Programmed necrosis induced by asbestos in human mesothelial cells causes high-mobility group box 1 protein release and resultant inflammation.

Asbestos carcinogenesis has been linked to the release of cytokines and mutagenic reactive oxygen species (ROS) from inflammatory cells. Asbestos is cytotoxic to human mesothelial cells (HM), which appears counterintuitive for a carcinogen. We show that asbestos-induced HM cell death is a regulated form of necrosis that links to carcinogenesis. Asbestos-exposed HM activate poly(ADP-ribose) polymerase, secrete H(2)O(2), deplete ATP, and translocate high-mobility group box 1 protein (HMGB1) from the nucleus to the cytoplasm, and into the extracellular space. The release of HMGB1 induces macrophages to secrete TNF-alpha, which protects HM from asbestos-induced cell death and triggers a chronic inflammatory response; both favor HM transformation. In both mice and hamsters injected with asbestos, HMGB1 was specifically detected in the nuclei, cytoplasm, and extracellular space of mesothelial and inflammatory cells around asbestos deposits. TNF-alpha was coexpressed in the same areas. HMGB1 levels in asbestos-exposed individuals were significantly higher than in nonexposed controls (P < 0.0001). Our findings identify the release of HMGB1 as a critical initial step in the pathogenesis of asbestos-related disease, and provide mechanistic links between asbestos-induced cell death, chronic inflammation, and carcinogenesis. Chemopreventive approaches aimed at inhibiting the chronic inflammatory response, and especially blocking HMGB1, may decrease the risk of malignant mesothelioma among asbestos-exposed cohorts.

Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H2O2 generation.

The release of H(2)O(2) from alveolar macrophages has been linked to the development of pulmonary fibrosis, but little is known about its source or mechanism of production. We found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H(2)O(2) and have high expression of Cu,Zn-superoxide dismutase (SOD). Because Cu,Zn-SOD is found in the mitochondrial intermembrane space (IMS), we hypothesized that mitochondrial Cu,Zn-SOD-mediated H(2)O(2) generation contributed to pulmonary fibrosis. Asbestos-induced translocation of Cu,Zn-SOD to the IMS was unique to macrophages and dependent on functional mitochondrial respiration and the presence of at least one of the conserved cysteines required for disulfide bond formation. These conserved cysteine residues were also necessary for enzyme activation and H(2)O(2) generation. Cu,Zn-SOD-mediated H(2)O(2) generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant in that Cu,Zn-SOD(-/-) mice generated significantly less H(2)O(2) and had less oxidant stress in bronchoalveolar lavage fluid and lung parenchyma. Furthermore, Cu,Zn-SOD(-/-) mice did not develop pulmonary fibrosis, and knockdown of Cu,Zn-SOD in monocytes attenuated collagen I deposition by lung fibroblasts. Our findings demonstrate a novel mechanism for the pathogenesis of pulmonary fibrosis where the antioxidant enzyme Cu,Zn-SOD translocates to the mitochondrial IMS to increase H(2)O(2) generation in alveolar macrophages.

Asbestos exposure affects poly(ADP-ribose) polymerase-1 activity: role in asbestos-induced carcinogenesis.

Asbestos is known to induce malignant mesothelioma (MM) and other asbestos-related diseases. It is directly genotoxic by inducing DNA strand breaks and cytotoxic by promoting apoptosis in lung target cells. Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear zinc-finger protein with a function as a DNA damage sensor. To determine whether PARP1 is involved in asbestos-induced carcinogenesis, PARP1 expression and activity as well as DNA damage and repair were evaluated in circulating cells of asbestos-exposed subjects, MM patients and age-matched controls. PARP1 expression and activity were also evaluated in pleural biopsies of MM patients and compared with normal tissue. Accumulation of the pre-mutagenic 8-hydroxy-2'-deoxyguanosine and elevated PARP1 expression were found both in asbestos-exposed subjects and MM patients. Although PARP1 was highly expressed, its activity was relatively low. Low DNA repair efficiency was observed in lymphocytes from MM patients. High expression of PARP1 associated with low PARP activity was also found in MM biopsies. To mimic PARP1 dysfunction, PARP1 expression and activity were induced in immortalised mesothelial cells by their exposure to asbestos in the presence of a PARP1 inhibitor, which resulted in transformation of the cells. We propose that exposure to asbestos inhibits the PARP1 activity possibly resulting in higher DNA instability, thus causing malignant transformation.

