Chao-Ning Zhang, Jin-Tian Li,*

1Traditional Chinese Medicine Clinical College.2Key Laboratory of Ministry of Education for Dunhuang Medicine and Transformation, Gansu University of traditional Chinese Medicine, Lanzhou 730000, China.

Abstract

Keywords:Radiation, Bystander effect, Gap junction intercellular communication,Reactive oxygen species

Introduction

The conventional concept holds that the radiation damage effects such as DNA damage, cell proliferation,and apoptosis, chromosome mutations, and formation of canceret al.come from radiation target effect.That is the damage effects induced by the cells of direct exposure to radiation instead of the cells that are not directly exposed to radiation.However, with the research development of radiation biology, a large number of researches in recent 20 years show that different types of ionizing radiation both have non-target effects namely radiation-induced bystander effect in different cells [1-2].The radiation-induced bystander effect is the phenomenon that the cells which are not directly exposed to radiation have identical or similar biological reactions with the cells of direct exposure to radiation.The biological reactions mainly include abnormal gene expression, gene mutation, DNA damage and repair, chromosome breaking and mutation,cell proliferation and apoptosis, inflammatory response,the transformation of tumor and formation of cancer and so on [3-4].

The radiation-induced bystander effect was already found in early clinical researches.After Parsons and others gave radiotherapy to the children who have chronic granulocytic leukemia in 1954, they found marrow of sternum was damaged [5].Littlefield and others found that there had multiple chromosomal aberrations such as chromosome breakage, chromatids and double centromere fractures in the normal cells which are not irradiated in the plasma of patients receiving high dose radiotherapy in 1969 [6].Demise and others researched the plasma of the Marshall Islanders after radiation exposure and found that the incidence of chromosomal aberrations of the normal lymphocyte culture containing radiated plasma is double compared with the control group [7].

However, until 1992, Nagasawa and his colleagues found that sister-chromatid exchange was induced in 30% to 50% of the other cells which were not exposed to the radiation while only 0.1% to 1% of the Chinese hamster ovary cells were radiated by α-particle [8].It indicated those cells which were not directly exposed to radiation also responded to radiation, and the radiation-induced bystander effect came into being.The bystander effect was just given great importance by the researchers so far.It has become a hotspot and frontier subject in the field of radiation biology and radiation medicine at present.

Study on radiation-induced bystander effect in vitro

The radiation-induced bystander effect is confirmed in the cell experiment in vitro culture.Yokota Y and others studied bystander suicide in human fibroblasts radiated by 60Coγ ray and carbon ion with co-culture and found the generation of nitric oxide was the major determinants of γ-ray and carbon ion radiation-induced bystander effects [9].Turchan WTet al.and others found bystander suicide effect was induced in A549 and MIA PaCa-2 human tumor cells by direct irradiated human endothelial progenitor cells with co-culture and the effect was transmitted by tumor necrosis factor α(TNF-α) and transforming growth factor (TGF-β) [10].Ghosh S and others studied human lung cancer cell A549 radiated by 2Gy60Coγ ray with the combination of medium transfer experiment and co-culture.It showed that the survival rate of the direct irradiation cells and not directly irradiated cells declined and the counts of γ-H2AX and p-ATM foci increased.And that radiation-induced bystander effect was confirmed in different types of cells [11].Tu W and others studied the relation of cell cycle and radiation-induced bystander effect of lung carcinoma A549 cell radiated by carbon ion with medium transfer experiment and found radiation-induced bystander effect was most significant in G(2)-M period [12].Mother [13] and Yang [14]confirmed the objective existence of radiation-induced bystander effect with medium transfer experiment.

However, the study on the radiation-induced bystander effect in vitro has particular limitations.Compared with one in vivo, the study is short of whole signal transduction pathway and effective immune response.Moreover, both of them play a vital role in signal transduction of radiation-induced bystander effect and cancer formation.Radiotherapy is the main treatment means of the malignant tumor.However,radial may damage the normal tissue around the tumor even cause second cancer during the radiotherapy process.So the study of the radiation-induced bystander effect in vivo is of great importance to the mechanism of bystander effect and tumor radiotherapy.

