Chemiluminescent Detection of Induced Reactive Oxygen Metabolite Production of Human Polymorphonuclear Leucocytes by Anthophyllite Asbestos

Transcription

1 Environmental Research Section A 88, 36}40 (2002) doi: /enrs , available online at on Chemiluminescent Detection of Induced Reactive Oxygen Metabolite Production of Human Polymorphonuclear Leucocytes by Anthophyllite Asbestos Toyoto Iwata*, Norihiko Kohyama-, and Eiji Yano* *Department of Public Health, Teikyo University School of Medicine, 11-1, Kaga 2-chome, Itabashi-ku, Tokyo , Japan; and -Division of Work Environment Evaluation, National Institute of Industrial Health, 21-1, Nagao 6- chome, Kawasaki, Kanagawa , Japan Received August 2, 2001 Incidences of lung cancer and pleural plaque have been reported in relation to exposure to anthophyllite asbestos. To investigate the pathogenic mechanisms of anthophyllite, chemiluminescence (CL) detection of reactive oxygen metabolite (ROM) generation of human polymorphonuclear leucocytes (PMN) stimulated by anthophyllite asbestos was determined and compared with that of other asbestos and mineral Aber samples. When anthophyllite Aber sample was mixed with the luminol-primed PMN, high levels of CL which exhibited a speciac time course characterized by two separate peaks were induced. The CL induced by anthophyllite sample was greater than that induced by chrysotile, crocidolite, and amosite asbestos. We further investigated the two peaks of CL using speciac inhibitors of signal transduction mechanisms. The two peaks of CL by anthophyllite sample were different in sensitivity to cytochalasin B and genistein; the former relates to the cytoskeleton-dependent mechanism and the latter has been shown to inhibit tyrosine kinase, which resides in the pathway to cause PMN activation. The strong ROM reaction of PMN by anthophyllite suggests that the surface characteristics of the Aber may participate in the pathogenic mechanisms of anthophyllite asbestos Elsevier Science INTRODUCTION It has been hypothesized that the carcinogenicity of mineral 7bers is dependent on 7ber shape, dimension, and durability (Stanton et al., 1981), though 1 To whom correspondence should be addressed. Fax: # eyano@med.teikyo-u.ac.jp. 36 physicochemical properties related to 7ber type and surface characteristics are also considered to play a role (Mossman et al., 1996). To analyze the factors and biochemical mechanisms related to carcinogenesis, it is necessary to re7ne our observations on the cellular and molecular interactions of 7bers, comparing asbestos 7bers with various characteristics. Anthophyllite asbestos has been reported to produce a high risk of lung cancer among a Finnish anthophyllite miner cohort (Meurman et al., 1994; Karjalainen et al., 1994). In addition to carcinogenicity, more than a thousand residents of a Japanese town, where an anthophyllite mine and mill had operated, have been shown to have pleural plaques (Hiraoka et al., 1998; Murai et al., 1997). To obtain an insight into the mechanism of 7ber carcinogenicity, we have examined the biological potency and differential reactivity of anthophyllite and other amphiboles and serpentine asbestos by studying its interaction with human blood polymorphonuclear leukocytes (PMN). PMN and macrophages produce reactive oxygen metabolites (ROM) when stimulated by chemotactic factors or other particulate agents, including asbestos. ROM production can be observed with luminoldependent chemiluninescence (CL) (Ishizaki et al., 1994, 1997). The signal of chemotactic factor, namely formyl methionyl-leucyl-phenylalanine (fmlp), is transduced to cause NADPH oxidase complex activation of the respiratory burst and associated superoxide production. The signal transduction occurs through a complex activation system involving intracellular Ca 2#, protein kinase C, protein tyrosine kinase, and phosphatidylinosital 3-kinase (Yan and Novak, 1999; Dusi et al., 1996; Arcaro and Wymann, 1993). Other systems such as /02 $ Elsevier Science All rights reserved.

