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The Time Dependence of Bystander Responses Induced by Iron-Ion Radiation inNormal Human Skin FibroblastsAuthor(s): Hongying Yang, Vered Anzenberg, Kathryn D. HeldSource: Radiation Research, 168(3):292-298. 2007.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR0864.1URL: http://www.bioone.org/doi/full/10.1667/RR0864.1

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RADIATION RESEARCH 168, 292–298 (2007)0033-7587/07 $15.00� 2007 by Radiation Research Society.All rights of reproduction in any form reserved.

The Time Dependence of Bystander Responses Induced by Iron-IonRadiation in Normal Human Skin Fibroblasts

Hongying Yang,1 Vered Anzenberg and Kathryn D. Held

Department of Radiation Oncology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114

Yang, H., Anzenberg, V. and Held, K. D. The Time Depen-dence of Bystander Responses Induced by Iron-Ion Radiationin Normal Human Skin Fibroblasts. Radiat. Res. 168, 292–298(2007).

Although bystander effects have been shown for some high-LET radiations, few studies have been done on bystander ef-fects induced by heavy-ion radiation. In this study, using aTranswell insert co-culture system, we have demonstratedthat irradiation with 1 GeV/nucleon iron ions can induce me-dium-mediated bystander effects in normal AG01522 humanfibroblasts. When irradiated and unirradiated bystander cellswere combined in shared medium immediately after irradia-tion, a two- to threefold increase in the percentage of bystand-er cells with �-H2AX foci occurred as early as 1 h after ir-radiation and lasted at least 24 h. There was a twofold in-crease in the formation of micronuclei in bystander cells whenthey were co-cultured with irradiated cells immediately or 1or 3 h after irradiation, but there was no bystander effectwhen the cells were co-cultured 6 h or later after irradiation.In addition, bystander micronucleus formation was observedeven when the bystander cells were co-cultured with irradi-ated cells for only 1 h. This indicates that the crucial signalingto bystander cells from irradiated cells occurs shortly afterirradiation. Moreover, both �-H2AX focus formation and mi-cronucleus formation in bystander cells were inhibited by theROS scavengers SOD or catalase or the NO scavenger PTIO.This suggests that ROS and NO play important roles in theinitiation of bystander effects. The results with iron ions weresimilar to those with X rays, suggesting that the bystanderresponses in this system are independent of LET. � 2007 by

Radiation Research Society

INTRODUCTION

Radiation-induced bystander effects, which were report-ed as early as 1922 (1), did not receive much attention untilthe 1990s. Since then, substantial evidence that supportsthe existence of bystander effects has accumulated, show-ing changes in gene expression (2–4), chromosome damage(4–6), mutation (7–9), cell killing (4, 10) and malignant

1 Address for correspondence: Department of Radiation Oncology,Massachusetts General Hospital East, Building 149, Charlestown, MA02129; e-mail: hyang5@partners.org.

transformation (11) in unirradiated cells that either are inproximity to irradiated cells or receive medium from or areco-cultured with irradiated cells. Although the underlyingmechanisms of the bystander effect are not well understood,some evidence suggests that gap junction-mediated inter-cellular communication (5, 12, 13) and/or soluble factorsreleased by irradiated cells such as cytokines (3), reactiveoxygen species (ROS) (2, 4, 14), and nitric oxide (NO) (15,16) are involved in different systems. The conventional par-adigms of radiation biology, such as target theory and thelinear, no-threshold hypothesis, are challenged by the ex-istence of bystander effects. Therefore, even though the im-portance of bystander effects in radiation-induced carcino-genesis and any possible role(s) for bystander effects inradiation therapy are not clear, the effects need to be con-sidered in radiation protection and radiation therapy.

