CIGARETTE SMOKE TOXICOLOGY

Introduction

The inhalation toxicity of tobacco smoke has become one of the
major public health problems of the 20th century. The chemical
complexity of tobacco smoke confounds the task of identifying its
toxic constituents. Tobacco smoke is comprised of thousands of
chemical components arising primarily from volatilization and
pyrolysis of the tobacco leaf (Stedman 1968; Green 1977). The
chemical gamut runs from traces of elemental metals, such as
cadmium, to nonvolatile whole tobacco leaf components that have
escaped degradation during the burning process (USDHHS 1981).
Approximately 90 percent of the individual constitutents are organic
compounds associated with both the particulate phase and the gas
phase (Guerin 1980). It is not surprising that chronic inhalational
exposure to this diverse mixture of potentially bioactive compounds
can evoke a wide variety of toxicologic responses. Over the years,
scientific and public concern has centered primarily on the carcino-
genic and atherogenic effects of tobacco smoke. In contrast, relative-
ly little is known about the involvement of tobacco smoke constitu-
ents in the pathogenesis of chronic obstructive lung disease (COLD)
(USDHHS 1981).

For the most part, smoke constituent toxicity studies, both
epidemiologic (Dean et al. 1977; Higenbottam et al. 1980) and
toxicologic (Walker et al. 1978; Lewis et al. 1979; Coggins et al. 19801,
have been confined to a comparison of the varying amounts of
particulate matter or tar delivered by smoke. In studies of this
nature, attempts have been made to distinguish between the relative
toxicities of the vapor phase and the particulate phase of tobacco
smoke. The general conclusion reached is that gas phase components
that penetrate to the small airways and alveoli may play a
significant role in the production of peripheral airway and parenchy-
ma1 diseases such as emphysema, whereas particulate phase compo-
nents that deposit in larger airways may play a role in the
development of disorders of the more proximal airways such as
chronic bronchitis (USDHHS 1981). This generalization may not
always hold, however. For example, in a review of the effects of
smoking on mucociliary clearance, Newhouse (1977) noted consider-
able disagreement among investigators with regard to whether the
vapor phase or the particulate phase was the major factor in smoke-
induced dysfunction of the mucociliary transport system. Also,
Coggins and associates (1980) observed an increase in both peripher-
al and central airway goblet cell number in rats after exposure to
tobacco smoke from which most of the vapor phase had been
removed. Cohen and James (1982) found that the level of oxidants in
tobacco smoke (oxidants have been implicated in the pathogenesis of

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emphysema) correlated with the amount of particulate matter in
smoke from various brands of cigarettes. At present, therefore,
attempts to associate a specific toxicologic response solely with
either the vapor phase or the particulate phase of tobacco smoke are
not recommended.

Because so little is known regal-ding the role of specific constitu-
ents or phases of tobacco smoke in the pathogenesis of COLD, this
section of the Report is organized on the basis of specific insults to
the respiratory system that may be brought on by exposure to whole
tobacco smoke and that may lead to structural and functional
changes within the lung. Included, as available information permits,
are data on the known contribution of individual smoke constituents
or phases to a specific insult. Human and animal studies are
described separately to provide a perspective on the extent to which
animal research has verified or extended clinical research and vice

versa.

Preliminary Considerations

Tobacco smoking is generally accepted as the major cause of
COLD (USDHEW 1979; USDHHS 1981). COLD is often subdivided
into three categories: (1) uncomplicated bronchitis, characterized by
mucus hypersecretion and cough, (2) chronic bronchitis with bronchi-
olar inflammation and obstruction of distal airways, and (3) pulmo-
nary emphysema, characterized by distal air space enlargement with
loss of alveolar interstitium. These three pathologic condit.ions are
often considered collectively within the context of COLD because
they can coexist in the lungs of smokers and because signs and
symptoms associated with one condition may presage the develop-
ment of another.

Effects on Airway Function and Ventilation
Human Studies

Cigarette smoke appears to have both chronic and acute effects on
airway function. In adults, smoking over a period of years leads to
narrowing of and histopathologic abnormalities in small airways
(Ingram and O'Cain 1971; C osio et al. 1980; Suzuki et al. 1983). Even
in teenagers, regular smoking for 1 to 5 years is sufficient to cause
demonstrable changes in tests of small airway function in some
smokers (Seely et al. 1971); the lungs of young cigarette smokers who
die suddenly show definite pathologic changes in the peripheral
airways (Niewoehner et al. 1974).

