Outline of Activities Under the Division of Biological and Medical Research

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DIVISION O F BIOLOGICAL AND MEDICAL RESEARCH Prepared for the meeting of the Advisory Committee i n Biology and Medicine at Argonne National Laboratory, March 13, 1953

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Since it will not be possible to present the work of the Division in detail at the advisory committee meeting, this brief outline has been prepared, mainly by the staff members concerned, to give an ove pall view of the directions in which research is being pursued. Details have been avoided, but the individual staff m e m b e r s will be glad to supplement this material with demonstrations and discussions. Certain “programmatic” areas of research will be discussed in more detail at the meeting. These include neutron toxicity, radium toxicity, and other internal radioelement toxicity. A chronic gamma r a y toxicity program has been planned and will be initiated in a few months; in the meantime a small program on soft X-ray effects on large animals will be carried out by the same staff. Outlines of these programs are included here.

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In addition t o overseeing the histologic and electron microscopic work of the Division this group is investigating the morphological and functional aspects of mesenchyme and effects of radiation on it, and has begun experiments on the cornea and intestinal epithelium. A submicroscopic network of solitary fibrils was shown to be a regular component of connective tissue, This was published last year. The formation of collagenous fibrils i n the regenerating tendon of the rat has been studied. The results are ready for publication. The formation of f i b r i l s in the regenerating tendon of the guinea pig is being studied under the conditions of Vitamin C deficiency and of recovery from experimental scurvy. The material from scorbutic guinea pigs also serves the purpose of deciding whether o r not the microscopic structure of the collagenous fib ril s undergoes changes due to the absence from the tissue of ascorbic acid. A reliable technique has been established for examining by the electron microscope the solution i n dilute acid and the reconstitution i n alkaline media of collagenous fibrils. constitution of fib ril s will be studied, The effect of ionizing radiation on the r e - The effect of nitrogen mustard and of ether on the mitotic activity in rat's corneal and intestinal epithelium is being studied at the present time. This group has placed m a j o r emphasis on the cytological effects of continuous irradiation of cells, utilizing tissue cultures, tumors, and mammalian tissues. The structure of the resting nucleus and effects on it of high intensity radiation a r e also being studied, The implications of the cytological effects of point source irradiation of tissue t o carcinogenesis by radioactive particles is of special interest to this group. Continuous irradiation of proliferating cells was shown t o have a n effect in many ways similar t o colchicine, suggesting that local spindle damage is an important radiation effect. Controlled continuous irradiation of certain tis sues (notably rapidly growing tumors) may, for several days, result in a normal rate of cell growth without cell division. Rates of synthesis of cellular compounds a r e to be investigated; and with similar preparations, the effect on cell differentiation will be studied. Immediate radiation effects on resting nuclei, probably associated with depolymerization and local nuclear damage, a r e under scr t'

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Turnover of white blood cells between blood a n d organs is being investigated by organ culture techniques. The cytological response of the mouse spleen and other organs to intermittent radiation has yielded unexpected findings which may be related to leukemogenesis. The group has collaborated with various other investigators in tissue culture experiments. The work of this group h a s to do with the late effects of absorbed radioelements, and falls i n the programmatic area. The present permissible values f o r plutonium depend largely on past work in this group. This group was established to investigate many unknown aspects of neutron toxicity and has used fission neutron and gamma irradiation, largely of mice. This program will also be outlined later. Chronic External Irradiation (E. Lorenz, Consultant) This group comprises a small staff associated with Dr. Lorenz at the National Cancer Institute, and was established to ensure close coordination of his work with hematological and biometric studies at Argonne. This group has made substantial contributions i n the areas of chronic gamma-ray effects and radiation protection by bone marrow. The work of this group has been carried out along a number of lines including both cellular and g r o s s mammalian physiology. Much of it has been oriented to radiation protection problems, and basically t o the question of factors influencing radiosensitivity. Studies with anaerobiosis and protective substances have done much t o support the in vivo importance of the reactions of irradiated water. These problems are being approached (1) by evaluating protective agents in terms of the many different radiation effects; (2) by studying relative effectiveness and additivity of protective factors with different qualities of radiation; and ( 3 ) by ascertaining influence of such factors as temperature and metabolic agents on protection. Much present evidence suggests that cysteine acts to

