Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV, LC/MS/MS and ESR

Abstract

To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron–sulphur (Fe−S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF_2α and 5-iPF_2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe–S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe–S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe–S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe–S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.

Keywords:

• tumour
• ESR
• Fe−S cluster
• ionizing radiation
• mitochondrial ETC
• semiquinone
• tumour

Acknowledgements

We would like to thank Editage (www.editage.jp) for English language editing.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This work was supported, in part, by Grant-in-Aids for Scientific Research from Japan Society for the Promotion of Science (grant Numbers 26461875, 17K10465 (TY), 17H03920 (OI)). The sponsors had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; nor the decision to submit the manuscript for publication.