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British Journal of Radiology (2007) 80, S7-S12
© 2007 British Institute of Radiology
doi: 10.1259/bjr/44550200
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Full paper

The early and initiation processes of radiation-induced bystander effects involved in the induction of DNA double strand breaks in non-irradiated cultures

W Han, PhD L Wu, PhD B Hu, PhD L Zhang, MSc S Chen, MSc L Bao, BSc Y Zhao, BSc A Xu, PhD and Z Yu, BSc

Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031, People's Republic of China

Correspondence: Lijun Wu, PO Box 1126, Hefei, Anhui 230031, PR China. E-mail: ljw{at}ipp.ac.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The initiation and the early process of bystander response induced by low dose {alpha}-particle irradiation are very important for understanding the mechanisms underlying the bystander response. Using a 1 cGy {alpha}-particle to irradiate 50% of the area of a rectangular mylar dish, time-dependent DNA double strand breaks (DSBs) were induced shortly after irradiation in AG1522 cells, located either in the irradiated area or in the non-irradiated bystander area, reaching a maximum 30 min post irradiation. Medium transfer experiments showed that the conditioned medium harvested from the irradiated culture induced excessive DNA DSBs in the medium recipient cells, and the DSB-inducing ability of the medium showed was time-dependent. The medium transfer results indicated that the soluble bystander signalling molecule(s) had been generated very soon (probably less than 2.5 min) after irradiation and exist continuously to 30 min although the production of signalling molecule(s) decreased after 10 min post irradiation. Pre-treatment with dimethyl sulphoxide (DMSO) eliminated the DNA DSB-inducing ability of the conditioned medium, as well as the formation of excessive DNA DSBs in both irradiated and non-irradiated bystander areas, indicating that reactive oxygen/nitrogen species etc. might be involved in these processes.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Radiation-induced bystander effect (RIBE) is defined as the induction of biological effects in cells that are not directly traversed by a charged particle, but are in close proximity to cells that are or that have received signals from these irradiated cells. The first ever report about RIBE was described by Nagasawa and Little [1]. An enhanced frequency of sister chromatid exchanges (SCE) was observed in 20–40% of Chinese hamster ovary cells when the culture was exposed to a low dose of {alpha}-particles such that only 1% of the cells' nuclei were expected to be traversed by a particle track. This has subsequently been confirmed by considerable evidence, showing the existence of RIBE in a variety of cell types of human and rodent origin and the induction of RIBE by many different radiation qualities [reviewed in 2]. It has been shown, for example, that irradiation of 10% of a confluent human hamster hybrid (AL) cell population with a single {alpha}-particle per nucleus results in a mutant yield similar to that observed when all the cells in the population are irradiated [3]. Bystander effects were also observed in non-{alpha}-particle studies. Mothersill and Seymour first demonstrated a highly significant reduction in cloning efficiency in both non-irradiated normal as well as malignant epithelial cell lines that had received media from 60Co-{gamma}-ray irradiated cultures [4]. These results suggested that irradiated cells secreted a cytotoxic factor into the culture medium that was capable of killing non-irradiated cells. Furthermore, transferring media from low linear energy transfer (LET)-irradiated cultures to non-irradiated cells leads to increased levels of various bystander effects, such as cell killing [5–7], neoplastic transformation [8] and genomic instability [9].

DNA double strand breaks (DSBs) are considered to be the most relevant lesion for the deleterious effects of ionizing radiation [10, 11]. The response to a single DSB is highly amplified and rapid (1 min) and initially involves the phosphorylation of ~2000 histone H2AX molecules surrounding the DSB [12, 13]. Recent studies have found that DSBs can be induced by low dose X-rays [14] or irradiation from the decay of 125I incorporated into cellular DNA [15], and have suggested that each {gamma}-H2AX focus represents an individual DSB. In a previous study, we reported that in situ visualization of DSBs could be used to assess the early stage event of irradiation-induced extranuclear/extracellular (bystander) effects [16]. Thus, the visualization of DSBs, which employs {gamma}-H2AX foci formation as a biomarker, might be a new and sensitive approach to assess the early initiation processes of RIBE, and might give some hints to explain how other damage endpoints are induced.

