RBE: mechanisms inferred from cytogenetics.

Cyclotron-accelerated heavy ion beams provide a fine degree of control over the physical parameters of radiation. Cytogenetics affords a view into the irradiated cell at the resolution of chromosomes. Combined they form a powerful means to probe the mechanisms of RBE. Cytogenetic studies with high energy heavy ion beams reveal three LET-dependent trends for 1) level of initial damage, 2) distribution of damage among cells, and 3) lesion severity. The number of initial breaks per unit dose increases from a low-LET plateau to a peak at approximately 180 keV/micrometer and declines thereafter. Overdispersion of breaks is significant above approximately 100 keV/micrometer. Lesion severity, indicated by the level of chromosomal fragments that have not restituted even after long repair times, increases with LET. Similar studies with very low energy 238Pu alpha particles (120 keV/micrometer) reveal higher levels of initial breakage per unit dose, fewer residual fragments and a higher level of misrepair when compared to high energy heavy ions at the same LET. These observations would suggest that track structure is an important factor in genetic damage in addition to LET.     

Experimental models for cellular radiation targets: LET, RBE and radioprotectors.

The topics of this presentation are: a brief review of the early research, the ideas it stimulated and the ways they are used in current efforts to explain cellular radiosensitivity; an analysis of the strengths and weaknesses of two experimental models used in vitro for simulating the induction of double strand breaks (DSB) and single strand breaks (SSB) in situ. Note that when alkali is used to denature cellular DNA for the determination of strand breaks, both overt SSB and the SSB that result from DSB in the denaturation process are recorded as total strand breaks (TSB). All information is taken from published literature.     

Repair of DNA double-strand breaks and its effect on RBE.

DNA double-strand breaks (DSB) are induced linearly with absorbed dose both for sparsely and densely ionizing radiations. By enzymatic repair the linear relationship between the number of DSB and absorbed dose is converted into a non linear one. Furthermore, the RBE-values of high LET radiations for residual DSB increase with increasing amount of DSB repair especially in the low dose range. Unrepaired and/or misrepaired DSB are supposed to be responsible for chromosomal aberrations, cell killing, oncogenic cell transformation and gene mutation. At low doses, for these endpoints much higher RBE-values than those for initial DSB are observed. However, with increasing doses the RBE-values for these endpoints approach those for initial DSB. These observations are likely to be interpreted using the following two parameters of the energy deposition structure: 1. The distribution of clusters with respect to their size at the nm-scale and to the number of ionizations per cluster (cluster distribution). 2. The distribution of distances between clusters of definite size and with definite number of ionizations (distance distribution of clusters). For the induction of DSB solely the ionization density in clusters of nm-dimensions (i.e. the cluster distribution) is important. For unrepaired or misrepaired DSB (responsible for chromosome aberrations, cell killing, oncogenic cell transformation and gene mutation) both the cluster distribution and the distance distribution of clusters are relevant. At low doses the distance distribution of clusters along a single particle track determines the RBE-value. However, with increasing dose the distribution of clusters produced by all particles traversing the cell nucleus becomes increasingly determinant. Here, solely the cluster distribution is important as it is the case for the induction of DSB.     

Monte Carlo track structure studies of energy deposition and calculation of initial DSB and RBE.

Estimation of exposure due to environmental and other sources of radiations of high-LET and low-LET is of interest in radiobiology and radiation protection for risk assessment. To account for the differences in effectiveness of different types of radiations various parameters have been used. However, the relative inadequacy of the commonly used parameters, including dose, fluence, linear energy transfer, lineal energy, specific energy and quality factor, has been made manifest by the biological importance of the microscopic track structure and primary modes of interaction. Monte Carlo track structure simulations have been used to calculate the frequency of energy deposition by radiations of high- and low-LET in target sizes similar to DNA and higher order genomic structure. Tracks of monoenergetic heavy ions and electrons were constructed by following the molecular interaction-by-interaction histories of the particles down to 10 eV. Subsequently, geometrical models of these assumed biological targets were randomly exposed to the radiation tracks and the frequency of energy depositions obtained were normalized to unit dose in unit density liquid water (l0(3) kg m-3). From these data and a more sophisticated model of the DNA, absolute yields of both single- and double-strand breaks expressed in number of breaks per dalton per Gray were obtained and compared with the measured yields. The relative biological effectiveness (RBE) for energy depositions in cylindrical targets has been calculated using 100 keV electrons as the reference radiation assuming the electron track-ends contribution is similar to that in 250 kV X-ray or Co60 gamma-ray irradiations.     

