The Role of Oxygen Free Radicals

in Occupational and Environmental
Lung Diseases

Val Vallyathan and Xianglin Shi

Health Effects Laboratory Division, National Institute for Occupational
Safety and Health, Morgantown, West Virginia

Oxygen free radicals and their metabolites, collectively described as reactive oxygen species
(ROS), have been implicated in the pathogenesis of many diseases. The pulmonary system is
particularly vulnerable to ROS-induced injury because of its continuous exposure to toxic pollutants
from a wide variety of sources in the ambient air. Additionally, lungs are exposed systemically to
ROS generated from xenobiotic compounds and endogenous sources. This review describes the
sources of endogenous and exogenous ROS generation in the lung. Special emphasis is given to
major sources of ROS in occupational and environmental exposures to asbestos, crystalline silica,
coal, chromium, herbicides, bleomycin, and cigarette smoke. ROS-induced lung injury at different
target levels may contribute to similar patterns of cell injury and alterations at the molecular level
by initiation, propagation, and autocatalytic chain reactions. Intracellular signalling, activation
and inactivation of enzymes, stimulation, secretion, and release of proinflammatory cytokines,
chemokines, and nuclear factor activation and alterations are also common events. Understanding
the interactions of these intricate mechanistic events is important in the prevention and amelioration
of lung injury that results from acute and chronic exposures to toxins in ambient air. - Environ
Health Perspect 1 05(Suppl 1 :1 65-177 (1997)

Key words: asbestosis, coal workers' pneumoconiosis, environmental lung diseases, occupa-
tional lung diseases, oxygen free radicals, reactive oxygen species, silicosis

Introduction

Free radicals are defined as atoms or
molecules with one or more unpaired
electrons. Oxygen-centered free radicals are
those in which an unpaired electron is on an
oxygen atom. While carbon- and nitrogen-
centered free radicals are also important in
biology and are reactive to living cells,
oxygen-centered free radicals play a central
role in the pathogenesis of many pul-
monary diseases. Various biological systems
naturally generate oxygen free radicals and
they are also produced in these systems
from exposure to exogenous substances.
One of the most common and important
oxygen free radicals is the superoxide anion
(02'-), which can be dismutated to form
H202 and the highly reactive hydroxyl

radical (OH) in the presence of Fe2+ and
other trace metals.

Occupational and environmental lung
diseases are part of a heterogenous group
of diseases that result from exposure to
chemicals, gases, fumes, organics, miner-
als, and man-made dust; they can be tran-
sient, acute, or chronic. These diseases can
be defined as those resulting from inhala-
tion of physical, chemical, or gaseous
agents in the ambient air or from expo-
sure when these agents are taken up by
the body through other routes of entry.
Occupational and environmental lung dis-
eases are invariably attributable to specific
etiologic factors entering the body. Many
of these diseases have unique mechanistic

Manuscript received 6 June 1996; manuscript accepted 23 August 1996.

Address correspondence to Dr. V. Vallyathan, Pathology and Physiology Research Branch, HELD, National
Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505. Telephone: (304)
285-5770. Fax: (304) 285-5938. E-mail: vavl@niordsl.em.cdc.gov

Abbreviations used: ADP, adenosine 5'-diphosphate; AP-1, activator protein-1; ATP, adenosine triphosphate;
CWP, coal workers' pneumoconiosis; DABCO, 1,4-diazabicyclo[2,2,2]octane; dG, 2'-deoxyguanine; DMPO, 5,5'-
dimethyl-1-pyrroline-N-oxide; DPTA, diethylenetriamine pentaacetic acid; ESR, electron spin resonance; GS.,
glutathione thiyl radical; GSH, glutathione; iNOS, inducible form of NOS; LDH, lactate dehydrogenase; LPS,
lipopolysaccharide; MIP-1 a, macrophage inflammatory protein; NADPH, reduced nicotinamide adenine dinu-
cleotide phosphate; NF-icB, nuclear transcription factor; NO, nitric oxide; NOS, nitric oxide synthase; 02',
superoxide anion; *OH, hydroxyl radical; 8-OHdG, 8-hydroxy-2-deoxyguanosine; ROS, reactive oxygen species;
SOD, superoxide dismutase; TNF-OC, tumor necrosis factor.

processes that can be traced back in the
disease pathogenesis.

Considerable evidence has emerged in
recent years implicating a central role for
oxygen free radicals in the initiation of cel-
lular injury that leads to the development
of several lung diseases (1-3). It is well
established that oxygen free radicals and
their metabolites-collectively called reac-
tive oxygen species (ROS)-can induce
direct cell injury, which may trigger a cas-
cade of radical reactions promoting the
disease process. Furthermore, excessive
generation of ROS may lead to: the stimu-
lation of the inflammatory process; secre-
tion of chemotactic factors, growth factors,
proteolytic enzymes, lipoxygenases, and
cycloxygenases; inactivation of antiprote-
olytic enzymes; and activation of onco-
genes and transcription factors (1-8).

Within the lungs, ROS are generated
from many exogenous and endogenous
sources in biological reactions. Exogenous
sources of oxygen free radicals include
tobacco smoke, toxic gases, vapors, chemi-
cals, dust partides, and ambient air contain-
ing toxins. Industrial operations, congested
urban environments, and automobile emis-
sions can produce oxidative stress. The adult
human lung has a large respiratory volume
(-500-600 liters of air/hr) with a large sur-
face area (-75-85 mi2) and a profuse blood
supply. As a result, exposure to toxic pollu-
tants present in the ambient air over an
extended period of time could lead to
significant toxic insults, even when the pol-
lutants are at low levels. The lungs are also
systemically exposed to many toxic com-
pounds and xenobiotics that enter through
the gastrointestinal route.

This review focuses on the different
sources of exogenous and endogenous oxy-
gen free radicals that lead to ROS genera-
tion in the lung and the current role of
these sources in tissue injury and disease.
Although the role of antioxidant defenses is
important in the pathogenesis of the dis-
ease processes, it will not be discussed in
this review because this subject has been
reviewed elsewhere (9-12).

Endogenous Sources

Oxygen free radicals are constantly gener-
ated in living cells and are usually benefi-
cial to the metabolic processes. Major sites
of endogenous generation of ROS in the
biological system are within the mitochon-
dria, microsomes, endoplasmic reticulum,
phagocytic cells, endothelial cells, and

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165

VALLYATHAN AND SHI

nuclei. Because lipids, DNA, proteins, and
enzymes are susceptible to oxidative damage
by the endogenous generation of ROS, a
well-balanced antioxidant defense system
protects against such damage.
Mitochondria

Among the various organelles in the cell,
mitochondria are a major site of ROS gen-
eration during normal metabolic processes.
Cellular oxidative phosphorylation results
in the univalent reduction of oxygen and
the generation of ROS. However, a major-
ity of the univalent reduction of oxygen
results in the formation of water through
the mitochondrial cytochrome oxidase sys-
tem without the generation of ROS (13).
In addition, several other enzymic reactions
in the mitochondria may also lead to the
univalent or divalent reduction of oxygen
to produce O2'- or H202 (14). Xanthine
oxidase, for example, can undergo redox
cycling to produce 02'- and H202 during
the conversion of hypoxanthine to xanthine
before converting to uric acid. Similarly,
aldehyde oxidase could produce H202 and
02'- by its group-specific activity to aldehy-
dic substrate. A monoamine oxidase present
in the outer membrane of mitochondria
produces H202 during the conversion of
5-hydroxytryptamine to 5- hydroxyindol
acetic acid. Sequential reductions of flavo-
protein, ubiquinone, and cytochromes by
one-electron transfer reactions often result
in the generation of ROS. The cyto-
chromes present in the cell usually reoxi-
dize the ubisemiquinones generated in
these reductions; however, ubisemiqui-
nones may also interact with cytochromes
to generate 02--- The autooxidation of
reduced flavoprotein by dehydrogenase is
another source of O2- in mitochondria. To
protect the cellular integrity from damage
by 02-, mitochondria possess an abundant
supply of superoxide dismutase (SOD) and
vitamin E. These antioxidants function as
chain terminators of lipid peroxidation
(15). In addition, cytochrome oxidase is
also abundant in mitochondria to negate
oxygen radical-induced lung injury.
Microsomes

