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Serial Review: Role of Reactive Oxygen and Nitrogen Species (ROS/RNS)in Lung Injury and Diseases

Guest Editor: Brook T. Mossman

REACTIVE OXYGEN SPECIES (ROS) AND REACTIVE NITROGEN SPECIES(RNS) GENERATION BY SILICA IN INFLAMMATION AND FIBROSIS

BICE FUBINI* and ANDREA HUBBARD

*Department of Chemistry IFM and Interdepartmental Center G. Scansetti for Studies on Asbestos and other Toxic Particulates,University of Torino, Torino, Italy; and Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, USA

(Received16 December2002; Revised24 February 2003; Accepted28 February 2003)

AbstractExposure to particulate silica (most crystalline polymorphs) causes a persistent inflammation sustained bythe release of oxidants in the alveolar space. Reactive oxygen species (ROS), which include hydroxyl radical, superoxide

anion, hydrogen peroxide, and singlet oxygen, are generated not only at the particle surface, but also by phagocytic cells

attempting to digest the silica particle. Two distinct kinds of surface centerssilica-based surface radicals and poorly

coordinated iron ionsgenerate O2 and HO in aqueous solution via different mechanisms. Crystalline silica is also

a potent stimulant of the respiratory burst in phagocytic cells with increased oxygen consumption and production of O ,

H2O2, and NO leading to acute inflammation and HO generation in the lung. Oxidative stress elicited by crystalline

silica is also evidenced by increased expression of antioxidant enzymes such as manganese superoxide dismutase

(Mn-SOD) and glutathione peroxidase, and the enzyme inducible nitric oxide synthase (iNOS). Generation of oxidants

by crystalline silica particles and by silica-activated cells results in cell and lung injury, activation of cell signaling

pathways to include MAPK/ERK kinase (MEK), and extracellular signal-regulated kinase (ERK) phosphorylation,

increased expression of inflammatory cytokines (e.g., tumor necrosis factor [TNF], interleukin-1 [IL-1]), and

activation of specific transcription factors (e.g., NFB, AP-1). Silica can also initiate apoptosis in response to oxygen-

and nitrogen-based free radicals, leading to mitochondrial dysfunction, increased gene expression of death receptors,

and/or their ligands (TNF, Fas ligand [FasL]). 2003 Elsevier Inc.

KeywordsSilica, Cell activation, Apoptosis, Free radicals, Surface radicals

HEALTH EFFECTS OF SILICA-GENERATED ROS/RNS

It has been known since ancient times that inhaled crys-

talline silica particles causesilicosis, a severe lung pneu-

moconiosis. Exposure to silica has also been associated

with the development of several autoimmune diseases,

such as systemic sclerosis, rheumatoid arthritis, lupus,

and chronic renal disease, whereas some crystalline sil-

ica polymorphs may cause lung cancer [1].The mecha-

nism of action at the molecular level is still unclear and

it is uncertain if any single mechanism underlies all the

This article is part of a series of reviews on Role of Reactive

Oxygen and Nitrogen Species (ROS/RNS) in Lung Injury and Diseas-es. The full list of papers may be found on the homepage of the

journal.Bice Fubini was educated at the University of Torino (Italy) where

she is currently Professor of General and Inorganic Chemistry in theFaculty of Pharmacy and the Head of an Interdepartmental Center forStudies on Asbestos and other Toxic Particulates. She specialized insolid state and surface chemistry in Torino and at the University ofBath (UK). In the past 20 years she has developed studies on thechemical basis of the toxicity of solid materials, which is presently hermain research interest. She authored a large number of research papers,book chapters, and reviews, mostly on the toxicity of mineral dusts. Shehas been invited to several consensus workshops in this field; reportsinclude contributions to IARC (International Agency for Research onCancer) monographs and to ECVAM (European Centre for the Vali-dation of Alternative Methods).

Andrea Hubbard received her PhD in Immunology from the Uni-versity of Tennessee Center for the Health Sciences in 1980 andconducted postdoctoral training at the Medical College of Wisconsin(19801983) and University of Arizona (19831988). She has been atthe University of Connecticut since 1988 and is currently AssociateProfessor in the Department of Pharmaceutical Sciences. Her researchinterests have focused on the molecular regulation of inflammatorygene expression and apoptosis in response to particle induced lunginjury.

