Dr. Sergie Grando

Project Title: Receptor-Mediated Mechanisms of Tobacco-Induced Lung Oncogenesis

University of California at Irvine , Irvine , CA

Grant Award: $485,000

Duration: February 2008 - March 2011

Abstract:

General hypotheses being tested: While en-route to/from its pleasure targets in the central nervous system, nicotine can directly act upon lung cells, substituting local hormone acetylcholine at the nicotinic class of acetylcholine receptors present in lung cells. The proposed studies will determine if specific drugs that can abolish an ability of cigarette smoke, nicotine and its carcinogenic derivatives termed "nitrosamines" to inhibit the oncogenic transformation cultured lung cells and lung tumor development in the stain A/J mice, an established animal model for studying tumor-producing effects of cigarette smoke and chemical carcinogens.

This project directly addresses an important medical problem: chemoprevention of lung cancers in current and former first- and secondhand tobacco smokers. A test cholinergic antagonist will be considered as a potentially useful chemopreventive agent if the treatment of cultured cells and A/J mice significantly reduces the oncogenic activity of nitrosamines in vitro and lung tumor incidence and/or multiplicity in vivo. Learning the pharmacology of nicotine toxicity will help design an efficient prevention program wherein at least some hazardous effects of smoking in quitters are counteracted by a drug, acting as a nicotine antidote. Given an expanding usage of nicotine replacement products, it will be important to determine if pharmacologic doses of nicotine exhibit any tumorigenic activity in A/J mice. Collectively, the results of the proposed studies will contribute to a better understanding of the biochemical pathways mediating tobacco carcinogenesis to diminish the risk of lung cancer development, and open a door for new approaches to clinical solutions to ameliorate oncogenic effects and intercede in disease pathways.

This is a multipurpose, comprehensive study addressing urgent problems of contemporary clinical medicine and environmental toxicology in order to improve understanding of the biochemical pathways of tobacco-related lung cancer mediated by nicotine and its derivatives. The conceptual framework, design, methods, and analyses of this proposal were developed based on the studies successfully completed by us in the past. Previously completed experiments identified potential problems, and suggested alternative approaches. The projected studies will provide crucial information for identifying the focus of future research toward elucidating the role of specific nicotinic receptor subtypes in tobacco-related carcinogenesis. This project employs and further develops the novel concept of receptor-mediated action of carcinogenic nitrosamines placing cellular nicotinic receptors in the center of the pathophysiologic loop. The obtained results will help elucidate the receptor-mediated mechanisms of the initiation and progression of smoking-induced lung cancers. Thus, funding of this proposal will lay a ground work for future research aimed at further elucidation of the genetic and epigenetic aspects of receptor-mediated effects of nicotine and nitrosamines on malignant transformation of respiratory cells, and tumor cell growth. Reaching the research goals will advance scientific knowledge about the role of pulmonary nicotinic receptors in lung cancer development, help develop a chemoprevention program for people exposed to environmental tobacco smoke at home or workplace, and allow preclinical testing of cholinergic drugs to prevent the development of second primary tumors often seen in patients treated for lung cancer.

Dr. Irfan Rahman

Project Title: Molecular Mechanisms of Cigarette Smoke-mediated Lung Inflammation in COPD

University of Rochester Medical Center , Rochester , NY

Grant Award: $435,000

Duration: February 2008 - March 2011

Abstract:

Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause of chronic morbidity and mortality in the United States . It is a disabling condition associated with progressive breathlessness. There is no specific treatment available to stop the progression of this disease. Smoking is the main etiological factor, however, the enigma is that only 15-20% of smokers develop the disease. This in turn reflects a lack of understanding of the specific cellular and biochemical pathways triggered in the lung by tobacco smoke. The reason why only a proportion of cigarette smokers are susceptible and develop COPD is unclear at present. Cigarette smoke-triggered inflammation plays a central role in the development of COPD by a mechanism mediated via oxidant-induced enhanced pro-inflammatory gene transcription. However, little is known about the biochemical and molecular mechanism whereby cigarette smoke-mediated oxidative stress triggers lung inflammation. Histone acetylation and deacetylation comprise a key regulator of the specificity and duration of gene transcription. Alteration in the overall balance between nuclear histone acetylation: deacetylation (chromatin remodeling), and/or in acetylation patterns at specific promoters can result in aberrant transcription of pro-inflammatory genes in the lungs. The hypothesis to be tested in this proposal is that cigarette smoke and its constituents alters histone acetylation and deacetylation by p38 MAPK, mitogen and stress kinase 1 (MSK1), NF-kappaB-inducing kinase (NIK) and NF-kappaB signaling pathways via ROS-dependent mechanism leading to enhanced pro-inflammatory gene transcription in macrophages and in mouse lungs exposed to cigarette smoke. Increased histone acetylation is the susceptible factor for abnormal inflammation in COPD. We propose to test this hypothesis in vitro in monocytes/macrophages and in vivo in mouse lungs exposed to cigarette smoke. We focus our work on macrophages because alveolar macrophages are known to be the main orchestrators of the inflammatory response seen in lungs of patients with COPD. To test this hypothesis three specific aims are proposed: Aim 1: To determine the molecular redox signaling mechanisms of histone acetylation and deacetylation in response to cigarette smoke and its toxic constitutents in vitro in macrophages and development of mouse model of cigarettes smoke exposure to study oxidative stress and inflammatory response in the lungs. Aim 2: To study the inhibition of reactive oxygen species release by the use of NADPH oxidase knock-out (KO) mice (p47phox-/- and gp91phox-/-), and NIK and MSK1 KO as well as chemical inhibition of p38 MAP, NIK, MSK1 and IkappaB kinase to attenuate cigarette smoke-induced histone acetylation and pro-inflammatory cytokine release in macrophages and in lungs of mice. Aim 3: To understand the molecular mechanisms of transcriptional signaling by studying NIK and MSK1 activation, p65/RelA-NF-kappaB acetylation and chromatin remodeling (histone acetylation, CBP and deacetylation-sirtuin) in resected lungs (specifically in tissue macrophages) obtained from smokers with and without COPD. These studies will permit identification of the underlying biochemical and molecular mechanisms whereby cigarette smoke and its toxic constituents (aldehydes/ROS) triggers cell signaling to cause abnormal lung inflammation. In this way, specific reactive components of cigarette smoke will be identified. The findings in resected human lungs from smokers with and without COPD will determine the role of histone acetylation: deacetylation, particularly sirtuin1 (an anti-inflammatory and anti-aging deacetylase) in the predisposition and susceptibility to accelerated decline in lung function or disease progression. This study will also establish an in vivo pre-clinical animal model of cigarette smoke exposure. Understanding the molecular mechanisms of lung inflammation in response to smoking and its toxicants could lead to targeted pharmacological interventions in the treatment of COPD.

Dr. Timothy O'Connor

Project Title: TSNAs, Smokeless Tobacco, DNA Damage, Mutations, and Cancer

Beckman Research Institute of the City of Hope , Duarte , CA

Grant Award: $385,000

Duration: January 2007 - July 2010

Abstract:

Smokeless tobacco (ST) is linked to oral cavity cancer and other cancers. Recently, tobacco marketing campaigns have pressed new ST products whose mutagenicities remain undefined. Cancer progressively develops passing from DNA damage, to unrepaired adducts, to mutations, and finally the accumulation of mutations in critical genes results in the development of malignancy. Tobacco-specific nitrosamines (TSNAs) including, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, nicotine nitrosamino ketone (NNK) and nitrosonornicotine (NNN), nitrosoanabasine (NAB) and nitrosoanatabine (NAT) are considered among the most mutagenic components of ST that can lead to cancer, yet data strongly support only NNK and NNN as carcinogenic. The levels of TSNAs in ST depend on the curing process and vary in different countries. However, to unravel the TSNA contribution to mutations from ST we first must understand how these compounds damage DNA at specific sites, how that damage is repaired, what mutations are formed, and the relation between those different factors. Therefore, we will examine those parameters for four TSNAs- NNK, NNN, NAB, and NAT in the same in vitro mammalian cell system. The Big Blue mouse embryonic fibroblast (BBMEF) system has 40-80 tandem copies of the lambda LIZ bacteriophage genome imbedded in genomic DNA. The cII transgene will be examined for DNA damage and repair at nucleotide resolution by the TSNAs in the first two specific aims using ligation-mediated PCR (LMPCR). In aim III, the BBMEF cells will be damaged using the TSNAs and allowed to replicate following damage. The lambda LIZ will be recovered and screened for mutations in bacteria. From the muation data, we will obtain the percentage of cells that have mutations following TSNA exposure (mutation frequencies), the types of mutations formed (mutation signatures), and finally the mutation map at individual nucleotide positions. Since tumors often manifest specific mutations based on nucleotide positions, this will establish a link between the three processes of DNA damage, repair, and mutagenesis that is a model for the initial steps of tumor formation. Moreover, these processes will be followed in the same cells. In aim IV, we will use the knowledge obtained from the first three aims to monitor DNA damage, repair, and mutation in a number of commercial ST products from Asia, Northern Europe, and North America . Extracting ST, we can measure the same parameters for ST extracts using the BBMEF model system as for the TSNAs. New commercial ST products from the United States will be compared to older ST products and those found internationally to determine differences in damage, mutagenicity, and signatures for mutations by ST products. From these investigations, we will evaluate the contribution of TSNAs to mutagenesis of ST products and assess differences in mutagenicity found for ST products. The direct link between mutagenicity and carcinogenicity means that the data obtained will have applicability in the estimation of the risk to human health. We anticipate that the mutagenicity of the individual TSNAs and the ST products will furnish researchers and the general public with a better understanding of the exposure risks of ST use.

