New insights into the cytotoxicity of mineral fibres: A combined time-lapse video microscopy and in vitro assays study of chrysotile, crocidolite, and erionite fibres

Although in the last 30 years mineral fibres have been the subject of intensive toxicological studies, the actual mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this scenario, our work focuses on the monitoring of the very early steps of the interaction between chrysotile, crocidolite and erionite fibres (classified as carcinogenic by the International Agency for Research on Cancer) and macrophages using time-lapse video microscopy coupled with in vitro assays (i.e. LDH cytotoxicity, MTT viability, ROS generation and annexin-FITC/PI apoptosis). All tests were performed on the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1), after acute exposure (8 h) to 25 μg/mL of mineral fibres. Erionite fibres exhibit early toxicity effects while the cytotoxicity induced by chrysotile and crocidolite fibres occurs with a slight delay (ca. 2 h). In concert with literature data, the toxicity of chrysotile and crocidolite is linked to their ability to stimulate Reactive Oxygen Species (ROS) production. ROS are generated by M0-THP-1 cells as a result of frustrated phagocytosis induced by the long asbestos fibres, or produced by asbestos through the redox-active Fe on the fibre surface and metals released into the cell medium as a consequence of the partial dissolution of the fibres. Erionite fibres are able to induce an intracellular ROS increase but this contribution is significantly lower than both crocidolite and chrysotile stimuli. In the short period, crocidolite and chrysotile trigger significant apoptotic phenomena in M0-THP-1 cells while fibrous erionite is associated with early cytotoxicity and probably necrotic-like effect. Because the erionite fibres are short in length, frustrated phagocytosis plays a limited role in the acute toxicity of this fibre. Investigations focused on the intracellular concentrations of Na⁺ and Ca²⁺ as a result of cell-fibre interaction suggest an alternative mechanism by which fibrous erionite may induces cell injury and cell death. During phagocytosis erionite fibres quick exchange their extra-framework Na⁺ with the ions present in the cytosol of M0-THP-1 cells leading to the dysregulation of ionic homeostasis, cell swelling and cell lysis. At the same time, engulfed erionite fibres can reduce the level of cytosolic Ca²⁺ and interfere with endoplasmic reticulum-mitochondria crosstalk causing a delay in M0-THP-1 induction of apoptosis.