The acute response to cigarette smoke has been reported to involve
large airways, small airways, or both. Costello and associates (1975),
in a study of asymptomatic smokers and nonsmokers, found that

427


tests of small airway function (maximum expiratory flow volume
curves, closing volume, and frequency dependence of compliance)
were unaltered after smoking one cigarette; however, specific
airways conductance, a measure of large airway function, fell
significantly in both groups. Essentially the same results were
obtained by Gelb and associates (1979), who reported a decrease in
airways conductance but little or no change in volume of isoflow
(another test of small airway function) in healthy nonsmokers after
smoking one cigarette. Likewise, McCarthy and colleagues (1976),
using several different tests, found no evidence for an acute effect pf
intensive cigarette smoking on small airways, but did demonstrate
increased large airways resistance. The decrease in conductance
caused by cigarette smoke has been shown to occur within 7 or 8
seconds of a single inhalation (Rees et al. 1982), and filtration seems
to reduce the degree of bronchoconstriction (Da Silva and Hamosh
1980). Irritant effects of tobacco smoke are not limited to the
particulate phase, because exposure to oxides of nitrogen at levels
present in cigarette smoke also can precipitate acute bronchospasm
(Tate 1977). Nicotine does not appear to be responsible for the acute
bronchoconstriction that accompanies cigarette smoking (Nadel and
Comroe 1961).

Zuskin and coworkers (1974) showed that in healthy human
subjects, smoking one or two cigarettes decreased flow rates on
maximum and partial flow-volume curves, and concluded that
smoking causes acute narrowing of small airways. Da Silva and
Hamosh (1973) and Sobol and colleagues (1977) measured airways
conductance, as well as maximum mid-expiratory flow rates, and
concluded that both large and small airways are probably affected by
the acute inhalation of cigarette smoke.

Though the bronchoconstrictive response to cigarette smoke has
most often been attributed to a cholinergic reflex originating with
stimulation of irritant receptors in the airways, there are data
suggesting that histamine may also be involved. Walter and Walter
(1982b) found a significant increase in the number of degranulated
basophils in the blood of smokers 10 minutes after smoking
compared with just before smoking. There is also some evidence to
indicate that smokers may differ from nonsmokers in their respon-
siveness to inhaled histamine (Brown et al. 1977; Gerrard et al. 1980)
as well as methacholine (Malo et al. 1982; Kabiraj et al. 1982; Buczko
et al. 1984), but smoking immediately prior to an inhalation test does
not appear to affect bronchial responsiveness to either histamine or
methacholine (McIntyre et al. 1982).

Asthmatic subjects have a greater than normal susceptibility to
the bronchoconstrictive effects of cigarette smoke when the smoke is
actively inhaled. The question whether tobacco smoke plays a role in

428


allergic asthma has yet to be resolved completely I USDHEW 1979;
Shephard 1982; Burrows et al. 1984).

Animal Studies

Binns and Wilton (1978) studied the acute ventilatory response to
cigarette smoke in rats. They found that within the first 3 minutes
after initiation of smoke exposure, tidal volume fell to 80 percent of
the preexposure level, then rose to 160 percent after 9 minutes of
exposure; respiratory rate dropped to 40 percent of the preexposure
level within 1 minute and remained there for the duration of the
exposure period. There was no adaptation of the acute ventilatory
response after 4 weeks of daily smoke exposures. In a similar study,
Coggins and associates (1982) found that rats exposed to a relatively
low dose of cigarette smoke demonstrated a persistent depression in
tidal volume and breathing frequency, whereas animals exposed to a
relatively high dose exhibited an increase in tidal volume with no
change in frequency.