2 ROM PRODUCTION BY ANTHOPHYLLITE 37 cytoskeletons are known to modulate these processes. Cytochalasin B, a cytoskeleton polymerization inhibitor, in8uences ROM production (Yan and Novak, 1999). In the present study, the characteristics of asbestos-induced CL reaction of PMN were investigated using several inhibitors of signal transductions. Macrophages have been shown to use a signal transduction system very similar to that of chemotactic peptide (Roney and Holian, 1989; Lim et al., 1997), when stimulated by chrysotile, crocidolite, and amosite. The purpose of the present study was to determine the characteristic PMN CL reaction induced by anthophyllite asbestos in contrast with that by obtained with chrysotile, crocidolite, or amosite. The speci7c time course of the CL reaction by anthophyllite was further examined using inhibitors known to in8uence PMN signal transduction, thus providing additional information for understanding the mechanism of ROM production by anthophyllite. MATERIALS AND METHODS Mineral Fibers and Chemicals Chrysotile B, crocidolite, amosite, and anthophyllite were UICC (International Union Against Cancer) reference samples (Timbrell et al., 1968). Crystalline silica was a standard silica sample (min-u-sil). All mineral samples were suspended in Krebs}Ringer}Hepes buffer (ph 7.35) containing 25 mm Hepes, 120 mm NaCl, 5 mm KCl, 1.2 mm MgSO 4, 1.2 mm KH 2 PO 4, and 1.3 mm CaCl 2 to make a stock suspension of 1.6 mg/ml. Luminol stock solution (10 mm) in dimethyl sulfoxide (DMSO) was diluted 10 times with Krebs}Ringer}Hepes buffer before use as working solution. Cytochalasin B, genistein, wortmannnin, and staurosporin were obtained from Sigma. Chemiluminescence Measurement In a measurement tube, 10 μl of the luminol working solution, 500 μl of PMN stock suspension, and 250 μl of Krebs}Ringer}Hepes solution were mixed and preincubated at 37@C for 5 min. In the experiment using inhibitors, 10 μl of DMSO or DMSO solution of cytochalasin B, genistein, wortmannin, or staurosporin was added during this period. Concentrations of inhibitors are expressed as that of the 7nal observation mixture. To the preincubation mixture, 250 μl of mineral 7ber stock suspension was added and mixed. Changes in the luminol-dependent CL was observed in a Biolumat LB 9505 luminometer (Berthold) at 37@C. With the luminometer, six simultaneous measurements can be performed in parallel, and all comparisons were made using these six measurements. The reproducibility of the magnitude and chronological changes of the CL reaction was determined using a minimum of three separate experiments. A typical set of such CL traces is shown in every 7gure. RESULTS CL reactions by asbestos and crystalline silica are illustrated in Fig. 1. When a suspension of anthophyllite (0.4 mg/ml) was applied to the luminolprimed PMN, CL started to increase after a latent period of several seconds, with a sharp peak appearing at approximately 1 min followed by a broader peak at 5 min after the 7ber application. Crystalline Cell Preparations Plasma rich in white blood cells was obtained from heparinized human blood after sedimenting erythrocytes in 3% dextran (Pharmacia). The plasma was layered on Ficoll-Paque (Pharmacia) and centrifuged at 2000 rpm for 30 min. The precipitated cells were suspended in 156 mm NH 4 Cl containing 8 mm Na 2 CO 3 and 0.1 mm EDTA (ph 7.35) to remove remaining erythrocytes and washed twice with the Krebs}Ringer}Hepes solution without CaCl 2. Finally, the PMN were suspended in the Krebs}Ringer}Hepes solution containing CaCl 2 at cells/ml and stored on ice until use. FIG. 1. CL reaction by asbestos and crystalline silica. Suspension of anthophyllite (a), crystalline silica (b), crysotile B (c), amosite (d), crocidolite (e), or Krebs}Ringer}Hepes buffer alone (f) was added to the observation tube containing luminol-primed PMN suspension at a 7nal concentration of 0.4 mg/ml. CL (cpm) was traced over time after mineral suspension was applied.

3 38 IWATA, KOHYAMA, AND YANO FIG. 2. Effect of cytochalasin B on CL reaction by anthophyllite. Zero μg/ml (a), 0.32 μg/ml (b), 0.63 μg/ml (c), 1.25 μg/ml (d), 2.5 μg/ml (e), or 5 μg/ml (f) of cytochalasin B at 7nal concentration was added to the observation tube containing luminol-primed PMN suspension. CL (cpm) was traced over time after 0.4 mg/ml of anthophyllite suspension at 7nal concentration was applied, FIG. 4. Effect of staurosporin on CL reaction by anthophyllite. Zero μm (a), 0.25 μm (b), 0.5 μm (c), 1 μm (d), or 2 μm (e) of staurosporin at 7nal concentration was added to the observation tube containing luminol-primed PMN suspension. CL (cpm) was traced over time after 0.4 mg/ml of anthophyllite suspension at 7nal concentration was applied. silica also induced a similar initial high peak of CL, with a shoulder at 2}3 min, but failed to produce a distinct second peak. Chrysotile B, amosite, and crocidolite induced a broad peak of CL at approximately 4 min at a much lower level of CL than anthophyllite or crystalline silica; however, the chrysotile B-induced CL continued to increase even after 20 min. The effect of pretreatment of PMN by cytochalasin B, an inhibitor of cytoskeleton polymerization, on the induction of CL by anthophyllite was also examined (Fig. 2). At a dose of 0.3 μg/ml, cytochalasin B suppressed the 7rst peak of anthophyllite-induced CL, while the second broad peak remained almost unchanged. At the highest dose of cytochalasin B(5μg/ml) the rate of viable PMN was 96%, while the control was 98% by trypan blue exclusion test. Genistein, which inhibits tyrosine kinases, markedly suppressed the second peak of the anthophyllite-induced CL at the lowest dose of 3 μm but the FIG. 3. Effect of genistein on CL reaction by anthophyllite. Zero μm (a), 3.1 μm (b), 6.3 μm (c), 12.5 μm (d), 25 μm (e), or 50 μm (f) of genistein at 7nal concentration was added to the observation tube containing luminol-primed PMN suspension. CL (cpm) was traced over time after 0.4 mg/ml of anthophyllite suspension at 7nal concentration was applied. FIG. 5. Effect of wortmannin on CL reaction by anthophyllite. Zero nm (a), 12.5 nm (b), 25 nm (c), 50 nm (d), 100 nm (e), or 200 nm (f) of wortmannin at 7nal concentration was added to the observation tube containing luminol-primed PMN suspension. CL (cpm) was traced over time after 0.4 mg/ml of anthophyllite suspension at 7nal concentration was applied.

