A murine MODEL of e-cigarettes exposure to evaluate the carcinogenic effects of vaping on the bladder urothelium.
Authors: zambrano ibardo , Manocha Ujjawal , Gong Weida , Tsai Yi-Hsuan , Zhu Manhua , Herman Melissa A. , Bjurlin Marc , Kim William Y. 
Published: February 2022

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Background: Data evaluating the carcinogenic potential of e-cigarette (ECS) by-products on the bladder urothelium is scarce, and to date, no group has studied the direct impact of ECS on the urothelium at the genomic and epigenetic level. While it has been published that ECS exposure causes bladder hyperplasia in mice, no bladder tumors formed and the field is therefore lacking appropriate preclinical models to be able to study ECS related bladder cancer. Importantly, whole organ mapping studies of tumor bearing human bladders have identified DNA methylation field changes in “adjacent normal” urothelium as early events in bladder carcinogenesis. Whether ECS exposure results in early field alterations of DNA methylation in the urothelium is unknown. Methods: Mice (4 females, 4 males; 35 weeks old) were exposed to 12% nicotine (120mg/ml) in 1:1 polyethylene glycol: vegetable glycerol (PG/VG) or vehicle (PG/VG) daily: 3 hr sessions, 3 sec vape duration, 10 min interval between vapes. Nicotine and cotinine serum concentrations were measured at 3 hrs, and then at 5 days using LC-MS. Bladders were harvested after 4 weeks. DNA was extracted from microdissected urothelial layer. Reduced representation bisulfite sequencing (RRBS) was performed (Zymo Research) using 100ng of DNA input and paired-end chemistry. Alignment was performed using Bismark. Number of loci covered by each sample was determined and data was filtered to only include loci with > 10 reads. Differentially methylated loci were determined. Adjusted p-value < 0.01 was used as threshold to generate heatmaps. Gene Set Enrichment Analysis was performed to contextualize the DNA methylation changes. Results: Acute and repeated exposure resulted in significantly higher levels of nicotine and cotinine serum concentration in the nicotine group vs. the vehicle group without significant adverse events. No tumors were observed on gross visualization at the time of urothelium harvest. The number of loci in the RRBS libraries with > 10 reads ranged from 130,000 to 250,000. 53 loci were differentially methylated between nicotine and vehicle. These loci did not correspond to the top hyper/hypo methylated genes in premalignant bladder tissue from recently reported whole-cystectomy mapping. Conclusions: We established an effective and safe murine model of ECS exposure closely resembling the human experience of vaping. Significant changes of DNA methylation were observed in mice exposed to ECS vapor vs. a vehicle control after four weeks of daily exposure. Ongoing experiments include longer vaping exposures as well as comparison of RRBS to a recently developed commercial mouse methylarray. Understanding epigenetic and genomic changes as a result of ECS exposure in preclinical models can serve as a foundation for studies developing assays to identify ECS users whose bladders contain early field defects that may place them at higher risk for bladder cancer.