Electronic Cigarettes Induce Mitochondrial DNA Damage and Trigger TLR9 (Toll-Like Receptor 9)-Mediated Atherosclerosis.

OBJECTIVE: Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development. Approach and Results: Eight-week-old ApoE-/- mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ (C-C chemokine receptor type 2) classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages. CONCLUSIONS: E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.

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

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.

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

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.

Aldehydes are the predominant forces inducing DNA damage and inhibiting DNA repair in tobacco smoke carcinogenesis.

Tobacco smoke (TS) contains numerous cancer-causing agents, with polycyclic aromatic hydrocarbons (PAHs) and nitrosamines being most frequently cited as the major TS human cancer agents. Many lines of evidence seriously question this conclusion. To resolve this issue, we determined DNA adducts induced by the three major TS carcinogens: benzo(a)pyrene (BP), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanoe (NNK), and aldehydes in humans and mice. In mice, TS induces abundant aldehyde-induced γ-hydroxy-propano-deoxyguanosine (γ-OH-PdG) and α-methyl-γ-OH-PdG adducts in the lung and bladder, but not in the heart and liver. TS does not induce the BP- and NNK-DNA adducts in lung, heart, liver, and bladder. TS also reduces DNA repair activity and the abundance of repair proteins, XPC and OGG1/2, in lung tissues. These TS effects were greatly reduced by diet with polyphenols. We found that γ-OH-PdG and α-methyl-γ-OH-PdG are the major adducts formed in tobacco smokers' buccal cells as well as the normal lung tissues of tobacco-smoking lung cancer patients, but not in lung tissues of nonsmokers. However, the levels of BP- and NNK-DNA adducts are the same in lung tissues of smokers and nonsmokers. We found that while BP and NNK can induce BPDE-dG and O6-methyl-dG adducts in human lung and bladder epithelial cells, these inductions can be inhibited by acrolein. Acrolein also can reduce DNA repair activity and repair proteins. We propose a TS carcinogenesis paradigm. Aldehydes are major TS carcinogens exerting dominant effect: Aldehydes induce mutagenic PdG adducts, impair DNA repair functions, and inhibit many procarcinogens in TS from becoming DNA-damaging agents.

Cigarette smoke component acrolein modulates chromatin assembly by inhibiting histone acetylation.

Chromatin structure and gene expression are both regulated by nucleosome assembly. How environmental factors influence histone nuclear import and the nucleosome assembly pathway, leading to changes in chromatin organization and transcription, remains unknown. Acrolein (Acr) is an α,ß-unsaturated aldehyde, which is abundant in the environment, especially in cigarette smoke. It has recently been implicated as a potential major carcinogen of smoking-related lung cancer. Here we show that Acr forms adducts with histone proteins in vitro and in vivo and preferentially reacts with free histones rather than with nucleosomal histones. Cellular fractionation analyses reveal that Acr exposure specifically inhibits acetylations of N-terminal tails of cytosolic histones H3 and H4, modifications that are important for nuclear import and chromatin assembly. Notably, Acr exposure compromises the delivery of histone H3 into chromatin and increases chromatin accessibility. Moreover, changes in nucleosome occupancy at several genomic loci are correlated with transcriptional responses to Acr exposure. Our data provide new insights into mechanisms whereby environmental factors interact with the genome and influence genome function.

Effect of CpG methylation at different sequence context on acrolein- and BPDE-DNA binding and mutagenesis.

