Cigarette smoke induces nucleic-acid oxidation in lung fibroblasts

Abstract

Oxidative stress is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD), but the molecular pathway connecting oxidative damage to tissue destruction remains to be fully defined. We suggest that reactive oxygen species (ROS) oxidatively damage nucleic acids, and this effect requires multiple repair mechanisms, particularly base excision pathway components 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III homologue 1 (NTH1), and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), as well as the nucleic acid-binding protein, Y-box binding protein 1 (YB1). This study was therefore designed to define the levels of nucleic-acid oxidation and expression of genes involved in the repair of COPD and in corresponding models of this disease. We found significant oxidation of nucleic acids localized to alveolar lung fibroblasts, increased levels of OGG1 mRNA expression, and decreased concentrations of NTH1, SMUG1, and YB1 mRNA in lung samples from subjects with very severe COPD compared with little or no COPD. Mice exposed to cigarette smoke exhibited a time-dependent accumulation of nucleic-acid oxidation in alveolar fibroblasts, which was associated with an increase in OGG1 and YB1 mRNA concentrations. Similarly, human lung fibroblasts exposed to cigarette smoke extract exhibited ROS-dependent nucleic-acid oxidation. The short interfering RNA (siRNA)-dependent knockdown of OGG1 and YB1 expression increased nucleic-acid oxidation at the basal state and after exposure to cigarette smoke. Together, our results demonstrate ROS-dependent, cigarette smoke-induced nucleic-acid oxidation in alveolar fibroblasts, which may play a role in the pathogenesis of emphysema.

Figure 1.

Nucleic-acid oxidation and repair in chronic obstructive pulmonary disease (COPD). Immunohistochemistry for 8OHG/8OHdG was performed in lung samples obtained from (A) patients without COPD, (B) patients with stage 2 COPD, and (C) patients with stage 4 COPD. (D) Immunohistochemistry for vimentin was performed on serial sections in lung samples of stage 4 COPD. Positive staining appears in blue. Arrows indicate the colocalization of 8OHG/8OHdG and vimentin immunostaining in alveolar wall cells. Bars = 50 μm. (E) The mRNA expression of OGG1, NTH1, SMUG1, and YB1 relative to GAPDH was investigated in whole-lung total RNA samples from seven patients without COPD, six patients with stage 2–3 COPD, and six patients with stage 4 COPD. Differences between groups were determined using the Kruskal-Wallis test, and differences between two groups were analyzed using the Mann-Whitney U test. *P < 0.05, stage 4 COPD, compared with stages 2–3 and non-COPD.

Figure 2.

Nucleic-acid oxidation in alveolar wall cells in mice exposed to CS, with 8OHG/8OHdG immunostaining (blue) in C57BL/6 mice exposed to cigarette smoke (CS) for (A) 1 day, (B) 3 days, (C) 1 month, or (D) 6 months. (E) Control mice were exposed to filtered air for 6 months. Scale bars = 100 μm (A–E). (F) Percentage of 8OHG/8OHdG-positive alveolar cells at different time points of CS exposure. Results are from at least four mice per group. Mean ± SEM. *P < 0.05, 3-day and 1-month CS exposure compared with 1 day of CS exposure. **P < 0.01, 6-month CS exposure compared with 1 day, 3 days, and 1 month of CS exposure. Immunofluorescence for 8OHG/8OHdG is depicted in lung samples from mice exposed to filtered air (G, I) or CS (H, J) for 6 months. (K–N) Comparison between slides not pretreated (K) or pretreated with DNase (L), RNase (M), or both (N) before 8OHG/8OHdG immunostaining in mice exposed to CS for 6 months. Immunohistochemistry for 8OHG/8OHdG (O) and vimentin (P) is depicted in serial sections in lung samples from mice exposed to CS for 6 months. Arrows indicate colocalization of 8OHG/8OHdG and vimentin immunostaining in alveolar wall cells. Scale bars = 10 μm (K–P).

Figure 3.

The mRNA expression of OGG1 and YB1 in mice exposed to CS. The mRNA expression of (A) OGG1 and (B) YB1 were investigated in whole-lung total RNA samples from C57BL/6 mice exposed to CS for 3 days, 1 month, or 6 months, and compared with age-matched mice exposed to filtered air. Results are from at least four mice per group. Mean ± SEM. Mann-Whitney U test. *P < 0.05, 6-mo CS exposure compared with 3 days and 1 month of CS exposure.

Figure 4.

Nucleic-acid oxidation in human lung fibroblasts exposed to CSE in vitro. Immunohistochemistry for 8OHG/8OHdG is depicted in fibroblasts exposed to (A, B) media alone or (C, D) 20% CSE for 3 hours. Effects are depicted of pretreatments with (E, F) DNase, (G, H) RNase, and (I, J) RNase/DNase combined on 8OHG/8OHdG staining in fibroblasts exposed to 20% CSE for 3 hours. Positive staining appears in blue. Scale bar = 50 μm. Immunofluorescence is depicted for (K, M, O) 8OHG/8OHdG in fibroblasts exposed to (K, L) media alone or (M, N) 20% CSE for 3 hours or (O, P) 24 hours. Flow cytometry for 8OHG/8OHdG (Q) is depicted in fibroblasts exposed to media alone (gray line), or 20% CSE for 3 hours (black line) or 24 h (solid gray). All experiments were performed in triplicate in at least three independent experiments.

Figure 5.

Effects of antioxidants on CSE-induced nucleic-acid oxidation and apoptosis in human fibroblasts. Fibroblasts were treated with (A) media alone or with (B) 20% CSE for 24 hours in the absence or (C) presence of NAC or (D) DFX. Flow cytometry for 8OHG/8OHdG (E, F) was performed on fibroblasts treated with 20% CSE for 24 hours alone (solid gray) or concurrent with antioxidants NAC (black line) (F, H) or DFX (black line) (G, I). All experiments were performed in three independent experiments in triplicate.

Figure 6.

Effects of down-regulation of OGG1 and YB1 on CSE-induced nucleic-acid oxidation in human fibroblasts. Flow cytometry for 8OHG/8OHdG (A–D) is depicted in fibroblasts transfected with siOGG1 (black line) (A, B), with siYB1 (black line) (C, D), or with a scrambled siRNA (Scr) as negative control sample (solid gray). Fibroblasts were exposed to (A, C) media alone or (B, D) 20% CSE for 24 hours. (E) Fold-change in 8OHG/8OHdG mean fluorescence intensity (MFI) in fibroblasts transfected with Scr siRNA (gray bars), siOGG1 (open bars), or siYB1 (solid bars) was determined after exposure to media alone or to 20% CSE for 24 hours. All experiments were performed in three independent experiments in triplicate. Mean ± SEM. Mann-Whitney U test. *P < 0.05, siOGG1 and siYB1 compared with Scr. ^#P < 0.05, media alone compared with CSE.