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 (>1011 ph/s) at micrometric spatial resolution (1.5×4.0 μm2). 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 As2O3. 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.