Reactive Oxygen Species: Their Relation toPneumoconiosis and CarcinogenesisVal Vallyathan, Xianglin Shi, and Vincent CastranovaHealth Effects Laboratory Division, National Institute for OccupationalSafety and Health, Morgantown, West VirginiaOccupational exposures to mineral particles cause pneumoconiosis and other diseases, includingcancer. Recent studies have suggested that reactive oxygen species (ROS) may play a key role inthe mechanisms of disease initiation and progression following exposure to these particles. ROS-induced primary stimuli result in the increased secretion of proinflammatory cytokines and othermediators, promoting events that appear to be important in the progression of cell injury andpulmonary disease. We have provided evidence supporting the hypothesis that inhalation ofinsoluble particles such as asbestos, agricultural dusts, coal, crystalline silica, and inorganic dustcan be involved in facilitating multiple pathways for persistent generation of ROS, which may leadto a continuum of inflammation leading to progression of disease. This article briefly summarizessome of the recent findings from our laboratories with emphasis on the molecular events bywhich ROS are involved in promoting pneumoconiosis and carcinogenesis. Environ HealthPerspect 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

IntroductionReactive oxygen species (ROS) have beenimplicated 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 ofnitrogen ('NO, NO2). Nonradicals such ashydrogen peroxide (H202) and hypochlo-ride (HOCI) have also been implicated inthe pathogenesis of these diseases. A delicatebalance exists between the generation ofROS and antioxidants in health. Initiationof disease processes is usually associated withan imbalance caused by the excessive gener-ation of ROS, resulting in oxidative stress.Exposure to redox-active toxicants may leadto a shift in prooxidant levels associatedwith utilization of antioxidants and resultantoxidative stress and cell injury, leading topneumoconiosis 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, inorganicdusts, or metal ions, results in the persistentinflux of inflammatory cells into the lungs,promoting the generation of ROS, whichmay be associated with the increased inci-dence of pulmonary neoplasms (5,6).However, the mechanisms by which ROSpromote pneumoconiosis and carcinogene-sis are speculative. This review briefly sum-marizes some of the recent findings fromour laboratories, emphasizing the molecularevents by which ROS are involved in pro-moting pneumoconiosis and carcinogenesis.

Mechanisms of ReactiveOxygen Species GenerationDuring the process of phagocytosis ofinhaled particulate, 02- is generated and

its dismutation results in the production ofH202. In the presence of transition metalions, such as ferrous iron or cuprous ions,H202 is converted to the potent oxidizingradical, OH, through the Fenton reaction.There is overwhelming evidence thatinhalation of toxic occupational and envi-ronmental pollutants leads to excessive invitro and in vivo generation of OH radicalsduring phagocytosis (7-9). Such an over-whelming burst of ROS generation subse-quently triggers a severe inflammatorycellular reaction resulting in additional gen-eration of ROS. To examine this process ofphagocytosis and interaction of differentminerals with phagocytes in vitro and invivo, we have studied the effects of coalmine dust, crystalline silica, asbestos, agri-cultural dusts and other inorganic minerals.Figure 1 shows the relative peak heights ofelectron spin resonance (ESR) spectra as ameasure of ROS generation resulting frominteraction between polymorphonuclearleukocytes and various dusts. Figure 2shows results of in vitro exposure of alveolarmacrophages to various occupational dusts.These studies, using standardized respirableparticulates based on equal surface area,indicate that asbestos, coal mine dust, and

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This paper is based on a presentation at the Second International Meeting on Oxygen/Nitrogen Radicals andCellular Injury held 7-10 September 1997 in Durham, North Carolina. Manuscript received at EHP 19 December1997 accepted 6 April 1998.

Address correspondence to V. Vallyathan, PPRB/HELD/NIOSH, 1095 Willowdale Rd., Morgantown, VVN26505. 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 ofnitrogen; 'OH, hydroxyl radicals; 02'-, superoxide anion radicals; PBN, phenyl-N-tert nitrone; RO0, alkoxylradicals; ROO, peroxyl radicals; ROS, reactive oxygen species; RS5, thiyl radicals; SN50, cell-permeableinhibitory peptide; TNF-x, tumor necrosis factor alpha.

