Citrate Does Not Change Uranium Chemical Speciation in Cell Culture Medium but Increases Its Toxicity and Accumulation in NRK-52E Cells Marie Carrie`re,* Ce´line Thiebault, Sarah Milgram, Laure Avoscan, Olivier Proux, ² and Barbara Gouget Laboratoire Pierre Su¨e CEA CNRS UMR 9956, CEA Saclay, 91191 Gif sur YVette, France, and Laboratoire de Ge´ ophysique Interne et Tectonophysique, UMR CNRS/UniVersite´ Joseph Fourier, 1381 rue de la piscine, 38400 Saint-Martin-D’He` res, France ReceiVed August 23, 2006 Uranium (U), as a heavy metal, is a strong chemical toxicant, which induces the damage to proximal tubule kidney cells. In order to reproduce U toxicity in Vitro and to avoid precipitation, it is necessary to complex it with a strong ligand such as bicarbonate before dilution with cell culture medium. It was recently shown, in Vitro on the NRK-52E normal renal tubular epithelial cells, that citrate increased the toxicity of U(VI)-bicarbonate complexes. This property was attributed to a change in U speciation, characterized by the occurrence of U(VI)-citrate complexes, which were supposed to be more toxic than U(VI)-bicarbonate. Here, we present the results of extended X-ray absorption fine structure spectroscopy (EXAFS) analyses of the media that were used to expose cells in Vitro. Resulting data show that even when citrate is added to the exposure medium, the predominant species is U(VI)- bicarbonate. Nonetheless, citrate increases U(VI) toxicity and accelerates its intracellular accumulation kinetics, without inducing precipitation. This study emphasizes another parameter that modulates U(VI) toxicity for renal tubule cells and further characterizes the mechanisms of U(VI) toxicity. Introduction Uranium (U) is a widely spread heavy metal, naturally present in rocks and surface water. Its intensive use in industry has led to increasing questions about its potential toxicity in the case of accidental exposure of workers and populations. Biokinetics studies have demonstrated that 24 h after acute exposure, U(VI) is detected in the kidneys and bones, where it causes severe toxicity (1). A solution of 1 mM of U(VI) is stable in Vitro in the exposure medium (i.e., MEM 1 ) when it is previously complexed to 10 mM bicarbonate (U(VI)-bicarbonate) or 10 mM citrate (U(VI)- citrate) (2). These ligands were chosen because of their physiological relevance: blood contains 25 mM bicarbonate and 0.15 mM citrate (3, 4). Together with serum proteins (5-6), they are the essential ligands of U(VI) in physiological fluids. Citrate is a strong chelator of calcium, continuously reabsorbed by kidney proximal tubules. Its average secretion in the urine flow is 640 mg per day (7-9); it is, thus, permanently present at the vicinity of renal proximal tubule cells, where its concentration varies as a function of metabolism and physi- ological state. It was proven that 700 μM to 1 mM U is lethal to LLC-PK1 renal cells when prepared as U(VI)-bicarbonate but not as U(VI)-citrate (2). U(VI)-bicarbonate toxicity was assessed on various renal cell lines including NRK-52E, which was chosen for future studies because of its high sensitivity to U (10). Recently, concentrations of 200-600 μM of U(VI)-citrate were shown to induce NRK-52E dose-response cell death. The CI 50 index (U concentration causing the death of 50% of the cells), which was 600 μM for U(VI)-bicarbonate, fell to 300 μM when citrate was added to the exposure medium: U(VI)-citrate is more toxic than U(VI)-bicarbonate (10). According to computer assisted speciation modeling, during in Vitro exposure, i.e., after dilution in MEM, U(VI)-bicarbonate speciation is dominated by carbonated forms: around 60% of U is complexed as UO 2 (CO 3 ) 3 4- , 27% as UO 2 Ca(CO 3 ) 3 (aq), 7% as UO 2 Ca(CO 3 ) 3 2- , and 6% as UO 2 (CO 3 ) 2 2- . Other species, such as UO 2 PO 4 - or UO 2 HPO 4 (aq) complexes, are also present, but their concentra- tion is minor compared to the concentration of uranyl carbonate complexes. When cells are exposed to U(VI)-citrate, 100% of U was calculated to be in the UO 2 (CIT) 2 4- complex form (10). It was, thus, postulated that the UO 2 (CIT) 2 4- complex was more toxic than uranyl carbonate complexes. When cells are exposed to U(VI)-bicarbonate, it intensively accumulates in the cyto- plasm (11), partly via the sodium-dependent phosphate cotrans- porter (12). Inside the cell, it localizes in lysosomes, where it coprecipitates with cell phosphates as pH drops, leading to the appearance of urchin-like electron-dense structures in the cell cytoplasm (2) and to major cell perturbations, particularly in the expression of genes implicated in signal transduction, trafficking, and calcium pathway (13). Here, we present the results of direct speciation analyses (EXAFS) of U(VI)-bicarbonate and U(VI)-citrate diluted in MEM. These solutions were then used to expose NRK-52E cells in order to evaluate citrate influence on U(VI) toxicity, cell accumulation, and repartition. This study permits to further * To whom correspondence should be addressed. Tel: +33 1 69 08 52 35. Fax: +33 1 69 08 69 23. E-mail: marie.carriere@cea.fr. ² UMR CNRS/Universite´ Joseph Fourier. 1 Abbreviations: ICP-MS, inductively coupled plasma-mass spectros- copy; EXAFS, extended X-ray absorption fine structure spectroscopy; FAME, French absorption spectroscopy beamline in material and environ- mental sciences; SEDEM, software package for EXAFS data extraction and modelling; SEM and TEM, scanning and transmission electron microscopy; EDS, energy dispersive spectroscopy; BCA, bicinchoninic acid; MEM, minimum essential medium; DMEM, Dulbecco’s modified Eagle medium; MTT, methyl thiazol tetrazolium. 1637 Chem. Res. Toxicol. 2006, 19, 1637-1642 10.1021/tx060206z CCC: $33.50 © 2006 American Chemical Society Published on Web 12/18/2006