One of the concerns of NASA is the biological effectsof the highly charged, energetic (HZE) particles and pro-tons that are encountered by astronauts in space. In addi-tion, compared to conventional radiation therapy with Xrays or � rays, heavy-ion radiotherapy has several advan-tages such as excellent dose distribution, a smaller depen-dence on oxygen concentration, reduction of cellular repair,less variation in sensitivity with the cell cycle, and highrelative biological effectiveness (17). Charged-particle ther-apy, such as proton therapy and carbon-ion therapy, is beingused to treat patients. Therefore, it is necessary to knowwhether heavy ions cause bystander responses. To date, theexperimental evidence for bystander responses has been ob-tained with both high-LET radiation such as � particles (2,5, 8) and low-LET radiation such as X rays and � rays (4,10, 12, 18). However, there are limited experimental datafor high-LET charged heavy ions (15, 19). In this study,we used a Transwell insert co-culture system to detect by-stander effects induced in normal human skin fibroblasts by1 GeV/nucleon iron-ion radiation (LET of 151 keV/�m).The bystander responses were measured as micronucleus(MN) formation and �-H2AX focus formation. The resultsshow that iron-ions induce medium-mediated bystander ef-fects in human skin fibroblasts, and ROS and NO are in-volved in the bystander signaling. Moreover, it was foundto be crucial to initiate co-culture of the irradiated and un-irradiated cells at an early time after irradiation to induce

293TIME DEPENDENCE OF BYSTANDER EFFECTS

bystander responses. Compared to the pattern of bystanderresponses induced by 250 kVp X rays (LET of 3 keV/�m),the results suggest that the bystander effects in this cellsystem are independent of LET.

MATERIALS AND METHODS

Cell Culture and Co-culture System

The AG01522 human diploid skin fibroblast cells were obtained fromthe Genetic Cell Repository at the Coriell Institute for Medical Research(Camden, NJ). The cells were grown at 37�C in a humidified atmosphereof 95% air and 5% CO2 with alpha-modified MEM (Sigma, St. Louis,MO) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan,UT), 100 �g/ml streptomycin and 100 U/ml penicillin.

A Transwell insert co-culture system was used to study medium-me-diated bystander effects as described previously (4). The bottom of theinsert dish (Falcon) is a membrane with an effective diameter of 23.1mm and with 1.0-�m pores at a density of 1.6 � 106/cm2 to allow thepassage of solutes in the medium but not the cells. The distance from themembrane of the insert dish to the bottom of the well of the companionplate (Falcon) is 0.9 mm. Glass cover slips with a diameter of 18 mm,which do not obviously impede medium diffusion, were used to growcells for the immunofluorescence assay. One day before irradiation, 4 �104–1 � 105 cells, depending on the end point and time, from a confluentculture were seeded on a glass cover slip in an insert dish to be bystandercells, and 1.3 � 105 cells were seeded in a companion well of a six-wellcompanion plate to be irradiated cells. There was no significant differencein the bystander effect when the bystander cell numbers varied from 1� 104 to 8 � 104 for MN formation or from 5 � 104 to 1 � 105 for�-H2AX focus induction. There was also no difference in the bystandereffect if the number of irradiated cells varied from 5 � 104/well to 2 �105/well. However, when the number was decreased to 1 � 104/well or2 � 104/well, the bystander effect was smaller than when the cell numberwas above 5 � 104/well but was still significantly greater than controllevels (data not shown).

Cell Irradiation, Co-culture and Treatment

Iron-ion (1 GeV/nucleon; LET 151 keV/�m) irradiation was conductedat the NASA Space Radiation Laboratory (NSRL) at Brookhaven Na-tional Laboratory (BNL) at dose rates of 0.1–2 Gy/min, depending onthe dose. Samples were placed in the plateau region of the Bragg curveand irradiated at room temperature. Dosimetry was performed by theNSRL physics staff. Since the heavy-ion beam at NSRL is horizontal,medium in the six-well plates was aspirated off the cells immediatelybefore iron-ion irradiation. It generally took no more than 5 min forsamples to be aspirated, transported to the target room, irradiated andreturned to the cell culture room. X irradiation was performed at Mas-sachusetts General Hospital using a Siemens Stabilipan 2 X-ray generatoroperated at 250 kVp, 12 mA with a dose rate of 2.1 Gy/min. Immediatelyafter irradiation with either iron ions or X rays, fresh medium was addedto the irradiated cells in the wells, inserts containing unirradiated cellswere put into the wells, and the plates were returned to the 37�C incu-bator. The irradiated and bystander cells were co-cultured until the timeat which cells were fixed for studies of different end points. The controlsamples were treated in the same way but were not irradiated. In someexperiments, ROS scavengers, Cu-ZnSOD (EMS Biosciences, San Diego,CA) and catalase (EMS Biosciences), or a NO scavenger, 2-(4-carboxy-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO; Molec-ular Probes, Eugene, OR), were added to the co-culture system imme-diately after irradiation to give a final concentration of 500 U/ml, 8 �103 U/ml and 20 �M, respectively.

