Heavy iron radiation offer significant advantages for the treatment of deep-seated local tumors in comparison to conventional megavolt photon therapy. For a plethora of malignancies,current radiotherapy using photons or protons yields marginal benefits in local control and survival. In the last decade,heavy-ion radiotherapy facilities have slowly been constructed in Europe and Asia,demonstrating favorable results for many of the malignancies for which conventional radiotherapy yields poor results. However,for a radiobiological perspective,the mechanism whereby heavy ion works in overcoming radio-resistance and the mechanism whereby radiotherapy combined with systemic therapies extends local control and survival are not yet fully understood. Space radiation,comprised of energetic protons and heavy nuclei,has been shown to cause distinct biological damage compared with radiation on earth,leading to large uncertainties in the projection of cancer and other health risks,and obscuring evaluation of the effectiveness of possible countermeasures. Research in the field of biological effects of heavy charged particles is needed for both heavy-ion therapy and protection from the exposure to galactic cosmic radiation in long-term manned space missions. This article reviews the progress in the research of the biological effects of heavy-ion radiation in space exploration and radiotherapy,and the open questions that remain to be addressed.

High-linear energy transfer (LET) heavy-ion radiation is generally characterized by an energy deposition peak (Bragg peak) at the end of its range,which provides good dose localization in critical cancer tissues^[1] and increases relative biological effectiveness (RBE) in killing cells within the peak^[2]. Meanwhile,carbon ion depth-dose distributions exhibit a "tail" of particle fragments created by fragmentation of carbon ions in the primary beam due to nuclear interactions. This tail contains high-LET components and continues for a distance into normal tissues distal to the target volume^[3]. In addition,the Bragg peak is also tissue dependent. In the relative biological effectiveness of carbon uncertainty expanded Bragg peak (approximately 1.5-3.4) can lead to large changes in biological dose in the target volume delivery. Thus,the role of individual susceptibility in therapy and risk of radiation exposure is obviously a major topic in radiation research. 2 Biological effects of heavy ions in space radiation 2.1 Characteristics of space radiation

The space environment is characterized by the presence of microgravity and space radiation. Space radiation consists of galactic cosmic rays,solar particles,and geomagnetically trapped particles. Galactic cosmic rays consisted mainly of protons (90%) but also included a small component of high-energy and high-2 (HZE) particles (α-particles,9%) and heavy ion (1%)^[4],which owing to their high biological effectiveness,contributing a significant fraction of the effective exposure dose received during space missions^[5]. Cucinotta et al^[6] calculated every cell nucleus in an astronaut's body which would be hit by a proton or a secondary electron every few days and by an HZE particle about once a month during a mission to Mars.

Radiation present in the space environment contains many components,including low-dose radiation,low dose-rate radiation,long-term exposure,high-LET particles,and interactions with microgravity. Compared with low-LET radiation X- or γ-ray irradiation,the RBE of heavy-ion radiation is higher and causes a larger amount of DNA damage.

Targeted effects: ionizing radiation directly damages the DNA in the short term. Compared with X- or γ-ray irradiation,high-LET heavy-ion radiation induced a more complex DNA damage (i.e. clusters containing mixtures of two or more various types of damage such as single- and double-strand breakages)^[7] and chromosome exchanges^[8],which led to more severe biological consequences,including cell death,mutation,and transformation. Furthermore,cell distribution in the cell cycle had no significant effects on radio-sensitivity when using heavy-ion radiation. Owing to the cell cycle distribution independence,heavy ions could potentially result in increased cell death of both slowly proliferating tumors and normal tissues,which could decrease therapeutic gain^[9].

Non-targeted effects: a large amount of recent evidence supports the non-targeted effects of exposure to ionizing radiation,for example,the "bystander effect". These non-hit responding cells are thus "bystanders" of either directly hit cells or energy depositions in extracellular medium. Thus,non-irradiation cells are affected by their neighboring irradiation cells through cell-to-cell junctions and by radicals generated by radiation.

The bystander effect should not be neglected in the space environment because heavy ions such asα-particles and carbon-ion beams were demonstrated to induce the bystander effect in chromatin damage through cell-to-cell junctions^[10] and in experiments observing cell proliferation and micronuclei mediated by nitric oxide^{[11, 12]}. In addition,irradiation with high-energy heavy ion can induce chromosomal aberrations in bystander cells,which could go on to manifest genomic instability. Ionizing radiation-induced genomic instability was hypothesized to be a key event in radiation-induced carcinogenesis^[13]. From these studies,heavy ion might induce carcinogenesis by indirect biological effects.