The Nalp3 inflammasome is essential for the development of silicosis.

Inhalation of crystalline silica and asbestos is known to cause the progressive pulmonary fibrotic disorders silicosis and asbestosis, respectively. Although alveolar macrophages are believed to initiate these inflammatory responses, the mechanism by which this occurs has been unclear. Here we show that the inflammatory response and subsequent development of pulmonary fibrosis after inhalation of silica is dependent on the Nalp3 inflammasome. Stimulation of macrophages with silica results in the activation of caspase-1 in a Nalp3-dependent manner. Macrophages deficient in components of the Nalp3 inflammasome were incapable of secreting the proinflammatory cytokines interleukin (IL)-1beta and IL-18 in response to silica. Similarly, asbestos was capable of activating caspase-1 in a Nalp3-dependent manner. Activation of the Nalp3 inflammasome by silica required both an efflux of intracellular potassium and the generation of reactive oxygen species. This study demonstrates a key role for the Nalp3 inflammasome in the pathogenesis of pneumoconiosis.

DNA double-strand breaks by asbestos, silica, and titanium dioxide: possible biomarker of carcinogenic potential?

DNA double-strand breaks (DSBs) can result in cell death or genetic alterations when cells are subjected to radiation, exposure to toxins, or other environmental stresses. A complex DNA-damage-response pathway is activated to repair the damage, and the inability to repair these breaks can lead to carcinogenesis. One of the earliest responses to DNA DSBs is the phosphorylation of a histone, H2AX, at serine 139 (gamma-H2AX), which can be detected by a fluorescent antibody. A study was undertaken to compare the induction of DNA DSBs in normal (small airway epithelial) cells and cancer cells (A549) after exposure to asbestos (crocidolite), a proven carcinogen, silica, a suspected carcinogen, and titanium dioxide (TiO(2)), an inert particle recently reported to be carcinogenic in animals. The results indicate that crocidolite induced greater DNA DSBs than silica and TiO(2), regardless of cell type. DNA DSBs caused by crocidolite were higher in normal cells than in cancer cells. Silica and TiO(2) induced higher DNA DSBs in cancer cells than in normal cells. The production of reactive oxygen species was found to be highest in cells exposed to crocidolite, followed, in potency, by silica and TiO(2). The generation of reactive oxygen species was higher in normal cells than in cancer cells. Cell viability assay indicated that crocidolite caused the greatest cytotoxicity in both cell types. Apoptosis, measured by caspase 3/7 and poly (ADP-Ribose) polymerase activation, was highest in crocidolite-exposed cells, followed by TiO(2) and silica. The results of this study indicate that crocidolite has a greater carcinogenic potential than silica and TiO(2), judged by its ability to cause sustained genomic instability in normal lung cells.

Macrophage plasminogen activator: induction by asbestos is blocked by anti-inflammatory steroids.

Intraperitoneal injection of asbestos fibres into mice induces the formation of exudates containing macrophages that produce plasminogen activator. Like-wise, in vitro addition of asbestos to macrophage cultures stimulates plasminogen activator secretion; the synthesis and secretion of lysozyme and lysosomal enzymes are not changed under these conditions. The enhanced secretion of plasminogen activator by macrophages exposed to asbestos is suppressed by low concentrations of anti-inflammatory steroids.

Activated cAMP response element binding protein is overexpressed in human mesotheliomas and inhibits apoptosis.

Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CREB target genes (c-FOS, EGR-1, MKP1, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for MM therapy.

Differences in gene expression profiles from asbestos-treated SPARC-null and wild-type mouse lungs.

