Detection of in vivo DNA damage induced by very low doses of mainstream and sidestream smoke extracts using a novel assay

Background: Mainstream (MS) smoke, the main smoke inhaled by active smokers, and sidestream (SS) smoke, the main component of secondhand smoke, induce a wide range of DNA lesions. Owing to technical limitations, the in vivo levels of tobacco-induced DNA damage are unknown. Recently, the authors developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to quantify endogenous and induced DNA damage.

Purpose: To quantify the in vivo levels of DNA damage induced by MS and SS smoke extracts in human cells using PADDA and define the strand-specific patterns of DNA damage and repair following exposure to diverse doses of MS and SS smoke.

Methods: Human epithelial cells were exposed to escalating doses of hydrogen peroxide (H2O2), MS, or SS smoke. TP53 gene DNA damage was quantified using PADDA at various time points. DNA double-strand breaks were detected by immunofluorescence analysis of phosphorylated histone H2AX (γ-H2AX). Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Data were collected and analyzed by t-test in 2012-2014.

Results: A dose-dependent increase in DNA damage was detected in vivo with increasing doses of H2O2, MS, and SS smoke. Even 1 hour of exposure to very low doses of MS or SS smoke resulted in significant DNA damage (p<0.01). MS and SS smoke induced distinctive strand-specific patterns of DNA damage and DNA repair kinetics.

Conclusions: Very low concentrations of MS and SS smoke induce significant DNA damage in human cells. Application of PADDA to population studies has major potential to establish biomarkers of susceptibility to tobacco-induced diseases.