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Pulmonary Toxicology and Epidemiology
Tobacco Smoke
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 and 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 and 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.
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