Differences in radiation response between cells in S-phase and non-S-phase cells of the granulocyte/macrophage progenitor (GM-CFC) compartment.

Studies were performed to investigate the radiation response of granulocyte/macrophage progenitor cells from canine bone marrow in different proliferative states, and in which way it will change if the S-phase cells are eliminated from the irradiated populations. To obtain progenitor cells of different proliferative states, bone marrow cell suspensions were kept in liquid cultures for 1 or 3 days in the presence of colony stimulating activity. Radiation dose response curves were determined (a) for the total population of progenitor cells under normal conditions (fraction of cells in S-phase 35%), (b) in a state of rapid cycling (fraction of S-phase cells 53% to 57%), and (c) after sterilization of S-phase cells by pretreatment with 3H-thymidine. The rapidly proliferating progenitor cells showed a strong decrease in their radiosensitivity (D0 = 0.84 Gy) within the first day in suspension culture when compared to the normal population (D0 = 0.50 Gy). The cell populations from which the S-phase cells had been eliminated were found more sensitive than the respective total populations (D0 values in the range from 0.44 Gy to 0.50 Gy). The D0 values for the S-phase cells were between 0.57 Gy and 1.13 Gy depending on the proliferative state of the cell populations. These data indicate that granulocyte/macrophage progenitor cells during progression through the S-phase become less radiosensitive than they are in other phases of the cell cycle.

Responses to accelerated heavy ions of spores of Bacillus subtilis of different repair capacity.

Inactivation, mutagenesis of histidine reversion and the involvement of DNA repair were studied in spores of Bacillus subtilis irradiated with heavy ions at LBL, Berkeley and GSI, Darmstadt. Five groups of ions (from boron to uranium) were used with residual energies from 0.2 MeV/u up to 18.6 MeV/u; in addition, carbon ions were used with a residual energy of 120 MeV/u. Action cross sections of both inactivation and mutagenesis show a similar dependence on ion mass and energy: for lighter ions (Z less than or equal to 10), the lethal response is nearly energy independent (Z = 10) or decreasing with energy (Z less than or equal to 6); these light ions, up to 18.6 MeV/u, induce hardly any mutations. For heavier ions (Z greater than or equal to 26), the lethal as well as the mutagenic responses increase with ion mass and energy up to a maximum or saturation. The efficiency of DNA repair to improve survival and the mutagenic efficiency per lethal event, both, increase with ion energy up to a saturation value which, depending on strain and endpoint, either roughly coincides with the X-ray value or is smaller than that after X-ray treatment. For repair based on recombination events, the increase in the survival effects with ion energy is more pronounced than for that based on repair replication. At energies of 1 MeV/u or below, neither DNA repair nor mutation induction appear to be significant. The results support previous suggestions on the importance of the radial distribution of the energy around the ion track in biological action cross section and the evidence that the entire core of the spore represents the sensitive site in responses to heavy ions.

Hematological effects of unilateral and bilateral exposures of dogs to 300-kVp X rays.

Accidental exposures to ionizing radiation from external sources usually result in an inhomogeneous dose distribution rather than a homogeneous total-body irradiation (TBI). To study the hematological effects of an inhomogeneous dose distribution, dogs were unilaterally exposed to a beam of 300 kVp X rays (HVL = 3.8 mm Cu) with their left side directed to the source. The entrance and exit surface doses were 3.8 Gy and 0.9 Gy, respectively. Dose measurements performed in bone marrow spaces of various bones revealed a maximum of 3.1 Gy in the head of the left humerus and a minimum of 0.9 Gy in the right iliac crest. Based on survival for granulocyte-macrophage progenitor cells (GM-CFC) determined in different bone marrow sites 24 h after the exposure, the dose-dependent reduction ranged from 0.44 to 16% of the control values. The regeneration of the GM-CFC compartments in the various bone marrow spaces showed patterns which were independent of each other up to Day 28. Values were normal again at Day 125 after exposure. For comparative purposes, three dogs were exposed bilaterally to achieve a homogeneous dose distribution. They received a TBI of 2.4 Gy, which according to previous calculations should have caused the same systemic damage to the GM-CFC compartment as the unilateral exposure. The peripheral blood cell changes, including the GM-CFC, and the colony stimulating activity in the serum showed a similar pattern for both exposures. These findings support the hypothesis that the overall survival fraction of progenitor cells in the bone marrow is the main determinant of the blood cell changes, independent of the anatomical distribution.

In vitro studies of the sensitivity of canine bone-marrow erythroid burst-forming units (BFU-E) and fibroblast colony-forming units (CFU-F) to X-irradiation.

The radiosensitivity of the early erythroid progenitor cells (BFU-E) and the progenitor cells of the stroma (CFU-F) in canine bone marrow was studied under steady-state conditions by in vitro irradiation with 280 kV X-rays. The dose-effect relationship for colony formation was determined for BFU-E obtained from the iliac crest marrow, and for CFU-F in bone marrow collected from the iliac crest and the humerus of adult beagles. The BFU-E were adequately stimulated with serum from lethally irradiated dogs to obtain a source of BPA (burst-promoting activity). The BFU-E proved to be extremely radiosensitive, and the survival curve was exponential (D0 = 15.3 +/- 1.8 cGy). We showed that buffy-coat leukocytes separated from bone marrow leukocytes obtained by aspiration were an optimum source of CFU-F. A curve was fitted to the data obtained for CFU-F obtained from the iliac crest or the humerus, resulting in D0 = 241 +/- 38 cGy and an extrapolation number n = 1.38 +/- 0.62 or D0 = 261 +/- 40 cGy and n = 1.04 +/- 0.42, respectively. According to these findings, and other published data, we conclude that the canine bone marrow BFU-E are presently the most radiosensitive hemopoietic cells detected among all hemopoietic cells of different mammals.