Address correspondence to: Dr. Bice Fubini, Dipartimento diChimica IFM, Facolta di Farmacia, University of Torino, Via P. Giuria7, Torino 10125, Italy; Tel: 39 (011) 670-7566; Fax: 39 (011)670-7855; E-Mail: [email protected].

Free Radical Biology & Medicine, Vol. 34, No. 12, pp. 15071516, 2003Copyright 2003 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/03/$see front matter

doi:10.1016/S0891-5849(03)00149-7

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above-mentioned diseases. However, the severe inflam-

mation, following exposure to silica particles, appears to

be a common initiating step. A large body of experimen-

tal work in the past 20 years, reported in recent reviews

and books [1 4], has evidenced two main points: the

crucial role played by the particle surface in triggering

the adverse biological response and the extreme variabil-

ity in the pathogenic potential among different sources of

particulate silica[5,6].Variability is not only the conse-

quence of the various existing forms of silica crystal-

line, vitreous, amorphous/natural, synthetic/mineral, bio-

genic [1] but is also due to (i) surface properties

determined by the history of the dust, (ii) several kinds of

surface features implicated in the mechanism of action,

and (iii) multiple particle/biological matter interactions

taking place in subsequent stages of the body reaction tosilica.

Figure 1 illustrates the cellular responses in the

lungs elicited by silica exposure. Once in the alveolar

space the particle may react with extracellular matter

(step 1) and be engulfed by alveolar macrophages

(AMs), which clear the particles out of the lungs (step

2). Depending upon the surface characteristics of the

particle itself this clearance process may either suc-

ceed (step 3) or fail (step 4). In the latter case mac-

rophages will become activated at the cellular and

molecular level with the activation of transcription

factors and the release of ROS and RNS, chemotactic

factors, lytic enzymes, cytokines, and growth factors,

with eventual cell death (necrosis/apoptosis), releasing

the particle. Subsequent ingestion-reingestion cycles

accompanied by a continuous recruitment of AM, neu-

trophils (PMN), and lymphocytes are the cause of the

sustained and chronic inflammation elicited by silica.

Target cells such as bronchiolar and alveolar epithelial

cells will then be affected by both AM products (step

5) and the extracellular particle itself (step 6), again

resulting in activation and/or cell death. Particle-de-

rived ROS may also react with cell-derived ROS and

RNS (7), yielding new toxic moieties, e.g., peroxyni-

trite (ONOO) from nitric oxide (NO) and superoxide

anion (O2) (step 7) [6]. Free radicals and ROS playa key role in steps 1, 5, 6, and 7, whereas the distri-

bution of silanols (SiOH) at the surface, which govern

hydrophilicity and adsorption processes [6], are

mostly related to steps 2, 3, and 4.

Surface modifying agents, including the historical anti-

silicotic drugs [2,3] polyvinylpyridine-N-oxide (PVPNO)

and aluminum lactate, recently revisited, inhibit most

adverse reactions to silica in vivo and also decrease the

generation of ROS and DNA damage caused by silica

[79] by selectively blunting surface active sites. Any

Fig. 1. Silica-induced cellular responses. (1) Interaction with extracellular matter; (2) phagocytosis by alveolar macrophages (AM); (3)

clearance; (4) macrophage activation and death; (5) response by target cells to AM products; (6) direct action of the particle on targetcells; (7) generation of additional ROS/RNS species.

1508 B. FUBINI and A. HUBBARD

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surface masking agent, like PVPNO, acting on silanols

and/or coating the reactive surface, will facilitate clear-

ance (Fig. 1, step 2), thus decreasing all effects due to

macrophage activation (see below), both in vitro and in

vivo. PVPNO, however, also appears to scavenge parti-

cle-generated hydroxyl radicals [10,11]. While new re-

search is needed to identify which surface properties areimplicated in each biological effect, these recent publi-

cations clearly confirm the crucial role played by the

particle surface in the overall pathogenicity.

In conclusion, there are two main sources of ROS

contributing to the adverse reactions to silica: particle-

generated free radicals and ROS, acting on cells and

extracellular components and cell (AM and PMN)-gen-

erated ROS and RNS.

