Pulmonary Toxicology and Epidemiology
Chemoprevention of Tobacco-Smoke Induced Lung Cancer (Hanspeter Witschi).
We completed two more studies on possible chemopreventive agents in the strain A mouse lung model system of tobacco smoke-induced lung cancer. In collaboration with the laboratory of Dr. Carroll E. Cross, UCDMC, we measured the concentrations of the putative chemopreventive agent beta carotene in plasma and lung tissues of mice fed various concentrations of the antioxidant. This was necessary because mice are notoriously poor absorbers of beta carotene. Beta carotene, at one time, seemed to be an ideal chemopreventive agent, particularly against lung cancer. Beta carotene is one of many carotenoids, pigments that occur in abundance in nature and are responsible for the colors of many fruits such as citrus fruits, tomatoes, and paprika. In other plants and vegetables that are rich in carotenoids, their color is often masked by chlorophyll. Some 30 years ago, it was found, in several epidemiological studies, that negative associations existed between estimated intakes of vitamin A or of beta carotene (provitamin A) and the risk of developing cancer at various sites. Epidemiological studies seemed to provide compelling reasons for the use of beta carotene as a chemopreventive agent. A large number of laboratory experiments, practically all of them carried out with cell and organ cultures, seemed to provide a sound mechanistic basis for the anticipated effect. Animal experiments, particularly colon, liver and skin tumor models, were suggestive for a beneficial effect. Large scale clinical trials with beta carotene were conducted. Their aim was to examine whether dietary beta carotene supplements would reduce the risk of lung cancer in smokers. The results were not as expected. Rather than protecting smokers against lung cancer, it was found that beta carotene, in active smokers, increased the risk. The effect was so obvious that one of the clinical trials had to be halted before its scheduled end. In our experiment, we fed mice concentrations of beta carotene ranging from 50 to 5000 ppm of beta carotene either during smoke exposure or after exposure to tobacco smoke. We did not see an effect. We also failed to see a protective or enhancing effect on lung tumor development when the mice were exposed for a full 9 months to tobacco smoke and fed a concentration of 0.5% beta carotene throughout. The antioxidant did not afford protection against tumor development, but also did not enhance tumor development, as was seen in the human clinical trials. In our most recent experiments we now have obtained evidence that beta carotene did indeed accumulate in measurable amounts in the plasma and tissues of the mice exposed to tobacco smoke. We now can positively conclude that beta carotene does not modify tumor development in mice exposed to tobacco smoke.
In another experiment we fed mice the Bowman-Birk protease inhibitor as a putative chemopreventive agent. We had shown earlier that intraperitoneal injections of this chemopreventive agent protected mice against the carcinogenic activity of either urethane or 3-methylcholanthrene. In the present experiment, mice were fed a concentrate of the protease inhibitor in the diet at a concentration of 1% while being exposed to tobacco smoke. Again, we failed to see an effect. These experiments strongly suggest that putative chemopreventive agents be evaluated in an animal model before costly and lengthy clinical trials are undertaken.
Finally we examined whether expression of cyclin D1/2 could serve as a marker of chemopreventive action. It has been speculated by others that agents that interfere with the expression of cyclin D, a cell cycle checkpoint protein, might have chemopreventive action. In our experiments we did find that agents that are effective in protecting animals against the carcinogenic action of tobacco smoke (dexamethasone and myoinositol) do, indeed, produce a substantial reduction of the expression of cyclin D1/2 in lung nuclear extracts. On the other hand, agents that are ineffective, such as phenethyl isothiocyanate, aspirin, d-limonene, Bowman Birk protease inhibitor or N-acetylcysteine, do not affect cyclin expression. It should therefore be possible to use this particular marker as a bookmarker of effect for chemopreventive agents.
Influence of Exposure to Environmental Tobacco Smoke on Perinatal Lung Development (Kent Pinkerton, Ann Bonham, Alan Buckpitt, Jesse Joad, Alice Tarantal).