Microsomes are the second major source of
endogenous ROS generation in the cell.
During normal electron transport reac-
tions, they produce 02-- and H202. In
addition, autooxidation of cytochrome P450
and oxidation of NADPH by NADPH
dehydrogenase are the two major sources of
O2'- production. Endoplasmic reticulum
contains several mixed-function oxidases

targeted to oxidize xenobiotics entering the
lungs. Oxidation of relatively inert sub-
stances can also enhance the production of
ROS. Activation of nucleophiles through
reduction by the flavin monooxygenase sys-
tem is another potential source of ROS
generation in the microsomes (15).

A well-balanced SOD system in micro-
somes converts O2- to H202 and prevents
potential damage by ROS. The rate of
H202 production is also influenced by gly-
colate oxidase, D-amino acid oxidase, and
urate oxidase in the microsomes. In hyper-
oxia, lung microsomes produce 85% of the
total H202.
EAizymes

Several enzymes generate O2'- in the cell
(14). Xanthine dehydrogenase normally
present in the tissues can oxidize xanthine
or hypoxanthine to uric acid and transfer
electrons to NAD+. In oxygen-deprived
and -disrupted tissues, the oxidation of-SH
groups or proteolysis by Ca2+-stimulated
protease converts the xanthine dehydroge-
nase to xanthine oxidase. In hypoxia, the
oxidation of xanthine and hypoxanthine by
xanthine oxidase results in the generation of
02-, which leads to cell injury (8,14,15).

Indole amine dioxygenase, another
enzyme commonly present in tissues except
the liver, is involved in the generation of
02'- This enzyme can cleave serotonin,
tryptophan, and other related compounds
and generate O2'- in the catalytic process.
Administration of bacterial endotoxin or
presence of a viral infection is associated
with an increase in pulmonary indole
amine dioxygenase, causing an increased
generation of 02>

The tryptophan dehydrogenase present
in the liver can also generate 02-- by its
specific reactions with tryptophan. Aldehyde
oxidase can react with a variety of substrates
in the liver to produce 02>- Several other
enzymes, including galactose oxidase,
monoamine oxidase, cycloxygenase, and
lipoxygenase also can produce 02'- (15).
Phagocytes

Phagocytes are the well recognized and
uniquely endowed cells with the potential
to generate ROS when they encounter
microorganisms, inhaled particles, or other
stimuli (16-18). The activation of phago-
cytes triggers a respiratory burst, which is
characterized by an increase in oxygen
uptake, glucose metabolism, and utilization
of NADPH. A plasma membrane-bound
oxidase catalyzes this reaction, leading to
the generation of ROS (19-21). The O2'

generated is dismutated to H202, which
generates OH through Fenton or Fenton-
like reactions.
Metal Ions

It is generally believed that transition metals
play an important role in the generation of
ROS. Among these metal ions, iron is the
most intensively studied. The steps in iron-
mediated free radical generation (22-24)
are summarized below:

02+ cellular reductants-> 02' + H202
Fe(III) + 02' - Fe(II) + 02
2 02--+2H+->O02+H202

Fe(II) + H202 -- Fe(III) + OH + OH-

Fe(II)/Fe(III)  -OH   OH-
O2_+ H202        --*02+     H+0

It can be noted from these reactions
that Fe(II) and Fe(III) function as catalysts
and there is no net change in the oxidation
state. Thus, a trace amount of iron is capa-
ble of generating a significant amount of
OH radicals.

Iron can facilitate the decomposition of
lipid peroxides to aldehydes and hydrocar-
bons. It is known that the addition of iron
promotes the nonenzymatic oxidation of
epinephrine and glutathione to 02- and
H202. The majority of the iron in the
body is bound to hemoglobin, myoglobin,
cytochromes, enzymes, the transport pro-
tein transferrin, lactoferrin stored as fer-
ritin, and hemosiderin. However, many
biological reducing agents, such as ascor-
bate, cysteine, and reduced flavin, can pro-
mote the release of iron from ferritin.
Transferritin in the blood is usually loaded
to about 30% capacity so that free iron in
the plasma is maintained at a very low
level. A cellular store of iron is usually not
available in free form to mediate oxidative
damage through a Fenton reaction in vivo
unless the iron is detached from protein. A
drop in pH, such as that which occurs in
phagocytes by the rupture of phago-
lysosmes, may favor the detachment of iron
from protein. Therefore, under normal
circumstances many endogenous ligands
prevent participation of iron and other
common transition metals in the generation
of ROS in living cells.

When proteins are loaded incorrectly or
when chelating agents such as adenosine
triphosphate (ATP), adenosine 5'-diphos-
phate (ADP), citrate, or acidic pH are pre-
sent, iron will become detached and
promote enhanced OH radical generation
(25,26). 02-- has the potential to release
iron from lactoferrin, saturated transferrin,

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OXYGEN RADICALS AND LUNG DISEASE

and ferritin in a catalytically active form
(27-29). These reactions are important in
situations, such as inflammation, that
increase the generation of O2'- (20).

In addition to iron, other transition
metals such as As(V), Be(II), Cd(II),
Co(II), Cu(II), Hg(II), Pb(II), and Ni(II)
are known to promote free radical reac-
tions. The oxidative role of transition
metals provides growing evidence of metal-
induced carcinogenesis. Metals are capable
of binding in vivo with the cell nucleus
providing site-specific -OH generation and
DNA damage. An example is the presence
of Cu(II) in association with guanines in
DNA and the site-specific oxidative dam-
age of DNA. The binding of metals to
DNA results in several promutagenic alter-
ations in DNA, including DNA strand
breaks, inter- and intramolecular cross-
linking of DNA and proteins, dearrange-
ments, modifications, and depurination
(30). Metals may also contribute to the
underlying pathogenic mechanism by
promoting inflammation, inhibiting anti-
oxidant defenses, inhibiting DNA repair,
and enhancing lipid peroxidation (30).
Metals may also bind to nuclear RNA
and gene regulatory proteins producing
finger-loops (31).

Exogenous Sources

Because of constant exposure to ambient air
containing toxic particulates and oxidant
gases such as nitrogen oxide and ozone, the
lungs are more susceptible to oxidant injury
than any other organ in the body. Further-
more, the lungs are also highly perfused
with a copious supply of blood, which
makes them more vulnerable to many
xenobiotics such as bleomycin, the herbi-
cides paraquat and diaquat, semiquinones,
and certain drugs.
Asbestos

Asbestos is a family of inorganic minerals
that cause pulmonary fibrosis (asbestosis),
malignant mesotheliomas, lung cancer,
and other pleural diseases (32,33). Based
on its physicochemical characteristics, the
family of asbestos minerals is subdivided
into serpentines and amphiboles. Chrysotile
is the most common serpentine asbestos
and accounts for more than 90% of the
world's production and industrial use.
Chrysotile fibrils consist of concentric
cylindrical tubes made of silicate, mag-
nesium, and a small amount of iron.
Amphiboles are of lesser commercial
importance and are distinctly different in
physicochemical characteristics. They

generally contain several cations and greater
concentrations of iron (32,34).

