Extremely low priming doses of X radiation induce an adaptive response for chromosomal inversions in pKZ1 mouse prostate

An adaptive response is a response to a stress such as radiation exposure that results in a lower than expected biological response. We describe an adaptive response to X radiation in mouse prostate using the pKZ1 chromosomal inversion assay. pKZ1 mice were treated with a priming dose of 0.001, 0.01, 1 or 10 mGy followed 4 h later by a 1000-mGy challenge dose. All priming doses caused a similar reduction in inversions compared to the 1000-mGy group, supporting the hypothesis that the adaptive response is the result of an on/off mechanism. The adaptive response was induced by a priming dose of 0.001 mGy, which is three orders of magnitude lower than has been reported previously. The adaptive responses completely protected against the inversions that would have been induced by a single 1000-mGy dose as well as against a proportion of spontaneous background inversions. The distribution of inversions across prostate gland cross sections after priming plus challenge irradiation suggested that adaptive responses were predominantly due to reduced low-dose radiation-induced inversions rather than to reduced high-dose radiation-induced inversions. This study used radiation doses relevant to human exposure.     

DNA double-strand breaks in human lymphocytes after single irradiation by low doses of pulsed X-rays: non-linear dose-response relationship

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. A pulsed mode of irradiation is even more problematic in terms of predicting the radiation-induced response in cells. Thus, the aim of this paper was to study and analyze the effects of dose and frequency of pulsed X-rays on the frequency of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes in vitro. Analysis of radiation-induced gammaH2AX and 53BP1 repair foci was used to assess the DNA damage in these cells. The dose-response curve of radiation-induced foci of both proteins has shown deviations from linearity to a higher effect in the 12-32 mGy dose range and a lower effect at 72 mGy. The dose-response curve was linear at doses higher than 100 mGy. The number of radiation-induced gammaH2AX and 53BP1 foci depended on the frequency of X-ray pulses: the highest effect was registered at 13 pulses per second. Moreover, slower repair kinetics was observed for those foci induced by very low doses with a nonlinear dose-response relationship.     

Persistence of DNA double-strand breaks in normal human cells induced by radiation-induced bystander effect

Our previous study suggested that the DNA double-strand breaks (DSBs) induced by very low X-ray doses are largely due to bystander effects. The aim of this study was to verify whether DSBs created by radiation-induced bystander effects are likely to be repaired. We examined the generation of DSBs in cells by enumeration of phosphorylated ataxia telangiectasia mutated (ATM) foci, which are correlated with DSB repair, in normal human fibroblast cells (MRC-5) after X irradiation at doses ranging from 1 to 1000 mGy. At 24 h after irradiation, 100% (1.2 mGy), 58% (20 mGy), 12% (200 mGy) and 8.5% (1000 mGy) of the initial number of phosphorylated ATM foci were detected. The number of phosphorylated ATM foci in MRC-5 cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; phosphorylated ATM foci were not observed at 5 h (20 mGy) or 24 h (200 mGy) postirradiation. We also counted the number of phosphorylated ATM foci in MRC-5 cells cocultured with MRC-5 cells irradiated with 20 mGy. After 48 h of coculture, 81% of the initial numbers of phosphorylated ATM foci remained. These findings suggest that DSBs induced by the radiation-induced bystander effect persist for long periods, whereas DSBs induced by direct radiation effects are repaired relatively quickly.     

Effect of low-dose radiation on repair of DNA and chromosome damage

In this report results of studies on the effect of different doses of low LET (linear energy transfer) radiations on the unscheduled DNA synthesis (UDS) and DNA polymerase activity as well as the induction of adaptive response in bone marrow cells (BMC) by low dose radiation were presented. It was found that whole-body irradiation (WBI) with X-ray doses above 0.5 Gy caused a dose-dependent depression of both UD5 and DNA polymerase activity, while low dose radiation below 250 mGy could stimulate the DNA repair synthesis and the enzyme activity. WBI of mice with low doses of X-rays in the range of 2-100 mGy at a dose rate of 57.3 mGy per minute induced an adaptive response in the BMC expressed as a reduction of chromosome aberrations following a second exposure to a larger dose (0.65 mGy). It was demonstrated that the magnitude of the adaptive response seemed to be inversely related to the induction dose. The possibility of induction of adaptive response in GO phase of the cell cycle and the possibility of a second induction of the adaptive response were discussed.     

