^99mTcO₄⁻-, Auger-Mediated Thyroid Stunning: Dosimetric Requirements and Associated Molecular Events

<div><p>Low-energy Auger and conversion electrons deposit their energy in a very small volume (a few nm³) around the site of emission. From a radiotoxicological point of view the effects of low-energy electrons on normal tissues are largely unknown, understudied, and generally assumed to be negligible. In this context, the discovery that the low-energy electron emitter, ^99mTc, can induce stunning on primary thyrocytes <i>in vitro</i>, at low absorbed doses, is intriguing. Extrapolated <i>in vivo</i>, this observation suggests that a radioisotope as commonly used in nuclear medicine as ^99mTc may significantly influence thyroid physiology. The aims of this study were to determine whether ^99mTc pertechnetate (^99mTcO₄⁻) is capable of inducing thyroid stunning <i>in vivo</i>, to evaluate the absorbed dose of ^99mTcO₄⁻ required to induce this stunning, and to analyze the biological events associated/concomitant with this effect. Our results show that ^99mTcO₄⁻–mediated thyroid stunning can be observed <i>in vivo</i> in mouse thyroid. The threshold of the absorbed dose in the thyroid required to obtain a significant stunning effect is in the range of 20 Gy. This effect is associated with a reduced level of functional Na/I symporter (NIS) protein, with no significant cell death. It is reversible within a few days. At the cellular and molecular levels, a decrease in NIS mRNA, the generation of double-strand DNA breaks, and the activation of the p53 pathway are observed. Low-energy electrons emitted by ^99mTc can, therefore, induce thyroid stunning <i>in vivo</i> in mice, if it is exposed to an absorbed dose of at least 20 Gy, a level unlikely to be encountered in clinical practice. Nevertheless this report presents an unexpected effect of low-energy electrons on a normal tissue <i>in vivo</i>, and provides a unique experimental setup to understand the fine molecular mechanisms involved in their biological effects.</p></div

Longitudinal follow up of ^99mTcO₄⁻-mediated thyroid stunning by SPECT/CT.

<p>^99mTcO₄⁻ uptake in the thyroid was measured by SPECT/CT in 12 mice at day 0 (D0) and again at day 1 (D1, n = 3), day 2 (D2, n = 3), day 4 (D4, n = 3), or day 8 (D8, n = 3). The second scan was performed by injecting a dose of 50 MBq ^99mTcO₄⁻. (A) Representative SPECT/CT images of ^99mTcO₄⁻ uptake by the thyroid. The images are normalized (100%) to the voxel with the highest activity in the series. (B) Thyroid uptake presented as percentage of the value at day 0± SD. (*: p<0.05; ***: p<0.001).</p

Changes in thyroid uptake 24^99mTcO₄⁻.

<p>Mice were injected with various amounts of ^99mTcO₄⁻ (25 to 150 MBq). Twenty-four hours later, a standard dose of 50 MBq ^99mTcO₄⁻ was injected to determine the capacity of the thyroid to take up the radioisotope. The figure represents the relationship between the initial absorbed dose to the thyroid (in Gy) and the percent change in ^99mTcO₄⁻ uptake 24 hours later.</p

γH2AX foci in thyroids and salivary glands of mice 24 hours after administration of ^99mTcO₄⁻.

<p>Mice were injected with various activities of ^99mTcO₄⁻ and culled 24 hours later for biopsy collection. (A) Western blotting of protein homogenates obtained from mouse thyroids subjected to different levels of irradiation. Results represent the normalized increase in H2AX phosphorylation level compared with the control condition. Protein quantification was performed with image J software. (B) Representative sections of thyroids or salivary glands of control mice or mice exposed to 30 Gy ^99mTcO₄⁻ were stained with a γH2AX- or NIS-specific antibody. Arrows represent foci of DNA double-strand break repair (γH2AX-positive nuclei). The figures presented are representative of 2 and 6 independent experiments for Western blot (A) and immunohistochemistry (B), respectively.</p

^99mTcO₄⁻-SPECT/CT imaging and absorbed dose to the thyroid.

<p>(A) Volume-rendered, fused SPECT/CT images in anterior and lateral views demonstrating the thyroid lobes (red) and the salivary glands (blue-green), 60 minutes after ^99mTcO₄⁻ administration. (B) Kinetics of ^99mTcO₄⁻ activity in the thyroids of four Balb/c mice fed with a normal iodide diet (open squares) or with a low iodide diet (filled squares), obtained upon injection of 150 MBq of ^99mTcO₄⁻. The values presented are not corrected for decay. (C) Correspondence between thyroid activity (MBq) at 60 minutes post injection and absorbed dose to the thyroid (Gy) at 24 hours. (D) Absorbed dose to the thyroid at 24 hours in mice injected with 10 MBq (circles, n = 4), 25 MBq (triangles, n = 9), 100 MBq (squares, n = 9) or 150 MBq (diamonds, n = 29) ^99mTcO₄⁻.</p

Expression of NIS mRNA and protein in thyroids of mice 24^99mTcO₄⁻.

<p>(A) Quantitative RT-PCR analyses of NIS expression in control thyroids or thyroids exposed to 5 Gy, 20 Gy or 30 Gy. The means (n = 4 mice/group) are presented (horizontal bars) as well as each individual point. (**: p<0.01; ***: p<0.001). (B) Quantitative RT-PCR analyses of NIS expression in control thyroids or thyroids exposed to decayed ^99mTcO₄⁻ (⁹⁹Tc on the figure) or 30 Gy of ^99mTcO₄⁻ (^99mTcO₄⁻ on the figure). The means (n = 3 to 4 mice/group) are presented (horizontal bars) as well as each individual point. (C) Western blot analyses of NIS expression levels assessed on membrane preparations from thyroids of control mice or from thyroids extracted 1 and 5 days after exposure to 30 Gy ^99mTcO₄⁻. The immunoblot shown is representative of three independent experiments.</p