The role of SPARC in the in vivo lung response to crocidolite asbestos was addressed by instillation of crocidolite asbestos in a series of wild-type or SPARC-null mice. Animals were sacrificed at one week, one month, and three months post-instillation to assess the impact of SPARC on multiple stages in the development of fibrosis. RNA was harvested from 10 animals/time point, pooled, and used to probe a mouse array containing approximately 10,000 probes. Gene expression data were analyzed for fold change, and for broader functional group alterations. As expected, the one-week time point displayed alterations in genes involved in immune recognition, energy utilization, and growth factor production. Later time points showed expression alterations for genes involved in protein degradation, Wnt receptor signaling, membrane protein activity, and transport. Molecules in the Wnt pathway have been implicated in bone growth, mediation of fibroblast activity, and have been directly linked to SPARC regulation.

Kinetics of CHO A L mutant expression after treatment with gamma radiation, EMS, and asbestos.

The flow cytometry mutation assay (FCMA) uses hybrid CHO A(L) cells to measure mutations of the cd59 gene located on human chromosome 11 by the absence of fluorochrome-conjugated antibody binding to the CD59 surface antigen. Mutant expression peaks between 6 and 12 days, then decreases to a stable plateau, instead of a constant mutant fraction obtained by clonogenic assays. To evaluate this variable mutant expression time, cells were treated with radiation, EMS or asbestos and cell proliferation and survival were measured at times leading up to peak mutant expression. Potential doubling time (T(pot)) values increased by at least 75% for each agent by 3 h after treatment but returned to control levels after only 3 days. Survival returned to 90% of control within a week, close to the peak expression day for all three agents. The survival of CD59(-) cells sorted on the peak day of expression was roughly half that of CD59(+) cells. Cloned EMS-treated CD59(-) cells had a doubling time of 16.7 vs. 14.1 h for CD59(+) cells. Triple mutants (CD59(-)/CD44(-)/CD90(-)) were preferentially lost from the population over time, while the proportion of CD59(-)/CD90(-) mutants increased. In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth.

SV40 oncoproteins enhance asbestos-induced DNA double-strand breaks and abrogate senescence in murine mesothelial cells.

SV40 virus has emerged as a potential cofactor with asbestos in the development of diffuse malignant mesothelioma, but its precise role in the pathogenesis of this tumor is unclear. SV40 large T antigen is known to inactivate cellular proteins involved in DNA damage and senescence, including p53 and pRb. We hypothesize that SV40 oncoproteins will sensitize mesothelial cells to DNA damage induced by asbestos or chemotherapeutic agents. SV40 oncoprotein expression in murine mesothelial cell lines enhanced spontaneous and asbestos-induced double-strand breaks, indicated by gamma-H2AX foci, and potentiated micronucleus formation. Mesothelial cells exposed to asbestos or bleomycin for 96 h acquired senescent-like morphology and displayed elevated senescence-associated beta-galactosidase activity, reduced bromodeoxyuridine (BrdUrd) incorporation, and reduced colony formation. SV40 oncoprotein expression abrogated the senescent phenotype, and transfected cell lines showed an increase in both BrdUrd incorporation and colony formation after prolonged DNA damage. Murine mesothelial cell lines lacking wild-type p53 due to a point mutation or gene rearrangement also failed to senesce in response to asbestos or chemotherapeutic agents. In addition, stress-induced senescence in human mesothelial cell lines was impaired by SV40 oncoprotein expression (MeT-5A), p53 small interfering RNA, or spontaneous p53 mutation (REN). These studies suggest that exposure to DNA-damaging agents can induce senescence in both murine and human mesothelioma cell lines and suggest a major, although not exclusive, role for p53 in this response. SV40 virus may contribute to mesothelioma progression by impairing stress-induced senescence, in part through p53 inactivation, thereby favoring survival and proliferation of mesothelial cells that have sustained DNA damage.

Conditioned medium from asbestos-exposed fibroblasts affects proliferation and invasion of lung cancer cell lines.

