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Chem. Res. Toxicol. 2007, 20, 724-733

Articles Genotoxic Mechanisms of Asbestos Fibers: Role of Extranuclear Targets An Xu,†,‡ Xuelian Huang,§ Yu-Chin Lien,† Lingzhi Bao,‡ Zengliang Yu,‡ and Tom K. Hei*,†,§ Center for Radiological Research, College of Physicians & Surgeons, and Department of EnVironmental Health Sciences, Mailman School of Public Health, Columbia UniVersity, New York, NewYork 10032, and Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui, Peoples’s Republic of China ReceiVed December 29, 2006

Asbestos fibers are carcinogenic to both humans and experimental animals. The continued discoveries of exposure routes whereby the general public is exposed to asbestos suggest a long-term, low-dose exposure for a large number of people. However, the mechanisms by which asbestos induces malignancy are not entirely understood. In previous studies, we have shown that asbestos is an effective gene and chromosomal mutagen when assayed using the highly sensitive AL mutation assay and that the mutagenicity is mediated by reactive oxygen species. The objective of the present study is to determine the origin of these radical species, particularly reactive nitrogen species, in fiber mutagenesis. Using the radical probe 5′,6′-chloromethyl-2′,7′-dihydroxyphenoxazine diacetate to trap reactive radical species, we showed that crocidolite increased the levels of oxyradicals in cytoplasts, in the absence of the nucleus, in a dosedependent manner, which was reduced significantly by cotreatment with the radical scavenger dimethyl sulfoxide. Treatment of enucleated cells with crocidolite asbestos followed by rescue fusion using karyoplasts from control cells resulted in significant mutant induction, indicating that the nuclearcytoplasmic interaction is necessary for fiber mutagenesis. Using the fluorescent probe 2,3-diaminonaphthotriazole, crocidolite fibers were shown to induce a dose-dependent increase of nitric oxide production, which was suppressed significantly by concurrent treatment with the nitric oxide synthase inhibitor, NGmethyl-L-arginine (L-NMMA). Similarly, there was a dose-dependent decrease in the mutation yield induced by crocidolites in the presence of graded doses of L-NMMA. These data showed that extranuclear targets play an essential role in the initiation of oxidative damage that mediates fiber mutagenesis in mammalian cells. Introduction Asbestos is a naturally occurring hydrated fibrous silicate and has been used in more than 3000 products by virtue of its high tensile strength, relative resistance to acid and temperature, varying textures, and degrees of flexibility. While the use of asbestos has been restricted in much of the developed world, consumption is growing in Asia, Latin America, and the Commonwealth of Independent States (1). Asbestos fibers have been found to be ubiquitous in the environment. Individuals can be exposed to asbestos in different nonoccupational circumstances such as living with asbestos workers and exposure to fiber-laced work clothes, environmental exposure in the neighborhood of industrial sources (asbestos mines and mills and asbestos processing plants), passive exposure in buildings containing asbestos, and natural environmental exposure to geological sources (2, 3). The fact that asbestos has been used extensively in industry and the continued discovery of asbestos * To whom correspondence should be addressed. Tel: 212-305-8462. Fax: 212-305-3229. E-mail: [email protected]. † College of Physicians & Surgeons, Columbia University. ‡ Chinese Academy of Sciences. § Mailman School of Public Health, Columbia University.

exposure in the general population suggest a long-term, lowdose exposure for a large number of people for many years to come. Both experimental animals and epidemiologic data have shown that asbestos exposure is closely associated with the development of pulmonary fibrosis, bronchogenic carcinoma, and malignant mesotheliomas of the pleura and peritoneum. However, the mechanisms by which asbestos produces malignancy are not fully known. Various in ViVo and in Vitro studies have been widely used to assess the carcinogenic potential of asbestos fibers. Although there is considerable evidence that asbestos fibers induce chromosome aberrations, micronuclear formation, aneuploidy, and polyploid formation in mammaliancells (4-6), earlier attempts at defining the mutagenic potential of fibers at the HPRT, OUA, and HLA-A loci in a variety of treated cells have yielded largely negative results (79). Subsequent studies have suggested that this could be a result of multilocus deletions induced predominantly by asbestos that are not compatible with the survival of the mutants (10). In the past few years, several mutagenicity assays that are proficient in detecting either large deletions, homologous recombinations, or score mutants located on nonessential genes have been used

10.1021/tx600364d CCC: $37.00 © 2007 American Chemical Society Published on Web 04/21/2007