Childhood exposure to a variety of indoor air contaminants including environmental tobacco smoke (ETS) produces significant risks in asthma, airway hyper responsiveness, and other respiratory symptoms such as cough, wheeze, and mucus production. Epidemiological studies suggest that exposure to ETS during the perinatal period may have adverse effects on lung function which can persist into adulthood. It has been estimated in the United States alone 200,000 to 1,000,000 children with asthma will have their condition worsened by exposure to ETS (USEPA, 1992). However, the mechanisms leading to this process are unknown. During the past few years, we have established a state-of-the-art inhalation system to study the effects of exposure to ETS on perinatal lung development in a nonhuman primate, the Rhesus macaque monkey. Exposure to ETS has been done using aged and diluted sidestream cigarette smoke as a surrogate for ETS. Two chambers located at the California Regional Primate Research Center Inhalation Facility have been expressly designed and configured to create conditions for passive smoke exposure to monkeys during pregnancy and early postnatal development under carefully controlled conditions. These studies have demonstrated significant alterations in lung development following exposure to ETS. These effects include changes in immune effector and inflammatory cells in the lung air spaces, alterations in pulmonary and peripheral blood cytokines and neurotrophins, alterations in the innervation and epithelial composition of the trachea, and changes in the activity and distribution of pulmonary cytochrome P450 monooxygenases and glutathione-S-transferases. All these changes are evident by 2.5 months of age in infant Rhesus monkeys. These findings confirm that ETS exposure during perinatal development significantly affects the lungs of non-human primate infants. We hypothesize that these changes represent the initial steps in the genesis of an asthmatic-like condition solely due to perinatal exposure to ETS. We also hypothesize that critical windows of exposure are present during the perinatal period that will exacerbate this effect.
Mechanisms and Treatment of Tobacco Smoke-Induced Lung Lesions in Hypertensive Rats (Kent Pinkerton, Kevin Smith, Urmila Kodavanti, Ling Yi Chang, and James Crapo).
Changes in the lungs of cigarette smokers include inflammation, epithelial damage, and subsequent remodeling of the airways. It has been postulated that persistent inflammation of the airways may be a driving force to cause the epithelium to undergo changes leading to the loss of ciliated cells, hypersecretion of mucin and squamous cell metaplasia. Epithelial remodeling is likely to play a critical role in the development and progression of tobacco smoke-induced airway disease driven by persistent inflammation. However, the mechanisms leading to normal or defective repair of the airway epithelium and the role inflammation plays are as yet undetermined. Studies from our laboratory have demonstrated exposure of spontaneously hypertensive (SH) rats to tobacco smoke for 8 weeks is associated with persistent airway inflammation, mucous cell hyperplasia and squamous cell metaplasia. Associated with these changes is an alteration in the pulmonary vasculature seen as smooth muscle hypertrophy. These results suggest SH rats exposed to tobacco smoke may represent a novel model of tobacco smoke-induced lung inflammation and disease. In this project we will further develop the use of the SH rat as a potential model of smoke-induced inflammation.
Pulmonary pathobiology & Physiology
Health Effects of Airborne Particles and Gases (Kent Pinkerton, Ann Aust, and John Veranth).
Our goal in this project is to examine the mechanisms of particulate toxicity in the lungs of neonatal rats following short (3-day) and long-term (28-day) exposure to iron/soot or coal flyash particles in the presence or absence of ozone. We have examined how particles directly impact on epithelial cells of the neonatal airways, centriacinar regions, and alveoli. Since epithelial cells are the first cells in the respiratory tract to come into contact with inhaled particles, we hypothesize that damage to these cells can serve as a direct and highly sensitive measure of particle toxicity. We hypothesize that epithelial cells lining the transitional zone between the airways and gas exchange regions of the lungs (i.e., the central acinus) are particularly sensitive and play a key role in the initiation and progression of particle-induced pulmonary injury. We further hypothesize that epithelial-particle interactions initiate a cascade of events that underlie the adverse effects associated with inhaled particles. We hypothesize that particle toxicity (in the presence of a transition metal - iron) begins with the depletion of cellular glutathione levels in epithelial cells, thus accentuating cytotoxic events leading to cell death.
We have tested each of these hypotheses using novel approaches to examine epithelial cell structure and function throughout the airways and alveoli. State-of-the-art techniques to measure epithelial cytotoxicity, proliferation, anti-oxidant capacity and epithelial particle clearance have been used in whole animal inhalation studies as well as in a human epithelial cell culture system. The sensitivity of epithelial cells to particulate matter and/or ozone are currently being examined by airway location and generation within the tracheobronchial tree, central acinus and alveoli of young neonatal rats during critical windows of lung development. We assume patterns of particle deposition are pivotal to the degree of injury measured in the lungs. A systematic approach has been taken to expose newborn rats (6 hours/ day) to a precise concentration of experimentally generated iron/soot particles that could be present in ambient urban air. Exposure to particles and ozone will be short (3 day) and long-term (28 day) at a concentration that is environmentally relevant (total iron/soot concentration not to exceed 250 mg/m3).
