Mechanism of genotoxicity induced by targeted cytoplasmic irradiation.

BACKGROUND: Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown. METHODS: A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human-hamster hybrid (A(L)) cells. RESULTS: Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in A(L) cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of A(L) cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway. CONCLUSION: We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect.

A Horizontal Multi-Purpose Microbeam System.

A horizontal multi-purpose microbeam system with a single electrostatic quadruplet focusing lens has been developed at the Columbia University Radiological Research Accelerator Facility (RARAF). It is coupled with the RARAF 5.5 MV Singleton accelerator (High Voltage Engineering Europa, the Netherlands) and provides micrometer-size beam for single cell irradiation experiments. It is also used as the primary beam for a neutron microbeam and microPIXE (particle induced x-ray emission) experiment because of its high particle fluence. The optimization of this microbeam has been investigated with ray tracing simulations and the beam spot size has been verified by different measurements.

Testing the stand-alone microbeam at Columbia University.

The stand-alone microbeam at Columbia University presents a novel approach to biological microbeam irradiation studies. Foregoing a conventional accelerator as a source of energetic ions, a small, high-specific-activity, alpha emitter is used. Alpha particles emitted from this source are focused using a compound magnetic lens consisting of 24 permanent magnets arranged in two quadrupole triplets. Using a 'home made' 6.5 mCi polonium source, a 1 alpha particle s(-1), 10 microm diameter microbeam can, in principle, be realised. As the alpha source energy is constant, once the microbeam has been set up, no further adjustments are necessary apart from a periodic replacement of the source. The use of permanent magnets eliminates the need for bulky power supplies and cooling systems required by other types of ion lenses and greatly simplifies operation. It also makes the microbeam simple and cheap enough to be realised in any large lab. The Microbeam design as well as first tests of its performance, using an accelerator-based beam are presented here.

Construction of mouse phantoms from segmented CT scan data for radiation dosimetry studies.

We present the complete construction methodology for an anatomically accurate mouse phantom made using materials which mimic the characteristics of tissue, lung, and bone for radiation dosimetry studies. Phantoms were constructed using 2 mm thick slices of tissue equivalent material which was precision machined to clear regions for insertion of lung and bone equivalent material where appropriate. Images obtained using a 3D computed tomography (CT) scan clearly indicate regions of tissue, lung, and bone that match their position within the original mouse CT scan. Additionally, radiographic films are used with the phantom to demonstrate dose mapping capabilities. The construction methodology presented here can be quickly and easily adapted to create a phantom of any specific small animal given a segmented CT scan of the animal. These physical phantoms are a useful tool to examine individual organ dose and dosimetry within mouse systems that are complicated by density inhomogeneity due to bone and lung regions.

A Mouse Ear Model for Bystander Studies Induced by Microbeam Irradiation.

Radiation-induced bystander effects have been observed in vitro and in cell and tissue culture models, however, there are few reported studies showing these effects in vivo. To our knowledge, this is the first reported study on bystander effects induced by microbeam irradiation in an intact living mammal. The mouse ear was used to investigate radiation-induced bystander effects in keratinocytes, utilizing a 3 MeV proton microbeam (LET 13.1 keV/µm) with a range in skin of about 135 µm. Using a custom-designed holder, the ear of an anesthetized C57BL/6J mouse was flattened by gentle suction and placed over the microbeam port to irradiate cells along a 35 µm wide, 6 mm long path. Immunohistochemical analysis of γ-H2AX foci formation in tissue sections revealed, compared to control tissue, proton-induced γ-H2AX foci formation in one of the two epidermal layers of the mouse ear. Strikingly, a higher number of cells than expected showed foci from direct irradiation effects. Although the proton-irradiated line was ~35 µm wide, the average width spanned by γ-H2AX-positive cells exceeded 150 µm. Cells adjacent to or in the epidermal layer opposite the γ-H2AX-positive region did not exhibit foci. These findings validate this mammalian model as a viable system for investigating radiation-induced bystander effects in an intact living organism.

Quantitative comparisons of continuous and pulsed low dose rate regimens in a model late-effect system.

