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

Nicotine is the major component of ECS (3). The majority (80%) of inhaled nicotine in smoke is quickly metabolized into cotinine, which is excreted into the bloodstream and subsequently into urine (14). Cotinine is generally believed to be nontoxic and noncarcinogenic (15); however, a small portion (<10%) of inhaled nicotine is believed to be metabolized into nitrosamines in vivo (16–18). Nitrosamines induce tumors in different organs in animal models (6, 19). Inhaled nitrosamines are metabolized into N-nitrosonornicotine (NNN) and nicotine-derived nitrosamine ketone (NNK). It has been proposed that NNK can be further metabolized and spontaneously degraded into methyldiazohydroxide (MDOH), pyridyl-butyl derivatives (PBDs), and formaldehyde, and that NNN degrade into hydroxyl or keto PBDs (20). While nicotine cannot bind to DNA directly, MDOH can methylate deoxyguanosines and thymidines in DNA (21). Although the fate of nitrosamine-induced formaldehyde and PBDs in vivo is less clear, both are capable of inducing DNA damage in vitro (22–25). Therefore, if ECS in fact is a carcinogen, it is likely that its carcinogenicity is derived from nitrosamines that are derived from the nitrosation of nicotine (5, 19, 21). Nitrosamines are potent carcinogens and it is generally believed that their carcinogenicity is via induction of methylation DNA damage (26, 27). As a step in examining the carcinogenicity of ECS, we determined whether ECS can induce O⁶-methyl-deoxuguanosine (O⁶-medG) adducts in lung, heart, liver, and bladder tissues of mice. Mice were exposed to ECS (10 mg/mL, 3 h/d, 5 d/wk) for 12 wk; the dose and duration equivalent in human terms to light E-cig smoking for 10 y. The results in Fig. 1 A and B, Fig. S1, and Table S1 show that ECS induced significant amounts of O⁶-medG adducts in the lung, bladder, and heart and that the level of O⁶-medG adducts in lung was three- to eightfold higher than in the bladder and heart. These results are consistent with the explanation that nicotine is metabolized into MDOH, which can methylate DNA (16, 20).

Recently, we found that aldehyde-derived cyclic 1,N²-propano-dG (PdG), including γ-OH-1,N²-PdG (γ-OH-PdG) and α-methyl-γ-OH-1,N²-PdG adducts, are the major DNA adducts in mouse models (28) induced by TS, which contains abundant nitrosamines and aldehydes (20). We therefore determined the extent of PdG formation in different organs of ECS-exposed mice using a PdG-specific antibody (28–30).

The results in Fig. 1 C and D show that ECS induced PdG adducts in the lung, bladder, and heart, and that the level of PdG in the lung is two- to threefold higher than in the bladder and heart. Moreover, the level of PdG is 25- to 60-fold higher than the level of O⁶-medG in lung, bladder, and heart tissues, indicating that induction of PdG is more efficient than induction of O⁶-medG by nicotine metabolic products and/or that O⁶-medG is more efficiently repaired in these organs. ECS, however, did not induce either O⁶-medG or PdG in liver DNA.

Due to the relatively minute amount of genomic DNA that is possible to isolate from mouse organs, in this case, specifically from bladder mucosa, which is only able to yield up to 2 μg of genomic DNA from each mouse, we used the sensitive ³²P-postlabeling thin layer chromatography (TLC)/HPLC method to identify the species of the PdG formed in lung and bladder tissues (13, 28, 31). The results in Fig. 1E show that the majority of PdG (>95%) formed in these tissues coelute with γ-OH-PdG adduct standards with a minor portion that coelute with α-OH-PdG standards.

