The Relative Biological Effectiveness of Radiations of Different Quality: Recommendations of the National Council on Radiation Protection and Measure (N C R P Report)

NCRP REPORT No. 104

THE RELATIVE BIOLOGICAL EFFECTIVENESS OF RADIATIONS OF DIFFERENT QUALITY Recorr~niendationsof the NATIONAL COUNCIL O N RADIATION PROTECTION AND MEASUREMENTS

Issued December 15, 1990 National Council on Radiation Protection and Measurements 7910 WOODMONT AVENUE 1 Bethesda, MD 20814

LEGAL NOTICE This report was prepared by the National Council on Radiation Protection and Measurements (NCRP). The Council strives to provide accurate, complete and useful information in its reports. However, neither the NCRP, the members of NCRP, other persona contributing to or misting in the preparation of this report, nor any person actingon the behalf of any of these parties: (a) makes any warranty or representation, express or implied, with respect to the accuracy, completeness or usefulness of the information contained in this report, or that the use of any information, method or pmceas diacloaed in this report may not infringe on privately owned rights; or (b) assume8 any liability with respect to the use of, or for damages resulting from the use of any information, method or process disclosed in this report, under the Civil Rights Act of 1964, Section 701 et seq. as amended 42 U.S.C. Section 2 W e et seq. (Title VII) or any o t k r statutory or common law theory governing liability.

Library of Con-

Cataloging-in-PublicatianDate

National Council on Radiation Protection and Measurements. The relative biological effectiveness of radiations of different quality : recommendations of the National Council on Radiation Protection and Managementa. p. cm.-(NCRP report : no. 104) "Ieeued December 15, 1990." Includes bibliographical references. ISBN 0-929600-12-6 :$22.00 (eat.) 1. Radiation-Physiological effect. 2. Relative biological effectiveness (Radiobiology) I. Title. II. Series. IDNLM: 1. Dose-Response Relationship, Radiation. 2. Radiation Effects. 3. Radiation Protection-standards. 4. Relative Biological Effectiveness. WN 650 N279rbl QP82.2.R3N35 1990 59Y.01915--&20 DNLMIDLC for Library of Congress

Copyright O National Council on Radiation Protection and Measurements 1990 All rights reserved. This publication ie proteded by copyright. NOpart of this publication may be reproduced in any form or by any means, including photocopying, or utilized by any information storage and retrieval system without written permission h m the copyright owner, except for brief quotation in critical articles or reviews.

Preface The relative biological effectiveness of radiations of different quality is examined in detail in this report. The analyses were performed by Scientific Committee 40 of the NCRP which is charged with the responsibility for analysis and evaluation of radiobiological data relevant to radiation protection recommendations. This report is a follow-on to the previous report of Scientific Committee 40 on its evaluation of the effects of dose rate which was published in 1980 a s NCRP Report No. 64 entitled Influence ofDose And Its Distribution In Time On Dose-Response Relationships For Low LET Radiations. Initially, it had been planned that the effects of dose and dose rate and relative biological effectiveness of radiations of different quality would be addressed in the same report. However, later, the Committee recommended publishing two separate reports which resulted in the publication of NCRP Report No. 64 and this report in succession. The Committee intentionally has not made recommendations on how to use the available RBE data in defining a quality factor (Q) for any of the radiations studied. Rather, the Committee performed a comprehensive compilation of RBE data across many different biological systems for numerous radiations of different quality. The evaluation of this data with regard to recommendations for Q will be performed by or under the umbrella of Scientific Committee 1on Basic Protection Criteria. The International System of Units (SO is used in the report in accordance with the procedures set forth in NCRP Report No. 82 entitled, SZ Units In Radiation Protection And Measurements. Serving on Scientific Committee 40 during the prepartion of this report were:

Victor P. Bond, Chairman Brookhaven National Laboratory Upton, New York Seymour Abrahamson University of Wisconsin Madison, Wisconsin

Eric J. Hall Columbia University New York, New York

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1

PREFACE

John D. Boice, Jr. National Cancer Institute Bethesda, Maryland

George Hutchisona Harvard School of Public Health Boston, Massachusetts

R.J. Michael Fry Oak Ridge National Laboratory Oak Ridge, Tennessee Douglas Grahn Argonne National Laboratory Argonne, Illinois

Gayle Littlefield Oak Ridge National Laboratory Oak Ridge, Tennessee Charles W. Mays (deceased) National Cancer Institute Bethesda, Maryland

Peter Groer" Harold Smitha Oak Ridge National Laboratory Brookhaven National Laboratory Oak Ridge, Tennessee Upton, New York Robert Ullrich University of Texas Gaveston, Texas Consultant John F. Thomson Argonne National Laboratory Argonne, Illinois NCRP Secretariat-William M . Beckner 1982-1990 E. Ivan White 19NL1982 Thomas Fearon 1978-1980 The Council wishes to express its appreciation to the members of the committee and consultant to the Committee for their time and effort devoted to the preparation of this report.

Warren K. Sinclair President Bethesda, Maryland September 26, 1989

.The Committee was reconstituted in 1984. Completing their service at that time were P. Gmer, G. Hutchieon and H. Smith.

Contents Preface ........................................................................................ iii 1 Introduction .......................................................................... 1 1 1.1 Summary .......................................................................... 2 1.2 Scope of the Endeavor ..................................................... 1.3 Definitions of FU3E and Radiation Quality ................... 3 1.4 Background Information ................................................. 5 1.5 Alternatives to the RBE Concept .................................. 13 2 Cytogenetic Effects in Plant, Animal. and Human Cells ........................................................................................ 2.1 Introduction ..................................................................... 2.2 Cytogenetic Effects in Plants ......................................... 2.2.1 Environmental Effects .......................................... 2.2.2 RBE Versus Dose and Dose Rate ......................... 2.2.3 RBE Versus LET ................................................... 2.2.4 Tradescantia Studies ............................................. 2.2.5 Summary of Cytogenetic Effects in Plants ......... 2.3 Cytogenetic Effects in Human and Other Mammalian Cells .................................................................................. 2.3.1 DoseResponse Relationships for Low-LET Radiation ................................................................ 2.3.2 DoseResponse Relationships for High-LET Radiation ................................................................ 2.3.3 FU3E Values for Cytogenetic Effects in Mammalian Cells .................................................. 2.3.3.1 Variation of RBE with Radiation Dose ... 2.3.3.2 RBE, for Cytogenetic Aberrations in Human Cells After In Vitro Exposures to Radiations of Differing LET ............... 2.3.3.3 RBE Values for Chromosome Aberrations in Human Cells After I n Vim Exposure to High-LET Radiations ... 2.4 Summary .......................................................................... 3 Transformation and Mutation in Mammalian Cells h Vitro ....................................................................................... 3.1 Introduction ..................................................................... 3.2 Radiation Induced Oncogenic Transformation Assayed In Vitro ............................................................................. 3.2.1 Basic Techniques ..................................................

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.

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vi

I

CONTENTS

3.2.2 Data for Neutrons: Fresh Explants of Cells from

................................................. and Fission Neutrons ............................................

Hamster Embryos

3.2.3 Data for C, W1OT 4fr Cells with High Energy

3.2.4 RBE Data for Incorporated Radionuclides and

Alpha Particles in 3T3 Cells

................................

3.3 Radiation-Enhanced Viral Transformation .................. 3.3.1 Basic Technique .................................................... 3.3.2 Data ........................................................................ 3.4 Mutation Studies with Mammalian Cells in Culture .. 3.4.1 Basic Technique .................................................... 3.4.2 The Mechanism of the Hypoxanthine-Guanine

Phosphoribosyltramferase (HGPRT) System ......

3.4.3 Radiation-Induced Mutation Studies ................... 3.4.3.1 Chinese Hamster V79 Cells ..................... 3.4.3.2 Human Fibroblasts .................................... 3.4.4 Conclusions From the Mammalian Cell

Mutation Data

.......................................................

3.4.5 The Pros and Com of the HGPRT System and

New Developments

.

................................................

3.4.6 Other Mdel Systems for Mutation Studies ........ 3.4.7 Mutation and The Dose Rate Effect ....................

4 Hereditary Eff-I ......................................................... 4.1 Dominant Lethal Mutations ........................................... 4.1.1 Neutron Irradiation .............................................. 4.1.1.1 Effects on Male Mice ................................ 4.1.1.2 Effects on Female Mice ............................ 4.1.2 Alpha Particle Irradiation .................................... 4.1.2.1 EGcts on Male Mice' ................................ 4.1.2.2 Effects on Female Mice ............................ 4.2 Chromosome Aberrations and Reciprocal

Translocations Induced in Spermatogonia .................... 4.21 Neutron Irradiation .............................................. 4.2.2 Alpha Particle Irradiation .................................... 4.3 Effects on Cells in Meiosis ...................... . . . ............. 4.4 Abnormal Sperm Morphology ........................................ 4.5 Summary .......................................................................... 5 Hereditary Eff&II ........................................................ 5.1 Mammalian Germ Cell Mutagenesis ............................. 5.1.1 Specific Locus Mutations-Spermatogonia ......... 5.1.2 Specific Locus Mutations in Oocytes ................... 5.1.3 Mammalian Germ Cell Summary and Conclusions ............................................................ 5.2 Non-Mammalian Germ Cell Studies .............................

.

5.2.1 Drosophila ............................................................ 95 5.2.2 Silkworm Studies ................................................. 101 5.2.3 RBE Values For Interspecies Genetic-

.

Cytogenetic Endpoints

..........................................

6 Experimental Carcinogenesi~ExtemdHigh-LET

105

Radiation .............................................................................. 106 106 107 107 110 113 6.2.4 Dose-Rate Effects .................................................. 113 6.3 Epithelial Cell Tumors ................................................. 114 6.3.1 Ovarian Tumors ................................................... 114 6.3.2 Dose-Rate Effects .................................................. 115 6.4 LungTumors ................................................................... 117 6.4.1 Types of Lung Tumors .......................................... 117 6.4.2 Lung Adenoma ...................................................... 118 6.4.3 Lung Adenocarcinoma .......................................... 119 6.4.4 Fractionation and Dose-Rate Effects on Lung Tumors ................................................................... 122 6.6 Mammary Tumors ........................................................... 123 6.5.1 Mammary Adenocarcinomas ................................ 123 6.5.2 Dose-Rate and Fractionation Effeds on Mammary Tumors ................................................. 126 6.6 Harderian Gland ............................................................. 126 6.7 Tumorigenesis in Rats .................................................... 127 6.7.1 Mammary Tumors ................................................ 127 6.7.2 Fractionation and Protraction .............................. 133 6.7.3 Skin ........................................................................ 133 6.8 Studies in Other Species ................................................. 133 6.9 Dose Rate and Fractionation .......................................... 134 6.10 The Relationship of LET and RBE .............................. 135 6.11 Conclusions Concerning the Influence of Radiation Quality on Carcinogenesis ............................................ 139 7 Internal Emitters ................................................................. 142 7.1 RBE of Alpha-Particles Versus Beta Particles for Inducing Bone Sarcoma .................................................. 142 7.2 RBE of Fission Fragments Versus Alpha Particles for Inducing Bone Sarcoma ................................................. 148 7.3 RBE of Alpha Particles Versus Beta Particles or Gamma Rays for Inducing Liver Chromosome Aberrations ...................................................................... 149 7.4 Lung Cancer Toxicity Ratio from Alpha Versus Beta Particles ........................................................................... 149 6.1 Introduction ................................................................. 6.2 Leukemia in Mice ......................................................... 6.2.1 Myeloid Leukemia ................................................. 6.2.2 Thymic Lymphoma ............................................... 6.2.3 Other Lymphomas .................................................

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CONTENTS

7.5 Toxicity of Selected Radionuclides Relative to 226Ra ... 150 7.6 Summary of Internal Emitters ...................................... 151

. Life Shortening in Mice..

RBE ......................................... 152 Introduction ..................................................................... 152 Single Exposures ............................................................. 155 Short-Term Fractionated and Protracted Exposures ... 157 Duration-of-Life and Other Long-Term Fractionated or Protracted Exposures ................................................. 161 8.5 Discussion of Life Shortening in Mice ........................... 163 9 Discussion and Conclusions ............................................. 167 References .................................................................................. 171 The NCRP ................................................................................ 198 NCRP Publications ................................................................ 205 Index ........................................................................................... 215

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8.1 8.2 8.3 8.4

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1. Introduction 1.1 Summary

This report is a review of the literature relevant to the selection of relative biological effectiveness (RBE) values for use in arriving at values of the quality factor (Q). Emphasis is placed on responses to small (<0.2 Gy) absorbed doses, or on larger absorbed doses delivered at low rates, to insure relevance to the predominant dose and time pattern of exposure important in connection with radiation protection. The data available are, in general, insufficient in quantity and quality to permit accurate estimation of the RBE at the low dose and dose rates relevant to radiation protection. However, for a wide spectrum of biological endpoints, RBE varies as a function of radiation quality, dose, dose rate, and endpoint. Although exceptions exist, for most endpoints examined, ranging from cell inactivation to tumor induction in laboratory mammals, RBE values appear to increase as the dose decreases, and as the dose rate decreases. The maximized values of RBE determined in these regions are designated RBEM. In a wide variety of systems, the RBEMfor fast (fission) neutrons, with low doses and dose rates, appears to be of the order of 20 or more compared to moderately filtered 250 kVp x rays and 40 or more compared to higher energy gamma rays. These values, which are much larger than those observed with large doses delivered a t high dose rates, are due mainly, but not entirely, to a decrease in the slope of the curve for the low-LET reference radiation a t low dose. The above generalizations appear to apply to all levels of biological organization, not just in relatively simple cell systems in which it is possible to study a specific response scored in individual cells over a wide range of doses and dose rates, with minimal perturbation from competing effects. The large number of observations possible in some simpler systems permit a direct estimation of the shape and slope of the dose-respsnse curve even at relatively low doses and dose rates. However, with more complex systems in which fewer observations are possible and in which additional competing effects may alter the shape of the dose response curve even at relatively low doses, such determinations are much more difficult. Consequently, the estima-

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1. INTRODUCTION

tion of the slope for the low-dose region in many systems, and therefore the RBEMvalue, cannot be inferred reliably by interpolations involving data a t higher doses. As a consequence, it is often difficult to arrive at RBE, values derived from complex systems that might be considered to be suitable for application to human populations.

1.2 Scope of the Endeavor This report deals with the influence of radiation quality on doseresponse relationships (stochastic effects only), as indicated by experimentally determined values of RBE. With the exception of internal emitters, there are no data on which RBE for high-LET radiation for cancer can be estimated for humans. This is contrary to earlier expectations, i.e., those existing prior to the revised dose assessment for the atomic bomb survivors, a t which time it was thought that the neutron contribution to the dose in Hiroshima was sufficiently large to permit such values to be deduced. Thus, the focus of this report is on RBE data for lower organisms, to the almost total exclusion of RBE data for humans. No recommendations are made on values for Q to be used in radiation protection. This is because, even though RBE plays a large role in the determination of Q, several additional factors must be taken into account. The differences between the two concepts are emphasized below, by describing some of the value judgments, including choices of RBE values, which may enter into a determination of Q: (a) Effective dose equivalent for high-LET radiations, especially with internal emitters such as the alpha particles from radon, involves weighting factors (w,) in addition to RBE. Evaluation of these factors is not within the scope of this report. (b) The RBE of some radiations of different LET, but within the range of LET encompassed by the "reference" radiation (f3-, P+,e-', y rays and x rays), are significantly different from unity. (This may require a more precise definition of the reference radiation for use in radiation protection, and perhaps the use of fractional values of Q for some of these radiations.) (c) Experimentally determined RBE values vary markedly, depending on the biological specimen use, the endpoint of concern, and the conditions of exposure. Also, as implied in the definition of RBE, values may be quite specific for the conditions employed. Thus, for a determination of Q, a choice must be made of which RBE values may be most relevant to endpoints of concern in the context of radiation protection.

1.3 DEFINITIONS OF RBE AND

RADIATION QUALITY

/

3

(dl Values of RBE represent dimensionless ratios, by meam of which the absolute value of risk for any high-IXT radiation can be estimated if the risk coefficient for the reference low-LET radiation is known or assumed. In considering values of Q de novo, or possible changes in extant values, the risk coefficient for the reference radiation, and particularly its limits of error should be taken into account in deciding the weight that should be given to RBE in assigning values of Q. This consideration may be quite important in view of the recent reassessment of doses for the Japanese atomic bomb survivors. (el Dosimeter readings taken at the body surface usually differ appreciably from measured or calculated values for absorbed dose a t depths in tissue. The depth dose patterns for low-LET as opposed to high-LET radiations (e.g., fast neutrons), for man, are substantially different. Thus RBE values derived from radiobiological experiments, in which efforts are made to insure homogeneous irradiation, clearly would not apply directly to the inhomogeneous irradiation patterns in humans.

