Jayanth Ramadoss

Current fetal alcohol spectrum disorders (FASD) studies primarily focus on alcohol’s actions on the fetal brain although respiratory infections are a leading cause of morbidity/mortality in newborns. The limited studies examining the pulmonary adaptations in FASD demonstrate decreased surfactant protein A and alveolar macrophage phagocytosis, impaired differentiation, and increased risk of Group B streptococcal pneumonia with no study examining sexual dimorphism in adaptations. We hypothesized that developmental alcohol exposure in pregnancy will lead to sexually dimorphic fetal lung morphological and immune adaptations. Pregnant rats were orogastrically treated once daily with alcohol (4.5 g/kg, gestational day [GD] 4 to 10, peak BAC, 216 mg/dl; 6.0 g/kg, GD 11 to 20, peak BAC, 289 mg/dl) or 50% maltose dextrin (isocalorically matched pair-fed controls) to control for calories derived from ethanol. Male and female fetal lung RNA from a total of 20 dams were assessed using the TapeStation (Agilent) and Qubit RNA broad-range assay. Samples with RNA Integrity Numbers (RINs) > 8 were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), xGen Broad-range RNA Library Prep (IDT), and xGen Normalase UDI Primer Plate 2 (IDT). Final libraries were checked for quality and quantity by Qubit hsDNA and LabChip. The samples were sequenced on the Illumina NovaSeq S4 Paired-end 150 bp. Fetal lung tissue were analyzed for histopathological assessments. Mean fetal weight, crown-rump length and placental efficiency of the alcohol-administered rats were significantly lower (P < 0.05) than the pair-fed control pups. Differentially expressed genes indicated a sex-linked gene regulation dichotomy with a significantly higher number of genes altered in the female fetal lungs compared to the male. Network analysis plot of downregulated genes in the females exposed to alcohol in utero showed a negative impact on T cell activation and regulation, T cell differentiation, decrease in CD8⁺ T cell number etc. The most altered genes were Cd8b, Ccl25, Cd3e, Cd27, Cd247, Cd3d, Ccr9, Cd2, Cd8a and were decreased by a log2fold change of > 2 (P < 0.05) in the female fetal lungs. KEGG analyses showed that male and female fetal lungs had downregulated genes associated with development and mitosis, whereas the females alone showed dysregulation of T cell genes. Comparison of gross appearance and histopathologic morphology showed that the developing lungs of both male and female fetal pups, displayed stunted differentiation, were relatively hypoplastic, and displayed a diminution of alveolar size and air spaces. Similarly, in both sexes, decreased alveolar capillarization was also evident in the alcohol-exposed fetal lungs. These data provide novel information in a growing area focused on alcohol effects on the offspring lung and its influence on appropriate fetal/neonatal immune responses and highlights the importance of examining sexual dimorphism in developmental adaptations.

Objective: Over the past decade e-cigarette (e-cig) usage has become a growing public health concern, especially during pregnancy as many perceive e-cigs as a safer alternative to combustible cigarettes. Although human and animal models exhibit altered uterine artery Doppler velocimetry and waveform abnormalities following traditional smoking and buccal nicotine exposure, the effects of e-cig aerosols on pregnancy are incompletely researched and understood. We hypothesized that an altered uterine artery proteomic signature, accompanied by functional deficits, will be caused by e-cig vaping during pregnancy. Methods: In a validated timed pregnant Sprague-Dawley rat model system, a custom-engineered vaping system comprising a commercial atomizer and vape unit was utilized to generate and deliver e-cig aerosols that mimic the aerosols produced by commercial e-cigs, once daily, to pregnant dams. Results: Mean fetal weight, crown-rump length, and placental weight were significantly lower in the E-Cig group than those in the pair-fed Control group. Mass spectrometry followed by proteomic analysis detected a total of 2129 proteins; 36 significantly altered proteins were mostly related to immune system and vasodilation. Principal component analysis validated the protein signature. The uterine arteries of e-cig exposed rats demonstrated impaired concentration-dependent acetylcholine-induced uterine artery relaxation. Supplementation of N(ω)-nitro-L-arginine methyl ester (L-NAME) confirmed a role for the nitric oxide (NO) system. Immunofluorescence validated the localization to the uterine artery endothelium and the decreased levels of vasodilatory excitatory P-Ser¹¹⁷⁷ endothelial NO synthase. Conclusions: Identifying the pathway(s) involved in the pathogenesis of uterine artery dysfunction creates a potential for pinpointing antagonistic and/or reversal medications, thereby preventing or reducing e-cigarette mediated uteroplacental dysfunction and fetal growth restriction.

Fetal growth restriction is a hallmark of Fetal Alcohol Syndrome (FAS) and is accompanied by maternal uterine circulatory maladaptation. FAS is the most severe form of Fetal Alcohol Spectrum Disorder (FASD), a term for the range of conditions that can develop in a fetus when their pregnant mother consumes alcohol. Alcohol exerts specific direct effects on lipids that control fundamental developmental processes. We previously demonstrated that direct in vitro application of phosphatidic acid (PA, the simplest phospholipid and a direct target of alcohol exposure) to excised uterine arteries from alcohol-exposed rats improved vascular function, but it is unknown if PA can rescue end organ phenotypes in our FASD animal model. Pregnant Sprague-Dawley rats (n = 40 total dams) were gavaged daily from gestational day (GD) 5 to GD 19 with alcohol or maltose dextrin, with and without PA supplementation, for a total of four unique groups. To translate and assess the beneficial effects of PA, we hypothesized that in vivo administration of PA concomitant with chronic binge alcohol would reverse uterine artery dysfunction and fetal growth deficits in our FASD model. Mean fetal weights and placental efficiency were significantly lower in the binge alcohol group compared with those in the control (p < 0.05). However, these differences between the alcohol and the control groups were completely abolished by auxiliary in vivo PA administration with alcohol, indicating a reversal of the classic FAS growth restriction phenotype. Acetylcholine (ACh)-induced uterine artery relaxation was significantly impaired in the uterine arteries of chronic in vivo binge alcohol-administered rats compared to the controls (p < 0.05). Supplementation of PA in vivo throughout pregnancy reversed the alcohol-induced vasodilatory deficit; no differences were detected following in vivo PA administration between the pair-fed control and PA alcohol groups. Maximal ACh-induced vasodilation was significantly lower in the alcohol group compared to all the other treatments, including control, control PA, and alcohol PA groups (p < 0.05). When analyzing excitatory vasodilatory p1177-eNOS, alcohol-induced downregulation of p1177-eNOS was completely reversed following in vivo PA supplementation. In summary, these novel data utilize a specific alcohol target pathway (PA) to demonstrate a lipid-based preventive strategy and provide critical insights important for the development of translatable interventions.

