Measurement of oxidative DNA damage induced by mainstream cigarette smoke in cultured NCI-H292 human pulmonary carcinoma cells

Abstract

Cigarette smoke is a complex dynamic mixture of more than 4800 chemicals distributed between the particulate and vapour phases. It is widely acknowledged that cigarette smoke is capable of causing oxidative damage in DNA, either directly or through generation of reactive oxygen species. In this study, we have used a novel system for exposing cultured NCI-H292 human pulmonary carcinoma cells at the air–liquid interface, to investigate the potential effects of cigarette smoke on oxidative DNA damage by use of the modified comet assay with formamidopyrimidine N-glycosylase (FPG) and endonuclease III (Endo III) to reveal purine and pyrimidine lesions, respectively. The results demonstrate that NCI-H292 cells exposed to mainstream cigarette smoke from Kentucky reference-cigarettes show considerable DNA damage in terms of strand-breaks (SBs), alkali-labile sites (ALS) and oxidative DNA lesions. Initial measurements of FPG-sensitive oxidative lesions were confounded by high levels of SBs and ALS. Strand-breaks, but not oxidative lesions are repaired during a 20-h recovery period, which allows the semi-quantitative measurement of FPG-sensitive oxidative DNA lesions. We also conclude that mainstream cigarette smoke does not generate increased levels of Endo III-sensitive oxidative DNA lesions, or that they are not distinguishable from SBs and ALS in vitro in this system. Moreover, we demonstrate the viability and versatility of this exposure system in combination with in vitro techniques as an investigative tool for damage induced by cigarette smoke and smoke constituents.