Comparative Genotoxicity and Mutagenicity of Cigarette, Cigarillo, and Shisha Tobacco Products in Epithelial and Cardiac Cells.

Epidemiology studies link cigarillos and shisha tobacco (delivered through a hookah waterpipe) to increased risk for cardiopulmonary diseases. Here we performed a comparative chemical constituent analysis between 3 cigarettes, 3 cigarillos, and 8 shisha tobacco products. The potency for genotoxicity and oxidative stress of each product's generated total particulate matter (TPM) was also assessed using immortalized oral, lung, and cardiac cell lines to represent target tissues. Levels of the carcinogenic carbonyl formaldehyde were 32- to 95-fold greater, while acrolein was similar across the shisha aerosols generated by charcoal heating compared to cigarettes and cigarillos. Electric-mediated aerosol generation dramatically increased acrolein to levels exceeding those in cigarettes and cigarillos by up to 43-fold. Equivalent cytotoxic-mediated cell death and dose response for genotoxicity through induction of mutagenicity and DNA strand breaks was seen between cigarettes and cigarillos, while minimal to no effect was observed with shisha tobacco products. In contrast, increased potency of TPM from cigarillos compared to cigarettes for inducing oxidative stress via reactive oxygen radicals and lipid peroxidation across cell lines was evident, while positivity was seen for shisha tobacco products albeit at much lower levels. Together, these studies provide new insight into the potential harmful effects of cigarillos for causing tobacco-associated diseases. The high level of carbonyls in shisha products, that in turn is impacted by the heating mechanism, reside largely in the gas phase which will distribute throughout the respiratory tract and systemic circulation to likely increase genotoxic stress.

Cytotoxicity and Genotoxicity of E-Cigarette Generated Aerosols Containing Diverse Flavoring Products and Nicotine in Oral Epithelial Cell Lines.

Electronic cigarettes are the most commonly used nicotine containing product among teenagers. The oral epithelium is the first site of exposure and our recent work revealed considerable diversity among e-liquids for composition and level of chemical constituents that impact nicotine deposition in a human oral-trachea cast and affect the formation of reactive carbonyls. Here, we evaluate the dose response for cytotoxicity and genotoxicity of e-cigarette-generated aerosols from 10 diverse flavored e-liquid products with and without nicotine compared with unflavored in 3 immortalized oral epithelial cell lines. Three e-liquids, Blue Pucker, Love Potion, and Jamestown caused ≥20% cell toxicity assessed by the neutral red uptake assay. Nine products induced significant levels of oxidative stress up to 2.4-fold quantified by the ROS-Glo assay in at least 1 cell line, with dose response seen for Love Potion with and without nicotine across all cell lines. Lipid peroxidation detected by the thiobarbituric acid reactive substances assay was less common among products; however, dose response increases up to 12-fold were seen for individual cell lines. Micronuclei formation indicative of genotoxicity was increased up to 5-fold for some products. Blue Pucker was the most genotoxic e-liquid, inducing micronuclei across all cell lines irrespective of nicotine status. A potency score derived from all assays identified Blue Pucker and Love Potion as the most hazardous e-liquids. These in vitro acute exposure studies provide new insight about the potential for some flavored vaping products to induce significant levels of oxidative stress and genotoxicity.

EMT and stem cell-like properties associated with miR-205 and miR-200 epigenetic silencing are early manifestations during carcinogen-induced transformation of human lung epithelial cells.

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression, but its potential role during premalignant development has not been studied. Here, we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent, irreversible, and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven, initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA), miR-200b, miR-200c, and miR-205, which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low), CD133, and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT, stem-like, and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells.

Rosiglitazone prevents the progression of preinvasive lung cancer in a murine model.

There is a critical need to identify efficacious chemopreventive agents for lung cancer that can be taken chronically with no side effects and whose mechanisms of action do not involve genotoxicity that could drive, rather than impede, cancer progression. We evaluated the ability of a chemopreventive cocktail that included selenium (antioxidant), rosiglitazone (peroxisome proliferator-activated receptor gamma agonist), sodium phenylbutyrate or valproic acid (histone deacetylase inhibitors) and hydralazine (cytosine-demethylating agent) to prevent the progression of lung cancer in A/J mice treated with NNK. Agents were administered alone or in various combinations. Effects of the chemopreventive agents were quantified based on the proportion of hyperplasias and adenomas within the mouse lung. Significant effects on tumor progression were seen in all treatment groups that included rosiglitazone as reflected by a 47-57% increase in number of hyperplasias and a 10-30% decrease in adenomas. Cell proliferation was also reduced in these treatment groups by approximately 40%. Interestingly, while treatment with rosiglitazone alone did not significantly affect lesion size, striking effects were seen in the combination therapy group that included sodium phenylbutyrate, with the volume of hyperplasias and adenomas decreasing by 40 and 77%, respectively. These studies demonstrate for the first time that chronic in vivo administration of rosiglitazone, used in the management of diabetes mellitus, can significantly block the progression of premalignant lung cancer in the A/J mouse model.