Increasing popularity of electronic cigarettes (e-cigs), including among women of reproductive age, is attributed to its perceived safety compared to conventional tobacco. However, there is a major knowledge gap surrounding the effects of e-cig aerosols on pregnancy and fetal development. We aimed to evaluate the effects of vaping e-cigs during gestation on offspring growth and to asses if growth deficits are accompanied by altered maternal and fetal vascular hemodynamics. Sprague-Dawley dams were assigned to Pair-Fed Control, Pair-Fed Juice, or Juice +Nicotine groups, and then underwent either a prenatal or prenatal+postnatal exposure paradigm in a custom-engineered vaping system. Mass spectrometry identified major aerosolized constituents from e-cig vaping. The Juice+Nicotine group exhibited significantly decreased fetal weight and crown-rump length (↓46.56%, and ↓23.83%, respectively). Pre-and postnatal exposure to Juice+Nicotine resulted in decreased pup weight at postnatal day (PND) 4-10. Crown-rump length was decreased by 24.71% on PND 10. Blood flow in the Juice+Nicotine group was decreased in the maternal uterine and fetal umbilical circuits by 49.50% and 65.33%, respectively. We conclude that chronic exposure to e-cig aerosols containing nicotine during early development can have deleterious health effects on the exposed offspring. Vaping e-cigs containing nicotine during pregnancy leads to a reduction in offspring weight and crown-rump length, associated with a marked decrease in blood flow in both the maternal uterine and fetal umbilical circulation (a strong indicator of growth restriction). Thus, chronic exposure to e-cig aerosols containing nicotine can lead to potentially harmful developmental effects in early life.

Fetal alcohol spectrum disorders (FASD) describe neurodevelopmental deficits in children exposed to alcohol in utero. We hypothesized that gestational alcohol significantly alters fetal brain regional protein signature. Pregnant rats were binge-treated with alcohol or pair-fed and nutritionally-controlled. Mass spectrometry identified 1806, 2077, and 1456 quantifiable proteins in the fetal hippocampus, cortex, and cerebellum, respectively. A stronger effect of alcohol exposure on the hippocampal proteome was noted: over 600 hippocampal proteins were significantly (P < .05) altered, including annexin A2, nucleobindin-1, and glypican-4, regulators of cellular growth and developmental morphogenesis. In the cerebellum, cadherin-13, reticulocalbin-2, and ankyrin-2 (axonal growth regulators) were significantly (P < .05) altered; altered cortical proteins were involved in autophagy (endophilin-B1, synaptotagmin-1). Ingenuity analysis identified proteins involved in protein homeostasis, oxidative stress, mitochondrial dysfunction, and mTOR as major pathways in the cortex and hippocampus significantly (P < .05) affected by alcohol. Thus, neurodevelopmental protein changes may directly relate to FASD neuropathology.

Background Alcohol exposure during pregnancy results in an array of structural and functional abnormalities called Fetal Alcohol Spectrum Disorders (FASD). Alcohol dysregulates the exquisite coordination and regulation of gestational adaptations at the level of the uterine vasculature. We herein hypothesized that chronic binge-like alcohol impairs maternal uterine artery reactivity to vasoconstrictors and dilators and that alcohol-induced vascular dysfunction is dependent on the endothelium. Methods We utilized a once-daily binge alcohol (4.5 g/kg body weight) exposure paradigm (gestational day (GD) 7-17) in a pregnant rat model system and investigated primary uterine artery function in response to vasoconstrictors and vasodilators utilizing wire myography. Results Alcohol (peak blood alcohol concentration, 216 mg/dl) produced uterine vascular dysfunction in the absence of grossly observable growth deficits in maternal and fetal body weights, fetal crown-rump length and placental weight. Alcohol did not produce altered uterine vascular reactivity to α1 adrenergic agonist phenylephrine or the prostanoid thromboxane. However, alcohol specifically impaired endothelium-dependent acetylcholine (Ach)-mediated uterine artery vasodilation but exogenous endothelium-independent vasodilators like sodium nitroprusside exhibited no alcohol effect; Ach significantly decreased vessel relaxation (P=0.003; ↓pD2 (negative log molar Ach concentration producing the half maximum response), −7.004±0.215 vs. −6.310±0.208; EMax (maximal Ach response), 92% vs. 75%). Conclusion We conclude that moderate alcohol exposure impairs uterine vascular function in pregnant mothers. Alcohol specifically impairs endothelium-dependent agonist-induced uterine artery vasodilation. In summary, the maternal uterine compartment may play a significant role in the pathogenesis of FASD. Thus, the mechanistic targets of alcohol at the level of both the mother and the fetus need to be considered in order to develop effective therapeutic treatment strategies for FASD.

