Heavy ion induced DNA double strand breaks in cells of E. coli.

Vegetative cells of E. coli differing in their radiosensitivity have been used in heavy ion irradiation experiment. Besides inactivation measurements also the induction of DNA double strand breaks (DSB) have been measured using the method of pulse-field gel electrophoresis. This method allows to separate linear DNA with length up to 8 Mio base pairs. After irradiation with heavy ions we find a higher amount of low molecular weight fragments when compared to sparsely ionizing radiation. This agrees with the idea that heavy ions as a structured radiation have a high probability to induce more than one strand break in a DNA molecule if the particle hits the DNA. The amount of intact DNA remaining in the agarose plugs decreases exponentially for increasing radiation doses or particle fluences. From these curves cross sections for the induction of DSB after heavy ion irradiation have been determined. These results will be discussed in comparison to the results for cell survival.     

Effect of heavy ions on bacterial spores.

Inactivation of B. subtilis spores has been studied using accelerated He, C, N, O and Ne ions. The energy dependence of the inactivation cross sections for heavy ions was very weak and the mean cross sections for carbon ions (0.6-4.7 MeV/amu), nitrogen ions (0.6-4.1 MeV/amu), oxygen ions (0.8-1.1 MeV/amu), and neon ions (2.2-3.7 MeV/amu) were found to be about 0.22, 0.23, 0.26, and 0.33 micrometer2 , respectively. Analysis was carried out along lines similar to Katz's target theory but the parameters were chosen so that they have an experimental basis.     

Microdosimetric considerations of effects of heavy ions on microorganisms

An estimation of dose around the trajectory of an ion has been made by use of Tabata and Ito's energy deposition algorithm for electrons which takes into account transmission coefficient. The result of the calculation, as well as Butts and Katz's dose, is successfully applied to the interpretation of inactivation cross sections of vegetative cells of E. coli B_s−1, and E. coli B/r and of B. subtilis spores for He, C and N ions.     

Biological effects of heavy ions from the standpoint of target theory.

The biological effect of heavy ions is best described through the action cross section, as a function of the end-point of interest and the charge and speed of the ion. In track theory this is called the "ion-kill" cross section, for it is the effect produced by a single heavy ion and its delta rays. As with nuclear emulsions the biological track structure passes from the grain count regime to the track width regime to the thindown region with an increase in LET. With biological cells, as with any detector capable of storing sublethal damage, with low LET irradiation the action cross section (in the ion-kill mode) is increasingly obscured by the effect of "gamma-kill", by the influence of overlapping delta rays from neighboring heavy ions. Thus at low LET response is dominated by the gamma-kill mode, so that the RBE approaches 1. The theory requires 4 radiosensitivity parameters for biological detectors, extracted from survival curves at several high LET bombardments passing through the grain count regime, and at high doses. Once these are known the systematic response of biological detectors to all high LET bombardments can be unfolded separating ion kill from gamma kill, predicting the response to a mixed radiation environment, and predicting low dose response even at the level of a single heavy ion. Cell killing parameters are now available for a variety of cell lines. Newly added is a set of parameters for cell transformation.     

K-ionization and biological effect.

Experimental inactivation cross sections are compared to vacancy production in inner shell (K-shell) of so-called strategic C, N, O atoms of the DNA: in a trial calculation, atoms on the DNA backbone or just contiguous to it are considered as strategic. Various processes are considered: ionization and electron capture by the primary particle, ionization by secondary electrons. The last phenomenon is shown to be important for highly charged and medium velocity ions. A strong similarity is observed between variations versus LET of inactivation and K-vacancy cross sections, especially with regards to LET values for which they maximize: for K-ionization these maxima occur when the ion impact velocity roughly matches the K electron velocity. It is shown that the "K-hypothesis" allows a quantitative fit of experimental inactivation cross sections if a 4% mean lethal efficiency is attributed to ion-induced K-vacancies in strategic C, N, O atoms.     

Mutagenic effects of heavy ions in bacteria.

