DNA Methylation of iNOS, eNOS, TP53 and LINE-1 in Gingival Tissues of Cigarette Smokers, Heat-Not-Burn Smokers and Never Smokers: An Exploratory Study

Abstract

Objectives: In this exploratory study, the influence of traditional cigarette smoking and heat-not-burn device smoking (HnB) on DNA methylation of cancer-associated genes in smokers compared to never smokers was evaluated.

Materials & methods: Twenty-eight healthy individuals undergoing dental care were enrolled into three groups: (i) 10 traditional smokers, (ii) 8 HnB smokers, and (iii) 10 never smokers. Gingival biopsies were obtained, and DNA methylation analysis was performed by pyrosequencing for the cancer-associated genes: iNOS, eNOS and TP53. LINE-1 sequence was selected for genome-wide methylation readout.

Results: HnB smokers exhibited approximately a 13% higher iNOS methylation compared to both traditional smokers and never smokers (p = 0.004 in a crude analysis and p = 0.01 in a multivariable linear regression model adjusted for gender and age). However, HnB smoking did not influence the DNA methylation levels of TP53, eNOS, and LINE-1 in gingival tissues, as these were comparable to the levels observed in traditional smokers and never smokers.

Conclusions: HnB device smoking increased DNA methylation levels of iNOS gene, which might indicate a decreased iNOS expression in HnB smokers, compared to traditional smokers and never smokers. Implications of the observed iNOS methylation status in the development of oral cancer needs to be investigated in future studies.

Keywords: DNA methylation; LINE‐1 elements; TP53 gene; eNOS enzyme; iNOS enzyme; oral mucosa; smoking.

Figure 1

Box and whiskers plots showing methylation (%) of eNOS, iNOS, TP53 genes and LINE‐1 in 28 patients (10 never‐smokers, 8 Heat‐not‐Burn (HnB) smokers, and 10 smokers of traditional smokers. The box contains 50% of the patients; the white line within the box represents the median value; box and whiskers contain about 85% of patients.