ORIGINAL ARTICLE Hugh H. Harris Æ Aviva Levina Æ Carolyn T. Dillon Irma Mulyani Æ Barry Lai Æ Zhonghou Cai Peter A. Lay Time-dependent uptake, distribution and biotransformation of chromium(VI) in individual and bulk human lung cells: application of synchrotron radiation techniques Received: 8 September 2004 / Accepted: 22 November 2004 / Published online: 16 February 2005 Ó SBIC 2005 Abstract Chromium(VI) is a human carcinogen, pri- marily affecting the respiratory tract probably via active transport into cells, followed by the reduction to Cr(III) with the formation of DNA-damaging intermediates. Distribution of Cr and endogenous elements within A549 human lung adenocarcinoma epithelial cells, fol- lowing treatment with Cr(VI) (100 lM, 20 min or 4 h) were studied by synchrotron-radiation-induced X-ray emission (SRIXE) of single freeze-dried cells. After the 20-min treatment, Cr was confined to a small area of the cytoplasm and strongly co-localized with S, Cl, K, and Ca. After the 4-h treatment, Cr was distributed throughout the cell, with higher concentrations in the nucleus and the cytoplasmic membrane. This time- dependence corresponded to 100% or 0% clonogenic survival of the cells following the 20-min or 4-h treat- ments, respectively, and could potentially be explained by a new cellular protective mechanism. Such processes may also be important in reducing the potential hazards of Cr(III) dietary supplements, for which there is emerging evidence that they exert their anti-diabetic ef- fects via biological oxidation to Cr(VI). The predomi- nance of Cr(III) was confirmed by micro-XANES spectroscopy of intracellular Cr hotspots. X-ray absorption spectroscopy (XANES and EXAFS, using freeze-dried cells after the 0–4-h treatments) was used to gain insight into the chemical structures of Cr(III) complexes formed during the intracellular reduction of Cr(VI). The polynuclear nature of such complexes (probably with a combination of carboxylato and hy- droxo bridging groups and O-donor atoms of small peptides or proteins) was established by XAFS data analyses. Keywords Chromium Æ Cancer Æ Human cells Æ Synchrotron-radiation-induced X-ray emission Æ X-ray absorption spectroscopy Abbreviations ANBF: Australian National Beamline Facility Æ APS: Advanced Photon Source Æ cys: Cysteinato(2-) Æ DMEM: Dulbecco’s modified minimal essential medium Æ EPR: Electron paramagnetic resonance Æ EXAFS: Extended X-ray absorption fine structure Æ FT: Fourier-transform Æ GFAAS: Graphite- furnace atomic absorption spectroscopy Æ MS: Multiple scattering Æ PBS: Phosphate buffered saline Æ pic: Picolinato(1-) = 2-pyridinecarboxylato(1-) Æ SRIXE: Synchrotron-radiation-induced X-ray emission Æ SS: Single scattering Æ XANES: X-ray absorption near-edge structure Æ XAFS: X-ray absorption fine structure Æ XAS: X-ray absorption spectroscopy Introduction A link between the occupational exposure to Cr(VI) compounds and cancers of the respiratory tract is well- established [1]. A generally accepted mechanism of Cr(VI)-induced genotoxicity [2, 3] includes the active transport of Cr(VI) into cells (through anion channels for water-soluble chromates or by phagocytosis of Electronic Supplementary Material: Supplementary material is available for this article at http://dx.doi.org/10.1007/s00775-004- 0617-1 H. H. Harris Æ A. Levina Æ C. T. Dillon I. Mulyani Æ P. A. Lay (&) Centre for Heavy Metals Research, and Centre for Structural Biology and Structural Chemistry, School of Chemistry, University of Sydney, Sydney, NSW, 2006, Australia E-mail: p.lay@chem.usyd.edu.au Tel.: +61-2-93514269 Fax: +61-2-93513329 C. T. Dillon Australian Key Centre for Microscopy and Microanalysis, Electron Microscope Unit, Madsen Building, University of Sydney, Sydney, NSW, 2006, Australia B. Lai Æ Z. Cai Experimental Facilities Division, Argonne National Laboratory, Argonne, IL 60439, USA J Biol Inorg Chem (2005) 10: 105–118 DOI 10.1007/s00775-004-0617-1