Extranuclear Targets and Genotoxicity of Crocidolites

successfully to demonstrate the mutagenic potential of various fiber types, suggesting a close relationship between chromosomal abnormalities that have occurred frequently in humans and rodent cells exposed to fibers and carcinogenicity in ViVo (11-13). Furthermore, there is evidence that phagocytotic uptake of fibers plays an essential role in mediating asbestos toxicity and that fiber-mediated mutation yield in mammalian cells can be enhanced by buthionine-SR-sulfoximine, a competitive inhibitor of the enzyme γ-glutamyl cysteine synthetase, and can be inhibited by concurrent treatment with catalase and superoxide dismutase (SOD)1 (14-16). These findings indicate that oxyradicals may be the key determinants in asbestosinduced mutagenesis and carcinogenesis. However, the precise molecular pathways linking asbestos exposure with oxidative stress and genotoxicity are still not fully understood. Oxidative stress has been widely implicated as a mechanism of carcinogenesis, occurring through overproduction of reactive oxygen and nitrogen species under either endogenous or exogenous insults (17, 18). Reactive oxygen species (ROS) including superoxide anion (O2-•) and hydrogen peroxide (H2O2) originate not only from redox reactions catalyzed on the asbestos fiber surface but also from the incomplete phagocytosis of fibers in various cells (19). In the lung, there is evidence that O2-• can be generated from metal ions on the surface of fibers (notably ferrous ion) by electron-transfer reactions involving a single electron to molecular oxygen (20). Reactions between O2-• and H2O2 produce OH• by either the Fenton or the HaberWeiss iron-catalyzed reaction (21). Concurrent exposure to antioxidant enzymes such as SOD, catalase, and the radical scavengers, tempol and dimethylthiourea, have been shown to protect phagocytic cells, mesothelial cells, and human and rat lung epithelium cells against fiber toxicity (16, 22). Although the formation of OH• via the Fenton reaction is a likely source of asbestos-induced intracellular oxidant, especially in the presence of iron, other pathways involving reactive nitrogen species (RNS) may also be involved since OH• is so highly reactive that it can only diffuse a short distance from the site of formation (23). It has been reported that crocidolite exposure increases the synthesis of NO in human lung epithelial cells, murine mesothelial cells, glial cells, and alveolar macrophages (24-26). Furthermore, NO reacts with O2-• to form the more reactive nitrogen radicals such as peroxynitrite anions (ONOO•), which result in the nitration of proteins, hydroxylation or nitration of DNA, and mutations (27). Although it has been postulated that NO and ROS cooperate in mediating the cytotoxic and mutagenic effects of asbestos, the origin of these oxyradical species and the pathways involved in the generation of other secondary radical species remains to be illustrated. Crocidolite, containing up to 27% iron by weight, is one of the most potent carcinogenic forms of asbestos fibers. We have demonstrated previously that hydroxyl radicals (OH•), generated through a superoxide-mediated process involving H2O2, play an essential role in mediating the genotoxic effects of crocidolite (28). Although the source of these radicals is not known, it is likely to be the mitochondria, the power house of cells in which approximately 90% of inhaled oxygen is consumed (29). Whereas crocidolites have been shown to alter mitochondrial gene expression, modify membrane potential, and cause mitochondria DNA damage in various human cancer cells (30, 31), 1 Abbreviations: L-NMMA, NG-methyl-L-arginine; D-NMMA, NGmethyl-D-arginine; ROS, reactive oxygen species; RNS, reactive nitrogen species; DMSO, dimethyl sulfoxide; CM-H2DCFDA, 5′,6′-chloromethyl2′,7′-dihydroxyphenoxazine diacetate; OH•, hydroxyl radicals; NO, nitric oxide; ONOO•, peroxynitrite anions; H2O2, hydrogen peroxide; O2-•, superoxide anions; NOS, nitric oxide synthase; SOD, superoxide dismutase.

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the role of mitochondria as a genotoxic target of crocidolite has yet to be established. Using human-hamster hybrid (AL) cells and enucleation and fusion techniques, we show here that extranuclear targets play an essential role in the initiation of oxidative damage associated with fiber mutagenesis and that ONOO• are important mediators in the process.

