Some Biological Aspects of Experimental Radiology A Historical Review

Some Biological Aspects of Experimental Radiology A Historical Review

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F G SPEAR Strangeways Research Laboratory. Cambridge. England Page 2 I . Introduction ...................................................... 7 I1. Biological Response to Radiations ................................. 1. Acute and Chronic Effects of Irradiation ..................... 9 2. Latent Period ............................................... 11 3. Direct, Indirect. and Constitutional Effects ................... 12 a . Direct Effect ............................................ 12 13 b. Indirect Effect ........................................... 14 c. Constitutional Effect ..................................... 4. Some Obvious Biological Effects of Penetrating Rays .......... 14 14 a Mitosis ................................................... 16 b. Metabolism .............................................. 17 c. Motility ................................................. d . Mutation ................................................. 19 20 e. Malignancy ............................................... 21 I11. Variations in Response with Different Tissues ..................... 21 1. Choice of Material .......................................... 23 2. Biological Indicators ......................................... 23 a . Vegetable Cells and Tissues ............................. b . Bacteria, Protozoa, Fungi, Molds, and Yeasts ............. 26 c. Ova, Larvae, and Some Developing Forms ................. 29 32 d. Tissue Cultures ........................................... 34 e. Higher Organisms ........................................ 37 f . Human Tissues ........................................... 39 IV . Effect of Radiation on Generative Tissues ......................... 1. Generative Glands as Biological Indicators of Tissue Response 41 42 2. Classification and Selection of Material ....................... 42 a . Process of Maturation: Testis ............................. 43 b. Process of Maturation: Ovary ............................. 43 c. Effect of Radiation on Germ Cells ......................... d. Effect of Radiation on Cell Constituents : Chromosomal Struc45 tural Change ............................................. e. Heritable Changes Unaccompanied by Visible Structural 47 Alteration ................................................ f . Animal Experiments in Relation to Human Data ............. 49 51 3. Radiation Sensitivity ......................................... V . Radiation Physics and Radiation Chemistry in Relation to Radiobiology 52 54 1. Radiation Physics ........................................... 54 a. Dosimetry ................................................ 54 b. Mechanism of Action .................................... 55 c. Protection ................................................ 56 d. Permissible Dose ......................................... e. Dose Units and Relative Biological Efficiency............. 56

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2. Radiation Chemistry ......................................... a. Mode of Action of Radiation .............................. b. Chemical Protection ...................................... c. Radioisotopes ............................................. d. Beneficial Uses of Radioisotopes ........................... e. Deleterious Effects of Radioisotopes ....................... f. Detection of Radioisotopes ................................ VI. Conclusions ....................................................... VII. Acknowledgments ................................................. VIII. References ........................................................

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I. INTRODUCTION Much confusion of thought would be avoided if the historical approach to scientific learning were permitted to occupy a more prominent place in the curriculum. The boundless optimism of the research worker would be suitably curbed, and the prodigal exploitation of popular and ambitious speculations would be reduced. (F. J. Cole, 1944)

Sixty years ago (1896) medical and lay presses of the world were loudly acclaiming the “marvellous triumph of science which is reported from Vienna” (a mistake for Wiirtzburg) and discussing with various degrees of excitement the possible use to which the newly discovered Xrays might be put, especially in the surgical field (Gifford, 1896; Lancet, 18% ; Natwre, 1896). Today (1956) medical and lay presses of the world are loudly declaiming against the harmful effects of penetrating rays now that virtually all mankind has, in consequence of the advances in nuclear science, become exposed to radiation hazards (cf. London Times, 1955; Brit. J . Radiol., 1957b ; Reporter, 1957). The sixty years between these two obtrusions of ionizing radiation into public notice have seen the rise and development of radiation biology into an independent field for investigation, with a steady increasing literature which has now reached vast proportions. Among the plethora of biological effects of penetrating rays which have been reported, three groups of observations have been mainly responsible for the remarkable change in the general attitude to radiation between 18% and 1956. The first was the experimental production of radiation-induced cancer in mice by Clunet in 1910. At the time this did not attract the attention it deserved partly because of Clunet’s own attitude to his discovery (Mustacchi and Shimkin, 1956) but mainly perhaps because it occurred some eight years after the first report of a human cancer arising in X-ray-damaged tissues (Frieben,

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1902). Clunet's experiment, however, was the first of many others which showed that radiation was a carcinogenic agent, and it preceded by five years the publication of the discovery of chemical carcinogens (Yamagiwa and Ichikawa, 1918; cf. Cook and Kennaway, 1940). [Compared with many of these, however, radiation is a much less efficient agent (Glucksmann, 1951-1954; Boag and Glucksmann, 1956).] The second group of observations occurred almost midway in the period which concerns us. Muller (1927) and Stadler ( 1928a, b) independently proved quantitatively the mutagenic properties of penetrating rays and thereby opened up a whole new field of investigation now included in the term radiation genetics (Muller, 1930, 1951; Catcheside, 1947, pp. 66, 109). Outside its own immediate field, however, this discovery had an important influence on the theory of radiation action (cf. Lea, 1946, p. 126) ; within it, radiation has become such an indispensable tool for geneticists everywhere that the subject has now become an impressive branch of the science of heredity (Wallace, 1956). The third group of observations led 'to the gradual realization that at least for man the injurious effects of radiation could, in some individual instances, remain latent for a variable number of years and then give rise to harmful lesions (including malignant disease) even though the subject had not been exposed to any further irradiation in the meantime (March, 1944 ; Fillmore, 1952) and during the interval appeared to be in normal health (Hatcher, 1945; Jones, 1953; Lisco et d.,1947; Simpsdn et al., 1955). A sudden focusing of public attention on the harmful effects of radiation was brought about by the publication of an official document of the U. S. Atomic Energy Commission on the biological hazard occasioned by radioactive fallout from atomic explosions (U.S. Atomic Energy Commission, I955 ; Cronkite et al., 1956) and by popular accounts which appeared in the medical and lay press about the same time of radiation injuries received by the crew of the Japanese boat Fakztryu Mara (Brit. Med. J., 1955). Unconvinced by the later admission that the doses first calculated as already received from atom bomb testing were an overestimate (Brit. Med. J., 1956), an acutely apprehensive public both in the Old World and the New has pressed for a reassessment of the place of radiation in human society (Medical Research Council, 1956 ; National Academy of Science, 1956). The change of attitude has not, however, been merely a superficial alteration in the public reaction to radiological matters: it has affected the pattern of scientific research in this field as well. So long as belief in the wholly beneficial action of radiations (provided they were properly used) was firmly held, the purpose of a great deal of radiological research

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was to further the curative use of radiation in medicine (Freund, 1904). The realization that penetrating rays could have both mutagenic and carcinogenic effects gave great impetus to investigations and speculations concerning the mechanism of radiation action first within the individual cell, then at subcellular levels, and finally in terms of chemical complexes and even of single chemical radicals (see Magee et al., 1953). As attention progressively becomes more concentrated on fewer chemical processes within smaller portions of the irradiated cell, there is a tendency to lose sight of the great variety of biological responses which are presented within the field of radiation biology, all of which must eventually be recognized and allowed for if a comprehensive explanation of radiation action is to be obtained and protection from its harmful effects achieved. Many of these responses seem incompatible with any simple mechanistic explanation of radiation effect whether physical or chemical. It is a purpose of this review to recall some of this variety which still continues to thwart the most well-meaning attempts at simplification for theoretical convenience. Diagnostic radiology was the first and certainly the most spectacular application of X-rays, and it was quickly followed by suggestions for the use of penetrating radiation as a disinfecting agent (Lyon, 1896). This effort met with little success at the time but is interesting historically in view of recent developments in this field (Dysart et d.,1954) ; on the other hand, diagnostic radiology, which was originally given such an enthusiastic welcome, is now in some quarters being criticized for the disproportionate contribution its excessive use may make to mankind’s total dose of radiation (Osborn and Smith, 1956). Next came the application of penetrating rays to therapeutics. Once the (empirical) discovery of the curative action of X-radiation on skin carcinoma had been made (cf. Forssell, 1931) there could be no question of delaying the practice of radiotherapy until the field of radiation biology had been completely explored to provide an adequate scientific basis. The medical man who wishes to lessen the empiricism of his radiotherapeutic practice, however, may be well advised to consider the results of at least some of the basic research in radiation chemistry, radiation physics, and radiation biology [this is the order in which Freund (1904) places the disciplines concerned] before too vigorously applying the many new radiological tools now available in increasing quantities for the treatment of human disease. The history of neutron therapy in California is both a warning and a pointer (Stone, 1948). Besides the medical radiologists who in spite of some criticism (or “croakings,” as Freund described it) optimistically applied the new tool in surgery and medicine with some dramatic successes, the early investiga-

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tors included the industrialists who vigorously attacked the technical problems associated with tube design and who in the process of their pioneering work later provided so large a contingent of radiation martyrs (Strahlentherapie Suppl. 1936) ; unattached investigators pursuing knowledge for its own sake, prompted only by curiosity to observe the response,to radiation of the widest variety of material both living and inanimate ; and the theorists who by 1896 had begun to discuss possible mechanisms of the action of radiation on biological material (Thomson, 18%). Their theories usually applied to the material with which they were familiar-a restraint which some more modern writers seem to think superfluous. Together these groups of observers collected a formidable mass of heterogeneous and sometimes apparently conflicting information, at first almost too quickly for its significance to be adequately assessed. For nearly thirty years the gross effects of radiation were studied mainly in their qualitative aspect and usually involved a lethal action or major disturbance in form or function of the irradiated material (see Colwell and RUSS,1924). The adoption of an international unit for the measurement of penetrating rays in 1928 (Brit. I . Radiol., 1928) marked a great advance in the development of radiological research and opened the way to a quantitative approach to many radiation problems. Direct comparison between experiments in different and often widely separated laboratories became possible and invited attention (not always given!) to dose recording and some consideration of the biological significance of dose level. Radiation exposure initiates many abnormal and pathological processes some of which are induced a t high, and others at low, dose level (cf. Glucksmann, 1954). If the results of two experiments are to be compared, it is desirable that at least the general pattern of radiation effects shall be similar, and that the dose levels producing them be of the same order. In an attempt to simplify experimental conditions and facilitate quantrtative observations both on the biological and on the physical side, many workers have selected a particular material to serve as a standard reference for biological response. It is easy to compile, though tedious to read, a whole alphabet of indicators which have been used (the list is purely illustrative of variety and makes no attempt to be exhaustive)-algae, amoebae, annelids, Arbacia, Ascwis ; bacteria, bats, bean roots, blood cells, Bodo caudatus ; Calliphora, chick embryos, chromosomes, Colpidium colpoda ; Daphnia, Drosophila, ducklings, duckweed ; enzymes, Euglena; frogs ; goats, goldfish, grasses, grasshoppers, guinea pigs ; hairs, hamsters, hormones, Hydra ; Infusoria, intestinal cells ; Jensen’s rat sarcoma ; kidney ; lentil seeds, lily, locusts,

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lymphocytes, lymphoid tissue ; malignant cells, mice, monkeys, muscle ; nerve tissue, Neurospora, newts ; osteoblasts, ova, ovary, oxyrrhis ; peas, pigeons, pollen grains ; rabbits, rats, retina, Rhizopus ; salamanders, sea urchins, silkworms, skin, spermatozoa, swine ; tadpoles, tissue cultures, toads, tomato, trypanosomes, turtles ; uterus ; vegetable tissues, viruses, Vorticella ; wasps, weevils, wheat, Xelaopus, xylem ; yeast, zygotes, zymogen, zymose. So long as observation is restricted to a single biological indicator or to one particular tissue, the experimental investigations seem fairly straightforward. This is the reason, perhaps, why to the newcomer the problems of radiobiology appear at first sight to be comparatively simple to solve. It is so easy to use radiation as a tool for producing a given type of response and then to continue studying that particular response on the same material on the assumption that the results must have a general application. It is when the diverse reactions of different cells and tissues are compared and the variety of response considered that the complexities of the situation appear (see Chase, 1953 ; Quastler, 1953) ; and the greater the range of material, the more complex do the radiobiological problems become. The adaptability of living cells to changes in their environment, including those brought about by radiation, is an unpredictable and varying factor except in the special cases where no reparative mechanism operates. The newcomer must first decide what he wishes to study and his angle of approach. He must also be ready to admit the limitations of his basic training for the work he may wish to do and be ready to listen to those trained in other disciplines and to learn their language (Chase, 1953; Mayneord, 1945). Radiation biology with extensions into physics and chemistry is itself a “group enterprise with many servants in varied stations” such as genetics, cytology, embryology, and ontology, to name only a few (Weiss, 1953). It Kas become a frontier science, and a frontier is “a place where thought may have to be translated to be communicated” and where emotions often tend to run high and give a distorted emphasis (Brues, 1955). It is here that the historical approach may be helpful in giving to those on one side of the frontier a more comprehensive view of the accomplishments of those on the other; and it may assist the newcomer by recalling forgotten investigations to get a proper perspective of experimental radiology as a whole. Since the living cell is the operative unit when penetrating rays act on biological material, it seems reasonable to base this survey on a consideration of the response of living tissues to irradiation as revealed by achievements in the field of radiation histology and radiation cytology, and

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then to relate these achievements to contributions from some of the other disciplines concerned with radiobiology. To keep the review within manageable proportions, however, it will be restricted on the biological side mainly to the reactions of norm1 tissues to penetrating rays, with priority of consideration for those experimental results which seem to have some bearing on the response of human tissues to irradiation. On the physical side it is limited chiefly to papers concerned with the effects following external radiation from conventional machines with only brief reference to the use of high-energy apparatus or radioisotopes as sources of internal radiation. The distinction between internal and external sources of radiation is essentially one of an alteration in the physical fahors of irradiation and does not call for detailed consideration here.

11. BIOLOGICAL RESPONSE TO RADIATIONS We are at the moment in the position of a man who tries to elucidate the mechanism of a telephone exchange by throwing bricks into it and observing some of the results. (J. A. V. Butler, 1956)

It would be impractical in the space available here to keep the historical perspective throughout this review, but a brief summary of the growth and development of radiation biology may assist the reader at this point. Three distinct, though overlapping, phases may be distinguished. First came the exploratory phase, whefi qualitative observations were made on a wide range of animate and inanimate materials and the general effects of radiation determined. The lethal action of penetrating rays on living cells and tissues was demonstrated ; local injuries (radiation burns) were observed; inhibition of growth in both normal and malignant tissue was seen, and in the lower range of dosage abnormal development was obtained from exposure of both somatic and generative tissues. Occasionally certain late effects of radiation exposure were noted : some radiation burns were followed by malignancy and blood changes some time after exposure to radiation had ceased. In spite of these deleterious results the general attitude toward radiation throughout this period was one of hope that, once the correct method of handling the agent had been learned, all the injurious actions of the rays could be avoided and their use confined to beneficial purposes in medicine, surgery, and biology. This led to the phase of application in the field of medical diagnosis, in industry, and in treatment of skin diseases and malignant growths. Diagnostic radiology does not concern us here, and the industrial uses of penetrating rays are also outside the scope of this review. An interesting

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account, however, of the present-day uses of X-rays in industry was given by Schall (1952) in the 31st Silvanus Thompson Memorial Lecture. The therapeutic application of X-rays began in a purely empirical fashion in 18% (Grubbe, 1933), and of radium in 1901 (Becquerel and Curie, 1901). The foundations of a scientific approach were laid in 1906 (Wickham and Degrais, 1910) with the establishment of the first biological laboratory for the experimental study of radium therapy in Paris, where Dominici did pioneer work in what would now be called radiation physics. Wickham was the first to use filters to increase the radium-tissue distance and absorb the less penetrating types of ray, and Dominici developed filtration by lead and aluminum screens to obtain a pure “ultra-penetrating” y-ray (Barcot, 1919-1920). H e was among the first td adopt Wickham’s “crossfire” methods to increase the depth dose in the irradiation of deepseated tumors. About the same time Abbe, to obtain high intensity, inserted radium into a goiter, leaving the material in situ for 24 hours, with startlingly successful results which he reported six years later (Abbe, 1911). Meanwhile, a series of observations were made by Perthes (1904a) on the use of aluminum filters with X-radiation following the original use of this element to eliminate electrical effects believed to be the cause of X-ray dermatitis (Tesla, 18%). Perthes also made the first depth-dose measurements, though others independently made similar observations and applied the results in clinical therapy. Thus a rational basis for radiation therapy began to be laid, a variety of dosage units were devised, and radiation biology became an independent field for investigation. Improvements in the design of X-ray tubes (cf. Imboden, 1931) and of radium applicators followed, and the need for a standard international unit of dose measurement became urgent (RUSS,1923). On the biological side the selective action of radiation was recognized and also the fact that the same dose produced a different result according to the time and intensity factors employed in its delivery. Proliferating tissues were found to show a more marked reaction to radiation than those without dividing cells ; and a latent period, which varied for different types of response, was shown to elapse between exposure and the appearance of radiation effects. The third and modern period may be called the preventive phase. As radiation sources became more powerful and larger volumes of tissue were involved in any given exposure, the problem of total dose to normal organs of the body became an urgent matter for investigation. The effects of radiation therapy on the general health of the patient could be severe, and it became important to have “some means of assessing quickly and accurately the energy absorbed by any or all of the distant parts of the body”

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(Wood et al., 1938). Thus the idea of integral or body close was cleveloped, marking a further but by no means final stage in the development of radiation dosimetry (Mayneord, 1940, 1942). This helped, but did not eliminate the occurrence of radiation sickness which might be induced, in sensitive individuals, by heavy exposures under therapeutic conditions or by whole-body irradiation whether planned (Levitt, 1940) or accidental (Hempelmann et al., 1952). The syndrome was reproducible in animals and could therefore be studied under experimental conditions (Curtis, 1951 ; Quastler, 1956). The symptomatology varied according to the system of the body which reacted most violently to the irradiation ; in some cases disturbance in the central nervous system predominated, in others the intestinal canal, and in others the hemopoietic apparatus. Besides these acute effects of radiation, more insidious changes could be induced by exposure, which led to long-term effects unassociated in their early stages with any recognizable disturbance of form or of function. In addition to these radiation actions affecting individuals it was found that even more insidious changes could be produced in the generative tissues, which caused heritable changes in the off spring. This, coupled with the increasing manufacture of radioactive substances and test explosions of atomic devices, has stimulated efforts at classification of biological response and the design of experiments to determine the mechanism of the action of radiation, with a view to devising means of protection other than the purely physical measures of distance factor and screenage. If the fundamental mechanism of radiation action proved to be a simple process, the solution of these problems would be greatly facilitated. I n the present chapter we shall consider some of the generalizations already made and commonly accepted, and later in the review proceed to examine in greater detail radiation response among different types of material.