Study on radiation-induced bystander effect in vivo

The study of the radiation-induced bystander effect in vivo is that one organ or a part of the organ of rat or mouse and so on is radiated by ray and the rest of the body is shielded then the organs which are not directly exposed to radiation are observed whether radiation damage occurs or not.

The research results of Khan and his co-workers showed while the lower part of the lungs of SD rats was radiated by 60Coγ ray micronucleus formation was detected in the shielded upper lung tissue.When 70 percent of the lower part of the lung was radiated, DNA damage was more obvious in the upper lung tissue [15].While one side of the body of the mouse was radiated with 1Gy X-ray, DNA methyltransferase activity was detected at a one-centimeter distance of the shielded other side of the body.It indicated that the cause of radiation-induced bystander effect included epigenetic regulation [16].

Except for radiation-induced bystander effect at short range, radiation-induced bystander effect at long-distance in vivo was also found in many studies.Koturbash I found that the head of the rat was radiated by 20 Gy X-ray and epigenetic changes were detected in spleen organ in his further study [17].Mancuso M detected DNA double strands, apoptosis, and formation of cancer in epencephalon of the rats when the rest of their body was radiated, and their heads were shielded with face shield [18].Camphausen Ket al.transplant tumor in the midline of the back of the rats then the legs of the rats were radiated by 10Gy X-ray.They found the growth rate of the tumor was decreased, and the effect was interdicted by the inhibitor of P53 [19].

Radiation-induced bystander effect in vivo is related to sex.Researches showed that the head of the mouse was radiated, the level of apoptosis and microRNA expression in the spleen of the male mouse was lower than that of the female mouse, but the level of cell proliferation was higher than the female mouse.DNA single-strand breaks were even more serious, and it more tended to low DNA methylation level of the global genome.Comparison to the mouse whose gonad was not removed, DNA damage of the spleen of the female mouse whose gonad was removed increased when their head was radiated.While DNA damage of the spleen of the male mouse whose gonad was removed decreased.It indicated that the male mouse was more sensitive to radiation-induced bystander effects than the female mouse.Its mechanism remains further study [20-23].

Time effect of the radiation-induced bystander effect

The time effect of radiation-induced bystander effect has not a unified conclusion yet at present.It is related to the kind and dose of the ray as well as the type of the radiation cell.The study of L?brich M and his colleague showed the numbers of y-H2AX and 53BP1 foci declined to exceed 90 percent within 24 hours after radiation.It indicated that the damaging of DNA double-stranded fracture was repaired within 24 hours[24].Yang and others found 6MV X-ray induced the radiation-induced bystander effect of A549 cell, and the damaging effect was most serious at 72 hours [25].Tomita M found that the signal factors such as NO and NF-κB were detected in bystander cells at 6 hours after radiation [26].Dong and others radiated macrophage U937 with carbon ion and γ-ray, then cultivated U937 and un-irradiated HMy.Micronucleus formation was used to evaluate radiation-induced bystander effects.Moreover, they found the time effect of radiation-induced bystander effects was relative to linear energy transfer (LET) [27].

Research progress of the mechanism of the radiation-induced bystander effect

The role of gap junction intercellular communication (GJIC)

GJIC is a pipeline aggregated with nexin.As intercellular direct contact way, it almost exists in the cell membrane of all animals.Nexin is passively transferred among cells using protein passageway of the adjacent cell membrane and mediates signal transmission of radiation-induced bystander effects.After Azzam E his colleagues and I used the inhibitor of nexin in their study, radiation-induced bystander effects disappeared [28].Azzam [29] and Belchior [30] verified GJIC was the delivery passageway of the damage factors of radiation-induced bystander effects.There also had studies showing that the function of GJIC in radiation-induced bystander effects was related to the kind of the ray and the species of the cell.

