Radiological Research Accelerator Facility Research Articles

Effect of medium on chromatin damage in bystander mammalian cells.

In the present study, we examined the potential contribution of irradiated medium to the bystander effect using custom-made double-Mylar stainless steel rings. Exponentially growing human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-Mylar dishes 2-4 days before irradiation. One side (with or without cells) was irradiated with alpha particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facility of Columbia University. Since alpha particles can traverse only a very limited distance (around 23 microm in water), cells plated on the other side of a medium-filled Mylar dish will not be irradiated by the alpha particles. The results of the cytogenetic assay of unirradiated target cells that were attached to the top Mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium-filled chambers than with just medium alone. Furthermore, when the medium was transferred from these cell-irradiated dishes to fresh A(L) cell cultures, chromatid-type aberrations were produced in the unirradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain unidentified modulating factors secreted from the irradiated cells on the bottom Mylar layer into the medium induce chromatin damage in the unirradiated bystander cells.

Proposed laser ion source for the Columbia University microbeam

A laser ion source is scheduled as an upgrade for the 4 MV Van de Graaff accelerator at the Columbia University’s Radiological Research Accelerator Facility (RARAF). The source has been designed for use with the RARAF single-particle single-cell microbeam, though it will be used also for broad-beam irradiations. The operating principle, laser ablation, can produce heavy-ions with high charge states so that their energies will be high enough to provide sufficient range – at least 20 μm – for irradiating cells on a thin surface at atmospheric pressure. The laser ion source being implemented at RARAF consists of three main components: a high-power 100 Hz Nd:YAG laser, a source vacuum chamber and a 24° spherical electrostatic analyzer and a double focusing element with point-to-point focusing in both the horizontal and vertical planes. We expect that the laser ion source will enable us to use ions of sufficient range from hydrogen to iron, providing a range of linear energy transfer from about 10 to 4500 keV/μm.

Laser ion source development for the Columbia University microbeam

A design is given of a laser ion source for the 4.2 MV Van de Graaff accelerator at the Columbia University Radiological Research Accelerator Facility (RARAF). The source has been designed with application in mind for the RARAF single-particle single-cell microbeam, though it will also be used for broad-beam irradiations. The operating principle, laser ablation, can produce heavy ions with high charge states so that their energies will be high enough to provide sufficient range—at least 20 μm—for irradiating cells on a thin surface at atmospheric pressure. The laser ion source being implemented at RARAF is based on the laser operated ion source used by Hughes at the University of Arkansas and consists of three main components: laser generator, source vacuum chamber, and spherical electrostatic analyzer.

The Columbia University single-ion microbeam.

A single-ion microbeam facility has been constructed at the Columbia University Radiological Research Accelerator Facility. The system was designed to deliver defined numbers of helium or hydrogen ions produced by a van de Graaff accelerator, covering a range of LET from 30 to 220 keV/microm, into an area smaller than the nuclei of human cells growing in culture on thin plastic films. The beam is collimated by a pair of laser-drilled apertures that form the beam-line exit. An integrated computer control program locates the cells and positions them for irradiation. We present details of the microbeam facility including descriptions of the collimators, hardware, control program, and the various protocols available. Various contributions to targeting and positioning precision are discussed along with our plans for future developments. Beam time for outside users is often available (see www.raraf.org).

Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells.

Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

Neutron-energy-dependent cell survival and oncogenic transformation.

Both cell lethality and neoplastic transformation were assessed for C3H10T1/2 cells exposed to neutrons with energies from 0.040 to 13.7 MeV. Monoenergetic neutrons with energies from 0.23 to 13.7 MeV and two neutron energy spectra with average energies of 0.040 and 0.070 MeV were produced with a Van de Graaff accelerator at the Radiological Research Accelerator Facility (RARAF) in the Center for Radiological Research of Columbia University. For determination of relative biological effectiveness (RBE), cells were exposed to 250 kVp X rays. With exposures to 250 kVp X rays, both cell survival and radiation-induced oncogenic transformation were curvilinear. Irradiation of cells with neutrons at all energies resulted in linear responses as a function of dose for both biological endpoints. Results indicate a complex relationship between RBEm and neutron energy. For both survival and transformation, RBEm was greatest for cells exposed to 0.35 MeV neutrons. RBEm was significantly less at energies above or below 0.35 MeV. These results are consistent with microdosimetric expectation. These results are also compatible with current assessments of neutron radiation weighting factors for radiation protection purposes. Based on calculations of dose-averaged LET, 0.35 MeV neutrons have the greatest LET and therefore would be expected to be more biologically effective than neutrons of greater or lesser energies.