4 ROM PRODUCTION BY ANTHOPHYLLITE 39 7rst peak remained unaffected (Fig. 3). Trypan blue-excluded cells were 96% among PMN treated by 50 μm genistein when the anthophylliteinduced CL second peak was almost completely suppressed. Staurosporin, a protein kinase C inhibiter, at a low dose of 0.25 μm slightly increased the 7rst peak and decreased the second peak of anthophyllite-induced CL (Fig. 4). However, at a higher dose of 0.5 μm, the 7rst and second peaks were similarly decreased and both peaks were completely suppressed at the highest dose of 2 μm. Wortmannin, which inhibits PI 3-kinase, quickly suppressed the 7rst peak of anthophyllite-induced CL at 25 nm and was almost completely suppressed at 200 nm (Fig. 5). DISCUSSION Anthophyllite stimulated PMN to produce a biphasic and higher level of CL compared to chrysotile, crocidolite, and amosite. The 7rst CL peak induced by anthophyllite was suppressed by a low dose of cytochalasin B, and the second peaks was suppressed by a low dose of genistein. Doll et al. (1982) demonstrated that anthophyllite induced the greatest PMN CL reaction compared with crocidolite, amosite, and chrysotile, and the results of the present study con7rmed their observation. Although some researchers claimed that anthophyllite induced smaller amount of CL than crysotile and other asbestos (Hedenborg and Klockars, 1987; Klockars et al., 1990), the experimental conditions were not the same. The latter group did not preincubate the cell suspension with luminol before the application of the 7ber. Early interactions of anthophyllite and PMN might not be detected when luminol and anthophyllite are added sequentially. Under our experimental conditions, anthophyllite obtained from both Afghanistan and Yamaga (Japan) also induced similar levels and time course of CL (data not shown). ROM production from phagocytes is related to in8ammatory processes and ROM is thought to cause DNA damage (Korkina et al., 1992). Formation of pleural plaque (Hiraoka et al., 1998; Murai et al., 1997) and lung cancer (Meurman et al., 1994) may relate to the ROM production. Cytochalasin B, an inhibitor of cytoskeleton polymerization, is known to potentiate CL by fmlp and crocidolite (Ishizaki et al., 1997). Contrary to fmlp and crocidolite, CL induced by anthophyllite was suppressed by cytochalasin B. The mechanisms of anthophyllite and crocidolite to induce CL from PMN may not be identical. Cytochalasin B at a low dose suppressed the 7rst peak of CL, although it scarcely in8uenced the second peak. The 7rst CL peak seems to be induced with the process related to cytoskeletons, which is in8uenced by the low dose of cytochalasin B. The suppression of CL and associated respective speci7c enzymic inhibition by genistein, staurosporin, and wortmannin of tyrosine kinases, protein kinase C, and phosphatidylinositol 3-kinase comprise distinct signal transduction systems leading to ROM production by anthophyllite. No clear difference in sensitivities to staurosporin and wortmannin were observed in the two CL peaks induced by anthophyllite. However, in contrast to the 7rst CL peak, the second CL peak induced by anthophyllite was suppressed by a low dose of genistein, thus indicating its dependency on tyrosine kinase(s) activity. The difference in the inhibitor sensitivity may correspond to cellular compartment enzyme localization. This observation con7rms the results obtained with cytochalasin B in indicating that the two CL peaks induced by anthophyllite are generated by distinct biochemical mechanisms. In summary, we have demonstrated that anthophyllite induces a strong PMN CL reaction, the time course of which exhibits two distinct peaks, and that the two peaks differ in their respective sensitivities to various inhibitors. ACKNOWLEDGMENT We would like to express our appreciation for our former colleague Dr. Peter H. Evans (Department of Public Health, University of Glasgow, Scotland), valuable comments to this study. REFERENCES Arcaro, A., and Wymann, M. P. (1993). Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: The role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296, 297}301. Doll, N. J., Stankus, R. P., Goldbach, S., and Salvaggio, J. E. (1982). In vitro effect of asbestos 7bers on polymorphonuclear leukocyte function. Int. Arch. Allergy Appl. Immunol. 68, 17}21. Dusi, S., Della Bianca, V., Donini, M., Nadalini, K. A., and Rossi, F. (1996). Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: Its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton. J. Immunol. 157, 4615}4623. Harington, J. S., Allison, A. C., and Badami, D. V. (1975). Mineral 7bers: Chemical, physicochemical, and biological properties. Adv. Pharmacol. Chemother. 12, 291}402.

