According to classical theory of radiation biology,the main target of ionizing radiation damage is believed to be the DNA in the cell nucleus. However,the discovery of so-called non-target effects,such as radiation-induced genomic instability in the progeny of cells survived from irradiation and bystander effects on cells not directly exposed to radiation,has challenged the central dogma of radiation biology in recent years^{[1, 2, 3]}. Previous studies have demonstrated that extranuclear target also plays an important role in mediating the genotoxicity of ionizing radiation^[4],but the details of the cytoplasmic effects on genomic instability in hit cells is largely unexploited,mainly due to the technical difficulties in selectively targeting the cytoplasm without affecting the nucleus. The charged particle microbeam,which can selectively target the cytoplasm of individual cells with a defined number of α particle,provides a useful tool to investigate the incidence and mechanism of genomic instability and other chromosomal damages induced by extranuclear irradiation. By using this delicacy facility,it has been shown that irradiation targeting cytoplasma might result in mitochondrial fragmentation and attenuated respiratory chain function in irradiated human small airway epithelial (SAE) cells,indicating a potential role of mitochondria in mediating radiation damages in mammalian cells^[4].

There are a few reports that radiation hits on cell organelles other than the nucleus,and these extranuclear effects are not the nuclear response to radiation but are the direct subsequence of radiation on other organelles^[5]. Since mitochondria may account for up to 30% of the total cell volume,and mitochondria are the only organelles that contain extranuclear DNA,it is not surprising that ionizing radiation can induce various lesions in the circular mitochondrial DNA,such as strand breaks,base mismatches and large deletions,as observed in nuclear DNA. Therefore,mitochondria are likely to be another major target of ionizing radiation in addition to the nucleus.

As a part of the cell responses to ionizing radiation,alterations in mitochondrial functions have been observed,including variation in energy metabolism,gene expression and subcellular proteomics,increased oxidative stress and apoptosis,and shifted cell signaling^{[6, 7, 8]}. 1 Mitochondrial DNA is prone to radiation damage

Mitochondrial DNA contains only 13 genes coding for the subunits of the electron transport chain enzyme complexes (Complexes Ⅰ,Ⅱ,Ⅲ,and Ⅳ) and the adenosine triphosphate (ATP) synthase. These enzymes are important for respiration,ATP synthesis,and the regulation of many cellular pathways within the cell. Unlike the nuclear DNA,the mitochondrial DNA (mtDNA) lacks histone protection and an efficient DNA repair system,and hence is more prone to oxidative damage leading to genetic defects^{[9, 10]}.

Changes in mitochondrial DNA content can be used as a measurement of cell response to radiation. A normal mitochondrion contains multiple copies of its intact DNA,and a proper mtDNA copy number is important for normal cell function. An increase in mtDNA copy number after radiation is termed as 'mitochondrial polyploidization',which is believed to be a compensatory mechanism or an adaptive response in post-irradiated cells and malignantly transformed progeny that have survived from radiation exposure^{[9, 11]}. It has been reported that there was an average 2-fold increase in mtDNA copy number in the peripheral lymphocytes in leukemia patients 24 h after they received 4.5-9 Gy of total body X-ray irradiation^[12]. Mitochondrial DNA has also been shown to be more susceptible than its nuclear counterpart to ionizing radiation. In an in vitro study,150 Gy γ-ray irradiation on mitochondria isolated from rat livers resulted in a 6-fold higher amount of 8-hydroxydeoxyguanosine per unit mass in mtDNA than nuclear DNA from the same liver. A 2-fold increase in strand breaks could be detected in mitochondria compared to nuclear DNA after γ-ray irradiation. On the other hand,the amounts of lesions repaired in the nuclear and mitochondrial DNA were 80% and 25%,respectively^[13].

The association of the so-called 'common deletion' has been showed a linear increase by irradiation with doses ranged from 0.1 to 10 Gy after 72 h^[14]. A non-linear relationship between the common deletion level and the radiation dose was also observed in HPV-G cells,which showed a higher frequency at 0.005 Gy than that at 5 Gy 96 h after γ-ray irradiation^[15].

The relationship between the frequency of the common deletion and the human cancers induced by nuclear accidents has been also investigated. By comparison of the previous Chernobyl residents with the Japanese control subjects,a greater frequency of mitochondrial deletion was found in radiation-associated post-Chernobyl papillary thyroid carcinoma than in sporadic cases^[16]. Since mitochondrial DNA alterations are frequently found in various human cancers as well as in directly irradiated and in bystander cells,the study of targeting cytoplasma radiation on both mitochondrial and nuclear DNA damage will provide important information on radiation carcinogenesis.