Micronucleus Assay

The frequency of micronucleus formation was measured using the cy-tokinesis-block technique (20). Briefly, after irradiation, the inserts wereput into companion wells, and cytochalasin B (Sigma) was added to thecultures to a final concentration of 1.5 �g/ml. After 72 h, the cells werefixed with methanol:acetic acid (3:1, v/v). After air drying, the cells werestained with 4�,6�-diamidimo-2-phenylindole (DAPI) (Sigma) solution(10 �g/ml) and viewed under a fluorescence microscope. At least 500binucleate cells in at least 10 fields of view were examined. The cyto-chalasin B treatment resulted in 15–35% binucleate cell formation indirectly irradiated cells, depending on the dose, and about 30% binucleatecells in bystander cells. No significant differences were observed betweenX rays and iron ions.

For studies of the effect of time on MN formation in bystander cells,the unirradiated cells were put into co-culture with irradiated cells atdifferent times after irradiation, then fixed 72 h after the start of co-culture, or the unirradiated cells were put into co-culture with irradiatedcells immediately after irradiation and co-cultured for different times be-fore inserts were removed, and culturing in the absence of irradiated cellswas continued until fixation 72 h after irradiation.

Immunofluorescence for �-H2AX

At appropriate times after co-culture with irradiated cells, bystandercells were fixed in 3% paraformaldehyde for 30 min on ice, washed inPBS for 3 � 10 min, then permeabilized in 0.5% Triton X-100 solutionfor 15 min on ice. Cells were blocked with 10% goat serum for 1 h at37�C, then incubated with anti-phosphorylated histone H2AX antibody(Trevigen, Gaithersburg, MD) for 1 h at room temperature, washed inPBS, blocked again with 1% bovine serum albumen for 1 h at 37�C, andincubated with Alexa Fluor� 488 goat anti-rabbit IgG secondary antibody(Molecular Probes) for 45 min at room temperature. The cell monolayerswere washed at least five times with PBS, stained with 10 �g/ml DAPIfor 2 min, and mounted with FluoroGuard� Antifade reagent (Bio-Rad).At least 500 cells in at least 10 fields were examined. Cells with at leastfive foci were considered positive cells.

Statistical Analysis

All data presented in this paper are representative of at least threeseparate experiments, and the results are shown as means � standarderrors. Comparisons between treatment groups and controls were per-formed using the Student’s t test of SigmaPlot 2001 software. A P valueof �0.05 between groups was considered significant.

RESULTS

Iron Ions Induce Micronucleus Formation in BystanderCells

Our previous results have shown that after co-culturewith X-irradiated cells, the frequency of micronucleus for-mation in bystander AG01522 cells increased about twofoldcompared to the unirradiated controls (4). To investigatewhether heavy-ion radiation also causes bystander micro-nucleus formation, we performed similar experiments using1 GeV/nucleon iron ions. Figure 1 shows the production ofmicronuclei in both directly irradiated and bystander cellsafter exposure to iron ions. The data show that there was a1.4-fold increase in micronuclei in bystander cells exposedto 0.05 Gy iron ions and a twofold increase in cells exposedto 0.1 Gy, but there was no further increase with increasingdose to the irradiated cells up to 2 Gy. In cells directly

294 YANG, ANZENBERG AND HELD

FIG. 1. Dose response for induction of micronuclei in AG01522 cellsirradiated with 1 GeV/nucleon iron ions and unirradiated bystander cellsafter exposure of irradiated cells to iron ions or X rays. X-ray data(dashed line) are reproduced from Yang et al. (4). Results are means ofthree independent experiments � SE.