1.3 Definitions of RBE and Radiation Quality The iduence of radiation quality on biological systems i$ usually quantified in terms of RBE. The RBE is defined, for a specific radiation (A), as:

lWE(*)

= ,

Dose of reference radiation required to produce a specific level of response Dose of radiation A required to produce an equal response

(1.1)

with all physical and biological variables, except radiation quality, being held as constant as possible. This definition must be retained because it does not depend on the dose-responsefunctions for the two radiations being the same, or that each be a proportional relationship. The RBE ratio is dimensionless. The term RBE, (ICRP-ICRU, 1963) is used here to indicate that it was obtained from the ratio of the slopes of the initial portion of both response curves, known or assumed to be linear (i.e.,the ratio of the "a terms" of the two dose response curves, see Section 2). This would then reflect, presumably, the maximum RBE value (RBE,). However, the term is used for convenience, because comparatively rarely can one be sure RBEMis the quantity measured. Its use does not exclude the possibility that a dose rate lower than that used

would have resulted in a different slope. Nor does it exclude an initial slope of zero, particularly for RBE values derived from animal data and thus an RBEM that may be indeterminate. Although RBEM assumes an initial linear dose-response relationship, clearly one could have an RBE that is constant with dose, if the dose-response functions are the same shape. For radiation protection purposes, RBE is currently considered to be a fundion of radiation quality, expressed in terms of LET. More specifically, in many systems the RBE increases with LET to a maximum near an LET of 100 keV pm- ' and then declines. Ifthe absorbed dose-response curves are proportional, the RBE can be obtained from the ratio of the slope of the high-LET curve to that of the reference radiation, i.e., h m the ratio of the linear risk coefficients. Because the responses (R)in the risk coefficient are set equal, the ratio becomes that of the dose for the reference radiation divided by the dose for the "test" radiation. The term "radiation quality" as used in this report refers to the energy imparted to matter divided by the length of the track of the charged particle over which the energy is lost. Different quantities have been used that approximate this definition of radiation quality. Charged particles lose energy through "collisions" that may: (1) result in the production of ions; (2) produce photon radiation Cbremsstrahlung); and (3) result in nuclear interactions. The f i s t of these types of interactions is usually much more probable and is generally the only one that deposits energy locally, i.e., within the range of the charged particle. A non-stochastic quantity representing this mechanism of energy loss, and called the "linear collision stopping power" (i.e., the average energy lost from a moving charged particle by this mechanism per unit path length of the charged particle) is currently used as a measure of the radiation quality for radiation protection purposes. Linear collision stopping power is commonly referred to as linear energy transfer (LET).It is used as an indicator of the expected biological effectiveness of the radiation. Although LET is, currently assumed to be the variable on which RBE is dependent, difficulties have been encountered which have prompted discussion of alternative quantities that should be considered for future application. For instance, although the biological effectiveness usually increases with LET, this quantity does not indicate energy deposition. For example, one keV electrons have a collision stopping power of about 13 keV pm-l of water but each electron can only deposit one keV. Furthermore, for highenergy electrons, the energy loss may result in a high energy secondary electron that will deposit its energy at some distance from the position at which the energy was lost from the primary radiation.

1.4 BACKGROUNDINFORMATION

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5

Although use of a restricted stopping power or linear energy transfer (i.e., energy losses per unit path length involving only losses below some specified energy) would minimize the difficulty encountered with high energy secondary particles, it does not take care of the limited deposition problem. Other effects that limit the usefulness of linear energy transfer for specification of radiation quality and discussion of alternative parameters such as y, the linear energy, are given, eg., in ICRU Reports 16 and 36 (ICRU 1970; 1983).

1.4 Background Information Although a number of papers and reviews are available on the relative biological effectiveness (RBE)of radiations of different quality (e.g., UNSCEAR, 1977)) only two reviews (ICRP-ICRU, 1963; NCRP, 1967) are directly concerned with radiation quality in the context of radiation protection. In both reviews, the authors commented on the dimculty of dealing effectively with the influence of radiation quality on RBE. In the period of time since these reports were written, the radiobiological and philosophical developments that have ensued have only served to emphasize these concerns. More recently reviews of RBE material in the context of radiation protection have become more numerous, eg., Sinclair 1982,1985and ICRU, 1986. In order to make clearer the importance of the recent developments, the extensive and valuable concepts and information in these two reports (ICRU-ICRP, 1963; NCRP, 1967) are reviewed briefly. Significant changes since the publication of these reports are then summarized, and information supplementary to the reports added. Quantitative studies on the influence of radiation quality on RBE are usually done by determining the dose response curves over a wide range of doses, for radiations of different energy or type. As noted above, an RBE value can be determined from the ratio of the slopes of all or parts of the resulting curves. By normalizing to a single reference radiation, the value of the RBE provides a quantitative index of the effectiveness per unit of absorbed dose of any given radiation. In radiobiology experimentation, the investigator is free to normalize to a radiation of choice, usually x or gamma radiation. In radiation protection, the reference radiations for the normalization are specified as those with an average LET of 3.5 keV pm-' or less (ICRP-ICRU, 1963). Radiation quality is of obvious and substantial importance not only in radiation protection, but in the development of models or

theories of the mechanisms of action of ionizing radiations (eg., Lea, 1955; Katz et al., 1972; Kellerer and Rossi, 1972; Kellerer, 1973; Leenhouta and Chadwick, 1978). Any poposed mechanism must be able to accommodate, if not explain, the observed effects of radiation quality on the response of biological systems to radiation exposure. In the 1963 "Report of the RBE Committee to the International Commission on Radiological Protection and the International Commission on Radiological Units and Measurements" (ICRP-ICRU, 1963),it was pointed out that relative biological effectiveness (RBE) is used in radiation protection in two ways: (1)To provide a means of determining occupational dose limits for high-LET radiation fkom accepted limits for low-LET radiation (and to allow the reverse procedure for certain bone-seeking radionuclides) and (2) to provide a means by which the doses of radiations of different quality might be added. The RBE was defined as the "inverse ratio of the doses of two kinds of radiation that produce the same biological effect." The report clearly differentiated the radiobiological concept of RBE from that of "quality factor," Q. Conceptually, Q has a meaning similar to RBE. However, a particular Q value applies only to radiation of a specifically designated linear energy transfer (LET)value. All actual radiations contain a mixture of LET values and the appropriateterm for this set of conditions for radiation protection is 8.

Where D (L) dL is the absorbed dose in the LET interval L to L + dL at the point of interest. Q (L) is the quality factor as a function of LET, and D is the absorbed dose at the point of interest. It was further recognized that, as indicated in Secion 1.1above, Q may not necessarily be identical to RBE. Thus while RBE values can be discussed in terms of radiobiological data and concepts, the concept of Q cannot be considered independently of the general philosophy that is to be applied to the derivation of dose limita for differentradiations in the context ofradiation protection. This means that factors and considerations in addition to RBE must be taken into account in arriving at values of Q. In dealing conceptually and practically with the limited data on RBE then available, particularly on the more relevant endpoints of mutagenesis and carcinogenesis, it was assumed that the dose response curve for high-LET radiation generally tended to be linear, at least at low doses, with some decrease in slope at higher doses

1.4 BACKGROUNDINFORMATION

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7

(curve n, Figure 1.1). This was generally interpreted as resulting from a single track ("single hitn)mechanism, such that the effect per unit dose was nearly independent of dose level and of dose rate. For the low-LET "reference" radiation, discussion was oriented largely around a quadratic function now frequently referred to as a "linear quadratic'" dose response curve (curve y,, Figure 1.1)because an initial linear component (curve y,) dominates a t low doses and dose rates, i.e.,

Dose

-

Fig. 1.1 Schematic dose-response curve. See text for explanation of y and n curves. The fractional response is that in ex= of the "spontaneoue* baseline incidence. 'Although the term "linear quadratic" ie a mathematical misnomer, it is ueeful in radiobiology because it the use of an additional parameter to provide a more detailed characterization of the observed doae-response relationship. The additional parameter is commonly aupplied in terme of the 'crowover point" or specification of the degree of curvature, i.e.. the dose at which the contributions b the response by the liand higherArtlei t e r n are equal. A small value of doee a t which the croesover point occurs indicates that the function should be indistinguishable from a quadratic for which the coefficient of the linear term is m; a large value indicatea that the initial part of the curve may not be distinguishable h m a linear reeponse. The linear quadratic formulation haa been used extensively in the literature, eg., see NCRP report No. 61 (NCRP, 1980).

where I is incidence, D is dose, and a and p are constants. The linear or "aD" component of the low-LET curve, interpreted as resulting from a single track mechanism, was thought to be due almost entirely to the "high-LET regions" a t the ends of the electron particle tracks. Strong evidence for this has come b m the study of the effects of ultrasoft x rays, i.e., 0 to 3 keV x rays (Goodhead, 1977; Goodhead and Thacker, 1977; Goodhead et al., 1979; Cox etal., 1977a). Thus, as the dose and/or dose rate was reduced, the dose squared term contributed less and less so that, ultimately, the lowLET radiation appeared to act like a "bigh-LET" radiation (curve y,, Figure 1.1). The slope of this linear component of the total dose response curve was of course expected to be largely independent of dose, dose rate and fractionation. With higher LET radiations, the initial linear term often extends to higher doses than seen with low-LET radiation. Frequently it is as difficult to demonstrate a quadratic term with high-LET radiation, as it is to demonstrate the initial linear term with low-LET radiations. On the basis of the linear-quadratic model, for low LET radiation, the RJ3E derived from data obtained at high dose rates would be expected, in many if not most systems, to be dependent on dose, with a sharp increase in RBE as the dose decreases (see Figure 1.1). With decreasing dose rate, the slope of the high-LET curve would be expected to change only minimally. With low-LET radiation, however, a t very low doses or with higher doses a t low dose rates, (or with a very high degree of fractionation), the curve would ultimately be expected to become linear with a slope equal to that of the linear component of the linear-quadratic dose response curve (curve v2,Figure 1.1).Thus, with the limiting conditions of very low dose andlor any dose a t very low dose rates, the limiting RBE should be equal to the slope of the high-LET dose response relationship, divided by the slope of the linear term of the linearquadratic dose-response relationship. It was also made clear in ICRP-ICRU, 1963 that most of the increase in RBE a t low doses was due to a decrease in slope of the low-LET curve as the dose decreases. In ICRP-ICRU, 1963,the comparison low LETor "reference7'radiation was designated as x rays, gamma rays, electrons, and positrons of any specific ionization, and an RJ3E of unity was assigned to any radiation having an average LET in water of 3.5 keV pm-I or less. RBE values relative to this "reference" were then tabulated for a variety of LET values and biological endpoints, as a basis for deriving the effect or risk per unit of absorbed dose of any high LET radiation,

1.4 BACKGROUND INFORMATION

1

9

relative to the risk per unit dose of the standard low LET radiation at low doses and dose rates. However, relatively few data were available on experimental and human carcinogenesis,and quantification of the risk of cminogenesis had not entered definitively into radiation protection philosophy. The report discussed the difficulties of dealing with different biological effeds and different accompanying RBE values, and of deriving Q as a function of LET on the basis of these data. The report dealt almost entirely with external radiations, with minimal reference to LET effects in the context of internal emitters. The concept of the dose equivalent (DE), now symbolized by H, was defined as the product of the absorbed dose D, the quality factor (QF) now symbolized by Q, a dose distribution factor (DF), and other necessary modifying factors, The product of D F and other modifying factors was later called N. Dose equivalent was later defined (ICRU, 1971; 1973) as a physical quantity having the same dimensions as physical dose (energy per unit mass), with units of sieved. The dose equivalent was thus numerically equal to the absorbed dose multiplied by the quality factor and appropriate modifying factors. At this time, H = DQ and N is assigned a value of one for all applications. The earlier report (ICRP-ICRU, 1963), in essence, recognized the use of dose equivalent as a convenient administrative approach to "adding up" amounts of different radiations having different effectiveness values per unit of absorbed dose. The report also recognized, however, that approaches other than the use of dose equivalent were possible (eg., the system could be extended to allow the conversion of absorbed doses for different quality radiations directly into increments ofrisk that would be additive, thus obviating any requirement for Q or dose equivalent). The merits of such an approach have been discussed (Bond, 1979; 1982; Bond, et al., 1985). However, until the risk coefficients are actually known for radiations that differ substantially in biological effectiveness, it will not be practical to implement a radiation protection system based on risk alone. The 1967 report of the Subcommittee on Relative Biological Effectiveness of the NCRP, Dose Effect Modibing Factors in Radiation Protection (NCRP, 1967) also contains extensive data on the variation ofRBE with LET in a variety of systems. Thisreport first pointed out the substantial importance of including in the definition of the reference radiation the stipulation that this radiation must be applied uniformly to the entire body. They then considered variables other than LET, and suggested numerical values of fadors for partial

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1

1. INTRODUCTION

body radiation (PBR) and a penetration factor (PF).It was also stressed that "dose equivalent," rather than being regarded as a physical unit with the same dimensions as absorbed dose, could equally well and should be regarded as involving equivalence in terms of the incidence of the biological effect of interest, or the probability of that biological effed. "Dose equivalent" would not be a physical quantity but rather have units of expected excess incidence or average risk. The conversion factor from dose to risk would be equal numerically to the slope of the dose response curve and would be assigned the appropriate units of "expected excess incidence", or mean risk, per unit absorbed dose. With whole body irradiation, the RBE would be the ratio of doses of the reference and test radiation for a given response in the same tissue. However, for use of a PBR factor, under which circumstances the risks from different tissues are being compared, only a risk, and not a "physical unit" definition of dose equivalent could apply. This gave rise to the concept that has since been defined as "effective dose equivalent." Suggestions were made in the report for providing quantitative estimates of average risk per unit dose for internal and external radiation, allowing in principle the adding up of incremental average risk from all types of radiation exposure. Many of the concepts put forth were incorporated into ICRP philosophy and practice (ICRP, 1977).It was also stressed that the concept and values of dose-effect modifying factors such as RBE represent a useful, but not a mandatory or the only approach, for use in radiation protection philosophy and practice. Neither of the above discussed reports on RBE recommended changes in the LET-RBE (and by extension, LET-Q), relationship then in use. These relationships are provided in Table 1.1.

TABLE1.1-Auemge LET-RBI3 relattonahips in water (from ZCRP-ZCRU, 1963) Average LET in water &eV pm-')

Relative biol@cal effdvenees (RBE)

T h e reference radiations with an RBE of unity are x or gamma rap, electrons and positrons of any LET ( h mICRP-ICRU, 1963). bIfE, I0.03 MeV for internal 8-, BP,or e-I, then ICRP-ICRU, 1963 recommended an RBE of 1.7.

1.4

BACKGROUND INFORMATION

/

11

Since the above-discussed reports (ICRP-ICRU, 1963;NCRP, 1967) were issued, several significant developmentshave occurredthat can affect how information on the influence of LET, in a wide variety of biological systems, may be applied to the problems of radiation protection. The principal developments are as follows: (1) Carcinogenesis principally, and mutagenesis (both termed "stochastic effects," see ICRP, 1977; NCRP, 1987a)have been designated as the potential harm of key importance a t the low doses andlor dose rates of interest in radiation protection. "Non-stochastic" effects, eg., cataract formation, sterility, and other organ-impairing effects, may be important at higher doses. (2) Much additional data on the effects of low and high LET radiations on mutagenesis and carcinogenesis in experimental animals have become available. (3) Risk coefficients (slope of the dose response curve) have been introduced for the reference low LET radiation for carcinogenesis in humans, using data obtained generally at high doses and dose rates. Through a process of interpolation or "extrapolation," estimates have been made with respect to the applicability of such risk coefficients to radiation delivered at low dose and dose rates (UNSCEAR, 1977; NRC, 1975; Evans et al., 1985; NASINRC 1972; NAS/NRC 1980; NCRP, 1980; Preston and Pierce, 1988; UNSCEAR, 1988). (4) Risk coefficients for carcinogenesis and other responses have been introduced into radiation protection philosophy and practice by the ICRP (ICRP, 1977). These concepts have been further developed by the NCRP (NCRP, 1987a). (5) Weighting factors, w , have been introduced in ICRP Publication 26 (ICRP, 1977) and in NCRP Report No. 91 (NCRP, 1987a). Where applicable, these factors must be applied, as well as 8,to arrive a t the effective dose equivalent. The w~ is similar to the partial body factor (PBF) suggested in NCRP (1967). (6) It was thought for a time that dose effect curves in humans had become available for a high-LETexternal radiation (Mole, 1975; Baum, 1977; Rossi and Mays, 1978; McGregor et al., 1977). The dose effect curves were for human carcinogenesis for the neutron spectrum delivered by the atomic weapon at Hiroshima. However, as a result of later reassessment of the dosimetry in Hiroshima (Kerr, 1981; h e w e and Mendelsohn, 1981; Sinclair and Failla, 1981; Bond and Thiessen, 1982, RERF,1987), it now appears that the neutron contribution to the total dose in Hiroshima was too small to permit such curves

12

/

1.