Materials and Methods Cell Culture. The human-hamster hybrid (AL) cells, containing a standard set of CHO-K1 chromosomes and a single copy of human chromosome 11, were used in these studies. These cells have an average diameter of ∼25 µm with an average nuclear cross-sectional area of 108 µm2. Chromosome 11 encodes cellular surface markers that render AL cells sensitive to killing by specific monoclonal antibodies in the presence of rabbit serum complement (HPR, Inc., Denver, PA). Antibody E7.1 specific to the CD59 antigen was produced from hybridoma culture as described (16, 28). Cells were maintained in Ham’s F-12 medium, supplemented with 8% heatinactivated fetal bovine serum, 25 µg/mL gentamicin, and 2× normal glycine (2 × 10-4 mol/L), at 37 °C in a humidified 5% CO2 incubator, and were passaged as described (32, 33). Preparation of Crocidolite Fibers. The International Union Against Cancer (UICC) standard reference crocidolite fibers (average length, 3.2 ( 1.0 µm; average diameter, 0.22 ( 0.01 µm) were used in these studies. The fibers were prepared as described (10, 28). Briefly, samples of fibers were weighted and suspended in distilled water. The fiber suspension was triturated 6-8 times with a 20 gauge syringe needle. A stock solution of the fibers was sterilized by autoclaving and mixed to ensure a uniform suspension before being diluted with tissue culture medium for cell treatment. Crocidolite Fibers and Chemical Treatment. Working concentrations of fiber containing media were prepared by diluting the stock with complete F-12 medium. Enucleated cytoplasts were treated with graded doses of crocidolite fibers for various periods of time. After treatment, cells were washed twice with plain F-12 medium and processed for the various end points. In experiments involving the nitric oxide synthase (NOS) inhibitor, both the active NG-methyl-L-arginine (L-NMMA) and the inactive enantiomer NGmethyl-D-arginine (D-NMMA; Molecular Probes, Inc., Eugene, OR), dissolved in distilled water (10 mM stock) and filter sterilized, were added to the cultures 24 h before crocidolite fiber treatment and remained in the medium or buffer throughout the treatment period. Enucleation of AL Cells. Exponentially growing AL cells (approximately 2 × 105 cells per dish) grown in either 35 mm diameter tissue culture plastic dishes or 35 mm glass bottom microwell dishes (DTC3 dishes, BiopTechs, Butler, PA) were enucleated as described (32, 34). Cells were treated with cytochalasin B (1 µg/mL) for 60 min, and the dishes containing the cells were centrifuged upside down inside a 250 mL Sorvall centrifuge bottle (Nalge-Nunc International, Binghamton, NY) using a GSA rotor at 6700 rpm at 37 °C for 20 min. After centrifugation, the cultures were washed three times with complete medium to remove excess cytochalasin B. The efficiency of enucleation was evaluated by the absence of nuclear staining with a 50 nM solution of Hoechst 33342 (Polysciences, Warington, PA) for 20 min. The enucleated cells were allowed to recover by incubation at 37 °C for 30 min before being subjected to crocidolite treatment for a period of 3-4 h, as described above. Preparation of Karyoplasts from AL Cells. The nuclei centrifuged out of the AL cells, containing a small amount of cytoplasm (6 µg/cm2 (p < 0.05). The fluorescent intensity in whole cells treated with a 6 µg/cm2 dose of crocidolites was about three-fold higher than the background. Similarly, enucleated cells treated with crocidolite at a dose of 2 µg/cm2 (Figure 8B,D) were able to induce ONOO-• in the absence of nuclei. The average fluorescent intensity in crocidolite-treated cytoplasts was 2.4-fold above nontreated cytoplasts. It should be noted that the fluorescent intensity obtained with whole cells was 15-20% higher than that of cytoplasts due to lost volume during the enucleation process. Consequently, to equate the relative fluorescent signals in the cytoplasts with those of whole cells, the sensitivity of the fluorescent detector was increased in studies with cytoplasts. In the presence of L-NMMA, the fluorescent signals in both crocidolite-treated whole cells (Figure 7C) and enucleated cells (Figure 8C) were suppressed significantly (p < 0.05).

Extranuclear Targets and Genotoxicity of Crocidolites

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Figure 7. Generation of ONOO• in AL cells treated with graded doses of crocidolite in the presence or absence of either L-NMMA or D-NMMA at a dose of 15 µM. Representative fluorescence signals generated from composite images were obtained by confocal microscopy in AL cells preloaded with dihydrorhodamine 123 with or without subsequent crocidolite treatment at a dose of 6 µg/cm2. (A) Control AL cells treated with only the fluorescent probe, (B) 30 min after the addition of crocidolite, (C) treatment as in part B but with concurrent L-NMMA (600×), and (D) relative fluorescence intensity of ONOO• in AL cells as a function of crocidolite concentration with or without either L-NMMA or D-NMMA. The relative intensities are expressed in arbitrary units. Data were pooled from three independent experiments; bars, (SD.