Various mutagenic effects by heavy ions were studied in bacteria, irradiated at accelerators in Dubna, Prague, Berkeley or Darmstadt. Endpoints investigated are histidine reversion (B. subtilis, S. typhimurium), azide resistance (B. subtilis), mutation in the lactose operon (E. coli), SOS chromotest (E. coli) and lambda-prophage induction (E. coli). It was found that the cross sections of the different endpoints show a similar dependence on energy. For light ions (Z < or = 4) the cross section decreases with increasing energy. For ions of Z = 10, it is nearly independent of energy. For heavier ions (Z > or = 26) it increases with energy up to a maximum or saturation. The increment becomes steeper with increasing Z. This dependence on energy suggests a "mutagenic belt" inside the track that is restricted to an area where the density of departed energy is low enough not to kill the cell, but high enough to induce mutations.     

Cell inactivation, repair and mutation induction in bacteria after heavy ion exposure: results from experiments at accelerators and in space.

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.     

Mutagenic effects of heavy ions in bacteria.

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.     

Role of endogenous thiols in protection.

Aminothiols represent the most important group of radioprotective compounds. The most effective compounds administered at an optimal dose and time before irradiation are able to provide a protection in mice with a dose reduction factor (DRF) of about 2-2.5. The working mechanism can partly be explained as a scavenging process of radicals induced in water and partly as a chemical repair process of injured DNA. The endogenous aminothiol which has far-out the highest intracellular concentration is glutathione (GSH). The importance of intracellular GSH in determining cellular radiosensitivity has been shown by irradiating cells that had very low GSH levels. Such cells appear to have a high radiosensitivity, especially in hypoxic conditions. On the other hand, it has been demonstrated that induction of a high GSH level (100-200% above the normal level) provides only a small protection. In vitro experiments with DNA indicate that thiols with a high positive charge condense in the vicinity of DNA and are effective protectors, whereas thiols with a negative charge are kept away from it and are poor protectors. In comparison with the most effective exogenous aminothiols like cysteamine and WR1065, GSH is not an effective radioprotector. Putative explanations for this relatively poor protective ability of GSH are presented.     

Saturn's E ring

The E ring is a loose collection of debris orbiting Saturn well outside the classical ring system. Compared to the classical ring system, the E ring is extraordinarily tenuous but occupies an enormous volume. The optical depth has a pronounced maximum near the orbit of Enceladus, which is therefore widely assumed to be the source of ring material. Optical properties of the ring suggest a collection of micron-sized spheroids whose composition is generally supposed to be water ice in accordance with the assumed Enceladus source.Apart from its unusual physical properties, the very existence of the E ring as a permanent feature of the Saturn system is problematical. The particles of the ring are susceptible to erosion by ion sputtering and outward transport by ion drag. Lifetimes against either of these processes in the presence of observed ion populations are estimated to be no more than a few thousand years, and it is not clear that Enceladus can replenish the ring particles continuously on such a time scale. It has thus been suggested that the E ring is a transient feature of the Saturn system, resulting from an episodic disturbance of Enceladus (e.g., a sizeable meteoroid impact or tectonic event) in the recent past. In any case, the E ring presently provides a convenient natural probe of magnetospheric transport processes because it presents a marginally significant cross section for the absorption of magnetospherically-trapped particles traversing its volume.     

Nuclear fragmentation database for GCR transport code development

A critical need for NASA is the ability to accurately model the transport of heavy ions in the Galactic Cosmic Rays (GCR) through matter, including spacecraft walls, equipment racks, etc. Nuclear interactions are of great importance in the GCR transport problem, as they can cause fragmentation of the incoming ion into lighter ions. Since the radiation dose delivered by a particle is proportional to the square of (charge/velocity), fragmentation reduces the dose delivered by incident ions. The other mechanism by which dose can be reduced is ionization energy loss, which can lead to some particles stopping in the shielding. This is the conventional notion of shielding, but it is not applicable to human spaceflight since the particles in the GCR tend to be too energetic to be stopped in the relatively thin shielding that is possible within payload mass constraints. Our group has measured a large number of fragmentation cross sections, intended to be used as input to, or for validation of, NASA’s radiation transport models. A database containing over 200 charge-changing cross sections and over 2000 fragment production cross sections has been compiled. In this report, we examine in detail the contrast between fragment measurements at large acceptance and small acceptance. We use output from the PHITS Monte Carlo code to test our assumptions using as an example ⁴⁰Ar data (and simulated data) at a beam energy of 650 MeV/nucleon. We also present preliminary analysis in which isotopic resolution was attained for beryllium fragments produced by beams of ¹⁰B and ¹¹B. Future work on the experimental data set will focus on extracting and interpreting production cross sections for light fragments.     