Acute airway responses to cigarette smoke have not been studied
as extensively in animals as they have been in man. Experiments in
anesthetized dogs (Aviado and Palecek 1967), rabbits (Sellick and
Widdicombe 1971), and cats (Boushey et al. 1972) indicate that acute
smoke exposure elicits reflex bronchoconstriction. There also is
evidence from experiments with isolated monkey lungs that smoke
exposures stimulate histamine release (Walter and Walter 1982a).
Histamine appears to be responsible, at least in part, for mediating
.he increase in collateral resistance in dogs following administration
If cigarette smoke (Gertner et al. 1982).
The chronic effects of cigarette smoking on pulmonary function in
logs were studied by Park and coworkers (1977). Active inhalation of
100 and 200 puffs of diluted (1:4) smoke 5 days per week for periods
jf 6 months and 1 year, respectively, did not produce any noteworthy
:hanges in pulmonary function. The effects of chronic cigarette
,moke exposure on ventilation in rats were studied by Loscutoff and
*oworkers (1982). Animals exposed for up to 24 months to cigarette
<make containing various amounts of tar and nicotine were found to
rave higher tidal volumes and lower respiratory rates than sham-
zxposed animals. The most pronounced changes were seen in
nimals exposed to smoke from low tar, high nicotine cigarettes.
Iistopathologic examination of lungs taken from these animals
evealed primarily granulomatous lesions with no evidence of
mphysematous changes (Wehner et al. 1981). Roehrs and colleagues
1981) used operant conditioning techniques to get baboons to smoke
0 cigarettes per day for 3 years. Measurements of lung volumes,
ompliance, and expiratory flow showed no differences between
moking animals and sham animals, but airway reactivity to inhaled
lethacholine was decreased in animals that had smoked. Subse-

429


quently, Wallis and associates 11982) reported that both acute and
chronic Inhalation of' nicotine mimicked this effect of cigarette
smoke :in brc;llchisI reactivity in baboons.

Effects on Permeability of the Pulmonary Epithelium

The pl-i I monwry epithelium functions to protect underlying struc-
tures from injuricus agents deposited in the airway lumen. Cigarette
smoke has been shown to diminish this protective function by
increasing enithelial permeability in all regions of the tracheobron-
chial tree (Simani et al. 1974). In the airways, irritant receptors
located just beneath epithelial tight junctions are more accessible
following exposure to tobacco smoke. Stimulation of these receptors
is thought to initiate rapid changes in ventilation and to induce
bronchoconstriction (Widdicombe 1977). Likewise, mast cells, a
source of potent bronchoconstrictive mediators, become more accessi-
ble to inhaled toxicants after smoke exposure (Guerzon et al. 1979).
In the alveolar region, an increase in epithelial permeability may
promote the transfer of noxious smoke constituents and endogenous
proteases to the interstitium, thereby facilitating disruption of
alveolar septa.

Human Studies

Subepithelial structures of the lung are important targets for
smoke-induced injury, and research has shown that tobacco smoke
alters epithelial permeability to allow offending agents to gain access
to these structures. Minty and colleagues (1981) showed that,
compared with nonsmokers, smokers had significantly shorter half-
time lung clearance as measured with inhaled radiolabeled aerosols.
After cessation of smoking, half-time clearance increased, but at 21
days it was still significantly less than that reported in nonsmokers.
Using similar techniques, Kennedy and colleagues (1984) compared
pulmonary epithelial permeability and bronchial reactivity to in-
haled histamine in smokers and nonsmokers. These researchers
found increased permeability in smokers, but could find no evidence
of increased reactivity. Although the mechanism by which cigarette
smoke induces an increase in alveolar epithelial permeability is not
fully understood, it has been suggested that carbon monoxide may
play an important role, with possible additional contributions from
nicotine and oxides of nitrogen (Jones et al. 19801.