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Background Fetal alcohol spectrum disorders (FASD) describe many of the well-known neurodevelopmental deficits afflicting children exposed to alcohol in utero. The effects of alcohol on the maternal-fetal interface, especially the placenta, have been less explored. We herein hypothesized that chronic binge alcohol exposure during pregnancy significantly alters the placental protein profile in a rat FASD model. Methods Pregnant rats were orogastrically treated daily with alcohol (4.5 g/kg, gestational day (GD) 5–10; 6.0 g/kg, GD 11–19) or 50% maltose dextrin (isocalorically matched pair-fed controls). On GD 20, placentae were collected, flash frozen, and stored until tissues were homogenized. Protein lysates were denatured, reduced, captured on a 10 kDa spin filter and digested. Peptides were eluted, reconstituted, and analyzed by a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer. Results Mass spectrometry analysis identified 2,285 placental proteins based on normalized spectral counts and 2000 proteins by intensity based absolute quantification. 45 placental proteins were significantly (P<0.05) altered by gestational alcohol exposure by both quantification approaches. These included proteins directly related to alcohol metabolism; specific isoforms of alcohol dehydrogenase and aldehyde dehydrogenase were upregulated in the alcohol group. Ingenuity analysis identified ethanol degradation as the most significantly altered canonical pathway in placenta, and fetal/organ development as most altered function, with increased risk for metabolic, neurological, and cardiovascular diseases. Physiologic roles of the significantly altered proteins were related to early pregnancy adaptations, implantation, gestational diseases, fetal organ development, neurodevelopment, and immune functions. Conclusions We conclude that the placenta is a valuable organ not only to understand FASD etiology but it may also serve as a diagnostic tool to identify novel biomarkers for detecting the outcome of fetal alcohol exposure. Placental mass spectrometry analysis can offer sophisticated insights into identifying alcohol metabolism-related enzymes and regulators of fetal development.

This is the first study to demonstrate maternal binge alcohol consumption during pregnancy disrupts uterine artery vascular function via impairment of the eNOS vasodilatory system.

Background: Gestational alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), an array of cognitive, behavioral, and physical developmental impairments. Mammalian target of rapamycin (mTOR) plays a key role in regulating protein synthesis in response to neuronal activity, thereby modulating synaptic plasticity and long-term memory formation in the brain. Based on our previous quantitative mass spectrometry proteomic studies, we hypothesized that gestational chronic binge alcohol exposure alters mTOR signaling and downstream pathways in the fetal hippocampus.Methods: Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol (peak BAC, 216 mg/dl) from GD 5-10 and progressed to 6 g/kg alcohol (peak BAC, 289 mg/dl) from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin.Results: In the Alcohol group, following this exposure paradigm, fetal body weight and crownrump length were decreased. The phosphorylation level of mTOR (P-mTOR) in the fetal hippocampus was decreased in the Alcohol group compared with controls. Alcohol exposure resulted in dysregulation of fetal hippocampal mTORC1 signaling, as evidenced by an increase in total 4E-BP1 expression. Phosphorylation levels of 4E-BP1 and p70 S6K were also increased following alcohol exposure. P-mTOR and P-4E-BP1 were exclusively detected in the dentate gyrus and oriens layer of the fetal hippocampus, respectively. DEPTOR and RICTOR expression levels in the fetal hippocampus were increased; however, RAPTOR was not altered by chronic binge alcohol exposure.Conclusion: We conclude that chronic binge alcohol exposure during pregnancy alters mTORC1 signaling pathway in the fetal hippocampus. We conjecture that this dysregulation of mTOR protein expression, its activity, and downstream proteins may play a critical role in FASD neurobiological phenotypes.

Background: Phosphatidylethanol (PEth) is a promising biomarker for gestational alcohol exposure. Studies show PEth accumulation in maternal and fetal blood following alcohol exposure, however, distribution of specific PEth homologues (16:0/18:1, 16:0/18:2, 16:0/20:4) in maternal and fetal blood is unknown. Additionally, PEth levels in highly vulnerable FASD targets in maternal and fetal compartments remain unexplored. We hypothesized that all three major PEth homologues will be detectable in the maternal and fetal blood, the maternal uterine artery (a reproductive tissue that delivers oxygen and nutrients to feto-placental unit), and fetal brain regions following gestational binge alcohol exposure, and that homologue distribution profiles will be tissue-specific.Methods: Pregnant rats received once-daily oragastric gavage of alcohol (Alcohol; BAC 216 mg/ dl@4.5g/kg/day; BAC 289 mg/dl@6g/kg/day) or iso-caloric maltose-dextrin (Pair-fed control) from gestation day (GD) 5-20 or 21. Following chronic exposure, maternal and fetal tissues were analyzed for PEth homologue concentrations utilizing LC-MS/MS technology.Results: All three PEth homologues were detected in alcohol-exposed maternal blood, fetal blood, maternal uterine artery, and fetal brain regions (hippocampus, cerebral cortex, cerebellum). In both maternal and fetal blood, respectively, PEth 16:0/18:2 was more abundant compared to 16:0/18:1 (P<0.0001,~66%,↑;P=0.0159,20.4%↑) and 16:0/20:4 (P=0.0072,~25%↑;P=0.0187,19.4%↑). Maternal PEth 16:0/20:4 was ~42% higher than 16:0/18:1 (P=0.0015). Maternal PEth 16:0/18:2 and 16:0/20:4 were ~25%↑ and ~20%↑ higher than in fetal blood (P<0.05). No homologue differences were detected in the maternal uterine artery. In all fetal brain regions, PEth 16:0/18:1 was significantly higher (P<0.0001) than 16:0/18:2 (~48-78%↑) and 16:0/20:4 (~31-62%↑) concentrations. PEth 16:0/20:4 was ~18% higher than 16:0/18:1 (P<0.05) in the fetal hippocampus and cortex. Conclusion:All major PEth homologues were detected in maternal and fetal blood following chronic gestational binge alcohol exposure; homologue distribution profiles were tissue specific.