PARTICLE-GENERATED FREE RADICALS AND ROS

Radicals may be bound to the surface as surface

radicals [X(s)] or generated as free moieties in aqueous

suspensions of the particles via a surface reaction with a

solute target molecule as free radicals. The former may

act as a center where the latter is generated.

Dangling bonds and surface ROS on the fractured

silica surface

Freshly ground (fractured) dusts are more inflamma-

tory and fibrogenic than aged crystals in animal experi-

ments[2,12,13],and are responsible for acute disease in

humans[2]. The cause of acute silicosis, which affects

individuals involved in sandblasting, drilling, or grind-

ing, has been found in the unique properties of freshly

cleaved surfaces[13].This is due, in part, to the greater

generation of silica-derived free radicals in freshly

ground material where surface ROS (peroxides or hy-

droperoxides) are formed[14 17].

When silica is fractured, both homolytic (dangling

bondsSi , SiO) and heterolytic (Si, SiO) cleavage

of the silicon-oxygen bond takes place [3,16].Molec-

ular oxygen reacts at these sites originating several

surface bound ROSSiO2(s), SiO3

(s), Si

-O2

(s).

These forms, which impart a peculiar reactivity to

freshly fractured surfaces, are visible in the Electron

Paramagnetic Resonance (EPR) spectrum of the dust[3,15]. Although surface radicals do decay, traces are

still visible in aged dusts. Surface bound ROS arise

from the surface or subsurface layers and cracks in the

silica particle. Mild treatment in hydrofluoric acid,

which dissolves the outer layers of silica, fully elim-

inates their trace in the EPR spectrum [18]. Quantum

mechanical calculations have hypothesized that aged

dusts might also be activated by adsorption of HO,

with consequent destabilization of the subsurface Si-O

bonds and formation of SiO(s) [19].

Surface active centers in the mechanism(s) of

particle-generated free radicals

Aqueous suspensions of quartz generate H2O2, HO,

O, 1O2[19].Both H2O2in solution, or iron traces at the

particle surface enhance the HO yield (which is inhib-

ited by catalase), suggesting a Fenton mechanism

[20,21].The yield is also enhanced by a pretreatment inascorbic acid [22]. Experiments with pure quartz and

iron-deprived quartz dusts, however, have shown that

HO is also generated in the absence of trace iron[23].

Both silicon-based surface radicals and iron ions located

in a particular redox and coordinative position at the

surface are active centers for free radical release in

solution[24].

The mutual contribution of the two mechanisms will

depend upon grinding procedure, time elapsed after

grinding, and level of iron ions occupying the active

sites. As shown in Fig. 2, the two centers react via

different mechanisms. Iron centers will yield HO radi-cals via the Fenton reaction:

Fe2H2O2 3Fe3OHHO

or via the Haber-Weiss cycle in the presence of reduc-

tants, with the superoxide ion as an intermediate.

Fe3 reductant (n)(s) 3 Fe2

reductant (n

1)(s), n being the redox state of the reductant molecule.

Fe2(s)O2 3Fe3

(s)O2

O2

H2O3

HO2

OH

or O2

2H

e

3

H2O2

2HO23H2O2 O2O2

H2O2 3HO

OHO2

Several metabolites can act as the reductant species, such

as ascorbate, cysteine, and glutathione. Generation of

hydroxyl radicals by the Haber-Weiss cycle requires iron

in only catalytic (trace) amounts, and the turnover of free

radicals can overload the antioxidant defense mecha-

nisms of living cells.

Surface radicals, SiO(s), SiO2(s), SiO3

(s), Si

-O2

(s)

in water or in the presence of hydrogen peroxide, will

directly form the HO radical [20,24], following reac-

tions such as:

SiO(s)H2O3SiOH(s)HO in water, or

SiOO(s)H2O2 3SiOH(s)HOO2

SiO2

(s)H2O2 3 SiOH(s)HOO2

in contact with hydrogen peroxide.

Grinding procedures determine the kind and the abun-

dance of free radical generating centers. When quartz

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was ground with jars and balls of different material the

outcome was a remarkable difference in free radical

yield[25].Inhalation of freshly fractured quartz contam-

inated with trace levels of iron further enhanced cellular

response in rats, suggesting that quartz had become more

pathogenic[26].In workplace dusts metal contaminants

embedded in the silica framework, which are alwayspresent to a larger or lesser extent, will contribute, even

if in traces, to the variability of silica hazard and may

account for the controversial results of epidemiology

studies [1].

