Reactive Oxygen Species: Their Relation to
Pneumoconiosis and Carcinogenesis

Val Vallyathan, Xianglin Shi, and Vincent Castranova

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

Occupational exposures to mineral particles cause pneumoconiosis and other diseases, including
cancer. Recent studies have suggested that reactive oxygen species (ROS) may play a key role in
the mechanisms of disease initiation and progression following exposure to these particles. ROS-
induced primary stimuli result in the increased secretion of proinflammatory cytokines and other
mediators, promoting events that appear to be important in the progression of cell injury and
pulmonary disease. We have provided evidence supporting the hypothesis that inhalation of
insoluble particles such as asbestos, agricultural dusts, coal, crystalline silica, and inorganic dust
can be involved in facilitating multiple pathways for persistent generation of ROS, which may lead
to a continuum of inflammation leading to progression of disease. This article briefly summarizes
some of the recent findings from our laboratories with emphasis on the molecular events by
which ROS are involved in promoting pneumoconiosis and carcinogenesis. - Environ Health
Perspect 1 06(Suppl 5):1 151-1155 (1998). http://ehpnetl.niehs.nih.gov/docs/1998/Suppl-5/
1 151-1 155vallythan/abstract.html

Key words: reactive oxygen species, occupational dust, pneumoconiosis, lung cancer

Introduction

Reactive oxygen species (ROS) have been
implicated in the pathogenesis of pneumo-
coniosis and pulmonary carcinogenesis
(1-6). ROS include hydroxyl radicals
('OH), superoxide anion radicals (02),
peroxyl radicals (ROO'), alkoxyl radicals
(RO0), thiyl radicals (RS), and oxides of
nitrogen ('NO, NO2). Nonradicals such as
hydrogen peroxide (H202) and hypochlo-
ride (HOCI) have also been implicated in
the pathogenesis of these diseases. A delicate
balance exists between the generation of
ROS and antioxidants in health. Initiation
of disease processes is usually associated with
an imbalance caused by the excessive gener-
ation of ROS, resulting in oxidative stress.
Exposure to redox-active toxicants may lead
to a shift in prooxidant levels associated
with utilization of antioxidants and resultant
oxidative stress and cell injury, leading to
pneumoconiosis and carcinogenesis through

a series of progressive events. In addition,
chronic exposure to redox-active toxicants,
such as asbestos, crystalline silica, coal, ciga-
rette smoke, agricultural dusts, inorganic
dusts, or metal ions, results in the persistent
influx of inflammatory cells into the lungs,
promoting the generation of ROS, which
may be associated with the increased inci-
dence of pulmonary neoplasms (5,6).
However, the mechanisms by which ROS
promote pneumoconiosis and carcinogene-
sis are speculative. This review briefly sum-
marizes some of the recent findings from
our laboratories, emphasizing the molecular
events by which ROS are involved in pro-
moting pneumoconiosis and carcinogenesis.
Mechanisms of Reactive

Oxygen Species Generation

During the process of phagocytosis of
inhaled particulate, 02- is generated and

its dismutation results in the production of
H202. In the presence of transition metal
ions, such as ferrous iron or cuprous ions,
H202 is converted to the potent oxidizing
radical, OH, through the Fenton reaction.
There is overwhelming evidence that
inhalation of toxic occupational and envi-
ronmental pollutants leads to excessive in
vitro and in vivo generation of OH radicals
during phagocytosis (7-9). Such an over-
whelming burst of ROS generation subse-
quently triggers a severe inflammatory
cellular reaction resulting in additional gen-
eration of ROS. To examine this process of
phagocytosis and interaction of different
minerals with phagocytes in vitro and in
vivo, we have studied the effects of coal
mine dust, crystalline silica, asbestos, agri-
cultural dusts and other inorganic minerals.
Figure 1 shows the relative peak heights of
electron spin resonance (ESR) spectra as a
measure of ROS generation resulting from
interaction between polymorphonuclear
leukocytes and various dusts. Figure 2
shows results of in vitro exposure of alveolar
macrophages to various occupational dusts.
These studies, using standardized respirable
particulates based on equal surface area,
indicate that asbestos, coal mine dust, and

C*

c=
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2.0 -
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1.0-

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This paper is based on a presentation at the Second International Meeting on Oxygen/Nitrogen Radicals and
Cellular Injury held 7-10 September 1997 in Durham, North Carolina. Manuscript received at EHP 19 December
1997 accepted 6 April 1998.

