C. Gary Gairola

Cigarette Smoke, Inflammation, and Lung Injury: A Mechanistic Perspective

Inflammation is a common feature in the pathogenesis of cigarette smoke-associated diseases. The recruitment of inflammatory cells into the lung following cigarette smoke exposure presents a risk of tissue damage through the release of toxic mediators, including proteolytic enzymes and reactive oxygen species. This review represents a toxicological approach to investigation of cigarette smoke-induced lung injury, with a focus on laboratory studies and an emphasis on inflammatory mechanisms. The studies discussed in this review analyze the role of inflammation and inflammatory mediators in the development of injury. In cases where information relating to cigarette smoke is limited, examples are taken from other models of lung injury applicable to cigarette smoke. The primary aim of the review is to summarize published work so as to permit (1) an evaluation of chronic lung injury and inflammatory responses in animal models, (2) a discussion of inflammatory mediators in the development of chronic injury, and (3) identification of immunological mechanisms of injury. These studies discuss the currently understood roles of cytokines, cell adhesion molecules, and oxidative stress in inflammatory reactions and lung injury. A role for lipocortin 1 (annexin 1), a naturally occurring defense factor against inflammation, is discussed because of the possibility that impaired synthesis and degradation of lipocortin 1 will influence immune responses in animals exposed to cigarette smoke either by augmenting T helper cell Th1 response or by shifting Th1 to Th2 response. While Th1 augmentation will increase the risk for development of emphysema, Th1 to Th2 shift will favor development of asthma.

Sidestream cigarette smoke accelerates atherogenesis in apolipoprotein E−/− mice

Epidemiological studies have strongly implicated active and passive smoking with increased risk of cardiovascular diseases. The present study was performed to determine if exposure to sidestream cigarette smoke (SSCS), a surrogate of environmental tobacco smoke, promotes atherogenesis in a mouse model of human atherosclerosis. Female ApoE-deficient mice, maintained on a Western diet, were exposed to SSCS in a whole-body exposure chamber for a total of 6 h each day, 5 days a week for 7, 10 and 14 weeks. Animals exposed to filtered ambient air served as controls. Elevated concentrations of blood carboxyhemoglobin and pulmonary CYP1A1 ascertained effective exposure of animals to SSCS. There were no consistent changes in serum concentrations of cholesterol between control and SSCS-exposed mice. Morphometric assessment of grossly discernible lesions covering the intimal area of aorta showed remarkable increases in SSCS-exposed mice at all three exposure durations studied. Increases in the lesion area defined by en face measurements were accompanied by parallel increases in the levels of esterified and unesterified cholesterol in the aortic tissues of SSCS mice. These results clearly demonstrate promotion of atherosclerotic lesion development by tobacco smoke in an atherosclerosis-susceptible mouse model.

Genome based cell population heterogeneity promotes tumorigenicity: The evolutionary mechanism of cancer

Cancer progression represents an evolutionary process where overall genome level changes reflect system instability and serve as a driving force for evolving new systems. To illustrate this principle it must be demonstrated that karyotypic heterogeneity (population diversity) directly contributes to tumorigenicity. Five well characterized in vitro tumor progression models representing various types of cancers were selected for such an analysis. The tumorigenicity of each model has been linked to different molecular pathways, and there is no common molecular mechanism shared among them. According to our hypothesis that genome level heterogeneity is a key to cancer evolution, we expect to reveal that the common link of tumorigenicity between these diverse models is elevated genome diversity. Spectral karyotyping (SKY) was used to compare the degree of karyotypic heterogeneity displayed in various sublines of these five models. The cell population diversity was determined by scoring type and frequencies of clonal and non‐clonal chromosome aberrations (CCAs and NCCAs). The tumorigenicity of these models has been separately analyzed. As expected, the highest level of NCCAs was detected coupled with the strongest tumorigenicity among all models analyzed. The karyotypic heterogeneity of both benign hyperplastic lesions and premalignant dysplastic tissues were further analyzed to support this conclusion. This common link between elevated NCCAs and increased tumorigenicity suggests an evolutionary causative relationship between system instability, population diversity, and cancer evolution. This study reconciles the difference between evolutionary and molecular mechanisms of cancer and suggests that NCCAs can serve as a biomarker to monitor the probability of cancer progression. J. Cell. Physiol. 219: 288–300, 2009.

Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro.

