Dr. Howard Glauert

Project Title : Cigarette Smoke and Pancreatic Carcinogenesis

University of Kentucky , Lexington , KY

Grant Award: $325,000

Duration: March 2009 - March 2011

Abstract:

Pancreatic cancer is the 4th leading cause of death from cancer. It is estimated that about 37,680 people will develop pancreatic cancer and that 34,290 will die from it in 2008. The five-year survival rate for pancreatic cancer is only 5%, making it one of the deadliest forms of cancer. A number of risk factors have been established for pancreatic cancer, but the most established environmental risk factor is cigarette smoking. It is estimated that tobacco smoking increases the risk of pancreatic cancer by at least 2- to 3-fold. However, the molecular mechanisms by which cigarette smoke contributes to pancreatic cancer are largely unknown. Recently, a mouse model of experimental pancreatic carcinogenesis has been developed. This model closely resembles human pancreatic cancer. In this application, we propose to test the hypothesis that exposure to cigarette smoke accelerates pancreatic carcinogenesis in this mouse model. Mice will be exposed to cigarette smoke or ambient air (for controls) for 5 or 9 months and the development of pancreatic tumors and precancerous lesions in the pancreas will be determined. Mechanisms by which cigarette smoke may increase the development of cancer will also be studied. With these studies, we hope to establish an animal model of cigarette smoke-enhanced pancreatic cancer, which can then be used to examine the natural history of the disease under controlled conditions, as well as nutritional or other interventions that may protect against the development of pancreatic cancer.

Dr. Kelly BéruBé

Project Title: Toxicogenomics of mainstream tobacco smoke in the lung: an in vitro versus in vivo investigation

Cardiff University , Cardiff , Wales , United Kingdom

Grant Award: $92,815

Duration:

Abstract:

There is a causal association with smoking and development of tobacco-related diseases, but only as determined by epidemiological studies. Robust risk assessments are required that involve human, target organ-exposure to mainstream tobacco smoke (MSS) and genomic biomarkers for injury/disease pathways.

The EpiAirway human lung tissue model (ETM, MatTek Corp, USA ) is a validated alternative model for inhalation toxicology, at the in vitro level. Our previous work with this model used a holistic approach and acute-exposures to select tobacco smoke components (TSCs; nicotine, urethane, cadmium, formaldehyde) to establish: (1) key biological lung epithelial responses, (2) temporal variations in expression of gene functional families (metabolism, stress, cell cycle, apoptosis) and, (3) correlation of candidate proteins to understand the functional mechanisms of TSC-action. We have previously identified/validated ' intelligent biomarkers ' (IBs) of exposure and harm that were related to pro-inflammatory lung and heart diseases.

This project will focus on producing the robust data needed to identify truly functional intelligent biomarkers of exposure and harm to inhaled xenobiotics, in the human respiratory epithelia, using mainstream cigarette smoke. This work will be conducted at the in vitro level in conjunction with an in vivo murine model of cigarette smoke. Lung tissue from both air and cigarette smoke exposed animals will be used in a comparative toxicogenomics (micro-array technology) investigation to profile the human and mouse genes in control and smoke exposed-treated mice. Bioinformatics analysis will involve in vitro versus in vivo correlations, as well as communal functional pathways following exposure to smoke treatments. In addition, Q-PCR will be used to validate a set of candidate genes. Histological examination will be conducted to identify any phenotypical changes in the in vitro/in vivo lung tissues.

Dr. Edward Snell

Project Title: A structural understanding the molecular defense against Chronic Obstructive Pulmonary Disease

Hauptman-Woodward Medical Research Institute, Buffalo , NY

Grant Award: $75,000

Duration: February 2009 - August 2010

Abstract:

Over 90% of patients diagnosed with Chronic Obstructive Pulmonary Disease (COPD) have been chronic smokers, yet only 15-20% of smokers become limited by COPD. Extracellular superoxidedismutase (SOD3) is the body's defense against free radicals which form and attack the lung tissue of smokers. Studies show that a slight variation in the amino acid makeup of SOD3 may, through antioxidant or anti-inflammatory effects, confer a degree of resistance to the development of COPD.

We do not know what the structure of SOD3 looks like and therefore, how a variation in the amino acid makeup would change this structure conferring resistance to COPD. By determining the three-dimensional structure of native SOD3 and that of the variant, we can determine what this change does and from that, the mechanism behind the resistance to the development of COPD. Visualizing enzymes on the atomic scale cannot be accomplished by light microscopy - the atoms are smaller than the wavelength of the light. However we can use an established technique called X-ray crystallography. This uses a crystal of the protein and the diffraction property of the crystal to produce a pattern of spots caused by diffracted X-rays. Conceptually the arrangement of those spots relates to the arrangement of the atoms in the enzyme and the relative intensity relates to which atoms contribute to each spot. By using sophisticated mathematical methods we can compute the structure and determine the three dimensional layout of the enzyme.

Once we can visualize the enzyme on the atomic scale we can see how the structure is modified by the variation that confers resistance. We can examine that modification and develop a model of the mechanism which can then be verified. With an understanding of how natural resistance to COPD occurs we may be able to use this as a direction to develop pharmaceutical based treatment and even prevention.