Mutagenic effects of a single and an exact number of alpha particles in mammalian cells.

One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single alpha particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1- mutants induced in human-hamster hybrid (A(L)) cells by either a single or an exact number of alpha particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1,2,4, or 8 alpha particles at a linear energy transfer of 90 keV/microm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A(L) cells (survival fraction approximately 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10(5) survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single a particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.

The Biological Effectiveness of Radon-Progeny Alpha Particles. V. Comparison of Oncogenic Transformation by Accelerator-Produced Monoenergetic Alpha Particles and by Polyenergetic Alpha Particles from Radon Progeny

Generation of estimates of risk caused by exposure to radon in the home, either from miner data or from A-bomb data, requires several scaling factors such as for dose, dose rate and radiation quality, and possible synergisms. Such scaling factors are best developed from laboratory-based studies. Two possible sources of alpha particles for such studies are (1) a polyenergetic spectrum, generated directly by radon and its progeny, or (2) a series of monoenergetic alpha particles. We compare here the results of oncogenic transformation from studies using both systems. At the Columbia University Radiological Research Accelerator Facility (RARAF), C3H 10T1/2 cells were irradiated with alpha particles of various energies, with defined LETs from 70 to 200 keV/mum. At Pacific Northwest Laboratory, cells from the same stock were exposed to alpha particles from radon gas and its progeny, which were in equilibrium with the culture medium. There was good agreement between the results of oncogenic transformation experiments using the two different exposure systems. Apart from the experimental transformation frequencies themselves, such a comparison requires (1) reliable dosimetry at both facilities and (2) estimated LET distributions for the polyenergetic alpha-particle irradiator. Thus this good agreement gives some confirmation to the technique which is used to fold together oncogenic transformation rates from monoenergetic alpha particles to yield a predicted rate for a spectrum of alpha particles.

Cellular and Molecular Analysis of Mutagenesis Induced by Charged Particles of Defined Linear Energy Transfer

Mutation induction by charged particles of defined linear energy transfer (LET) and gamma rays was scored using human-hamster hybrid AL cells. The LET values for charged particles accelerated at the Radiological Research Accelerator Facility ranged from 10 keV/microm protons to 150 keV/microm 4He ions. The induced mutant fractions at both the S1 and HGPRT loci were dependent on the dose and LET. In addition, for each dose examined, the mutant yield at the S1 locus was 30-60 fold higher than at the corresponding HGPRT locus. To determine whether the mutation spectrum was comparably dependent on dose and LET, independent S1- and HGPRT- mutants induced by 150 keV/microm 4He ions and gamma rays were isolated, and their DNA was analyzed by both Southern blotting and multiplex PCR methods. While the majority of radiation-induced mutants showed deletions of varying sizes, the relative percentage of large deletions was found to be related to both the dose and LET of the radiation examined. Using a mutation system that can detect multilocus changes, results of the present study show that radiation-induced chromosomal loss can be in the millions of base pairs.

Single-particle irradiation of mammalian cells at the radiological research accelerator facility: induction of chromosomal changes

Ionizing radiations have been shown to be able to induce the death of mammalian cells and initiate mutagenic or carcinogenic change. While all three end points are related through chromosomal changes, the latter in particular is of profound concern to human populations. We have undertaken a series of studies wherein mammalian cells were irradiated with low fluences of charged particles (protons, deuterons, helium ions) of defined LET from 10 to about 200 keV per micrometer. Frequencies of induced chromosomal changes were related to fluence at each LET, such that the induction of chromosomal changes per cell per charged particle could be estimated. However, for human exposures from densely ionizing radiation, such as the alpha particles from radon daughters, effects are dominated by the traversal of cells by single particles. Conventional experiments inevitably result in cells being exposed to a distribution (Poisson) of particle traversals. As the effect is unlikely to be a linear function of the number of traversals, a preferred approach would be to irradiate cells with exactly one (or any known number) of particles. To this end we are developing a dedicated beam line (microbeam) on a 4.2 MV Van de Graaff accelerator such that individual particles will vertically traverse individual living mammalian cells positioned by a microscope-based imaging system under computer control. “Conventional” low-fluence and “single-particle” studies will be compared, allowing critical evaluations of the potential of individual high LET charged particles to initiate change. This will have particular relevance both to consideration of the human health risks of radon daughter alpha particles and of basic mechanisms of chromosome aberration formation.