Animal Studies

In studies of rabbit tracheal rings exposed in vitro, just a few puffs
of diluted cigarette smoke have caused ultrastructural changes in
tracheal epithelial cells and an increase in the size of intracellular
spaces, but junctional complexes between cells remain intact (Davies

430


and Kistler 1975). Boucher and associates (1980, found that, com-
pared with controls, guinea pigs exposed to 100 or more puffs of
cigarette smoke exhibited a significantly faster transfer rate for
horseradish peroxidase across tracheal epithelium. These animals
also demonstrated a progressive disruption of epithelial tight
junctions as a function of the dose of tobacco smoke. Hulbert and
associates (1981) reported that smoke-induced increases in guinea
pig airway permeability were transient, reaching maximum levels at
30 minutes after acute exposure to 100 puffs and returning to control
levels 12 hours later. Gordon and associates (19831 reported that
acute exposure (48 hours) of hamsters to NO:! caused a marked
increase in bronchiolar and alveolar epithelial permeability to
horseradish peroxidase. Restoration of the epithelial barrier was
noted 48 hours after exposure in these animals. Ranga and associates
(1980) demonstrated a similar increase in guinea pig tracheal
epithelial permeability upon exposure to NO- for 14 days.

Effects on Mucociliary Structure and Function

The mucociliary system provides the lung with one of its most
effective lines of defense against inhaled pollutants. Disruption of
this system enables pollutants to remain in contact with the
respirat,ory membranes for prolonged periods and increases the risk
of toxic damage. Tobacco smoke can adversely affect mucociliary
function by increasing the amount or viscosity of respiratory tract
secretions or by depressing ciliary activity directly (Newhouse 1977;
Wanner 1977).

Effects on Cells: Pulmonary Alveolar Macrophages and
Polymorphonuclear Leukocytes

Pulmonary emphysema is believed to result from the slow
degradation of the elastin framework of lung parenchymal tissue.
Degradation of e&tin is most likely initiated by elastolytic enzymes
released locally in the lung and not adequately inhibited by
endogenous antiproteases. Recent studies of the effects of cigarette
smoke on these cellular sources of elastolytic enzymes have provided
additional insights into the relationships between smoking and
pulmonary emphysema. (See chapter 53

Human Studies

Normally, pulmonary alveolar macrophages (PAMs) function as a
defense mechanism against particulate material deposited on the
respiratory surfaces of the lung. However, cigarette smoke can
induce a number of changes in PAMs that may promote excessive
degradation of native lung tissue. For example, PAMs from smokers


have elevated elastase levels, and these cells may secrete elastase in
vitro (Harris et al. 1975; Rodriguez et al. 1977; Hinman et al. 1980).
Further, PAMs from smokers have been shown in vitro and in vivo
to bind and internalize elastase released from polymorphonuclear
leukocytes (PMNs) (Campbell et al. 1979; White et al. 1982). It has
been suggested that during the phagocytosis of smoke particulates by
PAMs, elastolytic enzymes may be released into extracellular spaces
(Hocking and Golde 1979; Brain 1980; Kuhn and Senior 1978).
Numerous investigators have reported significantly greater yields of
PAMs from the lungs of smokers compared with nonsmokers (Green
et al. 1977; Roth et al. 1981; Hoidal and Niewoehner 1982). The
effects of smoking on PAM mobility are unclear. Some researchers
have reported a significant increase in chemotactic migration of
PAMs from smokers versus PAMs from nonsmokers (Warr and
Martin 19741, but others have been unable to observe such an effect
(Demarest et al. 1979).

Macrophages from smokers exhibit various morphologic, metabol-
ic, and functional abnormalities. Structural changes noted in the
PAMs of smokers include a slight increase in cellular diameter, the
presence of "smokers inclusions" consisting predominantly of kaolin-
ite particles (which have been shown to be cytotoxic to human PAMs
in vitro (Green et al. 1977)) and increased numbers of lysosomes and
phagolysosomes (Brody and Craighead 19751. Perturbations in sever-
al metabolic pathways have been observed in PAMs from smokers,
and acrolein in smoke has been implicated in this toxicity (Green et
al. 1977; Laviolette et al. 1981). The production of superoxide
radicals and hydrogen peroxide (H202), both of which inhibit lung
antiproteases, has been reported to be enhanced in the PAMs of
smokers (Hoidal et al. 19811. Smokers' PAMs have also been shown
to release chemotactic substances for PMNs (Gadek et al. 1978;
Hunninghake et al. 1980). Additionally, there is evidence suggesting
that PAMs from smokers secrete factors that promote the release of
elastases from PMNs (Cohen et al. 1982).