The toxicity, pathogenicity, and carcino-
genic potential of various types of asbestos
differ considerably because of their physical
and chemical properties. Laboratory studies
and epidemiologic studies on exposed popu-
lations indicate that fiber shape, fiber size,
surface area, surface charge, and biologic
durability are important determinants of
pulmonary response (32,33,35-38). The
morphology of asbestiform minerals with a
high length-to-breadth ratio (aspect ratio) is
generally thought to be a major factor in
the pulmonary pathogenicity and carcino-
genity of these minerals (39). However,
the mechanisms that cause these various
types of asbestos-associated diseases remain
speculative and other studies suggest that
none of these factors sufficiently account
for the divergent cellular events involved in
asbestos-related diseases (40).

Many investigators (33,40-43) have
suggested that ROS generation catalyzed
by asbestos minerals has an important role
in the early events of cell injury. The iron
present in asbestos fibers can promote the
generation of ROS during the process of
phagocytosis. The iron catalyzes the forma-
tion of OH radicals from H202, as ferrous
ions are oxidized by H202 to ferric ions.
This reaction is dependent on the availabil-
ity of H202. During phagocytosis of long
asbestos fibers by alveolar macrophages,
the sustained generation of 02-- leads to
an increased supply of H202, which leads
to a chain reaction of OH generation cat-
alyzed by iron present in the fibers. Higher
concentrations of elemental iron present in
the fibers enhance this potential signifi-
cantly. Amphiboles (amosite -34%, croci-
dolite -18%, tremolite -4%) generally
contain greater concentrations of iron than
serpentine (chrysotile -1-2%).

Weitzman and Graceffa (42) first sug-
gested the role of iron in asbestos toxicity
and pathogenicity by providing evidence of
OH radical generation. They showed that
amosite, crocidolite, and chrysotile asbestos
catalyze the generation of OH radicals
from H202 in the presence of the radical
spin trap 5,5-dimethyl-1-pyrroline-N-
oxide (DMPO). Weitzman and Graceffa
(42) observed a stronger electron spin
resonance (ESR) signal from amosite than
from crocidolite and chrysotile. The gen-
eration of OH radicals was strongly
inhibited in the presence of the iron chela-
tor desferroxamine, suggesting the impor-
tant role of iron in asbestos-generated
*OH radicals (43-45).

A variety of asbestos fibers and other
minerals in the presence of spin traps have
been used to study the mechanisms
involved in the *OH radical generation
from H202. Gulumian and Van Wyk (46)
reported that glass fibers containing lower
concentrations of iron than asbestos gener-
ated more "OH radicals from H202.
However, the iron present in glass fibers
was essential for the *OH generation, since
it was shown that the addition of iron
chelators desferroxamine or diethylenetri-
amine pentaacetic acid (DPTA) inhibited
-OH radical generation.

Fiber dimension and length correlate
with cytotoxicity and the induction of
mesothelioma (33,39). The observed bio-
logical activity of fibers could be due to the
surface phenomena of ROS generation.
Several in vitro and in vivo observations
support this hypothesis (45-48). When
chrysotile asbestos was heated, the toxicity
decreased, possibly because of loss of elec-
trons, which results in a decreased poten-
tial to generate ROS. X-irradiation restored
the potential to generate oxygen radicals.

Phagocytosis of asbestos activates and
enhances the release of significant amounts
of O2'- from alveolar macrophages and
neutrophils (40). Release of O2'- from
phagocytes occurs spontaneously after in
vitro and in vivo exposure to asbestos and
continues for several hours. Asbestos, which
is indestructible, may result in repeated
phagocytosis and continued O2'- produc-
tion. This increased generation of O2--
results in the dismutation and increased
formation of H202. The generation of
more potent oxidizing *OH radicals pro-
moted by the asbestos-bound transition
metals through a Fenton reaction is the cru-
cial reaction leading to cell injury. The
ability of -OH radicals to initiate and prop-
agate lipid peroxidation in cell membrane
phospholipids may lead to cell injury and
cell death. In addition to cell membrane
injury, ,OH radicals can disrupt mitochon-
drial integrity, cause DNA strand breaks,
and inactivate proteins.

In cellular injury, disruption of the
cell membrane is thought to occur from
ROS. A major indicator of cell mem-
brane pertuburation and functional status
is the cell viability index. Alveolar macro-
phages exposed to asbestos exhibit much
greater membrane perturbation than
those exposed to glass fibers of similar size
and dimension (49). Asbestos containing
greater concentrations of iron generally
induced significant functional impairment
in cell membranes.

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VALLYATHAN AND SHI

Lipid peroxidation is one of the most
well-recognized and widely studied bio-
logic interactions of -OH-induced meta-
bolic alteration (50-52). An increase in
lipid peroxidation products in tissues and a
rise in lipid peroxides in body fluids are
generally considered important indicators
of 'OH-associated pathological processes
(53). A sequence of molecular reactions
leading to the formation of peroxides by
the destruction of cell membrane phospho-
lipids is initiated by 'OH radicals. In the
primary reaction of 'OH with polyunsatu-
rated fatty acids in cell membranes, the
abstraction of a hydrogen atom produces a
carbon-centered free radical. These lipid
radicals then react with molecular oxygen
to form lipid peroxy radicals. The lipid
peroxy radical can abstract another hydro-
gen atom from another double bond, initi-
ating an autocatalytic process.

Gabor and Anca (54) first demonstrated
that asbestos can initiate lipid peroxidation
in a cell model of red blood cells. Gulumian
et al. (55,56) showed that crocidolite
asbestos enhanced lipid peroxidation in rat
lungs and liver microsomes. Using a cell-
free phospholipid emulsion, Weitzman and
Weitberg (52) monitored the production
of thiobarbituric acid reactive substances
by crocidolite, amosite, and chrysotile, all
treated and untreated with desferroxamine.
They showed that amosite and crocidolite
caused significant lipid peroxidation,
whereas chrysotile asbestos, containing the
least iron, induced the least lipid peroxi-
dation. The iron chelator desferroxamine
inhibited the ability of all three types of
asbestos to stimulate lipid peroxidation
(52). In a rat liver model, lipid peroxida-
tion was enhanced significantly by the syn-
ergistic action of NADPH and crocidolite
and chrysotile asbestos (57).

DNA is a crucial target molecule in a
cell confronted with direct ROS insults.
Generation of ROS in close proximity to
DNA can cause single or double strand
breaks. By assessing the degree of ethidium
bromide fluorescence, DNA strand breaks
in DNA mixtures exposed to heat and alka-
line denaturation can easily be detected.
Jackson et al. (58) demonstrated that cro-
cidolite asbestos and cigarette smoke syner-
gistically increased the amount of strand
breaks in isolated DNA. An oxidized base
product of direct oxygen radical attack on
DNA is 8-hydroxy-2'-deoxyguanosine
(8-OHdG) (59). Asbestos-induced oxida-
tive damage can be monitored by the direct
measurement of 8-OHdG in a cell-free sys-
tem using a sensitive high-pressure liquid

chromatography (HPLC) technique cou-
pled with electrochemical detection (60).
Measurement of unscheduled DNA syn-
thesis using autoradiographic techniques
can also be used to monitor oxidative DNA
damage. Libbus et al. (61) have shown that
rat embryo cells exposed to crocidolite
asbestos and mineral fibers produce oxida-
tive DNA damage. Asbestos has also been
shown to cause multiple chromosomal
alterations, including breaks and transloca-
tion. These changes include chromosome
number, aneuploidy, polyploidy, and
structural abnormalities (62).
Crystalline Silica

Siicosis is the oldest pneumoconiosis known
to be associated with exposure to crystalline
silica in occupations such as stone cutting,
quarrying, and mining (63). Exposure to
crystalline silica results in an initial dramatic
pulmonary inflammation and leads to a
debilitating pulmonary fibrosis. The early
cellular injury and tissue damage are consid-
ered important events in the development of
silicosis. Crystalline silica-induced activation
of pulmonary phagocytes is considered
important in the generation of oxidants that
in turn injure lung cells (40,64-67).

