⁺ homeostasis; and that EtOH and or NGF effects on Ca²⁺ homeostasis are developmentally regulated. Our results indicated that behaviorally relevant levels of EtOH caused a rapid transient increase in basal [Ca²⁺]i, whereas there was no effect of NGF on basal [Ca²⁺]i. Ethanol and NGF interacted, resulting in the lowering of [Ca²⁺]i. During stimulation with high K⁺, EtOH inhibited the change in [Ca²⁺]i. NGF partially ameliorated this effect of higher levels of EtOH, allowing [Ca²⁺]i to increase. NGF and the lowest level of EtOH potentiated the high K⁺ stimulated increase in [Ca²⁺]i. Ethanol and NGF effects on [Ca²⁺]i were different in the PO neurons compared with our previously published observations in E21 neurons. Therefore, these data suggest that EtOH neurotoxicity and NGF protection involve mechanisms that regulate neuronal Ca²⁺ homeostasis, and the magnitude of these effects depend on developmental stage.     

In Utero Ethanol Exposure Increases Proenkephalin, a Precursor of a Neuropeptide That Is Inhibitory to Neuronal Growth

BACKGROUND: One of the effects of fetal alcohol syndrome (FAS) is altered motor function. In an attempt to elucidate a potential cause of this ethanol-associated damage, we investigated the effects of in utero ethanol exposure on the production of proenkephalin (PE). METHODS: PE is the precursor of met- and leu-enkephalin, two neuropeptides that inhibit the proliferation of neurons and astrocytes. PE mRNA and PE peptide were measured in the striatum and nucleus accumbens because of the importance of these brain regions to motor function, their sensitivity to the effects of in utero ethanol exposure, and their high concentration of PE mRNA and PE peptide. RESULTS: The present studies demonstrated that in utero ethanol exposure is associated with increased PE mRNA in the striatum and nucleus accumbens. Of specific interest is the elevation in PE mRNA in the nucleus accumbens in 5-, 12-, and 19-day-old ethanol-exposed offspring and the fact that this change persists for at least 19 days after the last exposure to ethanol. Further studies of postnatal day 19 (PN19) offspring localized the abnormality in the nucleus accumbens to the core region, an area that contains enkephalinergic projections to another motor area-the substantia nigra. In the nucleus accumbens, the increased PE mRNA was associated with a greater proportion of PE-expressing neurons that have 41% to 60% of the cell area covered by grains associated with PE mRNA. CONCLUSIONS: Although we were not able to detect a change in the concentration of the PE peptide in the nucleus accumbens or striatum, we cannot rule out the possibility that the increase in PE mRNA was reflective of a functional abnormality.     

Ethanol Effects on Cultured Embryonic Hippocampal Neuronal Calcium Homeostasis Are Altered by Nerve Growth Factor

The neurotoxic effect of acute ethanol treatment (AET) may lead to an alteration in the regulation of calcium (Ca²⁺) homeostasis in hippocampal neurons. Ca²⁺ homeostasis could be affected by AET when neurons are at rest or after depolarizing activity during synaptic transmission. It has been shown that nerve growth factor (NGF) can ameloriate some types of neurotoxicity by stabilizing Ca²⁺ homeostasis. Previously, we observed that ethanol (EtOH) changed unstimulated (basal) and potassium (K⁺)-stimulated intracellular calcium ([Ca²⁺]ⁱ) in embryonic septohippocampal neurons (Webb et al., Brain Res. 729:176-189,1996). The purpose of the present study is to determine the effects of NGF and EtOH on neuronal Ca²⁺ homeostasis in cultured embryonic hippocampal neurons. The hypotheses tested were the following: EtOH alters Ca²⁺ homeostasis in hippocampal neurons; NGF modulates Ca²⁺ homeostasis in hippocampal neurons; and NGF treatment alters the effect of EtOH on [Ca²⁺]¹, in hippocampal neurons. Our results indicated that hippocampal neuronal cultures treated with EtOH had lower basal [Ca²⁺]¹ than untreated neurons. EtOH decreased K⁺-stimulated (30 mM KCl) changes in [Ca²⁺]¹ in a dose-dependent manner. During K⁺ stimulation, 20 ng/ml of NGF slowed and reduced the increase in [Ca²⁺]¹. Hippocampal neurons treated with NGF increased or did not change basal [Ca²⁺]¹ and did not change or increase K⁺-stimulated [Ca²⁺], in response to EtOH. These responses were dose-related and indicated that NGF could alter the response of hippocampal neurons to EtOH. In conclusion, AET results in the alteration of Ca²⁺ homeostasis in unstimulated and depolarized cultured embryonic hippocampal neurons. NGF and EtOH independently and collectively affected the regulation of Ca²⁺ homeostasis in this neuronal population. Changes in [Ca²⁺]¹ can disrupt normal cellular function and contribute to cell death. Therefore, alteration of Ca²⁺ homeostasis may be an underlying mechanism involved in EtOH toxicity. NGF may ameliorate the toxic effects of EtOH by regulating Ca²⁺ homeostasis.