We then determined the relationship of PdG and O⁶-medG formation in different organs of each animal. The results in Fig. 2A show that the levels of PdG and O⁶-medG in the same organs are positively related to each other. Thus, a lung tissue sample that had a high level of PdG also had a high level of O⁶-medG. The same relationship between PdG and O⁶-medG formation was found in the bladder and heart (Fig. 2A and Table S1). The results in Fig. 2B show that in the same mouse, the levels of PdG and O⁶-medG formation in different organs also have a positive correlation: Mice with a high level of PdG and O⁶-medG formation in the lung also had a high level of these DNA adducts in the bladder and heart (Fig. 2B and Table S1). Together, these results indicate that the formation of PdG and O⁶-medG DNA adducts in the lung, bladder, and heart tissue are the result of DNA damaging agents derived from ECS exposure, and raising the possibility that the ability for nicotine absorption and metabolism and DNA-repair activity of different organs determine their susceptibility to ECS-induced DNA adduct formation.

Recently, we have found that lung tissues of mice exposed to TS have lower DNA-repair activity and lower levels of DNA-repair proteins XPC and OGG1/2 and that aldehydes, such as acrolein, acetaldehyde, crotonaldehyde, and 4-hydroxy-2-nonenal, can modify DNA-repair proteins, causing the degradation of these repair proteins and impairing DNA-repair function (11, 12, 28). These findings raise the possibility that, via induction of aldehydes, ECS can impair DNA-repair functions. To test this possibility, we determined the effect of ECS on the activity of the two major DNA-repair mechanisms in mouse lung tissues: nucleotide excision repair (NER) and base excision repair (BER) (32). We adopted a well-established in vitro DNA damage-dependent repair synthesis assay, which requires only 10 μg of freshly prepared cell lysates (11, 13, 28). Since the amount of bladder mucosa collected from individual mice was minute, we were only able to determine DNA-repair activity in lung tissues (28). We used UV-irradiated DNA, which contains cyclobutane pyrimidine dimers as well as <6-4> photoproducts; Acr-modified DNA, which contains γ-OH-PdG; and H₂O₂-modified DNA, which contains 8-oxo-dG, as substrates (13, 28). It is well established that NER is the major mechanism that repairs cyclobutane pyrimidine dimers, <6-4> photoproducts, and γ-OH-PdG, and that BER is the major mechanism that repairs 8-oxo-dG (32, 33). Therefore, these two types of substrates allow us to determine the NER and BER activity in the cell lysates (11, 13). The results in Fig. 3 A and B and Fig. S2 show that both NER and BER activity in lung tissue of ECS-exposed mice are significantly lower than in lung tissue of filtered air (FA)-exposed mice.

We then determined the level of XPC and OGG1/2, the two crucial proteins, respectively, for NER and BER (34, 35). The results in Fig. 3C show that the level of XPC and OGG1/2 in lung tissues of ECS-exposed mice was significantly lower than in control mice. We further determined the relationship between DNA adduct formation and DNA-repair activity in lung tissues of FA- and ECS-exposed mice. Since NER is the major repair mechanism for bulky DNA damage such as γ-OH-PdG and photodimers (11, 33) and BER is a major repair mechanism for base damage (32), we compared BER activity with the level of O⁶-medG adducts and NER activity with the level of γ-OH-PdG adducts. The results in Fig. 3D show that NER and BER activity in lung tissue of different mice is inversely related to the level of γ-OH-PdG and O⁶-medG adducts, respectively. These results indicate that in lung tissue, NER and BER activities are crucial factors in determining the level of ECS-induced γ-OH-PdG and O⁶-medG DNA damage; mice that are more sensitive to ECS-induced DNA-repair inhibition accumulate more ECS-induced DNA damage in their lung and, perhaps, bladder and heart. It should be noted that in human cells, repair of O⁶-medG adducts is mainly carried out by O⁶-methylguanine DNA methyltransferase (MGMT) (36, 37). The positive relationship between BER activity and the O⁶-medG level in lung tissues of mice implies that ECS impairs BER enzymes as well as MGMT, and/or O⁶-medG is repaired by a BER mechanism in mice.