Adaptive response for chromosomal inversions in pKZ1 mouse prostate induced by low doses of X radiation delivered after a high dose

Adaptive responses are induced by stress such as X radiation and result in a lower than expected biological response. Two-dose adaptive response experiments typically involve a low priming dose followed by a subsequent high radiation dose. Here, we used a sensitive in vivo chromosomal inversion assay to demonstrate for the first time an adaptive response when a low dose (0.01-1 mGy) was given several hours after a high 1000-mGy radiation dose. The adaptive responses in this study were of similar magnitude to the two-dose adaptive responses previously observed in this test system when the low dose was given first. A chromosomal inversion adaptive response was also induced by two 1000-mGy doses and when a 1-mGy dose was preceded or followed by a dose of 0.01 mGy, but not by two 4000-mGy doses. This is also the first example of an adaptive response when both doses are low. Our data agree with previous reports of an on-off mechanism of adaptive response. The induction of an adaptive response by a low dose after a high damaging dose provides evidence that the mechanisms underlying radiation adaptive responses are not due to prevention of damage induced by the high dose but to modulation of the cellular response to this damage.     

DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects

Ojima, M., Ban, N. and Kai, M. DNA Double-Strand Breaks Induced by Very Low X-Ray Doses are Largely due to Bystander Effects. Radiat. Res. 170, 365-371 (2008).Phosphorylated ATM immunofluorescence staining was used to investigate the dose-response relationship for the number of DNA double-strand breaks (DSBs) induced in primary normal human fibroblasts irradiated with doses from 1.2 to 200 mGy. The induction of DSBs showed a supralinear dose-response relationship. Radiation-induced bystander effects may explain these findings. To test this hypothesis, the number of DSBs in cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; a supralinear dose-response relationship was not observed. Moreover, the number of DSBs obtained by subtracting the number of phosphorylated ATM foci in lindane-treated cells from the number of phosphorylated ATM foci in untreated cells was proportional to the dose at low doses (1.2-5 mGy) and was saturated at doses from 10-200 mGy. Thus the increase in the number of DSBs in the range of 1.2-5 mGy was largely due to radiation-induced bystander effects, while at doses >10 mGy, the DSBs may be induced mainly by dose-dependent direct radiation effects and partly by dose-independent radiation-induced bystander effects. The findings in our present study provide direct evidence of the dose-response relationship for radiation-induced bystander effects from broad-beam X rays.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.     

Dose responses for adaption to low doses of (60)Co gamma rays and (3)H beta particles in normal human fibroblasts

The dose response for adaption to radiation at low doses was compared in normal human fibroblasts (AG1522) exposed to either (60)Co gamma rays or (3)H beta particles. Cells were grown in culture to confluence and exposed at either 37 degrees C or 0 degrees C to (3)H beta-particle or (60)Co gamma-ray adapting doses ranging from 0.1 mGy to 500 mGy. These cells, and unexposed control cells, were allowed to adapt during a fixed 3-h, 37 degrees C incubation prior to a 4-Gy challenge dose of (60)Co gamma rays. Adaption was assessed by measuring micronucleus frequency in cytokinesis-blocked, binucleate cells. No adaption was detected in cells exposed to (60)Co gamma radiation at 37 degrees C after a dose of 0.1 mGy given at a low dose rate or to 500 mGy given at a high dose rate. However, low-dose-rate exposure (1-3 mGy/min) to any dose between 1 and 500 mGy from either radiation, delivered at either temperature, caused cells to adapt and reduced the micronucleus frequency that resulted from the subsequent 4-Gy exposure. Within this dose range, the magnitude of the reduction was the same, regardless of the dose or radiation type. These results demonstrate that doses as low as (on average) about one track per cell (1 mGy) produce the same maximum adaptive response as do doses that deposit many tracks per cell, and that the two radiations were not different in this regard. Exposure at a temperature where metabolic processes, including DNA repair, were inactive (0 degrees C) did not alter the result, indicating that the adaptive response is not sensitive to changes in the accumulation of DNA damage within this range. The results also show that the RBE for low doses of tritium beta-particle radiation is 1, using adaption as the end point.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.     

