Detection of extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine as a biomarker of oxidative damage in X-irradiated fibroblast cultures: optimization of analytical procedure

Abstract

We have developed a simple methodology, based on single-step solid-phase extraction followed by isocratic high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD), to determine extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in culture supernatants of normal human dermal fibroblasts. A standard addition method, using externally added 8-oxodG (0.5 and 1 pmol) was employed to eliminate matrix effects arising from the chemically complex, protein-rich medium. Secondly, applying this procedure to X-ray irradiated fibroblasts, we report a significant twofold increase in the levels of 8-oxodG at the radiobiologically relevant dose of 6 Gy. This suggests that extracellular 8-oxodG might be a useful biomarker for oxidative stress following moderate doses of X-irradiation.

Keywords::

• 8-oxo-7,8-dihydro-2′-deoxyguanosine
• oxidative stress
• radiobiology
• solid-phase extraction
• culture supernatant
• high-performance liquid chromatography coupled with electrochemical detection

Acknowledgements

This work was supported by the Ligue Nationale Contre le Cancer (Comité du Calvados). We thank the physicians and radiotherapists from the Centre de Lutte Contre le Cancer François Baclesse. Prof. K. Meflah, Director of the same Centre is also greatly acknowledged for his support. We gratefully acknowledge Dr Mark Evans (Dept. Cancer Studies & Molecular Medicine, University of Leicester) for sharing experience on HLB-SPE method. We thank Dr. Anuradha Alahari for help in writing the manuscript.

Declaration of interest

The authors report no declarations of interest.