As with PAMs, PMNs have been found in elevated numbers in the
lungs of smokers (Reynolds and Newball 1975; Hunninghake et al.
1980a; Hunninghake and Crystal 1983). Exposure of PMNs to
cigarette smoke condensate in vitro has been shown to promote the
release of elastolytic enzymes (Blue and Janoff 1978). Hutchison and
coworkers (1980) found that the particulate phase of cigarette smoke
stimulated the release of lysosomal enzymes from human PMNs, but
they did not quantitate this release specifically for elastases. A
recent study by Totti and colleagues (1984) showed that nicotine was
chemotactic for human PMNs and that it enhanced PMN responsive-
ness to other chemotactic factors. These results are in contrast with
a previous study by Bridges and coworkers (1977) showing that
nicotine, when used in higher concentrations than those employed

432


by Totti and colleagues, inhibited the chemotactic response of PMNs
to casein.

In a preliminary laboratory study, Janoff and colleagues (1983)
found that smokers have elevated levels of PMN elastase in their
lung fluids compared with nonsmokers. This finding is of particular
interest in that human PMN elastase has been shown to induce
emphysema in animals (Janoff et al. 1977; Senior et al. 1977; Snider
et al. 1984). There is also evidence that cigarette smoke alters PMN
metabolism in such a way as to favor the release of toxic oxygen
metabolites. PMNs from smokers with an elevated white blood cell
count show a marked increase in the release of super-oxide anions
compared with PMNs from nonsmokers or with PMNs from smokers
with a normal white cell count (Ludwig and Hoidal 1982). These
unstable oxygen metabolites have harmful effects on various cells
and tissues in vivo and in vitro (Sachs et al. 1978; Fridovich 1978),
and are capable of injuring phagocytes and promoting the release of
proteolytic enzymes (Hoidal and Niewoehner 1982). Oxidants derived
from PMNs are also capable of inactivating lung antiproteases in
vitro; this may be yet another mechanism by which smoke-affected
PMNs contribute to the development of emphysema (Zaslow et al.
1983).

Animal Studies

Laboratory animal studies of the effects of cigarette smoke on lung
free-cell population and integrity have yielded results similar to
those obtained from human studies. Recruitment of PAMs to the
lungs following cigarette smoke exposure has been demonstrated in
a number of animal species, including mice (Matulionis and Traurig
1977; Guarneri 1977); hamsters (Hoidal and Niewoehner 1982), and
monkeys (DeLucia and Bryant 1980). In the rat, PAM recruitment in
response to smoke exposure appears variable. In two relatively
similar studies, one group of investigators (Drath et al. 1978) found a
depression in the number of PAMs in the lungs of rats exposed to
smoke for 30 days, whereas another group (Walker et al. 1978)
reported a significant increase in the number of PAMs after 6 weeks
of smoke exposure.

Several authors have described the effects of cigarette smoke
exposure on the morphology of rat PAMs. Observed changes include
increases in PAM size, lipid vacuoles, and lysosomes, as well as the
presence of "smokers inclusions" (Walker et al. 1978; Davies et al.
1978; Lewis et al. 1979). Cigarette smoke exposure of mice induces a
similar pattern of morphological changes in PAMs (Matulionis 1977).

Alterations of PAM phagocytic capacity have been demonstrated
in animals exposed to cigarette smoke. Fogelmark and colleagues
(1980) reported a dose-related increase in the rate of phagocytosis of
fungal spores in vitro by PAMs from hamsters and rats that had

433


been exposed to cigarette smoke. The ability of rats to mobilize
PAMs in response to a bacterial challenge does not appear to be
altered by cigarette smoke exposure (Guarneri 1977), nor is there a
significant effect upon PAM phagocytosis of Staphylococcus aureus
in rats after 30 days of smoke exposure (Drath et al. 1981).
Macrophages from mice exposed to cigarette smoke for 4 weeks
secrete significantly higher amounts of elastase than PAMs from
controls. However, it is not known whether this effect is due to the
stimulation of resident macrophages or to the recruitment of a
highly exudative population of macrophages to the lungs (White et
al. 1979).