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

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VALLYATHAN ETAL

silica have substantial potential to generateROS (Figure 2). The results thereforesupport the hypothesis that dust-exposedphagocytes generate ROS, which canmediate lung injury.

Another mechanism of ROS generationby inhaled particulates is through surfaceredox reactions catalyzed by metal ions.Many ambient air pollutants, such asasbestos, inorganic minerals, synthetic min-erals, and agricultural dusts, have redoxpotential and can generate excessiveamounts of ROS. The ability of minerals togenerate ROS in noncellular systems isshown in Figure 3. This potential augmentsthe phagocytic generation of ROS andresults in further generation of oxidants.

Studies from our laboratories also haveprovided evidence for the increasedgeneration of surface-based ROS fromcrystalline silica and coal resulting from themechanical processes of grinding or frac-turing as occur in occupational settings(10-12). The surface-based radicals createdon the cleavage planes of crystalline silicareact with water to produce potent *OHradicals (Figure 4). Transition metal ionspresent on the surface of silica or withinbiological milieus promote this reaction.

Another pathway that promotes excessivegeneration of ROS is associated with thefibrous nature of some insoluble inorganicminerals. Asbestos (amphiboles) is the

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classic example of an indestructiblemineral promoting repeated frustratedphagocytosis. In addition to the indestruc-tible nature of asbestos, inhaled fibersoften are long enough (10-40 gm) so thatphagocytes, with an average diameter of 12to 18 ,um, cannot engulf them completely.This incomplete phagocytosis by alveolarmacrophages and the subsequent activationof other alveolar macrophages lead toexcessive generation ofROS (5,7,13-15).Targets and ConsequencesLipid Peroxidation

The unsaturated fatty acids of cellmembrane phospholipids are major targetsof the highly reactive *OH attack (16).The *OH radical can extract a hydrogenfrom membrane phospholipids producinglipid radicals, which in turn can react withmolecular oxygen to produce lipid peroxylradicals. This process is propagated result-ing in the destruction of membrane phos-pholipids and cell injury. It is postulatedthat lipid peroxidation plays a major rolein the pathogenesis of pneumoconiosis(5,10,11,17,18). Recent studies, using cell-free lipid peroxidation models, alveolarmacrophages, or whole lung slices, provideevidence that supports the basic mechanismof cell injury and its correlation to increasedgeneration ofROS from minerals (11).

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'?05 . SopdFigure 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 x106 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 equalsurface area. The PBN spin adducts of free radicalswere measured by ESR (7).

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Figure 3. Relative oxygen radical generation in thereactions 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 using5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spintrap (10). Each bar indicates the mean and standardderivation of three experiments.

Membrane InjuryMany animal studies and humaninvestigations have reported that bron-choalveolar lavage fluid (BALF) collectedafter exposure to occupational and envi-ronmental pollutants shows increasedevidence of oxidative injury (17,18).Extracellular release of proteases, lyticenzymes, ROS, and cytokines, up regula-tion and consumption of antioxidantenzymes, and amplification of inflamma-tory mediators and growth factors are themost frequent changes accompanyingoxidative injury. Repeated cycles of cellinjury associated with the release of inflam-matory mediators and ROS set the stagefor activation of fibroblastic proliferationand collagen formation, resulting inpulmonary fibrosis.

In vitro findings show that freshlyfractured crystalline silica is more cytotoxicand inflammatory than the aged silica andthat this activity is associated with theenhanced concentration of surface radicalson fresh cleavage planes (10,11). Results ofstudies with rats after inhalation of fresh oraged silica provided direct evidence thatfresh silica is more potent in increasinginflammation, causing lung injury and lipidperoxidation, inducing generation of ROS,

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

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and causing up regulation and consumptionof antioxidant enzymes (17,18).

CarcinogenesisROS induce point mutations andchromosomal aberrations in cells. ROS aregenerated by multiple pathways in thelungs in response to inhalation of toxicpollutants. Many of the toxic substancesinhaled contribute to oxidative alterationand modification, and they target specificattack of vital components of the cyto-plasm and nuclei. Such changes mayinclude ROS-induced DNA strand breaks,oxidative modification of DNA, basemodifications, 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 phagocytosedminerals can continue to cause damageto key cellular organelles and promotethese reactions.

