Study reveals e-cigarette users with limited smoking history show similar DNA alterations as smokers

By Dr. Priyom Bose, Ph.D.Reviewed by Danielle Ellis, B.Sc.Mar 25 2024

A recent Cancer Research study assessed the effect of tobacco smoking and electronic cigarette (e-cigarettes) use on DNA methylation changes associated with carcinogenesis.

Background

In comparison to tobacco smoking, the use of e-cigarettes and smokeless, non-combustible tobacco has often been considered to be less harmful. However, recent studies have highlighted some of the potential adverse effects linked to e-cigarette use, including DNA damage and endothelial dysfunction. Therefore, it is imperative to understand the molecular changes and their long-term effects on health.

E-cigarette use has been associated with similar biomarker changes to cigarette smoking. It is essential to identify biomarkers that indicate the risk of cancer. Some of the characteristic features that must be present in biomarkers are (a) modifiability through tobacco smoking and e-cigarette use, (b) lie in genes linked to carcinogenesis, (c) induce cancer progression in a premalignant lesion, and (d) reflective of long-term cancer risk in a surrogate tissue to aid non-invasive monitoring.

The epigenome is a set of chemical modifications of DNA or proteins linked to DNA. Many studies have elucidated the role of epigenomics in carcinogenesis. This information has helped shed light on the long-term impacts of tobacco smoking and e-cigarette use.

DNA methylation (DNAme) at the cytosine C-5 position is an epigenetic modification. Its alterations enriched in genes are associated with smoking-related diseases. Some epigenetic changes remain persistent even after smoking cessation. These biomarkers could be used as an indicator of lung cancer. 

Epithelial cells that are exposed (e.g., oral mucosa and lungs) or not (e.g., cervix) to smoking or e-cigarette use are the key cells of origin for tobacco-related malignancies. Furthermore, smoking-related DNAme changes found in buccal samples are primarily of epithelial origin.

About the study

The current study addressed the aforementioned issues to understand better tissue- and cell-specific epigenetic impacts of e-cigarette or tobacco use on DNAme. It used more than 3,500 cervical, buccal/saliva, or blood samples from immune and epithelial cells at directly and indirectly exposed sites. Additionally, a control sample set was used for validation.

This study is a part of the female cancer prediction using cervical omics to individualize screening and prevention (FORECEE) study. The participants came from five European countries, were between 18 and 86 years of age, and completed an epidemiological questionnaire. The effect of tobacco use on epithelial and immune cells was analyzed systematically using computational deconvolution and cell type-specific DNAme inference.

The effect of direct (proximal) and indirect (distal) exposure to the use of smoking, smokeless tobacco (e.g., snuffs), or e-cigarettes on epithelial and immune cells was assessed. Furthermore, whether these uses also affected lung cancer tissue and prognosis were evaluated. The evaluation of the biomarkers at the cell-level is a key contribution of this study, as the majority of existing studies, including those predicting lung cancer, have used blood samples.

Study findings

The cell-specific alterations following cigarette and e-cigarette use that are associated with carcinogenesis have been uncovered in this study. Smoking was found to elicit changes in protected stem and submucosal gland cells. Cigarette smoking affected epithelial hypoM and this change was found in both proximal and distal exposure. Furthermore, DNAme alterations linked to specific proximal epithelial hyperM and distal epithelial hyperM were also identified.

Smoking was seen to affect the myeloid more prominently than the lymphoid lineage. No significant genetic overlap linked with specific functions was observed in the samples obtained from five different sites. Mostly, epithelial hypoM sites were linked with detoxification responses, whereas proximal epithelial hyperM sites entailed DNA damage responses.

The smoking-related DNAme loci (CpGs) identified here were clustered into four functional group based on anatomical site and cell type. Loci hypermethylated in cheek cells of smokers associated with the NOTCH1/RUNX3/growth factor receptor signaling showed a higher level of methylation in progressing lung carcinoma in situ lesions and cancer tissue. Alarmingly, the aforementioned CpGs were also noted to be hypermethylated in e-cigarette users who reported a limited smoking history.

This study further highlighted a partial reversibility of smoking-induced epigenetic alterations in former tobacco smokers. This observation was based on the fact that epithelial hypoM could not be distinguished between ex-smokers and those who never smoked. This observation could imply that the hypermethylated cells disappeared due to cell death or the displacement of the methyl group in the living cell.

Smokeless tobacco induces similar changes in DNAme in the epithelial hypoM and proximal epithelial hyperM sets, as cigarette smoking. It must be noted that only cigarette smokers exhibited changes in DNAme at immune hypoM sites. Proximal epithelial hypermethylation was robustly associated with lung cancer progression and cervical cancer.

Conclusions

In sum, the results presented here shed light on cell type-specific epigenetic changes following cigarette smoking. Some of these changes, which could also predict lung cancer, are similar to e-cigarette users.

A key limitation of this study was the use of pathway analysis based on gene names, which limited the investigation to cis genes alone. In the future, scientists must perform multi-omics profiling to investigate the association between methylation changes and gene transcription function more comprehensively.