1. Acute and Chronic Effects of Irradiation It is now generally realized that throughout the whole of his evolutionary history man, like all living organisms, has been exposed to small but variable amounts of ionizing rays from his natural surroundings. It is also common knowledge that all living tissue is killed outright if exposed to large enough doses of penetrating irradiation. W e have yet to find at what intermediate points between these two extremes the action of radiation begins to embarrass the cell in one or more of the almost infinite variety of processes which distinguish it as a living organism. It has become a convention to describe irradiation exposures, and, by simple extension, the effects they produce, as acute and chronic but with-

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out any attempt to define the line of demarcation between the two. A typical acute exposure is one in which the material is exposed to a relatively high dose followed by early and severe biological consequences. Chronic exposure, on the other hand, is one given either continuously at low intensity or as an extended series of irregular exposures over a prolonged time. Under these conditions the biological response appears more slowly, is less severe at the onset, variable in its histological characteristics, and long-standing in duration. An acute exposure can be given to any type of biological material. Chronic exposure, in the usual sense of the term, is applicable only to certain kinds of complex biological material where the life span of the organism is sufficiently long. Exposure throughout its lifetime of a single paramecium gives little scope for studying chronic effects, though this could be done with a colony of these protozoa. Most of the early experiments dealt with acute effects following high dosage. Observations began at a dose level which produced easily recognizable effects, a method which had the disadvantage of concealing small radiation effects under the more massive action of heavy irradiation. A more rewarding procedure, but one less often practiced, is to begin with subthreshold dosage and gradually increase exposure until some recognizable change is observed (Spear, 1931). The dose can then be further increased and any additional response noted. By this means small radiation effects can be detected and a correlation between effect and increasing dose established (Gliicksmann, 1954). The chronic effects of radiation were first observed not on material used for irradiation but on some of the experimenters themselves and those engaged in the manufacture of radiation apparatus. Among the latter it was customary to use the hands (dorsal surface toward the tube) to test the penetrating qualities of the radiation emitted from their apparatus, and not a few suffered the consequences of these and other thoughtless practices (Colwell and RUSS,1934). The resulting chronic effects, like the acute, were initially destructive but might be followed by inadequate, abnormal, or excessive repair which with further exposure could lead to malignant changes and cancerous growth. On the hands this was usually a surface phenomenon (Harvey, 1942), but change could also occur at a depth, for example in bone or glands, or at a deeper level still, where it affected the blood-forming tissues with the production of anemia (rare) or of leukemia (Carman and Miller, 1924 ; Nielson, 1932). It then became evident that the appearance of malignant growth was not dependent on continued irradiation of an already injured tissue. Cancer might occasionally supervene after acute exposure in the absence of further irradiation. In an early series of thirty-five X-ray cases the average inter-

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val for cancer production (where it occurred) was seven years (Rolleston, 1930). Then Burrows reported (1928) a basal carcinoma of the scalp which appeared eighteen years after X-ray epilation for ringworm. Once this possibility of late carcinogenesis was recognized more careful observation revealed even longer periods, some tumors following acute, and others chronic, exposure. The terms “early” and “late” effects of the irradiation of complex tissues serve to distinguish those in which the continuity of events have been followed histologically from those in which a period of apparent normality intervenes before cancer develops. More detailed histological investigation of the sites of radiation scar tissue would seem to offer a promising and exciting field for future research to determine the changes which occur at cytological level between exposure and-the alternatives of recovery (partial or complete) and the occurrence of malignancy. With the facilities provided by modern physics equipment, long-term experiments on the effects of chronic exposure are now possible. Certain types of response, however, e.g. alteration in life span after exposure, can be determined only if large populations of animals are irradiated simultaneously under identical environmental conditions, and this involves extensive radiation sources which are available only in a few research centers (cf. Mole et d., 1957; Wilding et QZ., 1952). The problems of protection are more often those associated with chronic than with acute exposure, and their solution must await further work in this particular field of research. The incidence of leukemia or of cancer in individuals occupationally exposed to radiation in the past provides, in the absence of any corresponding controls, data of quite another kind which should be matter for separate assessment (Court-Brown and Doll, 1957; Abbatt, 1956).

2. Latent Period In contrast to the “immediate” action of penetrating rays in fogging a photographic plate or rendering air conductive, there is in most interactions between radiation and living matter an interval of varying length between exposure and any recognizable change in the material exposed. Bohn (1903) noted that the injury might not be apparent for some time and then become manifest quite suddenly in connection with some special physiological activity, and his observation has been frequently confirmed in subsequent experiments by others (cf. Strangeways and Hopwood, 1926). For descriptive purposes this interval between exposure and visible effect was called the latent period, by analogy with muscle stimulation. It is a loose term in radiation biology, however, and is measured in anything from a few minutes to many years. I t is used indiscrimi-

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nately both for describing Bohn’s period of relative biological inactivity and the modern radiation chemists’ chain of events initiated by energy absorbed in the tissue. The tendency nowadays is to accept the sequence as inevitable : “we have the primary ionisations, the chemical consequences and the biological events which follow” (Haddow, 1956). This is true, however, only under certain conditions related to dose level and type of radiation, Under other circumstances the effect of an exposure may be seen only if the cell adopts an appropriate line of behavior ; otherwise it may never be visible at all.

3. Direct, Indirect, and Constitzctional Effects a. Direct Effect. When isolated biological units are exposed to radiation, the action must be a direct one on individual cells as a whole, on cell constituents, or on the medium (if any) which surrounds them. Experiments on tissue cultures have shown that suitable media for cell growth are affected only by relatively enormous doses, and any action by this process can be neglected for the purposes of this review. The question as to which constituent part of a cell is most sensitive to penetrating rays has been the subject of long and fierce debate (Scott, 1937). Almost every visible component of the cell has been named as a possible primary site of radiation action from the cholesterol in the external limiting membrane (Roffo and Correa, 1924) to fluid portions of the nucleus (Failla and Sugiura, 1939). The development of radiation chemistry has now driven the searchers to deeper levels represented by nucleoproteins and macromolecules (Bacq and Alexander, 1955). This continuing urge to canalize the action of radiation along a single, or at least a main, channel seems odd in face of the variety of demonstrable biological reactions. It has been shown that any part of the cell may be affected by radiation. The nuclear structures, however, particularly the chromosomes at division, undergo such dramatic changes that the effects on cytoplasm and nonnuclear cell constituents are sometimes overshadowed or neglected, although they are often just as crucial for cell survival as nuclear damage and merit equally serious attention. Changes in the cytoplasm include, for example, vacuolation, lysis, and keratinization ; changes in mitochondria and in the Golgi apparatus ; pigmentation ; alteration in permeability, in ultraviolet absorption, in pH, and in viscosity; increase in cell size, alteration in secretary activity; effects on the mitotic spindle ; surface changes affecting cytoplasmic cleavage (and therefore the distribution of nuclear material between daughter cells), metabolic disturbances, and effects on enzymes. The “nuclear” changes-more familiar because more often figured in

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the literature-chiefly concern the chromosomes at division and include various structural abnormalities] breakage, fragmentation, lag in movement, bridges at anaphase, uneven division of nuclear material, clumping of chromosomes, abnormal precipitation of chromatic material, formation of micronuclei, and vacuolation. Thus there are many different ways by which radiation may cause cell injury or death, and several may be operating simultaneously in any given instance (Politzer, 1934, p. 175 ; cf. Ludford, 1951). b. Indirect E f e c t . When applied to higher animals, penetrating rays affect the blood-forming organs, the cellular composition of the peripheral blood, and the tissues of which the heart and blood vessels are composed. Such a “direct action” of radiation on blood vessels, however, has an obvious importance beyond any significance for the circulatory system itself, since interference with vascular supply produces destructive effects on any tissue deriving nourishment from that supply, apart from any direct action of radiation on the tissue itself. By a long-standing custom among radiation biologists, the term “indirect effect”’ has come to be restricted to all those tissue changes which result from radiation interference with the blood supply. The amount of damage seen in a tissue after injury to its normal blood supply will depend on the suddenness with which the supply is cut off and the rapidity with which a collateral circulation (if any) can be established. If the supply is completely cut off, then the resulting destruction is widespread and indiscriminately distributed over the area supplied by the damaged vessels-a picture which is in marked contrast to the strictly regional distribution of degeneration effected by a direct action of radiation at levels below the catastrophic. With small laboratory animals and single doses of radiation below loo0 r. the direct action of radiation on a tissue predominates. At this level, the circulation remains intact and assists recovery from the direct effects of exposure. Above loo0 r. the effects of radiation are a combination of direct and indirect actions (Lasnitzki, 1945, 1947). Some at least of the effects of radiation on the nervous system recently reported seem most likely to have been produced via vascular disturbance (Gerstner et al., 1955; Gerstner et d.,1955), though Bering and his colleagues believe the results of irradiation of brains of monkeys with an inserted Tale2 wire “were more than could be accounted for by vascular damage alone” (Bering et al., 1955). The doses required to produce an 1 Radiation chemists have adopted the same term to indicate the conveyance of energy to dissolved molecules by other entities (molecuIes, radicals, or free radicals) derived from the water in which the substance is dissolved.

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effect on adult nervous tissue are consistently high, and as much as 300,000 r. has been given. At such levels, so far beyond the destructive dose for vascular tissue, it seems pointless to attempt to discriminate between direct and indirect effects. c. Constitutional E f e c t . An extension of the indirect (biological) effect of radiation is observed when a large part of an animal or the whole body is irradiated. Alternatively, massive doses of radiation (50,CW r. ) given experimentally to a single organ, produce within 3 hours a degree of devastation in a distant organ, protected from the original radiation, which is quite extraordinary (Henshaw, 1944). The too-rapid discharge into the blood system of the dCbris of cell destruction after an exposure may produce toxic effects on any or every tissue of the body, at the same time causing various degrees of shock or radiation sickness. For any given dose, the larger the volume of tissue irradiated, the greater is the relative significance of this constitutional action of radiation compared with the direct and indirect effects (Hempelmann et al., 1952).

4 . Some Obvious Bio1ogica.l E f e c t s of Penetrating Rays When living tissues are exposed to penetrating rays, there are certain conspicuous results which are common to a wide range of experimental material. These depend on biological processes some of which are sensitive to radiation whereas others are highly resistant. I n some instances an apparent radioresistance has later been shown to be the result only of insufficiently sensitive methods for detecting changes which had been induced by exposure. I n others, a radiation-induced change observed in one species of animal has been missed in another because the material was not kept sufficiently long under observation. Among biological processes about which attempts have been made to “generalize,” the following may be briefly discussed. a. Mitosis. The action of radiation which has attracted most and perhaps excessive attention is its effect on cell division. The sensitivity of proliferating tissues to penetrating rays has often been demonstrated ; an exposure of only a few roentgens can be effective in preventing mitosis, at least temporarily (Carlson, 1950). Under some circumstances, on the other hand, dividing cells can show a remarkable resistance to radiatione.g. those in regenerating amphibian limb buds (Stone, 1932, 1933), in regenerating liver cells (Mee, 1956), and the mitosis in radioresistant malignant tumors (Gliicksmann and Spear, 1945). In these instances mitosis persists even after doses measured in thousands of roentgens. This problem is quite distinct from the question of whether radiation ever stimulates cell division ; in the animal kingdom the evidence seems against

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any direct stimulus to divide, but in response to injury a hyperplasia can occur which may accelerate certain biological activities, e.g. the growth of hair (Chase, 1954, 1958, also personal communication, 1957). Among radiosensitive proliferating tissues there are different mechanisms by which disturbance of cell division is brought about. Arrest of division or breakdown of the cell may occur in pre-prophase, at prophase, or at any of the classical phases of mitosis; mitosis may be disrupted by interference with spindle formation, by the occurrence of multipolar spindles, by destruction of the centrosome, by persistent adhesiveness of chromosomes, by mechanical difficulty at anaphase following chromosome aberrations, by alteration of the hyaline material of the nucleus, by changes in protoplasmic viscosity, by cytoplasmic and/or nuclear “explosion,” or by changes in the cytoplasm leading to failure of cleavage, to name only some of the possibilities. Although certain of these processes occur more frequently in some tissues than in others, several may be present together in the same material and even in the same cell, and some types of response may be correlated with dose level (Cox, 1931b). It is not enough to be familiar only‘with the tissue selected as an indicator ; some knowledge of the material to which extrapolation is to be made should also be acquired. Attempts to determine the relative radiosensitivity of resting and dividing cells and of the different phases of the mitotic process have been made on many different materials with a wide variety in the results. Most of the earlier observers agreed in regarding the most rapidly proliferating tissues as the most radiosensitive (Regaud and Blanc, 1% ; Krause and Ziegler, 1906). From this it was concluded that cells were most readily destroyed during mitosis. Lacassagne and Monod (1922) have in this connection discussed the significance of degenerate mitotic cells in the radiotherapeutic treatment of malignant disease. Observations on ova and tissues from cold-blooded animals supported this view (Grasnick, 1917; Holthusen, 1921 ; Alberti and Politzer, 1923, 1926), and efforts were then made to determine the most sensitive phase of division. The dividing ova of Ascaris were found to be at least eight times as vulnerable as resting ova, with metaphase the most vulnerable stage of division (Mottram, 1913). The Langendorffs (1931) showed that in the irradiated germ cells of the sea urchin the maximum injury takes place immediately after impregnation, decreases slowly, reaches a minimum during the metaphase, and increases again with anaphase. Using chick fibroblasts in vitro, Strangeways and Hopwood (1926) showed that the cells were most sensitive just before visible division, and their conclusions were supported by Kemp and Juul (1930), who also used tissue cultures (cf. Ancel and Vintemberger, 1925). According to Regaud (1922), cells

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are particularly sensitive just before, during, and just after cell division, but the most sensitive stages of all are prophase and anaphase. O n the other hand, Vintemberger (1928) X-rayed the eggs of Rana fusca and found telophase to be the most sensitive stage. The very careful investigation by Eker (1937) not only throws further light on this problem but suggests some possible reasons for the discordant results which have been reported hitherto by different observers. In the locust Eker distinguishes between two different actions of radiation ; (1) disturbance in the normal rhythmic behavior of the cellular tissue, and (2) the production of histological abnormalities in individual cells. Cells irradiated in the course of division complete the process, but it seems very probable that at the dose level used (500 r.) they do not complete more than one division subsequently. Exposure is followed by (1) a temporary holdup in the entry of cells into division ; (2) abnormal ana- and telophase figures with retardation of these phases immediately after irradiation ; (3) cessation of the holdup with a compensatory rise, first in prophase and then in other phases of division; (4) an abnormally rapid passage through resting and early prophase stages leading to a real increase in the mitotic count for a given period (cf. Tansley et al., 1937). The temporary block occurs in early prophase. This stage of division is of slow development and “may be supposed to be more marked in germ cells than in most other cells,” rendering possible “a particularly exact localisation of the block” (Eker, 1937, p. 79). If cells perish, they do so at this stage. Irregularities at anaphase result from disturbance of the mechanism concerned with the polar migration of daughter ‘chromosomes. Although this may lead to unequal distribution of chromatic material at telophase in affected cells, it cannot account quantitatively for the number of cells which are seen to degenerate in prophase. The disturbances seen at anaphase in Eker’s material are not indicative of the general cellular damage: “they must be deemed to be derangements running collateral with the essential injuries and are obviously of subordinate importance as regards the viability of the cells.” It is easy to see that, although the effect of radiation on individual cells in other species may be comparable, the consequences of a disturbance of the rhythmic behavior of the tisues as a whole may lead to very different results. In Eker’s material neither giant nor multinucleate cells appeared. Careful studies on the effect of radiation on sperniatogenic cells in the mouse have been made by Oakberg (1955a, b), who has demonstrated the breakdown that occurs early in mitosis resulting in a postirradiation lack of ana- and metaphase figures. b. Metabolism. In contrast with the radiosensitivity of many cell

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processes connected with division, those associated with metabolism were long regarded as radioresistant (Scott, 1937) with the possible exception of the respiratory mechanism (Colwell, 1935). Data on the effect of radiation on respiration have been obtained from the behavior of sum*ving (tumor) tissues irradiated in vitro (Crabtree and Cramer, 1934) , but in the absence of any histological examination correlation of results with in Vivo material are lacking. There is general agreement, however, that changes incidental to irradiation are dependent on the rate of metabolism and that increased metabolic activity after exposure enhances lethality (Patt and Brues, 1954a) , whereas depression of metabolic rate decreases the rate at which radiation damage develops. Varying the rate by temperature control introduces another variable, since temperature itself may alter both the quality and quantity of cellular activity, which in time may modify the response to radiation. The general conclusion that metabolic processes in the higher organisms were affected only by high radiation doses well above those producing recognizable change in biological behavior held until the indirect (chemical) action of radiation on dissolved substances in aqueous solution at quite low dose levels was demonstrated (Dale, 1947, 1952, p. 117) with the implication that similar processes might occur after irradiation in vivo (cf. Allsopp, 1951). Among the bacteria, however, there are some varieties which withstand doses of‘ several thousands of roentgens without any permanent effect on their growth rate (Rubin, 1954). Subsequent work in this field has shown that the doses required to produce biochemical changes vary over such a wide range (sometimes well above the lethal level for almost any biological tissue) that attempts to generalize are no less difficult here than elsewhere (Patt, 1954). Much needs to be done to determine to what extent in vitro observations are valid for the much more complicated conditions in vivo. “When you are working with the living cell, you have a situation in which your system can replace the inactivated units rapidly, and so you may have all kinds of radiation effects which are without any biological significance, and are not detectable unless you are able to set up special experimental situations to detect them , . . it is by no means certain that inactivation of enzymes is of any great importance in over-all radiobiological effects” (Mazia, 1953, p. 107). Too little is known about the biochemistry of the histological alterations already familiar in irradiated tissue. A much closer collaboration between chemists and biologists here would help toward the solution of this type of problem. c. Motility. Motility is a property common to many kinds of animal cells but unusual among vegetable tissues. It is highly resistant to radia-

