PROJECT SUMMARY Title: Exosomal epigenetic biomarkers associated with flavored electronic cigarette use in adults This project addresses two scientific interest areas as defined in RFA-OD-19-021: 1) Utilize both blood and urine specimens to analyze biomarkers of tobacco exposure not currently measured by other national studies; 2) Examine differences in exosomal epigenetic biomarkers associated with exposure to tobacco products. E-cigarette, a handheld electronic device simulating the feeling of tobacco smoking, gains global popularity during the past few years especially in youth and young adults, largely due to various e-liquid flavors. However, the health risks of e-cigarette use, especially long-term health risks, are unclear. E-liquid usually contains propylene glycol and/or vegetable glycerin as well as nicotine and flavoring chemicals. Our previous experimental studies on lung epithelial cells and monocytes exposed to different flavoring chemicals or e-cigarette flavors without nicotine have shown different cytotoxicity. Our preliminary data showed that there were significant differences in exosomal epigenetic biomarkers (microRNAs and long non-coding RNAs) between non-users and e-cigarette users using human plasma samples. However, how e-liquid flavors (flavoring chemicals) lead to the changes in exosomal epigenetic biomarkers during the e-cigarette initiation, exposure, and cessation is largely unknown. Considering the crucial regulatory functions of micoRNAs and long non-coding RNAs and their association with diseases, it is critical to evaluate their changes and associated biological pathways during the flavored e-cigarette initiation, exposure and cessation to help understand health risks associated with e-cigarette use. The overarching goal of the proposed study is to identify exosomal epigenetic biomarkers (including microRNAs and long non-coding RNAs) associated with flavored e-cigarettes. We have requested both longitudinal urine specimens (PATH Wave 1 study conducted from 2013 to 2014 and Wave 2 study conducted from 2014 to 2015) and cross-sectional blood specimen (PATH Wave 1 study) from Population Assessment of Tobacco and Health (PATH) Study biorepositories. The samples are available from PATH based upon the Biospecimen Availability Report (BAR ID: 2019-005-BAR-2) received from PATH Study Biospecimen Access Program. We will link these biospecimens with PATH survey data through unique subject IDs. To achieve our goal, we will examine both blood and urinary exosomal epigenetic biomarkers (microRNAs and long non-coding RNAs) and associated biological pathways related to flavored e-cigarettes use (such as fruit flavor) (Aim 1), as well as the within- subject alterations in exosomal epigenetic biomarkers and associated biological pathways during the e- cigarette initiation and cessation (Aim 2). In addition, based on the identified key miRNAs and lncRNAs from aim 1 and aim 2 comparisons, we will conduct innovative experiments. We w...