E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells

Abstract

E-cigarette smoke delivers stimulant nicotine as aerosol without tobacco or the burning process. It contains neither carcinogenic incomplete combustion byproducts nor tobacco nitrosamines, the nicotine nitrosation products. E-cigarettes are promoted as safe and have gained significant popularity. In this study, instead of detecting nitrosamines, we directly measured DNA damage induced by nitrosamines in different organs of E-cigarette smoke-exposed mice. We found mutagenic O⁶-methyldeoxyguanosines and γ-hydroxy-1,N² -propano-deoxyguanosines in the lung, bladder, and heart. DNA-repair activity and repair proteins XPC and OGG1/2 are significantly reduced in the lung. We found that nicotine and its metabolite, nicotine-derived nitrosamine ketone, can induce the same effects and enhance mutational susceptibility and tumorigenic transformation of cultured human bronchial epithelial and urothelial cells. These results indicate that nicotine nitrosation occurs in vivo in mice and that E-cigarette smoke is carcinogenic to the murine lung and bladder and harmful to the murine heart. It is therefore possible that E-cigarette smoke may contribute to lung and bladder cancer, as well as heart disease, in humans.

Keywords: DNA damage; DNA repair; E-cigarettes; cancer; lung–bladder–heart.

Fig. 1.

ECS induces γ-OH-PdG and O⁶-medG adducts in the lung, bladder and heart. Genomic DNA were isolated from different organs of mice exposed to FA or ECS as described in text. (A–D) O⁶-medG and PdG formed in the genomic DNA were detected by immunochemical methods (28). (A and C) Slot blot. (B and D) Quantification results. The bar represents the mean value. (E) Identification of γ-OH-PdG adducts formed in the genomic DNA of lung and bladder by the 2D-TLC (Upper) and then HPLC (Lower) (28). ST, PdG, or O⁶-medG standard DNA. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig. 2.

Relationship of ECS-induced PdG versus O⁶-medG formation in different organs of mice. The levels of PdG and O⁶-medG detected in different organs from mice exposed to FA and ECS were determined in Fig. 1. In A, O⁶-medG formation is plotted against PdG formation in each organ in mice exposed to ECS (red triangles) and FA (blue dots). In B, formation of PdG and O⁶-medG in the bladder, heart, and liver is plotted against PdG and O⁶-medG formation, respectively, in the lung of mice exposed to ECS and FA. Each symbol represents each individual mouse.

Fig. 3.

ECS reduces DNA-repair activity and XPC and OGG1/2 in the lung. Cell lysates were isolated from lung tissues of mice exposed to FA (n = 10) or to ECS (n = 10) the same as in Fig. 1. The NER and the BER activity in the cell lysates were determined by the in vitro DNA damage-dependent repair synthesis assay as described (13, 28). (A and B) Ethidium bromide-stained gels (Upper) and autoradiograms (Lower) are shown in Left. In Right, the radioactive counts in the autoradiograms were normalized to input DNA. The relative repair activity was calculated using the highest band as 100%. (C) Detection of XPC and OGG1/2 protein in lung tissues (n = 8) by Western blot (Left). Right graphs are quantifications of ECS effect on the abundance of XPC and OGG1/2. The bar represents the mean value. (D) The relationship between the level of PdG and O⁶-medG adduct and the NER and BER activity in lung tissues of FA- (black square) and ECS (red dot)-exposed mice.

Fig. 4.

Nicotine and NNK induce γ-OH-PdG and O⁶-medG in cultured human lung and bladder epithelial cells. Human lung epithelial (BEAS-2B) cells and urothelial (UROtsa) cells were treated with different concentrations of nicotine and NNK as described in text. O⁶-medG and PdG formed in the genomic DNA were determined as described in Fig. 1. (A) The DNA adducts were detected by immunochemical methods (13, 28). (B) The PdG adducts formed in the genomic DNA were further identified as γ-OH-PdG adducts by the ³²P postlabeling followed by 2D-TLC/HPLC method (13, 28).

Fig. 5.

Nicotine and NNK reduce DNA-repair activity and the level of repair proteins XPC and hOGG1/2 in cultured human lung and bladder epithelial cells. Cell-free cell lysates were isolated from human lung (BEAS-2B) and bladder epithelial (UROtsa) cells treated with different concentrations of nicotine and NNK 1 h at 37 °C. The NER and the BER activity in the cell lysates were determined by the in vitro DNA damage-dependent repair synthesis assay as described in Fig. 3. (A) Ethidium bromide-stained gels (Upper) and autoradiograms (Lower) are shown. (B) Quantifications results. The radioactive counts in the autoradiograms were normalized to input DNA. The relative repair activity was calculated using the control band as 100%. (C) The effect of nicotine and NNK treatment on abundance of XPC and hOGG1/2 in human lung and bladder urothelial cells were determined as described in Fig. 3.

Fig. 6.

Nicotine and NNK treatments enhance mutational susceptibility and cell transformation. Human lung and bladder epithelial cells (BEAS-2B and UROtsa) were treated with NNK (0.5 mM) and nicotine (25 mM for BEAS-2B cells, and 5 mM for UROtsa cells) for 1 h at 37 °C; these treatments render 50% cell killing. (A) UVC-irradiated (1,500 J/m²) or H₂O₂ modified (100 mM, 1 h at 37 °C) plasmid DNAs containing the supF gene were transfected into these cells, and the mutations in control, and nicotine- and NNK- treated cells were detected and quantified as previously described (13, 28). (B) Detection of anchorage-independent soft-agar growth. A total of 5,000 treated cells were seeded in a soft-agar plate. The method for anchorage-independent soft-agar growth is the same as previously described (28). Typical soft-agar growth plates stained with crystal violet were shown. (C) Quantifications of percent of control, nicotine, and NNK-treated cells formed colonies in soft-agar plates.