Cigarette
smoke contains thousands of chemicals, many of which may contribute to cytotoxicity and
carcinogenesis. Using assays detecting
DNA strand breaks (terminal
transferase dUTP nick end labeling [TUNEL]) and
DNA content (flow cytometry), we evaluated the genotoxic effect of cigarette
smoke extract (CSE) on human fetal lung fibroblasts (HFL-1) cultured in three-dimensional
collagen gels as well as in monolayer culture. When HFL-1 cells were exposed to CSE,
DNA strand breaks were detected in most, as determined by TUNEL. This effect was dependent on CSE concentration, duration of CSE exposure, and the density of HFL-1 cells cast into the
collagen gels.
Buthionine sulfoximine (BSO), an inhibitor of
glutathione synthesis, significantly increased DNA damage induced by 1% CSE, and
N-acetylcysteine, a
glutathione precursor, blocked 5% CSE from inducing DNA damage. After CSE exposure, most cells were TUNEL-positive, but
DNA quantification revealed no hypodiploid cells, indicating that apoptosis was not occurring during the CSE exposure. CSE-induced DNA damage was reversible, and cells proliferated when CSE was removed after 24 h exposure. These results demonstrate that cigarette
smoke can induce DNA damage in HFL-1 cells cultured in both three-dimensional
collagen gels and monolayer cultures, and that
oxidants likely play a role in this damage. Moreover, this DNA damage is reversible, with cells surviving and TUNEL positivity reversing when CSE is removed within 24 h.