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reduce apparent radiation dose by deletion of a proportional amount of the radiation effect. Cysteine appears to afford significant protection against fission neutrons; with the higher neutron flux to be obtained soon, it will be possible to study this more decisively and to evaluate anoxia as well. Cysteine protection cannot be interpreted altogether in t e r m s of immediate oxidative reactions, as indicated by experiments in the protection of thymocytes, where conditions during the 30 minutes after irradiation a r e critical for the degree of protection. In view of this and other in vitro findings, the Krebs ascites tumor will be used in future studies. At present, the growth constants of this tumor a r e being established and the mechanisms of ascites formation, as well as physiological effects of the tumor on the host, Radiation effects on hepatic function, utilizing metabolic t e s t s of liver functions in rats, and involving interference with the normal oxygen supply to the liver, a r e being investigated. The relation between blood cell production and destruction is under investigation by several approaches, i n the hope of discovering homeostatic mechanisms keeping them in balance, as it is thought that they may have important bearing on the pathogenesis and therapy of radiation injury. A t present, plasma protein fractions a r e being studied in animals subjected to irradiation and to leucocyte depletion by other means, and i n the latter case (depletion into intraperitoneally injected fluid) the balance between blood and ascitic fluid leucocytes will be studied i n relation to the bone marrow It has been found that tissue mast cells a r e widely disrupted and r e - duced in number after total-body irradiation. The relation of this phenomenon to blood coagulation changes is of interest. In addition, basic studies in the origin, functions, and control of these little-known cells are being carried out in vivo by direct observation, and in tissue culture. Dogs and rats a r e being utilized to study concomitantly radiation effects on blood sedimentation and on the several plasma protein fractions. The relation of the plasma proteins to plasma amino acid changes and to increased amounts of histamine and other factors, and to anemia and anorexia, In the light of present knowledge of circulatory phenomena i n irradiated animals, the cardiac output and related phenomena (oxygen consumption, peripheral vascular resistance, and cardiac function) a r e being examined throughout the course of the acute radiation syndrome.

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This group includes a number of independent investigators and, f r o m t i m e to time, graduate students, fellows, and visiting associates. Under this group such subjects as the toxicity of T20,the disposition of acutely inhaled C02, activation analysis in soft tissues, and theoretical considerations in isotope retention and carcionogenesis, have been studied. The pathogenesis of the acute radiation syndrome in birds is under investigation by D r . Stearner. This state involves derangements in uric acid excretion, is very sensitive to dose r a t e , and may be reproduced both by local kidney irradiation and extrarenal irradiation. By combining adrenalin with anoxia, a remarkable degree of protection has been obtained. The mechanism of the syndrome requires further study, correlation with histological findings and with the radiation syndrome i n other animals. The basic parameters of the carcinogenic process are almost unknown. They a r e being studied mainly i n relation to irradiation, but other carcinogenic stimuli will profit by similar technique. ' An unsuccessful attempt t o produce carcinogens in irradiated cholesterol (about lo9 rep) was completed, and other normal compounds a r e being treated i n the same way. The differences in radiosensitivity of tumors in vivo and in vitro have been explained on the basis of the influence of anoxia. Electron micrographs of human and animal kidneys have shown structure s in the glomeruli which probably have a n important role in glomerular filtration. Studies will be made on the appearance of injected uranium in those structures and in tubular cells. . Effects of developing immunity against heterologously transplanted tumors have been measured, and future studies are aimed at discovering the role of lymphocytes in this process. Utilizing fodder f r o m the biosynthesis group, attempts will be made to produce radiotoxic effects in m i c e with G l 4 . Attempts to do this with large amounts of bicarbonate we r e unsuc ce ssful . Growth and synthetic processes after sublethal X radiation are being studied in Chilomonas and Tetrahymena. Duration of cell division block is a function of dose; during this period, most synthetic processes a r e unimpaired. The period before recovery i s , with a given dose, equal to a constant number of control generations. Some isolated c e l l s yield clones with permanently reduced growth rates; the nature and incidence of these deficiencies is being studied.

