United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
SEPA
Research and Development
EPA-600/S1-81-052 Aug. 1981
Project Summary
New Approaches to
Quantitating the Pulmonary
Effects of Inhaled Pollutants
P. A. Bromberg, R. C. Boucher, Jr., M. Friedman, M. J. Hazucha, and R. L.
Pimmel
A variety of non-invasive and other
techniques was developed to study
effects of inhaled pollutants on the
lung. In the area of airway mechanics,
a diameter gauge was developed to
make continuous measurements of
large airways caliber. The gauge
provides an electrical output. Rapid
methods for non-invasively measuring
respiratory mechanics using forced
random noise excitation at the mouth
were developed and validated. The
resulting respiratory impedance data
were applied to appropriate models to
obtain values for parameters such as
"central" and "peripheral" airways
resistance.
In the area of respiratory epithelial
function, a non-traumatic technique
was developed to measure transepi-
thelial potential difference across
respiratory (nasal and airways) epithe-
lium. Trachea! epithelial permeability
was measured in vivo, demonstrating
increased permeability and decreased
perselectivity in guinea pigs exposed
to 4 ppm, 1 ppm and 0.3 ppm 03.
In the area of pulmonary vasculature,
a rapid non-invasive multi-gas re-
breathing technique was developed to
measure lung water and was used to
develop an 03-induced pulmonary
canine model of delayed pulmonary
edema using 1 ppm 03
This Project Summary was devel-
oped by EPA's Health Effects Re-
search Laboratory, Research Triangle
Park, NC, to announce key findings of
the research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back),
Introduction and Summary
The study focused on developing new
approaches—conceptual and method-
ologic—to the study of pulmonary
responses to pollutants, with an
emphasis on techniques with possible
human applications.
A non-invasive method of estimating
the mechanical impedance of the
respiratory system to air flow using
excitation with a forced random noise
signal applied via a mouthpiece (humans)
or endotracheal tube (animals) was
developed, validated, and integrated
into an on-line system using a mini-
computer. Useful data are obtainable
between 2 - 35 Hz in animals and 5 - 35
Hz in humans. The impedance vs.
frequency data were fitted to a series
resistance-compliance-inertance model
when the real component of the imped-
ance (effective resistance) was not
frequency dependent, and fitted to a
five-parameter model including two
resistances ("central" and "peripheral"),
two compliances ("shunt" and "per-
ipheral"), and an inertance when the
effective resistance showed negative
frequency dependence. Other, simpler,
approaches were also developed for
fractionating frequency-dependent ef-
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fective resistance into central and
peripheral components. All approaches
use algorithms which require less than
two minutes real time. The methods and
analyses were successfully applied to
various studies in animals, in young
children, asymptomatic cigarette
smokers and in patients with COPD, in
subjects with asthma before and after
brochodilator Rx, and in normals and
mild asthmatics with methacholine
challenge (studies carried out at EPA
labs). In general, the techniques have
been shown to be sensitive to presence
of early disease (e.g., asymptomatic
cigarette smokers) and to yield repro-
ducible impedance patterns. The anal-
ysis of frequency dependence of effective
resistance has produced novel ap-
proaches to studying the axial localiza-
tion of airway changes, and a good
correlation between the derived
parameter, effective resistance extra-
polated to a frequency of 1 Hz, and
plethysmographically measured Raw
has been demonstrated.
A novel method for directly and
continuously measuring the internal
diameter of large airways was developed
and evaluated. This device is positioned
within the lumen and converts internal
diameter into an electrical signal. It has
a flat frequency response from DC to 8
Hz and exhibits excellent stability and
signal to noise ratio. The device was
used to examine quantitatively the
smooth muscle response to regional
arterial perfusion of the canine trachea
with acetylcholine, and is currently
being used to investigate the effects of
zone inhalation on tracheal smooth
muscle responsiveness.
Many of these studies focused on
techniques designed to study the
physiological function of respiratory
epithelium in vivo and acute effects of
ozone inhalation on large airways
epithelium.
A technique was developed to mea-
sure the in vivo transepithelial electrical
potential difference (PD). A fluid-filled
perfused catheter lightly touching the
epithelium serves as a bridge to an agar
bridge to a calomel half cell. The
reference electrode is a #19, agar-filled
needle inserted into the subcutaneous
tissue. With proper filtering of the
signal, stable signals can be obtained.
The PD values are —30 mV (lumen
negative) in canine and human trachea;
they decrease as more distal bronchi
reaching a value of 5 - 10 mV in
segmental bronchi in both species.are
explored. Guinea pig trachea has a PD of
about 13 mV while rabbit trachea has
about 20 mV. More recently, human
nasal epithelial PD has been studied
and this technique will soon be applied
in a collaborative study of pollutant
inhalation with the EPA/HERL Clinical
Studies Group. Exposure of conscious
guinea pigs or of anesthetized intubated
dogs to 1 ppm 03 and 4 ppm 03 for 2 - 3
hours failed to cause any acute change
in tracheal PD values. Since PD repre-
sents the ratios between ion-transport
generated current (I) and the passive
electrical conductance (G) of the tissue,
the lack of PD allocation following 03
exposure does not prove that no change
in bioelectric properties has occurred.
In vivo permeability of respiratory
epithelium by measuring early appear-
ance rates in arterial blood of molecular
probes simultaneously instilled onto the
surface of guinea pig trachea via a fine
catheter was examined. The probes
used were 14C-mannitol (MW-180
daltons), 3H-dextran (MW-10,000 dal-
tons) and horseradish peroxidase (HRP)
(MW-40,000 daltons). Data indicate
substantial increase in airways epi-
thelial permeability and loss of perm-
selectivity immediately following con-
clusion of exposure to either 4 ppm or 1
ppm03 for three hours. The effect
persists at least twenty-four hours but is
absent at seven days after exposure.