FIG. 2. Micronucleus induction in bystander AG01522 cells as a func-tion of time of starting co-culture after irradiation. Panel A: Iron ions;panel B: X rays. Results are means of three independent experiments �SE. *P 0.05 compared to the corresponding unirradiated control.

irradiated with iron ions, the levels of micronuclei weresimilar to those in the bystander cells at the lowest doses,but they continued to increase with dose from 0.1–2 Gy.The bystander response induced with iron ions was similarin both the pattern of dose dependence and the magnitudeof the bystander effect to what we have reported previouslyfor X rays (dashed line in Fig. 1) (4).

Time Dependence of Micronucleus Formation inBystander Cells

Two sets of experiments were performed to determinethe time dependence of bystander signaling for micronu-cleus formation in bystander cells. In one experimental set,the bystander cells were co-cultured with irradiated cells atdifferent times after irradiation, then fixed for the micro-nucleus assay 72 h after the start of the co-culture. In thesecond set, the bystander cells were co-cultured with irra-diated cells immediately after irradiation, then separatedfrom the irradiated cells at different times and fixed for themicronucleus assay 72 h after irradiation.

Figure 2A shows the micronucleus induction in bystand-er cells when they were added to the co-cultures at theindicated times after irradiated cells were exposed to ironions. Compared to the corresponding unirradiated controls,MN formation increased about twofold (P � 0.05) in thebystander cells when they were co-cultured with irradiatedcells immediately or 1 or 3 h after irradiation. However,when the bystander cells were co-cultured with irradiatedcells 6 h or later after irradiation, no MN formation wasobserved in bystander cells. This suggests that the signaltransduction from irradiated cells to bystander cells occursa short time after irradiation. Consistent with this, Fig. 3Ashows that co-culturing with iron-ion-irradiated cells im-mediately after irradiation for only 1 h can induce a similarlevel of bystander MN formation to that seen when the co-culturing time is extended to 24 h. To investigate whetherthe time effect of bystander responses is dependent on LET,the same experiments were performed with X rays. Wefound that X rays induced bystander effects of the same

magnitude and with the same time pattern as iron-ion ra-diation (Fig. 2B and 3B). This indicates that induction ofthis bystander effect is independent of LET.

�-H2AX Focus Formation in Bystander Cells afterIron-Ion Irradiation

�-H2AX focus formation, a widely used marker of ion-izing radiation-induced DNA double-strand breaks (DSBs),has been demonstrated in bystander cells after exposure toX rays and � particles by us (4) and others (21). In thepresent studies, we investigated whether high-LET iron-ionradiation caused �-H2AX focus formation in bystandercells through medium-mediated mechanisms. Similar to ourprevious results with X rays, �-H2AX foci were induced inbystander cells by iron-ion radiation (Figs. 4 and 5). Thefraction of �-H2AX-positive bystander cells increased two-to threefold compared with the unirradiated controls; theincrease was independent of dose above 0.1 Gy iron ions.The increase in the fraction of �-H2AX-positive bystandercells was observed as early as 1 h after co-culturing with

295TIME DEPENDENCE OF BYSTANDER EFFECTS

FIG. 3. Micronucleus induction in bystander AG01522 cells as a func-tion of time that irradiated and unirradiated cells were in co-culture. Co-culture was initiated immediately after irradiation. Panel A: Iron ions;panel B: X rays. Results are means of three independent experiments �SE. *P 0.05 compared to the corresponding unirradiated control.

