ToxSci Advance Access originally published online on November 12, 2007
Toxicological Sciences 2008 101(2):254-262; doi:10.1093/toxsci/kfm266
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Role of the Sodium-Dependent Phosphate Cotransporters and Absorptive Endocytosis in the Uptake of Low Concentrations of Uranium and Its Toxicity at Higher Concentrations in LLC-PK1 Cells
* Institut de Radioprotection et de Sûreté Nucléaire, Laboratoire de Radiotoxicologie Expérimentale, BP-166, 26702 Pierrelatte Cedex, France
GEPPR, LSTE-EA 3672, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
CEA/DEN/Valrhô-Dir-CVAR, BP 17171, 30207 Bagnols sur Cèze Cedex, France
1 To whom correspondence should be addressed at the β-Cell Development and Function Group, School of Health and Biomedical Sciences, 2nd Floor, Hodgkin Building, Guy's Campus, King's College London, London SE1 1UL, UK. Fax: +44 (0) 20-7848-6280. E-mail: dany.muller{at}kcl.ac.uk.
Received July 31, 2007; accepted October 7, 2007
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Abstract |
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It has been suggested that uranium uptake and toxicity could be mediated by endocytosis and/or the type IIa sodium-dependent phosphate cotransporter (NaPi-IIa). The aim of this study was therefore to characterize in vitro the role of these two cellular mechanisms in the uptake and toxicity of low (200–3200nM) and high (0.5 and 0.8mM) concentrations of uranium, respectively. At low concentrations, uranium uptake in LLC-PK1 cells was saturable (Vmax = 3.09 ± 0.22 ng/mg protein) and characterized by a K0.5 of 1022 ± 63nM and a Hill coefficient of 3.0 ± 0.4. The potential involvement of endocytosis and NaPi-IIa in the uptake of uranium was assessed by the use of various drugs and culture conditions known to alter their relative activity, and 233uranium uptake was monitored. Interestingly, the inhibitory effect of colchicine, cytochalasin D, phorbol 12-myristate 13-acetate, and chlorpromazine on endocytosis was highly correlated with their effect on uranium uptake, a relationship that was not true when the NaPi-IIa transport system was studied. Whereas the competitive inhibition of the NaPi-IIa by phosphonoformic acid (PFA) significantly decreased uranium uptake, this effect was not reproduced when NaPi-IIa inhibition was mediated by the replacement of extracellular Na+ with N-methyl-D-glucamine. Uranium uptake was also not significantly altered when NaPi-IIa expression was stimulated in MDCK cells. More surprisingly, we observed by transmission electron microscopy that uranium cytotoxicity was dependent upon the extent of its intracellular precipitation, but not on its intracellular content, and was suppressed by PFA. In conclusion, our results suggest that low-dose uranium uptake is mainly mediated by absorptive endocytosis, and we propose PFA as a potential uranium chelator.
Key Words: uranium uptake; endocytosis; phosphonoformic acid; LLC-PK1; MDCK; uranium cytotoxicity.