Many tobacco-specific nitrosamines that result from the nitrosation of nicotine, such as NNN and NNK, are potent carcinogens and can induce cancer in different organs, including the lung (20, 21, 27). While NNK and NNN cannot covalently bind with DNA directly, it has been proposed that one of NNK’s metabolic products, MDOH, can interact with DNA to induce mutagenic O⁶-medG adducts (20, 21, 27). These results raise the possibility that ECS-induced O⁶-medG is due to the nitrosation of nicotine, and that NNK resulting from nicotine nitrosation then further transforms into MDOH in lung and bladder tissue (20). To test this possibility, we determined the DNA adducts induced by nicotine and NNK in cultured human bronchial epithelial and urothelial cells, and the effect of nicotine and NNK treatments on DNA repair, using the same methods indicated in Fig. 1. The results in Fig. 4 show that both nicotine and NNK can induce the same type of γ-OH-PdG adducts, and O⁶-medG adducts. Since it is well established that many aldehydes can induce cyclic PdG in cells (38–40), these results suggest that aldehydes as well as MDOH are NNK metabolites, which induce γ-OH-PdG and O⁶-medG.

We next determined the effects of nicotine and NNK treatment on DNA-repair activity and repair protein levels in human lung and bladder epithelial cells using the method described in Fig. 3. The results in Fig. 5 show that nicotine and NNK treatments not only inhibit NER and BER activities, they also reduce the protein levels of XPC and hOGG1/2. We found that these reductions of XPC and hOGG1/2 induced by nicotine and NNK can be prevented or attenuated by the proteasome and autophagosome inhibitors MG132, 3-methyladenine (3-MA), and lactacystin (Fig. S3) (13, 41–43). These results indicate that metabolites of nicotine and NNK can modify DNA-repair proteins and cause proteosomal and autophagosomal degradation of these proteins and that ECS’s effect on the inhibition of DNA-repair activity is via modifications and degradation of DNA-repair proteins by its metabolites.

Together, these results indicate that human bronchial epithelial and urothelial cells as well as lung, heart, and bladder tissues in the mouse are able to nitrosate nicotine and metabolize nitrosated nicotine into NNK and then MDOH and aldehydes. Furthermore, whereas MDOH induces O⁶-medG adducts, aldehydes not only can induce γ-OH-PdG, they also can inhibit DNA repair and cause repair protein degradation.

The aforementioned results demonstrate that ECS’s major component nicotine, via its metabolites, MDOH, and aldehydes, not only can induce mutagenic DNA adducts, but that they also can inhibit DNA repair in human lung and bladder epithelial cells. These results raise the possibility that ECS and its metabolites can function not only as mutagens but also as comutagens to enhance DNA damage-induced mutagenesis. To test this possibility, we determined the effect of these agents on cell mutation susceptibility on UV- and H₂O₂-induced DNA damage using the well-established supF mutation system (13). The results in Fig. 6A show that nicotine and NNK treatment in both human lung and bladder epithelial cells enhances the spontaneous mutation frequency as well as UV- and H₂O₂-induced mutation frequency by two- to fourfold. These results indicate that nicotine and NNK treatment sensitize these human cells to the extent that they are more susceptible to mutagenesis. We further tested the effect of these agents on induction of tumorigenic transformation using the anchorage-independent soft-agar growth assay (44, 45). The results in Fig. 6 B and C show that nicotine and NNK greatly induce soft-agar anchorage-independent growth of human lung and bladder cells, a necessary ability for tumorigenic cells (46–49).

Acr-dG monoclonal antibodies and plasmid pSP189 were prepared, as described (13, 41). Acr-dG antibodies are specific for PdG adducts including Acr-, HNE-, and crotonaldehyde (Cro)-dG (29). Antibodies for XPC, hOGG1/2 (cross reacts with mouse OGG1/2), α-tubulin, and mouse/rabbit IgG; enzymes, T4 kinase, protease K, nuclease P, and RNase A; and chemicals, acrolein, nicotine, and NNK were commercially available. Immortalized human lung (BEAS-2B) and bladder epithelial (UROtsa) cells were obtained from American Type Culture Collection and J.R. Masters, University College London, London. All animal procedures were approved by the Institutional Animal Care and Use Committee, New York University School of Medicine.

