Ex-vivo dose response characterization of the recently identified EDA2R gene after low level radiation exposures and comparison with FDXR gene expression and the γH2AX focus assay

Abstract

Objective

Recently, promising radiation-induced EDA2R gene expression (GE) changes after low level radiation could be shown. Stimulated by that, in this study, we intended to independently validate these findings and to further characterize dose-response relationships in comparison to FDXR and the γH2AX-DNA double-strand break (DSB) focus assay, since both assays are already widely used for biodosimetry purposes.

Materials and methods

Peripheral blood samples from six healthy human donors were irradiated ex vivo (dose: ranging from 2.6 to 49.7 mGy). Subsequently, the fold-differences relative to the sham irradiated reference group were calculated. Radiation-induced changes in GE of FDXR and EDA2R were examined using the quantitative real-time polymerase-chain-reaction (qRT-PCR). DSB foci were quantified in 100 γH2AX + 53BP1 immunostained cells employing fluorescence microscopy. Examinations were performed at single time points enabling sufficient detection of both endpoints.

Results

A significant increase in EDA2R GE relative to the unexposed control was observed in the range of 2.6 mGy (1.6-fold, p = .045) to 5.4 mGy (2.2-fold, p = .0002), whereas the copy numbers increased linearly up to 13.1-fold at 49.7 mGy. On the contrary, FDXR upregulation (2.2-fold) became significant after a 22.6 mGy exposure (p ≤ .02) and increased linearly up to 4-fold at 49.7 mGy. A significant increase in radiation-induced foci (relative to unexposed, RIF-fd) was observed after 11.3 mGy (RIF-fd: 1.5 ± 0.5, p ≤ .03), while the foci increased linearly up to 3-fold at 49.7 mGy. From this, the FDXR and RIF-fd slopes have shown comparability, while the EDA2R slope was five times higher. Nevertheless, the coefficient of variation (CV) of EDA2R was about 30% higher than for RIF-fd.

Conclusion

Higher radiation-induced EDA2R GE changes and a lower radiation detection level compared to RIF-fd and FDXR GE changes examined under optimal conditions ex vivo on human samples appear promising. Yet, our results represent just the beginning of further studies to be conducted in animal models for further time- and dose-dependent evaluation and additional examinations on radiologically examined patients to evaluate the impact of confounder, such as age, sex, social behavior, or diseases.

Keywords:

• qRT-PCR
• γ-H2AX DSB foci
• low-level radiation exposure
• biomarker
• EDA2R
• FDXR

Acknowledgments

We greatly appreciate Christian Siebenwirth for his advice on the handling of our institute’s X-ray tube in the low-dose range.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary Materials.

Additional information

Funding

The German Ministry of Defense funded the work.

Notes on contributors

Simone Schüle

Simone Schüle, MD, is a Post-Doctoral Researcher of Radiobiology and a resident in Radiology at the Bundeswehr Institute of Radiobiology, Munich, Germany.

Carsten Hackenbroch

Carsten Hackenbroch, MD, achieved his habilitation at Ulm University Hospital and is a radiology attending at the German Armed Forces Hospital of Ulm, Germany.

Meinrad Beer

Meinrad Beer, MD, is a Professor of Radiology and Head of the Radiology Department of Ulm University Hospital, Germany.

Razan Muhtadi

Razan Muhtadi, M.Sc., has a Master’s Degree in Radiation Biology and is a doctoral student at the Bundeswehr Institute of Radiobiology, Munich, Germany.

Cornelius Hermann

Cornelius Hermann, is a pharmacist and researcher in radiobiology at the Bundeswehr Institute of Radiobiology, Munich, Germany.

Samantha Stewart

Samantha Stewart, M.Sc., has a Master’s Degree in Radiation Biology and is a doctoral student at the Bundeswehr Institute of Radiobiology, Munich, Germany.

Daniel Schwanke

Daniel Schwanke, MD, is a doctoral researcher and a resident in Urology at the Bundeswehr Insitute of Radiobiology, Munich, Germany.

Patrick Ostheim

Patrick Ostheim, MD, is a Post-Doctoral Researcher of Radiobiology and a resident in Radiology at the Bundeswehr Institute of Radiobiology, Munich, Germany.

Matthias Port

Matthias Port, MD, is a Professor of Radiobiology and Internal Medicine and Head of the Bundeswehr Institute of Radiobiology, Munich, Germany.

Harry Scherthan

Harry Scherthan, PhD, is a Professor and Specialist in molecular cytology at the Bundeswehr Institute of Radiobiology, Munich, Germany.

Michael Abend

Michael Abend, MD, is a Professor of Radiobiology and Deputy Head of the Bundeswehr Institute of Radiobiology, Munich, Germany.