X-ray fluorescence imaging of uranium distribution in human dopaminergic cells

• Main Authors: Carmona, Asuncion, Somogyi, Andrea, Roudeau, Stephane, Porcaro, Francesco, Medjoubi, Kadda, Vidaud, Claude, Malard, Veronique, Bresson, Carole, Ortega, Richard
• Format: Conference Proceeding
• Language: English
• Subjects: Aggregates; Calcium phosphates; Cells (biology); Cytotoxicity; Dwell time; Endosomes; Freezing; Grain size distribution; Liquid nitrogen; Localization; Low concentrations; Lysosomes; Microscopy; Neurological diseases; Neurons; Neurotoxicity; Pixels; Proteins; Room temperature; Spatial resolution; Synchrotron radiation; Synchrotrons; Uranium; X ray imagery; X-ray fluorescence
• ISSN: 0094-243X; 1551-7616
• DOI: 10.1063/5.0168512

Uranium exposure can cause neurological disorders, even at low non-cytotoxic concentrations, especially towards the dopaminergic pathway. To date, the mechanisms of uranium neurotoxicity are still poorly understood. One way to deepen the understanding of uranium neurotoxicity is to determine its localization within neurons. However, mapping low concentrations of uranium in subcellular areas is challenging. We have overcome this analytical challenge by using the capabilities of Nanoscopium beamline at SOLEIL synchrotron. The beam, of 17.6 keV energy, was focused with a Kirkpatrick-Baez mirror-pair, providing a spatial resolution in both directions of 300 nm and keeping an incident flux of 1010 photon/s. Between 2 to 4 hours were required to image a single cell, maintaining a pixel size of 300 nm and a dwell time of 300 ms per pixel. The analyses were conducted at room temperature, under atmospheric conditions, and using two silicon drift detectors located at 120° with respect to the beam direction on both sides of the sample. Human dopaminergic SH-SY5Y cells were differentiated into mature neurons and continuously exposed for seven days to a non-cytotoxic concentration of 10 µM of natural uranium, under the uranyl-carbonato soluble form. Living cells were transfected with transitory commercial kits to express fluorescently-labeled proteins of interest. We observed by epi-fluorescence microscopy the localization of lysosomes, early/late endosomes, nucleus and fetuin-A, a protein known for its high affinity for uranium. Epi-fluorescence microscopy was performed in living cells and the samples were cryofixed immediately after, by plunge freezing in isopentane cooled with liquid nitrogen, and further freeze-dried. Synchrotron x-ray fluorescence imaging (SXRF) revealed the formation of submicron-sized uranium aggregates in the cytoplasm. Some uranium aggregates were colocalized with iron hot-spots, suggesting common metabolic storage pathways. A strict correlation between the distribution of uranium and fluorescently-labeled fetuin-A could not be evidenced. The intracellular distribution of uranium followed a similar localization pattern than lysosomes and late endosomes, characterized by an accumulation on the same regions of the cell and similar grain size distribution. We evaluated the size of these uranium-rich areas and found that their diameter could range from