Lung Cancer Pathogenesis Associated With Wood Smoke Exposure

Javier Delgado, MSc; Luis M. Martinez, MD; Therasa T. Sanchez, RN; Alejandra Ramirez, MD; Cecilia Iturria, MD; Georgina Gonzalez-Avila, MD, PhD

Disclosures

CHEST. 2005;128(1):124-131. 

In This Article

Materials and Methods

Blood samples were collected in 5-mL lithium heparin-coated tubes from 62 patients with primary lung cancer between March 2003 and July 2004. The diagnosis was established histologically by the analysis of tumor samples obtained by bronchoscopy or percutaneous needle biopsy, and/or sputum cytology. Patients were staged prior to treatment according to Mountain.[15] Lung cancer patients were classified into the following two groups: (1) lung cancer associated with tobacco (LCT), consisting of 6 women and 17 men who were current smokers for > 10 years (mean smoking history, 23.65 ± 15.5 pack-years; range, 11 to 56 pack-years); and (2) lung cancer associated with wood smoke (LCW), consisting of 22 women and 2 men who were not tobacco smokers but had been exposed to domestic wood smoke for a mean duration of 44 ± 17.5 years (195.4 ± 92.8 h/yr). These patients used traditional "three-stone" stoves in their kitchens without a chimney. The LCW patients were screened for additional carcinogens associated with occupational exposure or passive tobacco smoke. Fifteen lung cancer patients were not included in these groups: 3 were passive smokers, 3 were smokers exposed to wood smoke, and in 9 cases, no association could be established. Clinical data are given in Table 1 .

Nine smoker patients with COPD were also examined. COPD diagnosis was confirmed by medical history and the results of spirometry. American Thoracic Society criteria were used: history of productive cough for 3 consecutive months each year for the past 2 years, with an FEV 1 = 80% of the predictive value, an FEV 1 /FVC ratio = 70%, and a reversibility in FEV 1 = 10% after inhalation of 400 =g salbutamol ( Table 2 ).[16] This group consisted of five women and four men who were current smokers for > 10 years (mean smoking history, 27.95 ± 18.2 pack-years; range, 10 to 70 pack-years) without exposure to wood smoke. Subjects with a history of asthma, atopy, or allergy were excluded from the study. None of the COPD patients had emphysema detected on a CT scan. Nine healthy nonsmoker volunteers, five women and four men with normal spirometry values, with no signs of infective respiratory disease during the past 3 weeks, no exposure to wood smoke, and no history of asthma, atopy, or allergy were used as control subjects.

Venous blood samples from cancer, COPD, and control subjects were centrifuged, and plasma protein content was measured by the bicinchoninic acid protein assay (Pierce Chemical Company; Rockford, IL).[17] Samples were then stored at -70°C until used. Informed consent was obtained from each patient, and the protocol was approved by the local Ethical and Research Committees.

Presence of p53, phospho-p53 and MDM2 isoforms was examined in cancer and control samples. Cancer samples from patients with an advanced stage of the disease (stages IIIB and IV), were used for this assay. Electrophoresis was carried out using 15 µg of protein per lane in 8% sodium dodecyl sulfate polyacrylamide gels under reducing conditions with 5% 2-mer-captoethanol boiled during 5 min. Proteins were then transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk in 100 mmol/L Tris-HCl buffer, pH 7.5, with 150 mmol/L NaCl and 0.1% Tween 20 (Tris buffered saline solution plus Tween 20) for 1 h. At this time, membranes were incubated with the following monoclonal antibodies: 5 µg/mL anti-p53 (clone Pab 240), 5 µg/mL antiphospho-p53 (serine 392) [both from Calbiochem-Novabiochem International; San Diego, CA], and 1 µg/mL MDM2 (SMP 14; Santa Cruz Biotechnology; Santa Cruz, CA). The MDM2 antibody recognizes all MDM2 isoforms and MDM2 complexes. Unbound antibodies were removed by washing with TTBS buffer, and primary antibodies were detected (VectaStain ABC kit; Vector Laboratories; Burlingame, CA). Bands identified in the Western blot assay were analyzed by densitometry (Kodak Digital Science ID Image Analysis Software; Eastman Kodak; Rochester, NY), which measures the surface and intensity of bands. Results were expressed as densitometry units (DU).

Densitometry results were analyzed using the Mann-Whitney Utest, and expressed as mean = SD; p ≤ 0.05 was considered significant.

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