Address correspondence to V. Vallyathan, PPRB/HELD/NIOSH, 1095 Willowdale Rd., Morgantown, VVN
26505. Telephone: (304) 285-6056. Fax: (304) 285-5938. E-mail: vavl @cdc.gov

Abbreviations used: 8-OHdG, 8-hydroxy-2'-deoxyguanosine; BALF, bronchoalveolar lavage fluid; DMPO,
5,5- dimethyl-1-pyrroline Noxide; dG, guanine residues; DNA, deoxyribonucleic acid; ESR, electron spin reso-
nance; HPLC, high performance liquid chromatography; NF-KB, nuclear factor kappa B; 'NO, 'NO2, oxides of
nitrogen; 'OH, hydroxyl radicals; 02'-, superoxide anion radicals; PBN, phenyl-N-tert nitrone; RO0, alkoxyl
radicals; ROO, peroxyl radicals; ROS, reactive oxygen species; RS5, thiyl radicals; SN50, cell-permeable
inhibitory peptide; TNF-x, tumor necrosis factor alpha.

Figure 1. Oxygen radical generation from the interac-
tion of polymorphonuclear leukocytes (human) and
dusts. Phenyl-N-tert-nitrone (PBN) was used as a spin
trap. The concentrations of dusts are: aged silica, 250
pg/mI; amosite, 50 pg/mI; chrysotile , 50 pg/mI; croci-
dolite, 50 pg/mI; freshly fractured silica 250 pg/mI. The
PBN spin adducts of free radicals were measured by
ESR according to the method of Vallyathan et al. (7).
Each bar indicates the mean and standard derivation of
three experiments.

Environmental Health Perspectives * Vol 106, Supplement 5 * October 1998

1

tl51

VALLYATHAN ET AL

silica have substantial potential to generate
ROS (Figure 2). The results therefore
support the hypothesis that dust-exposed
phagocytes generate ROS, which can
mediate lung injury.

Another mechanism of ROS generation
by inhaled particulates is through surface
redox reactions catalyzed by metal ions.
Many ambient air pollutants, such as
asbestos, inorganic minerals, synthetic min-
erals, and agricultural dusts, have redox
potential and can generate excessive
amounts of ROS. The ability of minerals to
generate ROS in noncellular systems is
shown in Figure 3. This potential augments
the phagocytic generation of ROS and
results in further generation of oxidants.

Studies from our laboratories also have
provided evidence for the increased
generation of surface-based ROS from
crystalline silica and coal resulting from the
mechanical processes of grinding or frac-
turing as occur in occupational settings
(10-12). The surface-based radicals created
on the cleavage planes of crystalline silica
react with water to produce potent *OH
radicals (Figure 4). Transition metal ions
present on the surface of silica or within
biological milieus promote this reaction.

Another pathway that promotes excessive
generation of ROS is associated with the
fibrous nature of some insoluble inorganic
minerals. Asbestos (amphiboles) is the

8-
7.

6

.R

c

U)

0
.CD
Cl:

5-

4-

3-

2-

1-

o-

classic example of an indestructible
mineral promoting repeated frustrated
phagocytosis. In addition to the indestruc-
tible nature of asbestos, inhaled fibers
often are long enough (10-40 gm) so that
phagocytes, with an average diameter of 12
to 18 ,um, cannot engulf them completely.
This incomplete phagocytosis by alveolar
macrophages and the subsequent activation
of other alveolar macrophages lead to
excessive generation of ROS (5,7,13-15).

