Resources, Conservation and Recycling 27 (1999) 89–97

Pulmonary aspects of nitrogen dioxide toxicity

J.B. Buick a,*, R.C. Lowry a, T.R.A. Magee b

a
Department of Respiratory Medicine, Belfast City Hospital, Belfast, UK
b
Department of Chemical Engineering, The Queen’s Uni6ersity of Belfast, Belfast, UK

Abstract

Little is known of the precise location in the respiratory tract where different gaseous
pollutants are absorbed. Thus, a model airway was used to study the site and mechanism of
nitrogen dioxide (NO2) absorption. The model consisted of sections of perspex tube,
representing the conducting airways, connected in series to a larger compartment of variable
surface area, analogous to an ‘alveolar sink’. The inner wall of the tube was lined with a
non-woven polymer fabric, moistened with sterile water, to simulate the surface of the
tracheobronchial tree where secretions are greater than 90% water. NO2 gas in nitrogen was
passed through the system and its concentration monitored at various positions. It was found
that most of the gas reached the larger compartment, confirming that minimal absorption
occurs in the upper airways. Thus, NO2 penetrates the gas exchanging region of the lung. In
further experiments, a natural pulmonary surfactant (CUROSURF) was exposed to 5 ppm
NO2 in nitrogen and post-exposure samples were scanned by electron-spin resonance (ESR)
to detect the presence of free radicals. The results were negative. In an adjunct to this study,
an aqueous solution of CUROSURF was exposed to NO2 (100 ppm) for 48 h to determine
its effect on surface tension. Results have indicated that exposure to NO2 has a marginal
effect on the ability of CUROSURF to lower surface tension. © 1999 Elsevier Science B.V.
All rights reserved.

Keywords: Respiratory tract; Air pollution; Nitrogen dioxide

* Corresponding author.

0921-3449/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 1 - 3 4 4 9 ( 9 8 ) 0 0 0 8 8 - 3
90 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97

1. Introduction

1.1. Air pollution and respiratory disease

An association between tropospheric pollution and pulmonary disease has been

recognised from time immemorial. It is likely that pollution was first encountered
when open fires were lit in confined spaces during early civilisation. Hippocrates, in
the fifth century B.C., wrote (in his book Airs, Waters and Places) ‘‘Because all men
inhale the same wind, when air is infected with such pollutants that are hostile to
the human race, the men fall sick’’. Since then, there is ample anecdotal evidence to
establish a presumptive link between pollution and lung disease. However, there
now exists an almost universal perception that air pollution is essentially a
twentieth century phenomenon. It is estimated that 60–80% of the world popula-
tion breathe air of marginal or unacceptable quality [1].
Air enters the respiratory tract through the nose or mouth, and subsequently the
trachea via the pharynx and larynx. The trachea then branches left and right into
two main stem bronchi. Each bronchus bifurcates up to 22 times into smaller and
smaller airways, terminating in the alveolar region of the lung.
It is generally accepted that the lung is particularly vulnerable to damage by
extrinsic agents. In a unique experiment, Douglas and Coe [2] demonstrated that
the eyes were only slightly more sensitive than the lungs in terms of a response to
irritant gas exposure. The lungs directly interface with a potentially hostile environ-
ment. The large alveolar surface area is ventilated by approximately 10×103 l of
air daily which may be contaminated by a diverse range of gaseous or particulate
pollutants. The inhalation of these pollutants may have deleterious effects either in
the airways or the lung parenchyma and regional deposition is closely dependant on
the nature of the toxicant.
Increasing concern regarding the relationship between air pollution and health
has stimulated the medical and scientific communities to intensify research in this
area. The relationship between air pollution and respiratory disease is rarely
specific. Airborne pollutants are chemically very complex and exposure is usually to
a cocktail of pollutants. The large number of potential toxicants, coupled with the
fact that their concentrations may alter rapidly with changing climatic conditions,
renders proper study difficult.
Despite the accumulation of research material, much of it empirical, there are
differences in the interpretation of this data and concensus is often lacking. This is
exemplified by the generally negative recent British review of air pollution and
asthma and the much more positive US review of the same material [3].

