Reversible cigarette smoke extract-induced DNA damage in human lung fibroblasts.
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.
Kim
Huijung
H
Department of Internal Medicine, Seoul Adventist Hospital and WonKwang University Sanbon Medical Center, Korea.
Liu
Xiangde
X
Kobayashi
Tetsu
T
Conner
Heather
H
Kohyama
Tadashi
T
Wen
Fu-Qiang
FQ
Fang
Qiuhong
Q
Abe
Shinji
S
Bitterman
Peter
P
Rennard
Stephen I
SI
D000111:Acetylcysteine / Q000737:chemistry; D000818:Animals; D000975:Antioxidants / Q000737:chemistry; D017209:Apoptosis; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D008168:Lung / Q000166:cytology* / Q000187:drug effects*; D016877:Oxidants / Q000737:chemistry; D051381:Rats; D012907:Smoking / Q000009:adverse effects*; D013997:Time Factors
D000975:Antioxidants; D016877:Oxidants; D019328:Buthionine Sulfoximine; D003094:Collagen; D004247:DNA; D005978:Glutathione; D000111:Acetylcysteine