Acrolein (Acr), an α,ß-unsaturated aldehyde, is abundant in tobacco smoke and cooking and exhaust fumes. Acr induces mutagenic α- and γ- hydroxy-1,N(2)-cyclic propano-deoxyguanosine adducts in normal human bronchial epithelial cells. Our earlier work has found that Acr-induced DNA damage preferentially occurs at lung cancer p53 mutational hotspots that contain CpG sites and that methylation at CpG sites enhances Acr-DNA binding at these sites. Based on these results, we hypothesized that this enhancement of Acr-DNA binding leads to p53 mutational hotspots in lung cancer. In this study, using a shuttle vector supF system, we tested this hypothesis by determining the effect of CpG methylation on Acr-DNA binding and the mutations in human lung fibroblasts. We found that CpG methylation enhances Acr-induced mutations significantly. Although CpG methylation enhances Acr-DNA binging at all CpG sites, it enhances mutations at selective--TCGA--sites. Similarly, we found that CpG methylation enhances benzo(a)pyrene diol epoxide binding at all -CpG- sites. However, the methylated CpG sequences in which benzo(a)pyrene diol epoxide-induced mutations are enhanced are different from the CpG sequences in which Acr-induced mutations are enhanced. CpG methylation greatly increases Acr-induced G to T and G to A mutation frequency to levels similar to these types of mutations found in the CpG sites in the p53 gene in tobacco smoke-related lung cancer. These results indicate that both CpG sequence context and the chemical nature of the carcinogens are crucial factors for determining the effect of CpG methylation on mutagenesis.

Effect of carcinogenic acrolein on DNA repair and mutagenic susceptibility.

Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic α- and γ-hydroxy-1, N(2)-cyclic propano-2'-deoxyguanosine adducts (α-OH-Acr-dG and γ-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair and mismatch repair. Although Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2, and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity.

An ammonium bicarbonate-enhanced stable isotope dilution UHPLC-MS/MS method for sensitive and accurate quantification of acrolein-DNA adducts in human leukocytes.

Acrolein (Acr), a ubiquitous environmental pollutant, can react directly with genomic DNA to form mutagenic adducts without undergoing metabolic activation. To sensitively and accurately quantify Acr-DNA adducts (including structural isomers and stereoisomers) in human leukocytes, we developed an enhanced stable isotope dilution ultrahigh performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) method using ammonium bicarbonate (NH4HCO3), which is thermally unstable and degrades readily to carbon dioxide and ammonia in heated gas phase. Interestingly, ammonium bicarbonate (as an additive to the mobile phase) not only improves the protonation of AcrdG adducts but also suppresses the formation of MS signal-deteriorating metal-AcrdG complexes during electrospray ionization, leading to the enhancement of their MS detection by 2.3-8.7 times. In contrast, routinely used ammonium salts (ammonium acetate and ammonium formate) and formic acid do not show similar enhancement. The developed method is potentially useful for enhancing ESI-MS detection of other modified 2'-deoxyribonucleosides that have difficulty in protonation and may form excess metal complexes during electrospray ionization. The limits of detection (LODs, S/N = 3) are estimated to be about 40-80 amol. By the use of the developed method, we found that the Acr adducts of three nucleotides (dG, dA, and dC) can be detected in human leukocytes. In addition to the known γ-AcrdG, α-AcrdA is also identified as an Acr-adduct of high abundance (2.5-20 adducts per10(8) nts).

Melanocytes are deficient in repair of oxidative DNA damage and UV-induced photoproducts.

Melanomas occur mainly in sunlight-exposed skin. Xeroderma pigmentosum (XP) patients have 1,000-fold higher incidence of melanoma, suggesting that sunlight-induced "bulky" photoproducts are responsible for melanomagenesis. Sunlight induces a high level of reactive oxygen species in melanocytes (MCs); oxidative DNA damage (ODD) may thus also contribute to melanomagenesis, and XP gene products may participate in the repair of ODD. We examined the effects of melanin on UVA (320-400 nm) irradiation-induced ODD and UV photoproducts and the repair capacity in MC and XP cells for ODD and UV-induced photoproducts. Our findings indicate that UVA irradiation induces a significantly higher amount of formamidopyrimidine glycosylase-sensitive ODD in MCs than in normal human skin fibroblasts (NHSFs). In contrast, UVA irradiation induces an insignificant amount of UvrABC-sensitive sites in either of these two types of cells. We also found that, compared to NHSFs, MCs have a reduced repair capacity for ODD and photoproducts; H(2)O(2) modified- and UVC-irradiated DNAs induce a higher mutation frequency in MCs than in NHSFs; and, XP complementation group A (XPA), XP complementation group C, and XP complementation group G cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSFs. These results suggest that: (i) melanin sensitizes UVA in the induction of ODD but not bulky UV photoproducts; (ii) the high susceptibility to UVA-induced ODD and the reduced DNA repair capacity in MCs contribute to carcinogenesis; and (iii) the reduced repair capacity for ODD contributes to the high melanoma incidence in XP patients.