1.2. Nitrogen dioxide and respiratory disease

Nitrogen dioxide (NO2) is a primary gaseous pollutant. It is also implicated in

the production of smog and ozone via a series of complex photochemical interac-
tions. Approximately 550 million t are released to the atmosphere annually, 90% of
which are produced naturally. Of the remaining 10%, the main source is the
 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97 91

combustion of fossil fuels, both indoors (e.g. gas burners) and outdoors (e.g.
internal combustion engines). Other sources include chemical processes, electric and
gas welding, detonation of explosives and silage production.
The toxic potential of NO2 has been recognised for almost 100 years [4]. NO2 is
implicated in the pathogenesis of respiratory disease and inhalation of high
concentrations (i.e. a few hundred parts per million) is fatal. The first major
fatalities occurred in May 1928 at The Cleveland Clinic, Ohio, USA, when X-ray
films, coated with nitrocellulose, caught fire and exploded, releasing oxides of
nitrogen. This resulted in the death of 150 people and a further 100 were
hospitalised [5].
As early as 1914, Hayhurst and Scott reported the sudden death of four
individuals who had entered a silage tower [6] . Those deaths were wrongly
attributed to asphyxiation by CO2. In 1949, Peterson et al. demonstrated that lethal
concentrations of NO2 were present in freshly filled silos and this was the reason for
the fatalities [7]. Following the deaths of a number of farmers exposed to high
concentrations of NO2, Lowry and Schumann [8] introduced the name ‘silo-filler’s
disease’. This remains the most frequent cause of accidental death from NO2
inhalation. In a recent American review, Zwemer et al. [9] reported an annual
incidence of five cases per 100 000 workers at risk, and a mortality of 20%.
The pulmonary insult associated with NO2 inhalation is caused by alveolar
oedema. Oedema refers to an increase in the amount of extracellular fluid in an
organ or tissue and in the lung is detrimental to gas exchange. More recently,
however, free-radical formation was postulated as the causative mechanism. Free
radicals can be especially harmful when they attack the unsaturated fatty acids
present in the liquid component of cell membranes, a process known as lipid
peroxidation [10].
The toxicity of NO2 has been attributed to its capacity to act, either directly or
indirectly, as an oxidant. It has also free radical potential because it contains
unpaired electrons. Roehm et al. [11] have demonstrated that low concentrations of
NO2 (1.5 ppm) rapidly oxidise lipids, specifically methyl linoleate. Thus, NO2 is
implicated in lipid peroxidation and is thought to initiate this type of peroxidative
damage in the lung.
Most free radicals of biological significance have a short lifespan, of the order of
milliseconds or less, and the method most commonly employed for their detection
is electron-spin resonance (ESR) spectroscopy [12].

1.3. Physiology of respiration

The lungs have a single primary function: to subserve tissue respiration by

effecting the transfer of oxygen from the atmosphere to the blood with the
excretion of carbon dioxide occurring in the opposite direction. Gas exchange
occurs solely in the alveolar region of the respiratory tract. It is complex, involving
multi-layer diffusion through a heterogeneous mixture of substances, including a
surfactant film.
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There are two main types of alveolar cell; the type I and type II pneumocytes.
The alveoli are largely lined by squamous type I cells, interspersed with cuboidal
type II pneumocytes. Type I cells typically make up more than 95% of the alveolar
surface area forming a complete but very thin layer which serves to minimise the
barrier for gaseous diffusion. The type II cell covers approximately 5% of the
alveolar surface area but comprises approximately 60% of the total alveolar cell
population. Thus, type I and type II cells comprise 10 and 12%, respectively, of the
total lung cell population.
Pulmonary surfactant, a phospholipid-protein complex, is synthesised and
secreted by type II pneumocytes. Its primary function is to reduce surface tension
at the air/alveolar interface. It is accepted that the phospholipid, dipalmitoyl-phos-
phatidylcholine (DPPC) is the surfactant component which is responsible for the
reduction of alveolar surface tension.
It is possible that free radical formation may result from the reaction of NO2
with surfactant.