Protein Nitration in Crocidolite-Treated Cells. Nitrotyrosine is considered as a long-lived footprint of NO and ONOO-• (42). Although a weak immunoreaction was observed in untreated controls, there was a dose-dependent increase in the nitrotyrosine immunoreactivity in cells exposed to crocidolite (Figure 9). Crocidolite treatment induced the nitration of several proteins, most notably proteins of molecular masses ranging from 37 to 75 kDa (indicated by arrows in Figure 9A). The addition of L-NMMA to the crocidolite-treated group (6 µg/ cm2) reduced the level of protein modification by 37%.

Discussion Asbestos is one of the most pervasive environmental hazards in the world. It has been estimated that more than 30 million tons of asbestos in its various forms have been mined in the past century (1). Over 70% of the world production is used in Eastern Europe and Asia. The highest per capita consumption occurs in Russia, Kazakhstan, Belorussia, Kyrgysztan, and Thailand (over 2.0 kg/capita/year), whereas less than 0.1 kg/ capita/year consumption occurs in Western Europe and North

America. Asbestos exposure affects millions of workers worldwide in occupational settings not only during maintenance, repair, and replacement of asbestos-containing materials, but also through the exposure to fiber-containing building materials such as heating and ventilating systems, the effect extends to workers’ families and consumers in environmental settings. In developing countries, where environmental and occupational health regulations are scant or nonexistent, the incidence of asbestos-associated diseases is likely to continue to increase for decades to come (43). Crocidolite, one of the most carcinogenic forms of asbestos, is mutagenic in cultured mammalian cells when assayed using a system that can detect multilocus deletions. There is a 50fold increase in mutations at the CD59 locus when compared with the HPRT locus in crocidolite-treated AL cells (10). The oxidation of CM-H2DCFDA by ROS, as detected using confocal microscopy, provided strong evidence that asbestos induced a dose-dependent increase of ROS in single cells that could be inhibited by DMSO. Moreover, mutation spectrum analysis showed that the types of CD59- mutants induced by crocidolite

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Figure 8. Induction of ONOO• in cytoplasts treated with crocidolite at a dose of 2 µg/cm2 in the presence or absence of either L-NMMA or D-NMMA at a dose of 15 µM. Representative fluorescence signals generated from composite images were obtained by confocal microscopy in cytoplasts preloaded with dihydrorhodamine 123 with or without subsequent crocidolite treatment at a dose of 2 µg/cm2. (A) Enucleated cells treated with only the fluorescent probe, (B) 30 min after the addition of crocidolite, (C) treatment as in part B but with L-NMMA (600×) , and (D) relative fluorescence intensity of ONOO• in cytoplasts treated with crocidolite concentration with or without either L-NMMA or D-NMMA. The relative intensities are expressed in arbitrary units. Data were pooled from three independent experiments; bars, (SD.

Figure 9. Western blot analyses of nitrotyrosine-modified proteins in cells treated with graded doses of crocidolite with or without concurrent treatment of either L-NMMA or D-NMMA (15 µM). β-Actin was used as a protein loading control. The image was quantified by ImageJ and expressed as induced mean pixel intensities as compared with nontreated controls.

fibers were similar to those induced by equitoxic doses of H2O2, indicating that similar mutagenic mechanisms are involved between asbestos fibers and chemically generated ROS (28). These findings are consistent with the reports stipulating the

functional role of oxidants in mediating the biological effects of crocidolite by linking glutathione content, heme oxygenase, and oxidative DNA damage as the end points being examined (19, 22). However, these experiments did not specifically