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.     

Biological effects and adaptive response from single and repeated computed tomography scans in reticulocytes and bone marrow of C57BL/6 mice

This study investigated the biological effects and adaptive responses induced by single and repeated in vivo computed tomography (CT) scans. We postulated that, through the induction of low-level oxidative stress, repeated low-dose CT scans (20 mGy, 2 days/week, 10 weeks) could protect mice (C57BL/6) from acute effects of high-dose radiation (1 Gy, 2 Gy). The micronucleated reticulocyte (MN-RET) count increased linearly after exposure to single CT scans of doses ranging from 20 to 80 mGy (P = 0.033). Ten weeks of repeated CT scans (total dose 400 mGy) produced a slight reduction in spontaneous MN-RET levels relative to levels in sham CT-scanned mice (P = 0.04). Decreases of nearly 10% in γ-H2AX fluorescence levels were observed in the repeated CT-scanned mice after an in vitro challenge dose of 1 Gy (P = 0.017) and 2 Gy (P = 0.026). Spontaneous apoptosis levels (caspase 3 and 7 activation) were also significantly lower in the repeated CT-scanned mice than the sham CT-scanned mice (P < 0.01). In contrast, mice receiving only a single CT scan showed a 19% elevation in apoptosis (P < 0.02) and a 10% increase in γ-H2AX fluorescence levels after a 2-Gy challenge (P < 0.05) relative to sham CT controls. Overall, repeated CT scans seemed to confer resistance to larger doses in mice, whereas mice exposed to single CT scans exhibited transient genotoxicity, enhanced apoptosis, and characteristics of radiation sensitization.     

Mechanisms of apoptosis induction and cell cycle regulation in irradiated leukemia U937 cells and enhancement by arsenic trioxide

Apoptosis is a common mode of cell death after exposure of tumor cells to radiation and/or chemotherapy. The factors that determine the rate of induction of apoptosis are generally related to the functioning of cell cycle checkpoints. In the present study, we investigated the involvement of several genes in cell cycle redistribution and induction of apoptosis in U937 cells after low and high doses of radiation. Activation of CDC2 was observed after both low and high doses of radiation in U937 cells that underwent apoptosis. Expression of CDK2, CDC2 and cyclin A was induced rapidly in the process of radiation-induced apoptosis. In addition, we investigated the use of a clinically relevant dose of radiation to promote As2O3-induced apoptosis in U937 cells. We found that combining radiation and As2O3 may be a new and more effective means of cancer treatment.     

Ionizing radiation induces delayed hyperrecombination in Mammalian cells

Exposure to ionizing radiation can result in delayed effects that can be detected in the progeny of an irradiated cell multiple generations after the initial exposure. These effects are described under the rubric of radiation-induced genomic instability and encompass multiple genotoxic endpoints. We have developed a green fluorescence protein (GFP)-based assay and demonstrated that ionizing radiation induces genomic instability in human RKO-derived cells and in human hamster hybrid GM10115 cells, manifested as increased homologous recombination (HR). Up to 10% of cells cultured after irradiation produce mixed GFP(+/-) colonies indicative of delayed HR or, in the case of RKO-derived cells, mutation and deletion. Consistent with prior studies, delayed chromosomal instability correlated with delayed reproductive cell death. In contrast, cells displaying delayed HR showed no evidence of delayed reproductive cell death, and there was no correlation between delayed chromosomal instability and delayed HR, indicating that these forms of genome instability arise by distinct mechanisms. Because delayed hyperrecombination can be induced at doses of ionizing radiation that are not associated with significantly reduced cell viability, these data may have important implications for assessment of radiation risk and understanding the mechanisms of radiation carcinogenesis.     