In this paper, we studied the formation of DSBs induced in non-irradiated cells, which were either adjacent to irradiated cells or treated with the culture medium from irradiated cells at a time schedule post irradiation to pursue this response from the moment when irradiation just finished to when this response was saturated. Our results indicate that the bystander response of DSB induction could be initiated rapidly soon after low dose irradiation and the initiation process of RIBE involving the induction of DSBs in non-irradiated cultures. It was also found that, after the irradiation, the irradiated cells secreted the DSB-inducing factor(s) to the medium in a time-dependent manner, and the factor(s) might be associated with reactive oxygen/nitrogen species etc.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture and {alpha}-particle irradiation procedure
AG1522 normal human diploid skin fibroblasts, received as a gift from Dr Barry Michael (Gray Cancer Institute, UK), were maintained in {alpha}-Eagle's minimum essential medium (Gibco, Grand Island, NY) supplemented with 2.0 mM L-glutamine and 20% fetal bovine serum (FBS; Hyclone, Logan, UT) plus 100 µg ml–1 streptomycin and 100 U ml–1 penicillin (Gibco) at 37°C in a humidified 5% CO2 incubator. For irradiation, approximately 1x104 exponentially growing AG1522 cells in passage 12–15 were seeded into each specially designed rectangular dish (internal area 10x6 mm2) consisting of a 3.5 µm thick mylar film bottom onto which cells attached. The culture medium was replaced every 2 days until the cells developed into a confluent monolayer before irradiation. At that time, about 92% of the cells were in G0–G1 as determined by flow cytometry [17].

The average energy of {alpha}-particles derived from the 241Am irradiation source, measured at the cell surface, was 3.5 MeV, and the particles were delivered at a dose rate of 1.0 cGy s–1. To investigate the kinetics of the bystander effects induced in the cells of irradiated and non-irradiated areas, and for analysis of the time-dependent relationships, 50% of the rectangular dish was shielded with 100 µm thickness aluminium below the dish, and cells on the other 50% of the dish were irradiated with a dose of 1 cGy. The dishes were then moved to an incubator for the specified time before fixing and staining to assess the levels of DSB-positive cells. Control dishes went through the same irradiation procedure but were 100% shielded. After irradiation, all dishes were put back into the incubator for the specified time and then fixed for immunochemical staining.

Medium transfer experiments
To confirm whether the irradiated cells secreted the soluble transmissible factor(s) into the medium or whether the factor(s) resulted in the genetic damage in the non-irradiated cells, the medium transfer experiment was adopted in this study. After AG1522 cells in a rectangular dish at a confluent condition were 100% irradiated with 1 cGy {alpha}-particles and incubated for the specified time, the medium from the irradiated population was immediately collected [18, 19] and then transferred to a rectangular dish full of confluent AG1522 cells. Thereafter, the receptor cells were incubated for 30 min with the conditioned medium and then fixed for DSB immunolabelling. Medium from the 100% sham-irradiated dish was transferred to the receptor cells to serve as a control. In some experiments, only medium without cells was irradiated and then the medium was transferred.

Treatment with dimethyl sulphoxide
To determine the role of reactive oxygen species (ROS) in DSB induction in the non-irradiated cells, cells in irradiated and non-irradiated bystander regions were treated with 1% dimethyl sulphoxide (DMSO) 15 min before, during and 30 min after irradiation with 1 cGy {alpha}-particles. For the medium transfer study, the donor cells were treated with 1% DMSO 15 min before, during irradiation and for a specified time before transferring. After treatment, cells were fixed to visualize the DSBs as described below. The dose of DMSO used was effective and has been shown previously to be non-toxic and non-genotoxic to the cells under the conditions used in the present studies [20, 21].

Immunochemical staining of cells ({gamma}-H2AX) and DSB measurement
Immunochemical staining of cells was performed as described previously [22]. Briefly, at specified time after irradiation, the culture plates were removed from the incubator, washed with phosphate-buffered saline (PBS) three times, fixed in a 2% paraformaldehyde solution in PBS for 15 min at room temperature and then rinsed three times with PBS again. Prior to immunochemical staining, cells were incubated for 30 min in TNBS solution (PBS supplemented with 0.1% Triton X-100 and 1% FBS) to improve their permeability and then incubated with anti-{gamma}-H2AX antibody (Upstate Biotechnology, Lake Placid, NY) in PBS+ (PBS supplemented with 1% FBS) for 90 min, washed in TNBS for 3x5 min and incubated for 60 min in PBS+ containing the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (Sigma, St Louis, MO). After another wash with TNBS for 3x5 min, cells were counterstained with Hoechst 33342 (5 µg ml–1 for 20 min at room temperature). After washing again with TNBS, the stained cells were mounted in 50% glycerol–carbonate buffer (pH 9.5) for microscopy.

The rectangular mylar dishes containing the stained cells were placed into one 35 mm diameter glass-bottomed dish (glass thickness 0.17 mm; The Netherlands). Immunofluorescent images of cells in the irradiated and non-irradiated areas were captured with a confocal laser scanning microscope (Leica, TCS SP2 Bensheim, Germany). For quantitative analysis, the cells with {gamma}-H2AX foci were regarded as the positive cells and the fraction of positive cells was calculated (cells with DSBs/total cells) [23–25]. At least 1000 cells were counted in each sample.

