Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses

Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.     

Metabolomic response of human skin tissue to low dose ionizing radiation

Understanding how human organs respond to ionizing radiation (IR) at a systems biology level and identifying biomarkers for IR exposure at low doses can help provide a scientific basis for establishing radiation protection standards. Little is known regarding the physiological responses to low dose IR at the metabolite level, which represents the end-point of biochemical processes inside cells. Using a full thickness human skin tissue model and GC-MS-based metabolomic analysis, we examined the metabolic perturbations at three time points (3, 24 and 48 h) after exposure to 3, 10 and 200 cGy of X-rays. PLS-DA score plots revealed dose- and time-dependent clustering between sham and irradiated groups. Importantly, delayed metabolic responses were observed at low dose IR. When compared with the high dose at 200 cGy, a comparable number of significantly changed metabolites were detected 48 h after exposure to low doses (3 and 10 cGy) of irradiation. Biochemical pathway analysis showed perturbations to DNA/RNA damage and repair, lipid and energy metabolisms, even at low doses of IR.     

The shape of the dose-response curve for radiation-induced neoplastic transformation in vitro: evidence for an adaptive response against neoplastic transformation at low doses of low-LET radiation.

A dose-response curve for gamma-radiation-induced neoplastic transformation of HeLa x skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with (137)Cs gamma radiation and either plated immediately or held for 24 h at 37 degrees C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2(n), where n is the number of observations (5); i.e., 1/32 congruent with 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of gamma radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.     

Differential expression of oxidative stress and extracellular matrix remodeling genes in low- or high-dose-rate photon-irradiated skin

Changes in the expression of genes implicated in oxidative stress and in extracellular matrix (ECM) remodeling and selected protein expression profiles in mouse skin were examined after exposure to low-dose-rate or high-dose-rate photon irradiation. ICR mice received whole-body γ rays to total doses of 0, 0.25, 0.5 and 1 Gy at dose rates of 50 cGy/h or 50 cGy/min. Skin tissues were harvested for characterization at 4 h after irradiation. For oxidative stress after low-dose-rate exposure, 0.25, 0.5 and 1 Gy significantly altered 27, 23 and 25 genes, respectively, among 84 genes assessed (P < 0.05). At doses as low as 0.25 Gy, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly altered, with changes >2-fold compared to 0 Gy. For an ECM profile, 18-20 out of 84 genes were significantly up- or downregulated after low-dose-rate exposure. After high-dose-rate irradiation, of 84 genes associated with oxidative stress, 16, 22 and 22 genes were significantly affected after 0.25, 0.5 and 1 Gy, respectively. Compared to low-dose-rate radiation, high-dose-rate exposure resulted in different ECM gene expression profiles. The most striking changes after low-dose-rate or high-dose-rate exposure on ECM profiles were on genes encoding matrix metalloproteinases (MMPs), e.g., Mmp2 and Mmp15 for low dose rate and Mmp9 and Mmp11 for high dose rate. Immunostaining for MMP-2 and MMP-9 proteins showed radiation dose rate-dependent differences. These data revealed that exposure to low total doses with low-dose-rate or high-dose-rate photon radiation induced oxidative stress and ECM-associated alterations in gene expression profiles. The expression of many genes was differentially regulated by different total dose and/or dose-rate regimens.     

Genetic efficacy of low doses of ionizing radiation in chronically-irradiated small mammals

Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding reasons this discrepancy the comparative analysis of genetic efficiency of low dose chronic irradiation and the higher doses of acute irradiation was carried out with natural populations of bank vole which inhabited the two sites differing in ground of radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range 0.04-0.68 cGy, the main part of absorbed doses consisted of external radiation of animals exposed to 137Cs gamma-rays. Dose-effect equations for acute irradiation to 137Cs gamma-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, using regression coefficient beta and doubling dose estimation. For chronic exposure the doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. The radiosensitivity of bank vole chromosomes were shown is practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation a higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods.     

Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.     

