Evidence of isotopic fractionation of natural uranium in cultured human cells

Abstract

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the ²³⁵U isotope with regard to ²³⁸U. Efforts were made to develop and then validate a procedure for highly accurate n(²³⁸U)/n(²³⁵U) determinations in microsamples of cells. We found that intracellular U is enriched in ²³⁵U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO₂²⁺) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

Keywords: analytical procedure development; isotopic fractionation; microsamples; neuronal cells; uranium.

Fig. 1.

Theoretical speciation diagram of uranium in the exposure solutions at 10 µM U. Hsa stands for human serum albumin. Thermodynamic constants are from refs. – and . Only species with proportion higher than 2.5% are represented. Circles indicate the percentages found at the pH of exposure solutions (∼7.5). Calculations were performed with the PhreeqCI 3.0.6-7757 software (66).

Fig. 2.

Effect of the analytical procedure and sample matrix on δ²³⁸ values. The δ²³⁸ values shown correspond to the IRMM-184 mass bias control solutions nonsubjected to the analytical procedure (full triangles), the IRMM-184 procedure control solutions subjected to the analytical procedure (full diamonds), uranium-free culture medium spiked with IRMM-184 material (empty triangles), and uranium-free cell samples spiked with IRMM-184 material (empty squares). The δ²³⁸ values are calculated relative to the certified n(²³⁸U)/n(²³⁵U) for the IRMM-184 material.

Fig. 3.

The δ²³⁸ values for extracellular and intracellular uranium. Red triangles correspond to uranium in the exposure solutions ([U] = 10 μM), whereas blue squares are for uranium incorporated by SH-SY5Y cells. The δ²³⁸ values are calculated relative to the natural uranium stock solution. Error bars correspond to the expanded uncertainty (k = 2). Plain red and blue lines correspond to the mean of δ²³⁸ values obtained for extracellular and intracellular uranium, respectively, in seven independent experiments. Dashed lines represent the reproducibility of the δ²³⁸ values (2σ, n = 7). Values for extracellular and intracellular uranium were statistically different according to a t test (***P < 0.001).

Fig. 4.

Suggested incorporation pathways of uranium into human neuronal cells. Pathway A corresponds to the facilitated diffusion of Tris-carbonate uranyl species. Pathway B corresponds to a three-step incorporation process consisting of (step 1) the formation of a complex with a high-affinity uranium transport protein through the exchange of one or more carbonates; (step 2) the transport of uranyl through the transport protein; and (step 3) the binding of uranyl to intracellular metabolites/proteins.

Fig. S1.

Schematic diagram of the protocol used for sample preparation before MC-ICPMS measurements. Samples were either cell pellets exposed to 10 μM U or 2 mL of exposure solutions at 10 μM U. U-free spiked samples were either control cells nonexposed to U spiked with 900 ng of U from IRMM-184 material or 2 mL of culture medium spiked with 900 ng of U from IRMM-184 material. Procedure control solutions were 900-ng⋅mL⁻¹ U solutions prepared from IRMM-184 material in 3 M HNO₃.

Fig. S2.

Reproducibility (2 SD) of δ²³⁸ values at different signal levels for the less abundant isotope (²³⁵U). The experiments were performed with IRMM-184 mass bias control solutions. The number of replicates varied from 6 to 12. Full diamonds, conventional system; empty diamonds, microflow system.