Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

Abstract

Background: Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs.

Objective: The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells.

Methods: Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively.

Results: EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage.

Conclusions: Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public.

Fig 1. Dose-dependent increase in DNA damage in cells exposed to EC aerosol extracts.

UM-SCC-1 (A and B) and NuLi1 (C and D) cells were exposed for 1 h to increasing doses of NJOY (N18) or eGo (E18) and DNA damage quantified by q-PADDA within the transcribed (TS) and non-transcribed (NTS) strands of the TP53 gene. Data are represented as mean ± SEM. *p<0.05, **p<0.01.

Fig 2. Effect of nicotine on DNA damage levels.

UM-SCC-1 cells were exposed for 1 h to EC aerosol extracts obtained from e-liquids with different nicotine concentrations and DNA damage quantified by q-PADDA (A) or ELISA (B). Data are represented as mean ± SEM. *p<0.05.

Fig 3. DNA damage levels measured after long-term exposure to EC aerosol or MS smoke extracts.

UM-SCC-1 cells were exposed for 2 weeks to EC aerosol (N18 and E18) or MS smoke extracts and DNA damage quantified by q-PADDA (A) or ELISA (B). Data are represented as mean ± SEM. *p<0.05; **p<0.01.

Fig 4. Exposure to EC aerosol increases cellular ROS and decreases TAC.

UM-SCC-1 cells were exposed for 2 weeks to EC aerosol (N18 and E18) or MS smoke extracts and the cellular levels of ROS (A) and TAC (B) were measured using standard methods. Data are represented as mean ± SEM. *p<0.05.

Fig 5. Exposure to EC aerosol changes the expression of DNA repair proteins.

UM-SCC-1 and POE9n cells were exposed for 2 weeks to EC aerosol or MS smoke extracts and the levels of mRNA (A,B) and protein (C) were measured by real-time RT-PCR and western blot, respectively. Protein expression after chronic exposure to EC aerosol or MS smoke extracts was quantified and compared to non-exposed cells (D). Data are represented as mean ± SD. *p<0.05; **p<0.01.