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tion and is one of the last functions of the cell to be lost before the lethal dose is reached. I n many growth rate studies loss of motility is the criterion of death (Mayer, 1930), but in a series of delayed lethal dose experiments with tissue cultures it was found that mitotic activity always ceased some days before motility was lost (Spear, 1930). I t had been shown previously by Chambers and Russ (1912) that B. fyocyaneus were unable to form colonies after doses which failed to inhibit motility (cf. Bruynoghe and Mund, 1925), and inactivation of still-motile sperm cells is the characteristic of the Hertwig effect discussed later. With sufficient dosage all cells lose their ability to move, but the niechanism is not the same in all instances. Irradiation has a specific action on some of the Flagellata by paralyzing the flagellum without any general action on the organism as a whole. This occurs at a dose level which is relatively low compared with that affecting motility among non-flagellate protozoa where changes in viscosity affect locomotion. Schaudinn ( 1899) found that organisms with a more fluid protoplasm were more sensitive to radiation than those whose protoplasm was more viscid. Loss of amoeboid form after exposure may reduce a migratory cell to a passive condition in which its position is changed only by movement of the medium in which it lies. This may be accompanied by enlargement and rounding up of the cell (Lasnitzki, 1947). If the cell is not lying free, it may enlarge without rounding u p ; such cells were figured by Clunet as early as 1914. One theory of radiation action has been based on this phenomenon of cell enlargement (cf. Ellinger, 1941, p. 263). The ability of most cells to move even after heavy closes of radiation raises an important point of histological interpretation. I n many developing organs, where mitosis is localized in a germinative zone, cell division may be arrested at dose levels which still permit migration of these potential mitotic cells into areas of already differentiated cells (Spear and Gliicksmann, 1938). In unorganized tissue, on the other hand, the distribution of mitosis appears haphazard. In tissue cultures of this type it has been shown that the distribution is influenced by population density in relation to food supply (Willmer, 1935, p. 31). I n glandular tissue, the separation between differentiated and undifferentiated zones is not evident. Malignant tissues exhibit analogous though varying patterns of organization. The distribution of mitosis in anaplastic tumors resembles in its haphazard arrangement that seen in an unorganized tissue culture. In partially differentiated epithelial tumors, on the other hand, foci of dividing cells can be easily distinguished from areas of differentiation where no mitosis is seen; these in turn are often quite distinct from degenerate cell masses which contain neither dividing nor differentiating cells. If

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such tissue is examined under high magnification for evidence of radiationinduced changes in proliferating cells, then very careful selection of fields is essential for any quantitative observations to be made. When the selection is properly made, the results of examination of different areas show a surprising uniformity both qualitatively and quantitatively (Gliicksmann and Spear, 1945). d. Mutation. The earliest suggestion of heritable changes induced in plant material by radiation arose from Dauphin’s work (1904) on Mortierella in which alterations in the manner of growth and nutritional requirements of the mold colonies were observed after exposure. When the radiation was removed, “the spores flourished again.” Four years later Gager (1908) studied the effects of irradiating germ cells of Oenothera and noted somatic abnormalities not transmitted to the succeeding generation. Heritable wing changes were reported by Morgan (1911) in irradiated Drosophila, but many similar experiments by other workers gave negative results. Meantime Muller ( 1928a) was perfecting his quantitative methods for the measurement of spontaneous mutation in Drosophila. This done, he was ready to undertake a critical investigation with X-rays which gave spectacular results (1928b). It was found that X-rays were able to induce mutations in this organism which were indistinguishable from those that appeared spontaneously (cf. Catcheside, 1947). Thus Morgan’s conclusions were confirmed and a new field of investigation opened up. The 1911 paper is barely three pages in length-a scanty birth certificate for an infant who has shown such sturdy growth since ! Meanwhile Stadler ( 1928a, b, 1932), working independently on vegetable tissues (barley and maize and later on higher plants), demonstrated heritable changes as a result of exposure. In the higher plants these were associated with visible changes in the chromosomes and thus differed from Muller’s gene mutations which were unaccompanied by any visible alteration in chromosome structure or behavior (Muller, 1928b). It thus became convenient to distinguish (as far as this was possible) between genetic changes due to gene mutation on the one hand and to chromosomal rearrangement or other abnormality on the other. The latter have sometimes been referred to as chromosomal mutations, but this is a confusing term and should be dropped. Gene mutation and heritable effects due to chromosomal rearrangement constitute “a real qualitative distinction even though we must often remain in doubt as to which class a given change belongs in” (Muller, 1951). Once this action of radiation was recognized, large-scale efforts were made (‘to produce the maximum number of mutations. By working on these lines very important results were obtained, among others in the field

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of agriculture. Quantity and quality of crops were improved” (cf. Hevesy, 1956). Hevesy goes on to question whether “any geneticist envisaged in those days that the time might come when the main concern would not be to produce mutations by irradiation but protect against them.” Reports of reverse mutations were frequent (cf. Timofeeff-Ressovsky, 1932) which appeared to restore the status quo of the irradiated material, and hopes were high that among the mutations produced by radiation an increasing number would be beneficial to mankind-a revival of the old optimism of the early days in a new form (cf. Patterson and Muller, 1930). That mutations, of whatever origin, could be detrimental was of course known before Muller’s quantitative demonstration of the mutagenic action of radiation, and it was a question of time only for the realization that radiation-induced mutations were qualitatively indistinguishable from those which occurred naturally, or those produced by other agents. The reaction was to issue warnings in superlative terms (cf. Ellis, 1948) and to assume that every mutation, at least among the higher organism and especially man, must be considered as detrimental even if it was not definitely in the lethal or sublethal deleterious classifications. The mere suggestion to the contrary at the Geneva Conference of 1955 had repercussions on an international level (Carling, 1956). Quantitatively the production of mutations by radiation is said to be proportional to total dose received and independent of any other physical factor of irradiation. This means in theory that there is no threshold dose for the effect and that the result of all exposures is additive, though the matter is not yet beyond dispute (cf. Caspari and Stern, 1948; Russell, 1952). With acute exposure of mice to X-radiation, the linear relation between dose and effect for mutation does not seem to hold (Russell, 1956). There is general agreement, however, that mutations can be produced with very low dosage down to a level which approaches natural background (Uphoff and Stern, 1949). On the other hand, investigations on mutation are made on “resistant” organisms, e.g. bacteria, at dose levels measured in some thousands of roentgens (cf. Patt and Powers, 1956), so that the dose range employed in this work is about as extensive as that used in studies on mitosis. Whatever may be the chemical composition of the genes, at least they have very varying radiosensitivity. The effect of this variability in determining precisely what is involved in the term “genetic hazard,’’ especially among the higher animals, is still unknown. One of the most pressing needs in the estimation of human risk is for much more basic information on mutation rates irf mammals (cf. Russell, 1952). e. Malignancy. I t is interesting to speculate what would have been the

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effect on the development of radiology had the carcinogenic action of radiation been discovered prior to its curative effect. In the event, distinction was quickly drawn between those conditions which were conducive to cure and those which, by overdose, led either to the radiation burnthe “acute effect”-or if prolonged caused the so-called chronic radiodermatitis (Harvey, 1942). A dozen papers on the histological changes seen after radiation appeared between 1896 and the first report of malignant change in an area of damaged skin (Frieben, 1902). The first attempted (‘explanation” was that the occurrence was an illustration in the new field of penetrating rays of the Arndt-Schulz law, viz. that small doses of an agent stimulate or irritate, medium doses depress, and large ones kill. It would have been reassuring to have convincing evidence that only accidental, haphazard exposure of inadequately protected operators of X-ray machines ran the risk of malignant growth, and that the only result of adequately planned exposure of diseased individuals was a beneficial one. A lively controversy ensued of which Rolleston (1930) has given an admirable summary. The balance of evidence from laboratory and clinic was against the possibility of applying the Arndt-Schulz law to the action of radiation, and the occurrence of malignancy has gradually become dissociated from the manner in which the radiation reaches the tissues. It was seen more as a change in the character of the cells as a result of exposure, and this led to the idea of a radiation hazard analogous to that attaching to any other curative agent which carried with it certain dangerous potentialities (Gliicksmann, 1952). The magnitude of the hazard is still undetermined, though the risk of any mischance is lower in animals with a short life span than in those (including man) which are long-lived. On present evidence there would seem to be little contraindication to the continued use of penetrating rays as therapeutic agents in trained hands.

111. VARIATIONS IN RESPONSE WITH DIFFERENT TISSUES Much more information is needed about the where in the cell radiations act and when in the life history of the cell irradiation is effective. It may even be true, at this time, that we have more information about the how than we can handle. (Daniel Mazia, 1956, p. 143)

1. Choice of Material The aim of radiation biology is to investigate the changes in living cells, tissues, and organisms brought about by penetrating rays. For convenience of description, work is often classified quite arbitrarily under one of three headings : “applied research” if it offers an immediately practical scientific basis in place of existing empiricism; “basic research” if there is reason-

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able hope of eventual extended use; and “fundamental research‘’ for experiments which seem remote from practical use but are concerned with possible mechanisms underlying biological change. It is not always the specially designed experiment, however, which gives a direct answer, and what appears to be a “fundamental” investigation may turn out to be of great practical value, whereas an observation made in a medical department may be as essential to a theorist (if he could be made aware of it !) as work conducted in his own department. Progress in radiation biology is not advanced by this partition of the subject into degrees of practical usefulness ; it would profit more by further encouragement of personal and informal contacts between the individuals concerned and a classification of material used, rather than a grading of the investigations in terms of so-called fundamentals. That a distinction should be made between normal and pathological material when assessing the action of radiation is obvious and, with few exceptions,2 is generally recognized. Human malignant tissue has always been considered on its own merits, and classifications of tumors according to their radiosensitivity, usually measured in terms of macroscopic shrinkage. shortly after exposure to radiation, have been a feature of radiotherapeutic textbooks for many years (cf. Cade, 1940, p. 231). The greater variety of normal tissues presents more difficulty. Based on accumulated experience, appropriate tables of radiosensitivity are included for the guidance of radiotherapists, the criterion in this case being the liability to necrosis after heavy exposure (Desjardins, 1932). T o obtain more precise information (since the field of experiment is restricted where human tissues are concerned) it became desirable to find other tissues which could be substituted for them and still yield information of use to the radiotherapist. Monkeys would seem an appropriate first choice for a reliable indicator (Haigh and Paterson, 1956)) but laboratory convenience is usually better served by using small animals, which also have an economic advantage. Hence indicators have usually been chosen for their ease of handling and studied irrespective of any affinity to human material. This easily creates a state of mind which thinks in terms of a common mode of action of radiation and speaks authoritatively and comprehensively about the biological effect of radiation. Many experiments, however, have been made without any intention of extrapolation to human tissue. But once results are published any reader is 2 For example, observations on surviving material, i.e., slices of tissue kept at 37” C. in saline solution which are slowly degenerating and will eventually die irrespective of irradiation or any other experimental handling.

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free to use the data as he likes, and many do so in a manner quite out of accord with the original author’s intentions. Indicators vary in size and complexity from the burro (Wilding et al., 1952), weighing anything to a quarter of a ton, to viruses whose dimensions are measured in micromillimeters (Markham, 1946), and these diverse experimental objects have been used not only in attempts to provide a scientific basis for radiotherapeutic procedures but also for studies in radiosensitivity and to provide data on which hypotheses of radiation action may be based.

2. Biological Indicators Whatever the purpose of the experiment, it would seem desirable, at least initially, to discriminate between the following groups of indicators in their response to irradiation [The reaction of viruses to radiation is outside the scope of this review (see Luria, 1955; Pollard, 1953).] a. Vegetable cells and tissues.

b. Bacteria, protozoa, fungi, molds, and yeasts. c. Ova, larvae, and some developing forms : ( i ) amphibian, (ii) warm-blooded. d. Tissue cultures : (i) unorganized, (ii) organized, (iii) clone cultures. e. Higher organisms: (i) whole-body exposure, (ii) normal tissues and organs (somatic, generative, germ cells) ; see Section IV. f. Human tissues : (i) normal, (ii) pathological.

a. Vegetable Cells and Tissues. Many interesting observations have been made on the effect of penetrating rays on vegetable tissues in the form of seeds, in various stages of growth, on growing root tips, on budding twigs, and on the fully developed plant. Few reviews, however, have so far appeared on the subject other than two chapters in Duggar’s “Biological Effects of Radiation” (1936) on the effects of X-rays on green plants (Johnson, 1936) and the effects of radium on plants (Gager, 1936), and a treatise (in Russian) by Breslavets (1946). This is probably because of some closely analogous effects to those which the animal kingdom provides (cf. Colwell and RUSS,1924, p. 151). I n consequence vegetable tissues have often been used as convenient biological indicators for the study of some particular action of penetrating rays, on the assumption that the response is representative of a much wider range of material including animal tissues. Although this may be true for a few of the direct effects of radiation on individual cells, extrapolation from the vegetable to the animal kingdom should be made with caution, if at all, to conditions where the indirect and constitutional effects of radiation come into play. Experiments made early in the century demonstrated the growth restricting properties of radiation on Mortierella (Dauphin, 1904) and on mustard

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and cress, and a variety of chromosome aberrations in cells of the bean root exposed to y- and (3-rays (Koernicke, 1904). Most of the early work in this field was concerned with acute exposures given at a particular stage of development. More recent experiments have been made with chronic exposures which allow radiation to act during many or all of the stages of a plant’s development. These have shown that this type of exposure yields a more marked and more varied morphological response than acute exposure (Gunckel and Sparrow, 1954). Even the radiation from P32 in tracer amounts of 4 pc./l. can produce recognizable lesions in the shoot tip, though the root tip required four times this concentration before it showed injury (Mackie et al., 1952). Experiments with vegetable tissue have been concerned with : the physiological effects of radiation including observations on seed germination, growth rate, respiration, and plant movement ; the morphological effects on stems, leaves, and flowers ; the histological and cytological effects ; and the genetic effects of exposure. Though there is general agreement on the essentially destructive action of radiation on vegetable tissue at higher dose levels, much of the literature is very controversial, especially that dealing with the stimulating action of radiation in small doses on the growth of seedlings (Johnson, 1936). The case for a stimulating action on certain plants seems to be established, and the present position is well summed up by Gunckel and Sparrow: “It seems to us that results from one species or variety should not be applied to others and that different types of responses to the ionizing radiation are to be expected in different plants. The fact that a positive response is not universal should not be used to invalidate those cases where a stimulation has been reported.” Differences in response from plant to plant are striking. It is well known that chemical agents such as colchicine which prolong mitosis increase the frequency of radiation-induced chromosome aberrations in Tradescantia (Guyer and Claus, 1939; Koller, 1946). The opposite effect, however, is seen in the onion, in which mitosis is halted in early prophase (Brumfield, 1943). “It seems likely,” continue Gunckel and Sparrow, “that in the same plant the responses of the seedling stages compared to older parts may be almost as variable as the responses of different plants to X-rays (Johnson, 1928, 1948 ; Sparrow and Singleton, 1953 ; Sparrow and Christensen, 1953). This differential response might logically be due to some such scheme as that proposed by Quastler and Baer (1950), in which the growth process is divided into growth initiation and growth completion phases with the latter less radiosensitive. However, it should be emphasized that the problem of differential radiosensitivity is a difficult and complex one which is poorly understood.”

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The conditions under which the roots and the aerial parts of a plant grow are so different that different kinds of response would be expected. “The nature of the response, then, should not be thought of in the older sense . . . that more active cells are more vulnerable to radiation but in the sense of Henshaw and Francis (1935) that during the life cycle of a plant various internal systems or cell components are more easily modified by radiation than others and that these in turn can be modified by changes with external or internal plant environment. At different dosages or dose rates the response for a particular plant . . . may be death, complete growth inhibition, mild morphogenic abnormalities, or marked proliferations resulting in severely abnormal or deformed plant parts’’ (Gunckel and Sparrow, 1954, p. 253). Molish (1912) showed long ago how the effects of radiation on Syringa buds differed according to the month in which exposure was made. Mottram (1913) chose the root tips of seedling beans as experimental material “because in plants mitosis occurs almost entirely at night, making it possible to expose the cells to radium, on the one hand, during mitosis, and on the other whilst in a resting condition ; and further by observing the subsequent growth to compare the effects of the two exposures.” An extensive literature concerns the cytological and genetic effects of radiation on plants (cf. Catcheside, 1948). The general aspects of this subject are referred to in Section IV. The relationship between mitotic inhibition, chromosome breakage, and growth inhibition in the bean root has been considered by several authors, particularly by Gray and his colleagues (Gray and Read, 1950; Gray and Scholes, 1951 ; Thoday, 1951), who conclude that structural damage to chromosomes constitutes a major factor in growth inhibition. It would appear, however, that indirect physiological effects of radiation also contribute to the limitation of growth (Quastler et al., 1952). Experimental results vary both with tissue species and with type of radiation: thus in the bean root exposed to acute radiation the meristematic cells show greater injury, whereas in the tomato subjected to chronic exposure damage first appears in the vacuolated cells adjacent to the meristem proper (Gunckel and Sparrow, 1954). Working with the bean shoot Gordon (1955, 1956) has shown that Xradiation inhibits the final stage, an enzymatic oxidation, in the synthesis of the growth hormone, indoleacetic acid, with the result that the plants are dwarfed. This is clearly not a system involved in animal growth and may not even be an essential component in root elongation of the same plant, but the effect is an interesting example of a localized action of radiation on one part of a vegetable organism.

