Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation

Abstract

Second-hand smoke (SHS) is associated with 20-30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers.

Keywords: DNA damage and repair; acrolein and BPDE; anchorage independent growth; lung and bladder cancer; second-hand and side-stream smoke.

Figure 1. The acrolein-deoxyguanosine (Acr-dG) adduct is the major cyclic propano-DNA adduct formed in the lung tissues of mice exposed to side-stream smoke

Genomic DNAs were isolated from the lung tissues of mice exposed to filtered air (FA) and side-stream smoke (SSS) for 16 weeks. Lung genomic DNA from three randomly chosen mice from the same treatment group were pooled together as one set (a total of three sets) for PdG adduct analysis by the ³²P-post-labeling and 2D-TLC/HPLC method [20]. A. Left panels are typical 2D-TLC autoradiographs of FA and SSS samples. The spots with circles were eluted from the TLC plates and subjected to further analysis by HPLC. Right panels show typical HPLC elution profiles. The elution positions of the Acr-dG, HNE-dG, and Cro-dG adducts are indicated. PdG adducts formed in the lung genomic DNA were also analyzed by the immunochemical slot blot method, as described previously [18, 20]. B. A typical slot blot result. Left panel: fluorescent slot blot results. Right panel: input DNA stained by methylene blue. The genomic DNAs modified with different concentrations of Acr (Acr-0 mM to Acr-2 mM) were used as standards for quantitation. F-1 to F-10 represent genomic DNA isolated from lung tissues of mice exposed to filtered air for 16 weeks. S-1 to S-10 represent genomic DNA isolated from lung tissues of mice exposed to side-stream smoke for 16 weeks. The quantitative results of PdG formation in lung tissues are shown using both the 2D-TLC/HPLC method C. and the immunochemical method D. Bars represent the geometric median levels of PdG adducts. * represent P values of < 0.05.

Figure 2. Acr-dG adducts are induced in the BAL, lung, and bladder-mucosa but not in the heart and liver of mice exposed to side-stream smoke (SSS). In contrast, SSS does not affect the formation of BPDE-dG adducts in any of these tissues

Genomic DNAs isolated from BAL and the lung, heart, liver, and bladder mucosa from mice exposed to SSS [n = 10, except for BAL, lung, and bladder-mucosa from the 8-week exposure where n = 14] and filtered air (FA) [n = 10, except for BAL, lung, bladder mucosa from the 8-week exposure where n = 5] for 8 and 16 weeks were analyzed for the formation of cyclic PdG and BPDE-dG adducts by the immunochemical method described in Fig. 1 [18, 20]. Bars represent the geometric median levels of PdG A. and BPDE-dG B. adducts. * and ** represent P values of < 0.05 and < 0.01, respectively. Note: no statistical significance in PdG formation was found in 1) lung samples from mice with 8-week exposure of SSS and FA exposure (p = 0.9), 2) lung samples from mice exposed to FA for 8 weeks and 16 weeks (p = 0.26), and 3) bladder samples from mice exposed to FA for 8 weeks and 16 weeks (p = 0.12).

Figure 3. Side-stream smoke inhibits DNA repair

Cell lysates were isolated from lung tissues of mice (n = 6 for 8 weeks, n = 10 for 16 weeks) exposed to side-stream smoke (SSS) or filtered air (FA) for 8 (A & C) or 16 (B & C) weeks. A and B. The NER and BER capacities in these lysates were determined using the DNA-damage-dependent repair synthesis method as previously described [19, 20]. UV-irradiated and H₂O₂-modified DNA substrates were used to measure NER and BER capacity. Upper panel: input DNA stained using ethidium bromide. Lower panel: autoradiographic bands. The band intensity represents the level of DNA repair synthesis. Relative NER and BER capacities shown in C. were calculated based on comparison of the intensity of each band to the band with the highest intensity (arbitrary assigned with a value of 100). ** and *** represent P values of < 0.01 and < 0.001, respectively. Note: The NER and BER activity in the lung tissues of mice exposed to SSS were lower than the activity in the lung tissues of mice exposed to FA.

Figure 4. Side-stream smoke reduces the levels of XPC and hOGG1 repair proteins

XPC and hOGG1 in cell lysates isolated from lung tissues of mice exposed to side-stream smoke (SSS; n = 3 for 8 weeks, n = 5 for 16 weeks) or filtered air (FA; n = 3 for 8 weeks, n = 5 for 16 weeks) for 8 or 16 weeks were detected by the method previously described [18, 20]. The relative levels of XPC and hOGG1 normalized to α-tubulin are indicated. Note: The levels of XPC (a NER protein) and hOGG1 (a BER protein) in the lung tissues of mice exposed to SSS for 16 weeks are lower than in the lung tissues of mice exposed to FA (p = 0.0002 and p = 0.02, for XPC and hOGG1, respectively).

Figure 5. Acrolein induces tumorigenic transformation in human bronchial epithelial and urothelial cells

Immortalized human bronchial epithelial cells BEAS-2B and human urothelial cells UROtsa were treated with Acr (2.5 μM) for 1 h, 50,000 of treated cells and untreated were seeded (duplicated) in a 6-cm dish with soft-agar growth medium as described [56]. The ability to grow in soft-agar was assessed by the formation of number of colonies with more than 50 cells/colony [56]. A. Typical results of soft agar growth of Acr-treated BEAS-2B and UROtsa cells. B. Number of colony formed in the soft-agar medium in BEAS-2B and UROtsa cells with and without Acr treatment. * and ** represent P values of < 0.05 and < 0.01, respectively.