FIG. 4. In situ immunofluorescence detection of �-H2AX foci. PanelA: untreated control cells; original magnification 40�. Panel B: Directlyirradiated cells 1 h after 2 Gy iron-ion irradiation; original magnification40�. Panel C: Directly irradiated cells 24 h after 2 Gy iron ion irradia-tion; original magnification 100�. Panel D: Bystander cells 1 h after 2Gy iron-ion irradiation; original magnification 40�. Panel E: Bystandercells 24 h after 2 Gy iron-ion irradiation; original magnification 40�.Panel F: Bystander cells 24 h after 2 Gy iron-ion irradiation; originalmagnification 100�.

irradiated cells, the earliest time studied, and the increasedlevel of foci persisted at least 24 h after iron-ion irradiation(Figs. 4 and 5A). Comparison of the data in Fig. 5A withthose in Fig. 5B shows that �-H2AX foci in bystander cellsafter X irradiation has a pattern similar to that for iron-ionradiation. This suggests that �-H2AX focus formation inbystander cells is independent of LET. In addition, com-pared to the directly irradiated cells (Fig. 5C), which havea high yield of �-H2AX foci at early times after irradiationand return to control levels by 24 h, bystander cells havea different kinetics of disappearance of �-H2AX foci.

The Involvement of ROS

Since the bystander and irradiated cells in this investi-gation share medium but are not in contact, no gap junctioncommunication exists between them. Hence soluble factorsmust be responsible for the medium-mediated bystander ef-fects. Oxidative stress has been implicated in toxic effectsobserved in bystander cells in which � or -particle radi-ation was used (22–24). We have demonstrated the produc-tion of ROS in directly irradiated and bystander AG01522

cells after X irradiation and a reduction of micronucleusformation and �-H2AX focus formation in bystander cellsby adding the ROS scavengers Cu-ZnSOD and catalase (4).In this study, we examined the effect of the ROS scaven-gers, Cu-ZnSOD and catalase, and the nitric oxide (NO)scavenger c-PTIO on micronucleus and �-H2AX focus for-mation in bystander cells induced by iron-ion radiation.Scavengers were added to the culture immediately after ir-radiation at the time when the bystander and irradiated cellswere combined and remained in the cultures until the sam-ples were fixed either 2 h later to assess �-H2AX foci or72 h to assess micronuclei. Table 1 shows that all threescavengers eliminate the micronucleus induction and�-H2AX focus formation in bystander cells. Therefore,ROS and NO are involved in the iron-ion-induced bystand-er effects.

DISCUSSION

In this study, we found that iron-ion irradiation elicitedmedium-mediated bystander effects such as �-H2AX focusformation and micronucleus induction in normal humanskin fibroblasts (Figs. 1, 4 and 5). Although gap junctionintercellular communication, oxidative stress and solublefactors secreted by irradiated cells have been implicated asmediators of bystander responses, the underlying mecha-nisms are far from clear. One important question is howsoon after irradiation the bystander responses are initiated.An advantage of the insert co-culture system is that it al-lows one to address timing issues easily by altering the timeof addition of the unirradiated bystander cells to the shared

296 YANG, ANZENBERG AND HELD

FIG. 5. �-H2AX focus formation in bystander AG01522 cells as a func-tion of time that irradiated and unirradiated cells were in co-culture (co-culture was initiated immediately after irradiation), and directly irradiatedcells with iron ions or X rays. Panel A: �-H2AX focus formation in bystanderAG01522 cells as a function of time in co-culture with iron ion-irradiatedcells. Panel B: �-H2AX focus formation in bystander AG01522 cells as afunction of time in co-culture with X-irradiated cells. Panel C: �-H2AX focusformation in directly irradiated cells with 0.1 and 2 Gy of iron ions or Xrays. Results are means of three independent experiments � SE. *P 0.05compared to the corresponding unirradiated control.

medium after irradiation or altering the time of removal ofthe bystander cells from the shared medium. Using thisapproach, our results show that chromosomal damage mea-sured by MN formation can be induced in nonirradiatedbystander cells if they are put into co-cultures with irradi-ated cells within 3 h after irradiation. Our data further dem-onstrate that the MN formation in bystander cells can beinduced after they are co-cultured with irradiated cells foras little as 1 h if they are co-cultured with irradiated cellsimmediately after irradiation. This suggests that the signal-ing molecules secreted by irradiated cells at early timesafter irradiation are crucial to the initiation of bystanderresponses.