DNA Strand BreaksROS can attack deoxyribose, purine, andpyrimidine bases in DNA resulting inDNA strand breaks. DNA strand breaksinduced by 'OH cause deletions and point

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mutations, resulting in oncogene activation.We have studied DNA strand breaks afterexposure ofDNA to occupational and envi-ronmental pollutants and monitored thealterations by an alkaline unwinding assay.Figure 5 illustrates the relative measure ofdisruption ofDNA structure caused by cro-cidolite, amosite, and chrysotile asbestos.Chrysotile asbestos caused significant DNAstrand breaks, which were further enhancedby the addition of Fe(II) and H202. Theseincreased DNA strand breaks by chrysotilemay be caused by the large surface area ofchrysotile. These results show that all athese particles can induce direct DNAstrand breaks. DNA strand breaks inducedby silica particles were also studied to exam-ine the role of ROS (21). As shown inFigure 6, incubation of DNA with silicacaused significant DNA strand breaks.When the incubation was carried outunder argon, no DNA strands breaks wereobserved, which demonstrates the require-ment for molecular oxygen. Addition ofthe catalase inhibited DNA strand breaks.Because molecular oxygen is the precursorof 02- and H202, the results presented inFigure 6 further demonstrate that ROSplay a key role in the mechanism ofsilica-induced DNA strand breaks.

'OH-Induced DNAOxidative DamageHydroxylation 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 electrochemicaldetection, we measured significant 8-OHdG formation in the presence of iso-lated DNA and varying concentrations ofoccupational and environmental dusts.Antioxidants such as catalase and formateinhibited the 8-OHdG formation, demon-strating that 'OH radical-specific interac-tion was involved in this DNA basedamage. DNA base modifications causedby these changes could cause DNA mis-pairing, which could lead to point muta-tions and oncogene activation. Figure 7illustrates the results of these studies usingasbestos, coal, and crystalline silica.

Nuclear Factor Kappa B ActivationNuclear factor kappa B (NF-icB) activationis an important transcription factor andplays a critical role in inflammatory andimmune response, as well as activation ofoncogenes, cytokine receptors, cell adhesionmolecules, and growth factors (23,24).NF-KB is activated by ROS, cytokines,viruses, protein kinase C activators, andimmunological stimuli (25). Antioxidantssuch as N-acetylcysteine and pyrrolidine

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Figure 5. Percentage of DNA strand breaks afterexposure 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 ethidiumbromide reactive fluorescence was measured at anexcitation wavelength of 525 nm and emission wave-length of 600 nm.

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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 freshlyfractured silica particles with X Hind Ill-digested DNAin a phosphate-buffered solution, pH 7.4; lane 3, sameas lane 2 but incubation was carried out under argon;lane 4, same as lane 2 but with 7500 U/mI catalaseadded. The samples were incubated for 3 weeks.

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

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dithiocarbamate are reported to inhibit the NF-iKB activation by promoting theNF-KB activation. We have observed that dismutation of 02'- to generate more 'OHinteractions of many occupational and radicals. Thus, among these reactive oxy-environmental pollutants with alveolar gen species, 'OH radical plays a major rolemacrophages 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 regulateica 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-aica was effectively blocked by catalase or production and the role of NF-KB andthe 'OH-specific scavenger, sodium for- ROS. Crocidolite asbestos induced themate. NF-KB was also inhibited by the production of TNF-ax from an alveolarmetal chelator, deferoxamine, suggesting macrophage cell line and from primarythe involvement of the Fenton or Fenton- alveolar cells in dose- and time-dependentlike reaction in 'OH generation. On the manners. Maximal secretion was induced inother hand, superoxide dismutase increased 8 hr at a concentration of 50 pg/ml croci-

dolite (27). Inhibition of NF-KB by aninhibitor of NF-icB nuclear translocation(SN50, cell-permeable inhibitory peptide),or by sequence-specific oligonucleotidesdirected against the TNF-a binding site ofNF-KB, attenuated the effect of asbestos onTNF-x production. Gene transfectionassays with a plasmid construct containing

... TNF-ac binding sites of NF-icB linked to aluciferase reporter gene further showed thatasbestos induced transcriptional activationof NF-icB-dependent genes. This activationWas also inhibited by SN50. Treatment ofthe cells with oxygen radical scavengersinhibited 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, alsoinhibited both NF-KB activation andTNF-cx production. These results show

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ated free radical reactions.~~~~~..

