РАДИАЦИОННАЯ БИОЛОГИЯ. РАДИОЭКОЛОГИЯ, 2007, том 47, № 3, с. 302-306
PROCEEDINGS OF THE 4TH INTERNATIONAL WORKSHOP ON SPACE RADIATION RESEARCH = AND 17TH ANNUAL NASA SPACE RADIATION HEALTH INVESTIGATORS' WORKSHOP (Moscow-St.-Petersburg, June 5-9, 2006)
УДК 576.32/36
EFFECTS OF HEAVY IONS AND ENERGETIC PROTONS ON NORMAL HUMAN FIBROBLASTS
© 2007 H. Yang, V. Anzenberg, K. D. Held*
Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA
At the low particle fluences of radiation to which astronauts are exposed in space, "non-targeted" effects such as the bystander response may have increased significance. The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. The objectives of this study were to establish the responses of AG01522 diploid human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation. Using a clonogenic survival assay, relative biological effectiveness (RBE) values at 10% survival were 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. For induction of micronuclei (MN), compared to the low LET protons, Fe and Ti are very effective inducers of damage, although C ions are similar to protons. Using a transwell insert system in which irradiated and unirra-diated bystander cells share medium but are not touching each other, it was found that clonogenic survival in unirradiated bystander cells was decreased when irradiated cells were exposed to Fe ions or X-rays. The magnitude of the decrease in bystander survival was similar with both radiation types, reaching a plateau of about 80% survival at doses of about 0.5 Gy or larger.
Heavy ions, energetic protons, RBE, clonogenic survival, micronuclei formation.
In space, astronauts are exposed to a complex and unique radiation environment composed of trapped proton and electron radiation, galactic cosmic rays (GCR) consisting of energetic protons and heavy charged ions, and radiation from the sun, including solar particle events (SPEs), primarily protons. The mixture of the various components of space radiation to which an astronaut could be exposed depends on whether the mission is earth orbit, lunar surface or deep space, as well as factors such as shielding and solar cycle. It has been estimated that on a trip to Mars an astronaut would be exposed to about 250 |Gy d-1 of protons, the equivalent of 0.4 hits per cell nucleus per day, as well as 35 |Gy d-1 a-particles and 3 |Gy d-1 of high mass and energy (HZE) particles [1]. Although these fluences can result in appreciable cumulative doses to the astronauts during long-duration missions, the exposures are at low fluences, such that particle traversals through individual cells in an astronaut's body are well separated in tissue location and time. Under such conditions, "non-targeted" effects, including bystander responses, may have increased significance [2].
Corresponding address: Kathryn D. Held, Department of Radiation Oncology, Cox 302, Massachusetts General Hospital/Harvard Medical School, 55 Fruit Street, Boston, MA 02114 USA; phone: 617-726-8161; fax: 617-724-8320; e-mail: kheld@partners.org.
The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. Although in early studies the bystander effect was demonstrated using low fluences of a-particles [3], it has since been demonstrated also using particle micro-beams (e.g., [4, 5]) and a media-transfer approach (e.g., [6]). Endpoints used in bystander studies have included changes in gene expression, chromosome damage, mutagenesis, cell killing and malignant transformation (for reviews see [7-9]), most of these endpoints being expressions of DNA damage. Although initial studies of the bystander effects were conducted with high LET a-particles [3, 10-12], subsequent studies have also shown its occurrence after low LET y- and X-rays [6, 13, 14], but only a few studies have investigated its occurrence after heavy ions, such as encountered in space [15, 16] (Yang et al. submitted).
The objective of this study was to establish the responses of AG01522 normal human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation.
MATERIALS AND METHODS
Cell Culture. The AG01522 human diploid skin fibroblasts were obtained from the Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, NJ). The cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO2 with a-modified MEM (Sigma, St. Louis, MO) supplemented with 20% fetal bovine serum (Hyclone, Logan, UT), 100 |g/ml streptomycin and 100 units/ml penicillin. One day before irradiation, 4 x 104-1 x 105 and 1.3 x 105 cells from a confluent culture were seeded on glass coverslips in a transwell culture insert dish (Falcon) with a 4.2 cm2 growth area and in a companion well of a 6-well companion plate (Falcon) with a 9.6 cm2 growth area, respectively. The bottom of the insert dish is a membrane with 1.0 |m pores at a density of 1.6 x 106/cm2 to allow the passage of solutes in the medium, but not cells. The distance from the membrane of the insert dish to the bottom of the well of the companion plate is 0.9 mm.