�-H2AX foci were produced in bystander cells within 1h after being co-cultured with irradiated cells. Figure 5shows that with both iron ions and X rays, the increasedlevel of �-H2AX foci in bystander cells lasts at least 24 h,while the level of �-H2AX foci in directly irradiated cellsis maximal at 1 h, then decreases to basal levels by 24 hafter irradiation. One possible explanation is that differenttypes of DNA lesions are produced in bystander and irra-diated cells, and the lesions are repaired through differentpathways at different rates. While �-H2AX foci are com-monly used as a marker of DNA DSBs in irradiated cells,whether �-H2AX foci in bystander cells represent DSBs iscontroversial. The bystander cells containing �-H2AX focicould be in S phase, and �-H2AX foci are formed at stalledreplication forks (25). However, using PCNA as an S-phasemarker (26), we found that �-H2AX focus induction in by-stander cells was not restricted to PCNA-positive cells (datanot shown), suggesting that bystander �-H2AX focus in-duction is not dependent on S phase in this system. This isin contrast to the observation of Burdak-Rothkamm et al.(27) that �-H2AX focus formation in bystander cells wasrestricted to S-phase cells. The reason for the different re-sults is unclear. However, in agreement with the S-phaseindependence of the bystander response in our system, ourunpublished results also show that �-H2AX foci were in-duced in noncycling confluent bystander cells co-culturedwith irradiated cells. This suggested that perturbation ofreplicating cells is not likely to be involved. Moreover,�-H2AX foci have been found to co-localize with DNADSB repair proteins such as 53BP1, ATM and Mre11 inbystander cells, suggesting that the appearance of �-H2AXfoci in bystander cells indicates the existence of DNADSBs (21). Therefore, the presence of �-H2AX foci mightbe an indicator of DSBs in at least some bystander cells.

If one accepts that appearance of �-H2AX foci indicatesDSBs, iron-ion radiation caused DSBs in bystander cells asearly as 1 h after co-culturing with irradiated cells, and theDSBs were still detectable for at least 24 h (Figs. 4 and 5).This could be due to remaining unrepaired DSBs or to thepersistent generation of DSBs in bystander cells during theco-culture time. Micronuclei have been suggested to arisepredominantly from unrejoined DNA DSBs (28). Hence theunrepaired DSBs in bystander cells may explain the sub-

297TIME DEPENDENCE OF BYSTANDER EFFECTS

TABLE 1Effects of SOD, Catalase and c-PTIO on the Frequency of Micronucleus Induction and �-H2AX Focus

Formation in Bystander Cells after Iron-Ion Irradiation

Dose(Gy)

Binucleate cells with micronuclei (%)

No additive SOD-treated Catalase-treated c-PTIO-treated

�-H2AX-positive cells (%)

No additive SOD-treated Catalase-treated c-PTIO-treated

0 5.9 � 0.6 5.0 � 0.2 5.3 � 0.6 5.8 � 1.0 5.1 � 0.4 5.3 � 0.4 5.4 � 0.3 5.9 � 0.20.1 10.0 � 0.6** 6.3 � 0.2** 6.5 � 0.5** 7.8 � 0.5* 9.4 � 0.9* 5.9 � 0.7* 5.2 � 0.2* 5.8 � 0.1*2.0 10.5 � 0.3** 6.4 � 0.4** 7.2 � 0.2** 7.1 � 1.3* 11.7 � 0.1** 5.5 � 0.1** 5.6 � 0.3** 5.2 � 0.1**

Notes. Results are the means of three independent experiments � SE. * P 0.05, ** P 0.01 compared to the relative control.

stantial increase in the levels of micronucleated cells at latertimes.