The importance of the role of fibroblasts in cancer microenvironment is well-recognized. However, the relationship between fibroblasts and asbestos-induced lung cancer remains underexplored. To investigate the effect of the asbestos-related microenvironment on lung cancer progression, lung cancer cells (NCI-H358, Calu-3, and A549) were cultured in media derived from IMR-90 lung fibroblasts exposed to 50 mg/L asbestos (chrysotile, amosite, and crocidolite) for 24 h. The kinetics and migration of lung cancer cells in the presence of asbestos-exposed lung fibroblast media were monitored using a real-time cell analysis system. Proliferation and migration of A549 cells increased in the presence of media derived from asbestos-exposed lung fibroblasts than in the presence of media derived from normal lung fibroblasts. We observed no increase in proliferation and migration in lung cancer cells cultured in asbestos-exposed lung cancer cell medium. In contrast, increased proliferation and migration in lung cancer cells exposed to media from asbestos-exposed lung fibroblasts was observed for all types of asbestos. Media derived from lung fibroblasts exposed to other stressors, such as hydrogen peroxide and UV radiation didn't show as similar effect as asbestos exposure. An enzyme-linked immunosorbent assay (ELISA)-based cytokine array identified interleukin (IL)-6 and IL-8, which show pleiotropic regulatory effects on lung cancer cells, to be specifically produced in higher amounts by the three types of asbestos-exposed lung fibroblasts than normal lung fibroblasts. Thus, the present study demonstrated that interaction of lung fibroblasts with asbestos may support the growth and metastasis of lung cancer cells and that chrysotile exposure can lead to lung cancer similar to that caused by amphibole asbestos (amosite and crocidolite).

Asbestos redirects nitric oxide signaling through rapid catalytic conversion to nitrite.

Asbestos exposure is strongly associated with the development of malignant mesothelioma, yet the mechanistic basis of this observation has not been resolved. Carcinogenic transformation or tumor progression mediated by asbestos may be related to the generation of free radical species and perturbation of cell signaling and transcription factors. We report here that exposure of human mesothelioma or lung carcinoma cells to nitric oxide (NO) in the presence of crocidolite asbestos resulted in a marked decrease in intracellular nitrosation and diminished NO-induced posttranslational modifications of tumor-associated proteins (hypoxia-inducible factor-1alpha and p53). Crocidolite rapidly scavenged NO with concomitant conversion to nitrite (NO(2)(-)). Crocidolite also catalyzed the nitration of cellular proteins in the presence of NO(2)(-) and hydrogen peroxide. Nitrated protein adducts are a prominent feature of asbestos-induced lung injury. These data highlight the ability of asbestos to induce phenotypic cellular changes through two processes: (a) by directly reducing bioactive NO levels and preventing its subsequent interaction with target molecules and (b) by increasing oxidative damage and protein modifications through NO(2) production and 3-nitrotyrosine formation.

Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha-dependent mechanism in murine macrophages.

Macrophages participate in inflammatory and repair processes in part through the selective release of cytokines that contribute to tissue remodeling. Extracellular matrix components generated at inflammatory sites may influence tissue remodeling by effects on leukocyte adherence and local cytokine production. In murine bone marrow-derived macrophages, we found that soluble hyaluronic acid stimulated IL-1 beta, TNF alpha, and insulin-like growth factor-1 (IGF-1) mRNA transcript expression as well as IGF-1 protein synthesis. Monoclonal antibodies to the hyaluronic acid receptor CD44 blocked the effects of hyaluronic acid on IL-1 beta, TNF alpha, and IGF-1 expression. TNF alpha and IL-1 beta mRNA expression preceded IGF-1 protein synthesis, and TNF alpha, but not IL-1 beta, was found to directly stimulate IGF-1. Furthermore, IGF-1 induction was dependent on endogenous TNF alpha production since IGF-1 protein synthesis was inhibited in the presence of anti-TNF alpha antiserum. In addition, IL-1 beta was found to exert a regulatory role on IGF-1 production by enhancing the TNF alpha effect. IL-1 beta and TNF alpha mRNA transcript expression as well as IGF-1 protein synthesis were also stimulated by chrysotile asbestos. Anti-CD44 antibodies had no effect whereas anti-TNF alpha antiserum blocked asbestos-stimulated IGF-1 production. These results indicate that hyaluronate activation of CD44 induces cytokine expression and macrophage-derived IGF-1 production is dependent on TNF alpha expression.

Chrysotile asbestos upregulates gene expression and production of alpha-receptors for platelet-derived growth factor (PDGF-AA) on rat lung fibroblasts.

