Effects of various man-made mineral fibers on cell apoptosis and viability

Abstract

Evaluating the pathogenic potentials of man-made mineral fibers (MMMF) is an important task performed by the European Community. Noting that it has been proposed that the use of laboratory animals for scientific tests should be reduced or phased out, macrophages then become the cells of choice for conducting in vitro studies. We have evaluated the in vitro toxicity of six commercial stonewool fibers (A, B1, B2, C, D, and E) on U-937 cells. The physical interaction between U-937 cells and MMMF was observed using scanning electron microscopy, and the cytotoxicity was evaluated by studying cell viability using MTT assay and cell apoptosis with an ELISA detection kit. Scanning electron microscopy (SEM) analysis has shown that long fibers can be covered by several macrophages, and that a small fiber can be completely engulfed by one cell. With 50 microg/mL of MMMF, a decrease in cell viability appeared after seven days of incubation, whereas 200 microg/mL induced loss of viability and apoptosis after one day. Fiber D, comprising a high proportion of fibers >20 microm in length and a high concentration of MgO, induced the highest loss in viability and the highest rate of apoptosis compared to the other five fibers. Whether this toxic effect is related to either the physical characteristics of the fibers (such as length), or to the high concentration of magnesium is still to be determined. Because the results can be rapidly obtained, the proposed model is suitable for studying the toxicities of mineral components, even if the tested concentrations are far from the ones reached in the lung.

Macrophage Culture as a Suitable Paradigm for Evaluation of Synthetic Vitreous Fibers

“…Furthermore, human AMs could successfully engulf 20-µm glass fibers whereas incomplete phagocytosis by rat AMs occurred already with glass fibers ≥17 µm in length [53]. This was also observed within the human monocytic cell line U-937 [34]. According to Table 1, with in vitro studies, the percentage of fibers with length ≥20 µm varies from 0 to 67%.…”

“…The contact of fibers with the cell membrane leads to changes in the morphology of the cell surface such as blebs formation. However, it has been reported that SVFs, after phagocytosis, induce less morphological alterations of the cells [34][35][36]. The mechanisms of fiber clearance involve mainly mucociliary movement of the nasopharyngeal and tracheo-bronchial mucosa, phagocytosis in the alveoli with subsequent moving of phagocytic cells towards and along the mucociliary escalator, and transportation to the lymphatic and the lymphoid tissue.…”

“…In this sense, AMs and U-937 monocytic cell lines adhered to fibers. One cell can attach to several small size fibers, while long fibers were covered by many cells; this process increased over time [20,34,35,52]. The phagocytosis process was more frequent and more efficient in AMs than in peritoneal macrophages (PMs).…”

“…According to Table 1, with in vitro studies, the percentage of fibers with length ≥20 µm varies from 0 to 67%. With transmission electron microscopy, phagocytic vacuoles were observed without any cytoplasmic alteration [20,34,52].…”

“…To quantify apoptosis, electrophoretic detection of fragmented DNA may be performed after DNA extraction as well as detection of nucleosomes and fragmented DNA by enzyme-linked immunosorbent assay (ELISA) (Table 2). Apoptosis was induced solely for high fiber concentrations (200 µg/ml) corresponding approximately to 100 to 400 fibers/cell after a 1-day exposure of U-937 cells to high-aluminacontaining rock wool fibers [34]. In such experimental conditions, cells were overloaded because ingested volume of fibers was greater than 6% of macrophage volume [85][86][87].…”