Induction of high grade astrocytoma (HGA) by protons: molecular mechanisms and RBE considerations.

Protons of a specific energy, 55 MeV, have been found to induce primary high grade astrocytomas (HGA) in the Rhesus monkey (Macaca mulatta). Brain tumors of this type were not induced by protons of other energies (32-2,300 MeV). Induction of HGA has been identified in human patients who have had radiation therapy to the head. We believe that the induction of HGA in the monkey is a consequence of dose distribution, not some unique "toxic" property of protons. Comparison of the human experience with the monkey data indicates the RBE for induction of brain tumors to be about one. It is unlikely that protons cause an unusual change in oncogenic expression, as compared to conventional electromagnetic radiation.     

Quantitative interpretation of heavy ions effects: models for the biological effects of heavy ions.

Heavy ions are an important part of space radiation. Although they contribute only about 1 percent in number the fraction in terms of energy deposited is much higher. Also the quality of radiation is different from the other components since the LET is generally quite high. This poses the problem of Relative Biological Effectiveness (RBE). It is considerably more important in space than on earth because shielding measures are costly and sometimes not even feasible. Radiation hazards appear to be the limiting factor In long term space flights and their evaluation constitutes a major task. There is still no general agreement about RBE of earthbound radiation, and even less concerning the biological weighting of very heavy and very energetic ions in space. Because of the lack of experimental data--particularly for risk estimates in humans-- theoretical approaches may be very helpful in this respect and provide the only means to judge the radiation protection situation in outer space. In order to be useful careful checks of their consistency are necessary. This paper summarizes some of the more common approaches in a critical manner. The unhappy conclusion at the end will be that at present it is not possible to understand even heavy ion action on survival quantitatively with an acceptable precision.     

Radiation effects in space.

The radiation protection guidelines of the National Aeronautics and Space Administration (NASA) are under review by Scientific Committee 75 of the National Council Protection and Measurements. The re-evaluation of the current guidelines is necessary, first, because of the increase in information about radiation risks since 1970 when the original recommendations were made and second, the population at risk has changed. For example, women have joined the ranks of the astronauts. Two types of radiation, protons and heavy ions, are of particular concern in space. Unfortunately, there is less information about the effects on tissues and cancer by these radiations than by other radiations. The choice of Quality Factors (Q) for obtaining dose equivalents for these radiations, is an important aspect of the risk estimate for space travel. There are not sufficient data for the induction of late effects by either protons or by heavy ions. The current information suggests a RBE for the relative protons of about 1, whereas, a RBE of 20 for tumor induction by heavy ions, such as iron-56, appears appropriate. The recommendations for the dose equivalent career limits for skin and the lens of the eye have been reduced but the 30-day and annual limits have been raised.     

The amphibian egg as a model system for analyzing gravity effects.

Amphibian eggs provide several advantageous features as a model system for analyzing the effects of gravity on single cells. Those features include large size, readily tracked intracellular inclusions, and ease of experimental manipulation. Employing novel gravity orientation as a tool, a substantial data base is being developed. That information is being used to construct a 3-D model of the frog (Xenopus laevis) egg. Internal cytoplasmic organization (rather than surface features) are being emphasized. Several cytoplasmic compartments (domains) have been elucidated, and their behavior in inverted eggs monitored. They have been incorporated into the model, and serve as a point of departure for further inquiry and speculation.     

HZE effects on mammalian cells.

In track segment experiments cell survival and chromosome aberrations of mammalian cells have been measured for various heavy ion beams between helium and uranium in the energy range between 0.5 and 960 MeV/u, corresponding to a velocity range of 0.03 to 0.87 C, and an LET spectrum from 10 to 15 000 keV/micrometers. At low LET, the cross section (sigma) for cell killing increases with increasing LET and shows a common curve for all ions regardless of the atomic number. This indicates that in this region the track structure of the different ions is of only a minor influence, and it is rather the total energy transfer, which is important for cell killing. At higher LET values, deviations from a common sigma-LET curve can be observed which indicate a saturation effect. The saturation of the lighter ions occurs at lower LET values than for the heavier ions. These findings are also confirmed by the chromosome data, where the efficiency for the induction of chromosomal aberrations for high LET particles depends on the track structure and is nearly independent of LET. In the heavier beams (Z > or = 10) individual particles cause multiple chromosome breaks in mitotic cells.     