In vitro effects of silica-derived free radicals

Silica dusts to a greater or lesser extent are all cyto-

toxic to various types of cells, due, only in part, to ROS

[27]. In a systematic study of artificial crystalline silica

samples, differing only in their size and shape, cytotox-

icity appears primarily governed by the form of the

particle and the extent of exposed surface [28]. Thedistribution of silanols mainly determines the degree of

hydrophilicity, hence modulating cell toxicity[29].Con-

versely, ROS are the direct cause of DNA damage [30],

morphological transformations in cells[24,31],and lung

injury [13,32]. The transformation frequency of Syrian

hamster embryo (SHE) cells caused by modified quartz

dusts, all generated from the same original sample, cor-

relates with the amount of HO radicals released in the

presence of H2O2[24].Calcined diatomaceous earth also

fits the correlation in that HO appears responsible for the

early transformation effects in mammalian cells, eventu-

ally yielding malignancies.

CELL-GENERATED ROS AND RNS AND LUNG INJURY

Crystalline silica is a potent stimulant of the respira-

tory burst in phagocytic cells with increased oxygen

consumption, production of O, H2O2 [33], and NO

[34]. Bronchoalveolar lavage cells from silica-exposed

rats evidenced enhanced oxygen consumption, chemilu-

minescence, and H2O2 release in response to an in vitro

stimulation with unopsonized zymosan particles [35].

Schapira et al. [36]noted in rats that quartz exposure by

intratracheal injection elicited increased OH production

in lung tissue compared to rats receiving the nontoxic

titanium dioxide.

Fresh surfaces and trace iron enhance ROS genera-

tion. Exposure to freshly fractured quartz resulted in

enhanced lung injury and inflammation in rats [13].Surface associated iron also enhanced the ability of silica

to stimulate the respiratory burst by rat AM in vitro and

to elicit acute pulmonary inflammation in rats exposed

by intratracheal instillation[21].

Quartz instillation into rat lungs elicited increased

mRNA for inducible nitric oxide synthase (iNOS) in

alveolar macrophages [34]. An enhanced iNOS-depen-

dent formation of NO is also implicated in lung injury,

since the reaction of NO with O2 yields peroxynitrite,

also capable of causing cell damage [37].

Fig. 2. Free radical-generating surface centers (adapted from Fubini et al. [24]).


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Oxidative stress elicited by crystalline silica is also

evidenced by the increased expression of some antioxi-

dant enzymes. Holley et al. [38] demonstrated in the

lungs of rats exposed by inhalation to cristobalite-silica a

significant increase in Mn superoxide dismutase (SOD)

localized primarily to type II epithelial cells. Janssen et

al.[39]extended these observations by demonstrating inrats exposed to cristobalite a significant increase in

steady state levels of MnSOD and glutathione peroxidase

mRNA. Antioxidant defenses may also be depleted by

silica. A dose- and time-dependent decrease in intracel-

lular glutathione (GSH) was found in isolated rat AM

exposed to silica. The GSH precursor, n-acetylcysteine

(NAC), decreased silica-induced ROS formation as well

as changes in membrane permeability and DNA strand

breaks[40].

ROLE OF OXIDANTS IN CELL RESPONSES

FOLLOWING SILICA EXPOSURE

Generation of oxidants by crystalline silica particles

and by silica-activated cells (e.g., macrophages[35]and

epithelial cells [41]) can result in cell and lung injury,

activation of cell signaling pathways, increased expres-

sion of inflammatory cytokines, and activation of specific

transcription factors (Fig. 1)[42,43].In some cases spe-

cific antioxidants were employed in order to investigate

the nature of the ROS implicated. A reduced effect in the

presence of catalase (which eliminates H2O2) and in-

creased by SOD (which converts O2 into O2and H2O2)

indicated a critical role played by hydrogen peroxide. In

most cases, however, the nature of ROS responsible forthe effect could not be determined, suggesting participa-

tion by more than one species. When the potent iron

chelator desferrioxamine was administered to cells to-

gether with silica [20,21], toxic effects were reversed,

implicating a Fenton-driven mechanism, since O2 may

react with iron (see above) .