A number of metabolic abnormalities have been noted in PAMs
from smoke-exposed animals (Low et al. 1977). Among these is an
increase in oxidative metabolism resulting in an increased produc-
tion of superoxide anions. Hoidal and Niewoehner (1982) showed
that enhanced oxidative metabolism in hamster PAMs was dimin-
ished if the particulates were filtered from the smoke. However,
other workers (Drath et al. 1981) studying smoke enhancement of rat
PAM oxidative metabolism have attributed this effect to the vapor
phase of smoke. In another study of PAM oxidative metabolism in
rats exposed to cigarette smoke for 180. days (Huber et al. 1980), it
was reported that metabolism was activated after 30 days, and at the
same time PAM superoxide dismutase (an enzyme that detoxifies
super-oxide radical) activity was depressed by 30 percent.

Animal PAMs, like human PAMs, can secrete PMN-directed
chemotactic factor in response to various stimuli. For example,
Gadek and colleagues (1980) demonstrated that noninfectious partic-
ulate material stimulated the release of PMN-specific chemotactic
factor from guinea pig PAMs. Perhaps tobacco smoke particulates
might evoke a similar response.

Owing to the ease with which PMNs can be harvested from
peripheral blood, most research concerning the effects of tobacco
smoke on PMNs has been conducted using human cells. In one
laboratory study, hamsters exposed to cigarette smoke for 2, 8, and
20 hours showed a progressive recruitment of PMNs to the lungs.
Control saline aerosol and filtered smoke did not stimulate recruit-
ment of PMNs, suggesting that this effect of cigarette smoke resides
in the particulate phase (Kilburn and McKenzie 1975).

Effects on Protease Inhibitors

In addition to efforts to characterize the effects of tobacco smoke
on cellular sources of elastolytic enzymes, considerable research has
gone into delineating the effects of tobacco smoke on the protease
inhibitor defense mechanism in the lungs, While several protease
inhibitors have been identified, a,-protease inhibitor (a,Pi) is consid-

434


ered to be the most important in neutralizing the effects of elastase
(Gadek et al. 1981,. Chemical oxidants are known to inactivate a,Pi
and diminish its capacity to inhibit elastase both in vitro and in viva
tAbrams et al. 1980). Cigarette smoke is an abundant source of
chemical oxidants that can exert : he same effect on n,?i and thereb?
reduce lung defenses against endogenous rlzstases.

Human Studies

The toxic effect of tobacco smoke on proteave in!libitors has beei-,
demonstrated in a variety of experimental situatioz. Janoff and
Carp 11977) reported that tobacco smoke condensiit;: suppressed thk,
inhibitory action of humcn serum, J>urcficd tr,Pi, nnJ bronchopu!:::cJ-
n:;irv lavage fluids on both pctrcir~:~ :I::d ELI.:::!:; ;lastase. The
suppression of human serum e::istxtacl I,;ilib!tc>r>, r;,pa::ity 51; s:moke
condensate solutions in vitro has aisu been den-,-.:Ltl,;ltt-d bp others
~Ohlsson et al. 1980,.

Comparison of the protease inhibitory capacity ,)t` serum sarnples
from smokers and nonsmokers has revealed a significant depression
in smokers that is correlated with smoking history (Chowdhury
1981: Chowdhury et al. 1982). The latter st.udies also reported that
the depression of serum prot,ease inhibitor; was reiated to an effect
of smoke on the inhibitors per se. and not to a decrease in serum
antiprotease concentration. Still, the effect of tobacco smoke on
serum and lung lavage fluid antiprotease concenrr:~tion and activitj-
remains controversial. Several investigators have reported that
smokers have elevated serum protease inhibitor le\pels (Rees et al.
1975; Ashley et al. 1980); others (Olsen et al. 197.5: Warr et al. 19771,
like Chowdhury and colleagues, have shown no difference in serum
or lavage fluid protease inhibitor concentrations between smokers
and nonsmokers.

Gadek and associates (1979) compared cr,Pi activity of lung lavage
fluids taken from smokers and nonsmokers and found that smokers
had a twofold depression of functional a,Pi activity. The activity of
a,Pi in this study was tested against porcine pancreatic elastase. In a
similar study (Carp et al. 19821, in which human neutrophil elastase
was used, bronchoalveolar lavage fluids obtained from smokers had
40 percent less a,Pi activity than fluids from nonsmokers. However,
Stone and colleagues 11983) found that smokers' bronchoalveolar
lavage fluids did not exhibit decreased functional cr,Pi activity when
tested against either porcine pancreatic elastase or human neutro-
phi1 elastase, and suggested that increased elastaae derived from
neutrophils may be the main factor in the genesis of emphysema in
smokers.

