Reactive oxygen species: their relation to pneumoconiosis and carcinogenesis.

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.


Introduction
Reactive oxygen species (ROS) have been implicated in the pathogenesis of pneumoconiosis and pulmonary carcinogenesis (1)(2)(3)(4)(5)(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 hypochloride (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 generation 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, cigarette 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 incidence of pulmonary neoplasms (5,6). However, the mechanisms by which ROS promote pneumoconiosis and carcinogenesis are speculative. This review briefly summarizes some of the recent findings from our laboratories, emphasizing the molecular events by which ROS are involved in promoting pneumoconiosis and carcinogenesis.

Mechanisms of Reactive Oxygen Species Generation
During the process of phagocytosis of inhaled particulate, 02is 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 environmental pollutants leads to excessive in vitro and in vivo generation of OH radicals during phagocytosis (7)(8)(9). Such an overwhelming burst of ROS generation subsequently triggers a severe inflammatory cellular reaction resulting in additional generation 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, agricultural 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  8-hydroxy-2'-deoxyguanosine; BALF, bronchoalveolar lavage fluid; DMPO, 5,5-dimethyl-1-pyrroline Noxide; dG, guanine residues; DNA, deoxyribonucleic acid; ESR, electron spin resonance; 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. pg/mI; amosite, 50 pg/mI; chrysotile 50 pg/mI; crocidolite, 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 tl51 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 minerals, 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 fracturing as occur in occupational settings (10)(11)(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-

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 resulting in the destruction of membrane phospholipids 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 cellfree 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).

Membrane Injury
Many animal studies and human investigations have reported that bronchoalveolar lavage fluid (BALF) collected after exposure to occupational and environmental pollutants shows increased evidence of oxidative injury (17,18). Extracellular release of proteases, lytic enzymes, ROS, and cytokines, up regulation and consumption of antioxidant enzymes, and amplification of inflammatory mediators and growth factors are the most frequent changes accompanying oxidative injury. Repeated cycles of cell injury associated with the release of inflammatory 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 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 cytoplasm 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 inactivation of tumor suppressor genes (19,20). Persistent generation of ROS from indestructible or incompletely phagocytosed minerals can continue to cause damage to key cellular organelles and promote these reactions.  20 10 mutations, resulting in oncogene activation. We have studied DNA strand breaks after exposure of DNA to occupational and environmental pollutants and monitored the alterations by an alkaline unwinding assay. Figure 5 illustrates the relative measure of disruption of DNA structure caused by crocidolite, 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 examine 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 requirement 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.

DNA Strand
'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 chromatography (HPLC) with electrochemical detection, we measured significant 8-OHdG formation in the presence of isolated DNA and varying concentrations of occupational and environmental dusts. Antioxidants such as catalase and formate inhibited the 8-OHdG formation, demonstrating that 'OH radical-specific interaction was involved in this DNA base damage. DNA base modifications caused by these changes could cause DNA mispairing, which could lead to point mutations 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  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 oxyenvironmental 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 doseand 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 crocidolite (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 ... 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 ofp53 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.

TNF-ac binding sites of NF-icB linked to
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 suppressor 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 posttranslational 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 prooxidant 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 protooncogenes, c-jun and c-fos, is reported to have a key role in the induction of growth factor genes (33). We have obtained preliminary evidence suggesting the involvement 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 activation. 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 activation, 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/xanthine 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.

Conclusions
Our studies provide evidence that supports the hypothesis for ROS-induced pneumoconiosis 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 pneumoconiosis and carcinogenesis induced by mineral particles.