Increased Cell Death and Reduced Neural Crest Cell Numbers in Ethanol-Exposed Embryos: Partial Basis for the Fetal Alcohol Syndrome Phenotype

Fetal alcohol syndrome (FAS) is characterized by growth retardation, craniofacial malformations, and heart and neural defects; the cellular and molecular mechanism(s) responsible for ethanol's teratogenicity remains unknown. Although the phenotype suggests that prenatal ethanol exposure perturbs neural crest cell development, direct proof that these cells are an in utero target is still lacking. Previous research suggested that cranial neural crest cells are eliminated by ethanol-induced apoptosis. We tested this hypothesis using a chick embryo model of FAS. A single dose of ethanol, chosen to achieve a concentration of 35–42 mg/dl, was injected in ovo at gastrulation and resulted in growth retardation, craniofacial foreshortening, and disrupted hindbrain segmentation. Ethanol exposure enhanced cell death within areas populated by cranial neural crest cells, particularly in the hindbrain and craniofacial mesenchyme. In contrast, control embryos had limited cell death within these regions. Subsequent immunolabeling with neural crest cell-specific antibody revealed that ethanol treatment resulted in fewer neural crest cell numbers, whereas neural crest migration patterns were unaffected by ethanol. These results suggest that prenatal ethanol exposure leads to loss of cranial neural crest cells. Such a loss could result, in part, in the phenotype characteristic of FAS.     

Ethanol-induced cephalic apoptosis requires phospholipase C-dependent intracellular calcium signaling

BACKGROUND: Although the ability of ethanol to elicit neural crest cell apoptosis is well documented, the initial target of ethanol in these cells, and the biochemical pathway leading to their apoptosis, have yet to be determined. Recent work in preimplantation mouse embryos demonstrates that ethanol induces a phospholipase-C (PLC)-dependent calcium transient that mediates ethanol's effects. We tested whether a similar effect on calcium and PLC is involved in ethanol-induced neural crest apoptosis. METHODS: Chicken embryos were collected and loaded with Fluo-3-AM to assess the effects of ethanol on intracellular calcium levels. Pharmacological agents were used to determine the sources and mechanism of intracellular calcium increases. In separate experiments, embryos were treated in ovo with pharmacological modulators of calcium signaling prior to ethanol exposure, and resulting levels of cell death were assessed by using the vital dye acridine orange. RESULTS: Ethanol exposure caused a localized increase in intracellular calcium levels in embryonic neural folds within 15 sec of ethanol exposure. Ethanol-induced apoptosis was specifically blocked by chelation of intracellular calcium before ethanol exposure. Pretreatment with the PLC inhibitor U73122 blocked ethanol-induced apoptosis as well as the intracellular calcium transient. Depletion of extracellular calcium resulted in a partial block of ethanol-induced apoptosis. CONCLUSIONS: Ethanol exposure alters calcium signaling within the neurulation-stage chicken embryo in a PLC-dependent manner. Increases in intracellular calcium and PLC activity are necessary for ethanol's induction of apoptosis within cephalic populations. These effects likely represent an early and crucial event in the pathway leading to ethanol-induced cell death.     

Genetic influences on craniofacial outcome in an avian model of prenatal alcohol exposure