Smokers may have a functional deficiency in bronchial mucus
protease inhibitor (BMPi) activity. A comparison of BMPi obtained
from tracheal aspirates of smokers with BMPi from nonsmokers

435


revealed that smokers' BMPi was 20 percent less active against PMN
elastase than nonsmokers' BMPi iCarp and Janoff 1980a).

Specific cigarette smoke constituents that may be responsible for
the inactivation of lung protease inhibitors have not been identified.
Nicotine and acrolein were studied for their ability to suppress a,Pi
activity and were found to be ineffective (Janoff and Carp 1977).
Several studies have shown that oxidizing compounds such as
chloramine-T and N-chlorosuccinimide can oxidize methionine
groups on a,Pi and reduce its activity against porcine pancreatic and
human PMN elastases (Cohen 1979; Johnson and Travis 1979; Satoh
et al. 1979; Abrams et al. 1980; Beatty et al. 1980). This has led to the
current belief that oxidants in cigarette smoke may be involved in
the inactivation of protease inhibitors (Janoff et al. 1983). In addition
to free radicals (Pryor 19801, cigarette smoke contains oxides of
nitrogen possessing their own free radical properties and able to
react with olefins in the gas phase or with peroxides to generate
potent oxy-radicals (Dooley and Pryor 1982; Pryor et al. 1983).

As mentioned previously, smoke condensate solution suppresses
the elastase inhibitory capacity of serum a,Pi in vitro. This
suppression can be prevented by the incorporation of phenolic
antioxidants into the test media (Carp and Janoff 1978). Similarly,
BMPi suppression by smoke condensate can be prevented by
antioxidants (Carp and Janoff 1980a). Cohen and James (1982) used
odianisidine oxidation to quantify the levels of oxidants in tobacco
smoke condensates from various brands of cigarettes and found that
oxidant levels correlated with capacity to suppress a,Pi deactivation
of elastase. Further, this study provided evidence that peroxides and
superoxide anions were responsible for the loss of a,Pi activity,
because inclusion of catalase and superoxide dismutase in the test
system reduced smoke condensate effects on a,Pi activity. Bron-
choalveolar fluid from smokers contains some amount of oxidant-
inactivated a,Pi, as evidenced by the presence of methionine
sulfoxide residues (Carp et al. 1982).

In addition to the numerous oxidizing agents present in cigarette
smoke, byproducts of smoke-stimulated phagocyte metabolism repre-
sent another potential source of oxidants capable of inactivating
lung protease inhibitors. Carp and Janoff (1979) demonstrated that
phagocytosing human PMNs produce activated oxygen species that
diminish the elastase inhibitory capacity of human serum and pure
a,Pi in vitro. These workers presented evidence to show that
hydroxyl radicals resulting from the reaction between superoxide
anions and H,O, were responsible for this effect. The inactivation of
a,Pi by a myeloperoxidase-mediated reaction was also described in
the study, which concurs with other studies demonstrating that
purified myeloperoxidase, in conjunction with H,O, and a halide ion,
can inactivate a,Pi in vitro (Matheson et al. 1979, 1981). Further

436


work has shown that activation of human blood monocytes, PMNs,
and PAMs by use of a membrane-perturbing agent (as opposed to
phagocytosis) results in the release of superoxide anions and H,O,
and the suppression of serum elastase inhibitory capacity (Carp and
Janoff 1980b). Clark and colleagues (1981) have demonstrated that
the myeloperoxidase-H,02-halide system from chemically stimulated
PMNs oxidizes a,Pi in vitro. Similar evidence ascribing inactivation
of BMPi to the myeloperoxidase-H,O,-halide system has been
reported (Carp and Janoff 1980a). In the studies noted above,
phagocytederived oxidants were shown to be capable of inactivating
a,Pi when porcine pancreatic elastase was used as the substrate.
These findings were recently extended to include the more pathophys-
iologically relevant protease, human neutrophil elastase (Zaslow et
al. 1983). Little is known about in vivo inactivation of protease
inhibitors by phagocyte-derived oxidants, other than that inactive
a,Pi (in the oxidized state) has been found in the synovial fluid of
patients with inflamed joints (Wong and Travis 1980). The extent to
which oxidants from stimulated phagocytes play a role in the
suppression of lung a,Pi activity in smokers is at present unknown.

