Background: Individuals exposed to gestational stressors such as alcohol exhibit a spectrum of growth patterns, suggesting individualized responses to the stressors. We hypothesized that intrauterine growth responses to gestational alcohol are modified not only by the stressor's severity but by fetal sex and the placenta's adaptive capacity. Methods: Pregnant C57BL/6J mice were assigned to one of three groups. Group 1 consumed a normal protein diet (18% protein by weight) and received 4.5 g alcohol/kg body weight (NP-Alc-8) or isocaloric maltodextrin (NP-MD-8) daily from embryonic day (E) 8.5-E17.5. Group 2 consumed the same diet but received alcohol (NP-Alc-13) or maltodextrin (NP-MD-13) daily from E13.5-E17.5. Group 3 consumed the same diet but containing a lower protein content (12% protein by weight) from E0.5 and also received alcohol (LP-Alc-8) or maltodextrin (LP-MD-8) daily from E8.5-E17.5. Maternal, placental, and fetal outcomes were assessed on E17.5 using 2-way ANOVA or mixed linear model.

Background: Prenatal alcohol exposure causes distinctive craniofacial anomalies that arise, in part, from the apoptotic elimination of neural crest (NC) progenitors that form the face. This vulnerability of NC to alcohol is puzzling as they normally express the transcriptional repressor Snail1/2 (in chick Snai2), which suppresses apoptosis and promotes their migration. Here, we investigate alcohol's impact upon Snai2 function. Methods: Chick cranial NC cells were treated with acute alcohol (52 mM, 2 hr). We evaluated NC migration, gene expression, proliferation, and apoptosis thereafter. Results: Transient alcohol exposure induced Snai2 (191% ± 23%; p = .003) and stimulated NC migration (p = .0092). An alcohol-induced calcium transient mediated this Snai2 induction, and BAPTA-AM blocked whereas ionomycin mimicked these pro-migratory effects. Alcohol suppressed CyclinD1 protein content (59.1 ± 12%, p = .007) and NC proliferation (19.7 ± 5.8%, p < .001), but these Snai2-enriched cells still apoptosed in response to alcohol. This was explained because alcohol induced p53 (198 ± 29%, p = .023), and the p53 antagonist pifithrin-α prevented their apoptosis. Moreover, alcohol counteracted Snai2's pro-survival signals, and Bcl2 was repressed (68.5 ± 6.0% of controls, p = .016) and PUMA was not induced, while ATM (1.32-fold, p = .01) and PTEN (1.30-fold, p = .028) were elevated. Conclusions: Alcohol's calcium transient uncouples the Snai2/p53 regulatory loop that normally prevents apoptosis during EMT. This represents a novel pathway in alcohol's neurotoxicity, and complements demonstrations that alcohol suppresses PUMA in mouse NC. We propose that the NCs migratory behavior, and their requirement for Snai2/p53 co-expression, makes them vulnerable to stressors that dysregulate Snai2/p53 interactions, such as alcohol. K E Y W O R D Sapoptosis, cell migration, fetal alcohol spectrum disorder, neural crest, p53, Snai2

Objectives Both prenatal iron deficiency and prenatal alcohol exposure (PAE) decrease body weight, increase susceptibility to anxiety, and impair memory and learning in the offspring. PAE itself causes fetal iron deficiency, even when mothers consume sufficient iron. We hypothesized that iron supplementation in PAE pregnancies would not alter offspring growth but would improve measures of anxiety, learning, and memory. Methods Pregnant Long-Evans rats received 5 g/kg/day ethanol or isocaloric maltodextrin (split-dose) from gestational day (GD) 13.5–19.5 and received 6 mg/kg/day iron as ferric sulfate or water from GD12.5–19.5. Male and female offspring were weighed regularly after birth and underwent elevated plus maze (postnatal day (P) 27), open field (P28), novel object recognition (P29–30), T-maze (P32–40), and fear conditioning (P42–50) tests. Mixed model analysis was used to determine significance of the effects of PAE, iron, and sex. Results PAE reduced postnatal body growth in both male (P = 0.028) and female (P = 0.026) offspring. In both sexes, PAE interacted with age to affect growth (P &lt; 0.001) from P5 to P50. In males but not females, iron supplementation interacted with age (P = 0.044) and age and PAE (P = 0.045) to improve growth in PAE + iron males, such that their weights approached control weights by P50. On the elevated plus maze, iron supplementation, regardless of PAE or sex, increased time spent on the open arms by 39–118%, indicating reduced anxiety-like behavior (P = 0.030). On the open field test, time spent in the center was not affected by PAE, iron, or sex (P’s &gt; 0.200). On the novel object recognition test, at delays of both 1 and 24 hours, all groups (except MD + Iron Males with 24-hour delay) recognized the novel object better than chance (P’s &lt; 0.040), but recognition at both time delays was not affected by PAE, iron, or sex (P’s &gt; 0.120), showing that these did not affect recognition memory. On the T-maze and fear conditioning tests, which assess learning and memory, PAE, iron, and sex had no effect on the results (P’s &gt; 0.080). Conclusions This work is the first to investigate gestational iron supplementation as an intervention for alcohol-exposed pregnancies. These results suggest iron supplementation may improve outcomes such as growth in males and anxiety in both sexes in alcohol-exposed populations. Funding Sources NIAAA NIDDK.