Advances in Radiation Biology: Volume 8

Advances in Radiation Biology

Volume 8

John T. Lett

Department of Radiology and Radiation Biology, Colorado State University, Fort Collins, Colorado

Howard Adler

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee

ISSN  0065-3292

Volume 8 • Number Suppl. (PB) • 1979

Table of Contents

Cover image

Title page

Advisory Board

Contributors to This Volume

Copyright page

Contributors

Contents of Other Volumes

Volume 1

Volume 2

Volume 3

Volume 4

Volume 5

Volume 6

Volume 7

Aspects of OER and RBE Relevant to Neutron Therapy

Publisher Summary

I. Introduction

II. Early Studies

III. The Rationale for Fast Neutron Therapy

IV. The Oxygen Effect

V. Repair after X Rays and Neutrons

VI. Relative Biological Effectiveness (RBE) for Normal Tissues

VII. Mixtures of Neutrons and X Rays

VIII. Responses of Tumors

IX. Clinical Results

X. Conclusions

Present Status of the Proposed Use of Negative Pi Mesons in Radiotherapy

Publisher Summary

I. Introduction

II. The Pion Proposal

III. Single-Cell Survival

IV. Somatic-Cell Mutagenesis Studies

V. Multicellular Tumor Spheroid Studies

VI. Normal versus Tumor Tissue Responses in the Laboratory

VII. Normal versus Tumor Tissue Responses in the Clinic

VIII. Summary

Human Diseases Associated with Defective DNA Repair *

Publisher Summary

I. Introduction

II. Xeroderma Pigmentosum

III. Ataxia Telangiectasia (Louis–Bar Syndrome)

IV. Fanconi’s Anemia

V. The Hutchinson-Gilford Progeria Syndrome

VI. Bloom’s Syndrome

VII. Cockayne’s Syndrome

VIII. Down’s Syndrome (Trisomy 21)

IX. Retinoblastoma

X. Chronic Lymphocytic Leukemia

XI. Miscellaneous Human Diseases with Possible DNA Repair Defects

XII. Conclusions

The Induction of Molecular and Genetic Recombination in Eukaryotic Cells

Publisher Summary

I Introduction

II Meiotic Recombination in General

III Mitotic Recombination

IV Mechanisms of Induced Mitotic Recombination

V The Relation between Induced Recombination in Lower and Higher Eukaryotes

VI Concluding Comments

DNA Damage and Repair in Higher Plants1

Publisher Summary

I Introduction

II Some Features of Injury of Plants and Plant DNA by Radiation and Chemical Treatments

III Principal Ways of Repair of DNA Damage

IV Initial Approaches to the Study of Repair in Higher Plants

V Photoreactivation in Higher Plants

VI Excision Repair of Damage Induced in Plant DNA with UV-Irradiation

VII Repair of Damage Induced in Plant DNA with Ionizing Radiation

VIII Repair of Damage Induced in Plant DNA with Chemical Mutagens and Carcinogens

IX Inhibition of Repair Processes in Higher Plants

X Repair and Mutagenesis

XI Concluding Remarks

The Effect of Oxygen on the Repair of Radiation Damage by Cells and Tissues1

Publisher Summary

I. Introduction

II. Important Aspects of the Oxygen Effect

III. Repair in Vitro

IV. Repair in Vivo

V. Repair of Molecular Damage

Manifestations of Damage from Ionizing Radiation in Mammalian Cells in the Postirradiation Generations

Publisher Summary

I Introduction

II Cellular Effects

III Subcellular Effects: Chromosome Aberrations

IV Effects on the Synthesis of Macromolecules

V Relation of the Behavior of Cells in the Postirradiation Generations to Cell Death

Heritable Lesions Affecting Proliferation of Irradiated Mammalian Cells1

Publisher Summary

I Introduction

II Early Observations

III Basic Techniques in the Study of Heritable Lesions

IV Heritably Damaged Cells

V Induction of Heritable Lesions by Densely Ionizing Radiations

VI Induction of Heritable Lesions by Tritiated Compounds

VII The Oxygen Effect

VIII Induction of Heritable Lesions by Alkylating Agents and Other Factors

IX Observations in Vivo

X Heritable Lesions and Survival Determination

XI Late Recovery

XII Quantification of Heritably Damaged Cells on the Basis of Growth Observations

XIII Postirradiation Intrapopulation Disturbances and Recovery

XIV Conjectures as to the Nature of Heritable Lesions and the Mechanisms of Late Recovery

XV Significance for Radiotherapy

XVI Summary and Future Objectives

Tritium in the Environment1

Publisher Summary

I Introduction

II Physical Characteristics

III Sources of Tritium

IV World Tritium Inventory

V Metabolism of Tritium: Intake and Distribution

VI Isotopic Effects and Transmutation

VII Dosimetry of Tritium

VIII Population Exposure to Tritium and Related Risks

IX Summary

Subject Index

Erratum

Advisory Board

J.Z. Beer, J.A. Belli, B.A. Bridges, M.M. Elkind, P.C. Hanawalt, R.H. Haynes, O.F. Nygaard, S. Okada and M.R. Zelle

Contributors to This Volume

Janusz Z. Beer, A.L. Carsten, Ursula K. Ehmann, Stanley B. Field, Errol C. Friedberg, L.E. Hopwood, Shirley Hornsey, M.M. Kligerman, Cameron J. Koch, Albert P. Li, Michael A. Resnick, Valery N. Soyfer, L.J. Tolmach, Jon I. Williams and John M. Yuhas

Copyright page

COPYRIGHT © 1979, BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED.

NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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ISBN 0–12–035408–X

PRINTED IN THE UNITED STATES OF AMERICA

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Contributors

Janusz Z. Beer,     Department of Radiobiology and Health Protection, Institute of Nuclear Research, PL–03–195 Warszawa-Zerań, Poland (363)

A.L. Carsten,     Medical Department, Brookhaven National Laboratory, Upton, New York 11973 (419)

Ursula K. Ehmann,     Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 (85)

Stanley B. Field,     MRC Cyclotron Unit, Hammersmith Hospital, London W12 OHS, England (1)

Errol C. Friedberg,     Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 (85)

L.E. Hopwood,     Departments of Radiology and Pharmacology, The Medical College of Wisconsin, Milwaukee, Wisconsin (317)

Shirley Hornsey,     MRC Cyclotron Unit, Hammersmith Hospital, London W12 OHS, England (1)

M.M. Kligerman,     Cancer Research and Treatment Center and Department of Radiology, University of New Mexico, Albuquerque, New Mexico 87131 (51)

Cameron J. Koch,     Experimental Oncology Group, Ontario Cancer Treatment & Research Foundation, Victoria Hospital, London, Ontario, Canada (273)

Albert P. Li,     Cancer Research and Treatment Center and Department of Radiology, University of New Mexico, Albuquerque, New Mexico 87131 (51)

Michael A. Resnick*,     Department of Biochemistry, College of Medicine, East Tennessee State University, Johnson City, Tennessee 37601 (175)

Valery N. Soyfer,     Laboratory of Molecular Biology of Plants, Institute of Applied Molecular Biology and Genetics, Moscow, USSR (219)

L.J. Tolmach,     Departments of Radiology and Anatomy, Washington University School of Medicine, St. Louis, Missouri (317)

Jon I. Williams,     Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305 (85)

John M. Yuhas,     Cancer Research and Treatment Center and Department of Radiology, University of New Mexico, Albuquerque, New Mexico 87131 (51)

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

*Present address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle, North Carolina 27709.

Contents of Other Volumes

Volume 1

Recent Research on the Radiation Chemistry of Aqueous Solutions

Harold A. Schwarz

Physical Mechanisms in Photosynthesis

Gordon Tollin

Effects of Intracellular Irradiation with Tritium

Donald E. Wimber

Effects of Small Doses of Ionizing Radiations

Arne Forssberg

The Radiation Chemistry of Amino Acids

J. Liebster and J. Kopoldová

The Relative Roles of Ionization and Excitation Processes in the Radiation Inactivation of Enzymes

Leroy G. Augenstein, Tor Brustad, and Ronald Mason

Author Index–Subject Index

Volume 2

Reactivation after Photobiological Damages

Claud S. Rupert and Walter Harm

The Study of Labile States of Biological Molecules with Flash Photolysis

Leonard I. Grossweiner

Repair of Premutational Damage

R. F. Kimball

The Genetic Control of Radiation Sensitivity in Microorganisms

Howard I. Adler

A Physical Approach to the Visual Receptor Process

Barnett Rosenberg

The Role of Genetic Damage in Radiation-Induced Cell Lethality

D. R. Davies and H. J. Evans

Author Index—Subject Index

Volume 3

Low Energy Electron Mean Free Paths in Solids

R. H. Ritchie, F. W. Garber, M. Y. Nakai, and R. D. Birkhoff

The Molecular Biology of Photodynamic Action: Sensitized Photo-autoxidations in Biological Systems

John D. Spikes and Robert Livingston

Sensitization of Organisms to Radiation by Sulfhydryl-Binding Agents

Bryn A. Bridges

Biological Effects of Radioactive Decay: The Role of the Transmutation Effect

Robert E. Krisch and M. R. Zelle

Human Radiation Cytogenetics

Michael A. Bender

Reflections on Some Recent Progress in Human Radiobiology

C. C. Lushbaugh

Author Index—Subject Index

Volume 4

Repair Processes for Photochemical Damage in Mammalian Cells

J. E. Cleaver

Enzymes Involved in the Repair of DNA

Lawrence Grossman

Mutation Induction in Mice

A. G. Searle

Experimental Radiation Carcinogenesis

Harry E. Walburg, Jr.

Toxicology of Plutonium

W. J. Bair

Effects of Ionizing Radiation on Terrestrial Plant Communities

F. Ward Whicker and L. Fraley, Jr.

The Breakage-and-Reunion Theory and the Exchange Theory for Chromosomal Aberrations Induced by Ionizing Radiations: A Short History

S. H. Revell

Author Index–Subject Index

Volume 5

The Radiobiological Implications of Statistical Variations in Energy Deposition by Ionizing Radiations

Norman A. Baily and John E. Steigerwalt

Radiological Assessment of Nuclear Power Stations

Stephen V. Kaye and Paul S. Rohwer

Effects of Continuous Irradiation on Animal Populations

Frederick B. Turner

Radiation-Induced Life-Shortening and Premature Aging

H. E. Walburg, Jr.

Molecular Mechanisms of Radiation-Induced Damage to Nucleic Acids

John F. Ward

The Four R’s of Radiotherapy

H. Rodney Withers

Subject Index

Volume 6

Effects of Ionizing Radiation on Nucleic Acid Synthesis in Mammalian Cells

Ronald A. Walters and M. Duane Enger

UV Damage at the Transcriptional Level

Walter Sauerbier

Interrelationships between Ionizing Radiation, Protein Synthesis, and the Physiological Expressions of Radiation Damage