Targets and Consequences

Lipid Peroxidation

The unsaturated fatty acids of cell
membrane phospholipids are major targets
of the highly reactive *OH attack (16).
The *OH radical can extract a hydrogen
from membrane phospholipids producing
lipid radicals, which in turn can react with
molecular oxygen to produce lipid peroxyl
radicals. This process is propagated result-
ing in the destruction of membrane phos-
pholipids and cell injury. It is postulated
that lipid peroxidation plays a major role
in the pathogenesis of pneumoconiosis
(5,10,11,17,18). Recent studies, using cell-
free lipid peroxidation models, alveolar
macrophages, or whole lung slices, provide
evidence that supports the basic mechanism
of cell injury and its correlation to increased
generation of ROS from minerals (11).

2u

._

0)

.2

CD
CD
uJ

.0-  b   - p   gp   ., 'h   .,,

'?05  .   So pd

Figure 2. Oxygen radical generation from the interac-
tion of alveolar macrophage and dusts. Reaction mix-
tures in a total volume of 3.5 ml contained 1 ml 2.5 x
106 alveolar macrophages and 2 ml 0.1 M PBN and var-
ious dusts. Samples were incubated at 370C for 1 hr.
The concentrations of dusts are adjusted to the equal
surface area. The PBN spin adducts of free radicals
were measured by ESR (7).

Cp    -        o,   h

Figure 3. Relative oxygen radical generation in the
reactions of various dusts with H202. The concentra-
tions of H202 and dusts were 20 mM and 10 mg/mI.
The radical generation was measured by ESR using
5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin
trap (10). Each bar indicates the mean and standard
derivation of three experiments.

Membrane Injury

Many animal studies and human
investigations have reported that bron-
choalveolar lavage fluid (BALF) collected
after exposure to occupational and envi-
ronmental pollutants shows increased
evidence of oxidative injury (17,18).
Extracellular release of proteases, lytic
enzymes, ROS, and cytokines, up regula-
tion and consumption of antioxidant
enzymes, and amplification of inflamma-
tory mediators and growth factors are the
most frequent changes accompanying
oxidative injury. Repeated cycles of cell
injury associated with the release of inflam-
matory mediators and ROS set the stage
for activation of fibroblastic proliferation
and collagen formation, resulting in
pulmonary fibrosis.

In vitro findings show that freshly
fractured crystalline silica is more cytotoxic
and inflammatory than the aged silica and
that this activity is associated with the
enhanced concentration of surface radicals
on fresh cleavage planes (10,11). Results of
studies with rats after inhalation of fresh or
aged silica provided direct evidence that
fresh silica is more potent in increasing
inflammation, causing lung injury and lipid
peroxidation, inducing generation of ROS,

g=2.0015
A

5G
H

B

20G

H

Figure 4. (A) A typical ESR spectrum of freshly fractured
silica particles. The spectrum was assigned to silicon-
based free radicals. (B) ESR spectrum recorded 2 min
after mixing 100 mM DMPO with 10 mg/mI freshly frac-
tured silica particles. The spectrum was assigned to
DMPIO/PH adduct, demonstrating the formation of *OH
radicals. The ESR measurements were made according
to the method of Vallyathan et al. (10) and Shi et al. (11\.

Environmental Health Perspectives * Vol 106, Supplement 5 - October 1998

1152

REACTIVE OXYGEN SPECIES LUNG DISEASE

and causing up regulation and consumption
of antioxidant enzymes (17,18).
Carcinogenesis

ROS induce point mutations and
chromosomal aberrations in cells. ROS are
generated by multiple pathways in the
lungs in response to inhalation of toxic
pollutants. Many of the toxic substances
inhaled contribute to oxidative alteration
and modification, and they target specific
attack of vital components of the cyto-
plasm and nuclei. Such changes may
include ROS-induced DNA strand breaks,
oxidative modification of DNA, base
modifications, sequence changes, poly-
ADP-ribosylation, activation of kinases,
activation of protooncogenes, and inactiva-
tion of tumor suppressor genes (19,20).
Persistent generation of ROS from inde-
structible or incompletely phagocytosed
minerals can continue to cause damage
to key cellular organelles and promote
these reactions.

