NEIL2 protects against oxidative DNA damage induced by sidestream smoke in human cells - PubMed (original) (raw)

NEIL2 protects against oxidative DNA damage induced by sidestream smoke in human cells

Altaf H Sarker et al. PLoS One. 2014.

Abstract

Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. NEIL2 level is low in lung cancer tissues.

Tissue NEIL2 level is low in about 50% and high in about 8% of lung cancer cases compared to normal lung tissue, as measured by immunohistochemistry.

Figure 2

Figure 2. SSS increased ROS production in hPF.

Cells were preloaded with MitoSOX™ Red to visualize ROS production. (A) Control cells imaged at 360 min did not have red fluorescence. (B) At 360 min, SS smoke treated cells had red fluorescence indicative of ROS production. (C) Bar graph showing increased intensity of fluorescence in treated cells, but not in control cells, over 600 min. In Figure 2C, data are the means of two experiments; the control and treated groups had 84 and 99 cells, respectively.

Figure 3

Figure 3. Increased oxidative DNA damage in various human cells.

Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with Fpg and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).

Figure 4

Figure 4. Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24_HPRT_ gene using LA-QPCR.

(A) left: NEIL2 knockdown from hPF was tested by qRT-PCR; right: Western blots showing shRNA-mediated knockdown of NEIL2 in HEK293 cells expressing Flag-NEIL2. (B) Cultured hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control followed by DNA isolation and digestion with Fpg and Nei as described in Materials and Methods. The DNA was used as template for HPRT large and short fragment amplification by LA-QPCR assay. The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL 2 knockdown were statistically significant (P<0.05); (C) HEK293 cells with NEIL2 knockdown without (left) or with (right) SSS treatment followed by HPRT large fragment amplification by LA-QPCR.

Figure 5

Figure 5. Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24h in the human POLB gene using LA-QPCR.

(A) Cultured primary hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control on the left, followed by processing of the DNA with Fpg and Nei. The DNA was used for Polβ (POLB) large and short fragment amplification by LA-QPCR. Quantification was with ImageQuant (Molecular Dynamics). The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL2 knockdown were statistically significant (P<0.05).

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