1.4. Pre6ious experimental work

Davidson and Schroter [13] were among the first to develop an experimental
model airway for investigative studies. They have shown that the bronchial airways
of the human lung can be considered as an assembly of segments from infinitely
long, straight tubes with absorbing walls of finite thickness. Their findings, using
this model, were:
1. penetration was greater using low solubility gases;
2. gaseous uptake decreased with increasing flowrate.
Although the model represented the total volume of the lung bifurcations,
consideration of the extensive alveolar surface area was not represented. This large
surface is a vital factor in determining gas absorption.
Ichioka used a similar model to investigate the absorption of SO2 and NO2 [14].
Again he used a long straight tube to represent the upper airway which was lined
with moistened filter paper to simulate airway lining fluid. Downstream of the glass
tube section, the gas passed into two bubblers in series, representing the ‘alveolar
sink’. Ichioka’s work concluded that the site of NO2 absorption was in the lung
regions represented by the bubblers. Again, little consideration was given to the
increasing surface area, representing the depths of the lungs, in the experiments.
Ichioka’s results contradict those of Davidson and Schroter when he showed that
increased gas absorption was detected at higher flow rates.
Although these authors have shown that a model airway is a feasible method of
studying the uptake of gases of varying solubilities, relatively little data is available.
Thus, the objectives of this investigation were:
1. to determine the location within the lung where NO2 is absorbed using a model
airway and to assess the medical implications;
2. to try to prove the existence of free radicals and to determine their destructive
effects on the lung surfactant system;
3. to establish if exposure to NO2 affected the surface tension of surfactant.
 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97 93

2. Experimental

Modelling of any biological system necessitates simplification and abstraction.

The object is not to reproduce the in vivo structure in intricate detail, but to include
major aspects of the functional system that influence the processes being studied.
Thus, the airways were modelled as a simple straight tube leading to a region of
greatly increased surface area, analogous to an alveolar sink (Fig. 1).
The first section of the model airway was constructed from clear perspex tubing
and consisted of three segments, each 100 mm long with an internal diameter of 20
mm, which were lined with a non-woven polymer material, (one-way nappy liners;
Boots, Nottingham, UK), moistened with distilled water. Additional lengths of
tubing were connected between the segments to establish laminar flow. This section
of the equipment represented the upper respiratory tract with a comparable surface
area.
The second section consisted of a clear perspex cylinder, 300 mm long and 120
mm internal diameter, containing nickel gauge at the inlet to give a good dispersion
of the gas. Cylindrical pads of a fibre-bonded superabsorbent (A3/300; Total
Filtration, Cardiff, UK) were positioned at intervals inside the cylinder. Locking
rings were used to hold the discs in position. Each one represented a cross-sectional
area of 11.3×103 mm2. This section represented the elveoler region in the lung.
Drager Polytron measuring heads (Drager, Blyth, UK) were attached to the
model airway at different positions to measure NO2 concentrations. Their use in a
clinical setting has been evaluated by Mercier et al. [15] who concluded that they
provided a relatively cheap, accurate and acceptable system for measuring NO2
concentrations.
NO2 was obtained as cylinders of compressed gas (Air Products, Crewe, UK)
with nominal concentrations of 5 and 100 ppm in nitrogen. Quality control analysis
was carried out by the manufacturer and the cylinder contents were confirmed:

Fig. 1. Diagram of model lung.

94 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97

1. ppm NO2, 0.7 ppm NO, balance N2;