Chromosomal Changes per Cell Nucleus per Charged Particle

The pre-eminent role of the lineal energy density of ionising radiations in eliciting biological effects has been recognised for many years. The pre-eminent role of chromosomal changes in the manifestation of human cancers, of mutational and lethal changes has attained equivalent recognition in recent years. Chromosomal alterations can be observed at the single cell level, and in in vitro systems the effects of defined fluences of charged particles can be related to cellular nuclear cross sectional area and hence changes can be assessed on a per particle per cell nucleus basis. This approach transcends reliance on the concept of absorbed dose. Charged particles generated in the Van de Graaff accelerator of the Radiological Research Accelerator Facility (RARAF) have been used to irradiate mammalian cells attached to Mylar substrates. The linear energy transfer (LET) ranged from 10 to 130 keV.µm-1. All classes of chromosomal changes were assessed as a function of fluence and of LET. When the frequency of 'two-break' aberrations per charged particle per cell nucleus is plotted against LET a quadratic relationship results from 10 to 80 keV.µm-1 with indications of saturation at 130 keV.µm-1. Analysis of the frequencies and types of 'two-break' aberrations in conjunction with spatial chromosomal allocation in cell nuclei enables assessment of proximities for lesion interaction and emphasises the dynamism associated with charged particle effects in mammalian cells. Hazards associated with low fluences, i.e. single or low numbers of high LET particle traversals, as might be expected from radon daughter irradiations, will probably not be uniform over the 80-200 keV.µm-1 LET range and will also probably vary based on cell nucleus presentation to incoming particles.

Chromosomal Changes per Cell Nucleus per Charged Particle

Abstract The pre-eminent role of the lineal energy density of ionising radiations in eliciting biological effects has been recognised for many years. The pre-eminent role of chromosomal changes in the manifestation of human cancers, of mutational and lethal changes has attained equivalent recognition in recent years. Chromosomal alterations can be observed at the single cell level, and in in vitro systems the effects of defined fluences of charged particles can be related to cellular nuclear cross sectional area and hence changes can be assessed on a per particle per cell nucleus basis. This approach transcends reliance on the concept of absorbed dose. Charged particles generated in the Van de Graaff accelerator of the Radiological Research Accelerator Facility (RARAF) have been used to irradiate mammalian cells attached to Mylar substrates. The linear energy transfer (LET) ranged from 10 to 130 keV.µm-1. All classes of chromosomal changes were assessed as a function of fluence and of LET. When the frequency of 'two-break' aberrations per charged particle per cell nucleus is plotted against LET a quadratic relationship results from 10 to 80 keV.µm-1 with indications of saturation at 130 keV.µm-1. Analysis of the frequencies and types of 'two-break' aberrations in conjunction with spatial chromosomal allocation in cell nuclei enables assessment of proximities for lesion interaction and emphasises the dynamism associated with charged particle effects in mammalian cells. Hazards associated with low fluences, i.e. single or low numbers of high LET particle traversals, as might be expected from radon daughter irradiations, will probably not be uniform over the 80-200 keV.µm-1 LET range and will also probably vary based on cell nucleus presentation to incoming particles.

Neutron-Energy-Dependent Oncogenic Transformation of C3H 10% 1/2 Mouse Cells

The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.

Prediction and experimental confirmation of the response function for neutron detection using superheated drops.

Neutrons can be detected with a suspension of superheated drops in an immiscible and compliant holding medium. The mechanism of this radiation-induced bubble nucleation has been studied. A model to predict the minimum neutron energy required to nucleate a vapor bubble in superheated liquids has previously been reported (R.E. Apfel, S. C. Roy, and Y.-C. Lo, Phys. Rev. A 31, 3194 (1985)). Now, the energy-dependent response function for the detector has been calculated based on the model and a detailed consideration of the possible interactions of neutrons with superheated materials. The response of the detector has been measured with monoenergetic neutrons (from 0.025 eV to 14 MeV) at facilities of the National Bureau of Standards and the Radiological Research Accelerator Facility at the Nevis Laboratories of Columbia University. The experimental results agree reasonably well with that of the calculations.

