The Vitamin E Analog Gamma-Tocotrienol (GT3) Suppresses Radiation-Induced Cytogenetic Damage
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Rupak Pathak↗
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[Extend the Usefullness of Cytogenetic Methodology as a Research Technique and as a Biomedical Monitoring Procedure through the Utilization of Automatic Electronic Scanning and Computer Analysis of Chromosomes]
Automatic microscope and computer program for chromosome analysis
COMPUTER PROGRAM↗
Cytogenetic studies of blood, experiment M111
Analysis of the chromosome patterns of Skylab crewmen preflight and postflight recorded two types of defects: minor structural defects including chromatid breaks, chromosome breaks, deletions, and fragments were not significantly increased; structural rearrangements including dicentrics, exchange figures, inversions, and translocations occured more frequently. Data indicate that the flight itself in Skylab missions was not a contributing factor to these abberrations.
Lockhart, L. H.↗
Skylab: Cytogenetic studies of blood (experiment mill)
Preflight and postflight chromosomal analyses of space flight crews are presented. Special attention is given to findings suggestive of exposure to ionizing radiation. Information was obtained by study of persons receiving an external body source, such as, therapeutic dosage or those accidentally exposed. Others receiving radiation exposure from an internal source, such as the decay of radioisotopes administered for diagnosis or treatment, were also analyzed.
Lockhart, L. H.↗
Cytogenetic analyses of peripheral lymphocytes subjected to simulated solar flare radiation
Solar flare protons share many radiological health characteristics of the inner Van Allen Belt protons, and both types of radiation pose serious dangers to a number of missions planned. It is appropriate to evaluate crew dose determination procedures in terms of the type of radiation responsible for the major part of the projected exposure, i.e., protons in the neighborhood of 100 MeV. Monitoring chromosome abnormalities in peripheral lymphocytes is one method to determine an individual's accumulated radiation dosage. Cell culture and harvest is a relatively simple procedure and is well within the capabilities of a station health facility, but the evaluation of prepared microscopic slides is a time consuming and subjective procedure. This project is part of an effort to demonstrate the utility of automated image processing and evaluation procedures in expediting dose evaluation. The initial goal of this project is to produce a set of reference chromosome spreads produced from control lymphocytes and from lymphocytes exposed in whole blood to protons or gamma rays. The results of manual and automated aberration scoring will ultimately be compared to test for systematic differences between the two evaluation procedures and between the two radiation qualities. Proton irradiations are performed at the University of Texas Health Science Center at Houston Cyclotron Facility. Proton dosimetry is supplemented by TLD packets from and by assay of short-lived proton activation products in the irradiation blood samples.
Prichard, H. M.↗
In vitro cytogenetic studies of organic chemicals found as contaminants in spacecraft cabin atmospheres
Astronauts can be exposed during spaceflight to organic chemical contaminants in the spacecraft cabin atmosphere. Toxic exposures may cause lesions in the cellular DNA which are subsequently expressed as sister-chromatid exchanges (SCE). Analysis of SCE is a sensitive short-term assay technique to detect and quantitate exposures to DNA-damaging (mutagenic) substances. The increase in SCE incidence over baseline (control) levels is generally proportional to the concentration of the mutagen and to the duration of exposure. Dichloromethane (methylene chloride) was chosen for this study since it occurred as an atmospheric contaminant in ten of the first 12 STS flights, and has been reported to have toxic and mutagenic effects in various test systems. Glutaraldehyde was chosen because relatively few data are available on the toxicity or mutagenicity of this common biological fixative, which is carried on STS flights for use in biological experiments. The BHK-21 baby hamster kidney cell line was the in vitro test system used in this study. Neither dichloromethane (10 ppm to 500 ppm) nor glutaraldehyde (1 ppm to 10 ppm) increased SCE levels following 20-hour exposure of BHK-21 cells to the test chemicals.
