[CANCER RESEARCH 55, 2085-2089, May 15, 1995] Induction of c-fos and c-jun Proto-oncogene Expression by Asbestos Is Ameliorated by A^-Acetyl-L-Cysteine in Mesothelial Cells1 Yvonne M. W. Janssen, Nicholas H. Heintz, and Brooke T. Mossman2 Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405 ABSTRACT Asbestos fibers cause dose-dependent, persistent increases in iiiKNA levels of c-jun and c-fos proto-oncogenes in rat pleural mesothelial (RPM) cells, the progenitor cells of asbestos-induced mesothelioma (N. Heintz, Y. M. W. Janssen, and B. T. Mossman. Proc. Nati. Acad. Sci. USA, 90: 3299-3303, 1993). Here we report that addition of ¿V-acetyl-L-cysteine decreases asbestos-mediated induction of c-fos and c-jun niKNA levels in a dose-dependent fashion. Exposure of RPM cells to asbestos causes depletion of total cellular glutathione, a response that can be abolished by pretreatment with JV-acetyl-L-cysteine. Pretreatment of cells with buthi- onine sulfoximine, an agent which diminishes glutathione pools, increases the magnitude of induction of c-fos and c-jun mRNA by asbestos. To determine whether asbestos-induced effects on proto-oncogene expression could be attributed to extracellular generation of active oxygen species (AOS), RPM cells were exposed to H,O, or xanthine and xanthine oxidase, a generating system of AOS. These oxidant stresses did not decrease cellular glutathione levels nor alter mRNA levels of c-fos or c-jun. How ever, increased mRNA levels of manganese-containing Superoxide dis- mutase and heme oxygenase were observed, indicating that RPM cells respond to AOS by increased expression of genes encoding antioxidant enzymes. These data indicate that the signaling pathways leading to c-fos/c-jun proto-oncogene induction by asbestos are not triggered directly by formation of extracellular AOS. However, intracellular thiol levels appear to influence the expression of c-fux and c-jun, suggesting a redox- sensitive component in the signaling cascade which modulates gene ex pression of c-fos and c-jun by asbestos. INTRODUCTION Asbestos fibers are a heterogeneous family of naturally occurring mineral silicates which have been used in multiple industrial settings (1). Exposure to asbestos in unregulated workplaces has led to the development of malignant mesotheliomas and lung cancers. The mechanisms by which asbestos induces tumors are poorly understood. Our laboratory has focused on the molecular responses that occur in mesothelial and trachea! epithelial cells in vitro and in vivo after exposure to asbestos, because these cells are the progenitors of asbestos-induced mesotheliomas and bronchogenic carcinomas, respectively. c-fos and c-jun are early response genes associated with the tran sition of cells into S phase (2). Induction of c-fos and c-jun is accompanied by increased binding of the transcription factor, AP-1, a heterodimeric protein complex composed of c-fos and c-jun gene products, to DNA. Both crocidolite and chrysotile asbestos cause dose-dependent and persistent increases in the expression of c-fos and c-jun in RPM3 cells (3). In HTE cells, only c-jun expression is Received 8/16/94; accepted 3/14/95. The cosls of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by National Heart, Lung and Blood Institute Grant ML 39469 and National Institute of Environmental Health Services Grant ES 06499. 2 To whom requests for reprints should be addressed, at Department of Pathology, University of Vermont, Soule Medical Alumni Building. Burlington, VT 05405. 3 The abbreviations used are: RPM, rat pleural mesothelial; AOS, active oxygen species; BSO, buthionine sulfoximine; GSH, glutathione; HO, heme oxygenase; HTE, hamster trachéal epithelial; MnSOD, manganese-containing Superoxide dismutase; NAC, A/-acetyl-L-cysteine; TPA, 12-O-tetradecanoylphorbol-13-acetate; X/XO, xanthine plus xanthine oxidase. induced by asbestos fibers, but c-fos and c-jun can be induced by H2O2 as has been reported in other cell types (4). In contrast, alter ations in expression of these proto-oncogenes are not observed after exposure of RPM or HTE cells to nonfibrous and biologically inactive analogues of asbestos or to a variety of inert particles (5). A number of molecular and cellular events triggered by asbestos fibers appear to be mediated by AOS (6, 7). For example, AOS are involved in asbestos-induced cytotoxicity, inflammation, and the de velopment of asbestosis in rats (6). In addition, AOS are implicated in asbestos-mediated activation of ornithine decarboxylase, a rate-limit ing enzyme in the synthesis of polyamines which are necessary for cell proliferation (7). These data suggest that AOS may be related causally to some asbestos-induced cell-signaling events. Recent work by a number of investigators indicates that the redox status of cells may be important in the regulation of c-fos and c-jun gene expression. For instance, the redox status of c-Jun is important for DNA binding to the AP-1 consensus sequence (8-12). Under oxidative conditions, Jun-Jun homodimers and Fos-Jun heterodimers are unable to bind to DNA. However, upon addition of reducing agents, DNA binding is restored (8, 9, 11, 12). Physical interaction with the Ref-1 protein, which constitutes a major source of redox activity in mammalian cells, is likely to exert this redox regulation in vivo (8, 11, 12). Moreover, escape from redox control by mutating c-fos or c-jun to allow DNA binding under oxidative conditions contributes to cell transformation (9, 10, 12). In studies here, we determined the intracellular levels of GSH, a small tripeptide with antioxidant properties that is important for maintaining thiol levels (13), in RPM cells after exposure to asbestos or chemical-generating systems of AOS, including xanthine, X/XO, and H2O2. To determine whether thiol levels were important in induction of c-fos and c-jun by asbestos, we pretreated cells with the GSH precursor, NAC, to boost thiol levels, or BSO to deplete GSH. Our results demonstrate that exposure to asbestos depletes GSH levels in RPM cells, an effect abolished by NAC. In addition, NAC pre treatment abolished elevation in c-fos and c-jun mRNA levels after exposure to asbestos. These results suggest that intracellular thiol pools modulate c-fos and c-jun induction by asbestos. MATERIALS AND METHODS Chemicals and Asbestos. Reference samples of the National Institute of Environmental Health Science processed crocidolite asbestos [Na2(Fe3+)2(Fe2+)3SisO22(OH)2] were obtained from the Thermal Insula tion Manufacturers Association fiber repository (Littleton, CO). Xanthine and X/XO were purchased from Calbiochem (La Jolla, CA). Fetal bovine serum, HBSS, PBS, and Ham's F-12 medium were purchased from GIBCO (Grand Isle, NY). [a-32P)dATP (3000 Ci/mmol) was obtained from DuPont New England Nuclear (Boston, MA). Glutathione reducÃ-ase was purchased from Boehringer Mannheim (Indianapolis, IN). All other reagents were from Sigma Chemical Co. (St. Louis, MO). Cell Culture and Exposure to Test Agents. RPM cells were isolated and propagated as described previously (3, 5). Cells were routinely passaged in complete medium containing 10% fetal bovine serum, and confluent cells were switched to 2% serum-containing medium for 24 h prior to the addition of test agents. Asbestos fibers were suspended in HBSS at 1 mg/ml and triturated 8 times through a 22-gauge needle prior to their addition to medium at nontoxic concentrations (3). H2O2, xanthine (100 (XM),and xanthine oxidase (0.1 and 2085 Research. on December 10, 2015. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from