Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Summary

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

Abstract

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Introduction

Uranium is a naturally occurring radioactive element present in soils, air and water; as such, animals and humans are exposed to natural uranium in their diets. In addition to natural sources, uranium originates from anthropogenic activities, which increases its abundance in the environment. Uranium poses both chemical and radiological hazards. However, because natural uranium (which is an isotopic mixture containing 99.27% ²³⁸U, 0.72% ²³⁵U, and 0.006% ²³⁴U) has a low specific activity (25.10³ Bq.g^-1), its impacts on health are attributed to its chemical toxicity.

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Protocol

1. Preparation of Uranyl Acetate Solution

1. To prepare 2 mL of a 100 mM uranyl acetate solution, add 85 mg of uranyl acetate (UO₂(OCOCH₃)₂, 2H₂O; M = 424 g.mol^-1) in solid state to a 5 mL plastic tube.
2. Add 2 mL of distilled water to the plastic tube and fit it with a plastic stopper.
3. Shake the tube vigorously until the total dissolution of the solid. Put the solution into the fridge for 24 h.
   CAUTION: The manipulation of uranyl [U(VI)] presents some chemical and radiological hazards. All the samples should be prepared and characterized with ad hoc equipment in specific laborato....

Results

Tartrate-resistant acid phosphatase staining was used to visualize osteoclasts as large purple cells having 3 or more nuclei. Representative images of osteoclasts obtained from RAW 264.7 cells cultured in the presence of RANKL and uranyl ions are shown in Figure 1. Changes in number and size of osteoclasts in response to uranium are easily visible in composite images of whole wells and in enlarged pictures.

Discussion

As far as we know, this is the first time that a detailed procedure aiming to study the effect of natural uranium on bone resorbing cells is described. This approach will be useful to achieve a better understanding of uranium impact on bone physiology and may provide an interesting new tool for the screening of uranium chelators. Furthermore, we believe that the protocol described here could be applied to study the impact of other heavy metals on osteoclatogenesis.

It is known that uranyl is c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Lonza	BE12-604F
α-MEM	Lonza	BE12-169F
EMEM without phenol red	Lonza	12-668E
Water for cell culture	Lonza	BE17-724F
PBS	Sigma-Aldrich	D8537
Penicillin-Streptomycin solution	Sigma-Aldrich	P4333
L-Glutamine solution	Sigma-Aldrich	G7513
Trypan Blue Solution 0.4%	Sigma-Aldrich	T8154
HyClone fetal bovine serum	GE Life Sciences	SH30071.03
7.5% sodium bicarbonate aqueous solution	Sigma-Aldrich	S8761
Acid Phosphatase, Lekocyte (TRAP) kit	Sigma-Aldrich	387A
Thiazolyl Blue Tetrazolium Bromide (MTT) powder	Sigma-Aldrich	M5655
Dimethyl sulfoxide	Sigma-Aldrich	D5879
Alizarin Red S sodium salt, 1% w/v aq. sol.	Alfa Aeros	42746
Osteoassay bone resorption plates, 24 well plates	Corning Life Sciences	3987
Multiwell 24 well plates	Falcon	353504
Flask 75 cm2	Falcon	353133
Polypropylene Conical Tubes 50 ml	Falcon	352070
Cell scrapers 30 cm	TPP	90003