Possible role of lipid peroxidation in the induction of NF-kappa B and AP-1 in RFL-6 cells by crocidolite asbestos: evidence following protection by vitamin E.

Asbestos fibers cause persistent induction of the oxidative stress sensitive transcription factors nuclear factor kappa-B (NF-kappa B) and activator protein-1 (AP-1) in mammalian cells. These transcription factors play an important role in the regulation of cellular activity. Lipid peroxidation, mediated by reactive oxygen species, is thought to be a possible mechanism in the pathogenicity of asbestos fibers. These studies were designed to determine if crocidolite asbestos-induced lipid peroxidation plays a role in the mechanism of formation of NF-kappa B and AP-1. Treatment of a rat lung fibroblast cell line (RFL-6) with crocidolite asbestos in the presence and absence of the membrane antioxidant vitamin E decreased the levels of crocidolite-induced AP-1 and NF-kappa B to background levels. Preincubation of RFL-6 cells with 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, prior to exposure to crocidolite, abrogated crocidolite-induced NF-kappa B DNA-binding activity to background levels. Coincubation with indomethacin, a cyclooxygenase inhibitor, had no effect on NF-kappa B DNA-binding activity induced by crocidolite. However, nordihydroguaiaretic acid, a lipoxygenase inhibitor, decreased levels of NF-kappa B to background levels. This would suggest that lipoxygenase metabolites of arachidonic acid, produced following lipid peroxidation, are involved in the cellular signalling events to NF-kappa B transcription factor induction by asbestos.


Introduction
Occupational exposure to asbestos fibers has been linked to the development of pulmonary fibrosis, bronchogenic carcinomas, and malignant mesotheliomas of the pleura and peritoneum (1,2). The underlying mechanisms by which asbestos induces respiratory malignancies are unclear. In addition to size and fiber durability being important in fiber pathogenicity, asbestos fibers can generate reactive oxygen species (ROS) from redox reactions catalyzed on the fiber surface or from incomplete phagocytosis of the fibers. ROS can react with DNA, which can cause DNA strand breaks (3,4) and base modifications (5,6) as well as induce cellular oxidative stress in which antioxidant defenses are compromised. Recent studies by Heintz et al. (7) have shown that both crocidolite and chrysotile asbestos cause dose dependent and persistent increases in expression of cfos and c-jun in rat pleural mesothelial cells and c-jun in hamster tracheal epithelial cells. Both c-fos and c-jun are immediate early response genes associated with transition of the cells from the G1 stage of the cell cycle to S phase (8), and their induction may provide the molecular switch for cell proliferation. Induction of c-fos and c-jun is accompanied by increased binding of the transcription factor activator protein-I (AP-1), a homodimeric Uun-Jun) or heterodimeric (Fos-Jun) protein complex to DNA. In addition, recent studies have shown that crocidolite asbestos induces an increase in nuclear factor kappa B (NF-iKB) DNA binding and NF-KBdependent genes such as c-myc (9).
ROS can initiate peroxidation of membrane lipids and there is now considerable evidence that products of lipid peroxidation are involved in carcinogenesis (10). Studies in our laboratory (11) have shown that mineral fibers cause time-and dose-dependent induction of lipid peroxidation, as evidenced by increases in thiobarbituric acid reactive products, in a rat lung fibroblast cell line (RFL-6). Additionally, certain end products of this process bind to cellular DNA and this may be a mechanism in the genotoxic action of asbestos fibers (11). Recent studies have shown that H202-induced c-fos expression in rodent smooth muscle cells is mediated by products of lipid peroxidation, arachidonic acid (AA) in particular, and from metabolism of AA via the lipoxygenase pathway (12). Other studies have shown that fecapentaene-12, a potent mutagen in the colon, causes oxidative damage in HeLa cells (13) and induces c-jun via the cyclooxygenase component of this pathway (SM Plummer, personal communication). The involvement of the lipoxygenases and leukotriene biosynthesis or cyclooxygenases and prostaglandin biosynthesis in the signaling pathway, which lead to the induction of transcription factors by asbestos, has not been assessed. In this study we assessed the role of lipid peroxidation in asbestos-induced DNA binding of transcription factors AP-1 and NF-KB by incubating RFL-6 cells with crocidolite asbestos in the presence and absence of vitamin E, an inhibitor of lipid peroxidation (14). In addition, further mechanistic studies in RFL-6 cells on crocidolite-mediated NF-KB induction were undertaken by preincubation of cells with an inhibitor of AA metabolism, 5,8,11,1 4-eicosatetraynoic acid (ETYA) (15), or with potent inhibitors of cyclooxygenase and lipoxygenase pathways, indomethacin (16) and nordihydroguaiaretic acid (NDGA) (17), respectively, prior to asbestos exposure. We report here that lipoxygenase metabolites of AA, produced after lipid peroxidation, are involved in the induction of the transcription factor NF-KB by asbestos.