In the present study, we showed that a single-dose treatment of normal breast epithelial cell line, MCF10A, for 72 h with cigarette smoke condensate (CSC) resulted in a transformed phenotype. The anchorage-dependent growth of these cells was decreased due to increased cell cycle arrest in S–G2/M phase; however, the surviving cells developed resistance due to an increased Bcl-xL to Bax ratio. Levels of PCNA and gadd45 proteins – involved in DNA repair in response to genomic damage – were increased, suggesting that the cells were responding to CSC-induced genomic damage. The transformation of MCF10A cells was determined by their colony-forming efficiency in soft-agar in an anchorage-independent manner. CSC-treated MCF10A cells efficiently formed colonies in soft-agar. We then re-established cell lines from the soft-agar colonies and further examined the persistence of their transforming characteristics. The re-established cell lines, when plated after 17 passages without CSC treatment, still formed colonies in the soft-agar. An increased staining of neuropilin-1 (NRP-1) further showed a transformation characteristic of MCF10A cells treated with CSC. In summary, our results suggest that CSC is capable of transforming the MCF10A cells in vitro, supporting the role of cigarette smoking and increased risk for breast cancer.

Acute pulmonary response of mice to multi-wall carbon nanotubes

Widespread use of carbon nanotubes is predicted for future and concerns have been raised about their potential health effects. The present study determined the pulmonary response of mice to multi-wall carbon nanotubes (MWCNTs). The MWCNT suspension in sterile phosphate-buffered saline (PBS) was introduced into mice lungs by oropharyngeal aspiration. Female C57Bl mice were treated with either 20 or 40 μg of MWCNTs in 40 μl PBS and control groups received equal volume of PBS. From each group, half of the mice were euthanized at day 1 and the remaining half at day 7 post treatment. Bronchoalveolar lavage (BAL) fluids, serum, and lung tissue samples were analyzed for inflammatory and oxidative stress markers. The results showed significant cellular influx by a single exposure to MWCNTs. Yields of total cells and the number of polymorphonuclear leukocytes in BAL cells were significantly elevated in MWCNT-treated mice post-treatment days 1 and 7. Analysis of cell-free BAL fluids showed significantly increased levels of total proteins, lactate dehydrogenase, tumor necrosis factor-α, interleukin-1β, mucin, and surfactant protein-D (SP-D) in MWCNT-treated mice at day 1 post treatment. However, these biomarkers returned to basal levels by day 7 post exposure except mucin and SP-D. An increase in the urinary level of 8-hydroxy-2’-deoxyguanosine in mice treated with MWCNT suggested systemic oxidative stress. Western analysis of lung tissue showed decreased levels of extracellular superoxide dismutase (SOD) protein in MWCNT-treated mice but copper/zinc and manganese SOD remained unchanged. It is concluded that a single treatment of MWCNT is capable of inducing cytotoxic and inflammatory response in the lungs of mice.

Acute pulmonary effects of combined exposure to carbon nanotubes and ozone in mice.

Ozone (O3) is a well-investigated gaseous air pollutant known to produce acute and chronic toxicity in the respiratory system. Whether prior exposure to nanoparticles influences the toxicity of O3 has not been well investigated. To determine if there are toxicological interactions between particulate and gas exposures, we examined acute pulmonary effects of a 3-h ozone exposure (0.5 ppm) in female C57Bl mice that had been preexposed to a single dose of 20 μ g multiwall carbon nanotubes (CNT) by pharyngeal aspiration 12 h earlier. A total of four groups were compared: (1) PBS/air-control, (2) PBS/O3, (3) CNT/air, and (4) CNT/O3. Analyses of the bronchoalveolar lavage fluid (BALF) and lung tissue samples collected at 5 and 24 h post O3 exposure were performed for various markers of cytotoxicity and inflammation using standard enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures. The results showed a pronounced cellular response and increase in various cytotoxicity/inflammatory markers in the lungs of CNT-exposed mice. Ozone by itself produced minimal effects, but in CNT-exposed animals there was a significant increase in total brochoalveolar lavage (BAL) cells and polymorphonuclear leukocytes. Additionally, protein, lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and mucin levels in BALF at 5 and 24 h were higher in CNT-exposed animals than in corresponding air-exposed controls or animals exposed to O3 alone. A comparable increase over the controls was also observed in the CNT/O3 group, but neither an additive nor a synergistic interaction was observed in mice that received sequential exposure to CNT and ozone. In fact, some CNT-induced cytotoxic/inflammatory responses were attenuated in mice following exposure to both CNT and low levels of ozone. These results are contrary to enhanced responses that were anticipated and may represent the development of “cross-tolerance” reported by others for some sequentially administered pollutants.

Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17β-estradiol (E2), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E2 was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E2 within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer. Cancer Prev Res; 3(6); 707–17. ©2010 AACR.