Mutation Induction and Relative Biological Effectiveness of Neutrons in Mammalian Cells: Experimental Observations

The induction of mutation by graded doses of monoenergetic neutrons was examined using the human-hamster hybrid cell system. The AL cells, formed by fusion of human fibroblasts with the gly- A mutant of the Chinese hamster ovary cells, contain the standard set of hamster chromosomes plus a single human chromosome, number 11. These cells contain specific human cell surface antigens that render them sensitive to killing by specific antisera in the presence of complement. Mutant AL cells that have lost the surface markers, however, would survive and give rise to scorable colonies. The cells were irradiated with neutrons produced at the Radiological Research Accelerator Facility of Columbia University. Doses corresponding to low, moderate, and high cytotoxicities and in energies ranging from 0.33 to 14 MeV were used. Neutrons induced a dose-dependent cytotoxicity and mutation frequency in the AL cells. Over the range of doses examined, it was found that the mutagenesis induced by neutrons was energy-dependent and the frequencies were a curvilinear function of dose for both the a1 and a2 antigenic loci examined. In comparison to gamma rays, the relative biological effectiveness (RBE) for cell lethality at the 10% survival level ranged from 5.2 for 0.33 MeV to 1.8 for 14 MeV neutrons. The RBE for mutation induction at the a1 locus, however, ranged from 30 for 0.33 MeV to 4.2 for 14 MeV neutrons at or around the lowest levels of effect examined. Results of the present study demonstrated that neutrons, when measured under conditions which permit detection of a spectrum of gene and chromosomal mutations, in fact, are more efficient mutagens than previously thought.

Mutation induction by charged particles of defined linear energy transfer.

The mutagenic potential of charged particles of defined linear energy transfer (LET) was assessed using the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT) in primary human fibroblasts. Exponentially growing cultures of early passaged fibroblasts were grown as monolayers on thin mylar sheets and were irradiated with accelerated protons, deuterons or helium-3 ions. The mutation rates were compared with those generated by 137Cs gamma-rays. LET values for charged particles accelerated at the Radiological Research Accelerator Facility, using the track segment mode, ranged from 10 to 150 keV/micron. After irradiation, cells were trypsinized, subcultured and assayed for both cytotoxicity and 6-thioguanine resistance. For gamma-rays, and for the charged particles of lower LET, the dose-response curves for cell survival were characterized by a marked initial shoulder, but approximated to an exponential function of dose for higher LETs. Mutation frequencies, likewise, showed a direct correlation to LET over the dose range examined. Relative biological effectiveness (RBE) for mutagenesis, based on the initial slopes of the dose-response curves, ranged from 1.30 for 10 keV/micron protons to 9.40 for 150 keV/micron helium-3 ions. Results of the present studies indicate that high-LET radiations, apart from being efficient inducers of cell lethality, are even more efficient in mutation induction as compared to low-LET ionizing radiation. These data are consistent with results previously obtained with both rodent and human fibroblast cell lines.

Oncogenic transformation by charged particles of defined LET.

Neoplastic transformation incidence and cytotoxicity were scored in the C3H/10T1/2 cell system following irradiation with charged particles of defined linear energy transfer (LET) produced at the Radiological Research Accelerator Facility of Columbia University. Cells cultured in monolayers, attached to thin Mylar sheets, were irradiated with accelerated protons, deuterons or helium-3 ions, and the results compared with X-rays. Defined LET values obtained by using the track segment mode, ranged from 10 to 120 keV/microns. For X-rays, and for the charged particles of lower LET, the dose-response curves for cell survival have a marked initial shoulder. With increasing LET, there is a progressive decrease in the size of the shoulder and for the highest LETs, survival curves approximate an exponential function of dose. The transformation incidence, likewise, showed a direct correlation to LET over the dose range examined. The efficiencies of transformation, however, appeared to approach a plateau between 80 and 120 keV/micron. Transformation data were analyzed using a linear-quadratic function of dose for the transformation probability. This latter quantity, when analyzed using microdosimetric consideration, suggests that the target volume for this end point is of the order of micrometer.

