Reply to Li Volti et al.: E-cigarette smoke exposure and effect in mice and human cells

We wish to emphasize that in our study (1) the E-cigarette smoke (ECS) was generated by E-juice (nicotine,10 mg/mL) in an E-cigarette (E-cig) machine operated at 4.2 V, the same voltage as a commercial E-cig pen (2). The ECS concentration was 130 mg/m³ (measured) and the nicotine concentration in the aerosol was 1.3 mg/m³ (assuming nicotine concentration in aerosols is the same as in E-juice), which is 2.6-fold that used by Waldum et al. (3) for rat exposure. Mice were subject to whole-body exposure (4). Mouse experiments necessitate using longer exposure times (3 h/d, 12 wk) to allow measurement of ECS’s effect on DNA adduct formation and DNA repair activity. We believe that our ECS mouse exposure conditions are comparable to a human, light smoker’s ECS exposure.

We estimated the amount of nicotine inhaled by a mouse per 3-h period time is 112–323 μg/kg body weight. This calculation is based on breath rate: 80–230 times/min; breath volume: 0.15 mL; and nicotine concentration in aerosol: 1.3 mg/m³ (1, 2). If we assume that a light E-cig vaporer consumes roughly 10–20 mg nicotine per day, then the nicotine consumption per body weight by a human is 133–267 μg/kg body weight per day. For comparison purposes, we assume the human life expectancy is 100 y and mouse is 120 wk, which lead us to come up with the statement that the mice exposed to ECS under our conditions are equivalent to a light E-cig smoker who smokes E-cig for 10 y. During and after 12-wk exposure, mice were healthy (hair and activity are the same as control, no weight lost, and all survived.) Mice showed no apparent abnormality in cardiovascular and pulmonary function. Therefore, we believe mice are not “intoxicated” by the aerosol mass and its content, as assumed by Li Volti et al. (5).

We irradiated pUC18 DNA with 1,500 J/m² UVC to generate ∼50 photodimers per plasmid as substrates for in vitro DNA repair synthesis (6). Nowhere in all of our experiments were cells irradiated with UVC (1). The viability of the nicotine and nitrosamine ketone-treated human cells used for DNA adduct analysis, DNA repair activity measurement, and mutational susceptibility determination, is 90–100%, and for anchorage-independent growth assay is 50%.

We do not dispute the results from certain studies showing that ECS is less detrimental than tobacco smoke to respiratory function (7). This was not in the scope of our study. Our results unambiguously demonstrate that ECS can induce DNA damage in the lung, heart, and bladder, as well as inhibit DNA repair in mouse lung. Nicotine and nitrosamine ketone can induce DNA damage, inhibit DNA repair, enhance cell mutability, and yield tumorigenic cell transformation in cultured human lung and bladder cells (1). We believe it is our obligation to inform the public about these detrimental effects of ECS on the lung, heart, and bladder. In doing so, we hope to prevent nontobacco smokers, particularly young adults, from smoking E-cig because they assume E-cig smoke is less harmful and noncarcinogenic and to encourage E-cig smokers to quit vaping.