Hypomethylation of LINE-1 repeat elements and global loss of DNA hydroxymethylation in vapers and smokers

ABSTRACT

The outbreak of vaping-related severe lung injuries and deaths and the epidemic of teen vaping in the U.S. underscore the urgent need for determining the biological consequences of electronic cigarette (e-cig) use. We have investigated the association between vaping and epigenetic changes by quantifying DNA methylation levels in Long Interspersed Nucleotide Element 1 (LINE-1) and global DNA hydroxymethylation (5-hmC) levels and measuring the expression level of enzymes catalysing the respective processes in peripheral blood of exclusive vapers, smokers, and controls, matched for age, gender, and race (n = 45). Both vapers and smokers showed significant loss of methylation in LINE-1 repeat elements in comparison to controls (P = 0.00854 and P = 0.03078, respectively). Similarly, vapers and smokers had significant reductions in 5-hmC levels relative to controls (P = 0.04884 and P = 0.0035, respectively). Neither the LINE-1 methylation levels nor the global 5-hmC levels were different between vapers and smokers. There was a direct correlation between methylation levels in the LINE-1 elements and global 5-hmC levels in the study subjects (r = 0.31696, P = 0.03389). Inverse and statistically significant correlations were found between both the LINE-1 methylation levels and the global 5-hmC levels and various vaping/smoking metrics in the study subjects. There were modest but not statistically significant changes in transcription of DNA methyltransferases and ten-eleven translocation enzymes in both vapers and smokers relative to controls. Our findings support follow-up genome-wide investigations into the epigenetic effects of vaping, which may further clarify the health consequences of e-cig use.

Abbreviations

5-mC: 5-methylcytosine; 5-hmC: 5-hydroxymethylcytosine; 8-OHdG: 8-hydroxy-2ʹ-deoxyguanosine; ACTIN: actin beta; ANOVA: Analysis of Variance; BER: base excision repair; BMI: body mass index; CO: carbon monoxide; COHb: carboxyhaemoglobin; COBRA: combined bisulphite restriction analysis; COPD: chronic obstructive pulmonary disease; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase 3A; DNMT3B: DNA methyltransferase 3B; e-cigs: electronic cigarettes; ELISA: enzyme-linked immunosorbent assay; ENDS: electronic nicotine delivery systems; FDA: Food and Drug Administration; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LINE-1: Long Interspersed Nucleotide Element 1; PBS: phosphate-buffered saline; RFU: relative fluorescence units; RT-qPCR: quantitative reverse-transcription polymerase chain reaction; ROS: reactive oxygen species; SAM, S-adenosylmethionine; SE: standard error; TET1: ten-eleven translocation 1; TET2: ten-eleven translocation 2; TET3: ten-eleven translocation 3.

KEYWORDS:

• DNA methylation
• DNA hydroxymethylation
• electronic cigarettes (e-cigs)
• gene expression
• vaping

Acknowledgments

We would like to thank Debra E. Moreno for help with subject recruitment and enrollment and specimen collection and processing, and Niccolo Pabustan for assistance with statistical analysis. We would also like to acknowledge Dr. Seth Rubin and Dr. Michael Garbati for providing technical support for the LINE-1 DNA methylation detection assay. We are grateful to Edward Avol and Dr. Rob McConnell and Dr. Marry Ann Pentz and their staff at the USC Tobacco Center of Regulatory Science (TCORS) for facilitating subject recruitment. Also, we thank Dr. Lourdes Baez Conde for supporting our community engagement and communication plans. Special thanks to Andrew Zaw and Gregory Wilkerson for creating web forms for our online survey and providing technical support for maintenance of the database of responses. We are thankful to Dr. Alan Hiti and his staff at the USC-Norris Outpatient Clinical Laboratory for phlebotomy work.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author Contributions

AWC: Performed experiments and collected data, analysed data, Co-wrote draft on M&M; AC: Performed experiments and collected data, analysed data, Co-wrote draft on M&M; ST: Performed experiments and collected data, analysed data and interpreted the results; Co-wrote the manuscript; AB: conceived and designed the study, performed experiments and collected data, analyzed data and interpreted the results, wrote the manuscript.

Additional information

Funding

This work was supported by grants from the National Institute of Dental and Craniofacial Research of the National Institutes of Health (1R01DE026043 to AB) and the University of California Tobacco-Related Disease Research Program (TRDRP-26IR-0015 and TRDRP-28IR-0058 to AB and TRDRP-26IP-0051 to ST).