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Any interference with mitosis affects growth rate, whatever cellular mechanism may be involved, so that growth inhibition in two different materials is not necessarily brought about by identical means. Results which at first sight seem surprisingly similar can be obtained by a variety of agents acting on a diversity of material. It was in this way that the group of radiomimetic substances came to be recognized, but further research has now shown some of the different mechanisms by which the various agents act (Elson, 1955; Koller, 1954). In spite of the variety of response exhibited by vegetable tissues, work in this field has thrown light on what has come to be known as the “oxygen effect” which has application beyond the plant kingdom. Holthusen (whose classical work on the irradiation of Ascuris eggs will be referred to later) had noted the relative resistance of these eggs during anaerobiosis and attributed this result to the absence of cell division (cf. Holthusen, 1921 ; Dognon, 1925). Petry, who had already observed a correlation between dehydration of seeds and radiosensitivity (Petry, 1922), proceeded the following year to study the increased efficiency of irradiation when seeds and rootlets were exposed in the presence of oxygen (Petry, 1923). The subject was explored in further detail by Mottram (1935), who made a systematic study of the bean root, basing his investigations, however, on the observations of Crabtree and Cramer (1933, 1934) on tumor cells and not on Petry’s work, of which he was apparently unaware. Mottram thus began a series of experiments which, continued by Gray and his colleagues, has yielded much information on the behavior of root tips exposed to radiation under various physical and environmental circumstances (Gray and Read, 1942-1950; Thoday, 1951 ; Read, 1952). The oxygen effect has now been demonstrated in a wide variety of material and linked on the one hand with the influence of blood flow on tissue sensitivity first observed by Schwarz (1909) and subsequently studied among others by Carty ( 1930), Evans et ul., (1942), and Howard-Flanders and Wright (1955), and on the other hand with respiration (see Gray, 1956). The possible use of the oxygen effect in radiotherapy is now being investigated (Churchill-Davidson et al., 1955 Gray, 1957). The oxygen effect illustrates the action of a sensitizing agent applicable to tissues in both animal and vegetable kingdoms under certain conditions of irradiation. Many of the other actions of radiation on plants, however, have no analogous response among animals. b. Bacteria, Protozoa, Fungi, Molds, and Yeasts. Bacteria were among the first organisms to be irradiated (Minck, 1896), in the belief that X-rays (like sunlight, which was originally supposed to contain them) would prove to be a useful germicidal agent (Lyon, 18%). It was an un-

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fortunate choice of material owing to the radioresistance of bacteria as a class when exposed in vitro in contrast to the extreme sensitivity of some inflammatory conditions due to bacterial invasion (Baker and Freund, 1950). The relatively high doses which many pathogenic bacteria could withstand, however, turned the attention of investigators to less virulent types as more convenient material for laboratory experiment, either in the vegetative form or as bacterial spores, among which anthrax was observed to be highly resistant (Chambers and RUSS, 1912). These authors were the first to report an exponential survival curve for irradiated bacteria. They started a fashion, followed for many years, for determining the form of survival curves under varying physical conditions including the action of different types of radiation (Lea, 1946, p. 316). This produced a collection of graphs which include almost every conceivable kind of variation between exponential and sigmoid plots which led Lea (who himself did much work in this field ; Lea et ul., 1941) to complain of the discussions ad nauseam in the literature “whether the exponential survival curve obtained, e.g. with bacteria, proves the disinfection to be of the single-unit action type, opponents of this view preferring to ascribe the exponential curve to an extremely skew distribution of resistance. As long as the argument is based only on the shape of the survival curve, the conclusion must be largely subjective, since it depends on whether one regards the a priori improbability of the target theory to be greater or less than the a priori improbability of the distribution of resistance to radiation among the organisms being of the extremely skew type required. Under these circumstances additional criteria rather than further discussions are called for” (Lea, 1946, p. 77). Bacteria have been used for studies on the factors influencing radiosensitivity, for investigations on the physiological effects of radiation (Zelle and Hollaender, 1955), for observations on lethal dose, and for various biochemical investigations. I n this connection Pirie ( 1956) ((wonders whether one can equate bacteria that are extremely radioresistant with mammalian cells that are radiosensitive.” Bacteria are now being used on an increasing scale for genetical studies often at very high dose levels (cf. Spiegelman, 1956), and many of the experimental results have been used in establishing theories of the mechanism of radiation action. Unicellular organisms in great variety are convenient for studying the direct action of radiation on individual cells (Kimball, 1955). Each organism is a separate and independent unit, and there is no spread of any effect to adjacent cells. Each lives and dies in isolation, uninfluenced by any reaction to radiation of its neighbors (unless the accumulated products of cell degeneration become injurious or adversely affect the medium in

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which residual cells are growing). O n the other hand “recovery” in a population of irradiated unicellular organisms given sublethal doses is independent of any of those coordinated activities which operate in an organized tissue, and it can be modified only by appropriate alterations in the culture medium. Rate of recovery reflects the rate of proliferation among the survivors of an exposure, The slime molds are an exception. They begin as independent amoeba-like bodies which eventually coalesce into a “slug” of differentiated tissue which finally produces a spore-bearing organ. These molds seem to offer particularly interesting material for radiological experiment. From the radiological point of view unicellular organisms can be regarded as differentiated cells retaining a capacity for cell division ; they are in a sense mature germ cells which do not produce tissue; their offspring, like themselves, are separate and independent units. Schaudinn (1899) found that among individuals of the same species multinucleated forms were more easily affected than the mononucleated and that the parasitic varieties were more resistant than the others. If attention is confined to protozoa and the criteria of effect limited to lethal effects, the observer still finds an astonishing variety in the response to X-radiation ; e.g., certain ciliates disintegrate after a few seconds’ exposure, the larger and more sensitive specimens of Pelomyxa after a few minutes’ exposure, and Amoeba lucida after about 10 hours’ irradiation. On the other hand, the dose required to cause immediate death is over 500,000 r. for some paramecia which are among the most resistant organisms in this group. A delayed lethal effect follows much lower doses-of the order of 1 0 , W to 12,000 r. Death may occur without any attempt at division, at or near the first division, after several divisions, or in association with autogamy (Kimball, 1955). The injurious effects of radiation may be altered by changes in the culture medium (Giese and Heath, 1948). Cell division is retarded by exposure to sublethal doses of radiation (Giese, 1947). This retardation may last for several divisions, but the normal rate is usually recovered sooner or later. This action of radiation, however, is not invariable (Halberstaedter and Luntz, 1929 ; Halberstaedter and Back, 1942), nor is the rate or manner of recovery constant. Radiation may retard the first division after exposure; it may result in a long cessation of the process after two or three divisions have taken place; or it may slightly increase the interval between divisions for a number of generations. A most detailed investigation was made by Robertson with the flagellate Bod0 cawdatus which well illustrates the complexity of this comparatively simple organism’s reactions to ionizing radiation (Robertson, 1935).

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Irradiation of the giant multinuclear amoeba Chaos chaos has given some fascitlating results. It would be premature to judge from its name that irradiation caused its utter ruin. After exposure the organism may break into fragments, but the largest of these develop into clones of normal size, while medium-sized fragments produce clones of about half the “parent” size, which on further irradiation produce clones of still smaller organisms (Schaeffer, 1946). No mechanism has been suggested for this curious result. It is reminiscent of the most highly developed Q-boat of World War I where each fresh bombardment resulted in successively smaller but still efficient fighting craft. Of the subgroup including fungi, molds, and yeast, yeast is the most popular biological indicator, but even among different species of yeast there are differences of biological response. This material has been used for the study of so-called mutation effects of radiation and for investigations on which theories of radiation action have been based (Tobias, 1952, p. 357). Yeast has also been used in experiments on ploidy in relation to radiation response (Zirkle and Tobias, 1953). Polyploid individuals are fairly common in plants but rare in animals, since polyploids are sterile when crossed with diploids. If a polyploid arises in a diploid population, it can only reproduce parthenogenetically or by self-fertilization, a process common in plants but not in animals. Latarjet (1943) has reported an effect of temperature in the recovery of yeast cells not seen with bacterial cells when similarly treated. Reference to fungi and molds will be made in a later section. c. Ova, Larvae, and Some Developing Forms. Ova and larvae of the sea urchin were among the earliest biological material to be systematically studied for radiation effects (Bohn, 1903), but the physical factors of the exposures are not detailed. The early popularity of ova of Ascaris as a biological indicator is explained by the abundant supply (Perthes, 1904b). It is convenient but a coincidence that the mean lethal dose for Ascaris eggs is nearly the same as the average human erythema dose for y-rays, as it gives the radiation biologist a useful yardstick when working within the therapeutic range of dosage with different types of radiation which have no common physical unit of measurement. This does not, however, justify extrapolating the experimental results from the one tissue to the other. Mottram, in an often-quoted paper (1913), used the Ascaris egg in experiments designed to test the radiosensitivity of the various phases of mitosis and found metaphase to be the most vulnerable. This was later confirmed by Holthusen (1921), who also studied the influence of temperature and oxygen on the results of X-ray exposure. The results are not strictly comparable to other conditions, however, since Mottram used

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a mixed beam of p- and y-rays in contrast to the normal y- or X-rays used in modern therapy. H e does not hesitate to apply his results to contemporary radiotherapy (where p-rays were more often used than now), but neither do much later writers, whatever type of ray is involved. Yet the rapidly fatal action of P-rays which can act almost like a fixative agent is now well known (cf. Canti, 1928), and other observers with different material obtained results which indicate that the effects of p-rays on Ascaris are not common to other material and different physical conditions. The use of ova, larvae, and developing forms in determining the radiosensitivity of the different phases of mitosis has already been referred to and the variety of the response noted. X-radiation accelerates the mitotic process in the eggs of Planorbis. Richards (1914) and Packard (1916) observed the same result with eggs of Arbacia exposed to radium. In comparing the action of X-radiation on resting and dividing cells Holthusen (1921) showed that, although sensitivity was increased by the presence of oxygen, the oxygen consumption was constant throughout both cell processes. The increased radiosensitivity he observed during mitosis therefore could not be associated with increased metabolism at that period. H e correlated sensitivity with mitotic activity and resistance with inactivity resulting from anaerobiosis. The temperature effect in the sense of increased sensitivity with rise in temperature is shared by Drosophila (Packard, 1930) but not by frog eggs (Ancel and Vintemberger, 1927). Ascaris eggs are still popular material in radiation experiments concerned with cell division (cf. Bauer and Le Calvez’s observations on chromosomal aberrations, 1944 ; absence of chromosomal rearrangement, McClintock, 1939 ; recovery factors, Cook, 1939). Seide (1925) used these eggs in unsuccessful attempts to demonstrate a stimulating action of radiation. Packard (1931) has developed the use of Drosophila eggs as a measure of dose to a high degree. H e claims that “if a large number of Drosophila eggs is exposed to an X-ray beam of unknown intensity for 10 minutes and if, as a result, half the individuals fail to hatch, then 180 roentgen units have been delivered at the rate of 18 r/min.” The constancy with which such quantitative experiments yield the same result is perhaps one of the most striking features of this type of investigation. With Drosophila eggs the error is not more than 376, and this order of accuracy is obtained with other types of biological material under laboratory conditions. Scott (1934) favored the eggs of the common bluebottle as biological indicators, probably on account of the ease of supply and convenience of handling. When such isolated biological units are exposed the action of radiation is directly on individual cells and the total effect depends on and

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runs parallel with the dose of radiation as measured in air. This is a welcome simplification under the conditions specified but the results do not hold for more complicated tissues such as the human skin. Developing organisms at later stages of development constitute another special type of biological indicator where two different kinds of activity occur-the one, mitosis, being predominant at first but later gradually becoming subordinate to the other, differentiation. If radiation is restricted to the mitotic period, the result of exposure may be no more than a temporary reduction in proliferative activity followed by a compensatory excess which soon restores the population to its original numbers, a good example of a completely reversible tissue reaction (Wilson et al., 1935). If an excessive number of cells is destroyed by the exposure, the result may be a fully differentiated adult of unusually small size but otherwise fairly normal in appearance (Warren and Dixon, 1949). When, however, radiation is delivered to the developing organism after the process of differentiation has begun, grotesque consequences may be seen (Hertwig, 1911). The effects are not specific to radiation and are produced by other deleterious agents (Oak Ridge Symposium, 1953). At this stage of development there is no longer a common pool of multipotential cells ; each organ has its separate quota and reacts individually and at its own rate. Cell destruction now causes local and uneven deficiencies, leading to disproportion in size between the component parts of the organism. I t is important to realize that the scale on which mitosis and differentiation operate at this stage of development is unique in the organism’s life history and that the results of exposure to radiation during this period are not representative of that at any other stage of growth and development (cf. Russell and Russell, 1954). The effect of radiation on this delicate interplay between mitosis and differentiation can be studied on a smaller and in some ways a simpler scale in those organs where cell division persists throughout life-e.g. skin (Miescher, 1925) , testis (Eker, 1937) , and intestine (Lacassagne and Gricouroff, 1956a). Familiarity with the normal behavior of unirradiated tissue selected for experiment is essential for interpreting correctly the results of exposure. This is especially so in those developing organs where proliferating cells are segregated in germinative zones, since the ability of many cells to continue migration after an exposure which prevents division may have an important bearing on the final histological picture. Degenerate cells in a differentiated area may be only breaking down undifferentiated cells which have migrated to the area from a germinative zone (Spear and Glucksmann, 1941) and not degenerate differentiating cells for which they have apparently sometimes been mistaken (Rugh, 1954).

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There is another critical stage in some developing organisms which must be taken into account when they are used as biological indicators. This arises from the establishment, at a particular stage of development, of a coordinating system which may itself be radiosensitive. The development of the circulation in the chick may be taken as an example. Although the radiosensitivity of individual cells does not appear to differ appreciably when tested immediately before or soon after the establishment of the circulation (Spear, 1948, p. 310),the sensitivity of the animal as a whole is greatly affected as a result of damage to the vascular system per se. This was prettily demonstrated by Strangeways and Fell (1927), who explanted cells in vitro from chicks irradiated in ovo at various ages and showed how the viability of cells in the vascularized embryo was correlated with the length of interval between irradiation in ovo and explantation. This increased vulnerability of the chick as a whole has been quite wrongly quoted as demonstrating an exception to the “general rule” that animal cells decrease in sensitivity with increasing age. It also, perhaps, serves to show how dangerous it is to generalize. There is no simple correlation between age and sensitivity in the growing organism ; the result of any irradiation varies with the particular kind of biological activity taking place at the time of exposure. A further matter for note when developing forms are used in radiation experiments is the normal occurrence of morphogenetic cell degeneration at certain stages of development. In amphibian embryos this may be on quite a massive scale (Gliicksmann, 1940). This phenomenon not only provides quite suddenly many degenerate cells in particular organs, but it also affects the organism’s capabilities for disposing of cellular dPbris resulting from external injury including that caused by radiation (Spear and Glucksmann, 1938). Care is needed in any quantitative radiological experiments in which such material is used, since the number of degenerate cells produced and their rate of clearance vary with the general physiological condition of the animal. d . Tissue Cultures. Ever since Harrison (1907) succeeded in growing nerve tissue in lymph in a hanging drop preparation most branches of biological science have used the tissue culture method with varying degrees of success. Tissue cultivated in vitro shows two types of growth, usually called unorganized and organized. I n the simple hanging drop preparation unorganized growth is obtained by explanting a fragment of tissueusually embryonic-into a drop of culture medium on a cover slip which is then inverted and sealed down over a hollow ground slide. On incubation amoeboid cells wander from the cut edges of the explant into the medium where they divide actively, eventually forming a broad halo of new

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tissue around the original fragment (Loeb, 1911 ; Levi, 1934 ; Parker, 1938; Strangeways, 1924). In a fluid medium cell migration accounts for nearly half the total outgrowth (Willmer, 1933). Organized growth, on the other hand, corresponds more nearly to normal growth in the body. The tissue enlarges as a whole with less diffuse outwandering, the normal histological structure is preserved, and if the explant is at an early stage of development it usually continues to differentiate histologically and often anatomically also (Bloom, 1937 ; Fell, 1940, 1953 ; Gaillard, 1942). Organized growth frequently occurs in the interior of an explant while unorganized growth is taking place at the margin. As with any other biological indicator tissue cultures have their advantages and their limitations in radiological work. The chief value of unorganized cultures lies in their relative simplicity ; the population consists of so-called resting cells all of which are potential dividing cells among which mitotic figures are scattered, all growing under conditions which eliminate any complications due either to nerve or blood supply, or to the reaction of the organism as a whole. During the period of most active growth less than 5% of the cells are in actual division simultaneously, but even this proportion represents in a single culture a large number-sometimes hundreds-of dividing cells at any one time, and the number remains remarkably constant over a period of several hours (Spear, 1931). The growing tissue forms such a thin sheet that when it is exposed to radiation physical conditions are uniform throughout the material and it is possible to achieve great precision in both the biological and the radiological procedures. It is easy to examine cells in the living state, and permanent records of any changes induced by exposure can be made by means of photomicrography and microcinematography (Canti, 1928). Tissue cultures have been used in radiation biology for the study of the lethal effects of radiation (immediate and delayed), for *quantitative observations on the effect of radiation on mitosis, and for studies on the relative biological efficiency of different types of ray. They have proved particularly useful in experiments to determine the significance of alteration in such physical factors of irradiation as dose, dose rate, wavelength, and quality. Tissue culture should be regarded “as a valid and important accessory technique to work done in vivo, but it is only partially a substitute for such work. One of the greatest advantages of the tissue culture method, viz. the simplification of experimental conditions which it implies, is at the same time one of its greatest limitations. It is often objected that tissue culture is useless because we do not get the same results in vifro that we

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obtain in vivo. This is not really a legitimate criticism, since one of the principal objects in using the method is to eliminate some of the numerous unknown factors which so often hopelessly complicate the issue in vivo. Naturally, therefore, we seldom expect to obtain the same results in vitro that we get in vivo. It must always be remembered that work in vitro can often give us only one chapter of a story that may run into several volumes. Even one chapter, however, is not to be despised when so many of the other pages are illegible” (Fell, 1935). Cultures were first used for radiological experiment by Wood and Prime (1914) and by Price Jones and Mottram (1914), who exposed cultures of malignant cells to radium. These authors pointed out the significant contribution which cell migration makes to increase in the superficial area of a culture which can continue to spread after the inhibition of all cell division. Strangeways (1922) used tissue cultures of chick fibroblasts for his classical work on normal mitosis and later (Strangeways and Hopwood, 1926) showed how quantitative as well as qualitative radiological experiment could be made on this material. This work was considerably expanded after his death, when the results of a long series of observations were used as a basis for further radiation experiments, under comparable physical conditions, on more complex tissues exposed in vivo. These animal experiments in turn were used as preliminary studies to quantitative work with human malignant material exposed to radiation in the ordinary course of radiotherapeutic treatment (Spear, 1948). Two interesting modern developments in tissue culture technique are the production of mass cultures of various strains of normal and malignant cells (Gey and Gey, 1936) and the establishment of clones derived from single cells (Earle, 1951) ; this is a revival of earlier and less successful attempts to grow isolated cells (Fischer, 1923; Nature, 1924). Among other interesting results which have come from this work is the demonstration that strains grown for long periods so adapt themselves to abnormal conditions of growth that their physiology becomes very abnormal and the cells may take on a malignant character. Freshly isolated tissues, such as those which have commonly been used for radiological experiment, are much less atypical. The range and extent of tissue culture experiments which already fall within the radiological field are too vast for detailed review here. Fortunately the interested reader can extract the story for himself from the recently published and monumental bibliography of tissue culture (with later supplement) compiled by Murray and Kopech (1953). e. Higher Orgmisms. The size of the experimental animal plays a