Melanin decreases clastogenic effects of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response

Melanin’s influence on the chromosome aberration frequency induced by radiation in human lymphocytes and mouse bone marrow cells has been studied. We revealed earlier that melanin significantly decreases the frequencies of different radiation-induced mutations in animal germ cells. Melanin protection in somatic cells has been found to be less effective. The melanin effect in somatic cells depends on radiation dose: the lower the damage level, the better the melanin protection. In order to determine the influence of melanin at low radiation doses, the adaptive response was investigated in mouse bone marrow cells in vivo. The level of chromosome aberrations in these cells after fractionated irradiation of 0.2 Gy+1.5 Gy with a 4-h interval was about half that after a single dose of 1.7 Gy. If melanin was injected prior to irradiation, the aberration level decreased by a factor of about two in both cases. This observed result may be due to the potential radioprotective effect of melanin and to the absence of any adaptive response, whereas in the case of melanin application between the priming and challenge doses, the combined effect of the adaptive response as well as melanin protection resulted in a 4-fold decrease of chromosome aberrations. These results allow us to draw the following conclusions: adaptive response can be prevented by a radioprotector such as melanin, and melanin is capable of completely removing low-dose radiation effects. Received: 2 December 1998 / Accepted in revised form: 15 September 1999     

Activation of Bak and Bax through c-abl-protein kinase Cdelta-p38 MAPK signaling in response to ionizing radiation in human non-small cell lung cancer cells

Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCdelta-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cdelta (PKCdelta) or siRNA targeting of PKCdelta attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCdelta activation. Upon stimulation of cells with radiation, PKCdelta was phosphorylated dramatically on tyrosine. c-Abl-PKCdelta complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCdelta and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCdelta-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.     

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects

Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence alpha particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 +/- 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 x 10(-6) in unirradiated controls to 2.3 x 10(-5) (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.     

Activation of the DNA repair system in tissues of mice subjected to chronic gamma irradiation

Mice exposed to gamma-quanta during 47 and 82 days at a dose-rate of 1.3 mGy/h and cumulative doses of 1.45 and 2.54 Gy, respectively, were subsequently subjected to a single acute irradiation with a dose of 20 Gy. Repair of DNA damages induced by the acute exposure was shown to proceed in the brain, pulmonary and splenic tissues of chronically exposed mice more readily than in the tissues of mice not subjected to chronic irradiation. The data obtained indicate that the induced adaptive response activates DNA repair in tissues of mice exposed to long-term low-level radiation.     

Alterations in the rat forebrain apoptosis following exposure to ionizing radiation

Ionizing radiation commonly used in the radiotherapy of brain tumours can cause adverse side effects to surrounding normal brain tissue. The most significant response of adult brain to radiation damage is induction of apoptosis. The adult mammalian subventricular zone (SVZ) of the brain lateral ventricles (LV) and their subsequent lateral ventricular extension, the rostral migratory stream (RMS), is one of the few areas, which retains the ability to generate new neurons and glial cells throughout life. Taking into account the fact, that ionizing radiation is one of the strongest exogenous factors affecting cell proliferation, the aim of the present study was to investigate the occurrence of radiation-induced apoptosis in this neurogenic region. Adult male Wistar rats were investigated 1, 5 or 10 days after single whole-body gamma irradiation with the dose of 3 Gy. Apoptotic cell death was determined by in situ labelling of DNA nick ends (TUNEL) and fluorescence microscopy evaluation of TUNEL-positive cells. Considerable increase of apoptotic TUNEL-positive cells was observed 24 hrs after irradiation in caudal parts of RMS; i.e. in the vertical arm and elbow of RMS. Initial increase was followed by strong reduction of apoptosis in the RMS and by secondary over-accumulation of apoptotic cells in the animals that survived ten days after exposure. Results showed, that the proliferating population of cells, arisen in SVZ are highly sensitive to radiation-induced apoptosis. This observation should have implications for clinical radiotherapy to avoid complications in therapeutic brain irradiation.     

If bystander effects for apoptosis occur in spleen after low-dose irradiation in vivo then the magnitude of the effect falls within the range of normal homeostatic apoptosis

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by five- to sevenfold 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.