PURPOSE: There is increasing interest and usage of pulsed low dose rate (PDR) brachytherapy, in which a single source is shuttled through the catheters of an implant, typically for about 10 min each hour. This study was designed to compare the late effects produced in various PDR regimens with those from the corresponding continuous low dose rate (CLDR) regimens. METHODS AND MATERIALS: A model late-responding system was used, namely, cataract induction in the rat lens. This system has the advantage of being highly quantifiable. The rats eyes were exposed to a total dose of 15 Gy either continuously over 24 h, or with three different PDR regimens, all with the same total dose and overall time. We addressed three questions: (a) are late effects increased when a CLDR regimen is replaced with 10-min pulses repeated every hour? (b) Are late effects increased if hourly 10-min pulses are replaced with 10-min pulses repeated every 4 h? (c) Are late effects increased if 10-min pulses are replaced with 100-s pulses? RESULTS: We found that the four regimens under test, continuous, 10-min pulses each hour, 10-min pulses every 4 h, and 100-s pulses every hour, showed no significant differences in cataractogenic potential, as estimated with the Wilcoxon-Gehan test. Power tests indicated that the experimental design was adequate to detect relatively small differences in cataractogenicity between regimens. CONCLUSIONS: The equality of late effects from CLDR and PDR in these experiments must imply that sublethal damage repair is quite slow in this model late-responding system, in agreement with trends observed in the clinic for sublethal damage repair of late sequelae. Such trends would suggest that PDR is unlikely to produce significantly worse late effects than the corresponding CLDR regimen, which is in agreement with early clinical data using PDR. Caution, however, is strongly recommended.

The bystander effect in radiation oncogenesis: I. Transformation in C3H 10T1/2 cells in vitro can be initiated in the unirradiated neighbors of irradiated cells.

It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation. Recently, evidence has accumulated that in cell populations exposed to low doses of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. The end points observed include chromosome aberrations, mutations and gene expression. The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of alpha particles, and to assay for oncogenic transformation. The single-cell microbeam delivered no, one, two, four or eight alpha particles through the nuclei of all or just 10% of C3H 10T1/2 cells. We show that (a) more cells can be inactivated than were actually traversed by alpha particles and (b) when 10% of the cells on a dish are exposed to alpha particles, the resulting frequency of induced transformation is not less than that observed when every cell on the dish is exposed to the same number of alpha particles. These observations constitute evidence suggesting a bystander effect, i.e., that unirradiated cells are responding to damage induced in irradiated cells. This bystander effect in a biological system of relevance to carcinogenesis could have significant implications for risk estimation for low-dose radiation.

The inverse dose-rate effect for oncogenic transformation by charged particles is dependent on linear energy transfer.

Mouse C3H 10T1/2 cells were exposed to single or fractionated doses of charged particles of defined linear energy transfer (LET) from 25 to 200 keV/microns. Dose fractionation with prolonged time intervals enhanced the yield of transformed foci compared with a single acute dose for a range of LET values between 40 and 120 keV/microns. Radiations of lower or higher LET did not show the enhancement that is commonly referred to as the inverse dose-rate effect. The fractionation scheme that was used consisted of three dose fractions; the maximum enhancement of transformation occurred with an interval of 150 min between dose fractions. This inverse dose-rate effect, demonstrated for cycling cells in log phase, was not seen for cells in plateau phase.

Mechanistic considerations on the dose-rate/LET dependence of oncogenic transformation by ionizing radiations.

When exposure to densely ionizing radiation is protracted, the resulting biological effect is sometimes, but not always, enhanced for transformational end points, relative to acute exposure. A pattern has emerged as to the dependence of this effect on dose, dose rate, and radiation quality. Previous calculations indicated that the dose and dose-rate trends can be predicted by a model in which there is a period within the cell cycle of very high sensitivity to oncogenesis. Recent experiments indicate that the inverse dose-rate effect is significant over a very limited range of LETs--from about 30 to 130 keV/microns. We discuss such LET effects in the context of cell cycle-dependent models, and suggest that the effects are understandable on the basis of such models. In essence, the inverse dose-rate effect disappears at high LET because of a reduction in the number of cells being hit, and disappears at LETs below about 30 keV/microns because most of the dose is deposited at low specific energies, insufficient to produce the saturation effect which is central to the phenomenon. At even lower LETs, damage repair yields the familiar sparing associated with protraction of X- or gamma-ray doses.