Nancy L. Oleinick and Ronald C. Rustad

Role of DNA Repair in Cell Inactivation, Aging, and Transformation: A Selective Review, A Speculative Model

Jerry R. Williams

Response of Cells in Vitro and Tissues in Vivo to Hyperthermia and X-Irradiation

Donald E. Thrall, Leo E. Gerweck, Edward L. Gillette, and William C. Dewey

Experimental and Clinical Aspects of Hyperthermia Applied to the Treatment of Cancer with Special Reference to the Role of Ultrasonic and Microwave Heating

Isaac Har-Kedar and Norman M. Bleehen

Thermal Potentiation of Mammalian Cell Killing: Clues for Understanding and Potential for Tumor Therapy

Burt V. Bronk

Autologous Stem-Cell Banks for Restoring Hemopoiesis

Bill M. Nelson and Gould A. Andrews

Subject Index

Volume 7

Use of Purified Lesion-Recognizing Enzymes to Monitor DNA Repair in Vivo

Malcolm C. Paterson

The Crucial Role of DNA Double-Strand Breaks in Cellular Radiobiological Effects

H. P. Leenhouts and K. H. Chadwick

Evaluating the Effects of Ionizing Radiation on Aquatic Organisms

B. G. Blaylock and J. R. Trabalka

Applicability of Bacterial Models of DNA Repair and Recovery to UV-Irradiated Mammalian Cells

Roger R. Hewitt and Raymond E. Meyn

Electrophysiological Studies on Radiation-Induced Changes in the Adult Nervous System

Makoto Sato

The Repair of DNA Modified by Cytotoxic, Mutagenic, and Carcinogenic Chemicals

J. J. Roberts

Subject Index

Aspects of OER and RBE Relevant to Neutron Therapy

Stanley B. Field and Shirley Hornsey,     Mrc Cyclotron Unit Hammersmith Hospital London, England

Publisher Summary

This chapter presents a rationale for fast neuron therapy and illustrates the relationship between radiosensitivity and oxygen tension. It also discusses skin reaction in mouse tails and rats’ feet as a function of time after irradiation. The primary rationale for neutron therapy lies with the oxygen effect. Other factors, particularly the various repair processes, certainly influence relative biological effectiveness (RBE) values for tumors and normal tissues, but the knowledge to predict optimal treatment methods on this basis is not available. From the available clinical evidence, neutrons appear to be a safe method of treatment. In some cases, neutron therapy appears to be advantageous over X-rays, but further clinical work is required to firmly establish this impression. As most radiobiological factors—such as the effects of oxygen and dose fractionation—are substantially less with neutrons, once confidence has been fully established, neutrons should be clinically easier to use than X-rays. Also, radiobiological findings point to neutrons being more reliable in their effect on tumors. In view of the differences in RBE values obtained with different physical setups and dosimetry, it is still advisable for any center embarking on neutron therapy to measure the RBE for their beam for a few normal tissues at dose levels to be used in the clinic.

I Introduction

II Early Studies

III The Rationale for Fast Neutron Therapy

IV The Oxygen Effect

V Repair after X Rays and Neutrons

A Repopulation

B Repair of Sublethal Damage

C Repair of Potentially Lethal Damage (PLD)

D Slow Repair

VI Relative Biological Effectiveness (RBE) for Normal Tissues

A Skin

B Intestine

C Esophagus

D Lung

E Hemopoietic Tissue

F Nervous Tissue

G General Considerations

H Neutron Fractionation

I RBE for Different—Energy Neutrons

VII Mixtures of Neutrons and X Rays

VIII Responses of Tumors

IX Clinical Results

Randomized Clinical Trial

X Conclusions

References

I. Introduction

Photons impart their energy by means of secondary electrons which are generally lightly ionizing. In contrast, most of the ionization in tissue resulting from fast neutrons is by secondary knock-on protons, α particles, and heavier recoil nuclei which are far more densely ionizing. This difference in linear energy transfer (LET) between neutrons and photons results in different radiobiological properties, which may be useful in the treatment of cancer providing they are understood. Radiotherapy with fast neutrons was first attempted from 1938 to 1943. At that time the radiobiological information was both inadequate and contradictory. Furthermore, it provided no definite rationale for the use of neutrons in radiotherapy. Excess normal tissue damage was caused in this early trial, which resulted in no further attempts at neutron therapy for nearly 30 years.

It was the discovery of the oxygen effect which provided the stimulus to try again. Tumors were shown to contain hypoxic cells, and hypoxia was shown to protect less against neutrons than against x rays. The phenomenon of repair of sublethal damage was elucidated in the early 1960s, and its reduced effect after neutrons compared with x rays has provided a basis for explaining the early difficulties and an understanding of relative biological effectiveness (RBE) and its changes with fractionation.

II. Early Studies

The existence of the neutron was proposed by Rutherford in 1920. By 1932 the search for it was over, and within 4 years reports of neutron irradiation of experimental tumors began to appear. Dosimetry at that time was by means of a standard Victoreen condenser type R meter normally used to measure response with x rays (roentgens). The n unit was defined as that quantity of neutrons which discharged the meter to the same extent as I R of x rays. One n unit is equivalent to about 2.5 rads. The relative biological effectiveness of x rays and neutrons was measured in the thirties for several systems, and a summary is given in Table I. The RBE values measured generally ranged from about 0.8 to 5, but for tumors ranged from 2.4 to 3.2.