BACKGROUND: Understanding the basis for ethanol's teratogenic effects may inform the etiology of fetal alcohol syndrome. Here we investigate how genetic background and susceptibility to ethanol-induced neural crest apoptosis contribute to the distinctive craniofacial phenotype observed after prenatal alcohol exposure. METHODS: Nine different chick strains were exposed to ethanol at gastrulation. The sensitivity of these embryos to ethanol-induced neural crest apoptosis was reported elsewhere (Debelak and Smith, 2000). Here, these embryos were permitted to develop until embryonic day 10, when facial morphogenesis was largely complete, and cephalometric measurements were made on cleared skulls. Shifts in facial growth were correlated against the severity of ethanol-induced apoptosis in facial precursors. RESULTS: The facial shape produced by ethanol exposure was a function of the embryos' genetic strain. Three general responses were observed: apparent midfacial flattening (Babcock B300 x Hampshire Red, ISA-Babcock, HyLine W98, and HyLine W36 strains), overall facial expansion (Spafas and Babcock B300 strains), or overall facial hypoplasia (DeKalb strains). When dose and timing of exposure were held constant, the embryo's genetic background predicted the facial outcome. For ethanol-sensitive strains, apoptosis of facial precursor populations was required to produce the facial defects. That some strains had essentially normal faces despite extensive cell death indicated a capacity to recover from the earlier neural crest losses. CONCLUSIONS: We propose that ethanol's effects on craniofacial development are multifactoral, and these influences may include susceptibility to apoptosis, regenerative capacity, and compensatory outgrowth of the facial primordia. The embryo's genetic background may modulate these events. The high and low responder chick strains are useful tools to dissect these contributions.     

Stage-Dependent Effects of Ethanol on Cranial Neural Crest Cell Development: Partial Basis for the Phenotypic Variations Observed in Fetal Alcohol Syndrome

Fetal alcohol syndrome (FAS) is characterized by growth retardation, mental deficiencies, and numerous craniofacial and neuronal anomalies; the type and severity of these defects may be related to the time and dose of maternal ethanol exposure. Ethanol administered during presomitic stages results in the typical FAS craniofacial phenotype and is accompanied by a loss of cranial neural crest cells (CNCCs) through ethanol-induced cell death. However, the stage-specific effects of ethanol on the CNCC population is unknown. We examined the effects of ethanol on CNCC populations by treating in ovo chick embryos with a single ethanol dose (0.43 mmol/egg) at various stages of CNCC development, and corresponding to the first 3–4 weeks of human gestation. Ethanol treatment induced cell death and reduced CNCC populations in patterns consistent with observed dysmorphologies of CNCC-derived cranial structures. The precise population affected was dependent on the timing of ethanol exposure. Treatment at gastrulation or neurulation induced cell death and losses of CNCC populations, particularly those in rostral positions, and resulted in more severe craniofacial defects. In contrast, treatment at early somitic stages (4–16 somites) induced cell death, primarily within caudal CNCC populations, but resulted in less severe craniofacial defects, suggesting an increased capacity for recovery. These results suggest that there are distinct developmental windows during which the CNCCs may be particularly susceptible to ethanol-induced cell death. We conclude that ethanol exposure seems to affect specific events adversely during neural crest development. The timing of embryonic ethanol exposure relative to CNCC development could account, in part, for the heterogenous craniofacial defects observed in FAS.     

Genetic and developmental modulation of cardiac deficits in prenatal alcohol exposure

BACKGROUND: Increasing evidence demonstrates that genetic background is an important modulator of alcohol's effects on the developing fetus. Such effects are separable from maternal ethanol metabolism. Here, we study ethanol's effects on cardiogenesis in an avian model that shows strong cell death within neuronal and neural crest precursors following ethanol exposure. METHODS: The study design tested the hypothesis that ethanol-induced losses of cardiac neural crest populations would disrupt outflow tract development and thus contribute to the valvuloseptal deficits observed in prenatal alcohol exposure. Three chick strains were exposed to alcohol at gestational windows between gastrulation and early heart septation (day 3 incubation), and then hearts were examined at the completion of morphogenesis (day 10 incubation). RESULTS: Ethanol's impact on cardiac development was influenced by fetal genetics. The B300 x Hampshire Red cross exhibited pronounced cell death within cardiac neural crest populations but had normal development of the heart and aortic arches. Neural crest migration and differentiation into the distal outflow tract were also normal in these embryos, which suggested a capacity to repair earlier losses. The DeKalb White x Hampshire Red cross also did not exhibit cardiac defects. Hearts of the B300 strain had a unique phenotype with respect to ethanol exposure and exhibited a thin ventricular compact layer, dilatation, and reduced myosin/deoxyribonucleic acid and myosin/protein content, a phenotype that indicates disrupted myocardial maturation and inductive cues. The deficit was only observed when ethanol exposure occurred at stages 15 or 18 and apparently was independent of neural crest cell death. Such ventricular thinning might go undetected in the absence of extensive screening. CONCLUSIONS: Results add to the increasing evidence that genetic background strongly modulates the effects of prenatal alcohol exposure. The results also suggest that embryos have a varying capacity to repair and recover from earlier neural crest losses.     