Torres, J.↗
Radiation-Induced Cytogenetic Damage as a Predictor of Cancer Risk for Protons and Fe Ions
We have successfully completed the series of experiments planned for year 1 and the first part of year 2 measuring the induction of chromosome aberrations induced in multiple cell types by three model space radiations: Fe-ions, protons and photons. Most of these data have now been compiled and a significant part subjected to detailed data analyses, although continuing data analysis is an important part of our current and future efforts. These analyses are directed toward defining the patterns of chromosomal damage induction by the three radiations and the extent to which such patterns are dependent on the type of cell irradiated. Our studies show significant differences, both quantitatively and qualitatively, between response of different cell types to these radiations however there is an overall pattern that characterizes each type of radiation in most cell lines. Thus our data identifies general dose-response patterns for each radiation for induction of multiple types of chromosomal aberrations but also identifies significant differences in response between some cell types. Specifically, we observe significant resistance for induction of aberrations in rat mammary epithelial cells when they are irradiated in vivo and assayed in vitro. Further, we have observed some remarkable differences in susceptibility to certain radiation-induced aberrations in cells whose genome has been modulated for two cancer- relevant genes, TP53 and CDKNIA. This data, if confirmed, may represent the first evidence of gene-specific differences in cellular metabolism of damage induced by densely-ionizing radiation that confers substantial sensitivity to protons compared to photons.
Williams, Jerry R.↗
Persistence of Space Radiation-Induced Cytogenetic Damage in the Blood Lymphocytes of Astronauts and the Effects of Repeat Long Duration Space Missions
The yield of chromosome damage in astronauts blood lymphocytes has been shown to increase after long duration space missions of a few months or more. This provides a useful in vivo measurement of space radiation induced damage that takes into account individual radiosensitivity and considers the influence of microgravity and other stress conditions. We present our latest follow-up analyses of chromosome damage in astronauts blood lymphocytes assessed by fluorescence in situ hybridization (FISH) chromosome painting and collected at various times, from directly after return from space to several years after flight. For most individuals the analysis of individual time-courses for translocations revealed a temporal decline of yields with different half-lives. Dose was derived from frequencies of chromosome exchanges using preflight calibration curves, and estimates derived from samples collected a few days after return to earth lie within the range expected from physical dosimetry. However, a temporal decline in yields may indicate complications with the use of stable aberrations for retrospective dose reconstruction, and the differences in the decay time may reflect individual variability in risk from space radiation exposure. Limited data on three individuals who have participated in repeat long duration space flights indicates a lack of correlation between time in space and translocation yields, and show a possible adaptive response to space radiation exposure.
George, Kerry A.↗
Cancer Risk Estimates from Space Flight Estimated Using Yields of Chromosome Damage in Astronaut's Blood Lymphocytes
To date, cytogenetic damage has been assessed in blood lymphocytes from more than 30 astronauts before and after they participated in long-duration space missions of three months or more on board the International Space Station. Chromosome damage was assessed using fluorescence in situ hybridization whole chromosome analysis techniques. For all individuals, the frequency of chromosome damage measured within a month of return from space was higher than their preflight yield, and biodosimetry estimates were within the range expected from physical dosimetry. Follow up analyses have been performed on most of the astronauts at intervals ranging from around 6 months to many years after flight, and the cytogenetic effects of repeat long-duration missions have so far been assessed in four individuals. Chromosomal aberrations in peripheral blood lymphocytes have been validated as biomarkers of cancer risk and cytogenetic damage can therefore be used to characterize excess health risk incurred by individual crewmembers after their respective missions. Traditional risk assessment models are based on epidemiological data obtained on Earth in cohorts exposed predominantly to acute doses of gamma-rays, and the extrapolation to the space environment is highly problematic, involving very large uncertainties. Cytogenetic damage could play a key role in reducing uncertainty in risk estimation because it is incurred directly in the space environment, using specimens from the astronauts themselves. Relative cancer risks were estimated from the biodosimetry data using the quantitative approach derived from the European Study Group on Cytogenetic Biomarkers and Health database. Astronauts were categorized into low, medium, or high tertiles according to their yield of chromosome damage. Age adjusted tertile rankings were used to estimate cancer risk and results were compared with values obtained using traditional modeling approaches. Individual tertile rankings increased after space flight and analysis of follow up samples indicated that the tertile rankings remained in the high category for more than 50% of the individuals assessed so far. Crewmembers that shift and remain in the high category are projected to have increased life-time cancer risk.