Silicosis is manifested pathologically in
two distinct common entities, i.e., chronic
silicosis and acute silicosis (63). Chronic
silicosis occurs two or three decades after
first exposure in occupations such as quar-
rying, stone cutting, and mining and is
characterized by the development of con-
centric nodular lesions in the upper lung
lobes. With the conglomeration of nodular
lesions, chronic simple silicosis develops
into complicated progressive massive fibro-
sis. Acute silicosis, on the other hand, is
characterized by the rapid onset of diffuse
alveolar silicolipoproteinosis-within 1 to
3 years after initial exposure to crystalline
silica-in primary occupations such as
silica flour mill operations, surface mine
drilling, tunneling, sandblasting, and pot-
tery making. Acute silicosis is rapidly pro-
gressive, with a high mortality rate, and is
associated with occupations where fractur-
ing of the molecular structure of silicon
dioxide occurs (64-67). Mechanically
breaking a silicon dioxide structure produces
the open molecules Si and Si-O on frac-
tured cleavage planes (64-67). These mol-
ecules exist on freshly fractured crystalline
silica as surface free radicals.

Generation of Free Radicals by Silica.
Using ESR spectroscopy, Hochstrasser and
Antonini (68) first reported that the frac-
turing of single crystals of quartz under

ultrahigh vacuum (10-10 torr) produced
silicon-based radicals, which are stable for
months. When exposed to ambient air, the
concentrations of these radicals instanta-
neously decrease by a factor of five and then
decay, with a half-life of approximately 36
hr. Hochstrasser and Antonini (68) also
reported that the newly generated silica sur-
face is chemically reactive and is capable of
absorbing and reacting with various gases.
For example, reaction of Si' with CO2 gen-
erates the SiCOO' complex. The bonding
between the Si' and CO2 molecules is par-
tially covalent and partially ionic, as indi-
cated by Si29 hyperfine coupling. Later
studies show that grinding quartz crystal
generates not only Si- radicals but also SiO'

and SiOO- radicals (69,70). The ratio of
the concentrations of [SiO']:[SiOO'] on
the oxidized surface is almost 1:1.

Bolis et al. (70) analyzed the surface
properties of silica of different origins and
found some characteristics of ground quartz
dust that can be related to silica toxicity.
These characteristics suggested that radicals
produced by grinding may act as reactive
sites for the disassociation of water and the
oxidation of proline. They also suggested
that the free radicals generated by grinding
may react within the cell, causing cascade
reactions and releasing fibrogenic factor.

Fracturing crystalline silica, as in occu-
pational settings such as sandblasting, rock
drilling, tunneling, and silica flour mill
operations, results in the generation of free
radicals on the cleavage planes (64,65). It
was shown that surface radicals are com-
prised mainly of silicon-oxygen radicals
and that they decay with time. The relative
intensity of surface radicals increased signi-
ficantly with extended grinding and was a
reflection of the generation of greater num-
bers of surface radicals. The concentration
of surface radicals generated by mechanical
grinding decreases following first-order
kinetics, with a T1/2 of approximately
30 hr in air (64,65).

The silicon-oxygen radicals on the sur-
face of cleavage planes can react with aque-
ous media to generate the most toxic,
short-lived -OH radicals. The generation
of short-lived 'OH radicals has been
detected by ESR with the aid of the spin
trap DMPO (64,65). Freshly fractured
crystalline silica in the presence of DMPO
exhibited a significantly stronger ESR sig-
nal, with a typical 1:2:2:1 quartet pattern
that is characteristic of the DMPO-OH
adduct. Competitive ethanol trapping and
quenching by hydroxyl scavengers further
verified the -OH radical generation. The

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OXYGEN RADICALS AND LUNG DISEASE

ability of fractured crystalline silica to
react with aqueous media and generate
OH radicals also decreased with time,
after fracturing with a T1/2 of approxi-
mately 20 hr. The ESR signal intensity of
OH radicals produced from freshly frac-
tured crystalline silica decreased substan-
tially in the presence of catalase, SOD,
mannitol, and diethyl thiourea (64).

In our laboratory we have shown that
freshly fractured crystalline silica contain-
ing silicon-oxygen radicals is biologically
more reactive toward pulmonary cells than
is aged crystalline silica (64-67). Freshly
fractured crystalline silica exhibited greater
biologic toxicity, causing more cell mem-
brane damage (400%), increased leakage of
enzymes (47%), increased cell death (65%),
and more lipid peroxidation (300%) than
aged crystalline silica of similar size (66).
The ability of freshly fractured crystalline
silica to oxidize polyunsaturated fatty acids
strongly correlated with its ability to gener-
ate OH radicals (66). It is known that
OH radicals can cause lipid peroxidation
of the cell membrane (71). The surface-
based crystalline silica radicals were shown
to play a central role in the enhanced
inflammatory process in rats exposed to
freshly fractured crystalline silica (66).
Inhalation of freshly fractured crystalline
silica resulted in substantial pulmonary
inflammatory response, indicated by differ-
ential cell numbers in lavage fluid, com-
pared to animals exposed to aged silica (total
cells, 119%; macrophages, 81%; neu-
trophils, 96%; lymphocytes, 112%; red
blood cells, 253%) (66). Besides the
enhanced inflammatory response exhibited
in animals exposed to freshly fractured
crystalline silica, the animals also exhibited
substantial increases in the release of bio-
chemical markers of cytotoxicity and lung
injury. Additionally, they induced increased
lipid peroxidation, showed up-regulated
levels of antioxidant enzymes, and pro-
duced more ROS during phagocytosis
(66). Animals exposed to freshly fractured
crystalline silica exhibited 37% more lipid
peroxidation than animals exposed to
aged silica. Inhalation of freshly fractured
silica resulted in an increase in protein
(34%), albumin (13%), and N-acetylglu-
cosaminidase (260%) compared to animals
exposed to aged silica. Lactate dehydroge-
nase (LDH), a cytoplasmic enzyme, showed
a 340% increase in concentration and the
lysosomal enzyme showed a 255% increase
in animals exposed to freshly fractured silica
(66). By contrast, SOD and glutathione
peroxidase showed significant declines in

concentration in the animals exposed
to freshly fractured silica compared with
animals exposed to aged silica. In both
exposed groups the antioxidant enzyme
concentrations were significantly elevated
compared to those of controls, suggesting
an up-regulated antioxidant defense system
and its compromise in the animals exposed
to freshly fractured silica (66). Consistent
with these results, the bronchoalveolar
phagocytes showed a 35% increase in the
generation of ROS in animals exposed to
freshly fractured silica (64).