Health Effects of Concentrated Ambient Particles from the Central Valley of California (Research C) (Kent Pinkerton, Kevin Smith, and Costas Sioutas).
We have examined plausible mechanisms of particulate toxicity in the lungs of rats following short-term (3-day) exposure to concentrated ambient particles of the Fresno area during the fall and winter months when particle size and composition in this region of California have been dramatically different. We wish to examine the effects these particles exert on epithelial cells of the airways, centriacinar regions, and alveoli. State-of-the-art techniques to measure epithelial cytoxicity, proliferation, and DNA damage are currently being used. The sensitivity of epithelial cells to particulate matter is being determined by airway location and generation within the tracheobronchial tree of the rat. These studies utilize a new generation of recently developed portable Particle Concentrators. These technologies, known as the California Particle Concentrators (designed by Dr. Costas Sioutas, University of Southern California, School of Civil and Environmental Engineering), maintain concentrated particles in an airborne state and supply them to exposure chambers for laboratory animal inhalation studies.
Particle Toxicity and the Respiratory Bronchiole: Species Sensitivity (Kent Pinkerton, Dallas Hyde, and Charles Plopper).
The overall objective of this project is to examine the mechanisms of particle toxicity in the lungs of rats and monkeys following short-term, repeated (3-day) exposure to particles that directly impact on epithelial cells of the airways, respiratory bronchioles, and alveoli. We hypothesize that epithelial cells lining the transitional zone between the airways and gas exchange regions of the lungs (i.e. the respiratory bronchiole) are particularly sensitive and play a key role in the initiation and progression of particle-induced pulmonary injury. Epithelial-particle interactions are assumed to initiate a cascade of events that underlie the adverse effects associated with inhaled particles.
This project is designed to determine if exposure to ammonium nitrate (AN) and carbon (C), two common components found in California PM, will affect the respiratory tract of healthy adult rats. Sprague Dawley rats were exposed to filtered air (FA), PM (150 µg/m3 AN and 200 µg/m3 C), ozone (0.2 ppm) or PM plus ozone for 6 h/day for 3 days. Epithelial cell permeability and reduced gluthathione levels (GSH) were used as biomarkers of effects along the airways and lung parenchyma. Bronchoalveolar lavage (BAL) was also used to determine lung cellular responses following exposure. Cellular proliferation measured by bromodeoxyuridine (BrdU) uptake was examined within site-specific regions of the airways and lung parenchyma. Following particle exposure, epithelial cell permeability was markedly increased at airway bifurcations. GSH levels, although different for each anatomical site examined, were not significantly changed by exposure to PM and/or ozone compared with controls. BAL following exposure to PM demonstrated a 73% increase in number above control value. BrdU labeled epithelial cells were significantly increased two-fold above control values at airway bifurcations following exposure to PM and PM plus ozone compared with FA controls, but were unchanged along the airways. Interstitial cell labeling at airway bifurcations was also significantly increased following exposure to PM plus ozone. Significant increases in BrdU labeling within central acinar regions of the lungs were also noted for all treatments groups compared with FA controls. These findings suggest (1) AN and C cause injury to the lungs, (2) these effects are independent of ozone, and (3) airway bifurcations and central acini are important sites of injury to inhaled particulate matter.
Exposure to ammonium nitrate and carbon for 6 hours/day for 3 days was repeated using Rhesus monkeys. Exposure conditions were similar between monkeys and rats. Cellular proliferation measured by bromodeoxyuridine (BrdU) uptake was examined in the respiratory bronchioles of each monkey and found to be significantly increased following PM exposure (2.4 ± 0.4% in controls and 4.1 ± 1.2% in animals exposed to PM). These findings further substantiate a significant particle effect within critical site-specific regions of the lungs, across two different species, the rat and the monkey.
Interactive Effects of Environmental Air Pollutants on Lung Injury (Kent Pinkerton and Hanspeter Witschi).