Gravity effects on membrane processes.

Application of the Gouy-Chapman-Debye-Hückel (GCDH) theory to a model membrane in contact with electrolytes of various concentrations and composition predict density variations within an interfacial layer. Assuming that on cellular dimensions hydrodynamics can be applied (the objections are briefly discussed) two types of gravity effects can be defined, 1. convection along the surface of vertically oriented membranes and 2. surface potential variations by layer deformations at horizontally oriented membranes. Both effects should affect transport across the layer to the membrane surface and across the membrane. According to the theoretical predictions first experiments with gramicidin channels incorporated into artificial phosphatidylserine bilayer membranes show a significant difference in single channel currents in vertical and horizontal membranes. The complexity of biological membrane functions requires investigation of isolated membrane surface reactions and transport systems to study the gravisensitivity for each process separately.     

Teratogenic effects of gravitational changes.

In investigating the effect of gravitational changes on development, it is instructive to think of altered gravity (delta g) as a teratogen--that is, an environmental factor influencing development. Observed effects on skeletal development include: suppression of morphogenesis in centrifuged mouse limb buds; advanced fusion stages in centrifuged mouse palates; smaller crown rump lengths (CRL) and decreased number of pregnancies in centrifuged rats and mice; altered differentiation of growth plates in young growing rats in space; and decreased length of calcified long bone regions in fetal rats exposed to microgravity in utero. These studies show that delta g is able to alter development in vivo and in vitro and suggest that delta g operates, at least in part, at the cellular level.     

Gravity effects on biological systems.

Theoretical investigations of the membrane-solution interface predict different effects of gravity on vertically and horizontally oriented planar membranes. Single channel events of gramicidin incorporated into phosphatidylserine planar bilayer membranes were measured in 0.1 M KCl solution, pH 7, at room temperature. The potential difference across the membrane was set to +/- 70 mV. The mean channel current was observed to be about 20% higher in horizontally oriented membranes compared to vertical membranes. This is in good agreement with the theoretical considerations and demonstrates that gravity does affect membrane processes by interaction with the membrane-solution interface which is a ubiquitous structure in biological systems.     

Neuritogenesis: a model for space radiation effects on the central nervous system.

Pivotal to the astronauts' functional integrity and survival during long space flights are the strategies to deal with space radiations. The majority of the cellular studies in this area emphasize simple endpoints such as growth related events which, although useful to understand the nature of primary cell injury, have poor predictive value for extrapolation to more complex tissues such as the central nervous system (CNS). In order to assess the radiation damage on neural cell populations, we developed an in vitro model in which neuronal differentiation, neurite extension, and synaptogenesis occur under controlled conditions. The model exploits chick embryo neural explants to study the effects of radiations on neuritogenesis. In addition, neurobiological problems associated with long-term space flights are discussed.     

Experimental concept for examination of biological effects of magnetic field concealed by gravity.

Space is not only a place to study biological effects of gravity, but also provides unique opportunities to examine other environmental factors, where the biological actions are masked by gravity on the ground. Even the earth's magnetic field is steadily acting on living systems, and is known to influence many biological processes. A systematic survey and assessment of its action are difficult to conduct in the presence of dominant factors, such as gravity. Investigation of responses of biological systems against the combined environment of zero-gravity and zero-magnetic field might establish the baseline for the analysis of biological effects of magnetic factors. We propose, in this paper, an experimental concept in this context, together with a practical approach of the experiments, both in orbit and on the ground, with a thin magnetic shielding film. Plant epicotyl growth was taken as an exemplar index to evaluate technical and scientific feasibility of the proposed system concept.     

A potentialgradient-conductivity-scanner for the investigation of effects leading to buoyancy-driven convection on continuous-flow-electrophoresis.