These in vivo studies agree with in vitro
studies that confirm the presence of reac-
tive sites on the surface of freshly fractured
crystalline silica and the significant role
they may play in the pathogenesis of acute
silicosis (64). As reported by Shi et al.
(72), in addition to -OH radicals, freshly
fractured silica particles in aqueous suspen-
sion are also capable of generating superox-
ide (O?2-) radical and singlet (102) oxygen.
They have shown that 102 generated from
freshly fractured silica can cause hydroxyla-
tion of dG residue in DNA to generate
8-OhdG. The 102 generated by silica par-
ticles may provide another significant bio-
logical plausibility in the mechanism of
silica-induced cellular injury.

Silica-induced DNA Damage.
Monitoring DNA double-strand breaks,
Daniel et al. (73) have shown that silica
causes DNA double-strand breaks in vitro.
Chemical etching of silica particles with
hydrochloric acid to remove metal ion
impurities and reactive centers created by
fracturing the surface resulted in markedly
diminished DNA-damaging ability. The
DNA damage was blocked by catalase and
by the -OH radical scavenging agents
DMSO and sodium benzoate. The DNA
damage in this study was considered to be
due to the OH radicals. Shi et al. (72)
have demonstrated that freshly fractured
quartz can also cause DNA double-strand
breaks through free radical reactions. The
observation that quartz particles can cause
DNA damage via oxygen-dependent, free
radical-mediated reactions may significantly
contribute to our understanding of the mech-
anism of quartz-induced carcinogenesis,
especially at the initiation stage.

Silica-induced Activation of Nuclear
Transcription Factor-KB. It has been
shown that exposure of alveolar macro-
phages to silica particles initiates produc-
tion of inflammatory mediators and
cytokines from several earlier response
genes (74,75). A frequent regulatory event
in such a response is the deregulation of

mRNA synthesis by transcription activator
proteins. The activity of these proteins can
be induced by a variety of mechanisms, fre-
quently involving the control of their DNA
binding, nuclear uptake, or the assembly or
disassembly of protein subunits. Recently,
Chen et al. (76,77) demonstrated that silica
can efficiently induce activation of nuclear
transcription factor (NF)-KB in the mouse
macrophage cell line RAW 264.7 and that
silica displays different characteristics from
lipopolysaccharide (LPS) in the activation
of NF-KB, both with respect to NF-icB
subtype composition and to the signal trans-
duction pathway that leads to the activation
of NF-iB. Chen et al. (76,77) have also
shown that abrogation of NF-KB activa-
tion by inhibiting the protease dramatically
reduces the mRNA expression of cyclooxy-
genase II, inducible nitric oxide synthase,
tumor necrosis factor a, and interleukin-
la. These findings suggest that NF-icB
plays an important role in silica-induced
inflammatory mediator production in the
phagocytic cells.

While the mechanism of silica-induced
activation of NF-wB is unclear, most, if
not all, inducers of NF-iB seem to rely on
the production of ROS, as is evident from
the inhibitory effect of antioxidants on
induction of NF-KB by all inducers tested
thus far (78,79). Since silica particles are
able to generate ROS such as H202 and
OH, it is likely that the mechanism of
the NF-KB involves silica-mediated free
radical reactions.
Coa

Inhalation of coal mine dust leads to the
development of coal workers' pneumoco-
niosis (CWP), silicosis, chronic bronchitis,
emphysema, and Caplan's syndrome (80).
Many factors, such as rank of coal, method
of mining, geographic location, and con-
centrations of minerals in the coal mine
dust, have been shown to play a part in the
prevalence, severity, and type of disease. In
addition, other unknown factors or par-
tially characterized factors are also probably
important in the pathogenesis of coal
workers' pneumoconiosis (80,81).

Many studies have reported on the
presence of coal-based, carbon-centered
free radicals in coal (82-85). These studies
showed that free radical concentration
increased with the mechanical breaking of
chemical bonds and postulated that miners
breathing freshly mined coal are exposed to
more toxic coal dust containing free radicals
(84,85). However, the carbon-centered
coal radicals are generally considered stable

Environmental Health Perspectives * Vol 105, Supplement I * February 1997

169

VALLYATHAN AND SHI

and entrapped in the coal and thus may
not be available for biologic reactions.
Nevertheless, the concentration of stable
coal radicals in the lungs of miners has
shown good correlation with coal mining
tenure and disease severity (86,87). These
results cannot be fully explained without
additional knowledge of other factors,
including mobilization of trapped radicals,
presence of transitional metals, and half-
lives of mobilized radicals.

Recently, Dalal et al. (88) showed that
five coal mine dust samples studied for the
generation of -OH radicals from H202 dif-
fered markedly in their potential to gener-
ate -OH radicals. Although all the coal
samples generated -OH radicals in the
presence of H202, there was good correla-
tion between the prevalence of CWP and
OH radical generation in coal mine dust
from the same geographic location. Lipid
peroxidation potential and surface iron
concentration in the coal mine dust also
showed good correlation to -OH radical
generation. From these studies it was evi-
dent that surface iron in coal mine dust is
an important factor in the generation of
OH radicals, increased cellular injury, and
the prevalence of CWP in different coal
mining areas (88).

Focal emphysema is recognized as an
integral part of the primary CWP lesion
macule. The relationship between coal min-
ing and disabling emphysema is an impor-
tant health issue of considerable scientific
interest. In a recent study, Huang et al. (89)
examined the relationship between a-1-
antitrypsin inactivation by five coal mine
dust extracts in aqueous solutions and its
possible relation to emphysema in coal min-
ers. They reported that coal mine dust from
Vouters coal fields in France, with the high-
est prevalence of emphysema, produced
more ROS and inactivated a-1-antitrypsin.
Diesel

Diesel-powered automobiles, public trans-
portation, and heavy equipment have
become more and more commonplace in
the last three decades. With the increasing
number of diesel-powered automobiles on
the road, the health effects of diesel expo-
sure have become a major concern in
underdeveloped countries. This increased
use of diesel-powered vehicles has also
aroused major concern about the potential
health effects of their use in underground
coal mining. Diesel emissions contain
diverse, potentially toxic materials in the
form of gases, metals, chemicals, and par-
ticulate matter. Many of these complete

and incomplete products of combustion are
known to be genotoxic, cytotoxic, fibro-
genic, and/or carcinogenic (90-93). Diesel
engines produce much more particulate
emissions than conventional engines. These
particulate emissions consist of insoluble
carbonaceous partides covered with solvent-
extractable organic compounds. Diesel-
emission particles are submicroscopic in size
with a relatively high rate of pulmonary
deposition. It is estimated that diesel vehi-
cles on average release 1 g of particulate
matter into the ambient air with every 1 km
of distance operation.

Pulmonary toxicity, carcinogenicity,
and other chronic effects of diesel emis-
sions are mainly associated with absorbed
materials (polycyclic aromatic hydrocar-
bons, nitrosamines, quinones) on the sur-
face of particulates (94-96). Filtered diesel
emissions evoke only minimal pulmonary
changes and tumors in experimental ani-
mals (97). The reaction of diesel exhaust
with nitrogen dioxide is known to enhance
cytotoxicity and mutagenicity. Gu et al.
(98) showed that diesel emission particu-
lates are mutagenic in the Ames assay and
that they can induce unscheduled DNA
synthesis and damage.

