Cigarette Smoke Disrupts Mitochondria in Airway Cells but Leaves Mitochondrial DNA Methylation Largely Untouched

Cigarette smoke has long been known to batter the lungs, but scientists are still untangling exactly how its toxic chemistry derails the tiny power plants inside our airway cells. In a new study published in Epigenetics Communications, researchers at the University Medical Center Groningen set out to determine whether the mitochondrial dysfunction seen in chronic obstructive pulmonary disease, or COPD, is driven by epigenetic changes on the mitochondrial genome itself. Their carefully controlled experiments deliver a surprising verdict: while cigarette smoke extract clearly impairs mitochondrial respiration and transiently reshapes mitochondrial gene expression, it leaves mitochondrial DNA methylation almost entirely unchanged, suggesting that this epigenetic mark is not the central culprit in smoke-induced mitochondrial failure.

COPD is the third leading cause of death worldwide, a progressive disease in which chronic inflammation and tissue damage produce irreversible airflow limitation. Cigarette smoke, the dominant risk factor, contains more than 7,000 compounds, including reactive oxygen species such as superoxide and hydroxyl radicals. Although the gaseous phase of smoke cannot easily cross cell membranes, the lipophilic components captured in cigarette smoke extract, including aldehydes and polycyclic aromatic hydrocarbons, can penetrate cells and reduce mitochondrial membrane potential and ATP production. The resulting damage activates immune cells, amplifies lung tissue destruction, and promotes COPD in susceptible individuals. Yet not all smokers develop the disease, and mounting evidence points to epigenetic mechanisms, including DNA methylation, as key players in determining who succumbs.

Mitochondria carry their own small genome, separate from the DNA housed in the nucleus, and this circular genome lacks the protective histone proteins that organize nuclear DNA. That absence has led researchers to focus on methylation of the mitochondrial DNA itself as a potential regulatory layer. Mitochondrial gene expression changes have already been documented in smokers: a comparative analysis found that 32 of 37 mitochondrial genes were upregulated in smokers with normal lung function, and lung epithelial cells from smokers show increased mitochondrial DNA copy number correlated with nuclear DNA methylation. Mitochondrial DNA methylation differences have also been reported in aging, metabolic disorders, neurodegeneration, and cardiovascular disease, making the mitochondrial epigenome an attractive suspect in COPD pathogenesis.

To test that suspicion, the Groningen team, led by Lin Liang and Irene H. Heijink, exposed human bronchial epithelial BEAS-2B cells to gradually increasing concentrations of cigarette smoke extract over periods ranging from four weeks to 25 weeks. Using Seahorse extracellular flux analysis, they measured the oxygen consumption rate, the gold-standard readout of mitochondrial respiration. After ten weeks of exposure reaching 5% smoke extract, the cells displayed a striking loss of respiratory capacity: basal respiration, ATP-linked respiration, and maximal respiration all fell significantly compared with unexposed controls, confirming that prolonged smoke exposure genuinely cripples mitochondrial energy production.

The damaged mitochondria were accompanied by a transcriptional response. Expression of the protein-coding mitochondrial genes MT-CYB and MT-CO2, which encode components of respiratory chain complexes III and IV, rose significantly in cells exposed to 5% and 6% smoke extract, along with the rRNA gene MT-RNR1 at the 5% time point. Because mitochondrial DNA copy number remained stable throughout the 25-week exposure, the researchers interpret this upregulation as a compensatory boost in transcriptional activity as the cells struggle to maintain energy output. Intriguingly, the elevation vanished by the 25-week mark at 10% smoke extract, suggesting that once dysfunction becomes severe enough, the compensatory capacity of mitochondrial gene regulation is itself overwhelmed.

The critical question was whether these changes were written into the mitochondrial epigenome. Using pyrosequencing targeted to the D-loop, the non-coding region harboring the mitochondrial promoters, and to the MT-CYB and MT-CO2 genes, the team measured methylation at individual cytosines, including the non-CpG sites that can be methylated in mitochondria. They complemented this with liquid chromatography tandem mass spectrometry to quantify global mitochondrial DNA methylation. The result was resoundingly negative: despite dramatic functional impairment and gene expression shifts, methylation in the long-term exposed BEAS-2B cells showed no statistically significant changes at any assessed site or across the whole mitochondrial genome. Some cytosines shifted consistently, but by less than 1%, a magnitude the authors consider biologically negligible.

Short-term exposure told a similar story. A 24-hour bath in 10% smoke extract triggered a massive surge in the oxidative stress marker HMOX1, upregulated more than 200-fold, and significantly reduced mitochondrial DNA copy number in BEAS-2B cells, possibly reflecting smoke-damaged mitochondria being cleared by mitophagy. Yet mitochondrial gene expression and methylation held steady. In the 16HBE bronchial epithelial cell line, which more faithfully mirrors primary airway cells, neither copy number nor gene expression budged after 24-hour exposures, although mean methylation of MT-CYB and MT-CO2 rose modestly at the 15% dose, hinting at a dose-dependent and cell-type-specific response that disappeared at 20%, where cellular tolerance may be exceeded.

The team then extended the work to primary airway epithelial cells isolated from six COPD patients, all with severe GOLD stage IV disease, and six non-COPD donors. Here the findings were equally unambiguous: mitochondrial gene expression, copy number, and methylation were indistinguishable between COPD and control cells, and 24-hour smoke extract exposure failed to alter any of these measures in either group. The authors caution that the modest donor numbers and the lack of information on the control donors’ sex, age, and smoking status could obscure subtler differences, and that the submerged culture conditions used here cannot capture the effects of the gaseous phase of smoke, which air-liquid interface cultures would expose.

Taken together, the study delivers a sobering message for the mitochondrial epigenetics field: mitochondrial DNA methylation appears not to be a dominant driver of the pathological changes that cigarette smoke inflicts on airway epithelium. The smoke-induced mitochondrial dysfunction seen in COPD likely arises through other mechanisms, while mild shifts in mitochondrial gene expression may be consequences rather than causes. The authors point toward promising future directions, including the emerging role of N6-methyladenine, a different DNA modification shown to regulate mitochondrial transcription and replication and to accumulate in mitochondrial DNA during aging across species. As researchers refine methods for measuring mitochondrial methylation, accounting for artifacts such as incomplete bisulfite conversion, the Groningen results provide a rigorous negative control that will help steer COPD research toward the molecular mechanisms that truly matter.

Subject of Research: Effects of cigarette smoke extract on mitochondrial function, gene expression, and mitochondrial DNA methylation in airway epithelial cells in relation to COPD

Article Title: The effects of cigarette smoke extract on mitochondrial function, mitochondrial gene expression and mitochondrial DNA methylation in airway epithelial cells

Article References: Liang, L., Wang, L., Jonker, M. R., Kosse, W., Jellema, P. G., Rots, M. G., & Heijink, I. H. (2025). The effects of cigarette smoke extract on mitochondrial function, mitochondrial gene expression and mitochondrial DNA methylation in airway epithelial cells. Epigenetics Communications, 6(1), Article 1. https://doi.org/10.1186/s43682-025-00040-4

Image Credits: AI Generated

DOI: 10.1186/s43682-025-00040-4

Keywords: mitochondrial DNA methylation, mitochondrial dysfunction, COPD, cigarette smoke extract, airway epithelial cells, mitochondrial gene expression, epigenetics, D-loop, oxygen consumption rate, oxidative stress, MT-CYB, MT-CO2