Mitogen-activated protein kinase

Silica stimulates ROS production via flavoenzyme-

dependent mechanism in a rat fibroblast cell line (Rat2)

and activates MEK and ERK phosphorylation. This

phosphorylation could be attenuated by catalase, andenhanced by SOD, suggesting a role for silica-induced

H2O2 production[44].

NFB activation

Silica-induced oxidative stress can also activate spe-

cific transcription factors, including NFB and AP-1

[42,43]. The role of ROS in the activation of NFB

signal transduction was initially illustrated in cells

treated with the prooxidant H2O2, which resulted in the

activation of NFB, as demonstrated by electromobility

shift assay (EMSA)[45].Accordingly, in cell lines that

overexpress catalase, a treatment with H2O2 failed to

increase NFB[46].

In the mouse macrophage cell line, RAW 264.7,

quartz exposure elicited the activation of NFB 2 to 12 h

after exposure as detected by EMSA using consensus

sequences[47]. In this model, the presence of the anti-oxidant NAC did not affect silica-induced NFB activa-

tion. In subsequent studies, catalase, formate, and defer-

oxamine did inhibit NFB activation, whereas

superoxide dismutase (SOD) enhanced the activation,

suggesting that HO radicals rather than ROS in general

played a key role in silica-induced NFB activation[48].

In contrast, the presence of the inhibitor of iNOS, N-

monomethyl-L-arginine (NMMA), enhanced silica-in-

duced NFB activation, suggesting that NO participates

in negative feedback regulation of particle-induced

NFB activation[49]. In more recent work, bronchoal-

veolar lavage (BAL) cells (AMs; PMNs) from rats in-stilled with silica demonstrated enhanced NFB activa-

tion (EMSA analysis) through the 18 h time course

evaluated. Treatment with the antiinflammatory agent

dexamethasone decreased NFB activation and concom-

itantly decreased luminol-dependent chemiluminescence

in these phorbol myristate acetate (PMA)-stimulated

BAL cells[50].Recent work by Hubbard et al. [42]also

demonstrated silica-induced NFB-dependent gene ex-

pression in vivo through the use of luciferase reporter

mice exposed to an intratracheal instillation of quartz.

AP-1 activation

The protooncogenes c-fos and c-jun encode proteins

within the c-Jun and c-Fos families, which compose the

sequence species transcription factor, AP-1. AP-1 is dif-

ferentially regulated temporally during cell cycle pro-

gression and in response to many diverse stimuli. Oxi-

dants can also induce AP-1[51];however, unlike NFB,

AP-1 is also strongly induced by some antioxidants such

as pyrrolidine dithiocarbamate (PDTC) and n-acetyl cys-

teine (NAC)[52].

Using AP-1 luciferase reporter transgenic mice in-

stilled with silica, Ding et al. [53]noted increased lucif-

erase activity, indicating AP-1 activation in lung tissue3 d post exposure. In addition, they also determined that

silica exposure of a rat epithelial cell line (RLE) stably

transfected with an AP-1 luciferase reporter plasmid,

evidenced AP-1 activation. Hubbard et al. [43] con-

firmed and extended these observations in AP-1 lucif-

erase reporter transgenic mice exposed to silica by dem-

onstrating AP-1-driven gene expression in lung

macrophages and bronchiolar epithelial cells. Using a

nontransformed type II epithelial cell line exposed to

quartz, Shukla et al. [54]demonstrated a role for oxi-


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dants in such silica-induced cellular changes as JNK

activation, AP-1-dependent gene expression, and en-

hanced cell proliferation.