Dr. Judith Zelikoff

Project Title: Influence of Maternal Atopy/Sensitization on Cigarette Smoke-induced Effects on Offspring Airway Reactivity and Immune Responsiveness

New York University School of Medicine, Tuxedo, NY

Grant Award: $94,382

Duration: June 2007 - December 2009

Abstract:

The proposed 1 year study will test the hypothesis that maternal atopy/allergen sensitization exacerbates the effects of prenatal cigarette smoke on airway reactivity and related immunologic responses in the 5- and 15-wk-old normal, non-sensitized offspring. Two Specific Aims are proposed to: (1) assess how (and the extent to which) maternal atopy/sensitization modulates cigarette smoke-induced airway reactivity (as measured by non-specific bronchoprovocation challenge) in normal, unsensitized offspring; and, (2) using a dedicated subset of offspring, ascertain whether (and the degree to which) biological parameters important in mediating airway reactivity (i.e., lavage cell profiles, serum and lavage T-helper [Th] type-1 and -2 cytokine levels, serum antibody [IgE] levels, and lung tissue-associated eosinophil and mast cell numbers) are altered in the offspring following smoke exposure of atopic, pregnant females (compared to those effects seen in offspring from non-atopic, smoking mothers).

Technical Approach: The proposed study extends our current investigation by determining whether (and to what extent) maternal atopy/sensitization influences cigarette smoke-induced effects on offspring airway reactivity and immune responsiveness. Despite the fact that a large percentage of pregnant women who smoke are also atopic and that both factors represent strong independent risk factors for asthma/atopic disease in the child, studies examining the contribution that both factors together play in childhood atopic disease are severely lacking. Such studies could help define the relationship between several independent risk factors important for bringing about childhood airway disease (i.e., asthma) and cigarette smoke-induced mechanisms of pulmonary toxicity. The proposed studies will utilize the same mouse strain, exposure parameters, cigarette smoke concentration and immunologic endpoints as those employed in our ongoing studies. For these investigations, mice sensitized with ovalbumin (OVA) will be subsequently mated and the pregnant mice exposed (via whole-body inhalation) from day 4 of gestation to parturition to either mainstream cigarette smoke (at the same PM and CO concentrations used in our current studies, i.e., 16 mg m 3 and 25 ppm, respectively) or filtered air and effects on juvenile (5-wk-old) and adult (15 wk-old) offspring airway reactivity (using a whole body plethysmograph) and immune-related parameters will be evaluated. Results from these particular studies will be compared to those previously obtained using non-sensitized (non-atopic), pregnant dams (see above data and IFSH Progress Report). The studies described herein will involve exposure of atopic/sensitized, pregnant dams. Half of the atopic dams will serve as the filtered air controls and the remainder will be exposed to intact cigarette smoke. The number of gravid females within each exposure group will be determined by the number of mice that actually become pregnant after mating. Considering that =80% of the mated females are expected to become pregnant (based upon current studies), 32 mice in each of the proposed sets of assays (16 per each exposure group) should provide 11-13 dams per exposure group. Two randomly-selected pups (one male and one female) from each air- and cigarette smoke-exposed dam will be used to assess airway responsiveness at 5- and 15-wk of age; another set of 32 dams will be used to supply male and female pups at the same age for each of the ex vivo assays (Table 1, below). These latter offspring will all receive similar treatments as those used to measure airway responsiveness except that they will not undergo the final bronchoprovocation challenge (i.e., those used during plethesmography). This is necessary since the bronchoprovocation challenges could, potentially, interfere with the specific biochemical/immunological endpoints being evaluated. We do not anticipate any changes in gestational parameters as a result of sensitization (litter size usually between 9 - 11 pups and incidence of pregnancy >80% following a 4 day mating cycle). However, a pilot study using non-exposed mice to assess the effects of OVA sensitization on these same gestational parameters will be performed prior to the actual experiments. If maternal sensitization is found to alter incidence of pregnancy of litter size, then the number of animals mated will be adjusted appropriately.

Taken together, these studies address a critical question regarding the health impact of maternal cigarette smoking on childhood health. Specifically, the proposed studies will examine whether maternal atopy acts to exert a more pronounced effect of prenatal cigarette smoke exposure on offspring airway reactivity and the immune response, thus, predisposing even "low-risk" infants/children to subsequent sensitization and atopic disease (i.e., asthma).