Raymond E. Zirkle

Effects of ploidy and linear energy transfer on radiobiological survival curves.

Abstract The radiobiological influence of linear energy transfer (energy transferred per unit length of the track of an ionizing particle) was investigated with respect to inhibition of cell division in three microorganisms: a diploid strain of the yeast Saccharomyces cerevisiae; a haploid yeast derived form the diploid; and the unicellular green alga Stichococcus. Complete survival curves were obtained for each organism at each of nine different values of linear energy transfer (LET), ranging from 0.73 to 190 k.e.v. per micron of path in tissue. Most of these values of LET were obtained by using different portions of the paths of very speedy deuterons, accelerated helium nuclei, and natural α-particles in such fashion that, in any given exposure, the value of LET was essentially the same for all particles that traversed the cell sample. At all values of LET, the survival curves of the haploid yeast were exponential, while those of the diploid were strongly sigmoid and of a shape essentially independent of LET. The relative biological effectiveness (RBE) (a quantity inversely proportional to the 50 % survival dose) of the nine radiations for both yeasts was practically constant for LET values from 0.73 to 27 k.e.v./μ but increased by about a factor of four or five between 27 and 190 k.e.v./μ. The yeast survival curves are consistent with a theory that, in the haploid, cell division can be inhibited by inactivating any one of some tens of chromosomal sites by means of a single ionizing particle, whereas in the diploid it is necessary to inactivate both members of any one allelic pair of corresponding sites. The variation of RBE with LET is not consistent with simple target theory but is readily explained in terms of chemical intermediates which can diffuse from their places of origin in the ionization tracks to the chromosomal sites. The survival curve of Stichococcus was sigmoid, and the RBE increased by a factor of twelve as LET varied from 1.5 to 190 k.e.v./μ.

The role of the spleen in radiation injury.

Summary and Conclusions These hematologic and histologic data indicate that: 1) Severe anemia, leucopenia, and thrombocytopenia develop in mice after a single dose of 600 r whole-body X radiation. 2) Ectopic erythrocytopoiesis, in the lead-protected spleens of mice given 600 r whole-body X radiation (exclusive of spleens) compensates with such rapidity and so extensively for the destruction and interruption of this activity in the marrow spaces that no anemia of significance becomes apparent. Ectopic granulocytopoiesis and megakaryocytopoiesis in the lead-protected spleens compensates significantly but at a slower pace and less completely for the bone marrow destruction. 3) A marked and sustained decrease in the amount of lymphatic tissue is produced in the lead-protected spleens of animals given 600 r whole-body X radiation. This decrease in lymphatic tissue may perhaps be a result of (a) unsuccessful competition of the lymphatic tissue with the ectopic hematopoiesis for nutritional requirements, (b) actual indirect effect of radiation and (c) a differential humoral suppression from some unknown site. The rapidity with which erythrocytopoiesis transfers from the X-ray damaged bone marrow to the lead-protected spleen in the absence of anemia suggests that the mechanism of stimulation of erythrocytopoiesis under the conditions of this experiment may involve some factor or factors other than, or in addition to, the accepted hemoglobin-oxygen relationship. This technic permits more or less exclusive protection of the spleen or the appendix or other visceral tissues from irradiation while applying various dosages to the remainder of the body. It provides a method of studying potential sites and mechanism of the production of ectopic blood formation, possible secondary effects of radiation as well as offering possibilities for determining the potential role of such sites in immune reactions, in preventing or alleviating radiation-induced hemorrhagic phenomena and in the study of survival or recovery from radiation injury.

PARTIAL-CELL IRRADIATION

Techniques for partial-cell irradiation and the preparation of radiation sources for use both inside and outside the cell are described. Applications of partial-cell irradiation techniques in studies of normal cell functions and as a radiobiological tool to yield information about the mechanisms by which radiations produce their effects on living systems are discussed. Results are summarized for recent studies which employed this technique. (71 references.) (C.H.)

Relationships Between Chemical and Biological Effects of Ionizing Radiations

In radiobiology we are interested in chemical effects of ionizing radiations only in so far as they may be involved in biological effects.2 As background for discussion of this relationship, let us briefly review the general state of our knowledge (or ignorance) of mechanisms of radiobiological actions:1. The first event in such an action is, of course, the absorption of radiant energy by molecules in the biological object or its medium. The molecules which thus acquire energy in excess of normal are said to be activated. Some absorb so much energy that electrons are ejected; these molecules are said to be ionized. Others absorb less energy; electrons are merely shifted to other orbits; such molecules are said to be excited. The foregoing physical processes are at present fairly well understood, and many aspects of them can be described quantitatively.2. After the absorption of energy, our knowledge of the radiobiological action is practically zero until the biological effect occurs. This effect—depending...

DISAPPEARANCE OF SPINDLES AND PHRAGMOPLASTS AFTER MICROBEAM IRRADIATION OF CYTOPLASM

I n earlier publications1

Shrinking and Swelling after Alpha Irradiation of Various Parts of Large Erythrocytes.