INTRODUCTION

to be deduced with sufficient reliability (Preston et al., 1987 and Preston and Pierce, 1988). (7) Extensive data now indicate that while the effectiveness per unit of absorbed dose for low LET radiations (gamma rays, x rays, electrons, etc.) with average LET values within the standard range of 3.5 keV pm-I or less, differ only by perhaps 10 or 20 percent a t high doses and dose rates, the slopes of the linear or "aD component" of the overall dose-response curves can differ by factors of two, three, or more (Bond et al., 1978). (8) The influence of "cell killing" and other complicating factors on the dose response relationships for carcinogenesis,particularly for high LET radiations, has become more clearly appreciated (see Section 6). The implications of the above changes, particularly the development of risk coefficients for mutagenesis and carcinogenesis, are substantial, and may change appreciably the framework in which data on the influence of LET in a wide variety of biological systems and for a large number of endpoints may be used in the context of radiation protection. Earlier (ICRP-ICRU, 1963; NCRP, 1967), virtually no quantification of average risk in man was available or used directly for setting radiation protection limits. Hence, RBE values and their extension to Q represented generalized dimensionless ratios of the effectiveness per dose of radiations of different LET, with neither the numerator or denominator being quantified or quantifiable in terms of absolute risk to man for the relevant endpoints of mutagenesis and carcinogenesis. With the introduction of absolute values of incidences or average risk for the reference low LET radiation (eg., ICRP, 1977), the risk coefficient or slope of the dose effect curve was administratively defined for low LET radiation. Serious questions remain about which numerical values, or range of values of the risk coefficient should be used for the reference radiation at low doses and dose rates commensurate with those experienced in radiation protection (NAS/NRC, 1972; NAS/NRC, 1980, NCRP, 1975; NCRP, 1980, Preston and Pierce, 1988). Nonetheless, risk coefficients for both cancer and heritable effeds now appear in radiation protection documents, so that the amount of risk associated with annual dose limits can be calculated. Under the above conditions, the RBE and by extension the assigned values of Q, then become much more than simply a '%ee floating" ratio, related only loosely, if at all, to risk. The comparison radiation component of the relative biological effectiveness ratio (RBE) is now, in effect,fixed in absolute terms for the specificbiological endpoint(s) of interest. RBE then immediately defines values for the slope of the dose response curve for the corresponding high LET

1.5 ALTERNATIVES TO THE RBE C O N C W

/

13

radiation, for the relevant human endpoints. Thus,not only can the risk to be attached to limiting dose be defined for the comparison radiation (RBEof one), but for a given high LET radiation as well. In view of the RBE values of three or more between low-LET radiations that currently qualify as reference radiations, additional and narrower definition obviously would be desirable. However, any change in the reference radiation is inextricably linked to values for risk coefficients to be used in establishing limits for radiation exposure. In other words, such a specification rests on considerations beyond RBE, so that such definition is not provided in this report.

1.5 Alternatives to the RBE Concept Modem approaches to improving on LET as a measure of radiation quality date, effectively, from the introduction of microdosimetry, (Rossi and Rosenzweig, 1955). The approach introduced by the authors recognized the advantages of considering the amount of energy deposited in tissue volumes of subcellular dimensions, rather than in linear arrays. However, because these efforts remain in the development stage with respect to their possible role in applied radiation protection, they are not directly relevant to the present report. Accordingly only brief statements and references tracing development are supplied. The microdosimetricapproach made it possible to measure energy deposited in spherical volumes of tissue of subcellular dimensions, and its use to replace LET was developed by Rossi, (1959) Kellerer and Chrnelevsky, (1975). The possible role of the microdosimetric quantities lineal energy (y) and the specific energy (2) has been discussed by Rossi, 1959 and in ICRU Report No. 36 (ICRU, 1983) and in ICRU Report No. 40 a joint ICRP-ICRU document, (ICRU, 1986).An approach to characterizing biological response in terms of microdosimetric concepts was introduced as the dual action theory (Kellerer and Rossi, 1972). Cell responses as a function of microdosimetric quantities was presented by Bond and Colleagues (Bond, 1982; Bond et al., 1985; 1988).Attempts to wed microdosimetric and traditional dosimetry concepts were discussed by Feinendegen, et al., (1985) and Booz et al., (1988). Additional such efforts, with derivations of values of a "quality factor" for possible use in radiation protection were made by Zaider and Brenner (1985),the ICRU (1986) and Kellerer and Hahn, (1988).Additional biological justification for microdosimetric approaches came in the demonstrations that most human cancers are of single cell origin (Fialkow, 1967; 1977).

2. Cytogenetic Effects in Plant, Animal, and Human Cells2

2.1 Introduction

During the last four decades, the influence of LET on the induction of mutations and chromosome aberrations has been extensively investigated in both plant and animal cells. Several detailed reviews of earlier works are available (Giles 1954; Lea, 1955; ICRP-ICRU, 1963; Bender, 1970; ICRP, 1972). This section summarizes data on the effects of radiations of differing qualities in plants, and discusses recent evaluations of LET effects on cytogeneticendpoints in somatic cells of human and other mammalian species.

2.2

Cytogenetic Effects in Plants

Plant materials were used extensively in studies on the RBE of different spatial distributions of radiation during the 1960's and, although fewer publications appeared in the 1970's, the later reports included more complete data in the lower dose range and involved a more varied spectrum of neutron energies. The earlier results were summarized in ICRP Publication 18 (ICRP, 19721, (see also Smith,

T o r over 60 years, genetic endpoints have been scored in the somatic and germinal tissues of both plants and animals e x p d to ionizing radiations. Measures of genetic damage have not been limited strictly to heritable changes, in the claesical sense, because certain measures (chromosome aberrations scored in vitm as an example) have proven to be especially seneitive indicators of variation in effectiveness that is related to changea in dose, dose rate, and LET. Consequently, the reader will find information on the genetic effects of different radiation qualities in Sections 2, 3 , 4 and 6 of this report.

1972), and are presented here in Table 2.1 for fission neutrons, in Table 2.2 for monoenergetic and mixed spectrum neutrons, and in Table 2.3 for high-LET radiations produced by accelerated heavy ions. Dose average LET values are listed in Tables 2.2 and 2.3. In this Section, those effects classified as "cytogenetic," "mutagenic" or "tumorigenic" are discussed, omitting most of an exteneive literature on other somatic effects such as reduction in seed germination, plant height and fertility. All irradiations listed in Tables 2.1,2.2 and 2.3 were administered as acute single exposures apart from those of Ecochard (1970) and Neary et al. (1963) shown in Table 2.2. The low-LET radiations used for comparison with high-LET radiations were either 250 kVp x rays or gamma rays, the latter most frequently h m *Co but, in some cases 13'Cs. In Table 2.1 the fission neutrons were unmoditied or slightly degraded, with a spectrum of energies peaking a t 1 to 1.5 MeV. In Table 2.3 the high-LET radiations are mainly accelerated heavy ions (4He to "Ar); but also high energy negative T mesons and protons which gave RBE values above unity. These high RBE values probably result from the interactions of high-LET secondary particles resulting from nuclear disintegrations. The types of response used to measure the relative mutagenic effectiveness of high versus low-LET radiation have been scored in either the treated generation (R,) or its progeny (R2)generation. Those scored in the treated generation include chromosome and chromatid aberrations, as well as somatic mutations arising mainly from loss of a dominant marker in tissues of a heterozygous plant. Those scored in the progeny (RJinclude direct measures of germinal mutation at loci governing chlorophyll production. Wide varieties of plants have been employed as experimental materials. Some of the more frequently used species have been Zea mays (maize), Triticurn genus (wheat), Tradescantia genus (spiderwort), Oryur sativa (rice), Arabidopsis thcrlianu (a shortdife-cycle crucifer) and Nigella damascenu (devil-in-the-bush). Different apecies have different sensitivities to radiation, which has been correlated with the average chromosome or nuclear volume (Sparrow, et al., 1968).This variable contributes to the variation in the magnitude ofthe RBE. Plants have been irradiated at different stages ofdevelopment thus involving different tissues such as seed, inflorescence pollen and root tips. The stage or plant part irradiated does not consistently influence the magnitude of the RBE. 26.1 Environmental Effcects Seeds have been the preferred experimental plant materials since they can be subjected, without impairment of function, to a wide

TABLE2.1-RBE

Species

Wheat Triticum Wheat Triticum

Maize Zea mays

values for mutagenesis of fast ("fission") neutrons versus low-LETradiations on plant species inrrdiated as dry seeds

Criteria (Reaponme level, generation scored)

Chromosome Aberration8 (RJ Chlorophyll Mutations &) Mutation8 (lflf, R,) Mutations (Mf, R1) Albino mutations (1%. R.2) Chlorophyll Mutatione (I%, Rg) Total mutations

High-LET Radiation h e Dcaratea (Gy) (mGy min-9

26-103 1-20.6

N

Low-LET Radiation Doaeg

Type

(Gy)

Dose rates (mGy min-9

250 kVp x rays 30-1,000 10,000-25,000

a

0.12-2.6

mCoy rays

50-250

29,000

RBE Referenma

5-9 Bhatt, et al. (1961) 25 Mataumura (1966)

A

0

0.68 2.7 615

1,700 250 kVp x rays 51.5 1,700 250 kVp x rays 99.1 50-120 230 y rays

18,000 18,000 3,000

76 Smith, et al. (1968) 37 Smith, et al. (1968) 2 Stoilov (1968)

6-15

230

yrays

.50-120

3,000

3-7 Stoilw (1968)

6-15

230

y rays

50-120

3,000

9-15 Stoilov (1968)

270

29.160

(1% %)

Ambidopeis Single locus mutations 17.5 Thaliam (I%, R,) NigeUa Chromosome 0.2-1.5 Damascem Aberrations (R,) aInformation not available

iI

900-2.160 300

oC@ '

y rays

W7Csy rays

10-320

933

16 h j i i (1964a) 78 Moutschen, et al. (1969) Moutechen and MoutschenDahmen (1970)

! B

TABLE2.2-RBE values for mutagenesis of monoenergetic and mixed spectrum neutrons versus low-LET radiations in higher plants Hinh-LET radiations

Material aud sped-

Criteria (gweratim mned,

irradiated

response level)

8eedkMaize

Single loeus Mutatio~w(R,)

Zea muye

NeutmD

mmgy

(MeV)

LET w-')

(LaV

Dom Ranp (Gy)

Low-LET radiations

Dose Rate ( m e min-1)

Dose Range Type

250 kVp x rays 250 kVp x rays 250 kVp x ray 250 kVp x rays 250 kVp x rays

(Gy)

15-160 15-160 15-160 16160 16-160

Doss Rats (me

=in-I)

4,560-9,800 4,550-9,800 4,560-9,800 4,550-9,800 4,550-9,800

250 kVp x raya 14-140 4,550-16.760 250 kVp x rays 14-14 4,550-16,760 250 kVp x rays 14-140 4,550-16,760 SeedsNigelb

Chromosome

D m c e ~ Aberrations (R1)

Inflorescence (Stamen

Pink mutant eella (R1)

la7Csy rays *?Cs y raya lS7Csy rays lmCs y raya GP?Cs y rays

10-320 10-320 10-320 10-320 10-320

300 300 300 300 300

250 kVp x rays 0.1251.28

290

250 kVp x rays 0.17-6.7

1,000

250 kVp x rays 0.125 1.28

290

hairs) !rru&Smh

Pink mutant cell8 (R,)

RBE 100 88 81 69 69

Re-

Smith, et al. (1964) Smith (1967) Smith and Rosei (1966)

92 72 57 115 67 85 61 59

Moutachen, et al. (1969) Moutachen; MoutachenDahmen (1970) 13- Underbrink, et 31 al. (1970, 1971); 18- Daviee and 40 Bateman (1963) 10- Underbrink et al.(1971) 16

CYTOGENETIC EFFECTS

Seeds Ambidopeis Single locw Thaliana Mutation (Rl. 1%)

14

38

&44

74400

W s y rays

300-700

y ray8

2.9-14.5

1,670

15

Fqiii (1904b,

5

Fqiii (1969)

1969)

Pollen-make Zeo mays

Single k~cua Mutation (R,)

14

38

2.9-107

F h t Calls-

Chromatid Aberrations (R,)

14.1

38

0.16-0.48

38

0.75

130

250 kVp x ray8 14-140 4,550-16,760 49

-160

0.1-1.4

206

'"Cs y rays

Broad bean vicia f i

Seeds,Maize

Mutations/ 14.7 Gyx 10-alcell(R1) 8eedsnigdlo Chromoeome Thermal Damaacena Aberrations (R,)

1648

250 kVp x rays

a

3-6 Savage (1968)

Zea mays

Pollem-Cmab Specific locus Lyqnemicon Mutatione (RJ Eseulentum

aInformatimnot available

10-920

933

81

Smith and Roeei (1966) Moutaehen and MoutschenDahmen (1970)

6

$

$

0

Thermal

45

4.95

1.7

yrap

6-27

0.17

1.6-3.OEeochnrd(1970)

!

P 5!

TABLE2.3-RBE

d u e s for rnufugenesisfollowing irradiation of seeds with high-LET maktions produced by occeleruted htmy iuns H i g h - r n radiations

Speeia

Cribria (Ibrpansebvel, psratioo srmed)

Ambidopsie Single locue Thhna mutations (0.596, R,)

Partide

'He

'F aAr

Sileloeue 'He mutations (3%. 'He RI) 'Li 'T

'80 W e

ah Wheat T.

Mono-

Low-LET radiations

Doee

Chromeome a +Li Ab-tio~ (Rl)

Incident Energy

LET (keV

pn-1)

42 MeV 20 125 MeV 230 416 MeV 2,500 40 MeV 9 MeV 17 MeV 44 MeV 67 MeV 91 MeV 222 MeV (1.6)(0.9) MeV

18 74 172 409 752 1,030 1,890 -170

Dose

Range

Rates

(Gy)

(Gy min-1)

Range

Done Ratea

(Gy)

(Oy min-')

Doee

10-50 10-50 30-50

40-50 40-50 40-50

h '"Cs y rays la7Cay rays '3'Cs y r a p

60-750 9.5-56 9.5-55 9.5-68 18-67 53-214 53-256 0.11-0.8

40-50 40-50 40-50 40-50 40-50 40-50 40-50 0.037-0.4

250 kVp x ray8 250 kVp x rays 250 kVp x rays 250 kVp x rays 250 kVp x rays 250 kVp x r a p 250 kVp x ray8 I3?Csy rays

Maize Za -YE

Protons

28 GeV Secondary

1.6-140

131Csy rays

2.8GeV Secwdary 690 Particlea

Fujii (1969); Fujii, et

10 35

350-1,200 16-17 350-1,200 16-17 350-1,200 1 6 1 7 350-1,200 16-17 350-1.200 16-17 350-1,200 16-17 350-1,200 16-17 5-30 0.17

1.4 21

f96'7')9669

;;

Himno et al. ,1910,

5-30

0.0017

0.001

250 kVp x rays

14-140

5

3.33-296

250 kVp x rays

20-80

4.95

la7cs rays

10-30

0.93

Particles

NigelIa Cluoumome Rotona D a m a 8 c e ~ Aberrations (Itl)

References

270-470 0.0017 270-470 0.0017 270-470 0.0017

cornurn Chlorophyll a + Li (1.6)(0.9)MeV -170 Mutatione (RJ Single l m Meaona 8 GeV Secondary 13 Mutations (R,) Particles

\

RBE

l5

1.3 1.6 23 Mataumura et 01. (1963) 29

Micke et al. (1964a,b) Smith (1967) 3.5 Smith (1967); Smith et al. (1965, 1974a) 3 Moukhen, 3

et aI.

(1969)

'Idonnation not available

Y'

9I !A

C)

M

3

C)

3

2.2 CYTOGENETIC EFFECTS IN PLANTS

1

21

range of radiation doses, storage times and environmental conditions (temperature, moisture, atmospheric content, etc.). Environmental factors greatly influence the degree of response, especially to lowLET radiations, and particularly for seed irradiations where large differences in moisture or oxygen content may exist. For example, Smith and Combatti (1967)found, for a single locus somatic mutation in maize, that seeds of 6.7 percent moisture gave an RBE of fission neutrons versus 250 kVp x rays of 67, whereas after 36 hours of soaking prior to irradiation the RBE was reduced to five. This decrease in RBE was solely because of an increased effectiveness of the x irradiation. Since oxygen can greatly enhance the effectiveness of sparselyionizing radiations compared to densely ionizing radiations, its presence during or after irradiation results in lower RBE values. Under conditions of oxygen exclusion, for the same maize system referred to above, a maximum RBE of greater than 100, and possibly as high as 1,100, was found for fission neutrons versus gamma radiation (Conger and Carabia, 1972; Conger, 1975). With inflorescences of Tradescantia the effects of x rays versus 1.02 MeV neutrons on the frequency of induced pink mutant stamen hair cells under aerated versus hypoxic conditions are shown in Figure 2.1 (Underbrink and Sparrow, 1974). It is evident that the listing of RBE values in Tables 2.1. 2.2 and 2.3 has limited meaning unless the state of the plant materials and the environmental conditions under which the irradiations were carried out are specified. Although this information is not completely available for all the experiments listed in the tables, nevertheless, the irradiations were usually carried out in air and irradiated seeds were usually described as dormant and dry (around 10 to 20 percent moisture content).