2. ppm NO2, 0.8 ppm NO, balance N2.
For the absorption studies, the nappy liner was cut into rectangles (160×210
mm) and placed in sterile water for 15 min to encourage maximum ‘wetting’. After
the removal of excess water from the lining it was wrapped around a cylindrical,
glass ‘former’ which was inserted into the first section of the model airway. The
former was then carefully withdrawn leaving the liner in place.
The A3/300 media was cut into 120-mm discs and prepared in the same way
before being placed in the second section. NO2 gas, at 100 ppm, was mixed with
pure N2 through a Y-piece, using accurately controlled flowrates, to vary the NO2
concentration admitted to the model airway. Upstream of the Y-piece, the N2 was
passed through a Dreschel bottle containing sterile water in order to saturate the
gas mixture with moisture to prevent evaporative cooling in the model airway.
Flow was controlled and measured using rotameters (Platon, Basingstoke, UK). At
predetermined flowrates, the concentration of NO2 gas was measured at specific
sites using the Polytron heads and the readings were recorded at 1-min intervals.
The effects of three variables: flowrate of gas; time of exposure; and surface area
(i.e. number of discs) on the absorption of NO2 were investigated.
Secondly, the effect of NO2 exposure on pulmonary surfactant was studied as it
has been postulated that the toxic effect of NO2 can be ascribed to its oxidative and
reactive capacities resulting in the generation of free radicals. Thus, an experiment
was devised whereby a natural surfactant (CUROSURF; Serono Pharmaceuticals,
UK) was exposed to NO2 and the sample scanned using an ESR spectrometer. The
surfactant solution was frozen using liquid nitrogen, placed in the sample cavity of
the spectrometer and scanned to obtain a baseline from which deviations could be
observed. After calibration, fresh surfactant was placed in the sample cavity and
NO2 was bubbled into the solution, via a fine capillary, for 5 min and the sample
scanned. After this investigation was completed, fresh surfactant, which was not
exposed to NO2, was scanned under identical conditions.
In an adjunct to this study, an aqueous solution of the natural surfactant
(CUROSURF) was exposed to NO2 (100 ppm) for 48 h to determine its effect on
surface tension. Surfactant samples, at a concentration of 0.4 g/l, were placed in a
Dreschel bottle and the NO2 gas bubbled through the solution at a rate of one
bubble every 10 s in order to avoid frothing. The surface tension of each sample
was measured before and after exposure using a torsion balance. Samples of sterile
water, and surfactant not exposed to NO2 were used as controls.

3. Results and discussion

3.1. NO2 absorption in the model airway

Absorption of NO2 in the perspex tubing, i.e. the first section of the model
airway, was minimal and no distinction could be made between the respective
amounts in each of the lined segments. NO2 uptake in the perspex cylinder, i.e. the
 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97 95

Table 1
Fractional absorption of NO2 as a function of exposure timea

Exposure time (min) 1 5 10 15 20

Fractional absorption of NO2 0.94 0.77 0.64 0.62 0.62

a
Flow rate = 0.5 l/min. Enhanced surface area = 22.6×103 mm2.

second section of the model airway, was significant and varied with exposure time,
surface area and flowrate. This reflects the low solubility of the gas in aqueous
solution and suggests that during in vivo exposures, NO2 will penetrate the alveolar
region with little absorption in the bronchial tree.
This is supported by clinical findings which show that lung injury induced by
NO2 is largely observed at the terminal bronchioles and proximal alveolar region.
Type I alveolar epithelial cells are particularly vulnerable to damage by NO2.
Since most of the absorption occurred in the perspex cylinder, the following
results are examples which were obtained from this section of the model airway.

3.1.1. Effect of exposure time on NO2 absorption.

Table 1 shows that the greatest amount of absorption of NO2 occurs at the
beginning of the exposure time and then gradually reduces by about one-third
between 10 and 15 min.

3.1.2. Effect of gas flowrate on NO2 absorption

It can be observed from Table 2 that flowrate of gas has an effect on the uptake
of NO2, i.e. the lower the flowrate, the higher the fractional absorption.

3.1.3. Effect of surface area on NO2 absorption

Table 3 indicates that more absorption occurs with a greater surface area.
What are the pathophysiological implications of these results? Ventilation refers
to the quantity of air moving into and out of the lungs per unit time. In humans,
ventilation varies between 4 l min − 1 under sedentary conditions to 80 l min − 1 on
exercise. Physiological analysis of the results obtained suggest that NO2 intoxication
occurs quickly, and is greatest during resting breathing. Also, the alveolar region
with its vast surface area is most susceptible to injury from NO2 exposure. This is
supported by clinical findings in NO2 exposed patients in whom parenchyemal
changes predominate.

Table 2
Fractional absorption of NO2 as a function of gas flowratea

Gas flowrate (l/min) 0.5 1.0 2.0 4.0

Fractional absorption of NO2 0.90 0.78 0.56 0.45

a
Exposure time = 20 min. Enhanced surface area =67.8×103 mm2.
96 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97

Table 3
Fractional absorption of NO2 as a function of surface areaa

Cross-sectional area (mm2) 22.6×103 45.2×103 67.8×103 113×103

Fractional absorption of NO2 0.45 0.56 0.78 0.84

a
Flow rate = 1.0 l/min. Exposure time =20 min.