Embryonic cerebral cortical progenitors are resistant to apoptosis, but increase expression of suicide receptor DISC-complex genes and suppress autophagy following ethanol exposure

BACKGROUND: In utero exposure to ethanol can result in severe fetal brain defects. Previous studies showed that ethanol induces apoptosis in differentiated cortical neurons. However, we know little about ethanol's effects on proliferating embryonic cortical progenitors. This study investigated the impact of ethanol exposure on the Fas/Apo-1/CD95 suicide receptor pathway, and on the survival of proliferating cortical neuroepithelial progenitors. METHODS: Murine embryonic-derived primary cortical neuroepithelial cells were maintained as neurosphere cultures and exposed to a dose range of ethanol for periods ranging from 1 to 5 days. Programmed cell death was measured by 4 independent means (Annexin-V staining, caspase activation, DNA fragmentation, and autophagic vacuole formation). Surface Fas/Apo-1 suicide receptor expression was measured by flow cytometry. Expression of Fas/Apo-1-associated DISC-complex genes was measured by quantitative polymerase chain reaction. RESULTS: Ethanol exposure did not substantially increase apoptosis, necrosis, or surface Fas/Apo-1 expression. Moreover, ethanol significantly decreased caspase activation and autophagic activity. Finally, ethanol exposure induced mRNA expression of genes that constitute the death receptor complex. CONCLUSIONS: This study provides surprising evidence that ethanol does not induce either programmed cell death or necrosis of immature progenitors during neurogenesis, although ethanol may render neural progenitors susceptible to future apoptotic insults. Furthermore, our novel observation that ethanol suppresses autophagy is consistent with a hypothesis that ethanol promotes premature neural progenitor maturation. Taken together with our previous data regarding the role of the Fas/Apo-1 receptor in neural development, we conclude that ethanol disrupts basic proliferation and differentiation machinery rather than initiating cell death per se.     

Free Radicals and Ethanol-Induced Cytotoxicity in Neural Crest Cells

Associations between ethanol-induced cranial neural crest cell (NCC) damage in mammalian embryos and subsequent malformations as observed in human fetal alcohol syndrome have previously been illustrated. The vulnerability of NCCs to this teratogen may result, at least in part, from their sensitivity to free radical damage. To examine relationships between free radical generation and NCC cytotoxicity, primary culture of mouse NCCs was used. NCC viability was determined in both dose- and time-response studies involving ethanol exposure. After 48 hr of culture, cell viability was significantly diminished at all doses tested (i.e., 50,100,150, and 200 mM ethanol). At 100 mM ethanol (a dosage that is teratogenic in vivo and in whole embryo culture), cell viability decreased to ?50% of control values over the first 12 hr of culture, and decreased further, to ?20% by 48 hr. Using nitroblue tetrazolium as a probe, it was observed that exposure of NCCs to ethanol stimulated the production of superoxide anion radicals. Co-treatment of the ethanol-exposed NCCs with free radical scavengers including 300 units/ml of superoxide dismutase, catalase (500 units/ml), or ?-tocopherol (300 μM) significantly improved NCC viability. These results suggest that the ethanol-induced NCC injury is mediated, at least in part, through the generation of free radicals. To test this hypothesis further, NCCs were exposed in culture to xanthine/xanthine oxidase. Exogenous free radicals generated by the xanthine/xanthine oxidase system resulted in reduced NCC viability, the severity of which Increased in a time and enzyme concentration-related manner. Superoxide dismutase (300 units/ml) and catalase (500 units/ml) significantly reduced the effects of the xanthine/xanthine oxidase-generated free radicals on NCC viability. The similarity between the susceptibility of NCCs to ethanol and their susceptibility to exogenous free radicals in concert with the free radical scavenger-mediated amelioration of ethanol and exogenous free radical-induced NCC death strongly suggest that free radicals play a significant role in ethanol-induced NCC death.     