2.2.2 RBE Versus Dose and Dose Rate

The irradiations listed in Tables 2.1,2.2 and 2.3 were administered a t a wide variety of dose levels and dose rates. In order to summarize the effects of these variables on RBE, the results were grouped into the following categories for the high-LET radiations. Average

Average

Average

Number of

Dose

Dose Rate

Studies

RBE

16 5 23

12 ? 3 2026 66 + 5

High High

Low

High

Low

Any

\

lo-'

"3

I--

Zo' >a MI-

2 5 0 - kVp X RAYS

2

5s la2 I-A 3

z$

A

AERATED

X I

E

OPEN AIR (SPARROW. UNDERBRINK AND

1cj3

16~

0.001

0.01

0.1

I

1 0

100

(GY)

Fig.2.1 plot of induced pink mutant events in ~mdescantiafor 1.02M ~ neutrons V and 250 kVp x rays irradiated under air and nitrogen atmospheres. (Adapted from Underbrink and Sparrow, 1974).

A high dose was arbitrarily set at greater than 2 Gy delivered at a higher dose rate, and a low dose rate at less than 0.1 Gy min -'. The radiation dose levels for plants required to produce an effect are generally higher than with animal material, especially in seed irradiation. However, the results on plants are consistent with those on animals in showing that low doses give large RBE values while high doses give relatively lower RBE values.

2.2.3

RBE Versus LET

In order to obtain overall RBE values for plants that can be compared with those of other organisms, average RBE values were calculated from the data listed in the tables. The average of all RBE values given in Tables 2.1,2.2 and2.3 is 39. The average for fission neutrons is 29 (Table 2.1, see also Smith et al., 1974b), and for all other highLET radiations is 42 (Tables 2.2 and 2.3). For further comparisons, the information in the tables is grouped below, where possible, according to different ranges in keV pm-'.

2.2 CYTOGENETIC EFFECTS IN PLANTS

LET (keV ( ~ m - l )

Number of Studies

1

23

RBE (Mean)

The RBE values are lowest in the LET ranges of 7 to 22 keV pm-' and above 175 keV ~ m - while I they are maximal in the 2 to

175 keV p,m-' range. These results are in agreement with similar data on animals; however, the RBE values are generally higher than for most other organisms tested. The use of accelerated heavy ions has allowed a very wide range of LET-radiation to be explored. Hirono et al. (1970) exposed the meristem of Arabidopsis (a small genus of herbs) seeds to ions with track average LET ranging from 72 to 1890 keV p m - ' (4Heto and scored their effects on tumor induction, inhibition of growth and somatic mutation induction. The RBE value for each criterion of response was remarkably similar throughout the range of LET, showing a maximum at 72 to 174 keV pm-' (see Figure 2.2). The influence on RBE values for species with different nuclear and chromosomal volumes irradiated with 250 kVp x rays versus fission neutrons and scored for three different criteria of response is illustrated in Table 2.4. The dose rate for x rays was 17 Gy min-I and for fission neutrons was 0.28 to 1.78 Gy min-'. The RBE decreases with the increasing dose required to produce the D,, endpoint. Higher doses are needed in species with the smaller nuclear and chromosomal volumes and for end points that are less affected by radiation. For example, the RBE value for 50 percent seed set in Nigella damascena (black caraway) is 25.9, whereas it is only 7.3 for 50 percent plant survival in Lycopersicon escukntum (tomato) (Smith et al., 1970). 29.4

Tmdescantia Studies

In order to explore further the relation between RBE and neutron energy, Tradescantia (spiderworta) inflorescences were exposed to neutrons of twelve different energy levels ranging from 0.065 to 13.4 MeV (Underbrink and Sparrow, 1974)(see Figures 2.3 and 2.4). A series of doses was chosen to demonstrate the effect of this variable on RBE values in that the lowest doses were in the region where the x ray dose-response curve becomes linear, about 50 to 100 mGy and the higher doses are in the region dominated by the quadratic term in the linear quadratic equation:

z 35 n

t

*-a

TUMOR INDUCTION (63%)

--a DRY WGT: INHIBITION (30%)

t

+---o

SOMATIC MUTATION (3%)

LET (keVlpm IN MERISTEM 1- LOG SCALE Fig. 2.2 RBE valuea for accelerated heavy ions and 250 kVp x raya at different

LET values (shownon the abscissa) with respect to tumor induction, growth inhibition and induction of eomatic mutations in seeds ofAmbidopsis. (From Hirono d al., 1970.)

TABLE2.4-Dose in Gy and RBE values for plunts grown from seedP irradiated with x my$ (XIor fission neutrons WI). ( h m Smith et al., 1970). +%-=a

Nigella

Zea may8

Damawena 392-32.7

280-14.0

Volume (kma) nucleus-chromosome Criteria (Dm) X Seed set 36.3 Plant height 56.8 Survival 60.3

Nf 1.4 3.6 5.7

Lyaopereicon

Eseulentum

1104.6

RBE X N, RBE X Nf RBE 25.9 210.6 9.7 22.3 348.6 24.4 14.3 15.8 297.6 25.1 11.8 401.1 51.2 7.8 7.3 10.6 340.1 33.1 10.3 518.8 70.7

2.2 C ~ E N E T I EFFECTSINPLANTS C

I

25

m2

I = aD + (2.1) Dose-response curves for induced pink mutants for each energy are shown as double logarithmic plots in Figures 2.3 and 2.4. RBE values were determined a t three different positions along the doseresponse curves a t mutant event frequencies of 0.003,0.01, and 0.03 events per hair (Table 2.5). The maximum RBE value opposite 50 mGy on the x ray curve, occurs at a pink mutant event frequency of 0.003 events per hair (or below). For all neutron energies, RBE values decrease with increasing dose above 50 mGy of x rays and increasing mutant event f?equency. Maximum RBE values taken at 0.003 mutant events per hair (Table 2.5) are plotted against neutron energy in Figure 2.5. RBE

Fig. 2.3 Plots of induced pink mutant events per stamen hair in Tmdesanticr at different dbses of 250 kVp x rays and neutrons ranging in energy from 0.065 to 0.43 MeV. A m w s indicate region on dose-response curves at (and below) for which the B E value is maximum.(Adapted from Underbrink and Sparrow, 1974.)

26

1

2. CVToGENE'l'IC EFFECTS

Fig. 2.4 Plots of induced pink mutant events per stamen hair in T d s c a n t i a at different doses of 250 kVp x rays and neutrons ranging in energy from0.552 to 13.4 MeV.Arrows indicate region of the dose-response curves at (and below) which the RBE value is maximum. (Adaptad from Underbrink and S p m w , 1974.)

values as a function of neutron energy (and hence of spatial distribution of radiation) increases fiom 14.7 a t 0.065 MeV up to a peak of 47.6 at 0.43 MeV, and then decreases to 10.4 at 13.4 MeV. In the experiments discussed so far, 250 kVp x rays have been used as a standard for comparison with radiations of denser ionizing paths. For the more sparsely ionizing radiation of gamma rays, an RBE value of approximately 0.6 was obtained at low dose rates when x rays were used as the standard (Underbrink, et al., 1970). Highenergy muons (p- ) with LET of approximately 0.25 keV pm - ' were found to have an RBE value of 0.81 compared to 250 kVp x rays in maize (Micke et al., 1964a) and of 0.79 (Davies et al., 1963) and

2.2 CY'IOGENETIC EFPECTS IN PLANTS

27

1

TABLE 2.5--RBE ualues for pink mutant eventa in Tmdcscwrntia stamen hairs

following irradiation with n e u h n s of &us energies and 250 kVp x rays (adapted h r n Underbrink and Spanvw, 1974). Neutron Pink evente Doee ( ~ G Y ) Energy Per haiZ (MeV)

0.065 0.110 0.220 0.340 0.395 0.430 0.662 0.680 1.02 2.05 6.0

(minu control)

x ray

Neutron

0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003 0.01 0.03 0.003

50 120 270 50 120 270 50 120 270 50 120 270 50 120 270 50 120 270 50 120 270 60 120 270

3.4 11.0 33.0 2.3 7.6 23.0 1.9 6.4 19.0 1.5 5.4 16.0 1.15 3.8 11.5 1.05 3.7 11.0 1.5 4.9

50

120 270 50 120 270 60 120 270 50

14.5

1.6 5.4 16.5 2.7 9.0 27.0 3.3 11.0 34.0 3.7 12.0 37.0 4.8

RBE 14.7 10.9 8.2 21.7 15.8 11.7 26.3 18.8 14.2 33.3 22.2 16.9 43.5 31.6 23.5 47.6 32.4 24.6 33.3 24.5 18.6 31.3 22.2 16.4 18.5 13.3 10.0 15.2 10.9 7.9 13.5 10.0 7.3 10.4

'Values averaged &om total mutant events divided by totaletamen hairs. Data are cumulative for daye 7 through 16 post irradiation.

0.59 2 0.06 (McNulty et al., 1974) compared to gamma rays in Tmdescantia. 26.5 Summary of Cytogenetic Eficts in Plants

The data for plant material are consistent with those to be shown for animals in showing a dependence of RBE on spatial distribution

1

0

0.2

1

1

0.4

1

0.6

1

1

0.8

1

1.0

1

1

1.2

1

1.4

1

~

1.6

1

1.8

11

1 ~

1

~1

~

2.0 2 . 2 ' w

NEUTRON ENERGY MeV)

Fig. 26 Plot of the RBEy value for induced pint mutant events in Tmdescanhh at differentneutron energies. The peak ie reached at 0.43 MeV. (Adapted from Underbrink and Sparrow, 1974.)

of radiation; and that the RBE value is influenced by other factors as well, eg., dose level, environmental variables, criteria of response and genotype.

2.3 Cytogenetic Effects in Human and Other

Mammalian Cells Simple methods for the culture of peripheral blood lymphocytes for cytogenetic evaluation were f i s t introduced in 1960 (Moorhead et al., 1960).This technical breakthrough has allowed detailed study of the variation in effects in human and other mammalian cells as a function of the LET of the radiation used. The lymphocyte culture system has many advantages in quantifying the effects of various radiation qualities at the cellular level. Human or other mammalian whole blood can be exposed under controlled conditions of temperature, pH, and oxygen tension designed to mimic conditions of in vivo exposure. Because the small, immunocompetent lymphocytes in blood samples from adults are virtually all in the "resting" (GJ stage of the cell cycle, problems resulting from differential radiation sensitivity during different stages of the cell cycle are avoided. Likewise, by culturing lymphocytes in the presence of bromodeoxyuridine (BrdU) and employing epecial staining techniques, populations of metaphases that have been arrested in their f i s t in vitro mitosis aRer radiation exposure can be selected for evaluation. Scoring of radiation-induced chromosome aberrations in these first division metaphases thus provides quantitative estimates of the amount of damage induced a t the time of exposure since loss of aberration-bearing cells due to mitotic death has not yet occurred.

2.3 CYTOGENETIC EFFECTS IN MAhlMALIAN CELLS

1

29

This methodology has been used extensively in studies to determine the dose-response relationships for chromosome aberration induction after in vivo andlor in vitro exposures to high- and lowLET radiations. Evaluations in these cell systems have verified that the well-defined dependence of aberration frequency on LET, previously derived for cytogenetic aberration induction in plant cells, also occurs in somatic cells of humans and other mammals. 2.3.1 Dose-Response Relationships for Low -LET Radiation

The types of chromosome aberrations that are induced by high or low-LET radiation will depend on the stage of the cell cycle during which the exposure occurred. For lymphocytes irradiated during Go, these include terminal and interstitial deletions which result from breakages in a single chromosome, and exchange-type aberrations which result from breakage and rejoining of two or more separate chromosomes or chromosome segments. The dose response relationship for exchanges, such as dicentric chromosomes, is curvilinear in human and other mammalian cells exposed to moderate to high doses of low-LET radiation, delivered at high dose rates (see Figure 2.6). When the dose dependency for dicentrics is interpreted in terms of the quadratic dose response function (e.g., equation 1.2), the a or linear term defines the relative proportion of dicentrics induced by single radiation tracks, and is thus dose-rate independent. Conversely, the @, or.dose squared coefficient, defines the frequency of dicentrics that result from the interaction of lesions induced by two independent radiation tracks, and thus predominates at higher radiation doses and dose rates. Several studies have shown that the mean repair time for primary chromosome lesions that may interact to yield dicentrics is about two hours in human lymphocytes (Schmid, et al., 1976; Purrott and Reeder, 1976; Liniecki, et d.,1977; Virsik and Harder, 1980; Lloyd, et al., 1984a). It thus follows that the frequency of dicentrics induced in human cells by inter-track events will depend not only on dose magnitude and spatial juxtaposition of lesions, but also on the temporal distribution of dose, as has been extensively documented in numerous cell systems (see NCRP, 1980, for detailed discussion of dose rate effeds). Since the mid-1960's, dozens of studies have been conducted to determine the dose response parameters for chromosomeaberrations in human lymphocytes exposed to various qualities of low-LET radiation. In the followingparagraphs, a summary of data on the induction of dicentric or ring chromosomes after acute exposure of whole blood to doses ranging from 0.2 to 6 Gy is presented. All data were derived

0

5

1

2 Dose (Gyl

Fig. 2.6 Dose response relationship for the induction of dicentric chromosomes in human lymphocytes exposed to BOCo gamma rays at a rate of 0.5 Gy min-' (Adapted h m Littlefield et al.. 1989).

from early harvest lymphocyte cultures, and in many recent studies, evaluations were restricted to first division metaphases in BrdUsubstituted cultures. Dose effect coefficients were calculated using either maximum likelihood or least-squares curve fitting methods which may produce different estimates of the a and p coefficients (Lloyd and Edwards, 1983)and thus may contribute to some disparity in results reported by different laboratories. A synopsis of published data for the induction of dicentric chromosomes in human lymphocytes exposed to 180 to 250 kVp x rays is shown in Table 2.6. In 12 studies that did not employ BrdU methodology, estimates of a and p vary rather widely. The degree of interlaboratory variability in estimates of both coe5cients is considerably less in six recent reports in which dicentrics were evaluated in first division metaphases. The relative efficiencies of high energy x rays may be compared with similar data for human lymphocytes exposed to gamma radia-

2.3 CYTOGENETJC EFFECTS IN

CELLS

1

31

TABLE2.6ExampIes of dose response cwfiients (Y = a D + Da) for dicentric aberrations after acute egposure of human whole blood to 180 to 250 kVp x rays X-ray Dose range a + SE B + SE

(Energy) (Gy) ( X lo-=Gy-'1 ( X lo‘= G Y - ~ ) Refemnce 180 kVp 0.25-5 5.40 + 0.50 2.02 + 0.40 Todorov (1975) 180 kVp 0.11-0.57 4.63 + 0.59 - Ziemba-Zoktowska et al. (1980P Sasaki (1971)' 200 kVp 0 . 2 4 7.51 + - 7.11 2 200 kVp 0.48-3.84 3.66 + - 8.01 + Muramatsu and b y a m a (1977) 220 kVp 0.254 7.80 + 1.30 4.15 ? 0.34 Schmid et al. (1972) 220 kVp 0.25-4 7.90 2 0.40 5.36 2 0.22 Schmid et al. (1976) 220 kVp 0.5-5 4.04 + 0.30 5.98 0.17 Bauchinger (19Wb 220 kVp 0.25-3.75 4.34 2 0.81 6.55 2 0.39 Littlefield et al. (1989)b 220 kVp 0.05-0.50 4.15 + 0.65 - Wagner et al. (1983)b 250 kVp 0 . 5 4 9.1 -c 0.2 6.0 + .7 Brewen and Luippold (1971) 250 kVp 0.054 4.76 + 0.54 6.19 + 0.31 Lloyd et al. (1975) 250KVp 0.50-4 5.21 + 3.00 7.20 ? 1.11 Leonard et al. (1977) 250 kVp 0.05-0.50 3.7 + 0.6 - Kucerova et al. (1972). 250 kVp 0.05-0.60 4.83 + 0.74 - Vulpis et al. (1976) 250 kVp 0.003-0.05 2.38 + 1.22 - Lloyd (1988Y 250 kVp 1.00-5 3.75 + 2.92 7.24 2 0.85 Bajaktarovic and Savage (19€Qb 250 kVp 0.05-2 4.37 + 0.99 5.95 2 1.14 Fabry et al. (1985)b 250 kVp 0.05-6 3.64 + 0.53 6.67 2 0.22 Lloyd et al. (1986a)b .Includes centric rings. bData from scoring first division metaphases.

tion from 60Co in Table 2.7.As with x rays, narrower ranges of values for a and flcoefficients are observed when inter-laboratory comparisons are restricted to data h m studies that employed BrdU methodology. These show that the dose dependency for dicentrics induced by two independent tracks is quite similar for x rays and hlgh energy gamma rays (i.e., = 5 to 7 x lop2dicentrics cell-' Gy -?, as would be expected if aberrations that result from inter-track lesions are independent of LET. However, comparisons of estimates of a generally show that hard x rays are more efficient than gamma rays in inducing "one t r a c k aberrations, as would be predicted if the proportion of exchanges resulting from intra-track effects were LET dependent. Recently, four laboratories have compared dose response relationships for dicentrics in first division metaphases following exposures of human lymphocytes to high dose rate x rays and 60Cogamma rays (Table 2.8). Based on the ratios of the a coefficients for these two reference radiations, estimates of a limiting RBE (see Figure 1.2)of 1.5 to 3.8 may be made for hard x rays versus 60Cog a m m a rays. Two Studies have quantified dicentric induction in lymphocytes after exposures of whole blood to 5.7 keV (Edbeta radiation from

TABLE2.7-Examples Radiation

="coy ="coy ="Coy

="cw "Cw "Coy

of dose effect coefficients (Y = a D + p 02) for dicentric abermtions after acute exposure of human whole blood to gamma or beta mdiatwn I? range a 2 SE p -+ SE (MeV) (GY) (Ilo-' Gy-I) (X Gy-9 Reference Sasaki (1971)" Brewen et al. (1972) Lloyd et al. (1975) Liniecki et al. (1977) Bauchinger et al. (1979) Konecra and Kalina (1987)" Bauchinger (1984)b Fabry and Wambersie (1985)b Lloyd et al. (1986Ib Vulpis and Scarpa (1986)b Littlefield et al. (1989Ib Matsubara et d.(1988Ib

%

13"Csy

0.59, 0.47 0.67

0.5-4 0.05-4

3.18 4.33

+ 1.80 1.50

6.09 4.31

2 Ir:

0.72 0.42

Dufrain et al. (1980Ib Takahashi e l al. (1982)

3.05 2 12.0 + Bocian et d. (1978P 5.7 keV 0.28-2.5 5.7 keV 0.10-4.13 5.37 r 0.58 1.28 -C .32 Prosser et al. (1983) "Includes centric rings. bData from scoring first division metaphases or from cultures with less than five percent second division metaphases.

