Whole body exposure of mice to secondhand smoke induces dose-dependent and persistent promutagenic DNA adducts in the lung

Abstract

Secondhand smoke (SHS) exposure is a known risk factor for lung cancer in lifelong nonsmokers. However, the underlying mechanism of action of SHS in lung carcinogenesis remains elusive. We have investigated, using the (32)P-postlabeling assay, the genotoxic potential of SHS in vivo by determining the formation and kinetics of repair of DNA adducts in the lungs of mice exposed whole body to SHS for 2 or 4 months (5h/day, 5 days/week), and an ensuing one-month recovery period. We demonstrate that exposure of mice to SHS elicits a significant genotoxic response as reflected by the elevation of DNA adduct levels in the lungs of SHS-exposed animals. The increases in DNA adduct levels in the lungs of SHS-exposed mice are dose-dependent as they are related to the intensity and duration of SHS exposure. After one month of recovery in clean air, the levels of lung DNA adducts in the mice exposed for 4 months remain significantly higher than those in the mice exposed for 2 months (P<0.0005), levels in both groups being significantly elevated relative to controls (P<0.00001). Our experimental findings accord with the epidemiological data showing that exposure to smoke-derived carcinogens is a risk factor for lung cancer; not only does the magnitude of risk depend upon carcinogen dose, but it also becomes more irreversible with prolonged exposure. The confirmation of epidemiologic data by our experimental findings is of significance because it strengthens the case for the etiologic involvement of SHS in nonsmokers' lung cancer. Identifying the etiologic factors involved in the pathogenesis of lung cancer can help define future strategies for prevention, early detection, and treatment of this highly lethal malignancy.

Cell cycle alterations induced by urban PM2.5 in bronchial epithelial cells: characterization of the process and possible mechanisms involved

“…After 3 and 24 h exposure to PM organic extract and BaP (15 μM), cells were washed in PBS, scraped and stored at -80°C. DNA was isolated from cells by a standard phenol extraction method and DNA samples were analysed as described [78,79] with minor modifications. Briefly, DNA (4 μg) was digested with micrococcal nuclease (288 mU; Sigma, UK) and spleen phosphodiestase (1.2 mU; MP Biomedicals, UK), enriched and labelled as reported.…”

“…After chromatography, TLC sheets were scanned using a Packard Instant Imager (Dowers Grove, IL, USA) and DNA adduct levels (RAL, relative adduct labelling) were calculated from the adduct counts per minute (cpm), the specific activity of [γ- 32 P]ATP (Hartmann-Analytic, Germany) and the amount of DNA (pmol of DNA-P) used. As in prior studies, total DNA adduct levels were measured in the diagonal radioactive zone (DRZ) area of the TLC plates and were considered representative of PAH-DNA and other aromatic/hydrophobic adducts resistant to nuclease P1 digestion [78,79]. The method provides a summary measure of a complex mixture of adducts present in the postlabelling chromatograms.…”

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

“…Detailed information on the experimental treatment of Big Blue® mouse embryonic fibroblasts with UVB and the chronic exposure of Big Blue® mice to 4-ABP or SHS are provided in our previously published reports (11,13,20). Briefly, the UVB irradiation of Big Blue® mouse embryonic fibroblasts was performed in vitro at a single biologically relevant dose of 75.6 mJ/cm 2 , and under physiologic conditions (11).…”

“…In vivo , 4-ABP (Sigma-Aldrich Inc., Saint Louis, MO) was administered intraperitoneally to male adult Big Blue® mice on a weekly basis for a duration of 6 weeks at increasing doses of 25–100 mg/kg bw (13). The SHS treatment of male adult Big Blue® mice was performed in vivo in exposure chambers of a TE-10 smoking machine (Teague Enterprises, Davis, CA) for 5 hr/day, 5 days/week for a duration of 4 months (20). Subsequent to all experimental treatments, genomic DNA was isolated using a standard phenol extraction-based protocol (21).…”

Hepatic DNA damage in harbour porpoises (Phocoena phocoena) stranded along the English and Welsh coastlines

“…An external benzo[ a ]pyrene‐7,8‐dihydrodiol‐9,10‐epoxide‐(BPDE)‐modified DNA standard was used as a positive control. As in prior studies, total DNA adduct levels were measured in the diagonal radioactive zone (DRZ) area of the TLC plates and were considered representative of PAH‐DNA and other aromatic/hydrophobic adducts resistant to nuclease P 1 digestion [Tang et al ; Kim et al ; Long et al ]. The method provides a summary measure of a complex mixture of adducts present in the postlabeling chromatograms.…”