Effect of duration of maternal alcohol consumption on calcium metabolism and bone in the fetal rat

BACKGROUND: Prenatal ethanol exposure can retard fetal growth and delay skeletal development. Ethanol also impairs maternal calcium (Ca) homeostasis and this impairment could mediate some of ethanol's effects on the fetal skeleton. Our previous studies suggest that the duration of maternal ethanol consumption may be an important factor for determining the severity of ethanol's effects on Ca homeostasis and fetal skeletal development. The purpose of this study was, therefore, to determine the effect of the duration of maternal ethanol consumption on fetal growth and skeletal development and to investigate the possibility that ethanol's effects may be related to perturbations in fetal/maternal Ca homeostasis. METHODS: Rats were fed ethanol (36% ethanol-derived calories) in liquid diets for 3 weeks (days 1-21 of gestation) or 6 weeks (for 3 weeks before and throughout gestation). Fetuses were collected on day 21 of gestation, and body weight and length were measured. Fetuses were stained to determine the degree of skeletal ossification, and fetal blood was analyzed for ethanol, Ca (total and ionic Ca), albumin, parathyroid hormone (PTH), and osteocalcin. RESULTS: Maternal ethanol consumption decreased fetal growth and delayed fetal skeletal development. Although there was a trend for fetal body length and serum osteocalcin levels to be more severely affected with an increased duration of maternal ethanol consumption, duration had no effect on fetal body weight or skeletal ossification. Fetal Ca homeostasis was also affected by ethanol exposure, with fetal hypocalcemia apparent after 6 weeks of maternal ethanol intake. A significant inverse relationship was found between fetal blood Ca levels and blood alcohol concentration (BAC), suggesting that the severity of the fetal hypocalcemia may have been related to differences in fetal BAC, rather than duration of maternal ethanol intake. Fetal serum PTH levels did not differ significantly among treatment groups indicating that the fetal hypocalcemia was not caused by a decrease in PTH levels. CONCLUSIONS: Prenatal ethanol exposure impaired Ca homeostasis and skeletal development in the fetal rat. The severity of ethanol's effects was only marginally dependent on the duration of maternal ethanol consumption per se and seemed to be more related to the relative exposure of the fetus to ethanol (fetal BAC). The relationship between the ethanol-induced fetal hypocalcemia and skeletal effects remains to be determined.     

Maternal diabetes in the rat impairs the formation of neural-crest derived cranial nerve ganglia in the offspring

Aims/hypothesis Maternal diabetes mellitus increases the risk for fetal malformations. Several of these malformations are found in organs and tissues derived from the neural crest. Previous studies have shown changes in fetal organs of neural crest origin in experimental diabetes and changes in migration of neural crest cells exposed to high glucose in vitro.Methods We used whole-mount neurofilament staining of embryos from normal and diabetic mothers to investigate the development of cranial nerve ganglia. Neural tube explants were cultured in 10 and 40 mmol/l glucose and cell death and caspase activity was measured with flow cytometry.Results The development of cranial ganglia V, VII, VIII, IX and X was impaired in day 10–11 embryos of diabetic rats. There was also a higher rate of cell death of neural crest derived cells cultured in 40 mmol/l glucose for 20 h (35% compared to 12% in 10 mmol/l). However, exposure of cells to 40 mmol/l glucose in culture did not increase the activation of the cell death effector proteins-caspases-measured as cellular binding of the activated caspase marker VAD-FMK. This suggests that the cell death is not caused by caspase-dependent apoptosis or that the caspases are activated at an earlier stage.Conclusion/interpretation The development of neural crest-derived structures is disturbed already at the organogenic period in embryos of diabetic rats and this deteriorated development could be due to high-glucose induced increase in cell death of neural crest derived cells.Abbreviations N Embryo of non-diabetic mother - MD embryo of diabetic mother - FITC fluorescein isothiocyanate - VAD-FMK Val-Ala-Asp-fluoromethyl ketone     