Electronic cigarette (e-cig) use during pregnancy has become a major health concern in recent years and many view them as less harmful and may help quit or reduce combustible cigarettes. Implementing a state-of-the-art engineered vaping system, comprising an atomizer similar to those sold in vape shops, we aimed to utilize a translational e-cig inhalation delivery method to provide crucial information on the impact of prenatal e-cig aerosols on the developing brain hippocampal mTOR system in a rat model system. Gestational e-cig vaping significantly increased P-mTOR levels (p < 0.05) in the rat fetal hippocampi in the nicotine group (comprising of VG/PG + nicotine) compared to the control and the juice (comprising of VG/PG) groups. Total mTOR expression was not different among groups. Immunofluorescence imaging demonstrated P-mTOR was detected exclusively in the granule cells of the dentate gyrus of the fetal hippocampus. E-cig did not alter DEPTOR, but RAPTOR and RICTOR were higher (p < 0.05) in the Nicotine group. Gestational e-cig vaping with nicotine increased (p < 0.05) the activity and expression of 4EBP1, p70S6K, but decreased (p < 0.05) P-PKCα in the fetal hippocampi. In summary, dysregulation of mTORC1 and the related mTORC2, their activity, and downstream proteins together may play a critical role in e-cig-vaping-induced neurobiological phenotypes during development.

Alcohol exposure during gestation can lead to fetal alcohol spectrum disorders (FASD), an array of cognitive and physical developmental impairments. Over the past two and a half decades, Mammalian Target of Rapamycin (mTOR) has emerged at the nexus of many fields of study, and has recently been implicated in FASD etiology. mTOR plays an integral role in modulating anabolic and catabolic activities, including protein synthesis and autophagy. These processes are vital for proper development and can have long lasting effects following alcohol exposure, such as impaired hippocampal and synapse formation, reduced brain size, as well as cognitive, behavioral, and memory impairments. We highlight recent advances in the field of FASD, primarily with regard to animal model discoveries and discuss the interaction between alcohol and mTOR in the context of various tissues, including brain, placenta, bone, and muscle, with respect to developmental alcohol exposure paradigms. The current review focuses on novel FASD research within the context of the mTOR signaling and sheds light on mechanistic etiologies at various biological levels including molecular, cellular, and functional, across multiple stages of development and illuminates the dichotomy between the different mTOR complexes and their unique signaling roles.

Alcohol consumption has a close relationship with blood lipid levels in a nonpregnant state, with a myriad of effects on the liver; however, little is known about the interaction of alcohol and lipids in the context of fetal alcohol spectrum disorders (FASD). We herein aimed to determine the effect of alcohol on the lipid profile in a pregnant rat model, with a focus on FASD. Dry blood spots (50 µL) were obtained from rat maternal blood collected on gestational day (GD) 20, two hours after the last binge alcohol exposure (4.5 g/kg, GD 5–10; 6 g/kg, GD 11–20). The samples were then analyzed using high-throughput untargeted and targeted lipid profiling via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In untargeted lipidomics, 73 of 315 identified lipids were altered in the alcohol group compared to the pair-fed controls; 67 were downregulated and 6 were upregulated. In targeted analysis, 57 of the 260 studied lipid subspecies were altered, including Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Phosphatidic Acid (PA), Phosphatidylinositol (PI), and Phosphatidylserine (PS); 36 of these were downregulated and 21 lipid subspecies were upregulated. These findings suggest alcohol-induced dysregulation of lipids in the maternal blood of rats and provide novel insights into possible FASD mechanisms.

Fetal alcohol exposure can lead to a range of developmental disorders, including impaired fetal growth and development of multiple organ systems. These disorders are grouped under the term fetal alcohol spectrum disorders (FASDs). Adequate nutrition and a conducive intrauterine environment are essential for healthy fetal development. Nutrient deficiencies resulting from inadequate maternal nutrient ingestion may be compounded by alcohol-induced altered nutrient metabolism, placental clearance, and malabsorption. Alcohol-induced alteration of the intrauterine environment is the main source of developmental deficits and nutritional insufficiencies can worsen the effects on fetal development. In this review, we discuss studies examining the collective and interactive effects of nutrition (specifically iron, selenium, vitamin A, thiamine, zinc, folate, vitamin B₁₂, choline, and amino acids) relative to gestational alcohol consumption and its effects on fetal growth and development. We also summarize scientific reports that tested potential benefits of micronutrient supplementation in animal models of fetal alcohol spectrum disorders and in humans. In summary, the deleterious effects of alcohol exposure in relation to nutrient homeostasis further validate that avoidance of alcohol consumption during pregnancy is the most effective way to mitigate the teratogenic effects of alcohol.