Hematological effects in dogs after irradiation of the lower part of the body with a single myeloablative dose.

The lower body of dogs, containing approximately 30% of the total bone marrow, was exposed to 300 kV X-rays with a single myeloablative dose of 11.7 Gy, whereas the upper body was shielded by a lead box. The results of the present study are discussed in connection with recently published results obtained after irradiation of the upper body (UBI), containing approximately 70% of the total bone marrow mass. The main findings are as follows: (1) the nadir in the blood concentration of thrombocytes, lymphocytes, and granulocytes strongly depends on the volume of irradiated bone marrow; (2) apart from some quantitative differences, the time-related pattern of changes in the concentration of granulocyte/macrophage progenitor cells (GM-CFC) in irradiated and shielded bone marrow sites is very similar after irradiation of the lower part of the body (LBI) and UBI, i.e. is apparently independent of the relative amount of damaged bone marrow at volumes applied in the present models; (3) the concentration of GM-CFC in the blood after LBI shows a transient increase during the first phase of most rapid bone marrow GM-CFC regeneration, i.e. between day 7 and day 23; the magnitude of this transient increase obviously depends on the fraction of irradiated bone marrow.

Hematological effects in dogs after sequential irradiation of the upper and lower part of the body with single myeloablative doses.

The compensating mechanisms determining the tolerance of the hemopoietic system to sequential hemibody irradiation (HBI) with large single doses, the regeneration of the irradiated bone marrow and the long-term effects of such treatment were studied in dogs. The main emphasis was laid on the determination of the granulocyte/macrophage progenitor cells (GM-CFC) in the bone marrow and blood. The general pattern of events in the GM-CFC compartment after each exposure was similar. Irradiation with a dose of 11.7 Gy of the upper body (UBI), that involved the abrogation of approximately 70% of the total active marrow, was followed by an immediate increase in the proliferation and differentiation of GM-CFC in the protected bone marrow. Repopulation of the GM-CFC in the irradiated sites most probably due to seeding of hemopoietic cells from the protected marrow already became evident at day 7 after UBI. At day 56 after UBI, when the irradiation of the lower body (LBI) was performed, the GM-CFC had recovered to between 30 and 40% of their pre-treatment values. Despite this incomplete regeneration, the GM-CFC compartment responded to LBI in a similar way as the GM-CFC had in the protected (normal) marrow after UBI, i.e. by an increased proliferation for at least 21 days. Already at day 7, the bone marrow of the iliac crest that had been exposed to LBI showed a considerable number of GM-CFC. Within no more than 370 days all the bone marrow sites irradiated during either the first or the second treatment had regained their normal GM-CFC values.

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.

Growth of erythroid burst-forming units (BFU-E) in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs.

Erythroid burst-forming units (BFU-E) from canine bone marrow and peripheral blood could be grown in methylcellulose in the presence of an appropriate batch of fetal calf serum (FCS), transferrin, and erythropoietin (Epo). However, improved colony formation (size and number of bursts) was obtained when serum from total body irradiated dogs was present in the culture. This serum, obtained from dogs at day 9 after total body irradiation with a dose of 3.9 Gy, reduced markedly the Epo requirement of BFU-E. Furthermore, it allowed the omission of FCS from the culture medium if cholesterol and bovine serum albumin (BSA) were used as FCS substitutes. BFU-E concentrations were found to be rather different in the peripheral blood and in bone marrow samples from different sites (i.e., iliac crest, sternum, and humerus) of normal beagles. The studies further show that canine bone marrow BFU-E can be cryopreserved in liquid nitrogen.

Genetic response of bacterial spores to very heavy ions.

Using spores of two Bacillus subtilis strains differing in repair capacity, we have studied repair and mutation induction in the spores after irradiation with very heavy ions up to uranium with specific particle energies up to 18.6 MeV/u. The results indicate that repair and mutation induction after heavy ion irradiation are closely related to each other and that both phenomena strongly depend on the atomic number and specific energy of the ions. The effects are discussed in comparison with results obtained after X-irradiation.

Mutation induction in spores of Bacillus subtilis by accelerated very heavy ions.

Mutation induction (resistance to sodium azide) in spores of Bacillus subtilis was investigated after irradiation with heavy ions from Neon to Uranium with specific particle energies between 0.17 and 18.6 MeV/u. A strong dependence of the mutation induction cross section on particle charge and energy was observed. From the results it was concluded that mutation induction in bacterial spores by very heavy ions is mainly caused by secondary electrons.

Advanced biostack: experiment 1 ES 027 on Spacelab-1.

The radiobiological properties of the heavy ions of cosmic radiation were investigated on Spacelab 1 by use of biostacks, monolayers of biological test organisms sandwiched between thin foils of different types of nuclear track detectors. Biostacks were exposed to cosmic radiation at several locations with different shielding environments in the module and on the pallet. Evaluations of the physical and biological components of the experiment to date indicate that in general they survived the spaceflight in good condition. Dosimetric data are presented for the different shielding environments.

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.