\

h,

Q

a

! ! 3

2.3 CYTOGENETIC EFFECTS IN MAWULUN CELLS

1

33

TABLE2.GLow Dose RBE, for x rays versus gamma rays for dicentrics in human lymphocytes" Dose Radiation

Range (Gy) (Rate)

a (X

+ S.E. Gy-')

220 kVp X rays

0.5-4 (0.5 Gy min-')

4.04

"Co Y rays

0.5-4 (0.5 Gy min-l)

1.07 2 0.41

x rays

250 kVp

0.05-2 (1.0 Gy min-l)

4.37 -c 0.99

Wo Y rays

0.05-2 (acute)

2.97

?

0.80

250 kVp X rays

0.05-6 (1.0 Gy min-I)

3.64

+

0.53

"Co Y rays

0.05-6 (0.5 Gy min-l)

1.42

2

0.44

220 kVp

0.25-3.75 (0.5 Gy min-')

4.34

?

0.81

&

Limiting RBE (a&)

Reference

Bauchinger (1984)

0.3 3.8

Fabry et al. (1985) 1.5

Lloyd (1986a) 2.6

X rays

C @ o' 0.25-4 1.57 + 0.66 Y rays (0.5 Gy min-') "Data from scoring first division metaphases.

Littlefield et al. (1989) 2.8

the decay of tritium. As with other low-LET radiations, the observed frequencies of radiation-induced chromosome aberrations is highly dependent on dose rate ( h s s e r et al., 1983)and the dose. dependency for dicentrics produces a good fitto the linear quadratic dose response function, with estimates of a similar to those observed for hard x rays (Table 2.7). 2.3.2 Dose-Response Relationships for High-LET Radiation

As has been extensively documented in plants, as well as in animal systems, the yields of all types of chromosomes aberrations vary predominantly as a linear fbndion of dose in human lymphocytes exposed to various high-LET radiations. The observed linearity would, of course, be expected if aberrations induced by high-LET radiation were primarily a manifestation of intra-track effects produced by localized depositions of large amounts of energy along discrete track segments.

Examples of dose effect coefficients for dicentrics observed after in vitro exposures of human whole blood to fission spectrum neutrons generated by various reactors are shown in Table 2.9. Best fits to linear dose response functions have been reported for all reactor neutron beams with the exception of the recent data of Bauchinger et al., (1984b) obtained from irradiations of lymphocytes with mixed neutron-gamma rays generated by a reactor neutron therapy converter (RENT). In 9 of these 11reports, estimates of a for the mixed fission spectrum neutrons differed by less than a fador of two (i.e., from 55 to 90 x dicentrics cell-' Gy-I). The data of Sevankaev et al., (1979) for nearly monoenergetic neutrons show a dependence on neutron LET with maximum effects a t about 0.35 MeV (Lloyd and Edwards, 1983). When upper doses have exceeded one or more Gy, most authors have reported best fits to the linear quadratic function for dicentric induction in human lymphocytes by fast neutrons produced by accelerated deuterons on beryllium or tritium. However, as is shown in Table 2.10, the aberrations induced by single tracks predominate, as is demonstrated by the fact that a / P usually exceeds 3 to 4 Gy for the majority of the reported studies. Estimates of a coefficientsrange from 13.9 to 74.5 for aberration induction in human lymphocytes exposed to D-Be neutrons having average kinetic energies ranging from 2 to 21 MeV. On the whole, the efficiency of these neutrons in inducing aberrations in human lymphocytes shows a general inverse correlation with neutron energy. However, derivations of LET relationships are obviously confounded by differences between laboratories in estimates of the slope coefficientsfor neutrons having similar energies. Considering that different methods for curve fittings were used by different authors and that only a few of the data were derived from scoring exclusively first division metaphases, there is reasonably good agreement between laboratories in the estimates of a for dicentrics induced in human lymphocytes by 14 to 15 MeV D-T neutrons. As expected, the human cell data generally show that these high kinetic energy neutrons are less efficientthan mixed fission spectrum neutrons in inducing aberrations. When coefficients obtained by the same laboratory are compared, a is shown to increase as neutron energy decreases as is exemplified in Figure 2.7. The human lymphocyte data closely parallel recent findings in mammalian fibroblasts which have demonstrated that while different cell lines may exhibit differing radiation sensitivity, the dose dependency for dicentric induction shows a strict dependency on photon or neutron LET, or on average lineal energy (y) (Rossi, 1959; Zoetelief and Barendsen, 1983; and Roberts and Holt, 1985). One

TABLE2.9-Dose effect coefficients(Y Neutron

i?

Source IRT-2000

(MeV)

= aD

+ B De) for dicentric aberrations a*

tke Range (Gy)

acute exposures of

spectrum neutrons a + SE (x

lo-=Gy-'1

P (X

5

human whole blood to fission

SE Gy-a)

Reference

"fission" Todorov et al. (1973) Vulpis et al. (1978) 0.4 Biola et al. (1974) "fhion" Biola et al. (1974) "fission" "tission" Biola et al. (1974) Lloyd et al. (1976) 0.7 m 0.9 ~ l ' o et ~ dal. (1976) '3 Littlefield (1983P 1.28 Bauchinger etal.(1984a)b 2 RENT 1.6 Lloyd et al. (1978) 26ZCf 2.13 Zhang et al. (1982)P 2B2Cf 2.13 KG2.5 0.04 Sevankaev et al. (1979)' Sevankaev et al. (1979)' KG2.5 0.09 Sevankaev et al. (1979)' BR-10 0.35 Sevankaev et al. (1979)' BRlO 0.85 d 'Data h m scoring first division metaphases or from cultures with less than five percent second division metaphases. bIncludes centric rings. 'Maximum likelihood fit with POLYFlT program by Lloyd and Edwarde (1983).

APIRO Cmc Nereide Harmonie BEPO AWRE HPRR

1 8

f

TABLE2.10-Dose effect eoef%cients ( I ='a D Neutrona

E

Source

(MeV)

D-Be Am-Be D-Be D-Be D-Be D-Be DBe D-Be D-Be D-Be

h e rauge

(GY)

+

02) for dicenfric aberrations a@r acute exposures of human whole blood to fast neutrons of varying energies a 2 S.E. p * S.E. (X l o 4 ~ y - 1 1 (I GY-~) Reference Sasaki (1971)b Zhang et al. (1982Ib Biola et al. (1974) Matsubara et al. (1988)" Fabry et al. (1985)' Lloyd et al. (1976) Fabry et al. (1985P Muramatau and Mamy(1977) Bajaktarovic et al. (1980)' Fabry (1985)'

4.33 2 1.03 Zhou et al. (1979)b 0.15-6.6 22.10 + 2.30 D-T 14.0 3.71 a S d (1971Ib 25.00 .c D-T 14.1 0.124.5 9.44 -C 1.64 Sevankaev et al. (1979) 0.483.64 18.46 k 4.17 D-T 14.7 8.84 + 2.78 Lloyd et a1. (1976) 0.05-3.03 26.16 + 3.96 D-T 14.7 14.10 k 0.70 3.77 r 0.12 Bauchinger et d. (1975) D-T 15.0 0.313.75 11.9 2 2.0 Lloyd et al. (1984bIe 0.04-2.28 19.5 2 1.8 D-T 14.9 D-T 14.5 0.02-2.10 17.9 & 1.5 7.4 + 1.4 Bauchinger (1975a)' 19.4 + 1.8 DT 14.8 0.004-0.24 - 16.0 -C 8.0 ~ o h l - ~ ueti hcrl.~(1986)' aDeute.ro~accelerated on beryllium [D-Be, 1°Be (d,n) 9Bl, or tritium [D-T, 3H (d.n) 'He] targets except for Zhang et al., (1982). bInclude~ centric rings. 'Data from scoring first division m e t a p h a s or from cultures with l e s ~ than five percent second division metaphasee.

. P

P 54

0

m

7

R

3

2.3 CYTOGENETIC EFFECTS IN UAMMALIANCELLS

1

37

recent report provides evidence that the relative efficiency of neutrons in inducing aberrations in plateau phase V-79 cells does not continue to increase in the energy range below 100 keV (Roberts et al., 1987), suggesting that RBE decreases at lower neutron energies as has previously been reported in several other cell systems (see Figure 2.5).

Linear dose response relationships have also been observed after exposures of mammalian cells to various heavy ions and a particle emitting radionuclides. Dose response curves for chromosome exchange induction in CH2B2cells after in v i m exposures to various high-LET radiations are shown in Figure 2.8, and the fkequencies of aberrations observed in Chinese hamster liver cells after in vivo exposure to alpha, beta, gamma, and fission fiagment radiations h m internally deposited radionuclides are shown in Figure 2.9. Extensive data have not been published on the in vitro dose response relationships for chromosome aberration induction in human lymphocytes exposed to alpha particles; however, a few reports a r e available. Vulpis (1973) observed a dose dependent increase in cytogenetic aberrations in human lymphocytes exposed to alpha particles produced by the O ' B (n, a)'Li reaction and linear dose response relationships have been reported after exposure of human lymphocytes to alpha radiation from 242Cm(Edwards et al.,

Fie.2.8 Doee r e g p o w curve8 for the production of chromatid exchange8 in CHJ3, aRer exposure to various heavy ions and x rap (Adapted h m S w a r d et al.,

-118

1967).

2.3 CYTOGENETIC EFFECTS IN MAlVIMALZAN CELLS

1

39

241Am %o Acute 2 5 2 ~ Citrate f 60~o Protracted r 2 3 9 ~ uCitrate o 144Ce-144Pr Citrate

20

10

30

Dose (Gyl Fig. 2.9 Dose response curvea for the production of chromsorneaberrationsin liver of Chinese hamster after in vivo exposure to alpha, beta, gamma or fission w e n t radiation (Adapted h m Brooks, 1975).

19801, 2 s 9 Pcitrate ~ (Purrott et al., 1980) and 241Am(DuFrain et al., 1979). There is considerable disagreement among these data regarding the relative efficiency of alpha particles in inducing aberrations. Purrott et al., (1980)and Edwards et al., (1980)reported a coefficients of 37.5 and 28.6 x dicentrics cell-' Gy-I for dicentric induction in human lymphocytes exposed to 5 to 6 MeV alpha particles generated via decay of 239Puor "%rn, respectively. Conversely, DuFrain et al., (1979)reported an a coefficient of 490 x exchanges cell- ' Gy -'in lymphocytes exposed to 5.5 MeV alpha particles from %'Am. In this later experiment, lymphocytes were exposed in culture medium containing differing activities of americium citrate. The large discrepancy in estimates of a may possibly be due to problems

40

/

2. CYTOGENE'I'IC EFFECTS

in measuring dose to target lymphocytes when cells and radioisotopes are mixed in a suspension. Fisher and Hardy (1982) re-examined the data of DuFrain et al., (1979) using microdosimetric analyses. They concluded that lymphocyte doses must have been several fold greater than those originally calculated, possibly as a result of aggregation of lymphocytes and activity in a portion of the medium, or adherence of radioactivity to cell membranes. Different results have also been reported regarding the relative efficiency of high energy accelerated particles in inducing one-track aberrations in human lymphocytes. Takatsuji el al., (1983), and Takatsuji and Sasaki (1984) evaluated dicentrics induced following irradiation of a single layer of Golymphocytes with monoenergetic beams of 4.9 MeV protons or 23 MeV alpha particles (Table 2.11). A best fit to the linear quadratic dose response function was observed for the low energy protons whereas dicentric induction was adequately fit by a linear response for the more efficient alpha particles. These authors note that based on the theory of dual radiation action (Kellerer and Rossi, 1972), a is expected to be proportional to LET, whereas for these data, the ratio of a coefficients for accelerated alpha particles versus protons was 2.74, while their LET ratio was 3.75. They suggest that the differences in the observed ratios may be the result of an LET dependent modification of the a term, possibly resulting from the selective loss of cells with more damage at the high-LET. Relative to results that would have been expected based on the Takatsuji data and on differences in track segment LET of the accelerated particles employed in the two studies, considerably lower aberration yields per unit dose were observed by Edwards, et al., (19861, who evaluated dicentric induction following irradiation of thin samples of blood with monoenergetic 8.7 MeV protons and 23.5 MeV helium-3 ions in the track segment mode. Additional studies that employ accelerated monoenergetic particles will be required to resolve the observed differences in LET dependency for aberrations in human lymphocytes.

2.3.3 RBE Values for Cytogemtic Effects in Mammalian Cells 2.3.3.1 Variation of RBE With Radiation Dose. As demonstrated in the preceding Tables, the yields of chromosome aberrations are predominantly linear functions of dose for high-LET radiations, but vary as a curvilinear function of .dose after exposure to low-LET radiation delivered a t high doses and dose rates. It thus follows that the RBE for chromosome aberration induction in human lympho-

TABLE2.11-Examples Radiation

Accelerated Protone Accelerated Protons Accelerated

Protons Monoenergetic a Particles Helium-3 ions

of dose mpol~~e coef/icients (Y = a D + p 02) for dicentric induction orfter erposurv of lymphocytes ta accelerated protons or dpha particles ( f i m Edwards et al., 1985) E LET Dose range a + S.E. fj r S.E. (MeV) (Irev vn-') (Gy) (x Gy-') (x lo-¶G Y - ~ ) Reference

-

7.4 4.9

7.9

0.5-4 0.16-3.1

5.1 27.6

+ 4.6

0.53

-C

13.6

5

5.1

0.06-3.48

23.0

29.6

0.262.99

+ 0.75 75.5 + 9.6

23.5

22

0.06-2.82

39.4

+

1.8

-

0.07-1.6

37.5

+ 2.4

-

0.11-4.18

28.6

+ 1.5

-

4.4

5.1 6.1

50-500 140

-

Bocian et d. (1973)

Takatsdi et (11.

8.7

238pU

a Particles U2Cm a Particles

+-

2.8

(1983)

Edwarde et al.

5.8

-c

2.1

+ 4.8

0.6

(1986) Takateuji and Sasaki (1984) Edwards et al. (1986) Purrott et al. (1980) Edwards et al. (1980)

K

3 P 3

C)

J

3

a

5

C)

cytes would be highly dependent on radiation dose as has been demonstrated in numerous mammalian cell systems (for example see Brooks, 1975). A recent example of the variation in RBE values as a function of dose for human lymphocytes exposed to 6 MeV D-T neutrons vs T o gamma rays is illustrated in Figure 2.10, and the dose-RBE relationships for the induction of dicentrics by fission neutrons, 14.5 MeV D-T neutrons, and 220 kVp x rays are compared with V o gamma radiation in Figure 2.11. The latter data set of Bauchinger (1984b) are based on the scoring of a total of 80,000 first division lymphocyte metaphases from dose response curves generated in that laboratory. These data clearly demonstrate that the RBE of fast neutrons increases with decreasing dose and suggest that 220 kVp x rays are approximately twice as efficient as 'Wo gamma rays in inducing aberrations a t low radiation doses.

2.3.3.2 RBEMfor Cytogenetic Aberrations in Human Cells After In Vitro Exposures to Radiations of Differing LET. As discussed in Section 1, estimates of the limiting RBE, or RBE at minimum dose (i.e.,RBEM)may be obtained by dividing the coefficients of the linear

Fig. 410 Variation of RBE with radiation dose for dieenEric aberrationsin human' lymphocytes exposed to 6.0 MeV neutrons, reference radiation 60Co gamma radiation (adapted h m Mataubara et al., 1988).