Table I

Ratio of Roentgens of x Rays to n Units of Neutrons to Produce the Same Level of Damage in Various Biological Systems, and an Estimate of RBE a

aFrom Axelrod et al. (1941).

There was at that time no rationale for using neutrons to treat cancer, only an awareness that the RBE was variable from one biological system to another and the hope that it might be higher for tumors than for normal tissues. The first patient was treated on September 26, 1938, with neutrons produced by bombarding beryllium with 8-MeV deuterons from a 37-in. cyclotron at Berkeley, California [i.e., Ed = 8 MeV(Be)]. The dose rate at the treatment distance of 70 cm was about 8 rads/min. Only single treatments were given, the maximum dose being 275 n units, i.e., about 700 rads. A typical treatment took about an hour, although the machine was most unreliable. Twenty-four patients with 64 fields were treated in this way. Skin erythema and mucosal reactions were observed not to be excessive even in eight patients followed for more than a year, and tumor regression was sufficiently promising to recommend a thorough trial of this new method of treating cancer (Stone et al., 1940).

In December 1939 treatment began with a new 60-in. cyclotron. This machine produced 16-MeV deuterons, and the dose rate was improved to about 13 rads/min. Using single doses on the first patients, the higher-energy neutron beam was shown to be less effective than the earlier one, consistent with the experimental observations of Zirkle and Lampe (1938) and, later, of Gray and Read (1943). Despite this finding, Stone and Larkin (1942) were conservative in selecting the doses to be given and considered the ratio 6 R to 1 n unit as the appropriate conversion factor from x rays to neutrons. This equates with an RBE of about 2.5, not very different from that used nowadays with similar-energy machines (Catterall et al., 1971). Skin reactions were acceptable up to a total dose of about 1800 rads, given over a wide range of fractions and overall treatment times, which is just at the upper limit of acceptable doses given today.

Tumor response was variable, as would be expected from the treatment of very advanced lesions, but three tumors of the parotid were noted as having responded particularly well. However, the observations made on skin and mucosal reactions indicate that the dose levels used were generally high. Stone was clearly aware of this in the Janeway Memorial Lecture of 1947, in which he described the results of all patients treated from 1938 to 1943. He wrote, in the majority of cases some degree of epidermolysis was produced. Many radiobiologists considered that we overtreated rather than undertreated the patients. Many patients had previously been treated with x rays, but this was ignored in Stone’s analysis, from which he concluded that the late effects after neutrons were excessive relative to the early damage, in comparison with previous experience with x-ray therapy (Stone, 1948).

Stone’s conclusion had a profound effect on neutron therapy. The next patient treated was at Hammersmith Hospital in 1967, but by then a far greater understanding of the effects of fast neutrons and a rationale for their use in cancer therapy had been developed. Also a reassessment of Stone’s results has since established that the doses used were often too high and the late reactions were consistent with the early damage (Sheline et al., 1971).

III. The Rationale for Fast Neutron Therapy

A rationale for using fast neutrons to treat cancer was developed long after Stone’s trial. It rests primarily with the oxygen effect. Hypoxia protects a wide variety of biological and chemical systems against x rays. This was first clearly and quantitatively demonstrated by Gray et al. (1953), although the effect had already been known for many years. It is now firmly established that for hypoxic cells and tissues, the dose of x rays must be increased by a factor of 2.0 to 3 to cause the same level of damage as in well-oxygenated systems. Based on earlier work by Mottram, Gray et al. (1953) argued that within a tumor there would be small regions in which the gradient of oxygen tension falls sufficiently to cause necrotic focuses. It was calculated that the oxygen tension would be zero at about 150 µm from a capillary. Working on a tumor in situ, Scott confirmed the presence of hypoxic cells under normal conditions by improving the oxygen supply and increasing the tumor sensitivity to x rays (Gray et al., 1953). Further work on the relationship between tumor histology and the presence of hypoxic cells was carried out by Thomlinson and Gray (1955) on human lung carcinoma. They concluded that there was a critical radius of about 145 µm from blood vessels, within which there was no necrosis and beyond which there was only necrosis with no intact tumor cells (Fig. 1). It was argued that on the boundary between healthy and necrotic tissue there would be cells short of oxygen and therefore resistant to x-irradiation, but after irradiation there would be reduced oxygen demand elsewhere in the tumor, and these hypoxic cells could then receive adequate oxygen (and possibly other nutrients also) to enable them to survive and repopulate the tumor.

Fig. 1 The fall in oxygen tension with distance from capillaries and resulting necrosis. At a distance greater than 150 μm necrosis appears due to anoxia. With increasing separation of capillaries the necrotic zone increases, with bands of healthy tumor tissue about 150 µm thick bordering the stroma. [From Thomlinson and Gray (1955).]

It was therefore established in the fifties that tumors, because of the way they grow, might develop hypoxic cells, and methods to overcome their resistance to x rays were being sought. One suggestion was the use of hyperbaric oxygen. This was based on the relationship between radiosensitivity and oxygen concentration, as shown in Fig. 2. A given increase in oxygen concentration would increase the sensitivity of fairly well-oxygenated normal tissues, N, a small amount to N′, but that of poorly oxygenated tumor tissue from T to T′, i.e., hyperbaric oxygen would have a much greater effect on tumor than on normal tissues.