* it was reported that heterochromatic ultraviolet microbeam i r r a d i a t i ~ n ~ . ~ of a small portion of the cytoplasm of metaphase newt (Triturus) cells in tissue culture caused the spindle to diminish or, if exposure was sufficient, disappear. Since spindles were thus affected even when the site of bombardment was as far as 30 p away, it was concluded that the effect is probably mediated by a spindle “poison” produced photochemically from a precursor that is some constituent of normal cytoplasm. It is of some interest to know whether the precursor is present only in newt cells or is a widely distributed cellular substance. As an approach to this question, experiments were performed on developing sand-dollar eggs5 (R. B. Uretz and R. E. Zirkle, in preparation) and it was found that in the two-celled stage, with each cell showing a bright metaphase spindle by polarization microscopy, suitable UV microbeam irradiation in the cytoplasm of one cell caused the spindle birefringence* to disappear promptly in that cell but not in the other. Thus i t would appear that the precursor is some substance present both in vertebrates and in echinoderms. Recently these observations have been extended to the seed plants (R. H. Haynes and R. E. Zirkle, in preparation) by use of the Bajer preparation of endosperm cells of Haemanthus Kathurilzae.6 A typical result, as observed by polarization microscopy, is shown in FIGURE 1. A slightly flattened metaphase cell is shown in FIGURE la, the spindle axis extending vertically. The spindle is bright by polarization microscopy. I n FIGURE l b the site of bombardment is indicated by the circumscribing cross-hairs (phase microscopy). FIGURE l c was taken 7 min. after irradiation; the birefringence of the spindle is no longer visible. It therefore appears that the precursor probably has a wide distribution among taxonomic groups.

ACTION SPECTRA FOR MITOTIC SPINDLE DESTRUCTION AND ANAPHASE DELAY FOLLOWING IRRADIATION OF THE CYTOPLASM WITH AN ULTRAVIOLET MICROBEAM

Summary Erythrocytes from Amphiuma and from man were irradiated in vitro with polonium alpha particles (a) from an extended source (4 mm in diameter) and (b) with a small beam (20 μ in diameter). The cells first shrank, then swelled and hemolyzed. Local irradiation of a part of a single cell resulted in shrinkage only in the irradiated portion. This was followed by swelling of the whole cell and hemolysis.

Components of the acute lethal action of slow neutrons.

Abstract— Previous workers have reported that, in a variety of cells, ultraviolet microbeam irradiation of cytoplasm adjacent to the mitotic spindle causes the spindle to diminish or disappear and have postulated that this effect is mediated by a spindle “poison” produced photochemically from a cytoplasmic precursor. In the present work, a microbeam action spectrum for this effect has been obtained to aid in the identification of this mediator material. A small portion of the cytoplasm of Ambystoma(salamander) tissue‐culture cells was irradiated in early metaphase with ultraviolet microbeams of various wavelengths and intensities but of the same diameter (8 μ). Spindle destruction was scored by observing diminution of its image as seen with a polarization microscope. In addition, the time of anaphase was recorded so that an action spectrum for anaphase delay was also obtained. These two action spectra are quite similar and are nearly parallel to the absorption spectrum of a tyrosine‐containing protein. A few proteins are discussed which might mediate this effect.

The Plutonium Project

Early in the history of the Plutonium Project, months before the first chain reacting pile was built, it was realized that both fast and slow neutrons would leak from any operating pile of finite dimensions and thus constitute radiation hazards. Fast neutrons, of course, were not a new radiobiological agent in 1942; reports of their various biological effects had been accumulating since 1935. With slow neutrons, however, there had been no radiobiological experience, because the earlier sources of neutrons, such as cyclotrons, had not been suitable for such investigations. Certain it was that slow neutrons would have radiobiological action, because it was known that slow neutrons were captured by and would react with the nuclei of many of the kinds of atoms found in tissues; each of these nuclear reactions was known to produce ionizing particles by some means, direct or indirect, and in many cases a significant fraction of the ionizing energy of these particles was certain to be absorbed in the nearby tiss...

Irradiation of Parts of Individual Cells

THE Plutonium Project was assigned the task of making and purifying the artificial element plutonium for use in atomic bombs. The method of making plutonium utilized the nuclear reaction between neutrons and uranium238, the neutrons being excess from a so-called nuclear chain reaction maintained by other neutrons and uranium235. The uranium238 and uranium235 were not separated. They were in their natural mixed condition. The fact that the chain reaction was used meant that according to pre-war standards fantastic amounts of gamma rays and fast and slow neutrons would be emitted in a chain reactor of any considerable magnitude. We were familiar, to a certain extent, with these radiations before the war, but we had had no experience whatever with the large amounts anticipated in a chain reactor. In addition to the tremendous potential hazards due to the radiations emitted in the chain reaction, there were also enormous hazards to be anticipated in the purification of the plutonium, since plutonium was forme...