PDGF isoforms have been postulated to serve as mediators of fibroblast proliferation and chemotaxis during lung fibrogenesis induced by asbestos inhalation. We have studied the interaction of chrysotile asbestos fibers with rat lung fibroblasts (RLF) in vitro and the consequent changes in PDGF receptor mRNA expression, PDGF binding, and mitogenic activity of PDGF isoforms. Northern blot analysis revealed that mRNA for the PDGF-receptor alpha subtype (PDGF-R alpha) on RLF was upregulated after a 24-h exposure to asbestos in culture (0.5-15 micrograms fibers/cm2). [125I]PDGF-BB receptor assays showed that normal RLF possess mainly PDGF-R beta and a paucity of PDGF-R alpha. In agreement with the Northern data, saturation binding of [125I]PDGF-BB to RLF exposed to asbestos demonstrated an approximately 40% increase in binding sites accompanied by a twofold decrease in receptor affinity. Treating asbestos-exposed RLF with PDGF-AA, which binds only PDGF-R alpha, blocked the PDGF binding sites that were upregulated by fiber exposure. PDGF-AA had increased mitogenic potency for fiber-exposed RLF, but PDGF-BB was a less potent mitogen for these RLF. Nonfibrogenic carbonyl iron spheres induced similar changes in PDGF growth responses. These data show that inorganic particulates alter the PDGF-R alpha population on RLF without significant change in PDGF-R beta.

Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke.

The mechanism by which cigarette smoking and asbestos exposure synergistically increase the incidence of lung cancer is unknown. We hypothesized that cigarette smoke and asbestos might synergistically increase DNA damage. To test this hypothesis we exposed isolated bacteriophage PM2 DNA to cigarette smoke and/or asbestos, and assessed DNA strand breaks as an index of DNA damage. Our results supported our hypothesis. 78 +/- 12% of the DNA exposed to both cigarette smoke and asbestos developed strand breaks, while only 9.8 +/- 7.0 or 4.3 +/- 3.3% of the DNA exposed to cigarette smoke or asbestos, respectively, developed strand breaks under the conditions of the experiment. Our experimental evidence suggested that cigarette smoke and asbestos synergistically increased DNA damage by stimulating .OH formation. First, significant amounts of .OH were detected by electron paramagnetic resonance (EPR) in DNA mixtures containing both cigarette smoke and asbestos, but no .OH was detected in mixtures containing cigarette smoke alone or asbestos alone. Second, the .OH scavengers, dimethylsulfoxide (DMSO), mannitol, or Na benzoate decreased both .OH detection by EPR and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. Third, the H2O2 scavenger, catalase, and the iron chelators, 1,10-phenanthroline and desferrithiocin, decreased both .OH detection and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. These latter findings suggest that iron contained in asbestos may catalyze the formation of .OH from H2O2 generated by cigarette smoke. In summary, our study indicates that cigarette smoke and asbestos synergistically increase DNA damage and suggests that this synergism may involve .OH production.

Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung.

Asbestos is a known inflammatory, carcinogenic, and fibrotic agent, but the mechanisms leading to asbestos-induced lung diseases are unclear. Using a murine inhalation model of fibrogenesis, we show that asbestos causes significant increases in mRNA levels of lung matrix metalloproteinases (MMPs 12 and 13) and tissue inhibitor of metalloproteinases (TIMP1), as well as increased activities of MMP 2, 9, and 12 in bronchoalveolar lavage fluids (BALF). Asbestos-exposed PKCdelta knockout (PKCdelta-/-) mice exhibited decreased expression of lung MMP12 and MMP13 compared with asbestos-exposed wild-type mice. Studies using small molecule inhibitors in murine alveolar epithelial type II cells (C10) and primary lung fibroblasts confirmed that asbestos transcriptionally up-regulates MMPs via an EGFR (or other growth factor receptors)/PI3K/PKCdelta/ERK1/2 pathway. Moreover, use of a broad-spectrum MMP inhibitor showed that MMPs play an important role in further enhancing asbestos-induced signaling events by activating EGFR. These data reveal a potentially important link between asbestos signaling and integrity of the extracellular matrix (ECM) that likely contributes to asbestos-induced lung remodeling and diseases.

Mutagenesis by man-made mineral fibres in the lung of rats.

The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.