Cytokine expression

One outcome of transcription factor activation can be

increased cytokine expression, another event influencedby silica-induced ROS. Gossart et al.[55]noted that AM

lavaged from silica-exposed rats evidenced increased

zymosan- or phorbol ester (PMA)-triggered chemilumi-

nescence, as well as increased TNFmRNA expression

and protein secretion. Pretreatment of these animals with

a free radical scavenger (N-ter-butyl-phenylnitrone) re-

versed lung pathology, and decreased ROS and TNF

production from the AMs. In another study, pretreatment

in vitro of mouse macrophages (RAW 264.7) with anti-

oxidants (dimethyl sulfoxide [DMSO], glutathione

[GSH] or NAC) prior to exposure to cristobalite silica

significantly decreased TNF, mRNA, and protein pro-duction. Many of these same antioxidants also decreased

mRNA levels for macrophage inflammatory protein

(MIP-2, MIP-1) and monocyte chemotactic protein

(MCP)-1[56]in response to silica. The role of oxidants

in silica-induced cytokine expression has also been in-

vestigated in epithelial cells. For example, observations

were made in a murine lung epithelial (MLE)-15 cell line

exposed to cristobalite silica [57]. Again, various anti-

oxidants (extracellular GSH, DMSO, NAC, and buthi-

onine sulfoximine [BSO]) decreased TNF-induced and

cristobalite-induced mRNA expression of MIP-2 and

MCP-1. However, these antioxidants did not reduce sil-

ica-induced TNF mRNA expression. Using human lung

epithelial cells (A549) primed with TNF, Stringer and

Kobzik [58] demonstrated increased IL-8 production

with quartz. This enhanced cytokine response could be,

in part, attributed to ROS, since pretreatment with NAC

decreased this IL-8 production by approximately 50%.

Production of H2O2, generation of a chemotactic cyto-

kine (MIP-2), and activation of NFB by rat alveolar

type II epithelial cells was inhibited by the antioxidant

rotenone, suggesting the participation of mitochondria-

derived oxidants in these events [59].

APOPTOSIS

As afinal step in cell activation, ROS can also induce

apoptosis. For example, the induction of ROS with or

without depletion or administration of antioxidants leads

to apoptosis; many apoptosis regulating proteins act

through oxidant-antioxidant pathways.

Cellular mechanisms of silica-induced apoptosis

Although diverse stimuli can initiate apoptosis, simi-

lar biochemical and morphological alterations are ob-

served. One pathway is initiated by the activation of

death receptors, whereas a second pathway can be initi-

ated by the release of cytochrome c from the

mitochondria.

Silica has been documented to cause necrotic cell

death. However, substantial evidence exists in vitro and

in vivo that silica can also initiate apoptosis dependent,in part, on oxygen- and nitrogen-based free radicals.

Sarih et al. [60]were among the first to describe initia-

tion of silica-induced apoptosis in peritoneal macro-

phages isolated from mice. Treatment of these cells with

silica elicited apoptosis and inflammation, as demon-

strated by DNA laddering and nuclear morphology and

the release of IL-1. Iyer et al. [61] treated human

alveolar macrophages with silica for 6 or 24 h and

measured apoptosis using DNA laddering, nuclear mor-

phology, and levels of cytosolic histone-bound DNA

fragments. Only treatment with quartz, but not with

amorphous silica or titanium dioxide, elicited increased

measures of apoptosis. In vivo studies in rats [62] re-

vealed evidence of apoptosis in cells from bronchoalveo-

lar lavage of rats instilled 10 d prior with quartz. Apo-

ptotic cells were still apparent in granulomatous lesions

of these rats nearly 2 months later. Apoptosis was also

apparent by DNA laddering in the human epithelial cell

line A549 treated with silica, as well as in bronchoal-

veolar lavage cells from silica-instilled rats [63].

The participation of caspase activation in silica-in-

duced apoptosis has also been investigated both in vitro

and in vivo. In studies described above, Iyer et al. [61]

treated human AM with an inhibitor of caspases (Z-

VAD-FMK) and noted a decrease in both silica-induced

apoptosis and IL-1 release. These authors also con-

firmed the involvement of caspase 3 in silica-induced

apoptosis in human AM using a specific inhibitor (Z-

DEVD-FMK)[64].Silica also stimulated the activation

of caspases 1, 3, and 6 in the mouse alveolar macrophage

cell line, MHS[65].Preliminary work by Hubbard et al.