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significant part in the design of radiation experiments. With the higher animals the experimenter can choose whether exposure shall be partial3 or to the whole body. Whole-body irradiation is necessary to determine the dose levels for immediate and delayed lethal effects (Patt and Brues, 1954a, p. 930). Histological examination of the organs of animals killed by irradiation, however, has so far yielded few clues as to the cause of death. Although the dose required to produce an “immediately lethal” effect, i.e. death under the beam, is probably as high for man as for animals (cf. Spector, 1956), that needed for a delayed lethal effect (and the delay may be only a matter of a few days) is remarkably low and similar to the dose which affects mitotic activity without inflicting serious damage on blood vessels. It is difficult to see how suppression of mitosis per se in an adult could cause death in so short a time--“the organism looks so normal, except that it is dead”-and some action on the coordinating systems of the body seems a plausible explanation, though it is not known which system is affected and what the mechanism of blockage may be (Paterson, 1957; Medical Research Council, 1922). That certain organs show greater change after whole-body exposure than when irradiated locally is now established (cf. Halberstaedter and Ickowicz, 1947 ; Leblond and Segal, 1942) ; on the other hand local irradiation if sufficiently intense can affect a distant organ well outside the field of exposure (Henshaw, 1944). This suggests that the coordinating systems have an important role where wholebody exposure is involved, and among these the part played by the endocrine system is perhaps the least explored. The degree to which the hormonal system is developed may have some bearing on radiosensitivity in different species. Experiments with partial exposures, the rest of the body being shielded, distinguish the present group of indicators from most of those considered in the previous paragraphs. With partial exposure the irradiated material is surrounded by unirradiated tissue which contributes to the ultimate picture presented (in the case of human tissues, “ultimate” may be a period of thirty to forty years). Thus the histological picture at any given time after exposure is the resultant of initial injury plus whatever degree of repair or other change has been achieved, and it varies in important details with the length of the period elapsing between exposure and examination. At low dose levels the maximum effect of an irradiation is delayed as the dose increases. This has been observed for mitotic depression (Hertwig, 1920; cf. Alberti and Politzer, 1923, 1924) and for the wave of cell de3 The development of microbeams has now enabled specific parts of cells and unicellular organisms to be irradiated, but such apparatus is not yet generaIly available for radiobiological investigation (cf. Bloom et al., 1955 ; Zirkle, 1956).

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generation associated with the period of renewed mitotic activity (Tansley et al., 1937). Plotted on a single graph, the maximum degenerate cell count for a series of increasing but low doses appears as a set of ascending peaks along the time abscissa; the point of highest count gradually shifts to the right as the dose increases. At high dose levels, on the other hand, the degenerate cell counts may be at their maximum by the end of a long exposure and diminish with time after exposure (Lasnitzki, 1943). Experiments on laboratory animals, descriptions of which account for the bulk of radiological literature: can be roughly classified as follows (references are illustrative only) : 1. Those which concern the significance of the physical factors of radiation in determining the kind of biological response elicited : viz. variation as regards time and intensity factors (Scott, 1937 ; Loutit, 1956), spacing of radiation (Regaud and Ferroux, 1930; Ellinger, 1943, 1945), type of radiation employed and use of internal and external sources of radiation (Warren et al., 1950; Lamerton and Harris, 1951; U.S. Atomic Energy Commission, 1951 ). With the increasing variety of radiation beams provided by modern developments in physics, much work in this field concerns the relative biological efficiency of different ionizing radiations (Gray, 1946 ; Boag, 1953) and related theoretical considerations (Lea, 1946). 2. Those concerned with increasing the sensitivity (Patt and Brues, 1954a, p. 934) or the resistance of cells to irradiation (Barnes and Loutit, 1956). 3. Determinations of radiosensitivity for tissues in health and disease (cf. Ellinger, 1941). 4. Comparisons of the animal response with that of simpler biological material (Stoel, 1928; Tansley et al., 1937) and of human material, i.e. experiments designed to provide a scientific basic for radiotherapeutic practice (Spear, 1948 ; Hoecker, 1956). 5. Comparisons of the result of irradiating specific systems of the body with the result of whole-body exposure (Jacobson et al., 1949). 6. Studies on the effect of radiation on the coordinating systems of the body : vascular, nervous, secretory (Merwin et al., 1950; Warren, 1943a ; Betz, 1956). 7. Experiments concerned with the dividing, the nondividing, and the differentiating cell (Carlson, 1954). 8. Study of the radiosensitivity of the same organ in different species (Glucksmann et al., 1957, p. 527). 9. Experiments concerned with carcinogenesis (Glucksmann et al., 1957, p. 497). 4 In the Annual Review of Nuclear Science Gray (1956) cites nearly 400 radiobiological papers which were published in 1955 alone.

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10. Studies of radiation effects peculiar to the higher animals (Kaplan et ul., 1953; Kaplan, 1949; Patt and Brues, 1954a, b ; Brues and Sacher, 1952; Betz, 1956). f, Human Tissues. The need for the direct observation of normal human tissues in order to discover how they respond to radiation is at last becoming recognized, and this is perhaps best indicated by the time given to the discussion of human data in recent conferences on radiation biology (Cronkite et ul., 1956; Mitchell et al., 1956). Extrapolation from animal tissues to human is being viewed more critically today than has sometimes been the case previously. However nearly the radiosensitivity of an indicator approaches that of human body cells, it is unlikely to give exactly the same information as would be obtained from direct observations of human tissues even when the response is similar. Since the irradiation of every deep-seated tumor must involve exposure of skin, a great many observations have been made on this tissue in the course of radiotherapeutic work, but the value of these observations is limited by the rarity of any opportunity to obtain histological specimens and the fact that dose is of necessity determined by therapeutic requirement rather than experimental expediency (Ellinger, 1941) . Macroscopic change becomes recognizable only after much histological alteration has already taken place and serves only as a very rough indicator of biological response. Investigations of radiation-induced changes in the epidermis were made by Lion in 1901 and by Dalons and Lasserre in 1905 ; those on the dermis were begun as early as 1898 (Unna). Classical work in this field has been done by Miescher (1928) and by Schinz and Slotopolsky (1928); Lacassagne and Gricouroff have recently given a general review of cutaneous response ( 1956b). The inflammatory reaction and subsequent fibrosis (depending on dose level) have been investigated in some detail, but histological examination has seldom extended over a period of more than a few months. It now seems, however, that observations may have been abandoned much too soon. I t was a fortuitous circumstance which demonstrated the curative action (three years symptom-free) of radiation (Forssell, 1931) prior to the recognition of its carcinogenic properties (Frieben, 1902). It has taken a further fifty years, however, to show that these are not simply alternative actions of radiation, one sequence of events being wholly beneficial, the other wholly malignant, but that the same course of irradiation which eradicated a given type of tumor thirty years ago may be the cause of a different type of tumor originating in the field of irradiation after this long latent period. The occurrence is undoubtedly rare, but how rare will not be known until the understandable but nevertheless inexcusable reluctance fully to investigate the matter is overcome. Yet the

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fact must be fitted into the over-all picture of the effects of radiation on biological material alongside that other paradoxical observation that further radiation can be used successfully to treat a radiation-induced malignancy (Williams, 1938). No attempt will be made here to consider systematically the reaction of normal human tissues to penetrating rays. The response is dkii assumed from the results obtained from animal experiments where the differences seem mainly of degree (Patt and Brues, 1954b, p. 1005). There are qualitative differences, however, which in the absence of any experimental basis still remain unexplained. Thus Williams (1901, p. 453) called attention to the action of X-rays in relieving pain ; malignant hypertension may follow irradiation of the human kidney unaccompanied by any renal lesion, though a lesion may be present in some cases (Luxton, 1953; Wilson and Ledingham, 1956) ; the enlarged thymus gland in children may be affected by radiation doses of the order used in diagnostic radiology; and the highly developed endocrine system in man appears to be responsible for some reactions to radiation which as yet seem to have no exact parallel in lower organisms (Patt and Brues, 1954b, p. 989) ; Ellinger, 1954). This is perhaps most dramatically illustrated in the successful treatment of sterility in women (Kaplan, 1954) by multiple-field irradiation in doses similar to those which can cause sterility when directed to the healthy ovary. The effects of whole-body exposure have been studied in persons exposed to radiation as an occupational hazard (Court-Brown and Abbatt, 1955), in victims of atomic bombing (Kusano, 1953), and in casualties resulting from accidents in nuclear radiation laboratories (Hempelmann et al., 1952). The difference in radiosensitivity between diseased tissue and its normal prototype has often been'noted (Ellinger, 1954). Most of the clinical work done on malignant tissue has been concerned with attempts to compare its sensitivity with that of the normal adult or embryonic tissue from which it was derived and thus help the radiotherapeutic treatment of cancer. Much experimental research in the laboratory was originally undertaken to determine the process by which malignant tumors disappeared after successful irradiation. Histological examination of irradiated tumors presented such complex and bizarre appearances that efforts were made to obtain clues to the radiation action from simpler material provided by laboratory experiment (Lacassagne and Monod, 1922 ; Donaldson and Canti, 1923 ; Canti and Donaldson, 1926). Three main schools of thought existed, one maintaining that tumors disappeared by a process of degenerative mitosis, another that radiation had a sterilizing action on malignant cells by prevent-

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ing mitotic activity, and a third that all the effects of irradiation on tumor tissue acted via the blood supply. Although most of the experimental work devised to test these possibilities was done on materials other than human, a few investigators made histological studies of human biopsy material and obtained direct evidence of local changes produced by radiotherapy in the area to which it was directed (Glucksmann, 1941). Radiosensitivity as indicated by the rapid disappearance of the tumor soon after irradiation is by no means synonymous with radiocurability, that is, permanence of radiation effect (Cade, 1940, p. 234). Thus, although much emphasis is often placed on the marked changes produced in anaplastic tumors by radiation, several observers have pointed out that the differentiating tumors, which clinically seem to react to radiation more slowly, give on the whole a more satisfactory ultimate response (Dominici, 1909; Alter, 1920; Regaud, 1928; Phillips, 1931). In a differentiating tumor, many of the daughter cells resulting from cell division become sterile because they differentiate, although abnormally. In this connection, the fact that radiation can promote differentiation as well as injure proliferating cells is of some significance (Fukase, 1930; Finzi and Freund, 1943; Spear and Gliicksmann, 1941), since with suitable types of malignant tumor radiation may have a double curative action by mitotic inhibition and by sterilization. In the undifferentiated or anaplastic tumor, on the other hand, even a marked destruction of cells from heavy dosage may be followed by a recrudescence of the tumor from residual cells, incapable of sterilization by differentiation, which have survived the radiation. It must be recognized, however, that a tumor capable of responding to radiation by increased differentiation may be adversely affected by excessive exposures which interfere with, instead of promoting, this process. Overirradiated normal tissues show an increase in cell division and a decrease in cell differentiation which have sometimes resulted in radiation carcinomata (Laborde, 1931; Ross, 1932). Such growths can, however, be treated by further radiation, if it is so delivered that the proliferative tendencies of potential dividing cells are checked and the differentiation processes promoted (Williams, 1938). IV. EFFECT OF RADIATION ON GENERATIVE TISSUES Beyond our preoccupation with our special interests, as anatomists, bacteriologists, cytologists, dendrologists, endocrinologists, geneticists, histologists, immunologists, limnologists, mammalogists, neurologists, oncologists, physiologists, radiologists, serologists, taxonomists, virologists, and zoologists, we remain above all biologists, at feast in spirit and allegiance, if not in performance. (Paul Weiss, 1953)

The demonstration (Albers-Schonberg, 1903) that X-radiation had an

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adverse effect on the generative organs of animals was one of the earliest proofs that the new radiation could affect tissues beneath the skin. Frieben (1903) showed that sterility in rabbits and guinea pigs may follow exposure, and a similar result for human subjects was demonstrated the following year (Philipp, 1905). About the same time it was noticed that atrophic changes in the ovary were induced by exposure to X-rays (Halberstaedter, 1905 ; BergoniC et al., 1905). Careful systematic histological studies of irradiated rat testes were undertaken by BergoniC and Tribondeau (1904, 1905) and by Barrat and Arnold (1911) ; later Eker used the grasshopper testicle to investigate quantitatively the effects of radiation at various stages of the insect’s development as well as in adult life (Eker, 1937). More recently Oakberg (1955a,b) has worked on the mouse testis and published a review on the results of irradiating spefmatogonia. BergoniC and Tribondeau summarized all their observations on adult, embryonic, and malignant tissue, including both animal and human material in the following terms (translated) : “The sensitivity of cells to irradiation is in direct proportion to their reproductive activity and inversely proportional to their degree of differentiation” ( BergoniC and Tribondeau, 1906). The “law” is usually quoted with the last eight words omitted (cf. Boyd, 1953), but this is saying no more than is contained in Perthes’ earlier statement that cells are most sensitive to radiation during mitosis (Perthes, 1904b). More rarely the reference to cell division is left out and only differentiation mentioned (cf. Sullivan and Grosch, 1953). In either case the result is misleading, as the complete statement applied specifically to any tissue, generative or somatic, in which an orderly process of cell division and cell differentiation was taking place, and in which the fully differentiated cell was incapable of further division. Obviously, not all the cells in such a tissue differentiate, as some daughter cells must remain as potential dividing cells to maintain the supply of differentiating elements ; these potential dividing cells may show the effect of radiation by degenerating if and when mitosis is attempted (Gliicksmann and Spear, 1939). But since each cell presumably has the alternative of dividing or differentiating, it is misleading to judge response simply by mitotic degeneration because a cell which was hindered from entering division by radiation may differentiate instead ; the normal sebaceous gland cells of rodent skin react to radiation in this way (Chase, personal communication, 1957). Clunet (1910) was among the first to notice increased keratinization in tumor tissue after irradiation, though Dominici (1909) had, as already mentioned, pointed out that differentiating tumors in the long run responded better to radiotherapeutic treatment than anaplastic growths. The evidence that radiation can promote differentiation (cf. Cheval and Dustin, 1931)

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has been accumulating for nearly fifty years, but there is a curious reluctance in some quarters to accept the evidence (cf. Jolles and Koller, 1950). When differentiation occurs in this way, cell degeneration may be postponed for a long time. Nevertheless, the “law” of BergoniC and Tribondeau is a convenient summary within the limits of its field of application, a field which Dustin some years later (1929) endeavored to enlarge by suggesting the additional words : “La chromatine nuclkaire est d’autant plus sensible aux radiations ou aux poisons, qu’elle est proche, d‘une phase de condensation : chromosomiale (cinAse) ou prkpycnotique (thymocytes, lymphocytes)” (cf. Trowell, 1952).

1. Generative Glands as Biotogical Indicators of Tisszte Response Many other workers have demonstrated the convenience of the testicle for radiobiological investigation (cf. Regaud and Lacassagne, 1927 ; Gatenby and Wigoder, 1929). Its behavior and reaction to radiation are typical of a group of tissues where there is “a cell stock and lateral offspring which terminate in sterile [this word is used in a special sense here] elements that are destined either to be eliminated (spermatozoa, horny cells of the epidermis, muciparous cells of glandular epithelium, etc.) or to be absorbed (‘degenerative’ cells of neoplasms). The perpetuation of the cell species belongs to the stock cells. Radiosterilisation of a normal tissue . . , and definitive cure of a neoplastic tissue are the consequence of total destruction of the stock cells. It is sufficient that the death be limited to these cells: the direct destruction of the offspring (generally more radioresistant) is not of importance, since they are destined to disappear in the simple course of their natural evolution” (Regaud, 1928). Schinz subsequently grouped these tissues under the descriptive name of “molting tissues” (Schinz, quoted by Ellinger, 1941, p. 277), a term which the more recently developed exfoliative histology of cervical, pulmonary, and other forms of cavitary carcinoma has made even more appropriate (Vincent Memorial Laboratory, 1950; Osborn, 1953). Not all fully differentiated cells, however, are shed or undergo degeneration. For example, in the growth and development of the central nervous system the oldest cells remain as highly differentiated and functioning cells which persist throughout life. There is a difference to be noted between the epidermis and the seminal epithelium, both of which may be regarded as typical molting tissues. In the skin, cell division and differentiation occur simultaneously for indefinite periods, whereas in the seminal epithelium portions of the tissue are quiescent while others are functioning ; i.e. there is a spatial separation of active and resting areas. On the other hand, in epithelial glandular tissues, both

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normal and neoplastic, there are alternations in time between the function of multiplication and that of secretion. “I believe,” continues Regaud, “that an alternation of this sort exists not only in the lateral offspring, but also in the stock cells of these tissues. If this be the case we can understand how radiosterilisation of this stock may not be possible, or rather how it will necessitate a considerable radiation dose such as to produce a diffusely caustic effect” ( Regaud, 1928). So long as the irradiation is local and observations are confined to the exposed tissue soon after irradiation, the response of generative tissues differs in no exceptional or unique manner from that of any other tissue in which cell proliferation and cell differentiation are taking place simultaneously as parts of an ordered and finely balanced mechanism; the differences are those of degree rather than kind.