Ethanol triggers neural crest apoptosis through the selective activation of a pertussis toxin-sensitive G protein and a phospholipase Cbeta-dependent Ca2+ transient

BACKGROUND: Alcohol is a potent neurotoxin that triggers the selective apoptosis of neuronal populations in the developing fetus. For neural crest cells, clinically relevant ethanol levels (0.3%) rapidly elicit a phospholipase C (PLC)-dependent intracellular Ca2+ transient that is sufficient to activate apoptosis. We investigated the biochemical origins of this Ca2+ transient. METHODS: Three somite chick embryos (stage 8-) were pretreated with agonists and antagonists of PLC signaling pathways before ethanol challenge. The resulting intracellular Ca2+ release was quantified using Fluo-3; apoptosis was assessed using vital dyes. RESULTS: Pretreatment of embryos with PLC antagonists U73122 or ET-18-OCH3 confirmed that a phosphoinositide-specific PLC was required for both the ethanol-dependent Ca2+ transient and subsequent cell death. Ethanol rapidly elevated intracellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the rostral portion of the embryo that contains neural crest progenitors. The Ins(1,4,5)P3 receptor antagonist xestospongin C blocked the appearance of the ethanol-dependent Ca2+ transient. Pretreatment with the pan-Galpha protein antagonist GDPbetaS, but not with the tyrosine kinase antagonist genistein, suppressed ethanol's ability to elicit the Ca2+ transient, suggesting that a rise in PLC activity and Ins(1,4,5)P3 concentration originates from stimulation of heterotrimeric G proteins. To probe the identity of this G protein, embryos were treated with G protein antagonists. Pertussis toxin and NF023 suppressed the ethanol-induced Ca2+ transient and subsequent neural crest apoptosis, whereas suramin was weakly inhibitory. C3 exoenzyme was embryolethal over a wide concentration range, consistent with suggestions that Rho family GTPases participate in neural crest development. Galphai2 was identified by immunostaining in the neural crest cells. CONCLUSION: We propose a role for Galphai/o protein activation and subsequent interaction of Gbetagamma with PLCbeta in mediating the proapoptotic effects of ethanol upon the developing neural crest.     

Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoked apoptotic cell death. Incubation of granule cells with PACAP prevented ethanol-induced apoptosis, and this effect was not mimicked by vasoactive intestinal polypeptide, suggesting that PAC1 receptors are involved in the neurotrophic activity of PACAP. Ethanol exposure induced a strong increase of caspase-2, -3, -6, -8, and -9 activities, DNA fragmentation, and mitochondrial permeability. Cotreatment of granule cells with PACAP provoked a significant inhibition of all of the apoptotic markers investigated although the neurotrophic activity of PACAP could only be ascribed to inhibition of caspase-3 and -6 activities. These data demonstrate that PACAP is a potent protective agent against ethanol-induced neuronal cell death. The fact that PACAP prevented ethanol toxicity even when added 2 h after alcohol exposure, suggests that selective PACAP agonists could have potential therapeutic value for the treatment of fetal alcohol syndrome.     

In Utero Ethanol Exposure Causes Mitochondrial Dysfunction, Which Can Result in Apoptotic Cell Death in Fetal Brain: A Potential Role for 4-Hydroxynonenal