(unpublished results) has also detected silica-induced

caspase activation and apoptosis in vitro (mouse macro-

phage cell line) and in vivo (intratracheal instillation of

mice). Exposure of RAW 264.7 cells to silica caused

apoptosis 6 and 24 h later as measured by caspase 3

activity and annexin V staining for phosphatidyl serineexternalization. Caspase 3, but not caspase 1 activity was

also apparent in whole lung homogenates 3 d after in-

stillation of silica into mice, whereas both caspase 3 and

caspase 1 activity were increased 14 d after silica expo-

sure. Further studies by others then demonstrated a po-

tential role for ROS in silica-induced apoptosis. Results

demonstrated a temporal pattern of apoptotic events,

beginning with increased ROS formation followed by

activation of caspase 9 and caspase 3, PARP cleavage,

and DNA fragmentation. These silica-induced apoptotic


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events were significantly inhibited by a caspase 3 inhib-

itor (Z-DEVD-CHO), as well as by the antioxidant eb-

selen [66]. In addition to the participation of ROS in

silica-induced apoptosis, evidence documenting a role

for nitrogen-based free radicals is also indicated. Srivas-

tava et al. [67] found in IC21 mouse macrophages that

the iNOS inhibitor, N(G)-nitro-L-arginine methyl ester(L-NAME), as well as an IL-1 monoclonal antibody,

decreased apoptosis. These authors then confirmed in

vivo a role for iNOS and IL-1 in silica-induced apo-

ptosis using iNOS knockout mice or IL-1 knockout

mice; depletion of these mediators led to significantly

less apoptosis and pulmonary inflammation than ob-

served in wild-type mice.

Cellular mechanisms underlying the induction of ap-

optosis by silica have examined the role of Fas/Fas

ligand interactions. Borges et al. [68]present convincing

evidence for this Fas/FasL interaction in silica-induced

apoptosis and pulmonary inflammation. Using gld mice,deficient in FasL, the authors noted in response to silica

instillation, a marked decrease in neutrophil influx, pul-

monary inflammation, and TNF production. Silica-in-

duced FasL and to a lesser extent, Fas expression, was

detected in lung macrophages. And finally, administra-

tion in vivo of a neutralizing anti-Fas ligand antibody

blocked the development of silicosis in wild-type mice.

This same group has recently confirmed the role of

caspase activation in silica-induced pulmonary inflam-

mation and collagen deposition [69].

Biologic significance of silica-induced apoptosis

Thus apoptosis initiated by silica may be the result of

increased ROS production, leading to a mitochondrial

dysfunction, increased gene expression of death recep-

tors, and/or their ligands (TNF, FasL). The biologic

significance of silica-induced apoptosis in resolution of

these inflammatory lesions may be through the elimina-

tion of damaged or injured cells and the maintenance of

tissue homeostasis [66]. For example, phagocytosis of

apoptotic cells may downregulate NO production by

macrophages[70],thus contributing to decreased inflam-

mation. On the other hand, others have suggested a

proinflammatory role for this apoptotic process in attract-ing more alveolar macrophages into the airways to en-

gulf apoptotic leukocytes maintaining a relatively stable

level of these cells at the sites of inflammation[68,69].

Initial steps in this pathway may include increased pro-

duction of ROS leading to enhanced expression of FasL

(or Fas) on inflammatory leukocytes[71].Indeed, defer-

oxamine in vitro prevented FasL expression induced by

silica. These macrophages targeted for apoptosis by in-

creased expression of death receptors may then release

chemotactic factors for PMNs. PMNs in turn would

engulf dying cells and/or damage nearby epithelial and

parenchymal cells, thus perpetuating the apoptotic pro-

cess through the release of toxic ROS, hydrolytic en-

zymes, and cytokines.

ROLE OF OXIDANTS IN FIBROTIC RESPONSES

FOLLOWING SILICA EXPOSURE

The long-term consequence of exposure to silica and

the resulting generation of reactive oxygen/nitrogen pro-

duction is often pulmonary fibrosis. The relationship

between oxygen/nitrogen reactive metabolites and silica-

inducedfibrosis has been evaluated by studying the tem-

poral relationship between these events and by altering

the fibrotic response with antioxidants. It is clear from

these studies that oxygen- and nitrogen-based free radi-

cals generated from silica exposure are important initia-

tors of thefibrotic process. Porter et al.[72]noted in rats

exposed to quartz that NO-dependent chemilumines-cence and zymosan-stimulated chemiluminescence in la-