Oncogenic transformation following sequential irradiations with monoenergetic neutrons and X rays.

Mouse C3H 10T1/2 cells were exposed sequentially to low doses (0.1 and 0.3 Gy) of monoenergetic neutrons (0.35, 0.45, 5.9, and 13.7 MeV) and 250-kVp X rays (1 and 3 Gy). The incidences of oncogenic transformation in the cells exposed to neutrons followed by X rays indicated that the effects of the individual radiations were simply additive. This supports the contention that risks associated with the two different radiation modalities may be considered to be additive.

The bystander response in C3H 10T1/2 cells: the influence of cell-to-cell contact.

Although radiation-induced heritable damage in mammalian cells was thought to result from the direct interaction of radiation with DNA, it is now accepted that biological effects may occur in cells that were not themselves traversed by ionizing radiation but are close to those that were. However, little is known about the mechanism underlying such a bystander effect, although cell-to-cell communication is thought to be of importance. Previous work using the Columbia microbeam demonstrated a significant bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. The present study was undertaken to assess the importance of the degree of cell-to-cell contact at the time of irradiation on the magnitude of this bystander effect by varying the cell density. When 10% of cells were exposed to a range of 2-12 alpha particles, a significantly greater number of cells (P < 0.0001) were inactivated when cells were irradiated at high density (>90% in contact with neighbors) than at low density (<10% in contact). In addition, the oncogenic transformation frequency was significantly higher in high-density cultures (P < 0.0004). These results suggest that when a cell is hit by radiation, the transmission of the bystander signal through cell-to-cell contact is an important mediator of the effect, implicating the involvement of intracellular communication through gap junctions.

Quantitative assessment of the cataractogenic potential of very low doses of neutrons.

We report on the prevalence and relative biological effectiveness (RBE) for various stages of lens opacification in rats induced by very low doses (2 to 250 mGy) of medium-energy (440 keV) neutrons, compared to those for X rays. Neutron doses were delivered either in a single fraction or in four separate fractions and the irradiated animals were followed for over 100 weeks. At the highest observed dose (250 mGy) and at early observation times, there was evidence of an inverse dose-rate effect; i.e., a fractionated exposure was more potent than a single exposure. Neutron RBEs relative to X rays were estimated using a non-parametric technique. The results were only weakly dependent on time postirradiation. At 30 weeks, for example, 80% confidence intervals for the RBE of acutely delivered neutrons relative to X rays were 8-16 at 250 mGy, 10-20 at 50 mGy, 50-100 at 10 mGy and 250-500 at 2 mGy. The results are consistent with the estimated neutron RBEs in Japanese A-bomb survivors, though broad confidence bounds are present in the Japanese results. Our findings are also consistent with data reported earlier for cataractogenesis induced by heavy ions in rats, mice, and rabbits. We conclude from these results that, at very low doses (<10 mGy), the RBE for neutron-induced cataractogenesis is considerably larger than the RBE of 20 commonly used, and use of a significantly larger value for calculating equivalent dose would be prudent.

The effects of the temporal distribution of dose on oncogenic transformation by neutrons and charged particles of intermediate LET.

The effects of dose rate and dose fractionation on high-LET radiation-induced oncogenic transformation of C3H 10T1/2 cells were examined. Cells were irradiated with graded doses of 5.9-MeV monoenergetic neutrons administered either in single acute exposures (30 mGy/min) or extended over an 8-h period at low dose rates (from 0.21 to 1 mGy/min). Although cell survival studies showed no difference in effect with a change in radiation delivery rate, enhancement of oncogenic transformation occurred when the dose rate was reduced. When the neutron dose was divided into three fractions over 8 h, the biological effect was intermediate between that for the acute and that for the low-dose-rate exposures. Further irradiations were made using deuterons with an LET of 40 keV/microns. The dose-mean lineal energy was comparable to that measured for the 5.9-MeV monoenergetic neutrons. An inverse dose-rate/fractionation effect for the induction of transformation by high-LET deuterons was observed when the time between each of three fractions for a 0.3-Gy total dose was at least 45 min. No further enhancement was seen for longer dose fractionations, suggesting that very long protracted exposures of high-LET radiation would produce no additional enhancement.

