Perinatal exposure to smoking and parenteral nicotine has been linked to a high risk of asthma, not just in the
exposed offspring, but also transgenerationally in progeny who never encounter any form of nicotine at least as
far as the F3 generation. Use of electronic-cigarettes (e-cigs) by pregnant women has been increasing, largely
the result of the perception that vaping is relatively safe. This perception is suspected to be erroneous, but few
objective studies have evaluated the adverse health effects of vaping. Our investigation will address the effects
of vaping and flavorings used in vaping on development of asthma in progeny of mice exposed to e-cigs during
pregnancy. We also ask whether this risk is transgenerationally inherited, and how heritability might occur. Germ
cells are the only cells that are passed from one generation to the next, and have been shown to be exquisitely
sensitive to environmental insults. Based on these considerations and on our preliminary data, we hypothesize
that exposure to vaping disrupts the epigenetic machinery in germ cells, causing alterations of epigenetic marks
across the genome that leads to a transgenerationally heritable asthma phenotype. Using established models,
we will test this hypothesis in vivo in mice by determining whether e-cigs increase risk of asthma in: 1) offspring
of pregnant F0 mothers and 2) offspring of F1 and F2 mothers who were naïve to e-cig or nicotine exposure. We
will test whether vaporized nicotine (AIM 1A) and flavorings (AIM 1B) have independent effects on
transgenerational asthma risk, whether these risks are additive and whether they are exacerbated on allergen
sensitization (AIM 1C). Nicotine-induced asthma is associated with a marked alteration of lung fibroblast
phenotype, and we hypothesize that these alterations are reversible once the epigenome is restored towards
normal. Hence, in AIM 2, we will assess the effects of nicotine and e-cig flavorings on viability (AIM 2A) and
epigenetic memory (AIM 2B) of germ cells. Using multi-dimensional -omics tools (the Mergeomics platform)
developed by our Co-I Xia Yang (UCLA), we will analyze data generated on molecular pathways across different
data sets to determine how germ cell epigenetics are impacted by e-cigs with or without flavorings (AIM 2C).
Finally, studies proposed in AIM 2D will attempt to reverse epigenetic changes in fibroblasts isolated from F3
progeny produced by e-cigs using RNAi. Dependent variables will include expression of molecular phenotypic
markers that are the hallmarks of the asthmatic phenotype, and functional characteristics of fibroblasts isolated
from F3 progeny, compared with appropriate controls. We anticipate that studies in Aim 2 will show reproducible
alteration of the phenotype of lung fibroblasts that have differentiated from germ cells in mice that have either:
1) been directly exposed to e-cigs; or 2) never been exposed to nicotine in any form, but inherited the effects of
ancestral exposure to e-cigs. These studies will importantly advance our mechanistic knowledge of how vaping
might lead to persistent, transgenerationally-inherited risk of asthma in offspring. Further, our studies will help
inform regulatory policies concerning exposure to e-cig nicotine and flavorings from vaping.