Fig. 2 The relationship between radiosensitivity and oxygen tension. T and N represent the sensitivities of tumor and normal tissue. T′ and N′ represent their improved sensitivities for an increase in oxygen concentration. [Taken from suggestions of Gray et al. (1953).]

Another suggestion was the use of fast neutrons. It had been shown by Conger, using ascites tumor cells (Gray et al., 1953), that the oxygen effect was greatly reduced by using fast neutrons (Fig. 3). Thoday and Read (1949) had already shown a similar effect with α particles on bean roots. Therefore, relative to well-oxygenated normal tissues, neutrons should have a greater effect on hypoxic tumor cells.

Fig. 3 Comparison of the effects of oxygen tension on cytological damage induced in mouse ascites tumor cells by x rays and by neutrons. The oxygen effect is clearly smaller for neutrons [A. D. Conger in Gray et al. (1953)].

Do human tumors contain hypoxic cells, and if so, do they play a part in limiting the effectiveness of conventional treatments? This cannot be answered directly or definitively, but there is a body of circumstantial evidence pointing to hypoxia being important. The presence of hypoxic cells in a wide variety of experimental tumors is firmly established (see, for example, Kallman, 1972). This would seem to be beyond doubt, despite different experimental techniques giving widely different estimates of the proportion of cells which are hypoxic. However, in order that hypoxic cells may survive and repopulate tumors, they must reoxygenate. This may change resistant hypoxic cells into sensitive oxygenated cells during a course of fractionated radiation treatment. This was demonstrated experimentally by Thomlinson (1960), using the rat fibrosarcoma, RIB5. The phenomenon has been subsequently shown to occur in most, but not all, tumors in which it has been investigated. Some examples of the rate and extent of reoxygenation in experimental tumors are given in Fig. 4. Reoxygenation will overcome the problem of hypoxic cells so that the relative advantage of neutrons (or hyperbaric oxygen) will therefore be reduced. It is possible that fractionation in radiotherapy owes its success to reoxygenation as much as to improved normal tissue tolerance and may in some cases totally eliminate the problem of hypoxic cells.

Fig. 4 Reoxygenation of hypoxic cells in tumors after an initial dose of x rays. Figure 1 represents the proportion of hypoxic cells immediately after irradiation. It is seen that reoxygenation in the C3H mouse mammary carcinoma is more extensive than in any of the three sarcomas. [From Denekamp (1977b).]

In considering the effect of hypoxic cells in human tumors, it is pertinent to examine the clinical results of therapy specifically designed to eliminate them. Hyperbaric oxygen has been demonstrated as showing a genuine therapeutic advantage in some tumor types, especially in sites in the head and neck (Henk and Smith, 1977). These authors conclude that the oxygen effect has been shown to be a real factor in clinical radiotherapy. An alternative method of overcoming the problem of hypoxic cells is the use of drugs which specifically sensitize them. Urtsun et al. (1977) have used such a sensitizer (Metranidazole) and demonstrated improved survival of patients treated for glioblastoma, which strongly implies that hypoxic cells play a part in the response of this tumor to x rays. Dische (1978) has shown a similar improvement using Misonidazole on a range of tumors. Bush (1978) has analyzed cases of carcinoma of the cervix and shown that low hemoglobin levels at treatment correlate with a reduced probability of survival, again implying that hypoxic cells matter. Therefore indirect evidence does imply that hypoxic cells do limit tumor control in a course of x-ray therapy despite their reoxygenation, suggesting a use for fast neutrons.

Another possible reason for using neutrons is based on the differential repair of sublethal damage. This form of damage accumulation and repair is less after neutrons, but if the reduction were more in tumors than in normal tissues, then neutrons would be the treatment of choice. Alternatively, the reduction may be greater in normal tissues in some sites. In such cases x rays would be the treatment of choice. Clearly a detailed knowledge and understanding of the repair capacities after neutrons and x rays is important for optimal treatment planning. Repair of potentially lethal damage (PLD) seems to occur after x rays but not after neutrons (Shipley et al., 1975). Since this is predominantly a feature of proliferation-inhibited cells, this form of repair may occur to a greater extent in tumors than in normal tissues, so treatment with neutrons would be an advantage. However, more information on PLD in slowly dividing normal tissues is needed since this might be important in late normal tissue damage.

The oxygen effect and the various tumor repair processes are considered again in greater detail, but it is clear that there are sound reasons for believing that neutrons may be valuable in the treatment of at least some types of tumors.

IV. The Oxygen Effect

With few exceptions throughout radiobiology, cells and tissues irradiated in the presence of oxygen are more sensitive than those irradiated in its absence. It is generally accepted that oxygen sensitization occurs through the fixation of damage by the reaction of the free radicals formed in the target molecules with oxygen to form peroxy-radicals (Alexander and Charlesby, 1954; Alper, 1956; Howard-Flanders, 1960). This fixation reaction competes with repair reactions in which H atoms are transferred from naturally occurring reducing compounds, e.g., —SH compounds, within the cell (Adams, 1972). These reactions occur very quickly. Rapid mix studies with O2 and electron affinic sensitizers have identified a fast (< 1.5 msec) and a slower (> 1.5 msec) component in the decay of the oxygen-sensitive damage (Michael et al., 1978). With such a rapid decay of the oxygen-sensitive damage, for the oxygen to affect the level of radiation damage it must be present at the target or be very near during the irradiation.