vaged AMs, as well as NOx levels in the BAL, were

significantly elevated within days after initiation of ex-

posure. Pathological changes were observed several

months after initiation of exposure with intense iNOS

and nitrotyrosine staining localized to these areas of

granulomatous inflammation. Gossart et al.[55]demon-

strated in rats exposed to quartz by intratracheal instilla-

tion granulomatous inflammation 2 4 weeks later, which

was associated with zymosan- or PMA-triggered chemi-

luminescence production from lavaged AMs. Adminis-

tration of the spin trap reagent N-ter-butyl--phenylni-trone as an antioxidant reversed lung histopathological

changes and decreased stimulated chemiluminescence

from lavaged AMs. Using iNOS knockout mice exposed

by inhalation to silica, Srivastava et al. [67] noted sig-

nificantly fewer histopathological lesions after several

weeks of exposure.

CONCLUSIONS

Current findings suggest that reactive oxygen inter-

mediates are generated not only at the particle surface,

but also by phagocytic cells. There is sufficient evidencethat, in both cases, radical yield is largely dependent

upon the surface characteristics of each individual dust

specimen, which contributes to thevariability of quartz

hazard.

A quantitative measure of surface sites, active in free

radical generation, together with the evaluation of the

other properties involved in the various steps of the

pathogenic mechanism depicted inFig. 1,could allow a

ranking of different sources of silica dusts on the basis of

their potential pathogenicity. This should be the target to


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address new experimental research, in order to provide

regulatory agencies sound physico-chemical and toxico-

logical data. At the same time, many relationships be-

tween particle characteristics and cellular responses elic-

ited still need to be investigated or established. This can

be performed only by means of set model samples of

silica particles, each one differing from the other in onlyone property at a time.

One question appears unresolved. The reactive radical

species described in this review survive for only seconds

in biological systems, while in humans silicosis develops

over decades. Once the continuous cycle of macrophage

re-ingestion is established (see Fig. 1) the consequent

sustained and persistent inflammation may, at least

partly, account for the development offibrosis. The par-

ticle could act, in these conditions, as a catalytic center

where radicals are generated. This may occur either

because the surface active site itself is a catalytic center

or because at each ingestion re-ingestion cycle, whichinvolves a variation in pH and oxidants in the medium,

active sites are re-generated. Typically, one may easily

assume that iron traces of endogenous origin may be

deposited on the particle under these circumstances.

Moreover, cellular reactions triggered by radicals may

proceed in a cascade for prolonged periods. Reactive

oxygen and nitrogen intermediates can initiate changes

in cell function to include cell signaling pathways, tran-

scription factor activation, mediator release, apoptosis,

and compensatory cell proliferation. However, many un-

answered questions remain in examining the role of

oxygen- and nitrogen-derived free radicals in silica-in-duced lung injury and fibrosis. The relationship between

silica-induced apoptosis and inflammation, a field that

contradicts much of the current dogma on the biologic

significance of apoptosis, is of intense interest. Although

the role of oxidant generation in silica-induced lung

injury in animal models has been investigated, few stud-

ies in humans have been conducted to examine the role

of oxidants generated soon after exposure to the later

culminating events of pulmonary dysfunction and

fibrosis.

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ABBREVIATIONS

SiO2silica

ROSreactive oxygen species

RNSreactive nitrogen species

AMalveolar macrophages

PMNpolymorphonuclear leukocytes

PVPNOpolyvinylpyridine-N-oxide

EPR electron paramagnetic resonance

SHESyrian hamster embryo

MEKMAPK/ERK kinaseERK extracellular signal regulated kinase

AP-1activating protein 1

SODsuperoxide dismutase

PDTCpyrrolidine dithiocarbamate

EMSA electromobility shift assay

NMMAn-monomethyl-L-arginine

L-NAMEN(G)-nitro-L-arginine methyl ester

iNOSinducible nitric oxide synthase

PMAphorbol myristate acetate

TNFtumor necrosis factor

DMSO dimethyl sulfoxide

GSH glutathioneNACn-acetyl cysteine

MIP-1macrophage inflammatory protein-1

MIP-2macrophage inflammatory protein-2

MCP-1monocyte chemotactic protein 1

BSO buthionine sulfoximine

IL-8 interleukin 8


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