Physical and biological organ dosimetry analysis for international space station astronauts

In this study, we analyzed the biological and physical organ dose equivalents for International Space Station (ISS) astronauts. Individual physical dosimetry is difficult in space due to the complexity of the space radiation environment, which consists of protons, heavy ions and secondary neutrons, and the modification of these radiation types in tissue as well as limitations in dosimeter devices that can be worn for several months in outer space. Astronauts returning from missions to the ISS undergo biodosimetry assessment of chromosomal damage in lymphocyte cells using the multicolor fluorescence in situ hybridization (FISH) technique. Individual-based pre-flight dose responses for lymphocyte exposure in vitro to gamma rays were compared to those exposed to space radiation in vivo to determine an equivalent biological dose. We compared the ISS biodosimetry results, NASA's space radiation transport models of organ dose equivalents, and results from ISS and space shuttle phantom torso experiments. Physical and biological doses for 19 ISS astronauts yielded average effective doses and individual or population-based biological doses for the approximately 6-month missions of 72 mSv and 85 or 81 mGy-Eq, respectively. Analyses showed that 80% or more of organ dose equivalents on the ISS are from galactic cosmic rays and only a small contribution is from trapped protons and that GCR doses were decreased by the high level of solar activity in recent years. Comparisons of models to data showed that space radiation effective doses can be predicted to within about a +/-10% accuracy by space radiation transport models. Finally, effective dose estimates for all previous NASA missions are summarized.     

Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.     

Imaging molecular pathways: reporter genes

Molecular imaging is a rapidly advancing field that allows cancer biologists to look deeper into the complex inner workings of tumor cells, or whole tumors, in a non-invasive manner. In this review, we will summarize some recent advances that enable investigators to study various important biological processes in tumors in vivo. We will discuss novel imaging approaches that allow investigators to visualize and quantify molecular pathways, such as receptor tyrosine kinase activation, hypoxia signal transduction, apoptosis, and DNA double-strand breaks. Select examples of these applications will be discussed. Because of the limited scope of this review, we will only focus on natural reporters, such as bioluminescence and fluorescent proteins.     

Microbeam irradiation of C. elegans nematode in microfluidic channels

To perform high-throughput studies on the biological effects of ionizing radiation in vivo, we have implemented a microfluidic tool for microbeam irradiation of Caenorhabditis elegans. The device allows the immobilization of worms with minimal stress for a rapid and controlled microbeam irradiation of multiple samples in parallel. Adapted from an established design, our microfluidic clamp consists of 16 tapered channels with 10-μm-thin bottoms to ensure charged particle traversal. Worms are introduced into the microfluidic device through liquid flow between an inlet and an outlet, and the size of each microchannel guarantees that young adult worms are immobilized within minutes without the use of anesthesia. After site-specific irradiation with the microbeam, the worms can be released by reversing the flow direction in the clamp and collected for analysis of biological endpoints such as repair of radiation-induced DNA damage. For such studies, minimal sample manipulation and reduced use of drugs such as anesthetics that might interfere with normal physiological processes are preferable. By using our microfluidic device that allows simultaneous immobilization and imaging for irradiation of several whole living samples on a single clamp, here we show that 4.5-MeV proton microbeam irradiation induced DNA damage in wild-type C. elegans, as assessed by the formation of Rad51 foci that are essential for homologous repair of radiation-induced DNA damage.     

Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

DNA double-strand breaks (DSBs) are a serious threat to genome stability and cell viability. Although biological effects of low levels of radiation are not clear, the risks of low-dose radiation are of societal importance. Here, we directly monitored induction and repair of single DSBs and quantitatively analyzed the dynamics of interaction of DNA repair proteins at individual DSB sites in living cells using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a surrogate marker. The number of DSBs formed was linear with dose from 5 mGy to 1 Gy. The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses. The YFP-53BP1 foci are dynamic structures: 53BP1 rapidly and reversibly interacted at these DSB sites. The time frame of recruitment and affinity of 53BP1 for DSB sites were indistinguishable between low and high doses, providing mechanistic evidence for the similar DSB repair after low- and high-dose radiation. These findings have important implications for estimating the risk associated with low-dose radiation exposure on human health.     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.     

New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell gel electrophoresis, and fluorescence signals

The quantification of DNA damage, both in vivo and in vitro, can be very time consuming, since large amounts of samples need to be scored. Additional uncertainties may arise due to the lack of documentation or by scoring biases. Image analysis automation is a possible strategy to cope with these difficulties and to generate a new quality of reproducibility. In this communication we collected some recent results obtained with the automated scanning platform Metafer, covering applications that are being used in radiation research, biological dosimetry, DNA repair research and environmental mutagenesis studies. We can show that the automated scoring for dicentric chromosomes, for micronuclei, and for Comet assay cells produce reliable and reproducible results, which prove the usability of automated scanning in the above mentioned research fields.     

A comparison of gamma and neutron irradiation on Raji cells: effects on DNA damage, repair, cell cycle distribution and lethality.

The Comet assay (microgel electrophoresis) was used to study DNA damage in Raji cells, a B-lymphoblastoid cell line, after treatment with different doses of neutrons (0.5 to 16 Gy) or gamma rays (1.4 to 44.8 Gy). A better growth recovery was observed in cells after gamma-ray treatments compared with neutron treatments. The relative biological effectiveness (RBE) of neutron in cell killing was determined to be 2.5. Initially, the number of damaged cells per unit dose was approximately the same after neutron and gamma-ray irradiation. One hour after treatment, however, the number of normal cells per unit dose was much lower for neutrons than for gamma rays, suggesting a more efficient initial repair for gamma rays. Twenty-four hours after treatment, the numbers of damaged cells per unit dose of neutrons or gamma rays were again at comparable level. Cell cycle kinetic studies showed a strong G2/M arrest at equivalent unit dose (neutrons up to 8 Gy; gamma rays up to 5.6 Gy), suggesting a period in cell cycle for DNA repair. However, only cells treated with low doses (up to 2 Gy) seemed to be capable of returning into normal cell cycle within 4 days. For the highest dose of neutrons, decline in the number of normal cells seen at already 3 days after treatment was deeper compared with equivalent unit doses of gamma rays. Our present results support different mechanisms of action by these two irradiations and suggest the generation of locally multiply damaged sites (LMDS) for high linear energy transfer (LET) radiation which are known to be repaired at lower efficiency.     