Extranuclear Targets and Genotoxicity of Crocidolites

identify the origin of these radical species and if/whether secondary radical species were involved in the genotoxicity of crocidolite fibers. By using enucleation and cell fusion techniques, we have demonstrated that the nucleus is not necessarily the only and sufficient target for fiber-mediated genotoxicity. Crocidolite treatment of enucleated cytoplasts initiated oxyradical generation in a dose-dependent manner as demonstrated through the use of a well-established free radical probe, CM-H2DCFDA, that can be trapped in live cells and thereby provide reliable intracellular fluorescent signals (37). Moreover, treatment of enucleated cells with crocidolite followed by rescue fusion with karyoplasts from controls resulted in a significant increase of mutation yield, which was in turn dramatically decreased by DMSO. These results suggested that the cytoplasm plays a critical role in the initiation of oxidative damage, a finding consistent with previous observations in enucleated cells treated with arsenic and cells targeted by cytoplasmic irradiation with R-particles (32, 44). Nevertheless, it should be noted that due to the biopersistent properties of crocidolite fibers, crocidolitetreated cytoplasts were likely to contain some fiber remnants even after extensive washing (45). Consequently, the reaction between crocidolite fibers and cytoplasts was unlikely to be completely eliminated after cytoplasts were rescued by fusion with karyoplasts. Mitochondria, the cytoplasmic organelles that harbor the bulk of oxidative pathways, are the major source of the generation of ROS (29). There is evidence to show that mitochondrial membrane damage results in the leakage of electrons and ROS into the cytoplasm and in an increase of oxidative stress of the cells (31, 46, 47). However, the exact functional role of mitochondria as a genotoxic target of crocidolite fibers has not been well-established. Since the discovery in 1987 that the endothelium-derived relaxing factor was NO, a huge amount of research has revealed that NO acts as a mediator in a wide variety of physiologic and pathophysiologic conditions such as inflammatory diseases and cancer (48, 49). Cellular production of NO from L-arginine is catalyzed by three NOS enzymes, each encoded by distinct genes: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (50). Endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed, while inducible NOS (iNOS) expression is enhanced by various inflammatory cytokines. The immunohistochemical expression of iNOS was detected in 74 and 96% of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively (51). Because mitochondria constitute a major locus for the intracellular formation and reactions with NO (which travels significant distances from its point of origin due to its electrical neutrality and small size), it is likely that multiple radical species are involved in the genotoxic response of crocidolite (52, 53). NO reacts with O2-• and can be rapidly converted into more reactive nitrogen compounds such as ONOO• that can cause nitration of proteins, hydroxylation or nitration of DNA, and mutations (54, 55). There is evidence to indicate that NO is mutagenic in TK6 cells and causes A:T to G:C transversions in plasmids and C to T transitions in a bacterial system (56). Crocidolite exposure has been reported to stimulate the expression of NOS and increase the production of NO in several kinds of cell systems (25, 57). In the presence of the NOS inhibitor L-NMMA, the mutation yield induced by crocidolite in AL cells was dramatically decreased in a dose-dependent fashion, a result consistent with the findings in transgenic V79 cells (11). These results indicate a key role of NO in the mechanisms of asbestos-

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induced genotoxicity. L-NMMA is a competitive inhibitor of the enzyme NOS (41). In this study, L-NMMA was used in completed F12 medium containing ∼1.2 mM L-arginine. At the dosages of 5 and 15 µM that were used, L-NMMA was not expected to compete with L-arginine for NOS from a strictly chemical point of view. However, evidence has been shown of the presence of a transporter system that concentrates methylarginine within the cells such that the intracellular concentration of L-NMMA may exceed the extracellular concentration by more than five-fold (58). Although inhibition of NOS activity by L-NMMA is reversible by the addition of L-arginine, the stoichiometry in biological systems is not necessarily 1:1 and an excess of arginine is required to reverse inhibition caused by L-NMMA. In the presence of L-NMMA, there is evidence that the apparent Km of NOS for arginine increases (59). In addition to inhibiting NOS generation reversibly, there are data to indicate that L-NMMA may also inhibit NOS in an irreversible manner under certain conditions and with time (41, 60). Several recent reports have suggested that the activity of NOS can be inhibited by L-NMMA in bone marrow cells, human cholangiocarcinoma cells, Hep3B cells, and adult ventricular myocytes (61-66). Although asbestos is perhaps the best known industrial fiber, the mechanism of asbestos carcinogenesis has not been clearly understood. It is only relatively recent that the importance of oxidative stress induced by the inhalation of biopersistent asbestos has been recognized in the induction of cancer and other asbestos exposure-associated conditions such as fibrosis and immune system toxicity. Our present findings that extranuclear targets play an essential role in the initiation of oxidative damage in fiber mutagenesis in mammalian cells provide new insight to understand the carcinogenicity of asbestos fibers and to improve therapies of asbestos-related diseases. Acknowledgment. This work was supported in part by grants from the National Institutes of Health ES05786, Superfund Grant ES10349, NIEHS Center Grant ES09089, and National Nature Science Foundation of China 20322202. We thank Dr. Vladimir Ivanov and Dimitar Zlatev for critical reading of the manuscript and Dr. Gengyun Wen for his technical assistance with Western blotting.

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