The locus within the epithelium of
this increased permeability is unclear.
After 4 ppm 03, there are marked
changes in the ultrastructure of the
intercellular "tight" junctions shown by
freeze-cleave preparations of the tracheal
epithelium. Cellular extrusion was also
noted. These changes were not seen,
however, after the 1 ppm 03 exposure.
It would be valuable to study in a more
controlled fashion the relation of
epithelial bioelectric properties and
specific transepithelial ion fluxes to the
permeability alterations induced by 03.
A tubular (cylinder of trachea) version of
the Ussing preparation is being devel-
oped for this purpose.
A correlation of in vivo permeability
alterations to altered responsiveness of
the airways to bronchoconstrictor drug
challenge after 03 exposure is also
planned, to explore the hypothesis that
03-induced increased epithelial per-
meability is responsible for greater
access of inhaled aerosolized drugs to
effector sites in the airway wall.
This project included a study of 03-
induced pulmonary edema in dogs
using a multi-gas (C2H2, He and C180)
rebreathing technique to repetitively
measure lung water following the 03
exposure. It was demonstrated in
anesthetized, intubated dogs that after
inhalation of only 1.0 ppm 03 for three
hours, a significant increase in lung
water occurred at twenty-four hours but
was not present either at forty-eight
hours or immediately after the exposure.
The induction of delayed edema in this
model offers the opportunity to study
"early" events which may play a
pathogenetic role in the eventual
development of edema. Although statis-
tically significant reductions in cardiac
output were found acutely after exposure
to 0.3 ppm 03, we could not demonstrate
edema. The rebreathing measurements
of lung water were confirmed by wet/
dry lung weights whenever possible.
This system will be further explored by
adding an increase in pulmonary
microvascular pressure at twenty-four
hours after 03 exposure in order to test
the hypothesis that the alveolar-capillary
membranes are injured even at low
levels of 03 exposure but that the injury
remains sub-clinical and can be detected
by increasing filtration pressure. Lung
lymph flow and protein composition will
also be examined in these studies.
The rebreathing technique was suc-
cessfully applied to a study of effects of
0.75 ppm S02 inhalation on human
subjects. No changes in lung water,
DUCO or FRC were observed in this
EPA/HERL study conducted by the
Clinical Studies Branch.
Finally, a tendency was observed for
canine pulmonary vascular resistance
to be increased by 03 exposure. It was
hypothesized that (similar to the effect
of indomethacin, a prostaglandin syn-
thetase inhibitor) Os might be responsible
for inhibition of prostacyclin synthesis
by blood vessels and thus enhance the
normal pulmonary arteriolar constriction
to alveolar hypoxia. This was studied in
dogs and it was possible to demonstrate
significant enhancement of hypoxic
pulmonary vascular constriction by pre-
treatment with indomethacin. Similar
changes were seen after 03 exposure
but did not reach the p < .05 level oi
statistical significance. Nevertheless
studies of 03 interaction with arachi-
donate metabolism may be of interest.
Conclusions
Techniques for non-invasively study
ing and analyzing respiratory mechanic;
have been worked out and appliec
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successfully to a variety of experiments
using human subjects. Some of these
experiments were carried out in EPA's
HERL Human Studies facility.
A diameter gauge was developed that
is suitable for direct, continuous mea-
surement of caliber of large airways to
aid in studies of pollutant effects on
airways mechanics and hyperrespon-
siveness to bronchoconstrictor agents.
An atraumatic technique for measur-
ing the bioelectric properties of respira-
tory epithelium in vivo was developed
and used to study 03 inhalation effects
on the trachea of animal models. The
technique was extended to nasal epithe-
lium which is readily accessible in
human subjects.
Striking increases in permeability and
loss of perm-selectivity of guinea pig
tracheal epithelium were demonstrated
following exposure to either 1 ppm or 4
ppm 03. (A similar, but lesser, effect has
now been demonstrated for 0.3 ppm 03
exposure.) Similar techniques are being
considered for development for human
use.
A canine 03- induced pulmonary
edema model was characterized with as
little as 1.0 ppm 03 exposure. This model
will be used to study the interaction
between 03 exposure and elevated
pulmonary microvascular pressure
which would be relevant to 03 effects in
patients with heart failure or other
causes of elevated pulmonary capillary
pressure.
During the course of this work a non-
invasive, multi-gas rebreathing tech-
nique to repetitively measure Di_CO,
FRC and lung water was developed and
extensively validated. This technique
was successfully applied to an EPA/
HERL study of acute effects of inhala-
tion of 0.75 ppm SO2 in man.
There is evidence suggesting that
prior 03 exposure may enhance the
pulmonary arteriolar vasoconstrictive
response to alveolar hypoxia in dogs.
The potential interaction between 03
and arachidonate metabolism warrants
exploration.
P. A. Bromberg, R. C. Boucher, Jr., M. Friedman, M. J. fiazucha, andR, L Pimmel
are with the Division of Pulmonary Medicine, School of Medicine, University
of North Carolina, Chapel Hill, NC 27514.
William F. McDonnell is the EPA Project Officer (see below).
The complete report, entitled "New Approaches to Quantitating the Pulmonary
Effects of Inhaled Pollutants," {Order No PB 81-222 382; Cost: $6.50. subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
* US GOVERNMENT PRINTING OFFICE 1981 -757-012/7305
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