George, Kerry A.↗
Mammalian Chromosome Analysis and Sorting by Flow Cytometry
The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell.
59 BASIC BIOLOGICAL SCIENCES↗
Chromosome aberrations induced by high-LET radiations
Measurements of chromosome aberrations in peripheral blood lymphocytes are currently the most sensitive and reliable indicator of radiation exposure that can be used for biological dosimetry. This technique has been implemented recently to study radiation exposures incurred by astronauts during space flight, where a significant proportion of the dose is delivered by high-LET particle exposure. Traditional methods for the assessing of cytogenetic damage in mitotic cells collected at one time point after exposure may not be suitable for measuring high-LET radiation effects due to the drastic cell cycle perturbations and interphase cell death induced by this type of exposure. In this manuscript we review the recent advances in methodology used to study high-LET induced cytogenetic effects and evaluate the use of chemically-induced Premature Chromosome Condensation (PCC) as an alternative to metaphase analysis. Published data on the cytogenetic effects of in vitro exposures of high-LET radiation is reviewed, along with biodosimetry results from astronauts after short or long space missions.
ISS Project↗
The evolution of chromosomal instability in Chinese hamster cells: a changing picture?
PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.
Non-NASA Center↗
Radiation-induced instability and its relation to radiation carcinogenesis
PURPOSE: A model that identifies radiation-induced genetic instability as the earliest cellular event in the multi-step sequence leading to radiation-induced cancer was previously proposed. In this paper ongoing experiments are discussed which are designed to test this model and its predictions in mouse mammary epithelial cells. RESULTS: Several lines of evidence are presented that appear to support this model: first, the development of delayed mutations in p53 following irradiation in altered growth variants; secondly, the high frequencies for the induction of both instability and transformation following irradiation in mammary epithelial cells; and finally, the demonstration that susceptibility to the induction of cytogenetic instability is a heritable trait that correlates with susceptibility to transformation and radiation-induced mammary cancer. Mice resistant to transformation and mammary cancer development are also resistant to the development of instability after irradiation. In contrast, mice sensitive to transformation and cancer are also sensitive to the development of cytogenetic instability. CONCLUSIONS: Data from this laboratory and from the studies cited above suggest a specific, and perhaps unique, role for radiation-induced instability as a critical early event associated with initiation of the carcinogenic process.
Review↗
Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus
Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
Chromosomal instability induced by heavy ion irradiation
PURPOSE: To establish the dose-response relationship for the induction of chromosomal instability in GM10115 cells exposed to high-energy iron ions (1 GeV/nucleon, mean LET 146 keV/microm) and gold ions (11 GeV/nucleon, mean LET 1450 keV/microm). Past work has established that sparsely ionizing X-rays can induce a long-lived destabilization of chromosomes in a dose-dependent manner at an incidence of approximately 3% per gray. The present investigation assesses the capacity of High-Z and High-energy (HZE) particles to elicit this same endpoint. MATERIALS AND METHODS: Clonal populations derived from single progenitor cells surviving heavy-ion irradiation were analyzed cytogenetically to identify those clones showing a persistent destablization of chromosomes. RESULTS: Dose-response data, with a particular emphasis at low dose (< 1.0 Gy), indicate a frequency of approximately 4% per gray for the induction of chromosomal instability in clones derived from single progenitor cells surviving exposure to iron ions. The induction of chromosomal instability by gold ions was, however, less responsive to applied dose, as the observed incidence of this phenotype varied from 0 to 10% over 1-8 Gy. Both iron and gold ions gave dose-dependent increases in the yield of chromosomal aberrations (both chromosome- and chromatid-type) measured at the first mitosis following irradiation, as well as shoulderless survival curves having D0=0.87 and 1.1 Gy respectively. CONCLUSIONS: Based on the present dose-response data, the relative biological effectiveness of iron ions is 1.3 for the induction of chromosomal instability, and this indicates that heavy ions are only slightly more efficient than X-rays at eliciting this delayed phenotype.