Autsavapromporna and others found that GJIC played a transfer role in radiation-induced bystander effects of high LET rays such as carbon ion and silicon ion.For example, the inhibitor of GJIC could reduce the micronucleus formation rate of bystander cells and increase cell viability.However, it could not influence the micronucleus formation rate of the cells exposed to the low linear energy transfer rays.Also, as well it could not affect DNA damage induced by low LET rays[31-32].Desai and others found after dealing with the inhibitor of GJIC A549 cell radiated transferred the damage signal to the cells which were not radiated [33].However, as far as the protective effect transferring between A549 and W138 cell concerned, GJIC does not serve any function at all.

The role of cell medium

Cells which are not in immediate contact can also produce radiation-induced bystander effects.It indicates that except GJIC, there is an indirect pathway that transmits radiation damage.That is mediated transport.The role of cell medium in radiation-induced bystander effects is that the signal factors such as ROS, NO,cyclooxygenase-2 (COX-2), TNF-α and TGF-β1et al.released from radiation cells transmit radiation damage[34]

The role of reactive oxygen species（ROS）

ROS is a kind of metabolic substance containing oxygen that is more vivid than ground state oxygen in terms of chemical property in the biological body.Also,it is the generic terms of several compounds which have high chemical activity and contain oxygen in the molecular organization.It includes superoxide anion,peroxide, and hydroxyl radical.ROS is induced by physical and chemical factors of the outside world, such as ionizing radiation.When ROS is in low concentration, it can activate several signal molecules,for example, mitogen-activated protein kinase and tyrosine kinaseet al.and it is very important to individual survival.While ROS is in high concentration,it can induce apoptosis and cause cell death because it can make protein, lipid, and DNA irreversible damages.It can also cause cell death by regulating calcium ion channels [35-36].

In recent years, lots of studies indicate that ROS is related to radiation-induced bystander effects.Jiang and others find that the signal pathway of TGF-β1-miR-21-ROS plays an important role in the bystander effect of H1299 cell [37].Wang and his colleagues find that boosted ROS plays a dual function in the bystander effect.One is to cause micronucleus and another is to induce protective autophagy [37].Widel M and his colleagues find that the level of ROS of the cell and the bystander cell increases after human dermal fibroblast is radiated by ultraviolet ray [389].Xu W and others test DNA methylation in Arabidopsis thaliana to verify epigenetic alterations caused by radiation-induced bystander effect in plants and find that it can inhibit transcriptional activation of DNA after disposing of with ROS inhibitor dimethyl sulfoxide.It proves that ROS plays a regulating role in the epigenetic variation of radiation-induced bystander effect [39].

The role of nitric oxide（NO）

NO is an important signal factor of the systems of immune, cardiovascular, and nerve and produced by nitric oxide synthase subtypes activating arginine.The effects of NO are diversification.In physiological status,NO is an endothelium-derived relaxing factor and takes part in the blood supply of cell and tissue.In the pathological state, NO is a cytotoxic effector molecule and a unique signal molecule of an oxidation-reduction reaction.Strong oxidant produced by NO can make many important proteins or enzymes inactivate and DNA strand break.It can also destroy the mitochondrion structure and activate lipid peroxidation and induce apoptosis or necrosis during oxidative stress[40].

NO participates in radiation-induced bystander effect.NO can diffuse to the distance of several cells diameter across cytoplasm and plasmalemma because of its relative stability and hydrophobic property.So it can diffuse to the bystander effect cells without GJIC.Human pulmonary fibroblast is radiated by argon ion in a study, and the formation of foci is detected in bystander effect cells.However, it can be inhibited by the scavenger of NO.The death of germ cell induced by NO is found in the bystander effect.Yokota Y and others find that COX2 of the signal pathway of Akt-NF-κB plays an important role in heavy ion induced bystander effect caused by NO [41].The survival rate of the cell decreases radiated by γ ray and carbon ion, and it can be inhibited by scavenger of NO[42].Liu Y and his colleagues find that c-PTIO(scavenger of NO) can alleviate the bystander effects of NSCCs instead of CSCs and it indicates the signal effect of NO in bystander effects is relative to the subtype of the cell [43].When dealing with L-NAME(the inhibition of nitric oxide synthase) or SOD invivo,the DNA damage of the upper lung shielded is inhibited,and it indicates that ROS and NO are the primary reason for bystander effects [15].