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.

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).

The biological effectiveness of radon-progeny alpha particles. II. Oncogenic transformation as a function of linear energy transfer.

Epidemiological studies have established an association between exposure to radon and carcinoma of the lung. However, based on data for either lung cancer in uranium miners exposed to radon or bronchial epithelial carcinomas in Japanese A-bomb survivors, it has not been possible to assign estimates of risk of lung cancer for the general population exposed to radon in their homes. Based on past success with the excellent quantitative properties of the C3H 10T1/2 in vitro oncogenic transformation assay system, the relative biological effectiveness (RBE) for radiation-induced transformation for charged particles of defined LET has been determined. As the LET of the radiation was increased, the rate of induction of oncogenic transformation increased and the RBEm approached 20. At higher LETs, RBE dropped precipitously. The rapid drop in effectiveness for alpha particles with LETs between 120 and 265 keV/microns implies a lower quality factor than the 20-25 currently considered appropriate when estimating lung cancer mortality.

The radiation-induced bystander effect for clonogenic survival.

It has long been accepted that the radiation-induced heritable effects in mammalian cells are the result of direct DNA damage. Recent evidence, however, suggests that when a cell population is exposed to a low dose of alpha particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by alpha particles. Experiments involving the Columbia University microbeam, which allows a known fraction of cells to be traversed by a defined number of alpha particles, have demonstrated a bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. When 1 to 16 alpha particles were passed through the nuclei of 10% of a C3H 10T(1/2) cell population, more cells were unable to form colonies than were actually traversed by alpha particles. Both hit and non-hit cells contributed to the outcome of the experiments. The present work was undertaken to assess the bystander effect of radiation in only non-hit cells. For this purpose, Chinese hamster V79 cells transfected with hygromycin- or neomycin-resistance genes were used. V79 cells stably transfected with a hygromycin resistance gene and stained with a nuclear dye were irradiated with the charged-particle microbeam in the presence of neomycin-resistant cells. The biological effect was studied in the neomycin-resistant V79 cells after selective removal of the hit cells with geneticin treatment.

A practical target system for accelerator-based BNCT which may effectively double the dose rate.

A dose-limiting component of a proton accelerator-based source of epithermal neutrons is the neutron production target. Possible targets are lithium, producing high yield but having low melting point and thermal conductivity, and beryllium, presenting less engineering problems but a much smaller neutron yield. We propose that a hybrid Be-Li target would provide the best of both worlds, with the upstream beryllium component producing neutrons and providing containment to the lithium, and the downstream liquid lithium in turn producing further neutrons as well as cooling the beryllium. The engineering considerations associated with such a target system are within the range of current technology. Calculations suggest a yield of such a practical target that is at least double that from pure beryllium.

Routine screening mammography: how important is the radiation-risk side of the benefit-risk equation?

The potential radiation hazards associated with routine screening mammography, in terms of breast cancer induction, are discussed in the context of the potential benefits. The very low energy X-rays used in screening mammography (26-30 kVp) are expected to be more hazardous, per unit dose, than high-energy X- or gamma-rays, such as those to which A-bomb survivors (from which radiation risk estimates are derived) were exposed. Based on in vitro studies using oncogenic transformation and chromosome aberration end-points, as well as theoretical estimates, it seems likely that low doses of low-energy X-rays produce an increased risk per unit dose (compared with high energy photons) of about a factor of 2. Because of the low doses involved in screening mammography, the benefit-risk ratio for older women would still be expected to be large, though for younger women the increase in the estimated radiation risk suggests a somewhat later age than currently recommended--by about 5-10 years--at which to commence routine breast screening.

Radiation-induced bystander effect and adaptive response in mammalian cells.

Two conflicting phenomena, bystander effect and adaptive response, are important in determining the biological responses at low doses of radiation and have the potential to impact the shape of the dose-response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we show here that non-irradiated cells acquire mutagenesis through direct contact with cells whose nuclei have been traversed with a single alpha particle each. Pretreatment of cells with a low dose of X-rays four hours before alpha particle irradiation significantly decreased this bystander mutagenic response. Results from the present study address some of the fundamental issues regarding both the actual target and radiation dose effect and can contribute to our current understanding in radiation risk assessment.