With high LET radiation there is a decrease in the oxygen enhancement ratio (OER) as the LET of the radiation is increased. With mammalian cells the OER is reduced to 1 at a LET value of about 180 keV/µm (Fig. 5). Two mechanisms have been proposed for the reduction in OER associated with an increase in LET. The first is the interacting radical hypothesis proposed by Alper (1956), in which it is suggested that the probability of a fixation reaction occurring between radicals produced along a track will be increased with increasing ionization density, so the importance of interaction with oxygen will decrease as the ion density increases. The second proposed mechanism is the oxygen-in-the-track hypothesis (Shekhtman, 1960). This suggests that molecular oxygen, actually produced in the densely ionizing track, would effectively oxygenate cell targets at the time of irradiation (Neary, 1965). Molecular oxygen produced in particle tracks has recently been measured (Baverstock and Burns, 1976).

Fig. 5 Oxygen enhancement ratio for survival of human kidney cells as a function of LET (Barendsen et al., 1966; Barendsen, 1968).

Neutrons damage cells through a range of secondary charged particles which consequently have a wide range of LET. For fast neutrons in the clinically useful energy range, the secondary particles may usefully be thought of as falling into two groups: knock-on protons with LET values up to about 100 keV/µm, and α particles and heavy recoil nuclei with LET values well above 100 keV/µm. The profile of LET will depend on the neutron energy. It has been shown that for the α and heavy recoil particles produced by 14- to 15-MeV d-T neutrons or by cyclotron-produced Ed = 16 MeV(Be) neutrons, the OER is about 1 (Broerse et al. 1968; Bewley et al., 1974). Since the RBE for these high LET particles is relatively high, their low OER causes a significant reduction in the overall OER for the neutron radiation. The OER for the knock-on proton component of the dose is only slightly less than that observed for photons. As most of the dose contributed by the protons will be at a LET well under 100 keV/µm (Bewley, 1968; Wilson and Fields, 1970), this low OER agrees well with expectations from observations of OER with monoenergetic particle beams (Fig. 5).

It is important to know whether the OER varies significantly with neutron energy in the clinically useful energy range (mean neutron energy of greater than about 6 MeV). With increasing neutron energy there is a steady decline in RBE (see also Section VI, I), but this is not generally found to be true for the OER, which remains relatively constant (Hall et al., 1973, 1977a, b; Hall, 1975, 1977; Broerse and Barendsen, 1973; Gragg et al., 1976; Guichard et al., 1978) (Fig. 6). Harrison et al. (1975, 1976), however, using Vicia seedlings, did show a gradual reduction in the OER from a mean neutron energy of 15 to 50 MeV, which was significant at the extremes of energy used, so the question of a reduction in the OER at very high neutron energies is still open. At lower neutron energies there is a significant reduction in the OER (Hall et al., 1973; Gray et al., 1953; Hornsey et al., 1960).

Fig. 6 The relation between OER and RBE with neutron energy is shown for two cell lines in vitro: kidney cells of human origin (•) [from ) D’Arville and Hornsey (unpublished); (+) Guichard et al. ) Guichard et al. (1978b); (Δ) Hall et al. (1977a); (∇) Hall et al. ) Hall et al. ) Ngo et al. (1977a); (×) Railton et al. (1975)]. Neutron doses include the γ-ray contribution. Where ⁶⁰Co γ had been the original reference radiation, a factor of 0.9 was used to convert all RBE values relative to 250– to 300–kV x rays.

It is also important to know whether the OER for neutrons varies as the beam penetrates into the body. Physical measurements indicate only very small changes as neutron beams penetrate a phantom. Biological measurements by McNally and Bewley (1969) found no change in the OER; Berry (1971) found a decrease in the OER at depth; Nias et al. (1971) found no change; and Bewley et al. (1976) found a slight increase. It may be concluded that any changes are very small and clinically insignificant.

The sublethal damage (see Section V,B) caused by neutron irradiation of tissues is probably due to the low LET components of the dose in the neutron beam. Therefore for low doses (i.e., many fractions) the low LET components will be fairly ineffective, most of the damage resulting from the high LET components. At higher doses the relative contribution from the low LET component will be greater. (The argument is similar to the RBE being greater for low doses per fraction; see Section V,B.) At low doses, therefore, more of the damage will be due to high LET particles with a low OER than at higher doses. It may therefore be predicted that the OER will decrease with increasing neutron fractionation. This has recently been demonstrated experimentally (Hornsey et al., 1977a; Andreozzi et al., 1979) (Fig. 7).

Fig. 7 Skin reaction in mouse tails. The total dose of fast neutrons [E,•) ( Hornsey et al. 1977).

V. Repair after X Rays and Neutrons

There are a variety of known repair processes after irradiation and probably others yet to be discovered. The most important affecting tissue responses are: (1) repopulation of surviving cells; (2) repair of sublethal damage; (3) repair of potentially lethal damage; and (4) slow repair. They will be considered separately.

A. Repopulation

It is of great importance to establish whether or not repopulation is the same after comparable doses of x rays or fast neutrons for two reasons. One is that an analysis of RBE depends on there not being a difference, and the second is to obtain knowledge of the effect of repopulation on a fractionated course of radiotherapy.

