Direct speciation analysis of arsenic in sub-cellular compartments using micro-X-ray absorption spectroscopy

Identification of arsenic chemical species at a sub-cellular level is a key to understanding the mechanisms involved in arsenic toxicology and antitumor pharmacology. When performed with a microbeam, X-ray absorption near-edge structure (μ-XANES) enables the direct speciation analysis of arsenic in sub-cellular compartments avoiding cell fractionation and other preparation steps that might modify the chemical species. This methodology couples tracking of cellular organelles in a single cell by confocal or epifluorescence microscopy with local analysis of chemical species by μ-XANES. Here we report the results obtained with a μ-XANES experimental setup based on Kirkpatrick–Baez X-ray focusing optics that maintains high flux of incoming radiation (>10 11 ph/s) at micrometric spatial resolution (1.5×4.0 μm 2). This original experimental setup enabled the direct speciation analysis of arsenic in sub-cellular organelles with a 10 −15 g detection limit. μ-XANES shows that inorganic arsenite, As(OH) 3, is the main form of arsenic in the cytosol, nucleus, and mitochondrial network of cultured cancer cells exposed to As 2O 3. On the other hand, a predominance of As(III) species is observed in HepG2 cells exposed to As(OH) 3 with, in some cases, oxidation to a pentavalent form in nuclear structures of HepG2 cells. The observation of intra-nuclear mixed redox states suggests an inter-individual variability in a cell population that can only be evidenced with direct sub-cellular speciation analysis.