Internalization of Libby Amphibole Asbestos and Induction of Oxidative Stress in Murine Macrophages

Abstract

The community members of Libby, MT, have experienced significant asbestos exposure and developed numerous asbestos-related diseases including fibrosis and lung cancer due to an asbestos-contaminated vermiculite mine near the community. The form of asbestos in the contaminated vermiculite has been characterized in the amphibole family of fibers. However, the pathogenic effects of these fibers have not been previously characterized. The purpose of this study is to determine the cellular consequences of Libby amphibole exposure in macrophages compared to another well-characterized amphibole fiber; crocidolite asbestos. Our results indicate that Libby asbestos fibers are internalized by macrophages and localize to the cytoplasm and cytoplasmic vacuoles similar to crocidolite fibers. Libby asbestos fiber internalization generates a significant increase in intracellular reactive oxygen species (ROS) as determined by dichlorofluorescein diacetate and dihydroethidine fluorescence indicating that the superoxide anion is the major contributing ROS generated by Libby asbestos. Elevated superoxide levels in macrophages exposed to Libby asbestos coincide with a significant suppression of total superoxide dismutase activity. Both Libby and crocidolite asbestos generate oxidative stress in exposed macrophages by decreasing intracellular glutathione levels. Interestingly crocidolite asbestos, but not Libby asbestos, induces significant DNA damage in macrophages. This study provides evidence that the difference in the level of DNA damage observed between Libby and crocidolite asbestos may be a combined consequence of the distinct chemical compositions of each fiber as well as the activation of separate cellular pathways during asbestos exposure.

Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages

“…Exposure to Libby amphibole asbestos generates an increase in reactive oxygen species and a decrease in intracellular glutathione levels resulting in oxidative stress in murine macrophages (Blake et al 2007). Libby amphibole asbestos also directly affects human alveolar macrophages leading to an increase in lymphocyte derived cytokine production, which suggests that amphibole asbestos fibers can modify immune function by altering antigen presenting cell (APC) activity (Hamilton et al 2004).…”

“…We have previously reported that Libby 6-mix asbestos decreases cell viability in RAW264.7 cells after 24 h at a final concentration of 62.5 µg/cm 2 (Blake et al 2007). To determine whether the decrease in cell viability was a result of apoptosis, RAW264.7 cells were exposed to Libby 6-mix as previously described (Blake et al 2007) and apoptosis was quantified through Annexin V staining.…”

“…To determine whether the decrease in cell viability was a result of apoptosis, RAW264.7 cells were exposed to Libby 6-mix as previously described (Blake et al 2007) and apoptosis was quantified through Annexin V staining. Untreated control cells maintained a basal level of apoptosis of 3 to 5 percent (Fig.…”

“…Our study utilizes RAW264.7 cells, a phagocytic murine cell line with characteristics similar to alveolar macrophages (Xia et al 2006). We have previously shown that exposure to Libby amphibole asbestos induces oxidative stress in these cells (Blake et al 2007). The results of this study extend these findings and indicate that Libby asbestos induces apoptosis in macrophages leading to the redistribution of SSA/Ro52 to apoptotic blebs.…”

Separation and Characterization of Respirable Amphibole Fibers from Libby, Montana

“…According to Meeker et al (2003), most of the Libby particles display characteristics that are "intermediate between cleavage fragments and long flexible fibers." Nonetheless, this assemblage has been shown to induce oxidative stress as well as apoptosis in macrophages (Blake et al, 2007;Blake et al, 2008).Fiber dimension is widely considered to be the most important determinant of fiber pathogenicity in terms of cancer development (Oberdorster, 2000). Early work suggested that fibers longer than 8 μm were more potent inducers of mesothelioma than were shorter fibers (Stanton et al, 1981).…”