Carcinogenesis: epidemiological studies were limited in subjects exposed to heavy-ion. Increased cancer incidence was not observed in a cohort of astronauts,consistent with the low-dose exposure in low earth orbit,which was experienced so far by crews of the international space station and other space missions. Animal studies reported that the induction of the growth of different tumors in rodents and had been recently reviewed. For instance,Weil et al^[14] found that ²⁸Si or ⁵⁶Fe ion-irradiated mice had a much higher incidence of hepatocellular carcinoma than γ-ray irradiated mice. However,⁵⁶Fe ions did not appear to be substantially more effective than γ-ray irradiation for the induction of acute myeloid leukemia^[15]. Datta et al ^[16] found that heavy-ion radiation exposure triggered higher intestinal tumor frequency. Why are RBE so different? The reason is most likely the difference in nature of liquid and solid cancers. Although leukemia is strongly related to specific chromosomal aberrations,solid cancers are associated to genomic instability. Radiation can act as both an initiator and a promoter of the carcinogenic process. For liquid tumors,radiation could act as an initiator,whereas for solid tumors,it can act as a promoter. Although heavy ions are more effective than X-rays in the induction of chromosomal rearrangements,most of the aberrations are lethal,and the RBE drops to about 1 in the surviving population. Meanwhile,heavy ions were highly effective in the induction of inflammation,which promotes carcinogenesis^{[17, 18]}.

Non-cancer effects: recent epidemiological studies suggested that non-cancer late effects,particularly cardiovascular diseases,might contribute to health risks after exposure from moderate to low doses of ionizing radiation^[19]. The radiobiological mechanism of heavy ion towards these end points and tissues were scarcely known. The major degenerative late effects that could result from exposure to high-energy charged particles were acute and late damages to the central nervous system (CNS),cataract formation,cardiovascular diseases,and other diseases related to accelerated senescence,including digestive and respiratory diseases,infertility,and endocrine and immune system dysfunctions.

CNS complications: including necrosis and leukoencephalopathy,were reported in patients treated for brain tumors by using X-rays or heavy ions. The study showed that radiation-induced injury to normal neurons was much more severe after irradiation with X-rays than after heavy-ion beam irradiation. X-rays produced much more severe radiation-induced injuries to cortical neurons than heavy-ion beams,suggesting that heavy-ion beams has a biological advantage over X-rays in the protection of normal brain tissues^[20]. Apart from cataractogenesis,the uncertainty of other,more harmful,non-cancer late effects,especially cardiovascular diseases,was much higher. Recent clear epidemiological evidence indicated that high radiation doses induce late cardiovascular diseases. A clear correlation between radiation dose to the heart in breast cancer patients and late ischemic heart diseases had been recently shown in a cohort of Swedish and Danish female patients who were treated with radiotherapy for breast cancer^[21]. A-bomb survivors' data also support these results. However,whether this cardiovascular risk has a low-dose threshold is unclear. This is highly important for space exploration. Liu et al^[22] suggested that carbon ion irradiation at 1 Gy significantly suppresses angiogenesis in vitro by inhibiting endothelial cell invasion and tube formation. Generally,the epidemiological evidence of non-cancer late effects in radiation-exposed individuals is consistent with radiation-induced acceleration of the aging process. In fact,heavy-ion irradiation has been shown to accelerate age-related diseases such as endocrine dysfunctions,digestive and respiratory diseases,infertility,and impairment of immune system function^{[23, 24, 25, 26]}.

Complex biological effects: some space experiments suggested interactions between space radiation and microgravity. Abnormal differentiation was observed more frequently in one kind of insects,[QX(Y12#]Carausius morosus[QX)],under microgravity conditions compared with which subjected to one gravity force generated by a centrifuge in space^[27]. Recessive lethal mutations were induced by space radiation at low,supposedly non-effective dose in the progeny of fruit flies which were taken into space^[28]. These space experiments suggested the possibility that microgravity may have elevated the frequency of mutations induced by space radiation. Some possible explanations of the effect of microgravity have been suggested. One suggestion was that a microgravity environment may inhibit the repair of DNA damage induced by space radiation. Another was that metabolic changes induced by microgravity indirectly modify biological processes. For example,microgravity may lead to the accumulation of stress-related proteins that might modify cellular sensitivity to space radiation. This possibility is supported by investigations that observed accumulated heat shock protein 72 or p53 in the muscle and spleen of goldfish,and in the skin and muscle of rats examined after space flight^[29]. 3 The applications of heavy ion in radiotherapy

Statistics showed that patients with cancer at least 60% received radiotherapy during the course of their treatment. The primary principle of radiotherapy lay on the precise localization of sufficient dose in the target lesion while minimizing damage to the surrounding normal tissues. Heavy-ion beams with high LET exhibited more beneficial physical and biological performances than conventional X-rays,thus improving the potential of this type of radiotherapy in the treatment of cancer.