Statistical analysis
Statistical analysis was done on the means of the data obtained from at least three independent experiments. Two replicates were counted for each point in each experiment to determine the positive cell yield. All the results were presented as means ± standard deviation (SD). Significance was assessed using Student's t-test at p<0.05 or p<0.01.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The effect of radiation-induced DSBs in irradiated and non-irradiated regions correlated with time
The fraction of induced DSB-positive cells as a function of the post-irradiation incubation time is shown in Figure 1Go. The fraction of induced DSB-positive cells in irradiated and non-irradiated bystander regions is significantly higher than that in sham-irradiated controls at most time points examined (p<0.01 or p<0.05 as indicated) and could be induced as early as 2.5 min after irradiation. Furthermore, by 30 min post irradiation, the incidence of DSBs reaches a maximum for both the directly irradiated and bystander cells. With further increases in time, the fractions of positive cells in both irradiated and non-irradiated bystander regions gradually decreased.


Figure 1
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  		Figure 1. Bystander double strand break(DSB) induction response as a function of time in primary human fibroblasts. The fractions of DSB-positive cells were calculated 2.5, 5, 10, 15, 30, 40 and 60 min after irradiation with 1 cGy of {alpha}-particles. Data were pooled from three or four individual experiments. Error bars represent the standard deviations of the means. The symbols * and ** depict values that are statistically significant (p<0.05 and p<0.01, respectively) between the corresponding control and experimental groups.

 
Figure 1Go also shows that the induction of DSBs at each time point in irradiated region is 8–19% higher than that in the non-irradiated bystander area. Based on dosimetric calculation of 1 cGy {alpha}-particle delivery at the dose rate of 1.0 cGy s–1 and a measured nuclear cross-section of 163±5 µm2 for the fibroblasts, the percentage of cell nuclei estimated to be traversed by an average of one {alpha}-particle in the irradiated region is 9.2%. That is to say, the difference in DSB induction between irradiated and non-irradiated bystander regions is partly due to the percentage of cells hit directly by particles.

The conditioned medium from irradiated culture induced the DSBs in the medium recipient cells
Many investigations [reviewed in 2] have implied that the diffusible factors secreted by irradiated cells would interact with the bystander cells to produce damages. The conditioned medium harvested from the irradiated culture in our study induced a significant excess of DSB-positive cells in the receptor cells, and the DSB-inducing activity (DIA) of the harvested medium in medium receptor cells showed time dependence (Figure 2Go). At 2.5 min post-irradiation time, the distinct DIA of medium was observed and then increased rapidly. By 10 min after irradiation, the incidence of DSBs reached a maximum (30.5–16.4%). With further increases in time, the DIA gradually decreased. At 30 min post irradiation, the DIA was detected to be at a control level (17.6–16.4%, p = 0.83). The irradiated medium without cells did not show distinct DIA in the recipient cells at the time points of 5 min, 10 min, 20 min and 30 min post irradiation.


Figure 2
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  		Figure 2. Different double strand break(DSB)-inducing activity in medium recipient cells as a function of time after irradiation. The conditioned medium harvested from the irradiated culture induced a significant excess of DSB-positive cells and showed time dependence. The irradiated medium without cells did not show notable DSB-inducing activity. Data were pooled from three or four individual experiments. Error bars represent the standard deviations of the means. The symbols * and ** depict values that are statistically significant (p<0.05 and p<0.01, respectively) between the corresponding control and experimental groups.

 
Maximum induction of DSBs among irradiated, non-irradiated bystander regions and medium recipient cells and attenuation of DSB formation by DMSO treatment
As shown above, the maximum DSB induction among irradiated, non-irradiated bystander regions and the medium recipient cells was about 43.9%, 25.2% and 30.5%, respectively. For the irradiated and non-irradiated bystander regions, the highest DSB inductions emerged 30 min post irradiation, while in the medium transfer experiment, the DIA appeared in the medium collected 10 min after 1 cGy {alpha}-particle irradiation (Figure 3Go).


Figure 3
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  		Figure 3. The maximum induction of double strand breaks(DSBs) 30 min after irradiation with 1 cGy of {alpha}-particles in irradiated regions, non-irradiated regions and medium receptor cells. Treatment with or without 1% dimethyl sulphoxide (DMSO) showed that the free radical scavenger, DMSO, effectively inhibited the bystander response of DSB induction for all the groups. Data were pooled from three or four individual experiments. Error bars represent the standard deviations of the means. The symbol * depicts values that are statistically significant (p<0.01) between the DMSO-treated and untreated groups.