Influence of asbestos on the uptake of benzo(a)pyrene and DNA alkylation in hamster tracheal epithelial cells.

The objective of these experiments was to understand the mechanism of cocarcinogenicity of asbestos and polycyclic aromatic hydrocarbons. Benzo(a)pyrene [B(a)P] was coated onto crocidolite or chrysotile asbestos fibers, resuspended in serum-free medium, and added to cultures of hamster tracheal epithelial cells. The fibers markedly enhanced cell uptake of B(a)P. Although considerable metabolism occurred, approximately 40% of the applied B(a)P was retained by the cells after 8-hr incubation as opposed to 5% after incubation with B(a)P in the absence of asbestos. The hydrocarbon-containing medium was replaced by fresh medium. Four days later, approximately 3% of the B(a)P that had been applied when adsorbed to asbestos was still persistent in cells as compared to 0.5% in cells treated with B(a)P alone. DNA from hamster tracheal epithelial cells was purified, and the amount of B(a)P alkylation was assessed. At 8 hr, the extent of alkylation after treatment of the cells with either B(a)P or B(a)P:asbestos was similar. However, the retained unmetabolized B(a)P was subsequently metabolized and contributed to further alkylation so that the B(a)P-asbestos treated cells demonstrated considerably higher levels of alkylation throughout the 4-day posttreatment period. None of these effects was observed if asbestos was added 1 hr before the addition of B(a)P. The enhanced uptake of B(a)P and subsequent additional alkylation of DNA might represent a mechanism of asbestos-induced cocarcinogenesis.

Transcriptional activation of the proto-oncogene c-jun by asbestos and H2O2 is directly related to increased proliferation and transformation of tracheal epithelial cells.

Asbestos causes persistent increases in c-jun mRNA and AP-1 DNA binding activity in hamster tracheal epithelial (HTE) cells, the progenitor cell type of asbestos-induced bronchogenic carcinoma. Studies here were designed to determine mechanisms of c-jun induction by asbestos and the phenotypic consequences of Jun expression in HTE cells. To examine whether asbestos or H2O2 induced transcription of c-jun, we transiently transfected HTE cells with a plasmid containing a fragment of the c-jun promoter coupled to a luciferase reporter gene. In addition, c-jun was overexpressed in cells using a full-length human c-jun construct, and effects on proliferation and transformation were examined. HTE cells transfected with the jun-luciferase construct showed increased luciferase activity when exposed to crocidolite asbestos or H2O2. These results demonstrate that asbestos and H2O2 activate AP-1-dependent gene transcription. Overexpression of c-jun led to increased proliferation and enhanced ability of HTE cells to grow in soft agar, an indication of cellular transformation. Data suggest that overexpression of c-jun may contribute to asbestos and oxidant-induced proliferation and carcinogenesis.

Asbestos induces activator protein-1 transactivation in transgenic mice.

Activation of activator protein (AP-1) by crocidolite asbestos was examined in vitro in a JB6 P+ cell line stably transfected with AP-1-luciferase reporter plasmid and in vivo using AP-1-luciferase reporter transgenic mice. In in vitro studies, crocidolite asbestos caused a dose- and time-dependent induction of AP-1 activation in cultured JB6 cells. The elevated AP-1 activity persisted for at least 48 h. Crocidolite asbestos also induced AP-1 transactivation in the pulmonary and bronchial tissues of transgenic mice. AP-1 activation was observed at 2 days after intratracheal instillation of the mice with asbestos. At 3 days postexposure, AP-1 activation was elevated 10-fold in the lung tissue and 22-fold in bronchiolar tissue as compared with their controls. The induction of AP-1 activity by asbestos appeared to be mediated through the activation of mitogen-activated protein kinase family members, including extracellular signal-regulating protein kinase, Erk1 and Erk2. Aspirin inhibited asbestos-induced AP-1 activity in JB6 cells. Pretreatment of the mice with aspirin also inhibited asbestos-induced AP-1 activation in bronchiolar tissue. The data suggest that further investigation of the role of AP-1 activation in asbestos-induced cell proliferation and carcinogenesis is warranted. In addition, investigation of the potential therapeutic benefits of aspirin in the prevention/amelioration of asbestos-induced cancer is justified.