Oral cytology assessment by flow cytometry of DNA adducts, aneuploidy, proliferation and apoptosis shows differences between smokers and non-smokers

Abstract

Oral cytology and morphometric staining is used to identify malignant keratinocytes in oral premalignant or malignant lesions. To detect and to begin to assess changes in oral keratinocytes exposed to tobacco-derived carcinogens, which are at risk for malignant transformation, a novel method is required. The approach uses oral cytology harvested oral keratinocytes analyzed using flow cytometry (FC) for changes in DNA content, damage, cell cycle and apoptosis. Six smoker and six non-smoker oral keratinocytes were evaluated using flow cytometry in the form of laser scanning cytometry (LSC) and laser microdissection (LMD). Among smokers compared to non-smokers, the method detected and assessed DNA damage from tobacco smoke exposure quantifying an enhanced formation of DNA adducts, such as, 8-hydroxy-2′-deoxyguanine (8-OHdG) which creates oxidation lesions and benzo[a]pyrene(B[a]P), which produces a B[a]P)-N2-dG bulky adduct. Increased DNA content, aneuploidy, percentage of cells in synthesis (S) and G2+Mitosis (M), and apoptosis were recorded. Tissue and cell controls were used to verify these relationships. Data suggested healthy smokers were at increased risk for malignant transformation of oral keratinocytes because of the changes stated above. Using identical methods, keratinocytes exposed to the tobacco derived carcinogen, B[a]P parallel results obtained from smoke exposure indicating a direct link. Flow cytometric evaluation of oral cytology harvested keratinocytes can be used to measure exposure to tobacco carcinogens, and possibly establish a link to premalignant and malignant transformation before a lesion is noted.