 
Pre-treatment of cells with the free radical scavenger DMSO (1%) significantly reduced the fraction of DSB-positive cells in the irradiated areas, non-irradiated bystander areas and the medium recipient cells. In cells irradiated with a 1 cGy dose of {alpha}-particles, treatment with DMSO reduced the number of cells containing {gamma}-H2AX foci in irradiated areas from 43.9% to 23.5% (p<0.05). Similar results were obtained among cells in non-irradiated bystander regions (from 25.3% to 15.6%) and medium recipient cells (from 30.5% to 13.6%), respectively (p<0.05). The decrease in DSB-positive cells after treatment with DMSO suggested that ROS/reactive nitrogen species (RNS) etc. might play important roles in the early initiation processes of the bystander effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Recently, {gamma}-H2AX has been shown to be a fast and reliable marker for assessing the number of DSBs produced in a cell. More importantly, the existence of unrepaired DSBs in a cell might be further correlated with the formation of micronucleus (MN), chromosomal translocations, mutation and apoptosis [26–32]. Rogakou et al [12] indicated that the induction of {gamma}-H2AX occurred by 1 min after irradiation, with maximal amounts reached by 10 min and maintained for 20 min. Consistent with these studies, cells in irradiated areas and non-irradiated bystander areas in our study showed excessive DSB formation after 1 cGy {alpha}-particle irradiation. Both areas showed that DSBs can be induced soon after irradiation (2.5 min), and induction reached a maximum at 30 min, after which the percentage of DSB-positive cells decreased rapidly. These results suggested that DSB formation is an early step, not only for directly irradiated cells but also for radiation-induced bystander cells.

The role of soluble transmissible factor(s) released by irradiated cells that in turn induce effects in non-irradiated cells was supported not only by medium transfer experiments but also by irradiation of subconfluent cultures with {alpha}-particles (reviewed in [2]). It was found that, after irradiation, the target cells secreted soluble factor(s) into the medium, and this resulted in the induction of genetic damage such as sister chromatid exchanges, micronucleus formation, gene mutation and apoptosis in a significantly higher fraction of cells than in those whose nucleus was actually irradiated [33–35]. In our experiments, the medium from the irradiated cells showed a DIA in receptor cells, while the medium irradiated without cells did not. This result indicated that the DSB induction factor(s) is/are not generated from the irradiated medium but from the irradiated cells. Moreover, the percentage of DSB-positive cells in medium recipient cells kept increasing from 2.5 min to 10 min after irradiation and decreased thereafter. That is to say, DSB induction factor(s) can be generated quickly by radiation and do not exist for long in the medium (probably less than 30 min). However, the nature of the DSB induction factor(s) is not clear. It has been shown in many papers that ROS or RNS are very important for the transfer of radiation-induced bystander effects [36, 37]. Narayanan et al [21] demonstrated that directly irradiated (donor) HFL1 cells produced significant amounts of H2O2 and O2– in a dose-dependent manner at 30 min, 3 h and even at 24 h after irradiation. Seymour and colleagues also demonstrated that the signal induced oxidative stress in the non-irradiated cells [7, 38, 39]. In the present studies, the fact that treatment with 1% DMSO effectively inhibited or eliminated the DSB-inducing ability of conditioned medium, as well as the DSB induction in both irradiated and non-irradiated regions, implied that the DSB-inducing factor(s) might be signalling molecule(s) associated with ROS or RNS, such as nitric oxide. Leach et al [40] have demonstrated that irradiation stimulated a transient activation of nitric oxide synthesis with maximal activity at 5 min post irradiation and return to the basal level at 10 min.

Furthermore, although our medium transfer study indicated that the DSB-inducing factor(s) are secreted from the irradiated cells into the medium, and these factors generated the DSBs in the receptor cells, the question is whether these factors could diffuse far enough and then uniformly attack the cells in the non-irradiated bystander region. To be relevant in non-irradiated confluent bystander cells, RIBE should be induced by a complex process, including both gap junctional intercellular communication (GJMC) and ROS/RNS pathways. However, by comparing the maximum induction of DSBs between the non-irradiated bystander region and the medium recipient cells (Figure 3Go), it was shown that there was less DSB induction in non-irradiated bystander cells (25.3–14.6%) than in the medium recipient cells (30.5–16.4%). These results suggested that the cells in the non-irradiated bystander region might be affected by less soluble DSB-inducing factor(s); other signal transmitting modes, for example GJMC, might play an important role in DSB induction.


   Acknowledgments
 
The authors are grateful to Dr Haiying Hang for his technical assistance with DSB detection and Jinesh N Shah (Department of Radiation Oncology, Columbia University) for proofreading to make the language more complete and fluid. This work was funded by the National Nature Science Foundation of China under grant nos. 10225526, 30570435 and 30070192, and grant KSCX2-SW-324 from the Chinese Academy of Sciences.

Received for publication May 12, 2006. Revision received September 26, 2006. Accepted for publication February 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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