To determine the effects of aged and diluted sidestream cigarette smoke (ADSS) as a surrogate of environmental tobacco smoke (ETS) on ozone-induced lung injury, male B6C3F1 mice were exposed to (1) filtered air (FA), (2) ADSS, (3) ozone, or (4) ADSS followed by ozone (ADSS/ozone). Exposure to ADSS at 30 mg/m3 of total suspended particulates (TSP) for 6 hours/day for 3 days followed by exposure to ozone at 0.5 ppm for 24 hours was associated with a significant increase in the number of cells recovered by bronchoalveolar lavage (BAL) compared with exposure to ADSS alone or ozone alone. The proportion of neutrophils and lymphocytes as well as total protein level in BAL were also significantly elevated following ADSS/ozone exposure compared with all other groups. Within the centriacinar regions of the lungs, the percentage of proliferating cells identified by bromodeoxyuridine (BrdU) labeling was unchanged from control following exposure to ADSS alone, but was significantly elevated following exposure to ozone (280% of control) and further augmented in a statistically significant manner in mice exposed to ADSS/ozone (402% of control). Following exposure to ozone or ADSS/ozone, the ability of alveolar macrophages (AMs) to release interleukin (IL)-6 under lipopolysaccharide (LPS) stimulation was significantly decreased, while exposure to ADSS or ADSS/ozone caused a significantly increased release of tumor necrosis factor (TNF)-a from AMs under LPS stimulation. We conclude that ADSS exposure enhances the sensitivity of animals to ozone-induced lung injury.
Pulmonary Toxicology & Epidemiology
Mechanisms of Particulate Toxicity: Effects on the Respiratory System of Allergic Rats and Asthmatic Humans (Kent Pinkerton, John Balmes, Alan Buckpitt, Dallas Hyde, Lisa Miller, Charles Plopper, Ed Schelegle, and Colin Soloman).
Numerous epidemiological studies have presented mounting evidence of adverse health effects associated with exposure to ambient airborne particles. These health effects appear to be greatest among susceptible populations of individuals including children and those with pre-existing cardiopulmonary disease. The mechanisms by which these adverse health effects occur with exposure to particulate matter are not clear. Asthmatic individuals could be more susceptible to airborne particulate matter due to underlying changes in the respiratory system associated with this disease process.
The objectives of the project are to examine the functional and cellular responses of the respiratory system in asthmatic human volunteers and airway-sensitized rats to controlled short-term particulate exposure. In rodents we have examined airway function as well as epithelial and interstitial cell responses along the conducting airways following exposure to carbon and ammonium nitrate. Corollary studies performed in tissues obtained from the lungs of human volunteers exposed to these identical particles will allow us to better understand the relationship of particle exposure, airway inflammation, and cellular function with individuals who have asthma. The two-pronged approach of examining both animals with sensitized airways as well as human volunteers with known asthmatic conditions will provide us with a very powerful means of better understanding the potential mechanism of airborne particles on the respiratory system. Such information should provide critical data for better evaluating airborne particulate matter and in regulating these air quality issues for the benefit of all individuals.
This proposal involves investigators from UC Davis, UC San Francisco and UC Irvine. This collaborative research effort is coordinated by the California Air Resources Board. The collaborative nature of this effort effectively utilizes the specialized expertise offered by investigators in each of the three groups. This effort has been designed to systematically investigate the mechanisms of particulate toxicity associated with exposure to particulate matter found in ambient California air. Exposure conditions, tissue sample collection, biological assays, and data analyses will be coordinated and shared within the three groups.
Early Childhood Effects of Air Pollution (Irva Hertz-Picciotto, Miroslav Dostal, and Radim Sram).
This study is examining the effects of air pollution on early development and on the health of infants and young children. The study sample consists of a birth cohort from the Czech Republic representing deliveries in two districts from 1994-1999. One of the districts, Teplice, is a coal mining and manufacturing district that historically supplied energy to much of Czechoslovakia; its air pollution is known to be among the highest in Europe. The other district, Prachatice, is characterized by much lower levels of air pollution. The study takes advantage of an extensive research infrastructure within the Czech Republic, which includes ongoing air monitoring, biomarker and health studies. This infrastructure was developed for the Teplice Program, and has been in place for more than six years under the direction of scientists at the Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, located in Prague, Czech Republic. The study has the following objectives: a) to conduct data analysis on quantitative measures of air pollutants (PM10, PM2.5, PAH's, NOx, SO2) in relation to immune parameters at birth, including lymphocyte distributions and immunoglobulin concentrations in both maternal and cord blood; b) to collect data at 4.5 years of age from the pediatricians' records and from the families for ~600 births that occurred Oct 1996 through Dec 1999 (these data have already been collected for births in 1994 - Sept 1996); c) to continue the air monitoring in Prachatice, the district with low pollution, through Dec 2002, so as to provide measures of post-natal exposure to the age of three years for children born 1997-1999; d) to analyze quantitative data on PM10, PM2.5, PAH's, NOx, and SO2 in relation to child growth and morbidity to the age of three years, taking both the local exposure concentrations and the timing of exposures into account.