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Inactivation ofTumor Suppressor...,,,2, t.,:ihsi's. s9gSi'W:: F' Gene (p53)

Mutational inactivation of the tumorsuppressor gene is an important molecularalteration most frequently found in manytypes of cancers (28). It is estimated thatapproximately 50 to 60% of human lungcancers 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 cancercells and preneoplastic cells. Several types of

Figure 8. Induction of DNA binding activity of NF-KB DNA damage can activate p53, includingprotein by silica and the effect of catalase, superoxide double-strand breaks (28,29). Oxidativedismutase. sodium formate, and deferoxamine mea- signaling is involved in the regulation ofsured according to the method of Chen et al (25). The early changes in gene expression in a cellRAW 264.7 cells were incubated with stimuli for 6 hr. cycle during GO to G, phase transitionLane 1 untreated cells (5 x 106/ml), lane 2 cells + 100pg/mI silica; lane 3, cells + 100 pg/mI silica + 10,000 (30,31). It is known that ROS induces aunits/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 ofp53 and thepg/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 controllinggrowth inhibitory and apoptotic pathways.It is believed that p53 serves as a tumor sup-pressor by preventing the passage of geneticlesions in cells with DNA damage to a newgeneration of cells. It does this either byhalting cell division to allow for DNArepair or by inducing apoptosis of damagedcells. Mutational inactivation ofp53 is afrequent molecular alteration in humancancers, which indicates the importance ofthe p53 gene in human carcinogenesis(28,29). There are 10 cysteine residues inp53 protein. Redox regulation at a post-translational level often occurs by reductionor oxidation of a disulphide bond. Thereducing environment in cells is importantfor active p53 protein. Exposure of cells tosilica partides may convert the cells to a pro-oxidant state resulting from the increasedproduction of ROS or decreased expressionof antioxidant enzymes. This oxidizingenvironment may render wild-type p53conformational mutant and give rise to thesame biologic outcome as p53 mutation.However, information is still not availableregarding the effect of silica particles onp53 activity. This area deserves activeinvestigation regarding the mechanisms ofsilica-induced carcinogenesls.

AP-1 ActivationAP-1 is an important transcriptional factorinvolved in tumor promotion and itsactivation appears to play an importantrole in carcinogenesis. Its regulation of pro-tooncogenes, c-jun and c-fos, is reported tohave a key role in the induction of growthfactor genes (33). We have obtained pre-liminary evidence suggesting the involve-ment of ROS in AP-1 activation. Themechanism of ROS-induced activation ofAP-1 is an area of active research. Janssenet al. (33) suggested that alteration in thecellular thiol redox status is caused byoxidative stress and subsequent AP-1 acti-vation. In our preliminary studies, ROSgenerated during the interaction of dustssuch as crystalline silica and asbestosinduced the activation of AP-1 in luciferasereporter mouse epidermal cells (JB6P+).Superoxide and catalase inhibited this acti-vation, indicating the involvement of ROSin the activation, of AP-1. Studies fromother laboratories have shown that asbestosand ROS generated by the xanthine/xan-thine oxidase system and H202/Fe(II)induces the expression of c-fos and c-junearly response genes (34). Products of c-fosand c-jun protooncogenes interact toregulate gene expression.

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ConclusionsOur studies provide evidence that supportsthe hypothesis for ROS-induced pneumoco-niosis and carcinogenesis. Upon reactionwith water or H202, silica and other mineralparticles can generate ROS. ROS can alsobe generated by phagocytic cells stimulated

by these particles. ROS and other secondaryradicals generated by ROS can cause lipidperoxidation leading to membrane damageand DNA damage via strand breaks andbase modification. Through free radicalreactions, mineral particles activate nucleartranscription factors, enhance secretion of

growth factors, induce oncogene expression,and cause mutation of tumor suppressorgenes, resulting in proliferation of cells.These processes may be significantlyinvolved in the mechanisms of pneumo-coniosis and carcinogenesis induced bymineral particles.

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