The production of ROS has been shown in human cellsafter irradiation (4, 29, 30). Evidence for up-regulation ofoxidative metabolism in bystander cells has also been pub-lished (2, 4, 29). Therefore, the bystander effects might beattributed to the production of factors that lead to genera-tion of ROS in bystander cells. The data from the currentstudy show that iron-ion radiation-induced bystander re-sponses are inhibited by the ROS scavengers SOD or cat-alase or the NO scavenger c-PTIO. This suggests that su-peroxide, hydrogen peroxide and NO are involved in theiron-ion radiation-induced bystander effects. However, su-peroxide, hydrogen peroxide and NO themselves are notvery effective at causing DNA DSBs (31, 32). Thus thebystander �-H2AX and MN formation likely result fromdownstream reactions of , H2O2 and NO, perhaps from•�O2•OH produced by the Fenton reaction at the site of metalsin or near chromatin or from peroxynitrite. Some cytokinessuch as tumor growth factor 1 and interleukin 8, whichcan be produced through NO or ROS signaling (33, 34),have been suggested as candidate signaling factors involvedin bystander responses (35, 36). It is possible that an earlyproduction of ROS/NO in the irradiated cells as an oxida-tive stress response could trigger production of cytokinesthat in turn trigger production of more ROS/NO in the by-stander cells.

To date, investigations of the LET dependence of thebystander effects have been limited. In the current study,using the insert co-culture system, we compared the by-stander responses induced by 1 GeV/nucleon iron ions(LET of 151 keV/�m) and 250 kVp X rays (LET of 3 keV/�m). The data show that iron ions and X rays induce sim-ilar bystander responses in terms of magnitude and timingin AG01522 cells, although iron ions are more effective atinducing direct damage in these cells than X rays. Com-pared to X rays, the RBE for iron ions for 10% clonogenicsurvival in this cell line is about 2.5 (37). Since bystanderresponses are low-dose effects, they may make a consid-erable contribution to the overall radiation effects at lowdoses. In this case, at the same low dose, the relative con-tribution of bystander responses to the overall effect foriron ions could be lower than for X rays if the direct andbystander responses act independently of each other.

The LET-independent bystander response in our system

is in contrast to the results of Shao et al. (15). Using car-bon-ion beams having different LETs, they observed a sig-nificant LET dependence of bystander effects such as cellproliferation and MN induction in neoplastic human sali-vary gland (HSG) cells. High-LET radiation was more ef-fective at inducing bystander effects than low-LET radia-tion. One possible explanation for the discrepancy is that adifferent cell line was used in their study. The medium-mediated bystander responses in HSG cells depended onboth the dose and the LET of the radiation. This was attrib-uted to the dose and LET dependence of the generation ofNO from the irradiated HSG cells, which suggested thatNO played a critical role in inducing bystander effects inHSG cells. However, in AG01522 cells, even though theNO scavenger c-PTIO was found to inhibit the bystanderresponses, other factors such as ROS appeared to be in-volved. Moreover, the bystander effects induced inAG01522 cells are independent of dose above 0.1 Gy, nomatter which radiation type is used. This suggests at leastin part that the underlying mechanisms of bystander effectsin these two cell lines might be different.

In summary, this is the first demonstration that iron-ionradiation produces bystander responses such as �-H2AXfocus formation and MN induction in normal human fibro-blasts. The molecular signals sent to nonirradiated bystand-er cells by irradiated cells at short times after irradiationare critical to the induction of the bystander effects. ROSand NO participate in the signaling pathway for medium-mediated bystander responses. Iron ions and X rays inducebystander responses with similar magnitudes and similartime dependences.

ACKNOWLEDGMENTSThe authors acknowledge the excellent support from all the support

personnel at NSRL and in the Medical and Biology Departments ofBrookhaven National Laboratory. In particular, the authors thank Drs.Adam Rusek, I-Hung Chiang and Michael Sivertz for their invaluablephysics and dosimetry support at NSRL. This research was supported byNASA grant NAG-2-1642. VA received support from NIH training grantT32 CA009078 to the Harvard School of Public Health.

Received: November 20, 2006; accepted: April 10, 2007

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