The role of mtDNA in the cellular response to radiation has been studied by using the ρ⁰ cells generated by long-term,low dose treatment of ethidium bromide to remove mitochondrial DNA from a cell line. In a recent study on the role of mitochondrial function in mediating genomic instability induced by high LET radiation,telomerase-immortalized SAE cell lines with or without mtDNA have been used to show an increase in nuclear DNA oxidative damage as well as induction of micronucleus in wild type ρ⁺ SAE cells with cytoplasmic irradiation by high LET particles,concomitant of a significant increase of autophagy reversible at 48 h^[17]. However,the biological effects induced by cytoplasmic irradiation with high LET particles are only minimal in ρ⁰ SAE cells,with no obvious nuclear DNA oxidative damage or micronucleus induction. In addition,the NF-κB/iNOS signaling pathway is activated in ρ⁺ SAE cells but not in ρ⁰ SAE cells. These results clearly explicit the role of mitochondria in modulating DNA damage by high LET radiation delivered to the cytoplasm of mammalian cells^[17]. Nevertheless,since the absence of mitochondria DNA in ρ⁰ cells may induce changes in mitochondrial functions,such as oxygen consumption,mitochondrial membrane potential,nuclear-encoded mitochondrial protein expression,and cell signaling pathways,the parent cells and the ρ⁰ cells may differ in the radiation response. 2 Irradiation alters mitochondrial energy metabolism

The activity of enzymes which comprise of the electron transport chain,namely Complexes Ⅱ,Ⅲ,Ⅳ,and ATP synthase,may be altered upon irradiation. 2 Gy X-ray irradiation to C57BL/6 N mice induced a deactivation of Complex Ⅰ (32%) and Complex Ⅲ (11%),a decreased succinate driven respiratory capacity (13%),and an increased level of ROS^[18]. At much lower dose of 5 to 50 cGy,superoxide and hydrogen peroxide in Chinese hamster lung fibroblast cells were up-regulated with a higher radiosensitivity and a poorer survival rate^[19].

Because these enzymes are involved in electron transport and ATP synthesis,processes associated with OXPHOS are likely to be affected. Indeed,1 h after 8.4 Gy whole body X-ray irradiation,a 20% decrease in rat liver phosphorylation was detected. If the murine liver was directly irradiated with 5 to 20 Gy of γ-ray irradiation,a significant decrease in respiratory rates could be induced as the dose of radiation increased^[20]. 3 Irradiation induces mitochondrial oxidative stress

Ionizing radiation induces both intracellular and mitochondrial oxidative stress. Fluorescent assays have revealed that mitochondria are the major subcellular site of radiation-induced oxidative stress. Mitochondria produce most of the ROS such as superoxide under physiological and abnormal conditions,and this makes them constantly under high oxidative stress. It has been reported that the superoxide anion is a major cause of radiation-induced apoptosis in the macrophages of C3H mice^[21],and an elevation in mitochondrial superoxide radicals has been observed,suggesting that mitochondrion is one of the major sites of ROS production upon irradiation.

Manganese superoxide dismutase is an enzyme resided within mitochondria and is responsible for the dismutation of highly reactive superoxide to water and hydrogen peroxide. Upon irradiation,the activity and protein expression of this enzyme can be activated. On the other hand,overexpression of manganese superoxide dismutase in CHO cells may enhance cell survival after 1 to 11 Gy of γ-ray irradiation,suggesting its radio-protective role by ROS scavenging effect. In addition,transfection with manganese superoxide dismutase makes cells more radio-resistant,as shown by the lower level of apoptosis,mitochondrial ROS production,and membrane lipid peroxidation^[22]. These findings provide insights for future investigations of the potential use of manganese superoxide dismutase in radiation protection.

Signal propagation between mitochondria can amplify the oxidative effects of radiation. Mitochondria directly hit by ionizing radiation are subjected to a transition in their permeability,leading to Ca²⁺ ions release and then being taken up by adjacent mitochondria. Thus,the Ca²⁺ ion could act as a signaling molecule in a chain reaction among the mitochondria after irradiation,resulting in the propagation and amplification of the ROS,which is also a signal in communication between mitochondria. Radiation-induced ROS development and the subsequent mitochondrial permeability transition may spread to adjacent mitochondria mediated via the mitochondrial permeability transition pore in a mitochondrial-potential driven process. This process is named as 'mitochondrial ROS-induced ROS'^[23].

In addition to communication between mitochondrial,signals propagation can also occur between mitochondria and the cell nucleus. By a mitochondrial retrograde signaling pathway,changes in the functional state of mitochondria can be communicated to the cell nucleus to achieve an adaptive cell response to radiation stress. Using the nuclear 53BP1 foci as a marker of DNA damage,it was shown that a significant increase in the focus could be induced 3 h after the cytoplasm of HeLa cells was irradiated. However,such effect could be seen in just 5 min if the nucleus was radiation-targeted^[24].