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work on COz fixation on a long term basis which serves to determine safe a i r levels of C14. The present investigations have grown out of this and have to do in general with mammalian C 0 2 fixation. They include: The effect of dietary intake of amino acids on fixation of GOz into the essential amino acids; de novo biosynthesis of s e r i n e and glycine in ani malt issues ; and the biological symmetry of glycerol and related compounds. In addition, the chemical nature of bone carbonate is under investigation. Present experiments have to do with relative rates of uptake of P3‘ and C14 under various conditions of incubation in vitro. The investigations of this group a r e oriented mainly toward recognition of biological effects brought about by radiations o r radioactive elements whose physical o r chemical mode o r locus of interaction with matter is known, as contrasted to studies on systems affected by radiations whose immediate effects are less well known orare unknown. The interests are almost entirely on mechanisms of radiation effect, and microorganisms a r e utilized. Current investigations are: . 1. The nature of the radiosensitive site i n the m a h g reaction in Paramecium bursa ria. The action spectrum for increase in reactivity sugg e s t s a flavin as the initial photoreceptor. This hypothesis will be tested through the application of flavin analogs. The action of visible light on the mating substance itself will also be investigated $th the large spectrograph. 2. The biological effects of very long wave length X rays. Using Bacillus megaterium spores in the dried state we propose t o irradiate in vacuo and thus to deliver to these organiams very soft X r a y s which have well-known precise loci of action in matter. 3. Specific ionization effects in E. megaterium. As a companion to the above, the effectiveness of deuterons, neutrons, and alpha particles compared with X and y rays will be measured. 4. The mechanisms of action of methylated purines in inducing bact e r i a l mutation studied with C14 labeled substances. The possibility of the incorporation of the mutagen into nucleic acids is being investigated. 5. The effect of very low temperatures (25O absolute or less) is r e - ducing the effectiveness of X r a y s in inactivating dried T-1 bacteriophage, a n extension of the work which has shown that at 77OK inactivation is but half that observed a t 273OK. This finding has important implications in current

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6 . Physico-chemical separation of particles f r o m yeast cells containing the postulated cytoplasmic particle s carrying the adaptive enzyme Immunology (B. Jaroslow, graduate student) The project is concerned with the effect of X irradiation on the innate and acquired immunity of the mouse against Trypanosoma duttoni. The r e - sults up t o the present have shown that X irradiation with 550 r breaks down both acquired and innate immunity t o this parasite. It has been shown that the spleen plays a definite role in the maintenance of the level of immunity. It is planned to investigate the effects of irradiation at various times before and after infection and to study the results of different dose levels upon innate and acquired immunity. The cytological, physiological, and biochemical aspects of the effects of X irradiation a r e being studied concurrently, with grasshopper embryos a s experimental material. Because the development of the organism depends on enzymes and their spatial relationships, as well as on the division of labor involved in the differentiation of tissues, almost all of these experiments a r e being carried out in vivo. It was discovered that tissue differentiation in the embryos was the physiological function most susceptible to irradiation. Cell division and anabolism were next most affected, and energy metabolism o r catabolism was the function most resistant to exposure. When metabolic processes were depressed t o 680 times in AF under anaerobic conditions, the dosages required t o elicit equal damage at a high o r normal A F output, in air, required 10 times the dose of ionizing radiaThe nymph ovary is being used to study the RBE of X r a y s , y rays, The c u r r e n t work of this group is that of the preparation of compounds of highest possible purity in quantities sufficient f o r the use of the plant physiology group. Special techniques have had to be developed f o r the syntheses of indolyl pyruvic acid, indolyl lactic acid, indolyl acetaldehyde, and indolyl propyleneglycol because of the instability of the compounds. These materials a r e of interest because of their structural relationships t …