Ross et al. (99) characterized free radical
generation from diesel particulate using
ESR spectrometry. The free radical signals
were sensitive to oxygen, nitric oxide (NO),
nitrogen dioxide, and ultraviolet radiation.
Vogl and Elstner (100) showed that diesel
soot particulate can catalyze the release
of ethylene from a-keto-y-methylthio-
butyrate, which suggests of the release of
strong oxidants. They showed that the
photodynamic catalysis reaction can be
inhibited by radical scavengers (a-toco-
pherol, 100%; catalase, 82%; azide, 40%;
1 ,4-diazabicyclo[2,2,2]octane (DABCO),
83%). From these studies, they concluded
that diesel particulates produced H202 by
the catalyzed oxidation of cysteine and that
the depletion of cysteine enhances the pro-
duction of ROS. Sagai et al. (101) have
demonstrated that diesel exhaust particles
could produce O2- and "OH radicals in
vitro without any biological activating sys-
tems. In this reaction system, 02' and
*OH production was inhibited by the addi-
tion of superoxide dismutase and dimethyl-
sulfoxide, respectively. They suggest that
most parts of diesel exhaust particle toxic-
ity are due to ROS such as O2- and 'OH.
Nagashima et al. (102) showed that the
8-OHdG levels in mice lung DNA were
significantly increased by the administra-
tion of diesel exhaust particles. Diesel

exhaust particles generated oxidative DNA
damage, leading to genomic instability and
the formation of 8-OHdG in DNA, which
may be responsible for mutations leading
to lung tumorigenesis in mice (102).
Chromium

Many metals, including chromium, nickel,
cadmium arsenic, beryllium, and lead, are
known carcinogens in humans and animals.
The mechanism of metal carcinogenesis is
not fully understood, although increasing
evidence indicates that metal-mediated
ROS reactions may play an important role.
Since the basic mechanism of ROS produc-
tion and related cellular damage is similar
to many carcinogenic metals, we will only
discuss chromium.

Chromium(VI)-containing compounds
have been shown to be potentially geno-
toxic in a number of in vitro and in vivo
studies (103-105). However, the underly-
ing biochemical mechanism for the car-
cinogenicity is not fully understood. The
Cr(VI) anions do not interact with isolated
DNA. The reduction of Cr(VI) by cellular
reductant to lower oxidation states has
been considered an important step in the
mechanism of Cr(VI)-induced DNA dam-
age (106-108). Using ESR and spin trap-
ping, Shi and Dalal (109) detected the
formation of Cr(V) and glutathione (GSH)
thiyl radical (GS-) in the reduction of
Cr(VI) by GSH. An increase in the con-
centration of GSH enhanced the relative
yield of both Cr(V) and GS' radical. The
GSH radical may react with another thiol
molecule to generate an 02-- radical, lead-
ing to formation of H202. The biologically
generated Cr(V) complexes are able to gen-
erate -OH radical from H202 through a
Fenton-like reaction (110).

The *OH radicals generated by Cr(IV)-
and Cr(V)-mediated reactions can react
with 2'-deoxyguanine (dG) and DNA to
generate 8-OHdG (111,112). It may be
noted that 'OH radicals generated in the
reaction of H202 with some other metal
ions, such as Ni(II), copper, or zinc con-
taining superoxide dismutase, exhibit very
limited reactivity. For example, the -OH
radicals generated by these systems cannot
be scavenged by either ethanol or formate.
It has been suggested that the -OH radicals
are generated within the domain of certain
macromolecules and hence are not free to
exhibit significant reactivity. The 8-OHdG
generation from the reaction of Cr(V) with
H202 demonstrates that 'OH radicals gen-
erated by this reaction are free and do have
the potential to cause cellular damage.

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OXYGEN RADICALS AND LUNG DISEASE

Cr(V) species reportedly have been
generated in the reduction of Cr(VI) by
various biological systems, in particular,
microsomes, mitochondria, and ascorbate
(113). Recently, Cr(V) formation has
been observed from whole living mice
treated with Cr(VI) using L-band (1.2
GHz) ESR spectrometry (114,115). This
Cr(V) has been identified as Cr(V)-
NAD(P)H complex. This finding is very
important, since the reactivity of Cr(V)
toward H202 to generate OH radicals
depends upon the structure of the Cr(V)
species. For example, Cr(V)-NAD(P)H
complex, and not CrO83-, is able to gen-
erate OH from H202.
Bleomycin

Several drugs used in the treatment of
human cancer and other disease condi-
tions can undergo oxidation-reduction
reactions and cause oxidative damage.
Some compounds of interest are meperi-
dine (a heroin designer drug), haloperidol,
salsolinol, and bleomycin.

Bleomycin is a cytotoxic glycopeptide
with powerful antineoplastic properties
and is commonly used in the treatment of
lymphomas and testicular tumors (116).
Bleomycin drug therapy causes severe pul-
monary toxicity, complicated by interstitial
pneumonitis, which leads to irreversible
pulmonary fibrosis. An acute inflammation
characterizes the initial reaction to the drug
in the lung. Several risk factors are consid-
ered important in bleomycin therapy. They
indude the dosage, age of patient, and con-
comitant oxygen and radiation therapy.
The risk of increased bleomycin-induced
pulmonary injury through oxygen and radi-
ation therapy is attributed in part to their
ability to promote free radical generation
and ROS-induced injury (117-120).

Bleomycin-induced pulmonary toxicity
is thought to occur through the mecha-
nism of ROS (117-120). Intracellularly,
bleomycin-Fe(II) complexes are formed,
which in turn produce ROS, causing DNA
damage and pulmonary fibrosis. Bleomycin
inhibits DNA and protein synthesis.
Significant increases in DNA strand breaks,
fragmentation, and activation of DNA
polysynthase activity have been reported in
bleomycin-induced lung fibrosis in
experimental animals. The mechanism of
DNA damage is related to the structural
components of bleomycin. The bithiazole
component of bleomycin is reported to
intercalate into a DNA helix, separating
the strands and changing pyrimidine and
imidazole. This complex forms with iron

to create the potential oxidative sites on
the nucleotides (119-121).

Initial pulmonary response to bleomycin
therapy is characterized by an acute inflam-
matory reaction associated with edema.
Bleomycin-induced lung damage subse-
quently occurs through enhanced lipid per-
oxidation, DNA damage, loss of enzymes,
and defective protein synthesis. Two major
pathways of cell injury are thought to
occur in bleomycin toxicity: one is depen-
dent upon DNA damage and the other is
independent of DNA damage.
Herbicides

Commonly used herbicides such as
paraquat, diquat, and other related chemi-
cals can be reduced by cellular enzymes
and by redox cycling. The herbicides are
oxidized to ROS in the presence of molec-
ular oxygen. Smith (122) has reviewed the
mechanism of paraquat toxicity in the
lung. Paraquat reacts with NADPH and
P450 reductase in cells, causing an electron
reduction and resulting in the generation
of paraquat radicals. This reaction is per-
petuated by a cascade of radical reactions
and results in the generation of more
ROS. The organ-specific toxicity of the
lung to these herbicides is considered to
be associated with the high oxygen ten-
sion and site-specific accumulation of the
paraquat in the lung type I and type II
cells. This selective accumulation in the
epithelial cells is thought to be dependent
on a diamine/polyamine transport process
located in the cells. The toxicity of these
compounds in lung tissue can be dimin-
ished by lowering oxygen supply. Recently
it was shown that in paraquat-induced
lung injury, NO plays a mediator role in
pathogenesis (123).
Nitric Oxide