Low-dose radiation hypersensitivity is associated with p53-dependent apoptosis

Exposure to environmental radiation and the application of new clinical modalities, such as radioimmunotherapy, have heightened the need to understand cellular responses to low dose and low-dose rate ionizing radiation. Many tumor cell lines have been observed to exhibit a hypersensitivity to radiation doses <50 cGy, which manifests as a significant deviation from the clonogenic survival response predicted by a linear-quadratic fit to higher doses. However, the underlying processes for this phenomenon remain unclear. Using a gel microdrop/flow cytometry assay to monitor single cell proliferation at early times postirradiation, we examined the response of human A549 lung carcinoma, T98G glioma, and MCF7 breast carcinoma cell lines exposed to gamma radiation doses from 0 to 200 cGy delivered at 0.18 and 22 cGy/min. The A549 and T98G cells, but not MCF7 cells, showed the marked hypersensitivity at doses <50 cGy. To further characterize the low-dose hypersensitivity, we examined the influence of low-dose radiation on cell cycle status and apoptosis by assays for active caspase-3 and phosphatidylserine translocation (Annexin V binding). We observed that caspase-3 activation and Annexin V binding mirrored the proliferation curves for the cell lines. Furthermore, the low-dose hypersensitivity and Annexin V binding to irradiated A549 and T98G cells were eliminated by treating the cells with pifithrin, an inhibitor of p53. When p53-inactive cell lines (2800T skin fibroblasts and HCT116 colorectal carcinoma cells) were examined for similar patterns, we found that there was no hyperradiosensitivity and apoptosis was not detectable by Annexin V or caspase-3 assays. Our data therefore suggest that low-dose hypersensitivity is associated with p53-dependent apoptosis.     

Multifraction radiation response of mouse lung

The response of mouse lung to repeated doses of 60Co gamma-rays of as low as 115 cGy per fraction was measured using death from pneumonitis between 80 and 120 days after irradiation as the endpoint. A fractionation interval of 3 h was maintained for most regimens but in the longer experiments some 12 h intervals were introduced for logistic reasons. The longest overall duration (for a 43 fraction regimen) was 8 days. The total doses required to produce 50 per cent mortality increased continuously as dose/fraction was decreased, even from 160 to 115 cGy per fraction. Of clinical relevance, the steepness of the isoeffect curve over the dose range 115-500 cGy indicates that the lung shows greater sparing from dose fractionation than is characteristic of more rapidly-responding normal tissues, resembling, in this respect, other more slowly-responding tissues such as spinal cord. The plot of the reciprocal of the LD50 values as a function of dose per fraction was non-linear, suggesting that a linear quadratic dose response model may not be appropriate or that repair of cellular injury in lung is not complete in 3 h, or both.     

Differential responses of stress genes to low dose-rate gamma irradiation

In the past, most mechanistic studies of ionizing radiation response have employed very large doses, then extrapolated the results down to doses relevant to human exposure. It is becoming increasingly apparent, however, that this does not give an accurate or complete picture of the effects of most environmental exposures, which tend to be of low dose and protracted over time. We have initiated direct studies of low dose exposures, and using the relatively responsive ML-1 cell line, have shown that changes in gene expression can be triggered by doses of gamma-rays of 10 cGy and less in human cells. We have now extended these studies to investigate the effects on gene induction of reducing the rate of irradiation. In the ML-1 human myeloid leukemia cell line, we have found that reducing the dose rate over three orders of magnitude results in some protection against the induction of apoptosis, but still causes linear induction of the p53-regulated genes CDKN1A, GADD45A, and MDM2 between 2 and 50 cGy. Reducing the rate of exposure reduces the magnitude of induction of CDKN1A and GADD45A, but not the magnitude or duration of cell cycle delay. In contrast, MDM2 is induced to the same extent regardless of the rate of dose delivery. Microarray analysis has identified additional low dose-rate-inducible genes, and indicates the existence of two general classes of low dose-rate responders in ML-1. One group of genes is induced in a dose rate-dependent fashion, similar to GADD45A and CDKN1A. Functional annotation of this gene cluster indicates a preponderance of genes with known roles in apoptosis regulation. Similarly, a group of genes with dose rate-independent induction, such as seen for MDM2, was also identified. The majority of genes in this group are involved in cell cycle regulation. This apparent differential regulation of stress signaling pathways and outcomes in response to protracted radiation exposure has implications for carcinogenesis and risk assessment, and could not have been predicted from classical high dose studies.     

Adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture.

This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose-response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture. When the single-cell microbeam delivered from 1 to 12 alpha particles through the nuclei of 10% of C3H 10T(1/2) cells, more cells were inactivated than were actually traversed by alpha particles. The magnitude of this bystander effect increased with the number of particles per cell. An adaptive dose of 2 cGy of gamma rays, delivered 6 h beforehand, canceled out about half of the bystander effect produced by the alpha particles.     

Accumulation, activation and interindividual variation of the epidermal TP53 protein in response to ionizing radiation in organ cultured human skin

