Scientists have identified another way smoking impacts lung cancer development.

A team from Johns Hopkins Kimmel Cancer Center have discovered preliminary evidence in laboratory-growth human airway cells that cigarette smoke triggers epigenetic changes in the cells that are consistent with the early stages of lung cancer.

“Our study suggests that epigenetic changes to cells treated with cigarette smoke sensitize airway cells to genetic mutations known to cause lung cancers,” Dr. Stephen Baylin, the Virginia and D.K. Ludwig Professor for Cancer Research and process of oncology at the Johns Hopkins Kimmel Cancer Center, said in a statement.

Epigenetic processes—which can build over time and make the airway cells increasingly sensitive to responding to mutations that initiate cancer—are switches that control a gene’s potentially heritable levels of protein production without involving changes to the underlying structure of a gene’s DNA.

For example, methylation—where cells add tiny methyl chemical groups to a beginning region of a gene’s DNA sequence, often silencing the gene’s activation—is considered an epigenetic change.

While scientists have known some of the genetic triggers that drive lung cancer growth—including mutations in the KRAS gene, which are present in one-third of patients with smoking-related lung cancer—the timing of the changes were unknown.

The researchers created the effect of tobacco smoke on cells by growing bronchial cells in the lab, while bathing them with a liquid form of cigarette smoke daily for 15 months.

The scientists found that after 10 days of exposure an overall increase in DNA damage response to the reactive oxygen species within the cells.

The cells also saw a two-to-four-fold increase in the amount of an enzyme called EZH2—which works to dampen the expression of genes—between 10 days and three months.

After six months, the amount of EZH2 and DNMT1—a protein that also increased after exposure early in the study—tapered off in the cells exposed to the smoke, while the impact of the two methylation-regulating enzymes was still seen at 10 to 15 months, when scientists found decreased expression of hundreds of genes.

To test the timing of the changes, the researchers inserted mutations into the KRAS gene in the DNA of cells exposed to the cigarette smoke condensate for six months, as well as those exposed for 15 months, and found that the inserted mutation transformed cells into cancer in only the 15-month cells, where methylation was fully established but not in the six-month exposed cells.