The role of cytokines

The function of cytokines such as TGF-β1 is attached to importance from the growing related report of the radiation-induced bystander effect.Cytokines are solubility polypeptide.They play an important role in the process of inflammatory cell signal transduction.Radiation can induce vast cytokines and growth factors,for example, IL-1β, IL-6, and TGF-β1 [44].They come into being as paracrine of intercellular communication and give rise to the adjacent cells.Also, they can also influence cells in the distance with autocrine.So they play an important role in the transmission of damage signal of the radiation-induced bystander effect.The cytokines and some special receptors in the cell surface activate the related pathways.So the pathways induce intracellular signal cascade amplification and affect the role of the target cells [45].

Transforming growth factor β1 (TGF-β1) takes part in the response of radiation-induced bystander effects.Also, it is an important mediator and can increase the level of ROS and the amount of micronucleus.The inhibitor of it can decrease the damage of radiation-induced bystander effects [46].The study indicates that the expression level of TGF-β1and its receptor increase in the non-target tissue after radiation[47].Shao and others found that TGF-β1 is the downstream product of NO and plays an important role in the radiation-induced bystander effect [48].Gow M D and others found that TGF-β1 play a role in high-energy electrons radiation-induced bystander effect [49].TGF-β1 is involved in regulating radiation-induced bystander effects with its downstream signaling pathway.Yin and others found that mutual regulation of the signal pathway of TGF-β1/Smad2 and miR-21 is involved in α particle radiation-induced bystander effect.However, when it is radiated by X-ray radiation-induced bystander effect does not come into being in irradiation cells, because of lack of Smad2 and continuously decreased expression of miR-21.It indicates that the radiation-induced bystander effect mediated by the signal pathway of TGF-β1/Smad2 and miR-21 is related to ray quality [50].In addition to TGF-β1, IL-6, IL-8, and TNF-αare the important regulating factors of radiation-induced bystander effect[51].

TGF-β1 is an important chemotactic factor.It can make alveolar macrophage, neutrophils, and lymphocyte chemotactic and produce and release the cytokines such as IL-6 and IL-1.As a result,inflammation is induced [52].The research of M.A.Brach and others shows that the fibroblast can secrete IL-6 after radiotherapy [53].IL-6 is a multifunctional biological activity peptide.It can make inflammation cells mainly focusing on neutrophil granulocyte aggregate in some regions and produce reactive oxygen and cause tissue inflammation.The activated neutrophil granulocyte can secrete the inflammatory factors such as IL-1 and IL-8 again and form complicated inflammatory network regulation system [54].Radiation can activate mononuclear macrophage and release inflammatory factors such as IL-1 and TNF-α.TNF-α is cytokines belonged to TNF superfamily.It comes from the activated mononuclear macrophage,lymphocyte, cardiac muscle cell, endothelial cells, and fibroblast.It is a multifunctional cytotoxic cytokine.It can cause tissue damage and make the other cells produce some cytokines or inflammatory mediators[55].

The mediators can make tissue cells produce the next level of inflammatory mediators again.They can stimulate mononuclear macrophage and generate more TNF-α.The inflammatory mediators come into the blood circulation and damage vascular endothelial cell directly.They also damage remote organs.

The role of the pathway of mitogen-activated protein kinase (MAPK) and COX-2

Studies indicate there are four signal transduction pathways of MAPK superfamily in the mammal at present, the pathway of the extracellular signal-regulated kinase (ERK1/2), the pathway of c-Jun N-terminal kinase (JNK), the pathway of P38 kinase and ERKS/BMK1.The different pathway is activated by different factors.For example, JNK can be activated by tumor necrosis factor, IL-1, and every stress such as ionizing radiation.The activated JNK is related to apoptosis and necrosis.Also, ERK can be activated by growth factor and cytokines [56].Every pathway is activated and form different transduction way.Different transcription factors are activated and take part in different vital movements.They play an important role in regulating gene expression and inflammatory response.They also play a part in the response of controlling cell growth, proliferation, variation, and apoptosis [57].For example, activated JNK can increase the activity of transcription factor complex AP-1 and up-regulate the expression of pro-apoptotic protein P53.It also can act on mitochondria and make cytochrome C release.It can activate caspase-3 and induce apoptosis[58].