Currently,although local control with heavy ion is generally high,in most malignancies,radiotherapy must be combined with systemic therapies to control metastasis and increase survival. Combined radiotherapy and chemotherapy protocols are already used for many cancers.Barazzuol et al^[30] suggested that the histone deacetylase inhibitor could induce human GBM LN18 cells,which are more sensitive to carbon ions. Poly (ADP-ribose) polymerase-1 or DNA-dependent protein kinase catalytic subunit inhibition also enhanced sensitivity to carbon ions^{[31, 32]}. A recent study showed that heavy-ion irradiation could confer tumor rechallenge resistance,which was dependent on CD8⁺ T cells and influenced by NK cells in this system,showing synergy with dendritic cell treatment approaches^[33]. Furthermore,immunity-related effects were assumed to play a role in the abscopal effect,which was defined as shrinkage of metastatic lesions far from the irradiation field during radiotherapy for a primary malignancy^[34]. Ogata et al^[35] observed that carbon ions significantly reduced lung metastasis in a LM8 osteosarcoma mouse model and a squamous cell carcinoma model in immunocompetent C3H mice. Thus,the abscopal effect was enhanced by heavy-ion combined with immunotherapy. This was an encouraging outcome for future protocols that combines immunotherapy and heavy-ions.

Heavy ions have an advantage over X-rays in overcoming tumor resistance^[36]. Recently,the molecular aspects of tumor biology and its microenvironment have become of interest in investigations regarding radio-resistance to radiation (e.g. cancer stem cells CSCs,tumor microenvironment,and metabolism). CSCs had been shown to be chemoresistant and radioresistant,as compared to their well-differentiated counterparts. The use of heavy ion could overcome the radioresistance of CSCs owing to their higher RBE and increased LET,independent of hypoxia^[37]. However,no standard CSC marker has been established. Thus,if a consensual agreement of CSC identification can be achieved,it can be incorporated into the decision to use carbon ion therapy. In addition,radiotherapy has been focused mainly on killing cancer cells,and little attention has been paid to the process supporting tumor growth and metastasis,including angiogenesis. Liu et al^[22] suggested that carbon ion irradiation at 1 Gy significantly suppressed the process of angiogenesis in vitro by inhibiting endothelial cell invasion and tube formation. Further evidence suggested that heavy-ion radiotherapy might suppress the production of X-ray-induced angiogenesis mediators and thereby increase radio-sensitivity^[38]. In addition,tumor cells used the less-efficient glycolytic pathway for energy production regardless of oxygen tension. Targeting tumor glucose metabolism had been shown to be an effective means of overcoming radioresistance in many tumor histologies^{[39, 40]}.

For many years,studies have concentrated on RBE measurements of heavy ions for different end points,especially cell killing (for radiotherapy) and carcinogenesis (for late effects),achieving remarkable results. However,multiple problems with heavy ions still exist in radiotherapy and deep space exploration,as follows: (1) Epidemiological studies of long-term data regarding toxicity and secondary malignancy of heavy ions in space irradiation are scarce. (2) The maximum permissible dose of space radiation is not defined. (3) The mechanism study of heavy ions on space irradiation was superficial. (4) A significant cost is associated with construction and operation of heavy-ion facilities in radiotherapy. (5) Individual heterogeneity in terms of tumor and normal tissue responses toward heavy-ion irradiation is immense. (6) Representative biomarkers for rapid and sensitive detection of individual radio-sensitivity are required.

Therefore,technical improvement and developments are needed to make heavy-ion therapy less costly and more widely available. Moreover,integration of heavy ions into multimodal treatment with systemic approaches,together with surgery,should be considered in radiotherapy. In addition,the application of systemic research strategy and methods will open a new approach to the translation of the achievement in heavy-ion radiobiology research to the application of radiation medicine in emergency rescue,risk assessment,and radiation exposure protection. Currently,heavy-ion radiobiology research is entering a new phase. If the problems are resolved,it will open new applications for both cancer therapy and protection from radiation exposure in deep space.