One method of assessing repopulation is the timing of a reaction after radiation treatment. Skin reactions have been analyzed in this way. It is seen that after the peak has been reached, healing, presumably due to cell repopulation, is similar after the two types of radiation. This argument was first used for mouse skin (Denekamp et al., 1966), and applies to rat skin (Field et al., 1967, 1968), pig skin (Bewley et al., 1963), and human skin (Morgan, 1967; Field et al., 1976b; Catterall et al., 1971). An illustration is given in Fig. 8. Tumor regrowth rates are also similar after the two types of radiation, as shown by Field and Hornsey (1971), Fowler et al. (1973), and Howlett et al. (1975). Berry (1967) injected mice with known numbers of lymphoma cells after irradiation with either x rays or neutrons. Neutron-irradiated cells caused slightly later death of the animals, and it was suggested that this was due to a higher proportion of slow-growing clones. However, experiments to investigate clone size distributions after x rays or high LET radiations have not shown any such difference between their effects (Westra and Barendsen, 1966; Fakhri, 1967).

Fig. 8 Skin reaction in rats’ feet as a function of time after irradiation. The curves are identical for treatment with doses of x rays or neutrons, which produce the same average reaction.

An alternative method of investigating repopulation is to measure the dose required to overcome its effect. This has been done in skin after comparable fractionated regimes of x rays or fast neutrons, followed by a test dose of x rays. It was found that irradiation stimulated repopulation, but to a similar extent after the two radiations (Denekamp and Field, 1974; Denekamp, 1977a).

It therefore seems reasonable to conclude that there are no important differences in repopulation after x rays or fast neutrons.

B. Repair of Sublethal Damage

Survival curves for x rays usually have a shoulder region, indicating an accumulation of sublethal damage. Repair is indicated by restitution of the shoulder with a second irradiation, given some time after the first (Elkind and Sutton, 1960). In tissues this may be measured as D2 – D1, where D1 is the single dose and D2 is the dose in two fractions to achieve the same level of damage. With increasing LET the shoulder becomes reduced (Fig. 9) and repair between fractions is less. This difference is illustrated schematically for x rays and neutrons in Fig. 10.

Fig. 9 Survival of human cells after radiations of different LET. [From Barendsen et al. (1966).]

Fig. 10 Hypothetical survival curves and second-dose survival curves for neutrons and x rays. D2-D1 is greater for x rays than for neutrons, even allowing for the difference in RBE. The inset shows typical Elkind recovery curves, i.e., D2-D1 or D2/D1 or cell survival ratio, plotted as a function of time between fractions. The curve for x rays, at 37°C, frequently has peaks and troughs resulting from changes in sensitivity to x rays as the cells progress through their mitotic cycle after synchrony has been imposed by the first dose. [From Field (1976).]

A major effect of this difference between neutrons and x rays is that the relative biological effectiveness (RBE), i.e., the ratio of the dose of x rays to that of neutrons to produce the same effect, is dose dependent, increasing with decreasing dose per fraction. With fractionated irradiations the RBE will primarily depend on the dose per fraction, providing that any repopulation between fractions is the same after the two types of radiation. This does appear to be the case, as described in Section V, A. In practice the dose per fraction depends on the number of fractions, so in this way the RBE will depend on the fraction number. It is known that tissues exhibit a considerable degree of sparing due to sublethal damage, usually far more than cells in vitro. Therefore at low doses per fraction the RBE for tissues may have high values. This is discussed further in Section VI.

These RBE considerations were first tested in vivo using pig skin. It was shown that the RBE increased with increasing numbers of fractions (due to the decreasing dose per fraction) (Fig. 11). It was suggested that this increase in RBE might explain the severe reactions observed by Stone in the early trial, since the original dose calculations were based on information from single treatments, although various fraction regimes were to be used in the clinical trial. This is now considered to be a partial, but not the total, reason for Stone’s failure, a detailed review of which has been made by Sheline et al. (1971).

Fig. 11 RBE for pig skin as a function of the number of fractions ( Bewley et al., 1967).

Figure 12 indicates that recovery from sublethal damage may occur later for neutrons. There are other results consistent with this observation (e.g., Bewley et al., 1967; Field, 1976), but others which are not (Hornsey et al. 1977c; Gragg et al., 1977).

Fig. 12 Survival of cells grown as spheroids and exposed to two doses of γ rays of 950 rads each or two doses of neutrons of 521 rads each, separated by the indicated times. [From Durand and Olive (1977).]

C. Repair of Potentially Lethal Damage (PLD)

Repair of PLD is a term used to describe an increase in survival if cells are in (or held in) a noncycling state after irradiation. Thus for cells in vitro, survival estimated by immediate subculturing may be markedly increased by not subculturing for several hours. The same phenomenon has been demonstrated in tumors, where the assay involved removal of the tumor and testing for cell viability by cell growth either in culture dishes or in other animals (endpoint-dilution assay). By waiting several hours after irradiation before removing the tumor, survival levels may be increased relative to immediate removal. Whether or not this phenomenon is related to repair of sublethal damage is still debated, but there are differences between the two types of damage. Sublethal damage occurs between two dose fractions and is the repeat of the shoulder region of the cell survival, normally without a change in D0. PLD does not require a second dose but only time before allowing further cell division to occur and is normally manifest by an increase in D0.

Using Lewis lung tumors, Shipley et al. (1975) demonstrated repairs of PLD after low LET radiation but not after neutrons (Fig. 13). Hall and Kraljevic (1976) and Gragg et al. (1977) made similar observations on cells in vitro. The available evidence therefore suggests that this form of repair may occur after x rays but not after neutrons. Since some tumors may contain considerable numbers of cells which are deficient in various nutrients and not actively dividing, these cells may have the capacity for repair of PLD, but only after x rays. This difference will cause a sparing of x ray damage to tumors, but not neutron damage, i.e., the RBE will be greater for such tumors. The same could also be true for slowly dividing normal tissues. Just how general or important PLD is remains to be seen.

