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A transcriptionally induced antioxidant program is elicited in thyroid cells after exposure to hydrogen peroxide

ABSTRACT: Radiation is an established cause of thyroid cancer and growing evidence supports a role for H2O2 in spontaneous thyroid carcinogenesis. Little is known about the molecular programs activated by these agents in thyroid cells. We profiled the DNA damage response and cell death induced by γ-radiation (0.1–5Gy) and H2O2 (0.0025–0.3mM) in primary human thyroid cells and T-cells. While the two cell types had more comparable radiation responses, 3- to 10-fold more H2O2 was needed to induce detectable DNA damage in thyrocytes. At H2O2 and radiation doses incurring double-strand breaks (DSB), cell death occurred after 24hrs in T-cells, but not in thyrocytes. We next prepared thyroid and T-cells primary cultures from 8 donors operated for non-cancerous pathologies and profiled their genome-wide transcriptional response 4hr after: 1) exposure to 1 Gy radiation, 2) treatment with H2O2, or 3) no treatment. Two H2O2 doses were investigated, calibrated in each cell type as to elicit levels of single- and double-strand breaks equivalent to 1 Gy γ-radiation. The transcriptional responses of thyrocyte and T-cells to radiation were similar, involving DNA repair and cell death genes. In addition to this transcriptional program, H2O2 also upregulated antioxidant genes in thyrocytes, including glutathione peroxidases (GPx) at the DSB-inducing dose. By contrast, a transcriptional storm involving thousands of genes was raised in T-cells. Finally, we showed that GPx inhibition reduced the DNA damaging effect of H2O2 in thyrocytes. We conjecture that defects of anti- H2O2 protection could promote spontaneous thyroid cancers.

ORGANISM(S): Homo sapiens

PROVIDER: GSE39156 | GEO | 2013/05/03

SECONDARY ACCESSION(S): PRJNA170114

REPOSITORIES: GEO

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Project description:Radiation is an established cause of thyroid cancer and growing evidence supports a role for H2O2 in spontaneous thyroid carcinogenesis. Little is known about the molecular programs activated by these agents in thyroid cells. We profiled the DNA damage response and cell death induced by M-NM-3-radiation (0.1M-bM-^@M-^S5Gy) and H2O2 (0.0025M-bM-^@M-^S0.3mM) in primary human thyroid cells and T-cells. While the two cell types had more comparable radiation responses, 3- to 10-fold more H2O2 was needed to induce detectable DNA damage in thyrocytes. At H2O2 and radiation doses incurring double-strand breaks (DSB), cell death occurred after 24hrs in T-cells, but not in thyrocytes. We next prepared thyroid and T-cells primary cultures from 8 donors operated for non-cancerous pathologies and profiled their genome-wide transcriptional response 4hr after: 1) exposure to 1 Gy radiation, 2) treatment with H2O2, or 3) no treatment. Two H2O2 doses were investigated, calibrated in each cell type as to elicit levels of single- and double-strand breaks equivalent to 1 Gy M-NM-3-radiation. The transcriptional responses of thyrocyte and T-cells to radiation were similar, involving DNA repair and cell death genes. In addition to this transcriptional program, H2O2 also upregulated antioxidant genes in thyrocytes, including glutathione peroxidases (GPx) at the DSB-inducing dose. By contrast, a transcriptional storm involving thousands of genes was raised in T-cells. Finally, we showed that GPx inhibition reduced the DNA damaging effect of H2O2 in thyrocytes. We conjecture that defects of anti- H2O2 protection could promote spontaneous thyroid cancers. Here we characterize the response of thyrocytes to H2O2 from the perspective of DNA damage (SSB and DSB), cell death and genome-wide transcription. We adopted a dual comparative set up. First, in order to gain preliminary insights about which effects are specific to thyrocytes, all experiments were run in parallel in T-cells. Second, all experiments were replicated with radiation, providing a comparison with a proven etiological agent of PTCs. Hence, we investigated all 4 combinations of two factors: thyrocytes vs. T-cells and H2O2 vs. radiation. Cell lines divide indefinitively owning to basic dysfunctions of cell cycle controls. These have the potential to distort the stress response, DNA repair and cell death in particular. Hence, we worked exclusively on human primary cultures. The radiation and presumably also the H2O2 responses vary among individuals. We seek to average out individual effects by replicating the microarray experiments across the matched T-cells and thyrocytes of 8 donors.

Project description:Rationale: Auger electrons bear a low energy (0-10 keV) which is deposited in a very small volume (a few nm3) around the site of emission. From a radio-toxicological point of view, the effects of Auger electrons on normal tissues are largely unknown, understudied and generally assumed negligible. In this context, the discovery that the Auger electron emitter 99mTc can induce stunning on primary thyrocytes in vitro, at low doses, is intriguing. Extrapolated in vivo, this observation suggests that, a radioisotope as commonly used in nuclear medicine as 99mTc may significantly influence thyroid biology. The aim of the present study was to evaluate the absorbed dose of 99mTc pertechnetate (99mTcO4−) required to induce thyroid stunning in vivo and to analyse the biological events associated/concomitant with this effect. Methods: SPECT/CT imaging of thyroid uptake of 99mTcO4− was used to demonstrate stunning effect in mice and quantitative analysis of the images was used for dosimetric calculations. The biological effects associated with this phenomenon were evaluated by Western blotting, quantitative RT-PCR, histology, immuno-histochmistry and global gene expression pattern analysis. Results: Auger electrons-mediated thyroid stunning can be observed in vivo, in mouse thyroid. The threshold of the absorbed dose to the thyroid required to obtain a significant stunning effect is in the range of 20 Gy. This effect is associated with a reduction in the levels of functional Na/I symporter (NIS) protein without any significant cell death. It is reversible within days. At the cellular level, a decrease in NIS mRNA, the activation of the p53 pathway and the generation of double-strand DNA breaks can be observed. Conclusion: Auger electrons emitted by 99mTc can induce thyroid stunning in vivo, in mice but the thyroid needs to be exposed to a dose of at least 20 Gy, a dose unlikely to be encountered in the clinical practice. Nevertheless, this report represents, to our knowledge, the first example of a significant action of Auger electrons on a normal tissue, in vivo and provides a unique experimental set-up to understand the fine molecular mechanisms involved in the biological effects of Auger electrons. One color design, corresponding to 2 conditions performed on 4 mice: TcO4- (Tc)-treated versus Control (Ct) mice for a total of 8 samples.

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A transcriptionally induced antioxidant program is elicited in thyroid cells after exposure to hydrogen peroxide

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