BACKGROUND: In utero ethanol exposure causes abnormal fetal brain development that may partly be due to enhanced cell death. The mechanisms underlying this remain to be defined, but ethanol-induced oxidative stress may play a role. The following studies investigated the effects of short-term in utero ethanol exposure on fetal brain mitochondrial events that are known to elicit apoptotic cell death. Evidence is presented suggesting that 4-hydroxynonenal (HNE), a toxic product of lipid oxidation, is a causal factor in the observed mitochondrial damage. METHODS: Mitochondria were isolated from control and ethanol-exposed fetal brains (days 17 and 18 of gestation). Permeability transition was determined spectrophotometrically, and cytochrome c and apoptosis-inducing factor (AIF) release were assessed by Western blotting. Caspase-3 activity and DNA fragmentation were determined both as markers for mitochondrially mediated apoptosis and as consequences of cytochrome c and AIF release. RESULTS: Maternal ethanol intake caused an increase in mitochondrial permeability transition, and this was accompanied by cytochrome c and AIF release from fetal brain mitochondria that exceeded control values by 62 and 25%, respectively (p < 0.05). In utero ethanol exposure resulted in a 30% increase in caspase-3 activity and a 25% increase in DNA fragmentation (p < 0.05) in the fetal brain. HNE levels were increased by 23% (p < 0.05) in mitochondria by in vivo ethanol exposure. In vitro treatment of fetal brain mitochondria with HNE (25-100 microM) also caused increases in mitochondrial permeability transition, as well as dose-dependent releases of cytochrome c and AIF. CONCLUSIONS: These studies illustrate that in utero ethanol exposure can elicit a cascade of events in the fetal brain that are consistent with mitochondrially mediated apoptotic cell death. Additionally, the increase in mitochondrial content of HNE after ethanol intake and the ability of HNE added to fetal brain mitochondria to mimic these effects of in vivo ethanol exposure support a potential role for HNE in the proapoptotic responses to ethanol.     

Rapid induction of apoptosis in gastrulating mouse embryos by ethanol and its prevention by HB-EGF

BACKGROUND: Ethanol exposure during gastrulation and early neurulation induces apoptosis within certain embryonic cell populations, leading to craniofacial and neurological defects. There is currently little information about the initial kinetics of ethanol-induced apoptosis, and interest in the ability of endogenous survival factors to moderate apoptosis is growing. Ethanol alters intracellular signaling, leading to cell death in chick embryos, suggesting that apoptosis could occur rapidly and that signaling pathways activated by survival factors might reduce apoptosis. METHODS: Pregnant mice were intubated with 1, 2, or 4 g/kg ethanol on day 7.5 of embryogenesis (E7.5) 1, 3, or 6, hours before harvesting gastrulation-stage embryos. Control animals received maltose/dextran. Blood alcohol concentrations (BAC) were determined by gas chromatography. E7.5 embryos isolated from untreated dams were cultured in vitro for 1 or 3 hr with 0 or 400 mg% ethanol and 0 or 5 nM heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Apoptosis was quantified using fluorescence microscopy to detect annexin V binding and DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL)] in whole-mount or sectioned embryos. RESULTS: Both annexin V binding and TUNEL were elevated (p < 0.05) in embryos exposed in utero to 1 g/kg ethanol for 3 hours, increasing linearly with time and ethanol concentration. Apoptosis increased (p < 0.05) in all germ cell layers. Mice treated with 4 g/kg sustained BAC of 400 mg% for nearly 3 hours, significantly increasing apoptosis within the first hour. Cultured embryos exposed to 400 mg% ethanol displayed 2- to 3-fold more TUNEL than vehicle-treated embryos (p < 0.05); however, exogenous HB-EGF prevented apoptosis. CONCLUSIONS: Ethanol rapidly produced apoptosis in gastrulation-stage embryos, consistent with induction by intracellular signaling. The ethanol-induced apoptotic pathway was blocked by the endogenous survival factor, HB-EGF. Differences in the expression of survival factors within individual embryos could be partly responsible for variations in the teratogenic effects of ethanol among offspring exposed prenatally.     