The radiological research accelerator facility at Columbia University

The Radiological Research Accelerator Facility located at the Nevis Laboratories of Columbia University is a laboratory dedicated to bio-physics research. Its principal component is a 4 MeV Van de Graaff accelerator which produces protons, deuterons and other ions. The most important secondary radiation is neutrons of energies from about 100 keV to 18 MeV. In addition, diatomic and triatomic beams of protons and deuterons are employed in a special study. The biological effects of these radiations are compared with those of more conventional X-ray sources which are also available. The biological effects are correlated with physical aspects of the irradiation which include not only accurate measurements of the absorbed dose (the mean energy absorbed per unit mass of irradiated tissue) but also microdosimetry research which evaluates the distribution of energy at cellular and subcellular levels. Differences in these distributions can greatly affect the biological effectiveness of a given absorbed dose. The facility will be described, brief outlines of some of the biological and physical studies given and general implications of results discussed.

Differential RBE Values Obtained for Mammary Adenocarcinoma Tumor Cell Subpopulations after 14.8-MeV Neutron Irradiation

For tumor cell subpopulations which were isolated from a single mouse mammary adenocarcinoma were examined for their relative sensitivities to 250-kVp x irradiation and 14.8-MeV neutron irradiation. The sublines are designated 66, 67, 4.10, and 68H and differ significantly in their biological characteristics. Exponentially growing cells were exposed at the Radiological Research Accelerator Facility (RARAF) at Brookhaven National Laboratories, Upton, NY. The purpose of these studies was to compare the response of these cell lines to ionizing radiation, for high-linear-energy-transfer radiation as well as for low. The interest of such an intercomparison lies in the fact that these different cell lines, while closely related, were biologically distinguishable. Survival curve parameters obtained by fitting the single dose-response curves to a linear-quadratic equation using linear least-squares regression analysis gave values for sublines 66, 67, 4.10, and 68H, respectively, of: ..cap alpha../sub n/ (G/sub 8//sup -1/) = 0.00. 0.150, 0.041, and 0.182; ..cap alpha../sub x/ (G/sub 8//sup -1/) = 0.672, 0.845, 0.787, and 0.709; ..beta../sub x/ (G/sub 8//sup -2/) = 0.0462, 0.0345, 0.0576, and 0.0503; and ..beta../sub n/ (G/sub 8//sup -2/) = 0.0253, 0.0000, 0.0156, and 0.0666. Different relative biological effectiveness (RBE) values were obtained for sublines 66, 67, 4.10, and 68H ofmore » 4.0, 3.6, 3.9, and 2.7 at the 50% level of survival and 2.4, 2.4, 2.2, and 2.0 at the 10% level. Sublines 67 and 68H show responses which suggest a constant RBE at low values of dose, while sublines 66 and 4.10 do not. It is felt that these data illustrate the need to consider biological information as well as microdosimetric considerations in attempts to relate celluar inactivation responses to radiation quality. Further implications of these data in relation to the dual-action model of radiation inactivation are discussed.« less

Initial Changes in Cell Cycle Progression of Chinese Hamster V-79 Cells Induced by High-LET Charged Particles

Asynchronous Chinese hamster V-79 cells were pulse-labeled with tritiated thymidine; then mitotic cells were sequentially colcemid-accumulated after helium ion irradiation. Similar fluences of charged particles depositing intracellularly 63, 90, 128, and 138 ke V/μm were delivered at the Radiological Research Accelerator Facility at the Brookhaven National Laboratory. Nuclear cross-sectional areas were log-normally distributed with a mean value of $110 \mu {\rm m}^{2}$. An initial decline and subsequent recovery in frequencies of mitotic cells was dose, and to a lesser extent, LET dependent. Cells in S phase at the time of irradiation were dose dependently delayed and then proceeded into mitosis at the same rate as control cells. This was not found for G2 phase cells, which were delayed and progressively bypassed by S phase cells and probably G1 cells as both dose and LET increased. The passage of one high LET particle anywhere through a nucleus probably induces some delay response, while particles depos...