Protective role of manganese superoxide dismutase against cigarette smoke‐induced cytotoxicity

Free-radical-induced oxidative damage has been implicated as an important mechanism responsible for the toxicity of both active and passive smoking. Cigarette smoke contains short- and long-lived radicals and can stimulate cellular production of highly reactive oxygen species. One of the antioxidant enzymes that is protective against reactive oxygen-induced damage is manganese superoxide dismutase (MnSOD), which is located in the mitochondria of mammalian cells. The present study was conducted to examine the role of oxidative damage in cigarette smoke toxicity. A mouse fibroblast cell line (C3H10T1/2) and its MnSOD-transfected, enzymatically active clone, R2 cells, which possessed about five-fold greater MnSOD activity, were used to test the cytotoxicity of condensates from mainstream (MS-CSC) and sidestream (SS-CSC) cigarette smoke. Growth and respiration studies of the two test cell lines showed that the R2 cells grew to a higher cell density and exhibited greater oxygen uptake than the parent cells under normal growth conditions. Both smoke condensates were cytotoxic to test cells, but SS-CSC exhibited slightly greater toxicity, and R2 cells were significantly less susceptible to SS-CSC toxicity than the parent cells. SS-CSC caused a slightly greater inhibition of respiratory activity in parent cells than in R2 cells. These results suggest a significant contribution of oxidative damage in SS-CSC cytotoxicity.

Enhancement of pre-existing DNA adducts in rodents exposed to cigarette smoke

Exposure to tobacco smoke has been implicated in the increased incidence of cancer and cardiovascular diseases. This report describes various experimental studies in animals that were carried out to determine the ability of cigarette smoke to form DNA adducts and to define chromatographic nature of the major adducts. Tissues from rodents exposed to mainstream or sidestream cigarette smoke in nose-only and whole-body exposure systems, respectively, for different durations were analyzed for DNA adducts by 32P-postlabeling assay. The results showed essentially similar qualitative patterns in various respiratory (lung, trachea, larynx) and non-respiratory (heart, bladder) tissues of smoke-exposed rats. However, adduct pattern in the nasal mucosa was different. The mean total DNA adducts in various tissues expressed as per 1010 nucleotides exhibited the following order: heart (700)>lung (420)>trachea (170)>larynx (150)>bladder (50). Some qualitatively identical adducts were routinely detected in tissues from sham-treated rats but at greatly reduced levels (5- to 25-fold). The levels of lung DNA adducts increased with the duration of exposure up to 23 weeks and returned to control levels 19 weeks after the cessation of exposure. Species-related differences in adduct magnitude and patterns were observed among rats, mice and guinea pigs; mouse being the most sensitive to DNA damage and guinea pig the least sensitive. Whole-body exposure of rats to sidestream cigarette smoke also enhanced the pre-existing DNA adducts by several fold in different tissues. Selective chromatography, and extractability in butanol suggested lipophilic nature of smoke-associated DNA adducts, which were, however, recovered significantly better in nuclease P1 than butanol enrichment procedure. The major smoke-associated adducts were chromatographically different from any of the reference adducts of polycyclic aromatic hydrocarbons (PAHs) co-chromatographed with the smoke DNA samples. Because PAH-DNA adducts are recovered with equal efficiency by the two enrichment procedures, the above observations suggested that smoke-associated adducts are not related to typical PAHs, like benzo[a]pyrene. It is concluded that cigarette smoke increased the levels of pre-existing endogenous DNA adducts (the so-called I-compounds) in animal models and that these adducts are unrelated to those formed by typical PAHs.

Mitosis-arresting effects of cigarette smoke condensate on human lymphoid cell lines

Whole-smoke condensates from the University of Kentucky reference cigarettes induced partial mitotic arrest in 4 human lymphoid cell lines. Treatment of cells for 3 h with 100 and 200 micrograms of cigarette-smoke condensate/ml of culture medium increased the frequency of metaphases and decreased the proportion of anaphases in the treated cell populations. Cytoskeletal studies using antitubulin immunofluorescence techniques and transmission electron microscopic studies demonstrated that in early stages of mitosis the formation of aster and the separation of centrosomes in condensate-treated cells were comparable to those of untreated control cells, but the poleward migration of centrosomes was inhibited. Arrested metaphases revealed two centrosomes surrounded by aggregated chromosomes in the center of each cell but the structure of the centrioles, microtubules and the kinetochores appeared normal. The results demonstrate the presence of antimitotic compounds in the tobacco-smoke condensate.