Cell Irradiation, Co-culture and Treatment. Ion irradiation was conducted at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL) with the following ions: Fe (1 GeV/amu; LET of 151 keV/|m), Ti (1 GeV/amu; LET of 108 keV/|m), C (290 MeV/amu; LET of 13 keV/|m) and protons (1 GeV/amu; LET of 0.2 keV/|m). Dose rates were 0.1-2 Gy/min, depending on the dose and the ion species. Cell samples were placed in the plateau region of the Bragg curve and irradiated at room temperature. Dosimetry was performed by NSRL physics staff. As the ion beam at NSRL is horizontal, medium in the six-well plates was aspirated off the cells, using vacuum, immediately before irradiation. X-irradiation was performed at MGH using a Siemens Stabilipan 2 X-ray generator operated at 250 kVp, 20 mA with a dose rate of 2.1 Gy/min. Immediately after irradiation with either ions or X-rays, fresh medium was added to the irradiated cells in the wells, inserts containing unirradiated cells were put into the wells, and the plates were returned to the 37°C incubator. For the ion irradiations, it generally took no more than 5 min for samples to be aspirated, transported to the target room, irradiated and returned to the cell culture room. Control experiments with X-rays showed that there was no difference in cellular response to radiation in the absence or presence of medium during irradiation.
Micronucleus Assay. The frequency of micronuclei (MN) formation was measured using the cytokinesis-block technique [17]. Briefly, after irradiation, cytocha-lasin B (Sigma) was added to the cultures to the final concentration of 1.5 |g/ml. After 72 h, the cells were fixed with methanol: acetic acid (3:1, v/v). After air drying, the cells were stained with 4', 6'-diamidimo-2-phenylindole (DAPI) (Sigma) solution (10 |g/ml) and viewed under a fluorescence microscope. At least 500 binucleate cells in at least 10 view fields were examined.
Fig. 1. Dose response curves for the loss of clonogenic survival in AG01522 human fibroblasts exposed to 1 GeV/amu Fe ions, 1 GeV/amu Ti ions, 1 GeV/amu protons, 290 MeV/amu C ions or 250 kVp X-rays. Data points are means ±standard error from at least 3 independent experiments.
Statistical Analysis. All data are representative of at least 3 independent experiments, and the results are shown as means ± standard error. Comparisons between treatment groups and controls were performed using Student's Z-test of SigmaPlot 2001 software. A p value of <0.05 between groups was considered significant.
RESULTS
A number of investigations have shown that high LET radiations are more effective at causing a decrease in clonogenic cell survival than low LET X-rays (for reviews see [18, 19]). The data in Fig. 1 confirm this in the AG01522 fibroblasts used in this study. At 10% survival, the values for Relative Biological Effectiveness (RBE) are 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. The data in Fig. 2 show that when micronuclei formation is the endpoint, Fe and Ti ions are more effective than protons or C ions at causing damage; at 0.5 Gy, Fe and Ti induce greater than 3-fold more MN than do protons or C ions.
Using the transwell insert system, we have demonstrated previously that AG01522 fibroblasts show a bystander response in the form of decreased clonogenic survival after exposure to X-rays [14]. The primary
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% Binucleate cells with micronuclei
Dose, Gy
Fig. 2. Dose response curves for the induction of micronuclei in AG01522 human fibroblasts exposed to 1 GeV/amu Fe ions, 1 GeV/amu Ti ions, 290 MeV/amu C ions or 1 GeV/amu protons. Data points are means ± standard error from at least 3 independent experiments.
Cell surviving fraction
Dose, Gy
Fig. 3. Induction of a bystander response in the form of loss of clonogenic survival in unirradiated AG01522 fibroblasts in inserts sharing medium with cells irradiated with 1 GeV/amu Fe ions or 250 kVp X-rays. Data points are means ± standard error from at least 3 independent experiments. * - indicates p < 0.05 compared to control sharing medium with unirradiated cells.
goal of this study was an initial investigation of whether Fe ion irradiation can induce a bysta
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