Micro-Raman investigation of an in vitro THP-1 cellular model exposed to chrysotile fibres

In chrysotile, the most commercialized asbestos, Fe and other toxic metals are worth considering in its pathogenicity (Gualtieri et al., 2019); in fact, large amounts of Fe (>1000 ppm) and trace metals (Cr, Ni, Co, Mn, …) are also intimately associated with the raw material. Since both Fe and trace metals are usually isomorphous substituent for Mg in octahedral sites, their release into the lung environment is strictly connected to that of Mg, when fibres are dissolved by the macrophage phagocytosis (Pollastri et al., 2015). Thus, trace elements are potentially involved in the production of reactive oxygen species (ROS), in addition to iron (Bloise et al., 2016). In the frame of the project PRIN 2017 3X8WA4, in vitro THP1-cells exposed to chrysotile fibres (from Balangero, Italy), reporting the contribution of Fe in cells mortality, demonstrated the formation of aggregated structures of both fibrous and non-fibrous species, including Fe oxides and sulphides extensively characterized by micro-Raman analysis. In these clusters, dissolution mechanism needs further investigation, looking at the spatial distribution of metals in the biological system and to the molecular structure of the existing species. To this extent, synchrotron-based X-ray imaging and spectroscopy represent an emerging and effective tool for investigating biological systems at the sub-cellular level, able to clarify metals mobilization mechanism, highlighting both their intracellular spatial distribution and the changes in their valence state induced by the fibres-cells interaction (Bardelli et al, 2017; Cammisuli et al., 2018; Pascolo et al., 2013). A combined approach with AFM and X-ray fluorescence (XRF) elemental mapping with a sub-micrometre resolution has been applied on THP-1 cells after different exposure time to fibers (8h, 24 h, 96 h) at the NanoInnovation lab and TwinMic beamline of the Italian Elettra Synchrotron Facility in Trieste, to clarify the role of iron in the surface reactivity of the chrysotile asbestos fibres and other iron compounds, especially in the observed clusters; in addition, other elements such as Mghave been mapped to obtain important information on the fibres dissolution. On the same samples, at the ID21 beamline (ESRF, Grenoble, France), micro-XANES at the Fe K-edge provided fundamental details about the chemical states of Fe and the potential transformations of Fe sulphides/oxides in the organic environment. Micro-XANES at the Cr K-edge elucidated the possible Cr toxicity, discriminating Cr(III) - from the asbestos fibres - and potential Cr(VI) –as intracellular toxic species. Results have been compared with the ones obtained with oxidative stress analysis, genotoxic and DNA damage investigations, cellular toxicity and viability tests. The whole study represents an important step forward in the understanding of asbestos pathogenicity, especially in the role of iron and other toxic metals in the surface activity of the fibres.