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 PGF2α and 5-iPF2α-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.

• Authors: Kumiko Yamamoto, Yoshinori Ikenaka, Takahiro Ichise, Tomoki Bo, Mayumi Ishizuka, Hironobu Yasui, Wakako Hiraoka, Tohru Yamamori, Osamu Inanami
• Journal: Free radical research (Free Radic Res) Vol. 52 Issue 6 Pg. 648-660 (Jun 2018) ISSN: 1029-2470 [Electronic] England
• PMID: 29620489 (Publication Type: Journal Article)

Chemical References

• Arachidonic Acids
• Benzoquinones
• Iron-Sulfur Proteins
• Reactive Oxygen Species
• 8-epi-prostaglandin F2alpha
• semiquinone radicals
• Adenosine Monophosphate
• Adenosine Diphosphate
• Adenosine Triphosphate
• Dinoprost

Topics

• Adenosine Diphosphate (metabolism)
• Adenosine Monophosphate (metabolism)
• Adenosine Triphosphate (metabolism)
• Arachidonic Acids (metabolism)
• Benzoquinones (agonists, metabolism)
• Dinoprost (analogs & derivatives, metabolism)
• Electron Transport (radiation effects)
• Energy Metabolism (radiation effects)
• HeLa Cells
• Humans
• Iron-Sulfur Proteins (metabolism)
• Lipid Peroxidation (radiation effects)
• Membrane Potential, Mitochondrial (radiation effects)
• Mitochondria (metabolism, radiation effects)
• Oxidation-Reduction
• Oxygen Consumption (radiation effects)
• Reactive Oxygen Species (agonists, metabolism)
• Tandem Mass Spectrometry
• X-Rays