Sensitivity and tolerance to autonomic effects of ethanol in adolescent and adult rats during repeated vapor inhalation sessions

BACKGROUND: It is during adolescence that most drinkers initiate ethanol intake, with some of this use being excessive. One possible contributor to the increased ethanol consumption often seen during adolescence in humans and in various animal models is age differences in ethanol sensitivity and tolerance. The present study examined the impact of age on ethanol-related alterations in the autonomic nervous system. METHODS: Sensitivity to the initial ethanol challenge and chronic tolerance as well as acute and protracted withdrawal-like phenomena were assessed in male adolescent and adult Sprague-Dawley rats, using implanted telemetry probes with ethanol delivered via vapor inhalation. RESULTS: Both ages showed similar ethanol-induced tachycardia and activity suppression; however, adolescents were found to be more sensitive than adults to the hypothermic effect of ethanol, data opposite other results from our laboratory and elsewhere using intragastric intubations or intraperitoneal administrations of ethanol. Although little tolerance to ethanol's tachycardic or activity suppressant effects was seen after repeated ethanol inhalation sessions, chronic tolerance to ethanol's hypothermic effect developed faster in adults than in adolescents. A withdrawal-like syndrome, characterized by bradycardia and hypoactivity, typically emerged during the dark phase of the diurnal cycle after ethanol vapor exposure sessions. These effects were observed in animals of both ages, with the bradycardic effect more pronounced in adolescents. CONCLUSIONS: In contrast to results indicating that adolescents may be less sensitive than adults to ethanol's hypothermic effect when ethanol is administered via bolus injection/intubation, adolescents appear more sensitive and develop tolerance to ethanol's hypothermic effects more slowly than adults when ethanol is administered at a more moderate rate via vapor inhalation.     

Avian genetic background modulates the neural crest apoptosis induced by ethanol exposure

BACKGROUND: Ethanol-induced neural crest apoptosis likely contributes to the distinctive craniofacial phenotype that results from prenatal alcohol exposure. The mechanism responsible for this apoptosis is incompletely understood. A serendipitous change in poultry production flocks led to the discovery that, in chick, the embryo's genetic background modulates its susceptibility to ethanol-induced apoptosis. METHODS: We examined the level of ethanol-induced neural crest apoptosis in 11 chick layer strains or crosses, using acridine orange uptake. RESULTS: Holding the ethanol dose and exposure stage constant, strains were classified into very sensitive (Babcock ISA, HyLine W98, Babcock B300/Hampshire Red cross [BxHR]), moderately sensitive (Spafas, HyLine W36, Babcock B300), and nonresponsive (DeKalb White and Black, Shaver White and 2000, DcKalb White/Hampshire Red cross). Detailed examination of two susceptible strains (W98, BxHR) and a resistant strain (DcKalb White) revealed that the DeKalb's nonresponse was not caused by a shift in timing of apoptosis, or to a lower alcohol exposure at either time of injection or time of death. Strains had identical stage distributions at the time of injection and at apoptosis; housing and diet were held constant. CONCLUSIONS: Factors within the embryo and/or egg environment can affect the susceptibility to ethanol-induced apoptosis. These sensitive and resistant strains will be important tools to dissect the molecular mechanism of ethanol-induced apoptosis, and for understanding how these losses affect subsequent development.     

Ethanol Differentially Affects Metabolic and Mitotic Processes in Chick Embryonic Cells

Our laboratory has been investigating the mechanisms by which ethanol-induced growth inhibition occurs in a developing embryo, and our studies have focused on disruption of cellular signaling pathways. Previous work on ethanol-induced changes in signaling systems that regulate omithine decarboxylase activity indicated that the pathways containing protein kinase A, protein kinase C (PKC), and insulin-dependent tyrosine kinase were important for the control of omithine decarboxylase in chick embryonic cells. Herein, we report ethanol's effect on the regulation of glucose uptake and thymidine uptake by these same kinase pathways. A pronounced increase in glucose uptake was associated with PKC downregulation in both vehicle- and ethanol-exposed cells, with the larger increase occurring in ethanol-exposed cells. An increase in thymidine uptake was associated with an activation of all three kinases, as well as with downregulation of PKC. Because previous work on signaling pathways has looked for changes in the insulin signaling pathway, the work herein focuses on the signaling pathways involving protein kinase A and PKC. cAMP levels were increased by ethanol treatment, but the increase was relatively small. Analysis of changes in PKC activity induced by ethanol exposure showed a significant suppression of PKC activity in the ethanol-treated cells and suggested that, overall, ethanol treatment affects the regulation of glucose uptake in embryonic cells predominantly by PKC downregulation.     