2. Classification and Selection of Material In considering the effect of radiation on generative tissues, distinction should be made between the action of penetrating rays ( a ) on the process of maturation of the germ cells, ( b ) on the sperm and on the ovum, (c) on constituent parts of the germ cells. Some species of animals (and plants) are more convenient than others €or studying some particular aspect of radiation response. Selection may be a question of supply, ease of handling, particular histological characteristics, or even just convention, with the associated risk that the selected species may not be sufficiently representative to be a general indicator of response. a. Process of Maturation: Testis. I n the opinion of most observers, radiation exerts its maximum effect on the most actively proliferating cells, i.e. the spermatogonia (cf. Gatenby and Wigoder, 1929; Mohr, 1919) ; the result of exposure is a breakdown of mitotic cells and diminution in the number of cells entering division. The daughter cells already produced, however, complete their maturation to spermatocytes, spermatids, and finally spermatozoa, and the gland appears to be functioning normally for a time ; eventually the supply is exhausted and the animal becomes sterile. This may be only a temporary condition, because either the most primitive of all the generative cells (those in contact with the basement membrane of the seminiferous tubule) have greater powers of recovery than their, immediate progeny, or because spermatogonia are repopulated from nondividing cells that have survived exposure. Eker has shown that in the locust those spermatogonia are most sensitive which are the farthest removed (seventh to eighth generation) from the primitive cells, and that the

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generations then show a retrograde disappearance (Eker, 1937) with the three youngest generations surviving the doses used. b. Process of Maturation: Ovary. The generative cells of ovarian tissue are in general more sensitive to radiation than the corresponding cells of the testes, but otherwise the sequence of events after exposure is analogous to that of the male gland when allowance is made for the numerical differences between sperm and ova. In the female, however, the association of ovarian function with the menstrual cycle enables the effect of radiation on gland function, especially in the human subject, to be more conveniently judged. At first the effect of the rays was believed to be specific only in suppressing ovarian function, and irradiation was tried therapeutically to control excessive bleeding in women. Intensive study of the action of radiation on the human reproductive system and that of different kinds of animals fol!owed, usually with acute exposures (Warren, 1943b). It was then observed that bleeding sometimes followed small doses to the ovaries, and it was suggested that they might be of use in the treatment of those forms of sterility that are unassociated with anatomical abnormalities and are possibly due to functional disturbance either of the sex glands themselves or of the ductless glands associated with them (Rongy, 1924). It was soon found that radiation is one of a number of agents which may affect the functioning of the female generative apparatus by an action on the endocrine system of ductless glands, particularly the ovary and the pituitary. Many individuals now alive owe their existence to this action of radiation. To take only one series of cases, of the children of fifteen mothers so irradiated twenty-five years ago, twenty are known to have married and have to date produced fourteen children (nine boys and five girls, including one twin). All these grandchildren appear to be mentally and physically normal (Kaplan, 1940, 1954). Destruction of the greater part of the pituitary, e.g by disease (in women) or by experimental procedure (.in animals), leads to degenerative changes in the maturing cells of the ovary so that the individual becomes sterile. The organ itself, however, remains largely unimpaired, so that if the activity of the pituitary gland can be restored to the requisite level, the ovary is capable of functional recovery. The healthy pituitary, in contrast to the diseased gland (Ellinger, 1941), is relatively resistant to radiation, and the glandular cells can withstand doses sufficient to destroy certain types of intracranial tumor, even when the tumor involves the pituitary itself. c. Effect of Radiation on Germ Cells. The use of germ cells of higher organisms as indicators needs special consideration. As end products of the reproductive glands they are independent units and react to radiation

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like unicellular organisms in isolation. But as agents of zygote formation their response to radiation may have far-reaching effects not only on embryonic development but also on later generations. The assessment of radiation effect on germ cells in terms of the damage subsequently seen in developing embryonic tissues is convenient in genetic studies, but this is in no way analogous to the lethal action of radiation on somatic tissues. The amount of chromosome damage which somatic cells can withstand as compared with germ cells is striking and was elegantly demonstrated over twenty years ago by Politzer (1934) in his detailed account of chromosome injury and fragmentation by radiation. The viability of the offspring of radiation-injured somatic cells studied by long-term cultivation and observation of chromosome-deficient cells still remains an unexplored yet promising field of radiological research. The effect of radiation on the fully formed animal or human spermatozoa operates by a direct action. The ovum, on the other hand, is subject to indirect actions as well. Mature sperms are probably the most resistant cells of germinal tissue, though their chromosomes, on the other hand, appear more sensitive to radiation than the chromosomes of spermatogonia. The radiosensitivity of sperms, however, is not constant and varies with the length of time the sperms have been mature. Both sperm and ovum may be damaged directly by radiation, or the germ cell may be defective as a consequence of the persistence of injury done to the cells from which it was derived. It is this possibility which constitutes the dangers of conception before the onset of the “sterile period,” though it has been argued that the cells which mature after the period of temporary sterility have recovered from radiation effects and are unlikely to carry injurious changes to any progeny (see Gliicksmann, 1947). Among the most spectacular results of experiments with sperm and ova, were those obtained by Hertwig (1913), who found that irradiated frog spermatozoa (within certain dosage limits) were capable of fertilizing ova, but that gross abnormalities of embryonic development resulted. Higher dosage, however, was compatible with normal development. This paradoxical result could be explained by supposing that the spermatozoa receiving high dosage, though rendered sterile, had not lost their motility and therefore were able to penetrate the ovum (though not able to affect syngamy ) and initiate parthenogenetic development. Both phenomenon and explanation have been confirmed by later work (cf. Rugh, 1939), who exposed spermatozoa of R a m pipiens to doses of X-rays from 15 r. to 50,000 r. At l0,OOO r. only 1.6% of the embryos hatched, but at 50,000 r. the figure rose to 90%. These embryos were morphologically uniform and

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very similar to the haploids produced by other means; they were undoubtedly parthenogenetic. Experiments of this type are more difficult with higher animals, but there is evidence that at least the first stages of the Hertwig effect can be produced in mammals (Parkes, 1947). At this point it is perhaps worth inquiring whether a sperm rendered abnormal by the action of radiation on its ancestral cells has an equal chance of taking part in a fertilization as compared with unaffected fellow sperms with which it is associated. In man, of approximately 300 million sperms available for the fertilization of a single ovum it seems likely that only the very fittest survive the hazardous journey by which the ovum is reached. Drosophila, by contrast, with a sperni/ovum ratio of 16 to 1 and no journey to make, would seem to lack a possible protective factor which may be of some biological significance to higher animals and especially man (Gliicksmann, 1947). The continuous supply of spermatozoa in such large numbers is a further means of “protection.” d. Effect of Radiation on Cell Constituents: Chrowzosowzal Structural Change. Chromosomes may be injured as a result of severe damage to the cell as a whole, or to individual threads within an apparent normal cell. Between these two extremes there is a wide gradation of radiation effect. Schaudinn, as already mentioned, described in 1899 sudden and violent disintegration of irradiated protozoa, and many observers since have studied explosive cells in a variety of material exposed to radiation in high doses (Cox, 1931a ; Strangeways and Oakley, 1923 ; Lasnitzki, 1943). Whether the explosion is linked in any way with mitosis is not clear. Cox found that in irradiated tissue cultures the number of breaking-down cells was greater in proliferating than in nonproliferating tissue, but the cells are too disorganized to be classified in terms of mitotic activity at the time of exposure. Explosive cells, indistinguishable from those seen after irradiation, also occur very occasionally in unirradiated material (Morley, personal communication, 1956). Individual chromosomes within a cell can be affected by quite low doses of radiation (Carlson, 1941). Perthes reported chromosomal fragmentation in 1904, and this is one of the most common effects of radiation on proliferating cells. In somatic cells fragmentation may lead to the formation of micronuclei in daughter cells (Strangeways and Oakley, 1923; Politzer, 1934) without otherwise disturbing the process of division. In generative tissue the result of fragmentation is different as it may affect the succeeding generation (if any). In Eker’s material irradiated with 500 r. no multinucleate cells were observed though they were seen in analogous material exposed to small doses (Mohr, 1919). In Eker’s ex-

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periments irradiated spermatogonia completed one division before breakdown in the succeeding prophase (cf. La Cour, 1953; Goodspeed and Avery, 1930). The chromosomal aberrations caused by radiation are striking and grotesque (Cox, 1931a). They arise from at least two different actions of radiation : (1) A generalized surface stickiness, not specific to radiation, which results in chromosomes at metaphase adhering where they happen to touch, and in sister chromatids failing to separate completely at anaphase and thus giving rise to chromosome bridges. For this type of action, called the primary effect in the earlier literature, Lea has suggested the term physiological e f e c t (Lea, 1946, p. 192). Carlson (1956) suggests that more than one phenomenon may be included under this term, e.g. the subchromatid exchanges described by La Cour and Rutishauser ( 1954). (2) Structural alterations in individual chromosomes arising from one or more breaks, owing to a highly localized action of radiation. This has been called the secondary effect of radiation, but since it is due to a direct action of radiation on the chromosome thread, the term structural change as suggested by Lea seems more appropriate. The particular form of structural change which finally emerges depends on the position and number of the breaks and subsequent rearrangements of the fragments by processes of deletion, inversion, and interchange (Kaufmann, 1954, Sax, 1940). Wherever the chromosome mechanism is found, it would seem reasonable to suppose that changes induced by radiation in any one type of material may also be found in irradiated chromosomes of other types. Although all chromosome material can be classed as among the radiosensitive tissues, the response to radiation is by no means uniform (Lea, 1946, p. 189). Inversion and interchange, for example, are more common in the fly than in plant material (Muller, 1951, p. 153). As a result of studies on “a limited number of cytologically favourable materials,” Lea (1946, p. 1%) concluded that the physiological effects, but not the structural changes, are exhibited by the cells already in division at the time of irradiation, and that the structural changes, but not the physiological effects, are exhibited by the cells which enter division after the expiration of the period of reduced mitotic activity which follows irradiation. Structural change in a chromosome may lead to alteration in the linear arrangement of its genes, so giving rise to heritable changes in viable cell progeny A radiation-induced deletion may be so small as to escape detection ; nevertheless if the sequence of the normal gene arrangement is disturbed (position effect) alteration in heritable qualities may follow and there may be considerable difficulty in distinguishing this mechanism of

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radiation action from that giving rise to a “point mutation” unaccompanied by any structural change at all (Muller, 1951). The question whether certain loci in the chromosome are especially radiosensitive has been considered (Kaufmann, 1939; Sax, 1940) and the frequency of breaks in certain regions compared with the distribution obtained by using other injurious (e.g. chemical) agents (cf. Fahmy and Fahmy, 1954, 1956). Associated problems are (1) the question of radiosensitivity of the chromosome per se at different stages of the maturation of the germ cell of which it is a constituent (Sparrow, 1951), and (2) the radiosensitivity of the cells themselves at different stages of maturation (Regaud and Blanc, 1%; Murray, 1931 ; Oakberg, 1955a, b). e. Heritable Changes Unaccompanied by Visible Strwtural Alteration. This type of response to radiation, unrelated to individual cell behavior, lies outside the application of Bergonit and Tribondeau’s law, but not a little confusion exists in radiological literature, even today, by failure to recognize the difference, and the results of genetic experiments are still frequently but unwarrantably regarded as indications of the probable behavior of somatic tissues under irradiation. Many of the so-called “genetic effects” of irradiation are now known to be irreversible, and the consequence to the subsequent development of embryonic tissue may be serious. In a somatic tissue, on the other hand, individual cells are easily expendable without loss of function of the organ. As indicators of response to radiation, independently existing cells (protozoon or germ cell) and cells which are constituents of a complex tissue are in essentially different categories. It now seems virtually established that penetrating rays can induce heritable changes, unaccompanied by visible structural changes, in all animal species, in a great number of plants, and in many of the lower organisms as well. This similarity in the mechanism of heredity in widely differing biological material seems to invite extrapolation from one set of experiments to another. As a consequence the response of material easily obtained or convenient to handle is sometimes regarded as a standard which is representative of genetic material as a whole, and since there is a case for the genic nature of heredity in bacteria, these organisms have “become a very valuable experimental tool because of the relative ease of making quantitative studies of both spontaneous and induced mutations,” although they do not necessarily have a constant nuclear constitution (Zelle and Hollaender, 1955, p. 402 ; Spiegelman, 1956). Many studies of the so-called radiation-induced mutations in bacteria have been made both by a direct action of the rays and also by indirect action via the culture media. A point which is seldom (if ever) emphasized, however, is the high dose levels which are used in

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this type of experiment which may be anything up to scores of kiloroentgens. It is this sort of casual indifference to total dose which gives the ordinary biologist an uneasy feeling. Even allowing for all the technical difficulties of the investigation it seems questionable whether a dose which is lethal to half the population of asexually reproducing organisms produces the same kind of change in the survivors as is produced in germ cells of higher organisms by doses of a few roentgens. However interesting and important the radiation induction of bacterial mutations, in the present state of knowledge it would seem wise to be cautious in extrapolating the results to other kinds of material. Owing to their convenience fungi and molds have also been much used in work on the mechanism of mutation induction: by point mutations, position effect, mutation by breakage, or translocations (Muller, 1954) ; on factors promoting or hindering mutagenesis : oxygen, dryness, etc. (Stapleton et al., 1952) ; and even on the contribution of mutation production to cancer therapy. With plants there is a large field for exploiting the constructive uses of radiation-induced mutations, e.g. the four- to fivefold increase in yield of PeniciZZium which results from successive exposures (Hollaender, 1954). Although many of these mutational effects in a variety of material seem to have quantitative rather than qualitative differences, nevertheless as work on particular cell types becomes more precise it is the differences, rather than similarities, which become emphasized. In this connection the genetic peculiarities of Drosophila may be noted (Muller, 1954) in respect of the position effect, and the different rates of reverse mutations for different species of Neurospora (Giles, 1952). Any detailed consideration of the mutagenic action of radiation is outside the scope of this review, but one aspect of the problem does concern us. The direct effects of radiation on living cells include two essentially different types of action. On the one hand there is the type of response that requires a certain threshold dose before any biological effect can be recognized, and on the other hand a type of response that is directly proportional to the amount of radiation given, however small (in theory) that amount may be. The difference is often expressed graphically by two superimposed curves: one, a skewed S curve, represents the threshold type of reaction, and the other, a straight line, that of the nonthreshold type. In physical terms the latter suggests the probability that a single-event action, sornetimes called a “hit” or target action, is involved, and the former implies that the response depends on a two- or multiple-event action, which is sometimes interpreted in terms of cumulative dose (Hoffman and Reinhard, 1934; Lea, 1938).

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In general terms, the “hit” action is one in which there is no recovery factor, and injury once done is permanent and unalterable ; the threshold type of reaction is one in which recovery factors operate successfully until sufficient energy is accumulated to prevent the rate of repair from keeping pace with the speed at which injury is inflicted, and a recognizable abnormality results. If constituent parts of generative cells are among the most radiosensitive materials of the body and if the heritable changes (in contrast to somatic effects) produced in them by radiation are dependent on total dose only down to the lowest levels, then the amount of radiation additional to natural background, resulting from the medical, industrial, and military activities of mankind in the nuclear age, may soon become of biological significance. The question of the possible mutagenic action of cosmic radiation has been seriously discussed at intervals since 1928. Mutations have already been produced by absorbed radioisotopes (Powers, 1947). Radiation-induced mutations have come to be regarded as the classical example of the nonthreshold type of reaction. Under certain conditions, the production of chromosome aberrations may also be included in this group. I t is the potential genetic hazard in this sense which has focused public attention on radiation problems and promoted the recent attempts to obtain rational assessment of present-day risks from all types of naturally occurring and artificially produced penetrating rays (Russell, 1952). f. A n i d Experiments in Relation to Hunzan Data. The most casual review of the recorded observations on human material shows how inadequate are the data from which any final conclusions concerning this hazard can be drawn. On the physical side the chief lack is of precise information about dosage ; on the biological side results of long-term observations covering several generations can only be collected very slowly. The advantage of experiments on animals and plants is that the observer can arrange conditions which promise to yield the greatest amount of information under any given set of conditions. Further, the use of animals with a relatively short life span enables a sequence of generations to be observed in a comparatively brief time. The question arises as to what extent conclusions drawn from animal and plant experiments are applicable to human conditions, and opinion differs sharply on this point. For some aspects of the problem the only way to find out how radiation affects human tissues is to study those tissues directly; for other aspects, observations on animals and plants can give much useful information some of which can reasonably be supposed to have a bearing on what happens when corresponding cells in man are exposed to penetrating radiation. The differences as well as the similarities

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of form and function in different species must be taken into account. For example, although the development of spermatogenesis is, in essentials, similar in the fruit fly (Drosophila), rodents, and man, the process takes about 26 days in the mouse and rat and 15 to 19 days in Drosophila. But the short life of the adult male fly and the comparatively long duration of spermatogenesis necessitate an earlier beginning of spermatogenesis in the embryonic life of the fly than in that of mammals (Gliicksmann, 1947). In mammals the sperms, once formed, remain fertile for 21 days and mobile for about 42 days ; in the fly they remain fertile for the duration of the fly’s life. In the female organs the developmental process of the ovum lasts 28 days in women and 5 to 6 days in mice. I n the fly (Drosophila) a quantity of mature or nearly mature ova are found in each ovary. On their passage through special ducts these ova are fertilized by sperms stored in special receptacles so that fertilization is possible any time after mating. The fertilized eggs are then laid in suitable food material. Sensitivity of different tissues differs widely. Relative figures showing size of dose required to produce a given effect are recorded in the following table. X-RAYDOSAGE REQUIRED To INDUCESTERILITY ( GLUCKSMANN, 1947) “Permanent” sterility Man Mouse Drosophila Temporary sterility Man Mouse

Male

500-600 r. 1600-3000 r. 12,000 r. 250 r. (12 months) 800 r. ( 4 months)

Female 300-320 r. 800-1500 r. 5000 r. 170 r. (12-36 months)

Experiments on mice have demonstrated that a certain percentage of the off spring conceived before the onset of irradiation-induced sterility (“early conception”) of the parents show impaired fertility and give consistently reduced litter sizes. This “semisterility” was most frequently obtained in male mice whose testes were exposed to 800 r. of X-rays, and in females whose ovaries were exposed to doses of the order of 260 r. of X-rays (Snell, 1933, 1941). The offspring of the mice sired after the end of the period of temporary sterility (“late conception”), however, did not show any significant impairment of fertility or reduction in litter size in a series of 477 litters obtained from irradiated males, as compared with 472 control litters (Hertwig, 1938).