The recent studies indicate that the pathway of MAPK plays an important role in radiation-induced bystander effect.The experiment of Dong shows ERK plays an important part in radiation-induced bystander effect and regulates bystander effect by regulating the signal factors ROS and NO of bystander cell.Every stress, such as ionizing radiation can also induce COX-2.It can mediate inflammation and tumor.Moreover, it plays a critical role in signaling cascade of the radiation-induced bystander effect.It is the key factor of activating the pathway of MAPK [59].Every stress,such as ionizing radiation can also induce COX-2.It can mediate inflammation and tumor.Also, it plays a critical role in signaling cascade of the radiation-induced bystander effect.It is the key factor of activating the pathway of MAPK.The study indicates that when bystander cells were dealing with active inhibitor NS-398 of COX-2，the occurrence rate of the bystander effect is decreased [60].

Existing problems on the studies

The researches of the radiation-induced bystander effect mainly concentrate on biological effect and mechanism now.Its transfer ways involve gap junction intercellular communication and cell medium.However, many studies also indicate more factors take part in radiation-induced bystander effect and play an important role in bystander effect.However, further research on the exact mechanism and signal pathway is needed.

The studies of bystander effect mainly focus on cell system cultured in vitro.Its biological effects are proved with the methods of co-culture and medium transfer test.Also, its mechanism is discussed in quantity.However, the studies in vitro mainly focus on the single cell system, and it is difficult to separate the irradiation cells and un-irradiated cells.There are little relevant studies on multicellular system.Because of the lack of entire immune response and signal transmission environment, the discussion about its mechanism is very limited.

The studies in vivo verify that radiation-induced bystander effect is produced in adjacent tissues as well as the organs in the distance.Its transmission way is similar to one in vitro.Oxygen radical and cytokines play a pivotal role in radiation-induced bystander effect in vivo.However, it is unknown at present how the factors induce damage to the un-target organs.Also, it is a conundrum, whether more other factors join while the factors transfer damage signals.The studies in vivo are also restricted in experimental methods and duplicating model.It is not easy to study in animals and cannot be done directly in the human body.

The time effect on radiation induce bystander effect,and its specific basic mechanism is yet unknown now.The studies on radiation-induced bystander effect are almost all at the cellular level now, and ones in vivo are still needed to strengthen.The radiation-induced bystander effect is a recognized injuries effect.However,with the further research and discovery of adaptive response and rescue effect induced by low dose radiation, it is still needed to verify whether the radiation-induced bystander effect is damage effect or protective effect.

Discussion

Although radiation-induced bystander effect is a weak biological effect, its share of the damage induced by low dose radiation is large.Moreover, it can increase the radiation damage.What，s more, its biological effects are related to the genetic instability and gene mutation.So it makes the risk of cancer caused by low dose radiation higher than the forecast of linear without threshold model.

Except for low dose radiation in the environment,radiotherapy can also cause second cancer and damage the healthy tissues around the tumor.

The objective existence of radiation-induced bystander effect poses a challenge for traditional radiation biology theory and standard of security of radiation protection.It makes radiation protection more complicated.Moreover, it is necessary to consider the radiation-induced bystander effect while surveying risk and randomness of low dose radiation.Also, it is needed to assess the radiotherapy effect and delayed effect again to make specific defense scheme and strategy.

Cosmic radiation mainly consists of heavy ion, γ ray,and proton.Although the proportion of heavy ion is low,its lethality to the cell is far more than low linear energy transfer ray because of its biological properties such as high biological effects, sharp Bragg peak, and high linear energy transferet al.And heavy ion is the ideal ray of tumor radiotherapy nowadays.However, it can damage the healthy tissues around the tumor and induce bystander effects.Its protection is an urgent problem for clinical oncology [61, 62].

Our research group will study heavy ion radiation-induced bystander effect in rats and intervene with Gui Qi Yi Yuan Ointment.Also, the influence on the radiation-induced bystander effect will be detected.