5 40 IWATA, KOHYAMA, AND YANO Hedenborg, M., and Klockars, M. (1987). Production of reactive oxygen metabolities induced by asbestos 7bers in human polymorphonuclear leucocytes. J. Clin. Pathol. 40, 1189}1193. Hiraoka, T., Ohkura, M., Morinaga, K., Kohyama, N., Shimazu, K., and Ando, M. (1998). Anthophyllite exposure and endemic pleural plaques in Kumamoto, Japan. Scand, J. Work. Environ. Health. 24, 392}397. Ishizaki, T., Yano, E., Urano, N., and Evans, P. H. (1994). Crocidolite-induced reactive oxygen metabolites generation from human polymorphonuclear leukocytes. Environ. Res. 66, 208}216. Ishizaki, T., Yano, E., and Evans, P. H. (1997). Cellular mechanisms of reactive oxygen metabolite generation from human polymorphonuclear leukocytes induced by crocidolite asbestos. Environ. Res. 75, 135}140. Karjalainen, A., Meurman, L. O., and Pukkala, E. (1994). Four cases of mesothelioma among Finnish anthophyllite miners. Occup. Environ. Med. 51, 212}215. Klockars, M., Hedenborg, M., and Vanhala, E. (1990). Effect of two particle surface-modifying agents, polyvinylpyridine-noxide and carboxymethylcellulose, on the quartz and asbestos mineral 7ber-induced production of reactive oxygen metabolites by human polymorphonuclear leukocytes. Arch. Environ. Health. 45, 8}14. Korkina, L. G., Durnev, A. D., Suslova, T. B., Cheremisina, Z. P., Daugel-Dauge, N. O., and Afanas ev, I. B. (1992). Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: Suppression by oxygen radical scavengers. Mutat. Res. 265, 245}253. Lim, Y., Kim, S. H., Kim, K. A., Oh, M. W., and Lee, K. H. (1997). Involvement of protein kinase C, phospholipase C, and protein tyrosine kinase pathways in oxygen radical generation by asbestos-stimulated alveolar macrophage. Environ. Health. Perspect. 105 (Suppl. 5), 1325}1327. Meurman, L. O., Pukkalak, E., and Hakama, M. (1994). Incidence of cancer among anthophyllite asbestos miners in Finland. Occup. Environ. Med. 51, 421}425. Mossman, B. T., Kamp, D. W., and Weitzman, S. A. (1996). Mechanisms of carcinogenesis and clinical features of asbestosassociated cancers. Cancer Invest. 14, 466}480. Murai, Y., Kitagawa, M., and Hiraoka, T. (1997). Fiber analysis in lungs of residents of a Japanese town with endemic pleural plaques. Arch. Environ. Health. 52, 263}269. Roney, P. L., and Holian, A. (1989). Possible mechanism of chrysotile asbestos-stimulated superoxide anion production in guinea pig alveolar macrophages. Toxicol. Appl. Pharmacol. 100, 132}144. Stanton, M. F., Layard, M., Tegeris, A., Miller, E., May, M., Morgan, E., and Smith, A. (1981). Relation of particle dimension to carcinogenicity in amphibole asbestoses and other 7brous minerals. J. Natl. Cancer. Inst. 67, 965}975. Timbrell, V., Gibson, J. C., and Webster, I. (1968). UICC standard reference samples of asbestos. Int. J. Cancer 15, 406}408. Yan, S. R., and Novak, M. J. (1999). Diverse effects of neutrophil integrin occupation on respiratory burst activation. Cell. Immunol. 195, 119}126.