Inhalation of cigarette smoke induces regions of altered DNA methylation (RAMs) in SENCAR mouse lung

The development of early biomarkers, both of exposure and effect, would substantially improve science-based risk assessment with regard to cigarette smoke (CS)-associated toxicity. Altered DNA methylation, an epigenetic mechanism, is linked to CS-induced lung tumorigenesis. We have taken an unbiased approach (i.e. genomic regions are not pre-selected) to assess early methylation changes within lung DNA from female SENCAR mice treated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), and then exposed to air alone, or CS for 4 or 8 weeks. Regions of altered DNA methylation (RAMs) were detected in mice treated with DMBA alone, or DMBA + 0.16, 0.32 or 0.48 mg wet total particulate matter per liter (WTPM/L) CS, using methylation-sensitive restriction digestion, arbitrarily primed PCR and capillary electrophoresis. Comparison of the RAMs that formed in different treatment groups revealed: (1) RAMs which “carried forward” across time (i.e. occurred at both 4 and 8 weeks) in a particular dose group, in addition to unique RAMs observed only at 8 weeks, and (2) RAMs which “carried forward” across dose (i.e. occurred in at least 2 dose groups at a particular time point), in addition to unique RAMs observed only in 1 dose group. Furthermore, a subset of RAMs was observed, at both 4 and 8 weeks, in DMBA-treated and DMBA + CS-exposed groups; the presence of unique RAMs in the latter suggest that combined DMBA + CS treatment more than just “magnifies” a subset of cell populations bearing the methylation changes induced by DMBA alone. Importantly, only minimal histopathological changes were observed in the lungs of CS-treated mice. This study is the first to demonstrate changes in lung DNA methylation at early times following exposure to CS, e.g., prior to overt histopathology. Thus, altered methylation might serve as a biomarker of CS exposure, and, in light of the fact that methylation changes are linked to CS-induced lung tumorigenesis, might also be useful as biomarkers of effect.