DNA Strand Breaks

ROS can attack deoxyribose, purine, and
pyrimidine bases in DNA resulting in
DNA strand breaks. DNA strand breaks
induced by 'OH cause deletions and point

70
60

=
ci
to
CL
x
CD
hC

0
C.
U0

C2

40 '

30
20
10

mutations, resulting in oncogene activation.
We have studied DNA strand breaks after
exposure of DNA to occupational and envi-
ronmental pollutants and monitored the
alterations by an alkaline unwinding assay.
Figure 5 illustrates the relative measure of
disruption of DNA structure caused by cro-
cidolite, amosite, and chrysotile asbestos.
Chrysotile asbestos caused significant DNA
strand breaks, which were further enhanced
by the addition of Fe(II) and H202. These
increased DNA strand breaks by chrysotile
may be caused by the large surface area of
chrysotile. These results show that all a
these particles can induce direct DNA
strand breaks. DNA strand breaks induced
by silica particles were also studied to exam-
ine the role of ROS (21). As shown in
Figure 6, incubation of DNA with silica
caused significant DNA strand breaks.
When the incubation was carried out
under argon, no DNA strands breaks were
observed, which demonstrates the require-
ment for molecular oxygen. Addition of
the catalase inhibited DNA strand breaks.
Because molecular oxygen is the precursor
of 02- and H202, the results presented in
Figure 6 further demonstrate that ROS
play a key role in the mechanism of
silica-induced DNA strand breaks.

'OH-Induced DNA
Oxidative Damage

Hydroxylation of guanine residues (dG)
to produce 8-hydroxy-2'-deoxyguanosine
(8-OHdG) is the most common marker of
'OH radical-induced DNA damage (22).
Using high performance liquid chro-
matography (HPLC) with electrochemical
detection, we measured significant 8-
OHdG formation in the presence of iso-
lated DNA and varying concentrations of
occupational and environmental dusts.
Antioxidants such as catalase and formate
inhibited the 8-OHdG formation, demon-
strating that 'OH radical-specific interac-
tion was involved in this DNA base
damage. DNA base modifications caused
by these changes could cause DNA mis-
pairing, which could lead to point muta-
tions and oncogene activation. Figure 7
illustrates the results of these studies using
asbestos, coal, and crystalline silica.

Nuclear Factor Kappa B Activation

Nuclear factor kappa B (NF-icB) activation
is an important transcription factor and
plays a critical role in inflammatory and
immune response, as well as activation of
oncogenes, cytokine receptors, cell adhesion
molecules, and growth factors (23,24).
NF-KB is activated by ROS, cytokines,
viruses, protein kinase C activators, and
immunological stimuli (25). Antioxidants
such as N-acetylcysteine and pyrrolidine

40-

.to

CD
Q
CD
o
CD:
0r
a0
SD

Sr

Figure 5. Percentage of DNA strand breaks after
exposure to various dusts. Reaction mixtures con-

tained 4 pg PM2 bacteriophages DNA, 65 pM H202,

with dusts (100 pg) in a final volume of 500 pi.
Samples were incubated at 370C for 1 hr and ethidium
bromide reactive fluorescence was measured at an
excitation wavelength of 525 nm and emission wave-
length of 600 nm.

1                       3          4

Figure 6. DNA damage by freshly fractured silica parti-
cle measured according to the method of Shi et al. (21).
Lane ,1 untreated control DNA; lane 2, 10 mg/mI freshly
fractured silica particles with X Hind Ill-digested DNA
in a phosphate-buffered solution, pH 7.4; lane 3, same
as lane 2 but incubation was carried out under argon;
lane 4, same as lane 2 but with 7500 U/mI catalase
added. The samples were incubated for 3 weeks.