2.3 CYTOGENETIC EFFECTS IN MAMULMN CELIS

I

43

ttss~onnou trons

220 kV X-rays

%o

(0.017 Gy min-1)

Fig. PI1 b R B E relationship for induction of dicentrice by h i o n neutrons. 14.0 MeV neutrons and 220 kVp x rayscompared with W o gamma radiation (adapted fmm Bauchiner. 1984).

term of the high-LET dose response curve by that for the low LET dose response curve. In several reports, coefficients for dicentric induction in human lymphocytes exposed to high energy x or gamma rays and those derived from exposures to various high-LET radiations have been published by the same laboratories, though not necessarily in the same paper. By calculating ratios of these linear coefficients, RBE, values for dicentric induction in human lymphocytes can be derived for several different radiation qualities (Table 2.12). Relative to %o gamma radiation as a reference standard, RBE, values ranging from 34 to 96 have been reported for mixed fission spectrum neutrons having kinetic energies of 0.4 to 2.13 MeV. Vulpis et al., (1978) who evaluated data for dicentric induction in

TABLE 2.12-RBEM wlws for dicentric induction in human lympfiocytes exposed to high-LET versus low-LET mdiution a . x

x

Gamm3 ray

lo-*

ES

RBE,

-.

High LET radiations

x rays

Fission neutrons 25.6 (Ti88ion1') 89.6 (0.4 MeV) 83.5 (0.7 MeV) 72.8 (0.9 MeV) 79.7 (1.3 MeV) 36.9 (1.6 MeV) 60.0 (2.13 MeV)

Gamma -

96.3 53.2 46.4 50.8 34.4 38.2 53*9

Rsfemce

Todorov et al. (1977) Vulpis et al. (1978) Lloyd et al. (1976) Lloyd et al. (1978) Littlefield Bauchinger (1984b) Lloyd (1984b)

D-Be neutrons

4.37 4.76 7.51 4.76 4.04 4.37 3.66

4.20 2.97 1.57 0.91 1.57 1.07 2.97

-

74.5 55.9 41.6 47.8

(2.0 MeV) (6.0 MeV) (6.5 MeV) (7.6 MeV)

D-T neutrons 25.0 (14.0 MeV) 26.2 (14.7 MeV) 17.9 (14.5 MeV) 18.4 (14.0 MeV) 28.9 (15.0 MeV)

-

9.5

13.3 14.0

10.0

30.4

102.3"

19kP

3.3 5.5 4.3 4.2 7.9 5 2.80

27.5 16.6 16.7 6.2

-

17? 4'

~ a t a u b a r ael al. (1988) Fabry et al. (1985) Lloyd

Sasaki (1971) Lloyd et al. (1984b) Bauchinger (1984b) Fabry et 01. (1985) Muramatau et al. (1973)

9

TABLE 2.12-Continued a x

X ram

Gamma rays

High LET radiations

Beta PH) 5.4 (5.7 keV) a particles 37.5 (5.16 MeV) 28.6 (6.1 MeV)

X rays

(4.76)C 4.76

(1.57)' 1.57

Protonsb 27.1 (4.9 MeV) 4.4 (8.7 MeV)

(1.57)C 1.57

Other radiations 75.5 (23.0 MeVd 39.4 (23.5 MeV)*

'(unweighted mean). bMonoenergeticprotons. Coefficients of Edwards, (1986) employed in calculations. d23MeValpha particles O23.5 MeV =Heions

0

Gamma

Reference

Prosser et al. (1983) 7.9

23.9

6.0 -

18.2 -

5.7 -1 3k2"

2.7

7+la

(4.76)' 4.76

F'

RBEH

15.8

8.27

12 + 4"

Purrott et al. (1980) Edwarda et al. (1980)

2123' 17.3

Takatsuji et al. (1983)' Edwards et al. (1986)

10k 7. 48.1 25.1 37?11a

Takatauji and Sasaki (1984) Edwards et al. (1986)

d cl

46

1

2. CYTOGENETIC EFFECTS

lymphocytes exposed to highly efficient 0.4 MeV neutrons. These data produced the highest estimate of RBE for fission neutrons relative to gamma rays. However, it should be noted that the reported linear coefficient for 60Co gamma radiation is lower than that reported by several other laboratories, which accounts for a higher estimate of RBEM.Dose response data from the other five laboratories produce estimates of RBE, that range from 34 to 53 for fission neutrons versus W o gamma rays. When hard x rays (180to 250 kVp) are used as the reference radiation, much lower RBEMvalues are obtained, ranging from 9 to 18.6 in six of seven reported studies. Todorov et al., (1977)observed a particularly low value for the linear coefficient for the fission spectrum neutrons, and thus these data produce a very low estimate of RBEM. A single estimate of RBE is available for 2.3 MeV neutrons produced by bombarding beryllium with deuterium. Based on coefficients obtained by Sasaki (1971), an RBEMfor neutrons of 82 and about 10 were calculated for gamma and hard x rays, respectively. The wide differences in estimates of RBEMfor the two reference radiations result from the fact that Sasaki's estimates of the a terms foroC@ ' gamma rays and 200 kVp x rays differ by a factor of greater than seven fold, which is a more pronounced difference than has been observed by several other laboratories. Whether gamma rays or x rays are used as the reference radiation, RBEMfor 6.0 MeV neutrons (D-Be) and -14 MeV neutrons {D-T) tend to be lower than the RBEMreported for mixed fission spectrum neutrons, in line with their lower dose average LETS. The RBEMfor alpha particles produced by radioactive decay are in the range of 7 and 20 compared to x rays and gamma rays respectively. For accelerated monoenergetic protons, RBEMis dependent on LET, but estimates obtained by two laboratories differ by factors of about two to six. For all high-LET radiations, comparisons of unweighted means for the RBEMdata demonstrate an approximately three-fold higher estimate when 60Cogamma radiation is used as the reference radiation rather than x rays.

-

2.3.3.3 RBE Values for Chromosome Aberrations in Human Cells After In VivoExposure to High-LETRadiations. There are numerous reports in the literature of cytogenetic findings in cultured lymphocytes from persons having occupational, medical or accidental exposures to various qualities of penetrating low-LET radiations. These have verified that the frequencies of chromosome aberrations induced by in vivo exposures are qualitatively and quantitatively similar to those observed after in vitro exposures. Notable examples are evaluations of radiation-induced chromosome aberrations in

2.3 CYTOGENETIC EFFECTS IN MAMMALIAN CELLS

1

47

lymphocytes in nuclear dockyard workers (Evans, et al., 1979)and in workers at three separate nuclear establishments in the U.K. (Lloyd, et al., 1980). These persons had occupational exposures to primarily external gamma radiation, and accumulated doses of about 15 to 50 mSv per year, over a range of several years. When adjustments were made for lymphocyte lifespan, curvefittings demonstrated that exchange induction was adequately described by linear dose response relationships with slope coefficients of 2.3 and 2.2 dicentrics cell-' Gy - l for the two study cohorts. The findings in these two groups of workers are in excellent agreement with data from in vitro dose response studies (Table 2.7). Although many studies have been conducted, relatively less information is available on the dose dependency for aberration induction in lymphocytes from persons having recent exposures to various qualities of high-LET radiation. Extensive cytogenetic aberrations have been reported in cultured lymphocytes from persons accidentally exposed to mixed neutron and gamma radiations in criticality accidents (Bender and Gooch, 1962;Bender and Gooch, 1966;Jammet, et al., 1980). However, because of the uncertainty regarding dose, determinations of the relative contributions of the neutrons to the observed cytogenetic effect were not possible in these studies. Schmid, et al., (1980)reported that radiation-induced aberrations in lymphocytes showed a positive correlation with skin dose in 17 patients who received fractionated exposures to 14 MeV (D-T) neutrons as therapy for various malignant tumors, but poor fits were obtained when the data were fitted to various dose response hnctions. Of course, it is quite difficult to accurately estimate radiation dose to lymphocytes in persons having partial-body exposures to external radiations. In such situations, only those lymphocytes that were in transit through the various radiation fields would actually receive a radiation dose, and the relative proportion exposed would be quite variable between patients having tumors located a t different regions of the body. Because of the complexities in accurately determining the dose dependency for aberration induction, direct comparisons of such data with that derived from persons having homogenous whole-body exposures will not yield accurate information on the relative efficiency of neutrons in inducing aberrations in vivo. Similar problems involving non-homogeneous exposure of lymphocytes are encountered in cytogenetic evaluations in persons having measurable body burdens of internally deposited alpha particle emitting radionuclides. A review of published findings and a detailed discussion of the unique problems inherent in interpreting cytogenetic data from these patients has recently been published (Bender et al., 1988).Because of the localized deposition of isotopes in specific

organs of the body, the short track lengths of a particles in tissue, 1988, Littlefield and and other confounding variables (Bender et d., DuFrain, 1984, lymphocyte populations in internally contaminated persons are apt to receive highly non-uniform exposures to highLET tracks. Thus, although increased frequencies of cytogenetic aberrations have been observed in lymphocytes of persons having accidental internal contaminations with various radionuclides, and in worker populations who received occupational exposures that resulted in substantial body burdens, it is virtually impossible to establish a strict dose dependency for aberrations induced in vivo in these human populations. While it has been possible to obtain quantitative estimates of the influence of LET on the production of chromosome aberrations in experimental animal models (for example, see Figure 2.9), data from cytogenetic evaluations in lymphocytes of persons having internal contamination with particle-emitting radionuclides are not likely to lead to reliable estimates of RBE for human cells in vivo. 2.4 Summary

A number of generalized conclusions regarding RBE can be drawn from published data on various genetic or cytogenetic endpoints measured in plant, animal, or human cells exposed to radiations of differing LET. (1) For all measured endpoints, the dose response relationship is best described by the linear quadratic model when cells receive acute exposures to high dose rate, low-LET radiation. (2) Reduction in dose rate results in a diminution of cytogenetic effects &r exposure to a given dose of low-LET radiation. At very low levels of dose and a t low dose rates, chromosome exchange induction varies as a linear function of dose. (3) For all high-LET radiations, cytogenetic exchange induction varies predominantly as a linear function of dose, and is doserate independent. (4) Because of the differences in the shape of dose-responsecurves for high- and low-LET radiations, RBE values vary inversely as a function of dose. ( 5 ) For the majority of reported studies, RBE varies as a function of LET of the radiation used and peaks at about 0.4 MeV. (6) Low LET radiation: x rays and gamma rays differ in response, x rays being 2 to 3 times as effective as gamma rays.

3. Transformation and Mutation in Mammalian Cells In Vitro 3.1 Introduction

The effect of radiation quality on the incidence of mutations or transformations in mammalian cells in vitro has been studied with neutrons and charged particles of various energies. In general, highLET radiations are more efficient than low-LET radiations in inducing oncogenic transformation and mutation on a per dose or per level of survival basis. The data causing the most controversy involves dose fractionation or low dose-rate.

3.2 Radiation Induced Oncogenic Transformation Assayed

In Vitro 3.2.1 Basic Techniques

A limited number of experiments have been performed with neutronsand charged particles, using three in vim transformation assay systems. The f i s t involves short term explants derived from midterm embryos of Syrian hamsters. In this system, cells are seeded at relatively low density and allowed to grow for two weeks, by which time they form colonies which can be seen readily with the naked eye. Colonies of normal cells stain lightly, and exhibit contact inhibition. Transformed colonies, produced by chemical carcinogens or radiation, show a different characteristic pattern: the cells stain heavily, pile up and form a criss-cross pattem a t the periphery of the colony. The proportion of cells transformed can be scored as a function of dose; for a dose of 10 mGy of x rays, the transformation incidence is The system of the order of The spontaneous level is about has the advantage that it involves diploid cells, but the disadvantage that, since the culture is established from a minced embryo, it contains a mixture of cell types.

50

I

3. TRANSFORMATION AND MUTATION

The second in vitro transformation system used widely is the C,WlOT 112cell line originally isolated and characterized in the late Charles Heidelberger's laboratory. This established cell line was derived from fibroblasts taken from the prostate of a C3H mouse embryo. In this system, cells are seeded and irradiated a t low density and allowed to grow until the normal cells form a confluent sheet in the culture vessel, a t which point they exhibit contact inhibition and stop dividing. This takes about two weeks. The cultures are then kept for a further 4 weeks with regular refeedings. During this time transformed foci show up as darkly stained areas, with a characteristic swirling pattern, against a background of lightly stained contactinhibited normal cells. Again, the proportion of transformed foci can be scored as a function of dose. For cell passages between 8 and 15, the spontaneous transformation frequency is around but increases for later passages. A third quantitative system for scoring oncogenic transformation is the BALBI3T3 cell system, also originating from a mouse embryo. These cells show contact inhibition of growth and evidence of toxicity from known carcinogens in the form of morphological alterations. In this system, about lo4 cells are seeded per dish prior to treatment. Transformed colonies show up against a background of normal cells, and can be scored in as little as three weeks post-seeding. 3.2.2 Data for Neutrons: Fresh Explunts of Cells from Hamster

Embros The first reported transformation study with high-LET radiation was that of Borek et al. (1978). Cells derived from fresh explants of Syrian hamster embryos were irradiated with relatively large doses of 430 keV monoenergetic neutrons, as well as 250 kVp x rays. The dose range was extended downward in a later study (Hall et al., 1982). Transformations could be detected a t doses as low as 3 mGy of x rays and 1mGy of neutrons. The data are shown in Figure 3.1. The lines fitted to the data are smooth curves that represent no particular mathematical form for the dose-response relationship, except that the data are monobnically increasing. Up to 1Gy, the neutron data might be represented adequately by a straight line with a slope of unity on this double logarithmic plot, indicating a direct proportionality between transformation incidence and dose. For x rays, likewise, transformation incidence appears to be proportional to dose between 10 mGy and 1 Gy, although the possibility of h e structure in the relationship cannot be ruled out. For both x rays and neutrons, a maximum in transformation frequency is reached

3.2 INDUCED ONCOGENIC TRANSFO-TION

1

51

lo-' d d W

u z

->

Z 10"

3 V) \

V)

I-

5

z a

0

tiz a a

95 % confidence limits

I-

10-~

lo-*

lo-'

lo0

10'

DOSE (Gy) Fig. 3.1 Pooled data,for the hamster embryo cells, of the number of transformanta per surviving cell following irradiation with 250 kVp x-rays (full symbols) or 430 keV monoenergetic neutrons (open symbols) produced at the Radiological Research Accelerator Facility. The e m r bars indicate 95 percent confidence intervals for the estimated value. The e w e s ahould be regarded only as a m t h repre~ntationof the shape of the data with a minimum of parametric-related bias.(From Hall et al., 1982).

at higher doses. Neutrons are clearly more effective than x rays in both cell killing and the induction of transformations. When the RBE of neutrons as compared to x rays was calculated as a function of dose for both oncogenic transformation and cell sunrival, it indicated clearly that the RBE value changed with dose for both endpoints examined (Figure 3.2).

52

/

3. TRANSFORMATION AND MUTATION

-

1

7

HAMSTER EMBRYO CELLS' 4 3 0 keV neutrons vs. 7 250 kVp x rays

lo2,

.

W

m

E-

_

d

-

= 10' --

O 0

--

loo-.

1

*...**.

1

10-~

..II... lo-'

-

Q'o

....#.*I

lo0

.*.**,.I

10'

NEUTRON DOSE (Gy) Fig.3.2 The RBE of neutrons versua x rays for hamster embryo cells plotted as a function of neutmn dose. The vertical bare correspond to oncogenic transformation as an endpoint, and indicate RBE values excluded with 80 percent confidence. RBE values for cell survival (open circles) are alao ehown. (FromHall et al., 1982).

It should be noted a t this point that the same authors, using the same transformation assay system, showed an RBE of two for x rays versus gamma rays at doses of a few tens of mGy (Borek, et al. 1983). Therefore, in considering RBE values for higher-LET radiations at low doses, it is necessary to take into account whether the reference radiation is x or gamma radiation. 3.2.3 Data for C$UIOTII, Cells with High Energy and Fission Neutrons Using C3H/10T1/, cells, Han and Elkind (1979) investigated the effect of single and fractionated doses of both x rays and a hardened beam of fission spectrum neutrons (average energy & = 0.85 MeV, <4 percent y ray contamination) generated by the JANUS reactor. The proportion of tramformants per surviving cell was found to increase as an exponential function of dose for both 50 kVp x rays and JANUS neutrons, with the neutron curve being the steeper trans(Figure 3.3). Neutrons produce a maximum of about 6 x formants per surviving cell, reaching this level at doses in excess of 3 Gy. The maximum x-ray m or mat ion rate was about 3.5 x reached a t doses of about 6 Gy. The RBE value for transformation ia about 10 for a neutron dose of 0.2 to 0.5 Gy and falls to 2.6 a t 1.5 Gy.

1

3.2 INDUCED ONCOGENIC TRANSM)RMATION I

I

I

1

I

I

53

--

--

-

1

A

(

-

C

0

"

--~ /HI O T ~ 5OkVpX-RAYS JANUS NEUTRONS ---

-

l"'o

I

2

I

4

I

6

I

8

I

10

I

12

14

Fig. 3.3 Induction of transformation in C,H/1OT'I2cells by x rays or fission-spectnun neutrons. The points plotted are the mean of three to five replicate experiments, and the vertical bars represent the standard error (Adapted from Han and Elkind, 1979.)