Chromium (VI) induces both bulky DNA adducts and oxidative DNA damage at adenines and guanines in the p53 gene of human lung cells.

Chromium (VI) [Cr(VI)], a ubiquitous environmental carcinogen, is generally believed to induce mainly mutagenic binary and ternary Cr(III)-deoxyguanosine (dG)-DNA adducts in human cells. However, both adenine (A) and guanine (G) mutations are found in the p53 gene in Cr exposure-related lung cancer. Using UvrABC nuclease and formamidopyrimidine glycosylase (Fpg), and ligation-mediated PCR methods, we mapped the distribution of bulky DNA adducts (BDA) and oxidative DNA damage (ODD) in the p53 gene in Cr(VI)-treated human lung cells. We found that both BDA and ODD formed at 2'-deoxyadenosine (dA) and dG bases. To understand the causes for these Cr-induced DNA damages, we mapped the distribution of BDA adducts and ODD in the p53 gene DNA fragments induced by Cr(III), Cr(VI) and Cr(V), the three major cellular Cr forms. We found that (i) dA at -CA- is a major Cr(VI) binding site followed by -GG- and -G-. Cr(VI) does not bind to -GGG-, (ii) Cr(VI)-DNA binding specificity is distinctly different from the Cr(III)-DNA binding in which -GGG- and -GG- are preferential sites, (iii) Cr(V) binding sites include all of Cr(VI) and Cr(III)-DNA binding sites and (iv) Cr(VI) and Cr(V) induce Fpg-sensitive sites at -G-. Together, these results suggest that Cr(VI) induction of BDA and ODD at dA and dG residues is through Cr(V) intermediate. We propose that these Cr(VI)-induced BDA and ODD contribute to mutagenesis of the p53 gene that leads to lung carcinogenesis.

Urothelial tumor initiation requires deregulation of multiple signaling pathways: implications in target-based therapies.

Although formation of urothelial carcinoma of the bladder (UCB) requires multiple steps and proceeds along divergent pathways, the underlying genetic and molecular determinants for each step and pathway remain undefined. By developing transgenic mice expressing single or combinatorial genetic alterations in urothelium, we demonstrated here that overcoming oncogene-induced compensatory tumor barriers was critical for urothelial tumor initiation. Constitutively active Ha-ras (Ras*) elicited urothelial hyperplasia that was persistent and did not progress to tumors over a 10 months period. This resistance to tumorigenesis coincided with increased expression of p53 and all pRb family proteins. Expression of a Simian virus 40 T antigen (SV40T), which disables p53 and pRb family proteins, in urothelial cells expressing Ras* triggered early-onset, rapidly-growing and high-grade papillary UCB that strongly resembled the human counterpart (pTaG3). Urothelial cells expressing both Ras* and SV40T had defective G(1)/S checkpoint, elevated Ras-GTPase and hyperactivated AKT-mTOR signaling. Inhibition of the AKT-mTOR pathway with rapamycin significantly reduced the size of high-grade papillary UCB but hyperactivated mitogen-activated protein kinase (MAPK). Inhibition of AKT-mTOR, MAPK and STAT3 altogether resulted in much greater tumor reduction and longer survival than did inhibition of AKT-mTOR pathway alone. Our studies provide the first experimental evidence delineating the combinatorial genetic events required for initiating high-grade papillary UCB, a poorly defined and highly challenging clinical entity. Furthermore, they suggest that targeted therapy using a single agent such as rapamycin may not be highly effective in controlling high-grade UCB and that combination therapy employing inhibitors against multiple targets are more likely to achieve desirable therapeutic outcomes.

Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies.

Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 µg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model.

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5' incision on 1 strand and a 3' incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB+UvrC-DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway.

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Förster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates ("bubble" DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA(2)B and contact of the beta-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.

Structural or functional defects of PTEN in urothelial cells lacking P53 drive basal/squamous-subtype muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) exhibits strong inter- and intra-tumor heterogeneity that affects biological behaviors, therapeutic responses, and prognoses. Mutations that activate RTK-RAS-PI3K and inactivate P19-P53-P21 coexist in 60-70% of MIBC. By time-controlled ablation of Tp53 and Pten, singly or combined, in adult mouse urothelium, we found that Tp53 loss alone produced no abnormality. While Pten loss elicited hyperplasia, it synergized with Tp53 loss to trigger 100% penetrant MIBC that exhibited basal/squamous features that resembled its human counterpart. Furthermore, PTEN was inactivated in human MIBC cell lines and specimens primarily by hyperphosphorylation of the C-terminus. Mutated or tailless PTEN incapable of C-terminal phosphorylation demonstrated increased inhibition of proliferation and invasion than full-length PTEN in cultured MIBC cells. In xenograft and transgenic mice, tailless PTEN, but not full-length PTEN, prevented further growth in established tumors. Collectively, deficiencies of both PTEN and P53 drive basal/squamous subtype MIBC. PTEN is inactivated by C-terminal hyperphosphorylation, and this modification may serve as a biomarker for subtyping MIBC and predicting tumor progression. Tailless PTEN is a potential molecular therapeutic for tumors, such as bladder cancer (BC), that can be readily accessed.

PKM2 Is Essential for Bladder Cancer Growth and Maintenance.

Pyruvate kinase M2 (PKM2) has been shown to promote tumorigenesis by facilitating the Warburg effect and enhancing the activities of oncoproteins. However, this paradigm has recently been challenged by studies in which the absence of PKM2 failed to inhibit and instead accelerated tumorigenesis in mouse models. These results seem inconsistent with the fact that most human tumors overexpress PKM2. To further elucidate the role of PKM2 in tumorigenesis, we investigated the effect of PKM2 knockout in oncogenic HRAS-driven urothelial carcinoma. While PKM2 ablation in mouse urothelial cells did not affect tumor initiation, it impaired the growth and maintenance of HRAS-driven tumors. Chemical inhibition of PKM2 recapitulated these effects. Both conditions substantially reduced complex formation of PKM2 with STAT3, their nuclear translocation, and HIF1α- and VEGF-related angiogenesis. The reduction in nuclear STAT3 in the absence of PKM2 also correlated with decreased autophagy and increased apoptosis. Time-controlled, inducible PKM2 overexpression in simple urothelial hyperplasia did not trigger tumorigenesis, while overexpression of PKM2, but not PKM1, in nodular urothelial hyperplasia with angiogenesis strongly accelerated tumorigenesis. Finally, in human patients, PKM2 was overexpressed in low-grade nonmuscle-invasive and high-grade muscle-invasive bladder cancer. Based on these data, PKM2 is not required for tumor initiation but is essential for tumor growth and maintenance by enhancing angiogenesis and metabolic addiction. The PKM2-STAT3-HIF1α/VEGF signaling axis may play a critical role in bladder cancer and may serve as an actionable therapeutic target. SIGNIFICANCE: Genetic manipulation and pharmacologic inhibition of PKM2 in mouse urothelial lesions highlight its essential role in promoting angiogenesis and metabolic addiction, events indispensable for tumor growth and maintenance.

Nicotine dose-dependent epigenomic-wide DNA methylation changes in the mice with long-term electronic cigarette exposure.

Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.