Lethal and mutagenic bystander effects in human fibroblast cell cultures subjected to low-energy-carbon ions

Abstract

Purpose: We studied lethal and mutagenic bystander effects in normal human fibroblasts irradiated with low-energy-carbon ions.

Materials and methods: After cells reached confluence, cells were irradiated with initial energies of 6 MeV/n carbon ions. The residual energy and LET value were 4.6 MeV/n and 309 keV/µm. The doses used for survival and mutational studies were 0.082 and 0.16 Gy. Irradiation was carried out using 4 different irradiation conditions and plating conditions: (1) The entire cell area on the Mylar film was irradiated (We abbreviate as ‘all irradiation’); (2) Irradiated and unirradiated cells were pooled in a 1:1 ratio and plated as a single culture until the plating for lethal and mutagenic experiments (We abbreviate as ‘mixed population’); (3) Only half of the area on the Mylar film were irradiated using an ion-beam stopper (We abbreviate as ‘half irradiation’); and (4) Only half of the area of the cells were irradiated, and a specific inhibitor of gap junctions was added to the culture (We abbreviate as ‘half irradiation with inhibitor’). Cell samples were analyzed for lethal and mutagenic bystander effects, including a PCR evaluation of the mutation spectrum.

Results: The surviving fraction of all irradiation was the same as the half irradiation case. The surviving fractions of both mixed population and the half irradiation with inhibitor were the same level and higher than those of all irradiation and half irradiation. The mutation frequencies at the HPRT (the hypoxanthine-guanine phosphoribosyl transferase) locus of all irradiation and half irradiation were at the same level and were higher than those of mixed population and half irradiation with inhibitor, respectively.

Conclusion: There is evidence that the bystander effects for both lethality and mutagenicity occurred in the unirradiated half of the cells, in which only half of the cells were irradiated with the carbon ions. These results suggest that the bystander cellular effects via gap-junction-mediated cell-cell communication are induced by high-LET-carbon ions.

Keywords:

• Low-energy heavy ions
• bystander effects
• gap-junction mediated cell-cell communication
• cell death
• HPRT mutation

Acknowledgements

The authors would like to thank Drs. Teruaki Konishi, Kenichi Matsumoto, Chizuru Tsuruoka and Cuihua Liu of the National Institutes for Quantum and Radiological Science and Technology for their help in performing the irradiation and biological assay, and the staff of HIMAC for the operation of the heavy-ion experiment.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the research project with Heavy Ions at NIRS-HIMAC, Quantum Beam Technology Program of the Ministry of Education, Culture, Sports, Science and Technology and JSPS KAKENHI Grant Numbers JP24620014 and JP15H03598.

Notes on contributors

Masao Suzuki

Masao Suzuki is a researcher in the field of radiation biology. He is a member of Department of Basic Medical Sciences for Radiation Damages at National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science.

Nakahiro Yasuda

Nakahiro Yasuda is a researcher in the field of radiological sciences and radiation regulation. He is a professor of Research Institute of Nuclear Engineering, University of Fukui.

Hisashi Kitamura

Hisashi Kitamura is a researcher in the field of radiological sciences. He is a member of Department of Radiation Measurement and Dose Assessment, at National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science."