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.

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.

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.

Integrated interdisciplinary training in the radiological sciences.

The radiation sciences are increasingly interdisciplinary, both from the research and the clinical perspectives. Beyond clinical and research issues, there are very real issues of communication between scientists from different disciplines. It follows that there is an increasing need for interdisciplinary training courses in the radiological sciences. Training courses are common in biomedical academic and clinical environments, but are typically targeted to scientists in specific technical fields. In the era of multidisciplinary biomedical science, there is a need for highly integrated multidisciplinary training courses that are designed for, and are useful to, scientists who are from a mix of very different academic fields and backgrounds. We briefly describe our experiences running such an integrated training course for researchers in the field of biomedical radiation microbeams, and draw some conclusions about how such interdisciplinary training courses can best function. These conclusions should be applicable to many other areas of the radiological sciences. In summary, we found that it is highly beneficial to keep the scientists from the different disciplines together. In practice, this means not segregating the training course into sections specifically for biologists and sections specifically for physicists and engineers, but rather keeping the students together to attend the same lectures and hands-on studies throughout the course. This structure added value to the learning experience not only in terms of the cross fertilization of information and ideas between scientists from the different disciplines, but also in terms of reinforcing some basic concepts for scientists in their own discipline.

Design of a novel flow-and-shoot microbeam.

Presented here is a novel microbeam technology--the Flow-And-ShooT (FAST) microbeam--under development at RARAF. In this system, cells undergo controlled fluidic transport along a microfluidic channel intersecting the microbeam path. They are imaged and tracked in real-time, using a high-speed camera and dynamically targeted, using a magnetic Point and Shoot system. With the proposed FAST system, RARAF expects to reach a throughput of 100,000 cells per hour, which will allow increasing the throughput of experiments by at least one order of magnitude. The implementation of FAST will also allow the irradiation of non-adherent cells (e.g. lymphocytes), which is of great interest to many of the RARAF users. This study presents the design of a FAST microbeam and results of first tests of imaging and tracking as well as a discussion of the achievable throughput.

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.

Ion, X-ray, UV and Neutron Microbeam Systems for Cell Irradiation.

The array of microbeam cell-irradiation systems, available to users at the Radiological Research Accelerator Facility (RARAF), Center for Radiological Research, Columbia University, is expanding. The HVE 5MV Singletron particle accelerator at the facility provides particles to two focused ion microbeam lines: the sub-micron microbeam II and the permanent magnetic microbeam (PMM). Both the electrostatic quadrupole lenses on the microbeam II system and the magnetic quadrupole lenses on the PMM system are arranged as compound lenses consisting of two quadrupole triplets with "Russian" symmetry. Also, the RARAF accelerator is a source for a proton-induced x-ray microbeam (undergoing testing) and is projected to supply protons to a neutron microbeam based on the (7)Li(p, n)(7)Be nuclear reaction (under development). Leveraging from the multiphoton microscope technology integrated within the microbeam II endstation, a UV microspot irradiator - based on multiphoton excitation - is available for facility users. Highlights from radiation-biology demonstrations on single living mammalian cells are included in this review of microbeam systems for cell irradiation at RARAF.

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.

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.

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.

Genotoxic damage in non-irradiated cells: contribution from the bystander effect.

It has always been accepted dogma that the deleterious effects of ionising radiation such as mutagenesis and carcinogenesis are due mainly to direct damage to DNA. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, it is shown here that non-irradiated cells acquire the mutagenic phenotype through direct contact with cells whose nuclei are traversed with 2 alpha particles each. Pre-treatment of cells with lindane, a gap junction inhibitor, significantly decreased the mutant yield. Furthermore, when irradiated cells were mixed with control cells in a similar ratio as the in situ studies, no enhancement in bystander mutagenesis was detected. Our studies provide clear evidence that genotoxic damage can be induced in non-irradiated cells, and that gap junction mediated cell-cell communication plays a critical role in the bystander phenomenon.

Microbeams, microdosimetry and specific dose.

Dose and its usefulness as a single parameter to describe the amount of radiation absorbed are well established for most situations. The conditions where the concept of dose starts to break down are well known, mostly from the study of microdosimetry. For low doses of high LET radiation it is noted that the process of taking the limiting value of the energy absorbed within a test volume divided by the mass within that volume yields either zero or a relatively large value. The problem is further exacerbated with microbeam irradiations where the uniformity of the energy deposition is experimentally manipulated on the spatial scale of cells being irradiated. Booz introduced a quantity to deal with these problems: the unfortunately named 'mean specific energy in affected volumes'. This quantity multiplied by the probability that a test volume has received an energy deposit is equal to dose (in situations where dose can be defined). I propose that Booz's quantity be renamed 'specific dose', that is the mean energy deposited divided by the mass within a specified volume. If we believe for instance that the nucleus of a cell is the critical volume for biological effects, we can refer to the nuclear specific dose. A microbeam experiment wherein 10 per cent of the cell nuclei were targeted with 10 alpha particles would be described as delivering a nuclear specific dose of 1.6 Gy to 10 per cent of the population.

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.

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 risk to low fluences of alpha particles may be greater than we thought.

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or < or =20%) of the cells in a confluent population and irradiated their nuclei with exactly one alpha particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

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 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.

Induction of a bystander mutagenic effect of alpha particles in mammalian cells.

Ever since the discovery of X-rays was made by Röntgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A(L) cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 microM dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

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.