1. Human Lung Inflammation
It is widely recognized that chronic inflammation is associated with cancer. Moreover, studies of human beings and laboratory animals have shown that tobacco-associated inflammation is associated with lung cancer and non-neoplastic pulmonary diseases. The smoking and chewing of tobacco are associated with chronic inflammation and with both malignant and non-neoplastic diseases of the oral cavity.
Today, it is recognized that cigarette smoke is a complex aerosol consisting of more than 6,000 different compounds. An examination of the current literature, however, has failed to identify a correlation between chronic lung inflammation and any given chemical compound, or any given class of chemicals. In attempting to account for the mechanisms underlying the observed chronic inflammation, it has been theorized that the inflammation can be attributed to the total of diverse toxins, present as microparticulates (‘tar”) and gases in mainstream smoke. This postulate has not been proven, but is difficult to refute. Notwithstanding, it is notable that many people who are exposed daily to urban air pollution and occupation-associated microparticulates do not have chronic lung inflammation and do not develop lung cancer.
Our laboratory is testing the hypothesis that chronic inflammation of the lung and mouth may be attributed in part to the numerous and diverse microbes and microbial toxins that are present in smoking and smokeless tobacco products. Tobacco for smoking and smokeless tobacco products are cured. There are different types of tobaccos, and different curing processes. The curing conditions of high humidity, temperature, poor ventilation and darkness favor the proliferation of diverse bacteria and mold. In this context it is noteworthy that the tobacco in cigarettes has bacterial-derived endotoxins (e.g., lipopolysaccharides; LPS). LPS has been also shown to be present in tobacco smoke – both in mainstream and environmental smoke. LPS is a potent inducer of inflammation. It has been demonstrated that very small amounts of LPS (<50 μg) delivered into the lungs of healthy subjects induces pulmonary inflammation. Tobacco also contains mold, the mycotoxin aflatoxin, a known human Class I carcinogen has been identified in some tobacco samples. Tobacco is grown in more than 120 countries of the world. Many of these are developing countries with little or no regulatory authority. Popular US cigarettes are a blend of different types of imported tobacco.
Our study scheme is to measure the production of pro-inflammatory cytokines by human lung macrophages cluttered in the presence of particulates and eluates of cigarette tobacco and mainstream smoke. The assay cells are a relatively homogeneous population of macrophages that have been isolated from fresh surgically isolated human lung tissue that has been excised to remove a lung tumor. Production of the cytokines is measured with a high throughput microbead ligand-capture assay. The rationale for undertaking these studies is that it may provide insight as to the underlying cause of tobacco-associated chronic inflammation of the lung and oral cavity.
2. Buccal Cells
The port for cigarette smoke to enter the body is the mouth. Due to the relatively small surface area of the mouth, cells of the mouth are exposed to tobacco smoke carcinogens and toxins. The association of tobacco smoking with malignant and non-neoplastic diseases of the oral cavity is well known.
We have recently reported the results of a structured literature review that was undertaken to: (a) determine if human buccal (mouth) cell changes are associated with smoking and smokeless (“chewing”) tobacco; (b) tabulate different buccal cell alterations that have been reported; (c) delineate buccal cell assays that have been used successfully; (d) determine whether buccal cell changes correlate with oral cancer as defined in clinicopathologic investigations; and (e) assess the feasibility of developing a high throughput buccal cell assay for screening smokers for the early detection of oral cancer. The results of the studies established that diverse buccal cell changes are associated with smoking and smokeless tobacco. This review documents also that buccal cells have been collected in a non-invasive manner, and repetitively for serial studies, from different sites of the mouth (e.g., cheek, gum and tongue), and from normal tissue, pre-neoplastic lesions (leukoplakia) and malignant tumors. Tobacco-associated genetic mutations and non-genetic changes have been reported; a partial listing includes: (a) micronuclei; (b) bacterial adherence; (c) genetic mutations; (d) DNA polymorphism, (d) carcinogen-DNA adducts; and (e) chromosomal abnormalities. Clinical studies have correlated buccal cell changes with malignant tumors, and some oral oncologists have reported that the buccal cell changes are practical biomarkers. Summarily, the literature has established that buccal cells are useful not only for characterizing the molecular mechanisms underlying tobacco-associated oral cancers but also as exfoliative cells that express diverse changes that offer promise as candidate biomarkers for the early detection of oral cancer. It is envisioned, therefore, that cells of the mouth (buccal cells) will show progressive tobacco-induced changes, and that these alterations will precede and predict subsequent incipient (i.e., earlier than visible pathology) diseases of the lung. The hypotheses to be tested in the proposed investigation is that: (a) buccal cells will be collected in a non-invasive manner from different sites of the mouth (e.g., cheek, gum, tongue and palate) and isolated for diverse studies; (b) significant differences will be documented for the autofluorescence of buccal cells of smokers and non-smokers; (c) buccal cell autofluorescence will be measured quantitatively with a technically simple, expedient, reproducible, and high-throughput (> 20,000 cells/minute) test using a state-of-the art flow cytometer having three different lasers.
The specific aims are to: (1) establish the optimal procedures for collecting buccal cells; (2) define a standardized method to assay buccal cell fluorescence by FACS analysis with the intent of developing a test for future large-population studies; (3) compare the fluorescence of buccal cells of current smokers and never smokers; (4) analyze buccal cells of subjects following smoking cessation; (5) compare the autofluorescence of buccal cells harvested from lesions and normal sites of patients with leukoplakia; (6) characterize the fluorescence emission of buccal cells by spectral confocal laser scanning microscopy; and (7) define the utility of FACS analysis for measuring micronuclei based upon total cellular DNA content.
The rationale for undertaking the proposed studies is derived in part form the results obtained in pilot studies and widespread success achieved in using laser induced fluorescence emission (LIFE) bronchoscopy for detecting pre-cancerous lesions in the upper airways of long term-smokers.
The success of the proposed studies may provide a means for detecting cellular changes in subjects exposed to environmental tobacco smoke. It is envisioned and anticipated that the test will prove useful for screening a large population of smokers. High-risk individuals will be identified for monitoring; of these, some will be potential candidates for chemo-preventative treatment.