I

20 -
10 -

0-

Coal     Asbesos     Silica    No dust

Figure 7. Hydroxylation of DNA by silica, asbestos,
and coal dusts. A total of 200 pg/mI bovine DNA was
incubated with dust for 0.5 hr at 370C. The DNA was
digested by nuclease P' and then by alkyline phos-
phatase. The digested DNA was analyzed for 8-OHdG
formation using HPLC with electrochemical detection.
Each bar indicates the mean and standard derivation of
three experiments.

Environmental Health Perspectives a Vol 106, Supplement 5 * October 1998

E-

1153

VALLYATHAN ET AL.

dithiocarbamate are reported to inhibit     the NF-iKB activation by promoting the
NF-KB activation. We have observed that     dismutation of 02'- to generate more 'OH
interactions of many occupational and       radicals. Thus, among these reactive oxy-
environmental pollutants with alveolar      gen species, 'OH radical plays a major role
macrophages result in the activation of     in silica-induced NF-ICB activation (26).

NF-iKB. Results of NF-icB activation by sil-   Since NF-iKB is known to regulate
ica and its inhibition are presented in     tumor necrosis alpha (TNF-a) production,
Figure 8. NF-KB activation induced by sil-  we have measured asbestos-induced TNF-a
ica was effectively blocked by catalase or  production and the role of NF-KB and
the 'OH-specific scavenger, sodium for-     ROS. Crocidolite asbestos induced the
mate. NF-KB was also inhibited by the       production of TNF-ax from an alveolar
metal chelator, deferoxamine, suggesting    macrophage cell line and from primary
the involvement of the Fenton or Fenton-    alveolar cells in dose- and time-dependent
like reaction in 'OH generation. On the     manners. Maximal secretion was induced in
other hand, superoxide dismutase increased  8 hr at a concentration of 50 pg/ml croci-

dolite (27). Inhibition of NF-KB by an
inhibitor of NF-icB nuclear translocation
(SN50, cell-permeable inhibitory peptide),
or by sequence-specific oligonucleotides
directed against the TNF-a binding site of
NF-KB, attenuated the effect of asbestos on
TNF-x production. Gene transfection
assays with a plasmid construct containing
... ....          TNF-ac binding sites of NF-icB linked to a

luciferase reporter gene further showed that
asbestos induced transcriptional activation
of NF-icB-dependent genes. This activation

Was also inhibited by SN50. Treatment of

the cells with oxygen radical scavengers
inhibited both asbestos-induced NF-iKB
......... .. .........                 . .activation  and  TNF-ra  production.

Treatment of the cells with a metal chela-
tor, which blocks the metal-mediated gen-
eration of *OH  radicals from H202, also
inhibited both NF-KB activation and

TNF-cx production. These results show

* .':...! ...: ' '-. ........Ss . |*

.......ii.  .. .  ; that NF-i1B is involved in asbestos-induced

; ;  :k >i  ; i ;TNF-a production through metal-medi-

.. .....   .........   ... . .  ...   ...  .....,

ated free radical reactions.

m : i s;;: ~~~~~.. :s: ....   ..... .;

.~~~~~~~~~ ,.'     . .:. '.-,S . Les ::i>:l  .s. :i:s: ...

.....  ...   . ....... Inactivation of Tumor Suppressor

...,,,2, t.,:ihsi's.  s9g  Si 'W::  ......   ..  .....  ............ ''". *r

F'   Gene (p53)

Mutational inactivation of the tumor
suppressor gene is an important molecular
alteration most frequently found in many
types of cancers (28). It is estimated that
approximately 50 to 60% of human lung
cancers contain a mutated form of p53.
Mutated p53 has a longer half-life and is
1      2      3      4      5     6     present in higher concentrations in cancer

cells and preneoplastic cells. Several types of
Figure 8. Induction of DNA binding activity of NF-KB  DNA damage can activate p53, including
protein by silica and the effect of catalase, superoxide  double-strand breaks (28,29). Oxidative
dismutase. sodium formate, and deferoxamine mea-  signaling is involved in the regulation of
sured according to the method of Chen et al (25). The  early changes in gene expression in a cell
RAW 264.7 cells were incubated with stimuli for 6 hr.  cycle during GO to G, phase transition
Lane 1 untreated cells (5 x 106/ml), lane 2 cells + 100