Nerve Growth Factor and Basic Fibroblast Growth Factor Protect Rat Cerebellar Granule Cells in Culture against Ethanol-Induced Cell Death

Neuronal cell loss is one of the most debilitating effects of fetal ethanol exposure. Cultures of cerebellar granule cells are a useful model to investigate ethanol neurotoxicity, because ethanol depletes cell numbers in these cultures, which also occurs in vivo. The primary goal of the present study was to identify and characterize agents that can ameliorate the ethanol-induced cell death that occurs in this culture system. Growth factors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) can prevent neuronal degeneration after toxic insult in various experimental paradigms. These growth factors were investigated in the current study to determine whether or not they can mitigate ethanol-induced death of cerebellar granule cells in culture. Results indicate that NGF and bFGF significantly reduced the ethanol-induced cell loss. Both the NGF- and bFGF-mediated neuroprotection required protein and RNA synthesis, because actinomycin D (RNA synthesis inhibitor) and cyclohex-imide (protein synthesis inhibitor) blocked their neuroprotective effects. In addition to its neuroprotective effect, bFGF also had a neurotrophic effect and could enhance cell survival in the absence of ethanol exposure. NGF did not have a neurotrophic effect. Neither EGF nor IGF-I was neuroprotective, although the latter did have a substantial neurotrophic effect. In conclusion, bFGF and NGF have long been recognized for their role in enhancing neuronal cell survival and differentiation. This study suggests that these growth factors can also provide neuroprotection against ethanol-induced cell death.     

Structural constraints for alcohol-stimulated Ca2+ release in neural crest, and dual agonist/antagonist properties of n-octanol

BACKGROUND: Prenatal ethanol exposure is a leading cause of mental retardation. Alcohol damages susceptible neuronal populations through its alteration of signaling pathways that direct cellular activity and survival. In early neural crest cells, ethanol elicits an intracellular Ca2+ transient that is necessary and sufficient to cause apoptosis. We tested the hypothesis that ethanol's activity represents a saturable and selective effect of alcohols upon this pathway. METHODS: Fura-2-loaded chick embryos, at the 3-somite stage, were exposed to n-alcohols ranging in size from ethanol (C2) to decanol (C10). Thereafter, Ca2+ mobilization was measured using Fura-2 and ratiometric imaging. Apoptosis was assessed using acridine orange uptake. RESULTS: Ethanol caused the dose-dependent mobilization of intracellular Ca2+ within neural crest populations, with an EC50 of 52.0 mM. n-Alcohols displayed increasing potency for Ca2+ mobilization through pentanol. Hexanol and heptanol were inactive. Unexpectedly, micromolar n-octanol concentrations triggered significant Ca2+ release and apoptosis in a G-protein-dependent manner. Decanol was inactive. Coaddition of either octanol or decanol antagonized the ability of ethanol to stimulate Ca2+ release. CONCLUSIONS: The selective, saturable effect of n-alcohols upon Ca2+ mobilization in neural crest is consistent with a hypothesis that ethanol stimulates these signals through specific interaction with one or more alcohol-binding sites on a target protein. Octanol may overcome structural constraints imposed upon C6 and C7 in interacting with this protein target; alternatively, it may interact through a unique binding site.     

Ethanol-Induced Inhibition of Chick Brain Growth

Retarded fetal brain growth is associated with a high incidence of mental retardation among the offspring of chronic alcoholic mothers. Research using an embryonic chick model suggests that ethanol exposure suppresses fetal development including suppression of brain growth. Total brain cyclic AMP content and endogenous brain protein kinase specific activity are not altered by ethanol; however, ethanol exposure does significantly stimulate kinase catalytic activity measured in the presence of saturating amounts of exogenous cyclic AMP.