Evaluation of Sex Differences in DNA Methylation and Lung Tissue Gene Expression in a Mouse Model of E-cigarette Exposure
Authors: Chirumamilla Praveen , Rousselle Dustin , Sharma Shikha , Commodore Sarah , Silveyra Patricia 
Published: May 2024

Long-standing research shows that cigarette smoking is known to impact DNA methylation and promote severe lung disease - however, research on electronic nicotine delivery systems (ENDS) or e-cigarettes and their effect on methylation and expression of oxidative stress genes is limited. Furthermore, the component of sex as a variable for susceptibility to methylation changes and lung damage - specifically for females - is not well understood. Given the strikingly increased use of ENDS - especially among youth and young adults - it is critical that its mechanism and impacts become well-understood. This study analyzes the sex-specific associations that exist in DNA methylation and expression of specific genes using a mouse model of ENDS exposure. We hypothesize that e-cigarette exposure will result in a differential and sex-specific gene expression activation in lung tissue. Our experiment focused on six different genes - glutathione peroxidase 1 ( GPX1) & G protein coupled receptor 132 ( GPR132) (both previously shown to display sex-specific methylation patterns in DNA samples from smokers), Interleukin-5 ( IL-5), Interleukin-6 ( IL-6), & Interleukin-7 ( IL-7) (due to their significant role in inflammation), as well as Chemokine (C-X-C motif) ligand 1 ( CXCL1) (with its crucial role in recruiting immune cells at various tissues, including the lungs). Our experiment used adult (8-10 weeks old) male and female C57BL/6J mice. These were divided in four groups (n=6/group; 3 males and 3 females) and treated for 14 days, as follows; group I: no treatment (control), group II: 0% nicotine e-liquid [50% propylene glycol (PG) and 50% vegetable glycerin (VG)], group III: 3% nicotine & group IV: 6% nicotine in PG/VG using an inExpose full body chamber (SCIREQ); e-cigarette puff (70 ml, 3.3s, 1 or 2 puffs per min/30 min/day). Following exposure, lungs were harvested and DNA was extracted for future analysis with the EZ DNA Methylation Sample Direct Kit. Total RNA was also extracted with the Direct-zol kit (Zymo) and retro-transcribed with the High Capacity cDNA kit (Thermo). The expression of all genes was determined with a Taqman gene expression assay (Thermo) using a QuantStudio 5 Real-Time PCR system. Ct values were analyzed with the deltadeltaCt method, and statistical analyses were conducted in Prism. Our preliminary results indicate that e-cigarette exposure resulted in an increased, although not statistically significant, expression of GPR132 in males, but not females. However, a significant interaction of sex and exposure was found for GPR132 expression (two-way ANOVA, p<0.05), with control (group I) females displaying higher GPR132 lung tissue expression than males, and female mice exposed to 3% nicotine displaying lower GPR132 expression than males. No differences in GPX1 expression were found for either sex or treatment exposure. The expression of IL-5 in control (group 1) and 0% nicotine (group 2) varied by sex - expression was relatively high in control (group 1) females, but lower in 0% nicotine (group 2) females. By contrast, expression was relatively low in control (group 1) males, but greater in 0% nicotine (group 2) males. The expression of IL-6 in control (group 1) mice greatly varied by sex - expression in 0% nicotine (group 2) females was relatively higher compared to the expression in 0% nicotine (group 2) males. In females, expression was much lower in 0% (group 1), 3% (group 2), and 6% (group 3) mice. In males, however, the expression amongst all groups was not significantly different. The expression of IL-7 in 0% nicotine (group 2) mice varied by sex - expression, in 0% nicotine (group 2) females was relatively high (compared to the control (group 1) females), while expression, in 0% nicotine (group 2) males was relatively lower (against 0% nicotine (group 2) females). (and not significantly different when compared to control (group 1) males). In both females and males, an increase in CXCL1 Gene expression was found with 0% Nicotine (group 2) compared to the control (group 1) - with increased nicotine concentrations of 3% and 6% (groups 3 and 4, respectively), however, CXCL1 gene expression decreased. We conclude that e-cigarette exposure results in a differential and sex-specific gene expression activation in lung tissue. In looking to the future, several, closely-related matters are of paramount importance to investigate. First and foremost, our ongoing measurement and analysis of DNA methylation patterns in mice will help us to identify the effects of ENDS exposure in the male and female lung. Additionally, the relatively limited (and often conflicting) data on “flavored” e-cigarettes - which are marketed towards younger individuals - necessitates the need to study their effects on gene expression. Finally, beyond gene expression, a future, in-depth analysis on the mechanisms giving rise to the changes observed in gene expression - based on sex, exposure, or an interaction of sex and exposure - will be instrumental in bettering our (thus far) limited knowledge of e-cigarette exposure. We acknowledge the support from School of Public Health, Indiana University, Bloomington, Indiana, USA. This work was partially funded by the Offce of the Vice Provost of Research at Indiana University - Bloomington through the research Equipment Fund. Equipment used in this study was supported, in part, with funding from the Indiana Clinical and Translational Sciences Institute, funded in part by grant number UL1 TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.