These RBE values are similar to those found for cell killing. In the region of the plateau, RBE values cannot be estimated since the requirement of "equivalent dose response" is not satisfied when transformation is expressed in terms of transformants per surviving cell. Han and Elkind (1979) found little or no change in transformation frequency following neutron dose fractionation (two fractions over various times of up to 24 hrs. post-irradiation), compared with a significant reduction in transformation frequency per survivor

54

1

3. TRANSFORMATION AND MUTATION

following x-ray dose fractionation. It should be noted that these conclusions relate to relatively high dose levels, 1Gy or more for 50 kVp x rays and equivalent levels for neutrons. In a later study, Han and Elkind (1982) reported an enhancing effect of the tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on both x ray and fission-spectrum neutron induced transformation in C3H/1OT1I2cells. This enhancement in transformation frequencies by TPA at a concentration of 0.1 pg ml-' was found to be greater following neutron exposure than after x-ray treatment, particularly a t low dose levels. However, this synergistic enhancement became smaller as the dose increased, reaching a constant fador in the plateau region. Furthermore, TPA treatment caused the RBE value to increase rather than decrease with increasing dosages, indicating that the effect of TPA might not simply be promoting. Studies on the effects of dose rate of both fission spectrum neutrons and gamma rays over a wide range of doses on the oncogenictransformation of C3H/1OT1I2cells are of particular interest (Elkind et al., 1984). By exposing cells to either high or low dose rate JANUS neutrons, 0.38 Gy min-I versus 0.86 mGy min-I and of gamma rays from Corn, 1Gy min-I versus 1mGy min-I they found that, while lowering the dose rate reduced transformation frequency for gamma rays, for neutrons it increased the number of transformants per surviving cell. Figure 3.4 shows the family of transformation dose response curves over a wide dose range for both types of radiation. The overall consequence is that the RBE value for neutrons relative to gamma rays is much larger for protracted as compared to acute exposure. An RBE value of about 48 is obtained from the ratio of the initial slopes i.e., the slopes of the lines from the spontaneous incidence to the incidence a t the lowest dose used. The maximum RBE for low dose rate neutrons compared with fractionated gamma rays (data not shown) is about 70. Figure 3.5 shows comparable data for gamma rays and neutrons where dose protraction is accomplished by delivering the dose in multiple fractions instead of continuously at low dose-rate. Again dose protraction leads to a sparing of biological effect for gamma rays and an enhancement for neutrons, leading to an increased neutron RBE for protraded exposures. Some investigators have not found a similar enhancement of transformation incidence by protraction of dose of high LET radiation. Hieber et al. (1987) found no difference in transformation incidence when C3H/1OT1I2cells were irradiated with a-particles (LET about 150 keV p.m-') at high and low dose-rates, defined to be 0.2 Gy min-'

3.2 INDUCED ONCOGENIC TRANSFORMATION

/

55

Fig. 3.4 The dose-rate dependencies of the tramformation of CSH/1OT1I2cells exposed to 60Cogamma rays or to JANUS fission spectrum neutrons. The RBE values were determined from the ratio of gamma ray to neutron doses at each frequency level examined. (Adapted h m Elkind et d.,1984.)

and 0.8 to 2.5 mGy min-I respectively; the lowest dose used was 0.125 Gy. In a systematic series of experiments, Miller et aZ. 1988, looked at the effects of fractionation (five fractions over eight hours) on transformation incidence in C,H/lOT1/, cells induced by monoenergetic neutrons from 0.23 to 14MeV. The enhancement of transformation incidence resulting from fractionation was significant at the 95 percent confidence level for only one of the five neutron energies examined, and even then the effect was less than a factor of two, which is considerably smaller than that reported by Han et al., (1979).

DOSE (Gy)

Fig. 35 Transformation data for C,H/1OT1I2 cells exposed to gamma rays or Janua fission Bpectnun neutrons in single or multiple doaee. The inset shows the low dose region on an expanded ecale. (Adapted fmm Hill et al., 1982.)

It would appear therefore, that, if protraction of high LET dose over a period of time does enhance oncogenic transformation, the effect is strongly dose and LET dependent. Hall et al. (1982) examined the effects of high energy neutrons (produced by 35 MeV deuterons on beryllium) on oncogenic transformation of C,H/lOT1/, cells and compared the results obtained with those generated by exposure to 300 kVp x rays. Cells exposed to neutron doses ranging from 0.5 Gy to 3 Gy demonstrated a linear increase in transformation frequencies (Figure 3.6). At neutron doses between 0.02 Gy and 0.50 Gy a plateau was seen in the dose-response curve indicating a lack of proportionality a t such low dosages where not more than one charged particle traversal per nucleus was expected. Cells exposed to x rays, however, demonstrated a complex dose-response relationship. At doses above 1Gy, the curve demonstrated a quadratic transformation dependence on dose. Below 0.3 Gy the data were consistent with a slope of one implying direct proportionality to dose. No correlation between dose and response, however, could be seen in doses between 0.3 Gy and 1Gy. When the RBE values for cell killing and oncogenic transformation per neutron dose were plotted (Figure 3.7), it could be seen that a t any given

3.2 INDUCED ONCOGENIC TRANSFORMATION

/

57

5 35 MeV d ++Be

-

9 5 % confidence limits

-

DOSE (Gy) Fig. 3.6 Dose-response curve of C,H/lOT1/,cells exposed to neutrons produced by 35 MeV d+ + Be (open circles) and 300 kVp x rays (closed circles). The error bars indicate 95 percent confidence intervals. (Adapted from Hall et al., 1982.)

neutron dose, the RBE values for the two endpoints were similar. For transformation, the RBE increased with decreasing dose while the RBE value for survival remained fairly constant. Studies by Miller and Hall (1980), using a similar system, showed that the RBE value at high neutron doses was around 2.9 at 1.5 x transformants per surviving cell. Furthermore, when cells were exposed to neutron doses given either singly or in two equal fractions, they found no statistically significant difference in transformation frequencies between the two treatment protocols. Miller et al., (1989) investigated oncogenic transformation in CsH/ 10T1/, cells as a function of neutron energy covering the range from 0.23 to 14 MeV. The RBE values obtained were similar for cell lethality and for oncogenic transformation as can be seen from Figure 3.8. The data are consistent with the neutron energy dependent quality factor proposed by ICRU Report 40 (ICRU, 1986). The data for transformation show the same trend as earlier data for plant seedlings and cell lethality in established lines of cells in culture.

'O2: l d i ~ X o i i"'"' I

-- 35 MeV d + + neutrons

lo',

1 I

1111

--

Be

-----

VS.

--

--

300 kVp x - r a y s

0

loo

'

10-~ 10lo0 NEUTRON DOSE (Gy)

10'

Fig. 3.7 RBE analysis of neutrons versus x rays for C,H/1OT1l2celb plotted as a function of neutron dose. The vertical bars correspond to oncogenic transformation as an endpoint and indicate RBE values excluded with 80 percent confidence. RBE values for cell survival (open circles) were shown including twostandard deviations. (Adapted from Hall et al., 1982.)

3.2.4 RBE Data for Incorporated Radionuclides and Alpha Particles in 3T3 Cells.

Using the mouse BALBl3T3 cell system, LeMotte et al. (1982) compared the induction of lethality and transformation by 5-'%Iiododeoxyuridine and 3H-thymidine incorporation into cellular DNA. Cells were released from density inhibition by replating; incubated for time ranges from 8 to 24 hours with either 3H-thymidine which emits one low energy f3 particle, or 1261which releases simultaneous multiple low-energy Auger ele~trons.~ Over the whole range of doses used, the ability of the radionuclides to induce transformation correlated somewhat with their cytotoxic ability. Iodine-125 was found to be 12 to 16 times more cytotoxic than 3H-thymidine and was 25 times more efficient in inducing transformation. However, at or approaching zero cell killing doses, the transformation-survival curves demonstrated significant transformation above the normal T h e simultaneous emission of several shortrange Auger electrons represents a very high concentration of ionization a t the point of emission.

3.2 INDUCED ONCOGENIC TRANSFORMATION

1

59

a A Survival

a Transformation

Neutron Energy (he!) Fig. 3.8 RBE values for neutrons relative to gamma rays for various monoenergetic neutrons from 0.22 to 14 MeV. The biological endpoints scored were cell survival and oncogenic transformation. The RBE values used were calculated from the initial slopes of the neutron and gamma ray dose reeponse curves, i.e., RBE, values (From Miller et al., 1989.)

spontaneous value. This was particularly obvious in the case of 1261 and suggested that electron-emitters that were localized in the genome may have malignant potential far greater than expected from their lethal properties. In a subsequent study, Robertson et al. (1983) examined the effect of alpha particles and compared their effecta on survival and transformation to that of 220 kVp x rays. The RBE of alpha particles was found to be dose dependent; ranging from 6.2 at 80 percent survival to 2.5 at 5 percent survival. The Dowas 0.7 Gy for alpha particles as compared to 1.7 Gy for x rays. Transformation frequency was shown to increase exponentially with dose in the range examined, (0.25 to 2.5 Gy)without evidence for a plateau, as was found with x rays. When irradiated cultures were held in plateau phase from 12 to 220 hours to allow time for potential lethal damage repair (PLDR), cells treated with x rays showed a proportional decrease in transformation frequency as recovery time was increased. No such

60

1

3. TRANSFORMA'ITON AND MUTATION

decrease, however, was obsemed with alpha radiation (Figure 3.9). This dramatic difference in PLDR between x rays and alpha particles greatly complicates the concept of RBE, since, for a given cell line and given radiation qualities, the value of the RBE may fluctuate a X RAYS

o aPAClTlCLES

Fig. 3.9 Effects of potentially lethal damage repair (PLDR) on transformation fkquencies of 3T3 cells. Open circles represent observed transformation frequenciee for cells exposed to 2 Gy of alpha radiation and closed circles 4 Gy of x rays. The cells were subsequently allowed to repair for the times indicated. Data are pooled h m three experiments. Hatches representspontaneoustransformationFrequencies. (Fhm Robertson et al., 1983.)

3.2 INDUCED ONCOGENIC TRANSFORMATION

1

61

widely depending upon the conditions for repair of potential lethal damage. Lloyd et al. (1979a; 1979b)showed that mammalian cells in culture (C,HIIOTII,) can be transformed by 5.6 MeV alpha particles generated in a Tandem van de Graaff. Transformation frequency was found to increase rapidly with dose-approximately with the cube of the dose-reaching amaximum of about four percent of the surviving ~ , corresponds cells at a particle fluence of 1.5 to 2.5 x lo7~ m -which to an absorbed dose of 2 to 3.4 Gy. Although the absolute values for the transformation frequency varied with the experimental conditions, the general shape of the dose-response curve was the same. No transformations above background were observed for alpha particle - ~ Gy), which corresponds to fluences less than 6 x lo6 ~ r n (0.83 about 19 alpha particles on average passing through each flattened nucleus, which is somewhat greater than the average value of 14 representing the mean lethal dose. This lack of an observed effect, however, undoubtedly reflects the fact that the number of observations was small. No concomitant data were taken for x or gamma rays, so that estimates of either the RBE value or the variation of RBE with dose are not possible. Estimation of RBE between high- and low-&T radiations for both transformation and cytotoxicity were recently reported by Hei et al., (1988a). In their studies, neoplastic transformation incidence and cytotoxicity were scored in C3H/1OT1I2cells following irradiation with charged particles of defined LET. Protons, deuterons and 3H ions were accelerated with a 4 MV Van de GraafF accelerator with defined LET of 10 to 120 keV pm-I. Corresponding data obtained from 250 kVp x rays were used to calculate RBE values. For x rays, and for the charged particles of lower LET (10and 40 keV pm-I), the dose response curves for cell survival had a marked initial shoulder. With increasing LET, there was a progressive decrease in the size of the shoulder and for the highest LET studied (120 keV pm-I), survival curves approximated an exponential function of dose. The RBE values for cell survival based on the initial slopes for the four charged particles studied in increasing order of energies were 1.72, 2.28,6.30 and 7.22 respectively. The neoplastic transformation incidence, likewise, showed a direct correlation to LET over the dose range examined (Figure 3.10). The calculated RBE values for transformation at the level of 0.02 percent transformants per survivingcell for the various charged particles studied, in ascending order of LET were 1.09, 3.48, 8.89 and 17.4 respectively. Transformation data were analyzed using a linear-quadratic function of dose for the transformation probability. This latter quantity, when analyzed

I

I

C3H lOTll2 CELLS

1

I

1

1

I

i

1

0

1

2

Dose3 (Gv)

4

5

6

Fig. 3.10 Tramformanta per surviving cell produced by graded dosea of x rays or c h a r d particlea of defined LET. Error bars reureaent 95 mrcent confidenceintervals. (~daptedfrom Hei, et al., 1988a.)

using microdosimetric consideration, suggeets that the target volume for this end-point is of the order of micrometers cubed.

3.3 Radiation-Enhanced Viral ~ r d o m a t i o n

3.3.1 Basic Technique Yang et al. (1980) investigated the effectiveness of high-energy neon ions, accelerated in the BEVALAC, to enhance the viral transformation of mouse C,WIOT1/, cells. In this system, cells are grown into a confluent layer, uniformly arrested in the G,phase of the cell cycle, before being infected with Simian Virus (SV40).Cells are then trypsinized and the cell suspension irradiated with x rays or heavy ions, before being plated out to form colonies, some of which later can be identified morphologically as transformed.

3.3 RADIATION-ENHANCED VIRAL TRANSFORMATION

1

63

3.3.2 Data For x rays, the number of transformants per cell increased with dose to a maximum at 3 Gy, followed by a sharp decline at higher ~ doses. Neon particles, with a residual range of 12.7 g ~ m in-water, were more effective than x rays with the number of transformants reaching a peak at about 0.2 Gy before falling off. The RBE value for enhancing the frequency of transformation per sunriving cell appeared to vary with dose, being higher at lower doses. The RBE value for enhancement of viral transformation was about 1.84 for 6 Gy of x rays and 2.28 for 3 Gy of x rays. The lowest dose of x rays used was 1 Gy. At this level the RBE cannot be determined with much confidence, but it does not appear to be greater than two. In these same experiments, cell killing was scored for cells infected with virus and irradiated with x rays or neon ions. RBE values are similar to those observed with transformation as an endpoint, reaching a value of about two at an x ray dose of 1 Gy. This system shows clearly that ionizing radiation can act as an efficient co-carcinogen at the cellular level. In addition, the experiments are performed with cells arrested at one phase of the cycle. Unfortunately, the experiments were on too small a scale to allow any extrapolation to be made to lower doses, and even over the dose range used, the transformation data are so fragmentary and are characterized by such large standard errors that the shapes of the dose-response relationships are largely a matter of conjecture. In a subsequent report, Yang and Tobias (1984)found similar RBE values for heavy ions as a function of dose using the BALBl3T3 cell system. The number of transformanta per surviving cell increased with dose and this increment was curvilinear for both argon particles and x rays. At a dose of 3 Gy of x rays, the RBE values for enhancement of SV-40induced oncogenic transformation for argon particles was found to be about 1.6 for a residual range of 0.4 cm and 2.1 for residual range of 1.4 to 6 cm. At a dose of 1 Gy of x rays, the RBE value was three for all argon particles. In general, high LET radiation is more effective than x rays in enhancing viral transformation. As shown in Figure 3.11, the RBE value calculated for a single heavy-ion particle as a function of LET in enhancing SV-40 viral transformation increases rapidly with LET values up to 350 keV pm-'. Since high LET heavy-ion particles are also more effective in inducing double-strand DNA breaks than conventional x rays, these data indicate that DNA breakage may be an important lesion involved in the carcinogenic interaction between ionizing radiations and oncogenic viruses.

1

64

3. TRANSFORMATION AND MUTATION

LET lKtV/pml

Fig. 3.11 The relative biological effectiveness (RBE) for several heavy particles as a function of LET in enhancing SV-40 induced onwgenic transformation of BALBlc 3T3 cells. (Adapted from Yang and Tobias, 1984.)

3.4 Mutation Studies with mammalian cells in culture

3.4.1 Basic Technique

There is no information on the mutagenic effectiveness of various ionizing radiations for man and only limited information about radiations other than x and gamma rays for experimental animals. The most complete studies involving mutagenesis as a function of LET involve systems based on cells in culture. 3.4.2

The Mechanism of the Hypnthine-Guanine Phosphoribosyltmnsfemse (HGPRT) System

The induction of thioguanine (TG)resistant mutants in cultured mammalian cells has been used to quantify the mutagenic effects of various physical and chemical agents, and it has been argued that such mutations arise primarily as a result of true gene mutations.

However, because ionizing radiations induce TGresistant mutants with very low HGPRT activity, while failing to induce the ouabainresistant phenotype in mammalian cells, it has been suggested that radiation leads to gross genetic damage rather than to point mutations in structural genes. Cox and Masson (1979) produced convincing evidence in cultured human fibroblasts that gross structural changes involving the x chromosome are the genetic basis of a significant proportion of radiation-induced mutations to TG-resistance. 3.4.3

Radiation-Induced Mutation Studies

In a series of papers, the Harwell group (Cox et al., 1977b; Thackeret al., 1979; Cox et al., 1980) described the irradiation of the HF19 strain of freshly isolated human diploid lung fibroblasts, as well as established Chinese hamster V79 cells with helium, boron or nitrogen ions covering a wide range of LET values from 20 to 470 keV pn-'. The loss of reproductive integrity was scored by colony formation in the usual way, and mutation frequency was calculated as the number of mutants per viable cell.

