Evaluation of the role of mitochondria in the non-targeted effects of ionizing radiation using cybrid cellular models

Silvana Miranda,Jorge Lima,Valdemar Máximo,Anabela G Dias,Marcelo Correia,Paula Boaventura,Paula Soares,Ana Pestana,Joana Nunes

doi:10.1038/s41598-020-63011-w

Abstract

Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied. The mitochondrial role in orchestrating this biological response needs to be further explored. Cybrids (cytoplasmic hybrids) are useful cell models for studying the involvement of mitochondria in cellular processes. In the present study we used cybrid cell lines to investigate the role of mitochondria in the response to radiation exposure. Cybrid cell lines, derived from the osteosarcoma human cell line 143B, harboring, either wild-type mitochondrial DNA (Cy143Bwt), cells with mitochondria with mutated DNA that causes mitochondrial dysfunction (Cy143Bmut), as well as cells without mitochondrial DNA (mtDNA) (143B-Rho0), were irradiated with 0.2 Gy and 2.0 Gy. Evaluation of the non-targeted (or bystander) effects in non-irradiated cells were assessed by using conditioned media from the irradiated cells. DNA double stranded breaks were assessed with the γH2AX assay. Both directly irradiated cells and cells treated with the conditioned media, showed increased DNA damage. The effect of the irradiated cells media was different according to the cell line it derived from: from Cy143Bwt cells irradiated with 0.2 Gy (low dose) and from Cy143Bmut irradiated with 2.0 Gy (high dose) induced highest DNA damage. Notably, media obtained from cells without mtDNA, the143B-Rho0 cell line, produced no effect in DNA damage. These results point to a possible role of mitochondria in the radiation-induced non-targeted effects. Furthermore, it indicates that cybrid models are valuable tools for radiobiological studies.

Highlights

Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied
In line with previous data[34], sequencing analysis confirmed the presence of the A3243T tRNALeu(UUR) mutation in Cy143Bmut cells, with approximately 40% of heteroplasmy, while the Cy143Bwt cells did not show the mutation (Fig. 1A)
Data subjected to two-way ANOVA and posterior Bonferroni test. (C) Schematic representation of the protocol used for evaluation of the direct irradiation (D_IR) effects. (D)

Summary

Introduction

Radiobiology is moving towards a better understanding of the intercellular signaling that occurs upon radiation and how its effects relate to the dose applied. Media obtained from cells without mtDNA, the143B-Rho[0] cell line, produced no effect in DNA damage These results point to a possible role of mitochondria in the radiation-induced non-targeted effects. It indicates that cybrid models are valuable tools for radiobiological studies. A possible mechanism related to these effects would be intercellular signaling mediated by factors released from irradiated cells, which could trigger a response in neighboring cells[11]. Reactive oxygen species (ROS), important signal molecules and key players in cellular homeostasis[17], are another possibility for the signaling transduction[7] as well as oxidized DNA fragments[18] and cell free chromatin, shown to induce a response in non-irradiated cells via the NF-E2 related factor-2 (NRF2)[19]. Le et al verified that cells exposed to IR emit biophotons which incite the release of exosomes on the bystander cells[24]

Methods

Results

Discussion

Conclusion