pg/mI silica; lane 3, cells + 100 pg/mI silica + 10,000  (30,31). It is known that ROS induces a
units/mi catalase; lane 4, cells + 100 pg/mI silica +  G1 arrest in proliferating fibroblasts accom-
500 U/mI superoxide dismutase; lane 5, cells + 100  panied by the accumulations of p53 and the
pg/mI silica + 0.625 mM sodium formate; lane 6, cells  cdk inhibitor IWAFI/CIPI (32). p53 is a
+ 100 pg/mI silica + 1.5 mM deferoxamine.   transcriptional activator that up regulates

the expression of several genes controlling
growth inhibitory and apoptotic pathways.
It is believed that p53 serves as a tumor sup-
pressor by preventing the passage of genetic
lesions in cells with DNA damage to a new
generation of cells. It does this either by
halting cell division to allow for DNA
repair or by inducing apoptosis of damaged
cells. Mutational inactivation of p53 is a
frequent molecular alteration in human
cancers, which indicates the importance of
the p53 gene in human carcinogenesis
(28,29). There are 10 cysteine residues in
p53 protein. Redox regulation at a post-
translational level often occurs by reduction
or oxidation of a disulphide bond. The
reducing environment in cells is important
for active p53 protein. Exposure of cells to
silica partides may convert the cells to a pro-
oxidant state resulting from the increased
production of ROS or decreased expression
of antioxidant enzymes. This oxidizing
environment may render wild-type p53
conformational mutant and give rise to the
same biologic outcome as p53 mutation.
However, information is still not available
regarding the effect of silica particles on
p53 activity. This area deserves active
investigation regarding the mechanisms of
silica-induced carcinogenesls.
AP-1 Activation

AP-1 is an important transcriptional factor
involved in tumor promotion and its
activation appears to play an important
role in carcinogenesis. Its regulation of pro-
tooncogenes, c-jun and c-fos, is reported to
have a key role in the induction of growth
factor genes (33). We have obtained pre-
liminary evidence suggesting the involve-
ment of ROS in AP-1 activation. The
mechanism of ROS-induced activation of
AP-1 is an area of active research. Janssen
et al. (33) suggested that alteration in the
cellular thiol redox status is caused by
oxidative stress and subsequent AP-1 acti-
vation. In our preliminary studies, ROS
generated during the interaction of dusts
such as crystalline silica and asbestos
induced the activation of AP-1 in luciferase
reporter mouse epidermal cells (JB6P+).
Superoxide and catalase inhibited this acti-
vation, indicating the involvement of ROS
in the activation, of AP-1. Studies from
other laboratories have shown that asbestos
and ROS generated by the xanthine/xan-
thine oxidase system and H202/Fe(II)
induces the expression of c-fos and c-jun
early response genes (34). Products of c-fos
and c-jun protooncogenes interact to
regulate gene expression.

Environmental Health Perspectives * Vol 106, Supplement 5 * October 1998

1154

REACMIVE OXYGEN SPECIES LUNG DISEASE

Conclusions

Our studies provide evidence that supports
the hypothesis for ROS-induced pneumoco-
niosis and carcinogenesis. Upon reaction
with water or H202, silica and other mineral
particles can generate ROS. ROS can also
be generated by phagocytic cells stimulated

by these particles. ROS and other secondary
radicals generated by ROS can cause lipid
peroxidation leading to membrane damage
and DNA damage via strand breaks and
base modification. Through free radical
reactions, mineral particles activate nuclear
transcription factors, enhance secretion of

growth factors, induce oncogene expression,
and cause mutation of tumor suppressor
genes, resulting in proliferation of cells.
These processes may be significantly
involved in the mechanisms of pneumo-
coniosis and carcinogenesis induced by
mineral particles.

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