Fig. 13 Survival curves for tumor cells for neutrons or γ rays. For neutrons there was no difference between cells removed directly after treatment, or 4 or 8 hr later. For γ rays survival was clearly higher when cells were removed later. [From Shipley et al. (1975).]

D. Slow Repair

When two doses of x rays were given to mouse lung, there was an increase in the LD50 due to pulmonary damage with increasing separation of the two treatments (Fig. 14). During the first few hours after the initial dose this increase in the LD50 is attributed to repair of sublethal damage between the two fractions, but the further increase was considered to be due to some slow repair process. In similar experiments with neutrons, the initial repair of sublethal damage was observed (although, as expected, much reduced) but no further slow repair was observed (Field and Hornsey, 1977). This phenomenon is thought not to be due to cell repopulation for two reasons: first, because in other tissues repopulation is thought to be the same after neutrons and x rays; and second, because repopulation would, in a slowly dividing tissue such as the lung, not affect tissue tolerance for several weeks after irradiation. There may then be a wave of compensatory cell division following the expression of radiation damage, which would only then increase the tissue tolerance. This did not occur: The repair seemed to remove damage exponentially.

Fig. 14 The increase in dose for lung damage as a function of the time interval between two fractions of x rays or neutrons. [From Field and Hornsey (1977).]

Slow repair is about 100 times slower than repair of sublethal damage. It may be fixed at cell division, as in PLD, and therefore would only be observed in slowly dividing cells and tissues. It may be related to a type of repair of PLD described by Reinhold and Buisman (1975) and by Van den Brenk et al. (1974) for the vascular system. This latter repair process has a similar time course to that of slow repair in the lung. It has also been shown by Curtis (1967) that there is a disappearance of chromosome aberrations in liver irradiated with x rays, but not with neutrons, which may also be a manifestation of slow repair.

Thus the indications are that with x-irradiation, slow repair may occur in slowly dividing tissues, but slow repair is absent after neutrons. This has two consequences: (1) The RBE for slowly dividing tissues would increase with increasing overall treatment time. Such an increase has been observed for skin damage with fractionation over 6 months (Field, 1977) (Fig. 15). (2) There would be a greater degree of residual injury after neutrons than after x rays because of long-term repair occurring after x-ray damage but not after neutron damage. Denekamp (1977b) observed an increase in residual injury in mouse skin after neutrons compared with x rays, but the difference was not significant. Hendry et al. (1977), using necrosis in mouse tails as their endpoint, observed a much larger fraction of a prior neutron dose remembered than was found with x rays. Field et al. (1979) have also observed more residual injury after neutrons, using mice feet skin reactions and late deformities.

Fig. 15 The RBE for skin damage as a function of the dose per fraction for neutrons produced by 16–MeV deuterons on beryllium. H = human skin; P = pig skin; M = mouse skin; C = clone counts on mouse skin; R = rat skin. The subscripts refer to the number of fractions used (results from various authors). A separate curve is shown for mice feet given one fraction per week for 25 weeks and scored from 125 to 155 days. [From Field (1977).]

As slow repair occurs after x rays, then long fractionation regimes should spare slowly proliferating tissues, which presumably are those involved in late radiation reactions. Such a sparing of tissue damage would not occur with neutrons. Where there is no benefit from long treatment times with neutrons, there may be disadvantages, such as tumor proliferation during therapy. Thus it might advisable, on the basis of the present knowledge, not to use long fractionation regimes with neutrons.

VI. Relative Biological Effectiveness (RBE) for Normal Tissues

It is now well established that the survival of most types of mammalian cells after x- or γ-irradiation can be characterized by a shouldered survival curve, the shoulder representing the accumulation of recoverable sublethal damage. As explained earlier, the shoulder is less for high LET radiation, causing an increase in RBE with decreasing dose per fraction.

The relationship between RBE and dose per fraction for damage to a variety of normal tissues was analyzed by Field (1969a), who showed that the RBE was high at low doses per fraction and decreased as the dose per fraction increased. The curve drawn through these data was used by Sheline et al. (1971) to calculate the appropriate RBE values and thus the equivalent doses of x rays in a reassessment of Stone’s first neutron trial. With the accumulation of more data, it became apparent that the RBE was different for different tissues (Hornsey, 1970). This has been particularly clearly demonstrated with the Ed = 16 MeV(Be) beam from the Hammersmith cyclotron (giving neutrons with a mean energy of about 7.5 MeV), from which there are variations in RBE between tissues by almost a factor of 2 (Fig. 16). A similar variation in RBE for normal tissues has also been shown for neutrons of ~8 MeV mean energy from an Ed = 22 MeV(Be) beam. At higher neutron energies the variation in RBE is less (Hall, 1977). Differences in RBE have been mainly associated with differences in the degree of repair of sublethal damage (Broerse and Barendsen, 1973; Hornsey and Field, 1974).

Fig. 16 RBE for various normal tissues as a function of dose per fraction of fast neutrons produced by 16–MeV deuterons on beryllium (Hammersmith cyclotron).

We will now briefly review the RBE for a number of different tissues.

A. Skin

The RBE for damage to skin is shown in Fig. 15. The main curve applies to damage leading to erythema, desquamation, and regenerating clones of cells in four species: man, mouse, pig, and rat. The fact that the results from experimental animals and man fall on the same