Electronic-cigarette smoke induces lung adenocarcinoma and bladder urothelial hyperplasia in mice

Abstract

Electronic-cigarettes (E-cigs) are marketed as a safe alternative to tobacco to deliver the stimulant nicotine, and their use is gaining in popularity, particularly among the younger population. We recently showed that mice exposed to short-term (12 wk) E-cig smoke (ECS) sustained extensive DNA damage in lungs, heart, and bladder mucosa and diminished DNA repair in lungs. Nicotine and its nitrosation product, nicotine-derived nitrosamine ketone, cause the same deleterious effects in human lung epithelial and bladder urothelial cells. These findings raise the possibility that ECS is a lung and bladder carcinogen in addition to nicotine. Given the fact that E-cig use has become popular in the past decade, epidemiological data on the relationship between ECS and human cancer may not be known for a decade to come. In this study, the carcinogenicity of ECS was tested in mice. We found that mice exposed to ECS for 54 wk developed lung adenocarcinomas (9 of 40 mice, 22.5%) and bladder urothelial hyperplasia (23 of 40 mice, 57.5%). These lesions were extremely rare in mice exposed to vehicle control or filtered air. Current observations that ECS induces lung adenocarcinomas and bladder urothelial hyperplasia, combined with our previous findings that ECS induces DNA damage in the lungs and bladder and inhibits DNA repair in lung tissues, implicate ECS as a lung and potential bladder carcinogen in mice. While it is well established that tobacco smoke poses a huge threat to human health, whether ECS poses any threat to humans is not yet known and warrants careful investigation.

Keywords: DNA damage; DNA repair; bladder hyperplasia; electronic-cigarette; lung cancer.

Fig. 1.

ECS exposure induces lung tumor formation in mice. Mice were exposed to FA (n = 20) and aerosols generated by Veh (isopropylene glycol and vegetable glycerin at a 1:1 ratio, n = 20) and ECS (36 mg/mL nicotine in Veh, n = 45) for 4 h per day and 5 d per week for 54 wk as described in the main text. Surviving mice at the end of exposure are as follows: FA-exposed (n = 18), Veh-exposed (n = 18), and ECS-exposed (n = 40). All mice dying before the 54-wk exposure time were lung tumor-free. (A) Lung tumor tissues. Gross anatomy photographs (Left) of ECS-induced lung adenocarcinoma tissues (28-2, 28-4, 30-1, 30-2, 36-1, 38-2, 39-2, 39-5, 40-2) and a lung adenocarcinoma from an FA-exposed mouse (101-1), and histological slides of H & E staining of these lung adenocarcinomas (Center and Right, 100× and 400× magnification, respectively) are presented. (B) Normal lung tissue (Left, 100× magnification; Right, 400× magnification). Notes: (1) Veh exposure does not induce lung tumor. (2) Only a gross anatomy photograph of the lung tumor of ECS-exposed mouse 28-2 is shown.

Fig. 2.

ECS exposure induces bladder urothelial hyperplasia in mice. Bladder tissues were harvested from the same mice exposed to ECS, Veh, and FA for 54 wk as described in Fig. 1. The tissue slides were prepared for histology examination and stained by H & E or antibodies for proliferation markers MCM-2 and PCNA and basal cell marker KRT5 (200× magnification). (A) Typical staining result of bladder tissues of mice exposed to FA, Veh, and ECS. (B) Histogram presentation of bladder urothelial hyperplasia in mice exposed to FA (n = 17), Veh (n = 16), and ECS (n = 40). Notes: (1) While we were able to examine bladder tissue samples from all 40 ECS-exposed mice, during sample preparation, 1 bladder from FA-exposed mice and 2 from Veh-exposed mice were inadvertently destroyed. (2) The simple (ECS1 mouse) and nodular (ECS2 mouse) hyperplasia had markedly thickened urothelial layers and strong expression of MCM-2, PCNA, and KRT5 (with the latter indicating expansion of basal cells), compared with FA- and VEH-exposed mice, which had very thin urothelial layers with low expression of the proliferation markers.