Animal Studies

Although most of what is known about cigarette-smoke-induced
oxidant injury to lung protease inhibitors has been derived from
human studies, some work has gone into the effects of cigarette
smoke on lung protease inhibitors in laboratory animals. It has been
demonstrated that very brief exposure of rats to cigarette smoke can
cause a significant reduction in the elastase inhibitory capacity of
a,Pi obtained from lung lavage fluid (Janoff et al. 1979). Very likely
this toxic effect of cigarette smoke is caused by oxidant damage to
protease inhibitors, because treatment of the lavage fluid with a
reducing agent partially restored normal elastase inhibitory capaci-
ty and because animals rendered oxidant tolerant by preexposure to
ozone did not exhibit a significant reduction in a,Pi activity
following exposure to cigarette smoke.

Effects on Lung Tissue Repair Mechanisms

A preponderance of the research to elucidate mechanisms by
which cigarette smoking induces emphysema has focused on the
factors that initiate lung tissue degradation. Recent studies suggest,
however, that the increased risk of emphysema associated with
cigarette smoking may be due partially to the effects of smoke on
lung repair mechanisms.

437


Human Studies

For the most part, work concerning the effects of cigarette smoke
on lung repair mechanisms has been conducted in experimental
animals. It has been shown, however, that cigarette smoke contains
an inhibitor that can prevent the cross-linking of human fibrin
polymers and thereby impede normal tissue repair (Galanakis et al.
1982). Smoke and smoke constituents have also been shown to induce
membrane damage in human lung fibroblasts (Thelestam et al.
1980). Of 464 smoke constituents tested, approximately 25 percent
caused membrane damage. The most active constituents were
amines, strong acids, and alkylated phenols; nitriles and polycyclic
aromatic hydrocarbons were inactive.

Animal Studies

Cigarette smoke has been shown to affect elastin synthesis in vitro
and elastin repair in vivo. Laurent and coworkers (1983) determined
the effect of solutions of smoke condensate on elastogenesis in vitro
by measuring the formation of desmosine (one of the major cross-
linking amino acids of elastin) during conversion of tropoelastin to
elastin. Using a cell-free system of purified tropoelastin from chick
embryo aorta or porcine aorta and lysyl oxidase purified from chick
embryo or bovine lung, these investigators found that desmosine
synthesis was inhibited from 80 to 90 percent in the presence of an
aqueous solution of the gas phase of cigarette smoke. Elastin repair
in vivo has been reported to be retarded by tobacco smoke. Osman
and colleagues (1982) showed that hamsters with elastase-induced
lung injury resynthesized elastin at a reduced rate if they were
exposed to six or seven puffs of whole cigarette smoke hourly for 8
hours per day during the repair period.

Summary and Conclusions

1. The mass median aerodynamic diameter of the particles in
cigarette smoke has been measured to average approximately
0.46 pm, and particulate concentrations have been shown to
range from 0.3 x log to 3.3 x log per milliliter.
2. The particulate concentration of the smoke increases as the
cigarette is more completely smoked.
3. Particles in the size range of cigarette smoke will deposit both
in the airways and in alveoli; models predict that 30 to 40
percent of the particles within the size range present in
cigarette smoke will deposit in alveolar regions and 5 to 10
percent will deposit in the tracheobronchial region.
4. Acute exposure to cigarette smoke results in an increase in
airway resistance in both animals and humans.

438


5. Exposure to cigarette smoke results in an increase in pulmo-
nary epithelial permeability in both humans and animals.
6. Cigarette smoke has been shown to impair elastin synthesis in
vitro and elastin repair in vivo in experimental animals
(elastin is a vital structural element of pulmonary tissuei.

439


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