Non-NASA Center↗
X-ray Ptychography Imaging of Human Chromosomes After Low-dose Irradiation
Abstract Studies of the structural and functional role of chromosomes in cytogenetics have spanned more than 10 decades. In this work, we take advantage of the coherent X-rays available at the latest synchrotron sources to extract the individual masses of all 46 chromosomes of metaphase human B and T cells using hard X-ray ptychography. We have produced ‘X-ray karyotypes’ of both heavy metal–stained and unstained spreads to determine the gain or loss of genetic material upon low-level X-ray irradiation doses due to radiation damage. The experiments were performed at the I-13 beamline, Diamond Light Source, Didcot, UK, using the phase-sensitive X-ray ptychography method.
59 BASIC BIOLOGICAL SCIENCES↗
Non-DNA radiosensitive targets that initiate persistent behavioral deficits in rats exposed to space radiation
Predicting future CNS risks for astronauts during deep-space missions will rely substantially on ground-based rodent data with space-relevant ions and behaviors. For rats, the accumulated evidence indicates that less densely ionizing radiation, such as 4 He and 12 C ions, induce behavior deficits at lower doses than densely ionizing ions, such as 48 Ti and 56 Fe. However, this observation conflicts with standard somatic radiobiology, in which densely ionizing ions are generally more effective than less densely ionizing ions, and where the DNA/nucleus is the accepted target for radiation-induced tumorigenesis, cytogenetic aberrations, genetic mutations, and reproductive cell death. To gain deeper insight into the subcellular nature of the radiation targets for behavior risks, we compared the effects of dose, fluence, and linear energy transfer (LET) of 4 He and 56 Fe particles using existing datasets for four distinct behavioral outcomes in rats: elevated plus maze (EPM-anxiety), novel object recognition (NOR-memory), operant responding (OR-response to environmental stimuli), and attentional set-shifting (ATSET-cognitive flexibility). We confirmed that less densely ionizing particles (except protons) showed ~100-fold lower threshold doses than densely ionizing particles for behavioral deficits (0.1–1 cGy for 4 He vs. 15–100 cGy for 56 Fe). However, when analyzed by fluence the behavioral responses converged, indicating that 4 He and 56 Fe were equally effective on a per-track basis. When analyzed by LET, there were ~100-fold differences in the LET for maximum effectiveness for behavioral deficits and DNA endpoints (~1 vs ~100 keV/μm, respectively). These unique features of radiation-induced behavioral deficits (high sensitivity to particles in the 1-keV/μm range, insensitivity to protons in the 0.2 keV/μm range, and isofluence dependence for particles with LET>1 keV/μm) provide evidence in support of a new hypothesis of sub-micron sized radiosensitive targets for behavioral effects consistent with the thickness of plasma membranes and/or small subcellular structures, smaller than a whole synapse. Like our behavior findings, mouse immature oocyte killing which is known to have a plasma membrane target was also better explained by fluence, rather than dose. In contrast, fluence analyses for DNA/nuclear endpoints in somatic cells (e.g., tumor induction, chromosome aberrations) showed opposite results, suggesting that behavior targets are not DNA. Our findings raise questions regarding the identity of subcellular targets and the multi-cellular functional unit for behavior risks, low-dose susceptibility, and generalizability from rat to other species and astronauts.
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
Analysis of muntjac deer genome and chromatin architecture reveals rapid karyotype evolution
Abstract Closely related muntjac deer show striking karyotype differences. Here we describe chromosome-scale genome assemblies for Chinese and Indian muntjacs, Muntiacus reevesi (2 n = 46) and Muntiacus muntjak vaginalis (2 n = 6/7), and analyze their evolution and architecture. The genomes show extensive collinearity with each other and with other deer and cattle. We identified numerous fusion events unique to and shared by muntjacs relative to the cervid ancestor, confirming many cytogenetic observations with genome sequence. One of these M. muntjak fusions reversed an earlier fission in the cervid lineage. Comparative Hi-C analysis showed that the chromosome fusions on the M. muntjak lineage altered long-range, three-dimensional chromosome organization relative to M. reevesi in interphase nuclei including A/B compartment structure. This reshaping of multi-megabase contacts occurred without notable change in local chromatin compaction, even near fusion sites. A few genes involved in chromosome maintenance show evidence for rapid evolution, possibly associated with the dramatic changes in karyotype.
59 BASIC BIOLOGICAL SCIENCES↗