Nitric oxide is a colorless gas used in manu-
facture of nitric acid. It is also used commer-
cially as an inhibitor of free radical-induced
decomposition of organic compounds. NO
is a short-lived radical, highly reactive and
lipophilic in nature, with a very short life
of a few seconds (124). NO reacts with air
to produce nitrogen dioxide and nitrogen
tetroxide. Human exposure to NO results
in pulmonary inflammation culminating in
acute edema and a high risk of mortality.
NO is known to cause cell and DNA dam-
age and induction of cGMP (125-127). In
the cell, NO is oxidized to biologically
inert nitrates and nitrites in the presence of
02'- and H202. NO is capable of inducing
iron depletion from iron stores, which in

turn is correlated with the activation of
guanylate cyclase and inhibition of mito-
chondrial respiration. NO interacts with
oxyhemoglobin to form methemoglobin
and nitrate. NO has a great affinity for iron
and binds readily with iron-containing
proteins such as hemoglobin, myoglobin,
cytochrome c, and guanylyl cyclase. It is
also reported to interact with O2- to pro-
duce peroxynitrite, which in turn can cause
DNA damage and oxidation of thiols
(128,129). The reaction of NO with O2-,
generating peroxynitrite, is almost at diffu-
sion-controlled rate (k = 6.7 ? 0.9 x 109
M-Ys). Peroxynitrite can oxidize a variety
of biomolecules, including lipids, sulfides,
thiols, and abscorbate (129). In vitro
experiments have shown that peroxynitrite
at concentrations similar to those observed
in NO-exposed cells can inactivate mito-
chondrial electron transport and ATPase
(130). In other instances, NO may func-
tion as a scavenger for O2- and limit O2-
radical-induced damage (130).

NO is generated enzymatically in the
cells by NO synthases, which are homol-
ogous to cytochrome P450 reductase
(131). Many tissues contain this constitu-
tive enzyme, but in the activated alveolar
macrophage, an inducible enzyme indepen-
dent of Ca2+ is present. Cytokines are impor-
tant inducers of NO synthase. Macrophage
NO is cytotoxic to microbial cells and tumor
cells. NO synthase inhibition attenuates
immune complex-induced vascular injury
and inflammation (123,124).
Ozone

Ozone (03) is a faintly blue gas with a
peculiar chlorinelike smell. In commercial
operations, ozone is used in certain bleach-
ing processes and for sterilizing water and
purifying air. An electric discharge or the
slow burning of phosphorous can produce
ozone. Ozone is present in ambient air as a
normal constituent but in urban air, levels
above those recommended by National
Ambient Air Quality Standards are often
reported (131). Ozone is produced from
several photochemical reactions and from
the combustion of automobile fuels. NO
emission from automobiles is oxidized to
nitrogen dioxide which in turn can be pho-
tochemically oxidized to form ozone. Ozone
in the strastospere is important because it
protects the earth from sun's ultraviolet
radiation. Chemicals, chlorofluorocarbons,
nitrogen dioxide, combustion engines, and
several industrial processes destroy ozone by
converting the ozone to free radicals. Ozone
itself is not a free radical, but it is a highly

Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997

171

VALLYATHAN AND SHI

reactive oxidant gas. In aqueous media,
ozone decomposes to produce H2O2 and
02. Exposure to ozone is associated with
airway hyperresponsiveness, airway inflam-
mation, bronchoconstriction, pulmonary
function perturbations in exercise, immuno-
suppressive effects, enhanced susceptibility
to infection, and increased incidence of
melanoma (132-135). Ozone in combina-
tion with acid aerosols exacerbates these
pulmonary effects, resulting in increased
morbidity. Asthmatics are considered more
susceptible to the adverse respiratory effects
of ozone than nonasthmatics.

The mechanism of ozone-induced
damage to cells is caused by the generation
of free radicals (136). Ozone reacts with
water at an alkaline pH to produce -OH.
Interacting with biological molecules, ozone
can produce 02>- Ozone interacts with
olefin and glutathione to produce free radi-
cals. Kinetic studies with ozone exposure
have suggested that ozone is short-lived and
unable to cross the thick mucous blanket
and penetrate deep into lung cells (136).
Ozone can cause increased lipid peroxida-
tion. Exposure to ozone impairs pulmonary
clearance of asbestos and is known to acti-
vate or inactivate enzymes on the epithelial
surface. Ozone produces oxidative protein
damage in human plasma (137).
Radiation

Since the discovery of X-rays in 1886 and
their subsequent worldwide use in medi-
cine, many radiation-induced health effects
have been recognized in exposed popula-
tions. As early as 1897, radiation-induced
skin injuries were reported in workers using
X-rays (138). It was subsequently reported
that in experimental animals, irradiation
induced sterilization of gonads. Several
somatic and genetic mutations, including
cell death, are caused by ionizing radiation.
Ionizing radiation occurs mainly in two
types: as particulate radiation and as elec-
tromagnetic radiation. Particulate radiation
induces direct molecular disruption of elec-
trons, protons, neutrons, atoms, and parti-
cles. This type of radiation causes greater
injury per unit dose than electromagnetic
radiation. Particulate radiation can disrupt
atoms and molecules in the cell, producing
free radicals and ions by the direct effect.
DNA is the most vulnerable target of this
type of ionizing radiation in the cell.
Radiation-induced changes include DNA
strand breaks, base changes, bond cleavage
of sugars, degradation, and cross-linking
of DNA. Particulate radiation can also
cause mitosis, mutations, and chromosome

aberrations in the cell (139). Indirect effects
of radiation to the cell result in the radioly-
sis of cellular water and the formation of
free radicals.

The second type of ionizing radiation is
electromagnetic radiation, which is believed
to cause indirect heat-induced energy
absorption. Electromagnetic radiation
includes y-rays and X-rays and is usually pro-
duced from man-made nonionizing sources.
Common sources are power transmissions,
broadcast equipment, medical equipment,
and electromagnets (140). Ultraviolet radia-
tion in sunlight is in the electromagnetic
range of radiation. Ultraviolet radiation can
cause genetic mutations, DNA strand
breaks, and DNA cross-links. It is also
known to induce DNA lesions in the dimers
(cyclobutane pyrimidine) in human skin.
Ultraviolet radiation causes an increase in the
melanin pigmentation of skin, which may
absorb much of the radiation (141,142).

Radiation can produce developmental
abnormalities, cancer, cell killing, and
mutations in the DNA (139). Gonads in
the germinal epithelium of both sexes are
the most vulnerable cells to radiation.
Lungs, with their copious vascular supply,
are also very vulnerable to radiation injury,
resulting in radiation pneumonitis and
fibrosis. Radiation results from the physical
transfer of energy, causing the ionization of
atoms in the target. Two distinct mecha-
nisms are involved in radiation-induced
lung injuries. In classical radiation pneu-
monitis, a local field of irradiation is trig-
gered to develop pulmonary fibrosis
initiated by an acute inflammatory process
and cytokine production. Classical radia-
tion pneumonitis exhibits a dose-response
relationship with increasing morbidity and
mortality with higher doses. Clinical pneu-
monitis, on the other hand, is believed to
be induced by an immunologically medi-
ated process triggered by irradiation and
results in diffuse alveolitis characterized by
an infiltration of T-lymphocytes (143).
Carette Smoke