In this study, we examined effects of low-dose ionizing radiation on organ cultured human foreskin and, in particular, on the epidermis. Diagnostic, therapeutic, natural environmental and incidental exposures to moderate to low doses of radiation are inevitable and, although information on cultured cells continues to accumulate, little is known about the effects of low-dose radiation on human tissues. Our hypothesis is that ex vivo organ cultured foreskin is a simple and reliable model to study the biochemical effects of low-dose radiation exposure on skin. A model such as this will aid in the identification and quantification of low-dose radiation-induced changes in proteins in human skin and may be useful in the development of a precise, non-invasive, and reliable assay of exposure. In this work, several aspects of skin responses to culture conditions and radiation were examined. The responses of epidermal TP53 from organ cultured skin irradiated in medium with and without serum were found to be similar. TP53 levels in organ cultured neonatal foreskin epidermis were then examined for baseline TP53 expression. After an initial increase at 4 h, the TP53 D01 signal returned to low steady-state levels for at least 72 h. Irradiated skin samples from different individuals revealed variations in the TP53 D01 signal. The dose and temporal response of dermis and epidermis to radiation were examined by Western blotting from 0 to 24 h after exposure. After irradiation and incubation, the epidermis was removed and assayed by Western blotting and was found to have increases in the TP53 D01 epitope and the TP53 phosphoserine 15 (TP53-S15p) epitope that reached a maximum at about 3 h. In the epidermis, doses of 1-5 cGy of radiation were detectable with the TP53 D01, and CDKN1A antibodies and doses greater than 10 cGy were detectable with the TP53-S15p antibody. When the dermis was compared to epidermis, it was found that dermis had a smaller response to radiation and more phosphorylated TP53.     

In situ biological dose mapping estimates the radiation burden delivered to 'spared' tissue between synchrotron X-ray microbeam radiotherapy tracks

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise γ-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to 'spared' tissue regions between MRT tracks. Γ-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30-40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies.     

Effects of very low fluences of high-energy protons or iron ions on irradiated and bystander cells

In space, astronauts are exposed to radiation fields consisting of energetic protons and high atomic number, high-energy (HZE) particles at very low dose rates or fluences. Under these conditions, it is likely that, in addition to cells in an astronaut's body being traversed by ionizing radiation particles, unirradiated cells can also receive intercellular bystander signals from irradiated cells. Thus this study was designed to determine the dependence of DNA damage induction on dose at very low fluences of charged particles. Novel techniques to quantify particle fluence have been developed at the NASA Space Radiation Biology Laboratory (NSRL) at Brookhaven National Laboratory (BNL). The approach uses a large ionization chamber to visualize the radiation beam coupled with a scintillation counter to measure fluence. This development has allowed us to irradiate cells with 1 GeV/nucleon protons and iron ions at particle fluences as low as 200 particles/cm(2) and quantify biological responses. Our results show an increased fraction of cells with DNA damage in both the irradiated population and bystander cells sharing medium with irradiated cells after low fluences. The fraction of cells with damage, manifest as micronucleus formation and 53BP1 focus induction, is about 2-fold higher than background at doses as low as ～0.47 mGy iron ions (～0.02 iron ions/cell) or ～70 μGy protons (～2 protons/cell). In the irradiated population, irrespective of radiation type, the fraction of damaged cells is constant from the lowest damaging fluence to about 1 cGy, above which the fraction of damaged cells increases with dose. In the bystander population, the level of damage is the same as in the irradiated population up to 1 cGy, but it does not increase above that plateau level with increasing dose. The data suggest that at fluences of high-energy protons or iron ions less than about 5 cGy, the response in irradiated cell populations may be dominated by the bystander response.     

Broad modulation of gene expression in CD4+ lymphocyte subpopulations in response to low doses of ionizing radiation

Gruel, G., Voisin, P., Vaurijoux, A., Roch-Lefèvre, S., Gré goire, E., Maltère, P., Petat, C., Gidrol, X., Voisin, P. and Roy, L. Broad Modulation of Gene Expression in CD4(+) Lymphocyte Subpopulations in Response to Low Doses of Ionizing Radiation. Radiat. Res. 170, 335-344 (2008).To compare the responses of the different lymphocyte subtypes after an exposure of whole blood to low doses of ionizing radiation, we examined variations in gene expression in different lymphocyte subpopulations using microarray technology. Blood samples from five healthy donors were independently exposed to 0 (sham irradiation), 0.05 and 0.5 Gy of ionizing radiation. Three and 24 h after exposure, CD56(+), CD4(+) and CD8(+) cells were negatively isolated. RNA from each set of experimental conditions was competitively hybridized on 25k oligonucleotide microarrays. Modifications of gene expression were measured after both intervals and in all cell types. Twenty-four hours after exposure to 0.5 Gy, we observed an induction of the expression of BAX, PCNA, GADD45, DDB2 and CDKN1A. However, the numbers of modulated genes greatly differed between cell types. In particular, 3 h after exposure to doses as low as 0.05 Gy, the number of down-modulated genes was 10 times greater for CD4(+) cells than for all other cell types. Moreover, most of these repressed genes were taking part in the cell processes of protein biosynthesis and oxidative phosphorylation. The results suggest that several biological pathways in CD4(+) cells could be sensitive to low doses of radiation. Therefore, specifically studying CD4(+) cells could help to understand the mechanisms involved in low-dose response and allow their detection.