Continuous flow electrophoresis (CFE) is a mild and high-resolving method for the separation of sensitive biomolecules like peptides and proteins. The resolution is decreased by several effects, of which density gradients are important since they lead to buoyancy-driven convection. A tool for the measurement of physical quantities that cause the separation process, a potentialgradient/conductivity-scanner has been developed. With this PC-scanner consecutive potential- and concentration profiles can be measured inside the CFE-chamber. Local current densities and local joule heat yielding density gradients are calculated from the measurement data. With the PC-scanner the influence of gravity on separations by CFE can be evaluated more easily by the knowledge of the thermal and electrical conditions inside the separation slit.     

Microelectronics effects as seen on CRRES.

A MicroElectronics Test Package (MEP) measured total dose degradation and single event upsets (SEUs) on 60 device types on the Combined Release and Radiation Effects Satellite (CRRES) in an 18 degrees inclination orbit between 350 km and 36000 km from July 1990 to October 1991. Simultaneous measurements of the high energy particle environment were used to make a direct cause and effect comparison of the energetic particle backgrounds and microelectronic performance characteristics. The galactic cosmic ray background for the period of the CRRES mission was at a minimum. The SEUs experienced from the cosmic ray background were correspondingly few in number, but surprisingly produced an equal probability of upset over an L-shell range of 8.5 Earth radii (RE) down to less than 3.0 RE. Cosmic ray induced upset frequencies in proton sensitive chips were over 2 orders of magnitude lower than those produced by protons in the heart of the inner proton radiation belts. Multiple upsets, those produced when a single particle upsets more than one memory location, were just as common from protons as from cosmic rays.     

Combined effects of radiation and trauma.

Injuries, caused by both whole-body irradiation and wounds or burns, have been relatively little studied. Possibly because many investigators think that these injuries are just modified radiation-induced diseases for which the same treatment principles are valid. Other authors had the impression that, for instance, the radiation burn trauma is a new kind of disease which differs significantly from either radiation syndrome alone or from burn disease. There are many experimental data on animals which suggest that the pathology of combined injuries differs significantly from that of radiation-induced disease or of thermal or mechanical traumas. Wounds or burns which, in general, do not cause septicaemia could become entrance ports for bacteria when animals are exposed to whole-body irradiation. Thrombocytopenia is the reason for hemorrhages in wounds. The susceptibility to shock is increased considerably in combined injuries and the formation of callus in the bone fractures is significantly delayed. The healing of wounds and burns in the initial phase of the radiation syndrome does not always differ from healing in the non-irradiated organism. However, a few days or weeks later very serious wound infections and hemorrhages can occur. The additional injuries almost always worsen the development and prognosis of radiation-induced disease. The recommended treatment for combined injuries will differ in many respects from the treatment of wounds and burns or the radiation syndrome.     

Animal models for the study of the effects of spaceflight on the immune system.

Animal models have been used to determine the effects of spaceflight on the immune system. Rats and rhesus monkeys have been the primary animals used for actual space flight studies, but mice have also been utilized for studies in ground-based models. The primary ground based model used has been hindlimb unloading of rodents, which is similar to the chronic bed-rest model for humans. A variety of immune responses have been shown to be modified when animals are hindlimb unloaded. These results parallel those observed when animals are flown in space. In general, immune responses are depressed in animals maintained in the hindlimb unloading model or flown in space. These results raise the possibility that spaceflight could result in decreased resistance to infection in animals.     

A ground-based study for a shuttle BRIC experiment on gravity effects on gene expression.

A BRIC (Biological Research In a Canister) experiment to investigate the effects of reduced gravity at the molecular level using Arabidopsis has been initiated. In preparation for a space flight experiment, a series of ground-based studies were conducted. Results from these studies indicate that: 1) up to 20,000 seeds can be germinated on a 100 mm diameter Petri plate, 2) nylon membrane is the best surface for recovery of plant material after freezing, 3) depending on the age of the seedlings at the time of freezing, 20 to 40 g of tissue can be obtained from Petri plates that fit in a single canister; 4) tissue from one canister yields adequate amounts of RNA to perform differential display to isolate gravity-regulated genes. Our results indicate that the proposed BRIC experiment is feasible and can provide valuable information on the possible effects of microgravity on gene regulation.