Viral nanoparticles as tools for intravital vascular imaging

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.     

Versatility of thermoluminescence materials and radiation dosimetry – A review

Thermoluminescence (TL) materials exhibit a wide range of applications in different areas such as personal dosimetry, environmental dosimetry, medical research etc. Doping of different rare earth impurities in different hosts is responsible for changing the properties of materials useful for various applications in different fields. These materials can be irradiated by different types of beams such as γ‐rays, X‐rays, electrons, neutrons etc. Various radiation regimes, as well as their dose–response range, play an important role in thermoluminescence dosimetry. Several TL materials, such as glass, microcrystalline, nanostructured inorganic materials and recently developed materials, are reviewed and described in this article.     

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.     

In vivo recombination after chronic damage exposure falls to below spontaneous levels in "recombomice"

All forms of cancer are initiated by heritable changes in gene expression. Although point mutations have been studied extensively, much less is known about homologous recombination events, despite its role in causing sequence rearrangements that contribute to tumorigenesis. Although transgenic mice that permit detection of point mutations have provided a fundamental tool for studying point mutations in vivo, until recently, transgenic mice designed specifically to detect homologous recombination events in somatic tissues in vivo did not exist. We therefore created fluorescent yellow direct repeat mice, enabling automated detection of recombinant cells in vivo for the first time. Here, we show that an acute dose of ionizing radiation induces recombination in fluorescent yellow direct repeat mice, providing some of the first direct evidence that ionizing radiation induces homologous recombination in cutaneous tissues in vivo. In contrast, the same total dose of radiation given under chronic exposure conditions suppresses recombination to levels that are significantly below those of unexposed animals. In addition, global methylation is suppressed and key DNA repair proteins are induced in tissues from chronically irradiated animals (specifically AP endonuclease, polymerase beta, and Ku70). Thus, increased clearance of recombinogenic lesions may contribute to suppression of homologous recombination. Taken together, these studies show that fluorescent yellow direct repeat mice provide a rapid and powerful assay for studying the recombinogenic effects of both short-term and long-term exposure to DNA damage in vivo and reveal for the first time that exposure to ionizing radiation can have opposite effects on genomic stability depending on the duration of exposure.     

Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm² with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.     

Zinc sensing for cellular application

Numerous tools for Zn2+ sensing in living cells have become available in the past three years. Among them, fluorescence imaging using fluorescent sensor molecules has been the most popular approach. Some of these sensor molecules can be used to visualize Zn2+ in living cells. Some of the biological functions of Zn2+ have been clarified using these sensor molecules, especially in neuronal cells, which contain a high concentration of free Zn2+.     

Dual-Color imaging of nuclear division and mitotic spindle elongation in live cells of Aspergillus nidulans

We have developed a dual-color imaging system based on cyan fluorescent protein-labeled histone H2A and green fluorescent protein-labeled alpha tubulin to visualize DNA and spindles simultaneously in the same living cell of Aspergillus nidulans. This system allows new details of mitosis and nuclear movement to be revealed.     

Nonuniform irradiation of the canine intestine. II. Dosimetry

An experimental model has been developed for quantitative studies of radiobiological damage to the canine small intestine following partial-body nonuniform irradiation. Animals were irradiated with 60Co gamma rays to simulate the nonuniform irradiation which do occur in victims of radiation accidents. The model used a short source-to-surface distance for unilateral irradiations to produce a dose gradient of a factor of two laterally across the canine intestinal region. The remainder of the animal's body was shielded to prevent lethal damage to the bone marrow. In situ dosimetry measurements were made using thermoluminescent dosimeters to determine the radiation dose delivered as a function of position along a segment of the small intestine. This system made it possible to correlate the radiation dose delivered at a specific point along the small intestine with the macroscopic and microscopic appearance of the intestinal mucosa at that point, as determined by direct observation and biopsy using a fiberoptic endoscope. A key feature of this model is that dosimetry data for multiple sites, which receive a graded range of radiation doses, can be correlated with biological measurements to obtain a dose-response curve. This model is being used to evaluate the efficacy of new therapeutic procedures to improve survival following nonuniform irradiation.     

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.     

A hypothetical stochastic mechanism of radiation effects in single cells

This paper is a sequel to an earlier one (Neyman & Puri 1976), which deals with a hypothetical structural stochastic model of radiation effects in living cells. This model incorporates two important details of the mechanism overlooked in its mathematical treatment by the previous workers. The first is that the passage of a single "primary' radiation particle generates a "cluster' of secondaries which can produce "hits' that damage the living cells. The second detail concerns the time scales of radiation damage and of the subsequent repair. The events of arrival of a primary particle, its generation of secondary particles and their causing "hits' on the sensitive targets within the cells, all occur for all practical purposes instantly. On the other hand the subsequent changes in the damaged cell, such as repair, appear to require measurable amounts of time. While the biological and physical justifications for some of the underlying assumptions of the model were discussed in the previous paper referred to above, the present paper is concerned mainly with the mathematical details and also with how the model attempts to explain some of the empirical findings available in the literature.