Cigarette smoke-induced DNA damage and repair detected by the comet assay in HPV-transformed cervical cells

Abstract

Abstract. Human papillomavirus (HPV) is the causative factor in the development and progression of cervical cancers in >97% of the cases, although insufficient. Epidemiological studies suggest an elevated risk of cervical cancer for cigarette smokers; therefore, we examined cigarette smoke-induced DNA damage and repair in HPV16-transformed human ectocervical cells (ECT1/E6 E7). Cells were treated with cigarette smoke condensate (CSC) for 72 h to assess the formation of single-and double-strand DNA breaks, measured by alkaline and neutral single cell gel electrophoresis assays, respectively. The mean tail length of cells with single-strand breaks was increased by 1.8-, 2.7-and 3.7-fold (p<0.001) after treatment with 4, 8 and 12 μg/ml CSC, respectively. The tail length with double-strand breaks was also increased dose-dependently. These results were further supported by measurement of the mean tail moment: the increase in both single-and double-strand breaks were much more pronounced with increasing concentration of CSC, by up to 23.5-fold (p<0.0001 for both assays). To examine the DNA repair, cells were treated with CSC for 72 h, followed by CSC withdrawal and re-incubation of the cells with fresh medium for 24, 48, or 72 h. Both single-and double-strand DNA breaks were removed during the initial 24 h but no further removal of the damage was observed. Up to 80% of residual single-and double-strand DNA breaks (p<0.05) were found to persist at all CSC concentrations examined. Ellagic acid, a known antioxidant and free-radical scavenger, was found to significantly inhibit DNA breaks induced by CSC. Thus, free radicals may be a plausible source of CSCinduced DNA damage. These data show that CSC-mediated DNA strand breaks are highly persistent, and suggest that persistence of cigarette smoke-associated DNA damage in the presence of HPV infection may lead to increased mutations in cervical cells and ultimately higher cancer risk.

Chemopreventive Role of Apigenin against the Synergistic Carcinogenesis of Human Papillomavirus and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

“…In the present and our previous studies [21], we consistently observed that the level of total POB-DNA adducts in HPV-positive cells was significantly higher than that in HPV-negative cells, suggesting synergistic carcinogenesis of HPV and NNK. Moktar et al [45] reported that single-and double-strand DNA breaks induced by cigarette smoke condensate were dose-dependent and persistent in HPV-transformed human ectocervical cells, which gave support to our results. To prevent the synergistic DNA-damaging function of HPV and NNK, we tried to discover a natural product as a potential chemopreventive agent against the synergistic carcinogenesis of HPV and NNK.…”

High-Risk Human Papillomavirus and Tobacco Smoke Interactions in Epithelial Carcinogenesis

“…It has been demonstrated that HPV16 and 18 infected cells are capable of generating high levels of BaP metabolites, thus increasing adduct formation, DNA damage and probably, favoring the possibility of HPV integration into the host [138]. In accordance with this finding, HPV-transformed cervical cancer cells were highly susceptible to DNA damage promoted by CSE, detected by comet assay [139]. Interestingly, Wei et al found increased DNA mutations and double strand breaks in CIN I cells harboring episomal HPV after TS exposure [101].…”

Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques

“…Moktar et al, 2009 reported that cigarette smoke condensate-mediated DNA strand breaks are highly persistent, and suggest that persistence of cigarette smoke-associated DNA damage in the presence of HPV infection may lead to increased mutations in cervical cells and ultimately higher cancer risk [ 29 ], however, in our study the status of smoking was not ascertained.…”