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The different results obtained with early and late conception are explained by the elimination of the affected immature germ cells through a series of cell divisions which they have to undergo during spermato- and ovocytogenesis. This phenomenon has been termed “germinal selection.” It remains to be determined, however, whether this germinal selection eliminates all the descendants of affected undifferentiated germ cells. I t is feasible that cells carrying only slight abnormalities might be able to pass through the series of cell divisions and that these radiation effects might produce changes in the progeny. The fact that in Drosophila sperms are stored for relatively long periods of time makes it difficult to detect whether germinal selection occurs or not (cf. Mossige, 1955).

3. Radiation Sensitivity Radiosensitivity can be viewed from several aspects (cf. Sparrow, 1951). It may be assessed in terms of the amount of radiation required to produce lethal or other irreversible effects of radiation, or to bring about some nonlethal effect under specified conditions ; it may be studied in terms of the varying response obtained when different generative and somatic tissues are exposed to the same (sublethal) dose of radiation, or the same tissue is exposed to different types of ray; or it may be considered in relation to the changes in sensitivity exhibited by any given tissue or cell when irradiated in various physiological states. Examples of these various approaches have already been cited in this and the previous section. In an attempt to sum up it may be said in general that radiations are potentially injurious to the cells which absorb them, and that above the threshold dose the changes produced may be transitory (reversible) effects, or permanent (irreversible) effects, with an intermediate class of effect where the radiation changes disappear completely but leave the tissue in a state of lowered resistance to further radiation ; (conditioned reversible effect) (Ellinger, 1941). Experiments on complex animal tissues which have demonstrated a selective action of radiation have also shown that observations must be continued for a long period if the full effect of radiation is to be appreciated (1) in inducing initial destructive changes and (2) promoting a variety of reparatory reactions : making good, excess repair, and/or patching with simpler material, e.g. connective tissue, with resulting fibrosis either in a generalized form or confined to the vascular system and leading to vessel occlusion. It may be questioned whether tissues irradiated in vivo are ever completely restored to normal, and any residual defects are liable to initiate subsequently recognizable pathological change if the tissue is subjected to

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unusual stress or other untoward stimulus. It is also to be noted that when a large volume of tissue is irradiated the greatest biological change is not necessarily found at the points of highest dosage. On the other hand, no tissue is entirely invulnerable, and all organisms are killed if sufficiently large doses of radiation are given. When the question of biological response is considered in a more general sense, one of the most striking results of radiation experiments seem to be the enormous dose range involved, from reactions which are obtained after exposure to a few roentgens to those requiring millions. There is a ten-thousandfold difference, for example, between the extremes of sensitivity among different types of living cells when measured by the lethal effect, and the same organism may vary greatly in radiosensitivity at different stages of development. The reasons for such great differences in sensitivity to radiation are still quite unknown. All this makes radiosensitivity a difficult term to define precisely. A body cell whose behavior is capable of being altered by a given dose of radiation may be regarded as a radiosensitive cell under the given conditions, irrespective of whether the change in behavior is advantageous or otherwise to the organism as a whole. A particular form of behavior can sometimes be elicited when the right handling of radiation as a tool has been learned. For any given effect there would seem to be optimum physical conditions for producing it with the minimum expenditure of energy. When these have been discovered, it is still possible to increase or decrease the degree of response by various chemical or biological agents. Some of these will be included among the subjects discussed in the following section.

V. RADIATION PHYSICS A N D RADIATION CHEMISTRY I N RELATION TO RADIOBIOLOGY Are we justified in looking for a few key reactions, or are the effects [of radiation] rather the sum of a multitude of processes? (W. M. Dale, 1952, p. 186)

Radiation biology began in a physics laboratory, and one of the earliest explanations of the biological action of Roentgen's rays (the 's was dropped about the same time) was in terms of their chemical effects (Thomson, 1896). But although the three disciplines of chemistry, physics, and biology have been associated in this field of research from the earliest days, one or other of the three has at different times tended to predominate and to influence both the theory and practice of contemporary radiobiological procedure. Roentgen, and a little later Becquerel, provided investigators in many fields with two new tools which they were not slow to exploit, and modern

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physics has greatly increased the range and variety of radiations available to workers throughout the many subdivisions into which biology is now broken up. There are thus a number of specialist fields worked partly by those who are familiar with their biological material and partly-and to an increasing degree-by physicists and physical chemists who have moved from the main body of their own science into radiobiology where their knowledge is indispensable but their experience is limited as a result of both training and outlook (cf. Davson, 1953). “The conceptions of physics,” says Mayneord ( 1945) , “tend towards the static and universal ; those of biology towards the dynamic and individual. The physicist learns to deal with effects accomplished and finished with fairly clear comprehension of the chain of events between. The study of living organisms necessitates intrusion into a delicately-poised working mechanism which may react in unsuspected and disconcerting ways. There is apt to be a great gap of ignorance between the original stimulus and the resulting effect, with a consequent belief that the mechanism is much simpler and more amenable to mathematical analysis than is in fact the case. The physicist is prepared to admit variability, but has a feeling that proper statistical methods will lead to unerring conclusions. The biological experimenter (and good clinician) has to make many inspired guesses on most insufficient evidence, and sometimes needs a good deal of convincing as to its inadequacy.” Hence come the biologists’ quest for simple material-at least to start with-and the physicists’ almost passionate desire for simple explanations which serve to link as many unrelated phenomena as possible. “Cellular damage from exposure to X-rays,” runs a recent annotation, “is believed to be caused by two different processes-first by direct hits by the ionising particles on the chromosomes, and secondly, by ionization of the water contained in the cell with the production of very short lived oxidizing radicals which inactivate the cellular enzymes and may lead to cell death” (Brit. Med. J., 1957). As if this were all! One is reminded of the controversy between Koch and Virchow in the 1880’s over the significance of the tubercle bacillus. “There you have tuberculosis,” said Koch, in effect ; to which Virchow retorted that tuberculosis was something quite different, viz. a complicated and varied response of the body tissues to a deleterious stimulus from outside. In this instance the stimulus was of very small dimensions, but an analogy with radiation biology is not inapt. It is estimated that 1 r. of X-rays will affect 1 in about 1O1O molecules of irradiated tissue, “and a dose of 50 r. seems quite negligible from this point of view” (cf. Lea, 1946, p. 64). Yet it produces marked biological effects in many types of experimental material, with differing response according to the way

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the dose is administered. The most active drugs such as acetylcholine or the toxin of food poisoning (botulinus) are effective down to a dilution of about 1 in 10l2.

1 . Radiation Physics a. Dosimetry. The discovery of X-rays was made without any idea of a biological or any other application of the new radiation. When their biological effects had been recognized, the need for a universal dose unit was soon apparent, and one equally suited to experimental and clinical use was desirable. It was not until 1928, however, that international agreement was achieved, although the unit suggested by Villard in 1908 was essentially the same as the international roentgen agreed at the Stockholm Radiological Congress (Brit. J. RadioE., 1928). But this was only a first step ; radiation dosimetry is still a developing subject. b. Mechanism of Action. Meanwhile interest in the mechanism of the action of penetrating rays had grown especially, though not exclusively, among physicists. Among several early suggestions the two main ones were that (1) the energy of electrons liberated by the absorption of roentgen rays in living matter is translated into heat at isolated points within the cell which becomes visibly altered when from 1 to 10% of its molecules have been affected (Dessauer, 1923) ; (2) the energy required for biological effects originates from the state of excitation induced in protein molecules (Holthusen, 1924). The former and more favored idea was developed into the quantum hit theory by Blau and Altenburger (1923), modified by Crowther ( 1926), and supported by a number of subsequent investigators (cf. Holweck et aZ., 1929). A biological response was thought to follow the absorption of a minimum number of energy quanta in a specific and particularly “sensitive spot” within the cell, and in this form the idea has become known as the target theory (cf. Lea, 1946). It is interesting now to recall that some twelve years later Crowther (1938) himself stated that “the majority of upholders of the target theory would tend to look to chemical action as the first link in the chain of events” following exposure (cf. Guilleminot, 1910). The theory in respect both of its applications and of its limitations was developed by Lea (1946) some of whose admirers have tended to overstress the applications and minimize the limitations. Lea himself took pains to specify the types of biological response to which he thought the theory applied, though his interpretation of the results of some genetic experiments in terms of his version of the theory have been criticized (Muller, 1951) as being open to alternative explanation. By the time his book appeared, however, Lea had got beyond the somewhat limited concepts of the target theory, even where he thought it

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applicable, as was shown by the volume of orderly notes on radiation chemistry which he left behind and which give abundant evidence that he was, at the time of his death, thinking along new lines (Lea and Dainton, 1949; Spear, 1950). It is now generally accepted that the theory applies to particular types of lethal effect or to actions producing some irreversible change in a still viable cell. Data derived from genetic material had played a conspicuous part in the elaboration of the target theory of radiation and not least the implications of the mutagenic action of radiation. Here was a widely occurring type of biological response among lower organisms depending solely on total dose, independent of dose rate or fractionation, and for which, at least in theory, there was no threshold dose. But, as Muller himself has stressed (1939), “not all of the effects of radiation in killing organisms or disturbing their development are referable to changes either of the class of gene mutations or chromosome re-arrangement.” Nor has the linear relationship between dose and effect yet been completely established for many of the higher organisms. c. Protection. Although the theory had only limited application, the possible consequences of the mutagenic effects of exposure greatly stimulated physicists to search for ways and means of providing adequate protection from the harmful effects of radiation. The deleterious action of X-rays on somatic cells had, of course, been known for a long time, and the taking of precautionary measures against the harmful effects of radiation go back many years before the mutagenic action was proved (Muller, 1928b, c). I n his textbook published in 1901 Williams shows (p. 52) tube holders devised by Rollins for screening operators and patients from the soft, scattered X-radiation (cf. Rollins, 1903) which was the cause of so many X-ray burns among the early pioneers. I t is a far cry, however, from “numerous coats of white lead paint” which sufficed Dr. Rollins to the 6 to 10 feet of concrete now used to shield a moderately sized cyclotron (Gallop et al., 1957). Nor do workers nowadays have to plan in isolation. The first protection committee to operate on a national scale was formed in the United States in 1920: the British committee was set up the following year (see Brit. J. Radiol., 1953). The Stockholm Congress in 1928 appointed an International Commission on Radiological Protection (I.C.R.P.), and there is now in addition a committee of the United Nations which is concerned with the questions of protection on a world scale. The functions of the British committee were taken over in 1953 by a Protection Committee of the Medical Research Council with subcommittees corresponding to those of I.C.R.P. Thus although the monitoring service provided by radiation physics

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was in the first instance directed against injury to the somatic tissue as the result of overexposure, the degree to which protective measures should be taken is now being more and more influenced by the genetic effects of irradiation with the long-term carcinogenic action as an added spur where no genetic risk is involved or where ingestion of radioactive isotopes constitute an industrial or military hazard to any human population. d. Permissible Dose. Among the first duties of the I.C.R.P. (see Brit. J . Radiol., 1955) was to fix for each of the various types of external radiation a maximum permissible dose level (m.p.d. or q.p.1.) which “in the light of present knowledge is not expected to cause appreciable bodily injury to a person at any time during his lifetime.” The biological data for y- and X-rays far exceeds that for other types of radiation, and I.C.R.P. has fixed the m.p.d. for these two radiations at 0.3 r./week, (or 0.03 rad, or 30 millirem; see below), assuming an average specific ionization (in terms of ion pairs per micron of water) of 100 or less. Some efforts have recently been made to take account of excitation of molecules as well as ionization and to substitute linear energy transfer (LET) for average specific ionization. Since, however, in practice the values of LET are generally derived from the specific ionization in air, the distinction is essentially a formal one in the present state of affairs (Brit. J. Radiol., 1955, p. 18). e. Dose Units and Relative Biological Eficiency. To arrive at the m.p.d. for a type of radiation producing an average specific ionization in excess of 100 ion pairs per micron of water, it is necessary to consider another factor : the relative biological efficiency ( R B E ) of the new type of radiation as compared with X- or y-rays (cf. Boag, 1953; Zirkle, 1952, 1954). The RBE may be defined as the inverse ratio of the two doses required to produce the same biological effect. This, of course, involves a unit of dose measurement common to the two types of ray, and since the roentgen (r.) has only a limited range of application, a more flexible measure is required. This is found in a modification of Parker’s (1948) rep (roentgen-equivalent-physical), which though originally intended for corpuscular radiation only may now be defined as the radiological dose produced in tissue by radiation other than X- or y-rays which gives the same energy absorption in tissue as 1 r. of X- or y-rays (see Brit. J . Radiol., 1950). This absorption is about 93 ergs/g. (for water or wet tissue) and replaces the 84 ergs/g. of the original definition which referred to air. It was then considered more satisfactory to have a new unit of tissue dose which would be of the same order of magnitude as the rep but which would be independent of the roentgen, which is tied to ionization produced in air. This resulted in the rad (defined before it was given its present

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name!), which is the dose of any ionizing radiation such that the energy imparted to the tissue is 100 ergs/g. (1 rad corresponds roughly to 1.07 r. in soft tissue ; or 1 r. = 0.93 rad) . Since the purpose of most protection work has applications to man, a further special unit, the rem (roentgen-equivalent-man) , has been suggested for use where attempts are made to compare the effects of different types of radiation on human tissues. The rem (like the rep) is applied to types of rays other than X- or y-radiation, and 1 rem may be defined as that amount of radiation which produces the same biological effects as 1 rad of X-rays. Thus: dose in rem = dose in rads X RBE. Neither roentgen, rep, nor rem in its definition takes into account the amount of tissue irradiated, an omission first felt when apparatus was developed which involved large regions of the body in any single exposure (Wood et al., 1938). Among suggestions put forward to meet this situation, Mayneord (1940) proposed the gram-roentgen, defined as the energy lost in 1 g. of air by 1 rep of ionizing radiation. This unit is used to integrate the total energy dissipated in a mass or volume of tissue, the dose derived then being called the integral or volume dose. The same integral dose is delivered whether 100 g. of tissue is irradiated with 1 r. or 1 g. is exposed to 100 r. Although much remains to be done on the physical side in making the various units of dosage more precise, it must be admitted that RBE is a somewhat loose term except where it applies to very simple biological effects produced at low dose levels. Boag (1953) has summed up the position thus : “It is found, in general, that any simple biological effects produced by one type of ionizing radiation can also be produced by any other type. Thus, both X-rays and neutrons will produce chromosome . breaks and aberrations, and examination of a particular break will not reveal whether it was produced by X-rays or by neutrons. There will, however, be observable differences in the proportions of the different types of break which are found in the two cases, and these can be correlated with the known differences in the spatial distribution of the energy communicated to the material by the two types of radiation. “The foregoing statement is only true, however, of the simplest biological effects. When more complicated effects such as death of the organism, regression of a tumour, loss of weight, or reduced viability are considered, the pattern of response to X-rays may be very different from that evoked by neutrons. It is reasonable to suppose that such semiqualitative differences in response arise from competition between many different simple effects, some of which are produced more readily, but not exclusively by X-rays and others more readily but not exclusively by neutrons. When all are

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produced simultaneously in an organism its response-pattern will be determined by the relative magnitudes of the various effects, and their relative importance to the organism.” The biological problems involved are in some ways simpler under the conditions of chronic irradiation, which is the condition with which most protection investigations are concerned (cf. Spear, 1944 ; Mole et al., 1957 ; Spear and Tansley, 1944 ; Tansley et al., 1948). But even within the range of chronic exposures the RBE is not constant; the results of some experiments have suggested a rise in value as the dose rate is lowered. This rise in value is very marked for some isolated chemicals (the term should be RCE in this instance, Dale and Davies, 1956) but has not appeared in some recent experiments at Harwell on neutron effects on mice where no evidence of any increase in RBE has been found with increase in exposure time (Mole et al., 1957). The RBE may vary in the same animal according to the organ selected, the most sensitive being termed the critical tissue, or the particular biological response chosen as the criterion of effect. The variation may range from four- to tenfold or even more. Different strains of the same species of animal give different results, and this makes extrapolation from laboratory findings to human conditions of rather doubtful validity.