Results
Crocidolite asbestos stimulated both NF-icB and AP-1 transcription factor expression in both a time-and dose-dependent manner in RFL-6 cells, with maximum induction at 8 and 24 hr at 5 and 10 pg/cm2 (p< 0.05) (Figure 1). To examine whether lipid peroxidation mediated by asbestos is involved in the induction of transcription factors AP-1 and NF-icB, crocidolite asbestos was added to RFL-6 cells in the presence and absence of the membrane antioxidant vitamin E. The increase in both AP-1 and NF-iKB by crocidolite was ameliorated in the presence of vitamin E at both concentrations (p < 0.05) ( Figure 2). To examine the involvement of AA metabolism in NF-icB transcription factor induction by asbestos, we incubated RFL-6 cells with crocidolite in the presence and absence of ETYA, which is an inhibitor of AA metabolism. The levels of NF-iKB DNA binding by crocidolite in the presence of ETYA were reduced to background levels ( Table 1). To investigate further the involvement AA metabolism in NF-icB induction by asbestos, specifically either the cyclooxygenase or lipoxygenase components of this metabolic pathway, we incubated RFL-6 cells with crocidolite in the presence and absence of indomethacin, an inhibitor of the cyclooxygenase pathway (IC50 = 0.1 pm), or NDGA, an inhibitor of the lipoxygenase pathway (IC50 = 0.2 lpM for 5-lipoxygenase and 30 pM for 12-and 15-lipoxygenases). Treatment of cells with crocidolite in the presence of indomethacin did not alter NF-iKB DNA binding in RFL-6 cells ( Figure 3). However, in the presence of NDGA, the levels of NF-icB DNA binding induced by crocidolite were significantly reduced (p<0.05) compared to levels seen with crocidolite alone (Figure 4).

Discussion
There is strong evidence that signaling pathways are involved in the enhanced expression of oxidative stress-inducible genes such as c-fos, c-jun, and transcription factors AP-1 and NF-ICB (19,20). There is also data to support the initiation of these signaling events at the plasma membrane (21,22) that involve kinase cascades (21) and redox regulation (23). Because cellular membranes are an important target for asbestos-mediated ROS production, we analyzed whether damage to membrane lipids is Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997   crocidolite in the presence of the membrane antioxidant vitamin E, the levels of DNA binding of AP-1 and NF-KB were reduced to background levels. The lipophilic antioxidant vitamin E inhibits lipid peroxidation by scavenging lipid radicals and thereby halting the propagation of the process and preventing accumulation of lipid hydroperoxides that subsequently decompose to a wide range of end products, such as malondialdehyde, hexanal, and 4-hydroxynonenal (4-HNE) (24). 4-HNE can also mediate the activation of enzymes involved in signal transduction, such as adenylate cyclase and phosphatidyl inositol 4,5-biphosphate-phospholipase C in membrane preparations (25,26) and phospholipase D in cells (27). In addition, endogenous 4-HNE has the potential to activate these enzymes within these cells during oxidative stress because this and other aldehydes have half-lives longer than most ROS and can diffuse within or out of the cell (28). Besides the well characterized function of vitamin E as an antioxidant, alternative roles such as that of a membrane stabilizer and regulator of membrane fluidity have been proposed (29). In addition, vitamin E is an inhibitor of cellular 5-lipoxygenase activity (30), thus preventing breakdown of AA into active metabolites. This study investigated whether asbestos-mediated induction of the transcription factor NF-KB involves metabolites of AA as second messengers via the cyclooxygenase or lipoxygenase pathway. In enzymatic lipid peroxidation, oxidized fatty acids such as AA are released preferentially by phospholipase A2 from membrane lipids. Regardless of the source of its release, AA is highly relevant to signal transduction within the cell because it is a substrate for synthesis of eicosanoids, namely prostaglandins and leukotrienes, by cyclooxygenases and lipoxygenases, respectively. Little is known about the role of AA or its metabolites in asbestos-mediated gene transcription, but in recent studies, the induction of c-fos gene expression with agents such as tumor necrosis factor (31) or H202 (12) was mediated by the conversion of AA to a metabolite(s) via the lipoxygenase pathway. In these studies (12,31) cyclooxygenase inhibitors had no effect on the modulation of mRNA levels of c-fos. We showed for the first time that AA metabolites are involved in the cellular signaling events leading to NF-iKB induction by asbestos. In the present study, asbestos treatment of cells in the presence of ETYA, an inhibitor of AA metabolism (15), reduced NF-KB DNA binding to basal levels. In addition, with the use of inhibitors of the cyclooxygenase and lipoxygenase pathways, namely indomethacin (16) and NDGA (17), respectively, we demonstrated that lipoxygenase metabolites are involved in NF-icB induction by asbestos. In these studies, asbestos treatment of cells in the presence of NDGA reduced NF-cB DNA binding to basal levels. In addition, the concentration of NDGA (10 pM) that reduced transcription factor induction would suggest that it is the 5-lipoxygenase pathway that is being inhibited (12). In Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997 contrast, indomethacin had no effect on the crocidolite response in these cells.
In summary, this study has shown that lipid peroxidation is involved in asbestosmediated cellular signaling events, which lead to AP-1 and NF-KB transcription factor induction, after inhibition with the membrane antioxidant vitamin E. In addition, our results suggest that when RFL-6 cells are treated with crocidolite asbestos, AA is released following lipid peroxidation and processed via the lipoxygenase pathway to a metabolite or metabolites required for crocidolite-mediated NF-icB induction. Studies are in progress to determine which lipoxygenase product or products are involved in the cellular signaling pathways mediated by asbestos.