Dr. Judith Zelikoff

Project Title: Prenatal Exposure to Cigarette Smoke Alters Offspring Immune Mechanisms Associated with Anti-tumor Activity and Increased Cancer Risk

New York University , Tuxedo, NY

Grant Award: $80,000

Duration: March 2009 - September 2010

Abstract:

The major goal of this study is to determine how, and to what extent, in utero exposure to intact cigarette smoke (CS) or a single major constituent, nicotine (NT) alters immune surveillance, resulting in an increased risk for offspring to develop cancer later in life. As epidemiological and toxicological studies have confirmed that prenatal exposure to intact CS (or NT alone) can alter neonatal immune parameters which, in some cases, last well into adulthood, it is hypothesized that: increased tumor susceptibility in mouse offspring exposed in utero to CS is due, at least in part, to CS-induced alterations in T-lymphocyte-mediated immune anti-tumor mechanisms (cytotoxic T-lymphocytes and T-regulatory cells, in particular); and, observed effects on the fetal immune system are due specifically to CS-associated NT .

Using 5-wk-old male offspring from timed-pregnant B 6 C 3 F 1 mice exposed (from gestational day [GD] 1 until parturition) to either filtered air or unfractionated CS (15 mg total suspended particulates [TSP]/m 3 levels; equivalent to smoking <1 pack/d; based upon observed results, follow-on studies will use 7 or 30 mg TSP/m 3 levels) by inhalation, or to NT (via osmotic mini-pump at a dose equivalent to that in the inhaled CS), and then subsequently challenged with cultured EL4 lymphoma cells, three Specific Aims are identified:

1. To ascertain if cytotoxic T-lymphocyte (CTL) activity is altered by prenatal exposure to CS and/or nicotine. At 1, 8, and 15 d post-tumor cell challenge, offspring will be sacrificed and effects on splenic CTL tumoricidal activity determined both from recovered whole splenocytes and isolated CD8 + (CTL) cells.
2. To assess major cellular/molecular mechanism(s) responsible for any exposure-induced alterations in CTL function during tumor development. To define particular mechanisms by which CTL activity may have been altered, immune parameters will be examined, including: thymic and splenic T-lymphocyte sub-populations, with particular emphasis on T-suppressing, T-regulatory (Treg) cells; CTL-associated cytotoxic factors critical for tumor cell killing; and, T-helper type 1 and 2 (T H 1/2) cell activities/cytokines that impact on CTL function.
3. To determine the extent to which exposure-induced effects on CTL/Treg activity are responsible for reduced host resistance against transplanted tumor cells in the prenatally exposed offspring . To link in utero CS-induced effects on offspring CTL activity with increased tumor susceptibility, mice prenatally-exposed to either air, CS, or NT/saline will be injected with anti-CD8 (CTL)/anti-CD25 + FoxP3 + (Treg) monoclonal antibody prior to injection of the tumor cells, and tumor incidence/growth rate then compared among and between groups.

This project will focus on Specific Aim two to determine the mechanisms that prenatal exposure to CS acts to alter CTL activity during tumor development so as to increase childhood risk of later life cancer.

Dr. Christopher Heppel

Project Title: TSNA-related DNA adducts in pancreatic tissue from nonsmokers, smokers and snuffers

Walther Straub Institute of Pharmacology and Toxicology, Munich , Germany

Grant Award: $75,000

Duration:

Abstract:

In the public health community, smokeless tobacco use as a complete substitute for cigarette smoking or as a method of cessation is gaining support, but remains hotly debated. Currently, cigarette smoking is the leading cause of death in the U.S. , accounting for approximately one in six deaths and 5.5 million years of potential life lost. In snuffers using "low-nitrosamine" smokeless tobacco such as Swedish snus, the relative risk for diseases including cancer is considered to be at least 90% less than for cigarette smokers. One possible exemption is pancreas cancer for which recent epidemiological studies indicated a twofold increased risk in Swedish snusers compared to nonsmokers. Overall, cancer of the pancreas is the thirteenth most common type of cancer worldwide, with a 5-year survival rate of 4%. Risk factors are increasing age, diabetes mellitus, high intake of red meat and smoking. Among the many carcinogens taken up by both smokers and snuffers tobacco-specific nitrosamines (TSNA) are the only compounds known to induce pancreas tumors in animal models. Although highly plausible it has not yet been proven that TSNA are the carcinogens responsible for pancreas tumors in smokers and snuffers. This may be possible by showing an Increased binding of TSNA to DNA in smokers and snuffers. This binding leading to so-called DNA adducts is a first essential step in tumor development. Using highly advanced analytical techniques we are now able to determine DNA adducts of TSNA in human tissues. First results were equivocal showing some dependence on smoking and snuffing in oral mucosa but not no difference according to smoking in esophagus and lung from sudden death victims. In the present investigation TSNA adducts will be determined in pancreatic tissue of nonsmokers, smokers and snuffers obtained at autopsy of sudden death victims. Results of this study will allow a better understanding of tobacco-related pancreas tumors. In case of a positive correlation of TSNA adducts with snuffing and smoking every effort should be undertaken to reduce the concentration of TSNA in tobacco products as far as possible.