2. Radiation Chemistry In spite of the early suggestion of the probable significance of the chemical actions of penetrating rays in radiation biology, little progress in this direction was made for many years owing to the high dosage required to produce recognizable chemical change when living material was irradiated (cf. Scott, 1937). A partial decomposition of water by radium or radon had been demonstrated soon after Roentgen’s discovery (cf. Debierne, 1909), but the possible relevance of this type of action to biological tissues under irradiation was not generally recognized until pioneer work was done on the irradiation of chemicals in solution by Risse (1929, 1930) and Fricke (1934). Their work showed that besides any direct release of energy within solute molecules there was an “indirect” action of radiation via change in water molecules whence energy was transferred to molecules in soIution. Then Dale (1942, 1943a, b) succeeded in magnifying the results of exposure by using purified enzymes in aqueous solution and measuring the changes induced by radiation in terms of the loss of enzymatic activity on a substrate. In this way it was shown that chemical change could be effected by quite low doses certainly well within the therapeutic range (cf. Dale, 1947). Weiss (1944) suggested that the energy carrier concerned is a free hydroxyl radical. The reason for the earlier

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failures then became clear. The number of solute molecules decomposed by a given radiation dose depends on the concentration of activated solvent produced, not on the concentration of the solute, and will therefore be relatively small; hence the concentration of solute employed must be the smallest consistent with chemical analysis, in order that changes in it may be relatively large. I t was the widespread failure to recognize this which led to the supposition that significant changes could not be produced in vivo by doses within the therapeutic range (cf. Allsopp, 1944). The relevance of these chemical investigations to biological conditions now became apparent, and radiation chemistry received formal recognition at the Sixth International Congress of Radiology, on equal terms with radiation histology and radiation genetics (Allsopp, 1951). a. Mode of Ackion of Radiation. The problems involved in attempting to trace the sequence of events between radiation-induced chemical change and the observed alteration in cell behavior are still formidable, however (cf. Zirkle, 1952). There are two general methods of attack: One is to study the effects of radiation on cellular molecules or chemicals of biological importance in vitro and from the knowledge gained attempt to synthesize a picture of the processes likely to occur in vivo. The second method is to observe the effect of irradiating cells in vivo as a function of modifying conditions during the experimental procedure, in the hope of using such indirect data to infer the intermediate processes (Sparrow and Forro, 1953). To the biologist the latter alternative has the greater appeal; he prefers rather to keep his eye on the cell and correlate observed behavior with possible chemical change than to acquire chemical data and foist them on the cell to explain its action. This alternative has been tried SO often and failed. When just after the lipoid theory of narcosis was advanced (Meyer, 1899) it was discovered that radiation affected lipoids, many papers were published which attempted to explain the biological action of radiation in terms of an effect on lethicin and a little later on cholesterol (Schwarz, 1903; Roffo and Correa, 1924, 1929; cf. Keller and Weiss, 1950). Then other single substances, e.g. globulin, were suggested. But none of these suggestions survived, and the series of physical theories of radiation action-point heat (Dessauer, 1923), quantum hit (Blau and Altenburger, 1923), target hypothesis (cf. Lea, 1946), photochemical theory (Holthusen, 1921) etc., with or without modifications (cf. Jordan, 1938)-were put forward. Now it was the turn of chemistry again to supply a clue. The target theory in its then accepted form was clearly limited in application or needed considerable modification if it were to be applied to more than a few types of biological response. The theorists were quick to see how the local

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liberation of free radicals within a cell might provide an alternative to and a more flexible mechanism than quantum hits to explain some of the mechanisms of the action of radiation (cf. Gray, 1951, 1952), Interference with nucleic acid synthesis and enzyme activity were soon being investigated as possible “sites” for radiation action (cf. Furth and Upton, 1953), and for the delayed lethal action of radiation a particular radical was tentatively named (Alexander, 1953; Alexander e f ul., 1954). For the rest, radiation chemistry has offered a new approach. To get away from the rather rigid language of the older conceptions, Gray (1951) suggested the term monotopic for action in which the initial process is the dissipation of energy within a strictly localized region without regard to whether the energy is dissipated within a biological structure or in the immediately surrounding medium. This seems a desirable step to take toward unraveling the complicated series of events which occur during the somewhat inappropriately named “latent period.” But it still takes little account of the dynamics of the situation. W e may be fascinated by the modern techniques of radiation chemistry, impressed by the knowledge gained of changes occurring “in the first few microseconds of exposure,” convinced that potent chemical substances are produced in Vitro by the action of penetrating rays-but where biological material is involved there is still the living cell to be reckoned with. W e may ask what happens when a chemical is irradiated, not in bulk but in the tiny, scattered mitochondria which under the microscope may be “seen as bright threads which move slowly about the cell with worm-like movements. They are most conspicuous in the cell processes especially when these are being extended in the direction of advance of the cell when they may be seen to leave the neighborhood of the nucleus. Occasionally mitochondria may be seen to divide. Before this happens a mitochondrium instead of being irregularly curved and apparently lying loose and waving in the protoplasm becomes straight as if stretched. Thereupon it snaps across and the portion nearest the advancing end of the cell moves forward with comparative rapidity until it reaches the extremity of the cell process” (Canti, 1929). How does this modify the consequences of exposure? Is there not a safety factor in dispersion and perhaps more so when each dispersed unit is in active motion? b. Chemical Protection. Although further progress in the elucidation of these problems would help in devising chemicaI measures of protection against penetrating radiation, some advances in this direction have already been achieved. Oxidizing radicals (OH, HOa, and H 2 0 2 ) , which can be produced by a few hundred roentgens, are known to be capable of reacting with essential enzymes containing the -SH group and

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converting this to an inactive S-S form (Barron et al., 1949). By supplying from outside competitive substances capable of reacting with the liberated radicals Patt and his colleagues (1949) have attempted to combat the damaging effect of radiation. They have succeeded by this means in diminishing appreciably the mortality of irradiated mice by giving cysteine to the animals immediately before X-irradiation with an otherwise lethal dose. The precise mechanism of action, however, has still to be explained. A substance protective to one species of animal is not necessarily protective to another (Patt et al., 1950). A number of chemical and physical agents are now known to have a protective action ; among these may be mentioned glutathione (Patt et al., 1950))BAL, thiourea (Mole et al., 1950)) glucose (Baclesse and Loiseleur, 1947), ethanol (Paterson and Matthews, 1951), immunization as well as a previous exposure to p- or X-irradiation (Raper, 1947; Cronkite et al., 1950)) anoxia (Dowdy et al., 1950),or just partial shielding in otherwise whole-body irradiation (cf. Furth and Upton, 1953). Cysteine and glutathione are effective when given just prior to exposure; bone marrow and spleen are effective if administered not too long after irradiation. Some substances are protective to whole animals and to isolated cells ; others protect animals but not isolated cells, showing that the protection is sometimes systemic and unconnected with chemical radicals produced locally by irradiation (van Bekkum and de Groot, 1956). An interesting extension of the work on protective devices was made by Lorenz et al. (1951), who injected biological tissue into irradiated animals with promising results. Bone marrow, marrow cells (Nowell et al., 19563, bone chips (Lorenz and Congdon, 1954) , spleen (Jacobson, 1952 ; Barnes and Loutit, 1955), horse serum, estrogens, and adrenal cortical extract have all been shown to be capable of a protective action when injected sometimes as a “mush,” sometimes as viable cells, which, at least in the case of bone marrow, seem to act at times by a process of replacement therapy (Ford et ul., 1956). It is clear that a protective effect like the original radiation action can be obtained by several different processes ; they range from a modification in the primary chemical change induced by radiation in individual cells to an increased general resistance of the body as a whole. It should be noted that a protective action against acute lethal effects does not necessarily give protection against the more chronic sequelae due to irradiation, in so far as different mechanism may be involved in their development (Patt and Brues, 1954a). The modification of radiation effects can be in the direction either of a higher resistance to radiation or of an increase in radiosensitivity. Many

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noxious stimuli enhance susceptibility to radiation, as also does adrenal insufficiency, infection, trauma, exhaustion, a deficiency in either protein or vitamin intake, or an increase in oxygen tension. In the radiation treatment of malignant disease a sensitizing agent needs to be localized in the tumor if normal tissues are to be shielded from its action (cf. Mitchell, 1955). This localization has not yet been successfully achieved, and radiotherapeutic measures still depend largely on the differential action of radiation for their effect. The influence of hormones in the action of radiation is a field that is only beginning to be explored (Betz,

1956).

Most of the experiments on modification of radiation effects have been done with acute irradiations and under a variety of conditions of exposure. This sometimes makes the comparison of results from different laboratories difficult, since manipulation of the physical factors of radiation themselves may enhance, leave unchanged, or diminish the effect of a given total dose. The most effective use of sensitizers must await the determination of those physical factors of exposure which enable the greatest biological effect to be obtained with the minimum amount of radiation energy. c. Radioisotopes. Radiation chemistry and radiation physics are, in a sense, combined in the various applications to biology of the wide range of radioisotopes now available. Unstable versions of almost any element can be used in two different ways. They can be administered in quantities so small that their presence can be detected only by sensitive physical apparatus, and in sufficiently high dilution that no biological effects of the substance can be observed, at least in animals, with the means at present available. The progress of the radioactive atom through the organs of plant, animal, or man followed by these physical detectors yields information not otherwise obtainable. The method was used first by Hevesy in 1923 when he employed radium D as a tracer for its isotope lead, and later radium E for bismuth in his studies on plant metabolism. These substances are not, however, those normally used in plants and were admittedly foreign substances introduced into the plant’s economy for experimental purposes. The two great advantages of the artificially produced radioactivity is that substances can be selected which are natural to the plant’s metabolism, and that by “tagging” atoms of a substance natural to the plant or animal under observation it is possible to discriminate between the added compound and similar compounds already present in the system. This is a conspicuous advantage over ordinary methods of chemical and biochemical analysis where added substances are indistinguishable from those already present. I n larger amounts radioactive substances can be administered so that

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the radiation they emit produces definite biological changes-usually destructive ones such as the elimination of unwanted cells. The problem in this case is to localize the radioactive substance in the particular region required. The length of life of the artificial product is another limiting factor and shortens the list of usable substances. d . Beneficial Uses of Radioisotopes. The therapeutic administration of radioisotopes is a more difficult field of research than metabolic studies with tracer substances, and progress must of necessity be slower and less spectacular. The hope that by this means irradiation in substantial doses might be delivered to any point of the body has only been realized in two or three instances. Radiophosphorus was first used therapeutically in 1936, when it was found that its beneficial effect on certain blood diseases such as leukemia resembled that following X-radiation (Lawrence, 1940), with an end result at least as good as the X-radiation treatment and far less inconvenient for the patient. Neither is a certain cure, but considerable periods of relief are often obtained before the blood again shows abnormality. e. Deleterious Effects of Radioisotopes. As with external radiation there is another side to the picture. Uptake of any radioisotope in normal bone at certain sites may lead not only to undesirable changes in the bone tissue itself but to secondary but none the less highly significant biological consequences elsewhere. If absorption occurs in the proximity of bloodforming tissues there is danger of affecting the growth and differentiation of maturing blood cells which can lead to permanent disruption or, more rarely, destruction of the normal processes. The danger is negligible with tracers and can be avoided in the therapeutic administration of radioisotopes by the choice of appropriate substances. A very different situation arises, however, with long-lived isotopes accidentally or unintentionally absorbed into the body and which become deposited in bone. Radium was the first bone-seeker to become notorious owing to the disaster it produced among a group of luminous-dial painters who licked their brushes conlaining radium salts (Martland and Humphries, 1929; Hoecker and Roofe, 1951). The explosion of atomic weapons and devices produces at least two other long-lived bone-seekers, cesium and strontium. Compared with the natural background of radiation to which every living creature is inevitably exposed, the radiation due to strontium fallout is only a small fraction at the present rate of testing. But the amount present in biological materials is measurable and must be watched for any indications of a dangerous level being reached (cf. Booker et al., 1957). f. Detection of Radioisotopes. A conference, the first of its kind, was

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held in Leeds in 1956 to discuss means of measuring the radioactivity of the human body and of detecting the smallest possible increase that may occur from occupational or other causes (Brit. J. Radiol., 1957a). The instruments which have been devised for this purpose have already reached a fantastic degree of sensitivity. Although this is an impressive argument in favor of their still further development, it seems desirable to emphasize that the level of detection appears to be far below the concentration which can at present be considered dangerous. Values less than one-hundredth of that of the average natural background can now readily be measured. But it must be remembered that the intensity of this background varies from place to place in the world by a factor of 5 to 10, or, if exceptional areas are included, e.g. the areas of radioactive sands in Travancore, India, this figure may be raised to a level of 50 to 60. The uptake and distribution of strontium in bone under laboratory conditions is being very carefully studied (see Vaughan and Jowsey, 1956). The proportion of strontium retained in the body, the areas in which the element is laid down in bone, the rate of deposition and removal, and consequently the biological effects of the radiations emitted vary greatly. Although the uptake is greater in young animals than in old, the rate of absorption is not constantly related to age; it depends on the local metabolic activity in the bone at the site of absorption, and this varies inconstantly with age within the growing animals so far studied. The dosimetry of an isotope outside the body is easily determined. The dosimetry of radioactive substances within the body is difficult and entails a determination of its uptake, “turnover,” and elimination from the body as well as the location and distribution of the material among the various tissues while it is in the body. These losses and migrations, as was shown by Marinelli (1942) and by Evans and Quimby (1946), result in a “biological half-life” in some cases very different from and usually shorter than the physical half-life. This presents a problem in dosimetry of quite another order (Mayneord, 1950). VI.

CONCLUSIONS

We began this inquiry by noting the remarkable change between 18% and 1956 in the general attitude of both lay and scientific people to problems connected with the biological actions of penetrating radiations. W e went on to consider the variety of biological response among living tissues when exposed to the rays under different conditions; we have noted beneficial and deleterious effects of radiation on man; and we have discussed some of the suggestions which have been put forward concerning the mechanism of radiation action on plants and animals. No single theory so far pro-

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pounded is sufficient to explain all the observed facts. Only more intensive work can decide between rival theories or point the way to new concepts. Some like to form a hypothesis and devise experiments to test or disprove its validity. Others prefer to collect information about the response of living tissues to radiation and leave the theoretical considerations to follow. By whatever process they are obtained, new facts are needed today more than new theories. Modern civilization will come to depend to an everincreasing extent on the further use of radioisotopes with their emitted radiations and consequent dangers. But as Brues and Sacher (1952) have recently put it, “although we can say that essentially nothing we do is without hazard and that life is a succession of naive and qualitative risk calculations, it is in radiobiology that man has elected to attack such a problem squarely and is finding it necessary to evolve and put under scrutiny some new or neglected concepts. Whether this has had an irrational or rational origin is beside the point; if one is willing to be more inquisitive than irritable [italics mine] it is bound to become scientifically fruitful.”

VII. ACKNOWLEDGMENTS

I gratefully acknowledge the help and encouragement of my colleagues in the preparation of this review and particularly that of Dr. H. B. Fell, F.R.S., and Dr. A. Gliicksmann ; I am also grateful for help received from Dr. J. G. Torrey (University of California) and others who were consulted on matters within their own special interests. I wish to thank Miss Houghton for her part in preparing the manuscript for publication. VIII. REFERENCES Abou Ben Adhem’s name led all the rest . . . Prompting a thesis wildly theoreticalThat even recording angels find it best T o keep us alphabetical. [J.B.B., Punch 232, 699 (1957) 1 Abbatt, J. D. (1956) in “Progress in Radiobiology” (Mitchell, Holmes, and Smith, eds.), Section 11, p. 494. Oliver & Boyd, Edinburgh. Abbe, R. W. (1911) Ann. Surg. 45, 235. Albers-Schonberg, H. E. (1903) Miinch. med. Wochschr. 60, 1859. Alberti, W., and Politzer, G. (1923) Arch. mikroskop. Anat. u. Entwicklungsmech. 100, 83. Alberti, W., and Politzer, G. (1924) Arch. mikroskop. Anat. u. Entwicklungsmcch. 109, 284. Alberti, W., and Politzer, G. (1926) Strahlentherapk 21, 535. Alexander, P. (1953) Brit. J . Radiol. 26, 413. Alexander, P., Fox, M., and Hitch, S. F. (1954) Brit. J . Radiol. 27, 130. Allsopp, C. B. (1944) Trans. Faraday SOC.40,79.

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Allsopp, C. B. (1951) Brit. J. Radiol. U ,413. Alter, N.M. (1920) J. Med. Research 41,439. Ancel, P., and Vintemberger, P. (1925) Compt. rend. soc. biol. 92,986. Ancel, P., and Vintemberger, P. (1927) Compt. rend. soc. biol. 97,796. Baclesse, F., and Loiseleur, J. (1947) Compt. rend. sac. biol. 141, 743. Bacq, Z. M., and Alexander, P. (1955) “Fundamentals of Radiobiology.” Academic Press, New York. Baker, A. H., and Freund, F. (1950) Am. I. Roentgenol. Radium Therapy 64, 810. Barcot, J. (1919-1920) Arch. Radiol. Electrotherapy W ,343. Barnes, D. W.H., and Loutit, J. F. (1955) J. Natl. Cancer Inst. 15, 901. Barnes, D. W.H., and Loutit, J. F. (1956) Ciba Foundation Symposium, Ionizing Radiations and Cell Metabolism p. 140. Churchill, London. Barratt, J. 0. W., and Arnold, G. (1911) Arch. Zellforsch. 7, 242. Barron, E. S. G., Dickman, S., Muntz, J. A., and Singer, T. P. (1949) J. Gen. Physiol. 32, 537. Bauer, H., and Le Calvez, J. (1944) Chroniosoma 2, 593. Becquerel, H., and Curie, P. (1901) Comfit. rend. 132, 1289. Bekkum, D. W. van, and de Groot, J. (1956) in “Progress in Radiobiology” (Mitchell, Holmes, and Smith, eds.) , Section 5, p. 243. Oliver & Boyd, Edinburgh. Bergonit?, J., and Tribondeau, L. (1904) Compt. rend. soc. biol. 57, 400, 592 (rat tissue), 595 (human tissue). BergoniC, J., and Tribondeau, L. (1905) Compt. rend. soc. biol. 58, 156. BergoniC, J., and Tribondeau, L. (1906) Compt. rend. 189, 983. Bergoni6, J., Tribondeau, L., and Recamier, D. (1905) Compt. rend. SOC. biol.

68, 284.

Bering, B. A., Bailey, 0. T., Fowler, F. D., Dillard, P. H., and Ingraham, F. D. (1955) A m . J. Roentgenol. Radium Therapy Nuclear Med. 74,686. Betz, E. H. (1956) “Contribution ii 1’6tude du syndrome endocrinien provoquC par I’irradiation totale de l’organisme.” Masson, Paris. Blau, M., and Altenburger, K. (1923) Z . Physik. 12,315. Bloom, W. (1937) Phys. Rev. 17,589. Bloom, W., Zirkle, R. E., and Uretz, R. B. (1955) Ann. N.Y. Acad. Sci. 59, 503. Boag, J. W. (1953) Natl. Bur. Standards ( U . S . ) Publ. No. 2946. Boag, J. W., and Gliicksmann, A. (1956) in “Progress in Radiobiology” (Mitchell, Holmes, and Smith, eds.), Section 10,p. 476. Oliver & Boyd, Edinburgh. Bohn, G. (1903) Comfit. rend. soc. biol. 66, 1442, 1655 ; Compt. rend. 136, 1012, 1085. Booker, D. V., Bryant, F. J., Chamberlain, A. C., Morgan, A., and Spicer, G. S. (1957) Atomic Energy Research Establ. ( G . Brit.) Publ. No. HP/R 2182 (Harwell). Boyd, W. (1953) “Textbook of Pathology,” 6th ed., p. 245. Kimpton, London. Breslavets, L. P. (1946) Acad. Sci. U.S.S.R., quoted by Gunckel and Sparrow

(1954).

Brit. 1. Radiol. (1928) “Report of 2nd International Congress of Radiology, Stockholm,’’ 1, 363. Brit. J . Radiol. (1950) Supfil. 2. Brit. J . Radiol. (1953) Editorial 26, 553. Brit. J . Radiol. (1955) Supfil. 6. Brit. J. Radiol. (1957a) Suppl. 7. Brit. J . Radiol. (1957b) Editorial 30, 281 ; U.N.O. Statement, p. 282.

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