Twenty FVBN (Jackson Laboratory, Charles River) male mice were randomized into two groups, 10 each. Mice were exposed to ECS (10 mg/mL), 3 h/d, 5 d/wk, for 12 wk. ECS was generated by an E-cig machine, as previously described (64). An automated three-port E-cigarette aerosol generator (e∼Aerosols) was used to produce E-cigarette aerosols from NJOY top fill tanks (NJOY, Inc.) filled with 1.6 mL of e-juice with 10 mg/mL nicotine in a propylene glycol/vegetable glycerin mixture (50/50 by volume; MtBakerVapor MESA). Each day the tanks were filled with fresh e-juice from a stock mixture, and the voltage was adjusted to produce a consistent wattage (∼1.96 A at 4.2 V) for each tank. The puff aerosols were generated with charcoal and high-efficiency particulate filtered air using a rotorless and brushless diaphragm pump and a puff regime consisting of 35-mL puff volumes of 4-s duration at 30-s intervals. Each puff was mixed with filtered air before entering the exposure chamber (1 m³). Tanks were refilled with fresh e-juice at 1.5 h into the exposure period during the pause between puffs. Mass concentrations of the exposure atmospheres were monitored in real time using a DataRam4 (Thermo Fisher Scientific) and also determined gravimetrically by collecting particles on Teflon filters (Teflo, 2 mm pore size; Pall) weighed before and after sample collection using an electrobalance (MT-5; Metler).

Exponentially growing BEAS-2B and UROtsa were treated with different concentrations of nicotine (BEAS-2B: 0, 100, 200 µM; UROtsa: 0, 1, 2.5 µM), and NNK (BEAS-2B: 0, 100, 300, 1,000 µM; UROtsa: 0, 50, 100, 200 µM) for determination of DNA adduct and DNA-repair activity. For XPC and hOGG1/2 detection, BEAS-2B were treated with nicotine (0, 50, 100, 200 µM), and NNK (0, 500, 750, 1,000 µM) and UROtsa were treated with nicotine (0, 1, 2.5, 5 µM) and NNK (0, 100, 200, 400 µM) for 1 h at 37 °C. Genomic DNA and cell lysate isolation from these cells was the same as described (28).

Cyclic PdG and O⁶-medG adducts formed in the genomic DNA were determined by the immunochemical slot blot hybridization method using Acr-dG and O⁶-medG antibodies and quantum dot labeled second antibody, as described (13, 28). PdG adducts formed in cultured human cells, and mouse lung tissue were further analyzed by the ³²P postlabeling-2D-TLC/HPLC method, as previously described (28).

The DNA-repair activity was assessed by an in vitro DNA damage-dependent repair synthesis assay, as previously described (13).

The levels of XPC and OGG1/2 proteins in lung tissues of mice with and without ECS exposure, and in BEAS-2B and UROtsa cells treated with nicotine and NNK, were determined, as described (13).

Shuttle vector pSP189 plasmids, which contain the tyrosine suppressor tRNA coding gene the supF, were UV (1,500 J/m²) irradiated or modified with H₂O₂ (100 mM, 1 h at 37 °C), then transfected into cells with and without pretreated with nicotine and NNK for 1 h at 37 °C. Mutations in the supF mutations were detected, as previously described (13).

Lung (BEAS-2B) and bladder (UROtsa) epithelial cells were treated with NNK (0.5 mM) and nicotine (25 and 5 mM) for 1 h at 37 °C; these treatments rendered 50% cell killing. The method for anchorage-independent soft-agar growth is the same as previously described (28).

Statistical analysis and graphs were performed with Prism 6 (GraphPad) software. Two group comparisons were conducted with the unpaired, two-tailed Mann–Whitney u test or the unpaired, two-tailed t test with Welsh’s correction for unequal variances. A P value <0.05 was considered to be significant.