Nuclear fragmentation of high-energy heavy-ion beams in water.

As a part of the physical-technical program of the heavy-ion therapy project at GSI we have investigated the nuclear fragmentation of high-energy ion beams delivered by the heavy-ion synchrotron SIS, using water as a tissue-equivalent target. For a direct comparison of fragmentation properties, beams of 10B, 12C, 14N, and 16O were produced simultaneously as secondary beams from a primary 18O beam and separated in flight by magnetic beam analysis. The Z-distributions of beam fragments produced in the water target were measured via energy loss in a large ionisation chamber and a scintillator telescope. From these data we obtained both total and partial charge-changing cross sections. In addition we have performed Bragg measurements using two parallel-plate ionization chambers and a water target of variable length. The detailed shape of the measured Bragg curves and the measured cross sections are in good agreement with model calculations based on semi-empirical formulae.     

Cellular parameters for track structure modeling of radiation hazard in space.

Based on irradiation with 45 MeV/u N and B ions and with Co-60 gamma rays, cellular parameters of Katz's track structure model have been fitted for the survival of V79-379A Chinese hamster lung fibroblasts. Cellular parameters representing neoplastic transformations in C3H10T/1/2 cells after their irradiation with heavy ion beams, taken from earlier work, were also used to model the radiation hazard in deep space, following the system for evaluating, summing and reporting occupational exposures proposed in 1967 by a subcommittee of NCRP. We have performed model calculations of the number of transformations in surviving cells, after a given fluence of heavy charged particles of initial energy 500 MeV/u, penetrating thick layers of cells. We take the product of cell transformation and survival probabilities, calculated along the path lengths of charged particles using cellular survival and transformation parameters, to represent a quantity proportional to the "radiation risk factor" discussed in the NCRP document. The "synergistic" effect of simultaneous charged particle transfers is accounted for by the "track overlap" mode inherent in the model of Katz.     

Inactivation probability of heavy ion-irradiated Bacillus subtilis spores as a function of the radial distance to the particle's [correction of paricle's] trajectory.

The understanding of the radiobiological action of heavy ions requires the knowledge of the dependence of the inactivation probability on the distance between the particle's trajectory and the biological test organism (the impact parameter). Spores of Bacillus subtilis with a cytoplasmic core of about 0.22 micrometer cross section are suitable test objects for the study of this radial inactivation probability in its microscopic details. The spores are irradiated at low fluences of some 10(6) ions/cm2 with very heavy ions at different specific energies up to 10 MeV per atomic mass unit u while in fixed contact with visual nuclear track detectors. The methods are described by which the biological response of individual cells can be evaluated and the impact parameter be determined with an accuracy typically better than 0.2 micrometer. The results demonstrate that the common characteristics of inactivation, e.g., an effective range of inactivation extending to at least 3 micrometers, a nonmonotonic dependence of the inactivation probabilities on the radial distance, and the fact that the inactivation probability even for direct central hits on the cytoplasmic core is substantially below one, are nearly independent of the particle energy and type. The results are incompatible with the assumption that the radiobiological effectiveness can be attributed to the dose of secondary electrons as currently understood. They also demonstrate that the widely held notion of an "overkill" at low impact parameters does not apply for the spores even with the most densely ionizing ions.     

Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms.

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.     

Early and late mammalian responses to heavy charged particles.

This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles. RBE-LET relationships for these mammalian responses are compared with results from in vitro studies. The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture. Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture. Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure. Issues discussed focus on challenges associated with assessments of early and late effects of charged particles based on dose, RBE and LET, and with the concordance or discordance of results obtained with in vivo and in vitro model systems. Models for radiation damage/repair and misrepair should consider effects observed with in vivo as well as in vitro model systems.