The radiation-induced mitochondrial ROS has been suggested as the signal to mediate the communication between the mitochondria and the nucleus,as well as the cellular response to radiation exposure. It is well-known that radiation causes an acute,transient increase in intracellular oxidative stress. Recently,a number of studies have reported the persistent increase in oxidative stress after radiation exposure. A delayed elevation of mitochondrial ROS was observed in human fibroblast like cells 72 h after 2 to 6 Gy of γ-ray irradiation,and this elevation remained steady as long as 7 d post-irradiation^[25].

The persistent mitochondrial oxidative stress after the initial exposure is believed to be a result of permanent impairment of mitochondria after ionizing irradiation. As an evidence,the frequency of the common deletion in mtDNA has been increased until 63 d after 0.1 to 2 Gy of γ-ray irradiation in human fibroblast cells. The higher frequency of the common deletion has been accompanied by an increased mitochondrial content and micronuclei formation,and subsequently,nuclear damage,cellular transformation and tumor development may follow as a result. The long-term,persistent damage to the mitochondrial DNA has been defined as 'radiation-induced instability of the mitochondrial genome'^[14].

Along with the genomic instability,mitochondrial dysfunction is also observed in the progenies of the cells that have survived from ionizing radiation exposure,such as increased oxygen consumption,elevated Complex Ⅱ activity,higher intracellular ROS levels and lower manganese superoxide dismutase activity,as well as reduced Complex IV activity and hence a defective respiratory pathway^[26].

These studies suggest that mitochondrial dysfunction is a likely cause of radiation-induced genomic instability,which could propagate to cell progenies/offspring,leading to radiation later effects including cancer. A cohort study is expected to exam the possibility of trans-generational increase in cancer risk due to mitochondrial abnormality subsequent to ionizing radiation to the mother. However,it is still speculated whether ionizing radiation leads to the insertion of mitochondrial DNA fragments into the nuclear genome and whether such nuclear mitochondrial pseudogenes can disrupt the nuclear genome and contribute to an increase in cancer risk in radiation exposed individuals or in their offspring. 4 Mitochondria mediate radiation-induced adaptive response

Accumulating evidence links mitochondrial dynamics and metabolism with cell proliferation and cell cycle regulation,and suggests that mitochondria play an important role in radiation-induced adaptive response via the regulation of mitochondrial metabolism^[27]. Mitochondria affect cell fate by association of glycolysis and the pentose phosphate signaling pathways with cell cycle progression and apoptosis. A few recent studies have reported the role of Cyclin B1/CDK1 in low dose radiation-induced adaptive response^[28]. Via its cytoplasmic,nuclear and centrosomal localization,a subset of Cyclins and CDKs such as Cyclin B1/CDK1 synchronizes the crucial events of early mitosis,nuclear envelope breakdown and centrosome separation. It has been known that cell cycle progression depends on highly ordered events controlled by Cyclin B1/CDK1 complex in regulation of the cell entry into mitosis at the G₂/M border. Mitochondrial localization of Cyclin B1/CDK1 and its role in the integration of mitochondrial fission with the onset of G₂/M transition have been identified^[29],suggesting that Cyclin B1/CDK1 activity is involved in mitochondrial morphological dynamics,mitochondrial bioenergetics,and mitochondria-mediated resistance to ionizing radiation.

A conceptual mechanism is proposed to clarify radiation-induced cell adaptive response via the cell cycle regulator mediated mitochondrial activity. The nuclear events such as radiation-induced nuclear DNA damages and the cell division are linked with mitochondrial regulation,indicating a unique signaling network that appears to enable the mitochondria to sense and respond to radiation-induced DNA damages and repair. Since Cyclin B1/CDK1 is considered as one of the components of the mitochondrial protein influx which is delivered with mitochondrial chaperones to mitochondria and potentially cooperated with the nuclear protein influx,this hypothesis represents a paradigm for understanding nuclear-to-mitochondria and mitochondria-to-nuclear crosstalk in the adaptive response induced by ionizing radiation^[30].

Further investigations are needed to clarify the mechanisms of radiation-induced alterations in mitochondrial functions and their biological significance in basic research and clinical applications. For example,how the mitochondria and the cell nucleus are interacted in response [JP2]to ionizing radiation to induce short- and long-term radiation effects,and how exactly the different nuclear and mitochondrial encoded subunits of the electron transport chain coordinate to alter mitochondrial ROS production and thus contribute to genomic instability. The detailed information resulted from the studies of mitochondrial radiation biology will help to elucidate mechanisms underlying radiation oncogenesis as well as in application of mitochondria as potential targets for radiation protection.