(28) Flavored E-Cigarette Condensates Without Nicotine Elicit Greater Differences on the Trophoblast Cell Transcriptome

Margeaux W. Marbrey¹; Meera Khare¹; Rennica Huang¹

1. Department of Obstetrics & Gynecology, Division of Reproductive Sciences, Duke University School of Medicine, Durham, NC, USA

Abstract Text:

An estimated 15% of pregnant women use electronic cigarettes or e-cigarettes as a safer alternative to traditional cigarettes. However, the safety of e-cigarette use during pregnancy is understudied. E-cigarettes heat and aerosolize a base liquid made up of propylene glycol and vegetable glycerin with additives such as nicotine and flavoring. During early pregnancy, fetal-derived trophoblast cells migrate through the maternal tissues to remodel the vasculature and establish the early placenta. These trophoblast cells invade and secrete factors to facilitate this remodeling process consisting of activated invasion, angiogenesis, cytokine signaling, and recruitment of specialized immune cells. Impaired trophoblast cell signaling can lead to hypertensive pregnancy disorders such as preeclampsia. Recent studies suggest e-cigarettes can impair trophoblast tubal formation, proliferation, and chemokine signaling. Yet, these studies are limited as many of the e-liquid treatments were not physiological or relevant to current individual use. Furthermore, no studies have examined how the individual e-cigarette components contribute to trophoblast cell signaling. To address these concerns, we hypothesized e-cigarettes with increased additives exhibit greater toxicity to trophoblast cell signaling and subsequent placental development. To test this, we first established an in-house method to capture aerosolized e-cigarette condensate, physiologically replicating how condensates travel to lung alveolar tissues, become absorbed in the bloodstream, and accumulate in the placenta. We then treated immortalized trophoblast cells (HTR8/SVneo) with 1.5% saline or condensates derived from four e-cigarette liquids: base liquid, base liquid with 0.6% nicotine, base liquid with flavoring, base liquid with flavoring and 0.6% nicotine. RNA was isolated and bulk RNA sequencing was performed by the Duke University Sequencing and Genomics Technologies Core (n=3). Genome alignments, read quantification, and ANOVA comparisons were performed using Partek Flow. We determined 377 genes were changed with base liquid alone, 388 genes with base liquid with nicotine, 1,407 genes with base liquid with flavoring, and 1220 genes changed with base liquid with nicotine and flavoring with a p-value less than or equal to 0.05 and a fold change greater than 2.5 or less than -2.5. The most changed KEGG pathways included cytokine receptor interaction and neuroactive ligand receptor interaction. Follow up qRT-PCR validation (n=3) revealed changes in placental angiogenic factors including adrenomedullin (ADM) and placental growth factor (PGF) and genes involved in proliferation and the antioxidant response. Thus, we determined the base liquid with flavoring treatment exhibited more transcriptomic changes than without nicotine, suggesting e-cigarettes with flavoring can elicit worse outcomes than e-cigarettes containing nicotine. Future directions include examination of cytokine expression levels, oxidative stress accumulation, and trophoblast invasive properties for all e-cigarette treatment groups. These studies suggest use of any type of e-cigarette during pregnancy should be carefully considered. This work was supported by R00HD104900 to MWM.