Cigarette smoking is the single most
avoidable health hazard and is known to
cause many diseases, including cancers,
emphysema, and cardiovascular, cerebro-
vascular, and perinatal abnormalities. The
effects of cigarette smoking are synergistic
with several occupational and environmen-
tal hazards. An overwhelming majority of
lung cancers occur in cigarette smokers
(144). Cigarette smoke is a complex mix-
ture of more than 4000 substances (145).
Among the various substances identified are

nicotine, tar, and a gas phase containing
organic chemicals that are considered to be
the major elements to the pathogenesis of
diseases associated with cigarette smoke. In
the lungs, cigarette smoke can release
H202, which can diffuse into the nuclei of
cells and react with iron to produce -OH.
*OH causes DNA strand breaks and base
damage, which can lead to oncogene acti-
vation and tumor suppressor gene inactiva-
tion and thereby promote carcinogenesis.
In addition, many tumorigenic-reactive
electrophiles have been identified in ciga-
rette smoke. Upon oxidation, these reactive
substances can also form DNA adducts,
causing activation or inactivation of onco-
genes. Cigarette smoke contains tumor
promoters and tumor initiators. Current
evidence suggests that there are two sources
of free radicals generated from cigarette
smoking. Some groups of radicals are pre-
sent in the gas phase and another group is
present in the tar. Four types of radicals,
considered stable, have been identified and
reported present in the tar (146).
Mechanism of

Reactive Oxygen

Species-induced Disease

In the biological reactions of environmental
particulates and toxicants, involvement of
ROS is both direct and indirect (Figure 1).
Cell membrane damage is a common
phenomenon in all kinds of cellular injury.
In ROS-induced cellular injury, damage to
membranes of the cell and organelles
occurs as a result of direct interaction of
ROS or by the formation of peroxides and
their breakdown products. If not con-
trolled, this self-propagating reaction can
result in widespread damage of cellular
membranes. Injury to cell membranes
results in profound ionic alterations within
the cells and organelles.

In ROS-induced cellular injury, the
primary changes that occur are generally
confined to the cell membranes and mem-
branes of the organelles. Lysosomal mem-
branes are generally considered to be
resistant to ROS-induced lipid peroxida-
tion because of the differences in their
membrane lipids. This is apparent from
studies showing that lysosomal enzymes
such as N-acetyl-,B-D-glucosaminidase and
P-glucuronidase are not released from alve-
olar macrophages injured by the phago-
cytosis of a nontoxic ROS-generating dust
such as coal (147). On the other hand,
lysosomal membranes of alveolar macro-
phages exposed to crystalline silica or
asbestos are ruptured, causing the release of

Environmental Health Perspectives * Vol 105, Supplement I * February 1997

172

OXYGEN RADICALS AND LUNG DISEASE

Monocyte            Macrophage phagocytosise -n   _               no

respiratoryburst                         diesi

and 

Chemokines                  Feb                Feb

S                                         ~~~~~~~~~~~NO

_ Cytokines4 Neutrophil                                A

ROS

Lytic enzymes   omycin an
ElIstase       other drugs
g t i ;   Coflagenase
B-cell                                   Proteas

4~~~~~~~~~~~~~~LB

Lipi Pe
/2h 1z?                ImIelCell me

Rie In k
Coll death                 ~~~~Endonu4
ROS                        ~~~~~~~~DNA stn
Di \ af ;//        Cell;death                            Protein,

DNA bo|

$~~~~~~~~O                                             DNA fra,

IgE, IgM, IgA, lgG      FC=7              -ok-k                     nsh

janic minerals
el, coal, metals

cigarette smoke

Herbicides

02               b

'I I

id  Cigareftt smnoke

N02

03

Fibroblast proliferation

J_      _ ____

rs      o_     .   . -

O~~~~~P -w-
_ mo-W,molw~

>|Carcinogenesis          -.A

|Fbrognesis

Figure 1. Schematic representation of the formation of reactive oxygen species and their interrelationships with the sequence of major events involved in pulmonary injury.

Environmental Health Perspectives * Vol 105, Supplement I * February 1997

._ ___

173

VALLYATHAN AND SHI

lytic enzymes and cell death. If the toxicity
of the crystalline silica is prevented by coat-
ing the surface with a polymer (Prosil-28,
PCR Inc., Gainesville, FL), the lysosomal
damage is prevented. Therefore, it is appar-
ent that in ROS-induced cellular injury,
lysosomal leakage is a secondary event and
plays no important role in cell injury.

ROS are involved in the recruitment of
inflammatory cells into the lung through the
expression of chemoattractant proteins called
chemokines. Macrophage inflammatory pro-
tein (MIP-la) is chemotactic to monocytes,
neutrophils, eosinophils, basophils, and lym-
phocytes. Induction of MIP-la by the acti-
vated alveolar macrophages is thought to be
initiated by ROS. ROS can activate the
nuclear transcriptional regulatory factor
NF-iCB. This factor can bind to enhance
sequences of the promoter genes of inter-
leukins and enhance the expression of genes
to produce more cytokines. The enhanced
cytokine production results in a cascade of
biological reactions promoting the disease
process (Figure 1).

ROS also play an important role in the
regulation of gene transcription and signal
transduction pathways. For example, at
least two well-defined transcription factors,
NF-iKB and activator protein-1 (AP-1), are
regulated by the intracellular redox state.
NF-ICB and AP-1 are implicated in the
inducible expression of a variety of genes
involved in oxidative stress and cellular
response mechanisms. Among cellular

genes regulated by NF-icB are several
proinflammatory and cytotoxic cytokines,
including IL-2, IL-6, and TNF-a. Since
reactive stress has been shown to change
intracellular Ca(II) homeostatasis, cellular
redox state may modify more than just these
transcription factors. Such an effect may
exacerbate free radical reactions, activate
endonudease, and contribute to apoptosis.

ROS also effect the synthesis of NO,
which plays a pivotal role in the inflamma-
tion response process when macrophages
are activated by a proinflammatory stimu-
lus. Production of NO is dependent on the
activity of the enzyme nitric oxide synthase
(NOS). At least three distinct genes have
been described that encode different iso-
forms of NOS. Macrophages appear to be
the principal cellular source for the inducible
form of NOS (iNOS) activated in response
to an inflammatory event. Several binding
sites for transcriptional factors have been
identified in the promoter region of the
iNOS gene, including NF-icB and AP-1,
which are partly regulated by ROS. NF-cB
is believed to play a particularly important
role in the regulation of gene expression of
the iNOS isoform.

Conclusion

Lungs are vulnerable to endogenous and
exogenous sources of ROS insults. They
are well equipped with antioxidant
defenses to negate normal oxidative insults.
However, when the oxidative defenses are

overwhelmed by formidable oxidant influx,
injury results. ROS are frequently associ-
ated with many pulmonary diseases. They
are increasingly being recognized as media-
tors of early cell injury in lung diseases.
Inflammatory response associated with a
primary stimuli in the lungs results in the
generation of ROS and increased secretion
of cytokines such as y-interferon, IL-I j,
and TNF-a. These events appear to be
important in the progression of injury
and disease.

We have seen that several physical,
chemical, and gaseous agents produced in
workplaces and present in the environment
can damage cell membranes, lipids, pro-
teins, and genetic material. Pathological
changes observed for many pulmonary dis-
eases are unique and associated with an
inflammatory response. Depending on the
duration, dosage, and severity of toxic effect
on the cell, the cell injury may lead to a
disease process or functional adaptation.

Progress toward understanding the
interaction of several occupational and
environmental agents has been delayed
because of inadequate knowledge of inter-
actions, as well as skepticism. The unique
mechanistic reactions of cell membrane-
perturbation, lipid peroxidation, genetic
material damage, specific protein denatura-
tion, induction of up-regulated antioxidant
defenses, and calcium influx are common
pathophysiological changes found in ROS-
induced injuries.

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