3.2. Effect of NO2 exposure on pulmonary surfactant

3.2.1. Free radicals

The initial trace showed a deviation from the baseline at approximately 3420G
field setting. This region was more closely examined by increasing the system gain
to achieve a narrow scan and the trace obtained indicated the presence of a free
radical at 3420G. However, this was also observed in the control sample and was
not therefore consequent to any reaction between NO2 and surfactant. Thus, in the
absence of positive results we conclude that exposure of pulmonary surfactant to
NO2 does not generate free radicals. Their formation is probably due to chemical
reactions with cell membrane components.

3.2.2. Surface tension

The results shown in Table 4 indicate that exposure of pulmonary surfactant to
NO2 produced minimal change in surface tension indicating that the biophysical
properties of pulmonary surfactant, specifically its ability to reduce surface tension,
may be altered very slightly.

4. Conclusions

Nitrogen dioxide penetrates to the gas-exchange portion of the lung.

Nitrogen dioxide intoxication occurs quickly and is greatest during resting
breathing.
The vast surface area of the alveolar region of the lung is most susceptible to
injury from nitrogen dioxide exposure.

Table 4
Surface tension results of control samples and surfactant exposed to N2 and NO2 for 48 h

Sample Exposure Surface tension (pre-exposure), Surface tension (post-exposure),

nN/M nN/M

Sterile water – 72.5 –

Surfactant (con- – 37.3 –
trol)
Surfactant N2 37.3 37.0
Surfactant NO2 37.3 39.5
 J.B. Buick et al. / Resources, Conser6ation and Recycling 27 (1999) 89–97 97

Exposure of pulmonary surfactant to nitrogen dioxide does not generate free

radicals.
Exposure of pulmonary surfactant to nitrogen dioxide has a marginal effect on
its ability to lower surface tension.

References

[1] Hauchman FS. Air pollution and health in developing countries. Washington, DC: Agency for
International Development, Environmental Health Workshop, 1991:1 – 26.
[2] Douglas RB, Coe JE. The relative sensitivity of the human eye and lung to irritant gases. Ann
Occup Hyg 1987;31:265–7.
[3] Bates DV. Particulate air pollution. Thorax 1996;51:53 – 8.
[4] Wood FC. Poisoning by nitric acid fumes. Arch Int Med 1912;10:478 – 504.
[5] Anon. The hazard of toxic gases from combustion of roentgen-ray films. J Am Med Assoc
1929;92:1764–5.
[6] Hayhurst ER, Scott E. Four cases of sudden death in a silo. J Am Med Assoc 1914;63:1570 – 2.
[7] Peterson WH, Thomas RW, Anderson RF. Yellow gas from corn silage. Hoard’s Dairyman
1949;94:870–1.
[8] Lowry T, Schuman LM. Silo-filler’s disease — a syndrome caused by nitrogen dioxide. J Am Med
Assoc 1956;162:103–10.
[9] Zwemer FL, Pratt DS, May JT. Silo-filler’s disease in New York State. Am Rev Respir Dis
1992;146:650.
[10] Witschi HP, Brain JD. Handbook of experimental pharmacology, vol. 75. New York: Springer,
1985.
[11] Roehm JN, Hadley JG, Menzel DB. Antioxidants versus lung disease. Arch Intern Med
1971;128:88–93.
[12] Symons M. Chemical and biochemical aspects of electron-spin resonance spectroscopy. New York:
Reinhold, 1978.
[13] Davidson MR, Schroter RC. A theoretical model of absorption of gas by the bronchial wall. J
Fluid Mech 1983;129:313–35.
[14] Ichioka M. Model experiments on absorbability of the airway mucus membrane of SO2 and NO2
gases. Bull Tokyo Med 1972;19:361– 75.
[15] Mercier JC, Zupan V, Dehan M, Renaudin MH, Bouchet M, Raveau C. Device to monitor
concentration of inhaled nitric oxide. Lancet 1993;342:431 – 2.