Chinese Hamster V79 Cells. Using V79 Chinese hamster cells, and scoring cell inactivation, a considerable difference was observed in the effectiveness of helium and boron ions of similar LET, though, of course, the boron ions had a greater velocity. For cell killing, boron ions of 200 keV pm-l were about as effective as 90 keV pm-' helium ions but considerably more effective than 470 keV pm-I nitrogen ions. The mutation induction data, though less precise, showed a similar trend to survival response; radiations with LET in the range 90 to 200 keV pm-' were all of very similar effectiveness (see Figure 3.12). Unique RBE values cannot be given because of differences in the shape of the dose response curves a t different LET for both cell inactivation and mutation induction. In the case of mutation induction, the data points for the different LET radiations were fitted by the above mentioned linear quadratic, equation 2.1. The ratio of the alpha terms of the equations for the high and low LET curves give the "initial slope RBE" and is a measure of the difference in effectiveness of the radiations a t low doses. Other RBE estimates include the RBE calculated a t a given level of responsesuch as 10 percent survival and the comparable mutation frequency. While it must be emphasized that some of these RBE estimates are very inaccurate, the maximum RBE for mutation induction is two or more times that for inactivation, whichever method of RBE estimation is used. 3.4.3.1.

1

66

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3. TRANSFORMATION AND MUTATION I

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LET (keV pm-I ) Fig. 3.12 RBE-LETrelationships for Chinese hamster V79 cells. Open symbols represent mutation induction; closed symbols represent inactivation: 0, *Co ;rn 14Nions. (FromCox et al., 197%) gamma rays; 0, He ions; A, A '9o,

+

Human Fibroblasts. Human diploid fibroblasts often, though not always, exhibit unshouldered survival curves for x rays as well as for more densely ionizing radiations, allowing the calculation of unique values of the RBE. In the report of Cox et al. 197713, with cell killing as the endpoint, dose-response relationships of 3.4.3.2

3.4 MUTATION STUDIES

1

67

human fibroblasts were indistinguishable from exponential functions of dose, i.e. unshouldered, for all radiations studied. Helium ions showed increasing effectiveness of cell killing with LET from 20 to 90 keV p - l . Boron ions showed decreased effectiveness with increasing LET from 110 to 200 keV pn-l. Nitrogen ions of 470 keV pm-' were less effective than boron ions of 20 keV pn-l. The RBE values were not dose dependent. In contradistinction to the V79 data, which have separate RBE maximum values for helium and for ions of greater atomic number, human fibroblasts appear to give a single RBE maximum between LET of 90 to 110 keV pm-l. Dose-response relationships for mutation of human fibroblasts were approximately linear for all radiations. Helium ions showed increasing mutagenic effectiveness with increasing LET from 20 to 90 keV pm-l. Boron ions of increasing U T showed similar mutagenic effectiveness,while nitrogen ions of 470 keV pm-' were considerably less effective than boron ions. The RBE-LETrelationships for mutation induction have a humped form, similar to that observed for inactivation (see Figure 3.13).

Human Fibroblasts oo mutation lethality

0

10

I

50 100 200 LET (keV urn-')

i 500

Fig. 3.13 RBE values versus LET relationship for early passaged human fibroblasts irradiated with various ionizing radiations including protons, deuterons, helium-3ions, helium4 ions, boron-10 ions and nitrogen-14 ions at specified LET values. Closed symbols indicate data for a11 lethality and open symbols represent mutation data a t the HGPRT locus. Reaults presented are from Cox et al., 1977b, 0, (circles) and Hei et al.,1988b, 0, (squares). Law LET reference radiations include x rays (closed circles and squares) and gamma rays (open circles and squares).

.

68

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3. TRANSFORMATION AND MUTATION

However, while RBE values for mutation and inactivation differed only slightly for radiations with LET in the range of 20 to 50 keV pm-', at higher LET, RBE values for mutation were about twice those of the corresponding RBE for inactivation. The RBE maximum for mutation of human fibroblasts was in the range 90 to 200 keV pm-l; similar to that obtained for the same mutation in Chinese hamster V79 cells. The most important feature of the inactivation data is that early passage human diploid fibroblasts, which give unshouldered survival curves for low-LET radiations, show humped RBE versus LET relationships similar to those obtained for the inactivation of established lines of mammalian cells. More recent data for early passaged human fibroblasts comes from Hei et al. 1988b. Cells were irradiated with protons, deuterons or Helium-3 ions of defined LET in the track segment mode. The RBE continues to increase up to 150 keV p,m-l, and for the highest LET, the RBE for mutation is almost double that for cell lethality. These data are also shown in Figure 3.13.

3.4.4 Conclusions From the Mammalian Cell Mutation Data

The variation of RBE with LET for cellular inactivation and mutation induction are similar in both human and rodent cell types, with maxima in the LET range of 85 to 200 keV pm-l. In this LET range, the RBE for mutation induction was about twice that for inactivation. No current genetic technique for somatic cells can distinguish small deletions from point mutations. In man, however, the HGPRT gene is known to be located on the long arm of the x chromosome and gross genetic changes associated with loss of HGPRT may be detectable in mutants of cultured cells by chromosome banding techniques. Cox et al. (1977b) showed that visible aberrations occur in about 40 percent of HGPRT deficient mutants induced by nitrogen ions of high LET, while in contrast, x-chromosome aberrations have not been detected in either spontaneous or x-ray induced TG resistant mutants. A simple relationship exists between inactivation and mutation induction for low-LET radiations as proposed by Thacker and Cox (1975). The additional mechanism operative for more densely ionizing radiations could explain the higher maximum RBE for mutation induction relative to inactivation. These data suggest that with high LET radiation either an additional mutagenic mechanism may be involved or that more extensive chromosomal damages may be inflicted in the viable mutants.

The cultured human cells show a maximum RBE for mutation induction somewhat less than the current maximum Q value of 20. However, it should be stressed that the RBE values determined for mutation to HGPRT deficiency in cultured cells are probably averages for more than one type of genetic change. It is clear, therefore, that there is urgent need to study other mutations, comprising different combinations of genetic changes, when suitable experimental systems are developed. 3.4.5

The Pros and Cons of the HGPRT System and New Developments

The HGPRT system has been widely used to investigate and compare the mutagenic properties of a wide range of physical and chemical agents. Its chief advantage is that it works in relatively simple experiments that are not too cumbersome or expensive to perform. Its proponents also highlight that the system utilizes marnrnalianeven human cells. It suffers from the defect that i t has in common with essentially all screening systems, namely that the chromosomes in which the mutation is produced are also required for cell replication. There always remains the concern that, in the case of a mutagen that is also a powerful cytotoxic agent, potential mutants are lost before they can be expressed, because of cell killing. Detecting the absolute number of x-ray induced TG mutants in an irradiated culture is beset with a number of difficulties. The main source of error is associated with the use of a post-irradiation growth period in normal medium before mutants are selected by their ability to grow in medium containing thioguanine. This procedure is based on the assumption that induced genotypic mutants require a growth period in non-selective medium before they may be recognized as phenotypic mutants. This experimental procedure gives stable and repeatable induced mutation frequencies when doses are used that result in surviving fractions greater than 0.3.However, after larger doses, the induced mutation frequencies are not stable and usually decline after reaching their maxima. This suggests selective effects against the mutant subpopulation during post-irradiation growth. Such selection may be expected if mutants had non-lethal but deleterious lesions in excess of those present in non-mutant cells. It may be shown that, on average, radiation-induced mutants of cultured mammalian cells sustain a higher dose than the total population (Thacker et al., 1970;Goodhead et al., 1980).The excess average dose to mutants a t doses giving a surviving fraction of 0.3 were 15 percent for helium ions of 90 keV pm-I and 75 percent for nitrogen ions of

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TRANSFORMATION AND MUTATION

470 keV p,m-'. Because of their larger projected nuclear area and

greater radiosensitivity, the comparable figures for excess dose to mutants in human fibroblast cultures are 36 and 186percent, respectively. It is known that the loss of HGPRT activity alone is of little disadvantage to the cell, but if the damage leading to radiationinduced mutation were more extensive than alteration of a small part of the gene coding for HGPRT, then the induced mutants might be at a significant disadvantage relative to the rest of the cell population. The HGPRT gene is located on the x chromosome of many mammalian cells, including the Chinese hamster with an average size of 30 Kb. Since there is only one copy of this chromosome that is active within a cell, cells carrying extensive x chromosome damage may not compete well with non-mutant cells.

3.4.6 Other Model Systems for Mutation Studies

In an attempt to overcome the diaculties associated with the conventional HGPRT assay system, a number of other mutation models have been utilized to study the mutagenic effects of ionizing radiation. The AL human hamster hybrid cell system has received considerable attention since the report of Waldren et al. (1986)which suggested that the conventional HGPRT assay tends to underestimate the mutagenic effect of radiation by as much as 200 fold. Recent studies by Hei et al. (1988~)also reported a substantially higher mutation incidence in AL cells by monoenergetic neutrons than previously reported with the HGPRT assay. The A, cells contain all the Chinese hamster ovary cell chromosomes plus one human chmmosome. The single human chromosome codes for specific human cell surface antigens in the hamster cells that render them sensitive to killing by specific antisera in the presence of complement. The increased sensitivity of this assay is due to the larger genomic targets in the AL cells for mutation than in conventional systems, such as the HGPRT assay (Waldren et al., 1986). By placing the genes used to measure mutation on a chromosome that is not required for cell survival, a spectrum of mutagenic events, from single base changes to chromosome loss, become measurable. The ALsystem can measure both intragenic and chromosomal damage such as large deletions that are frequently produced by radiation and are likely to be lethal and missed in the standard assay. Thus the AL cell system may represent an upper limit for mutagenic effeds of various agents. Mutant AL cells that have lost the surface markers, however, would survive and give rise

to scoreable colonies. Mutagenic effeds of monoenergetic neutrons at the a, locus in the A, cells were examined. Doses corresponding to low, moderate and high cytotoxicitiea and in energies ranging from 0.33 to 14 MeV were used. Over the range of doses examined, it was found that the mutagenesis induced by neutrons was energydependent and the frequencies were a downward curvilinear function of dose (Figure 3.14).In comparison to gamma rays, the RBE for cell lethality at the 10 percent survival level ranged from 5.2 for 0.33 MeV to 1.8 for 14MeV neutrons (Figure 3.14).The RBE for mutation induction a t the a, locus, however, ranged from 30 for 0.33 MeV to 4.2 for 14 MeV neutrons at or around the lowest levels of effect examined, (Figures 3.14 and 3.15). Similar to the data for charged particles of defined LET, the neutron RBE for mutagenesis appears to be higher than that for cell lethality a t approximately the same dose. Other promising systems that have been developed allow the quantitative measurement of mutation in hemizygous genes. Evans et al.

'

0

1

2

3

4

DOSE (Gy) F3g. 3.14 Mutation induction at the a, locus in A, celle irradiated with graded do= of neutrons of various energies. Data obtained for gamma rays were included for comparisons. (Adapted h m Hei et al., 1988c.)

72

3. TRANSFORMATION AND MUTATION

1.0 0.1

1.0 10 Neutron Energies(MeV)

Fig. 3.15 RBE ae a function of neutron energy for mutation and cell survival in A, hamsterhuman hybrid cells containinghuman chromosome11.RBE was calculated at the 10 percent survival level for cell lethality and at the 0.1 percent level for mutation. ( h m Hei et al., 1988c.)

(1986) determined mutation frequencies in x-ray and chemically treated mouse G5178Y cells at the hemizygous thymidine kinase locus (Tk + I - ) and reported a significant increase in sensitivity of this assay system as compared to the monosomic x-linked HGPRT locus. Similar findings have also been reported by Little and coworkers with neutron and x-ray induced mutants in human lymphoblasts that are hernizygous a t the TK locus. They showed that x ray induced mutation at the hemizygous TK locus was six fold higher than the homozygous HGPRT locus in the same cell if incubation is sufficiently long to allow enumeration of the slow growing colonies (Yandell et al. 1986). 3.4.7

Mutation and The Dose Rate Effect

Limited data are available on the effect of dose fractionation or protracted exposure to densely ionizing radiation on mutagenesis.

3.4

MUTATION STUDIES

1

73

Recent studies by Hei et al., (1989), and by Kronenberg and Little (1987) have reported quantitatively similar enhancement in mutation frequencies .by protraded versus acute exposure to neutrons using two different mutation assay systems. While fractionation of monoenergentic neutron doses (five fractions over eight hours) resulted in no sparing effect on the survival of cells, Hei et d.found a two fold enhancement in mutation frequencies at the a, locus of the A, cells (Hei et al., 1989). This increase was more pronounced in the lower dose ranges for 6 MeV neutrons (Figure 3.16) and for several other neutron energies examined (0.33,2 and 14MeV). Quantitatively similar findings have also been represented recently by Kronenberg and Little with protracted (lessthan 20 mGy d-'1 versus acute exposure using fast neutrons at the TK locus in human lymphoblasts (Kronenberg and Little, 1988).

74

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3. TRANSFORMATION AND MlJTATION

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DOSE ( G ~ ) Fig. 3.16 Mutation induced at the a, locus in ALcells irradiated with graded doses of 6 MeV neutrons either singly or in five fractions over an eight hour period. (Adapted from Hei et al.. 1988c.)

4. Hereditary Effects-I 4.1 Dominant Lethal Mutations The measure of dominant lethal mutations has been widely used in assessing radiation effects for over forty years, because of its "technical simplicity and . . . parallelism (with) viable chromosome rearrangements" (Sankaranarayanan, 1982). The method has been used for comparative radiation studies and is used extensively in the field of chemical mutagenesis (Bateman and Epstein, 1971). The dominant lethal mutation induced in the germ cell leads to death of the conceptus or of the fetus before birth, usually at the time of implantation in the uterine wall in mammals. The mutation event involves any one or more of many kinds of chromosome or chromatid rearrangements that are incompatible with survival beyond the first six or seven cell divisions after conception (Russell, 1962). The dominant lethal test (DLT) basically consists of mating exposed males or females with unexposed partners at recorded intervals after or during exposure. If males are irradiated (the DLT has obvious advantages for testing exposed males) and bred with virgin females, the pregnant females are sacrificed at 12 to 17 days after conception, and the following items are recorded: number of corpora lutea, live fetuses, dead but fully formed fetuses, and early postimplantation deaths noted by the induced uterine wall reaction (deciduomata). The frequency of postimplantation mortality, measured as the proportion of live fetuses (LE) among total implants (IMP), is distributed among females accordingto Poisson statistics. The mutation rate, p, can, therefore, be derived directly from the first term of the Poisson distribution, e-', the probability of no lethal events, because

Some additional information can be gained with the inclusion of all losses after conception, by using the ratio of live embryos per pregnant female, or for preimplantation losses only by noting the difference between the number of implants and the number of corpora lutea. These measures are not reliable at doses above several

76

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4. HEREDITARY EFFECTS-I

Gy, delivered at high dose rates, due to the drop in sperm count by the 6th week (irradiated spermatogonia). The consequent increase in the occurrence of unfertilized ova inflates the dominant lethal mutation rate (Searle and Beechey, 1974a). An additional important feature concerns cell stage in gametogenesis, because not all stages are equally sensitive to the induction of lethals. Spermatids, for example, are 1.5 to 2 times more sensitive than are mature sperm. Spermatocyte sensitivity varies with dose but tends to be intermediate. Spermatogonia appear to be only about one-tenth as sensitive as meiotic and post-meiotic stages; however, the unbalanced or asymmetrical interchanges do not survive cell division and are eliminated before reaching the spermatocyte stage. Dominant lethals induced in spermatogonia largely reflect the frequency of balanced translocations. Unfortunately, not all experimental information is based upon a uniform protocol, so that some comparisons may be confounded with differences in cell stage sensitivities. A complication arises when dealing with continuous or periodic exposures. After five weeks of exposure, all postspermatogonial cell stages will have been exposed to the same cumulative dose and will have progressed through all the maturation stages so that any conceptions produced by males at this time and after will be at an equilibrium between the damage induced in these stages and the five week dose accumulation. From this time on, however, a small additional contribution will come from the irradiation of these mature germ cells while they were still spermatogonia. Termination of external exposures permits the separation of cell stage effects, but for studies involving the internally deposited radionuclides that remain in the gonads, it is never possible to sort out cell stage variables. Although there are occasional efforts to identify a so-called "primary dominant lethal" not associated with structural or numerical chromosome aberrations, these lethals probably do not contribute significantly to the total dominant lethal mutation rate. Lethals induced in spermatogonia are recognized to be due to the chromosomally unbalanced gametes segregating at meiosis from spermatocytes carrying a balanced translocation.

4.1.1.1 Effects on Male Mice. One of the first studies on the induction of mutations with high-LET radiations was performed in the late 1930s by Snell and Aebersold (1937) using cyclotron neutrons

to induce dominant lethal mutations in mice. No direct comparison was made at that time with a standard radiation quality. The first quantitative comparisons were those performed by Russell et al. (19541, wherein cyclotron neutrons, fission neutrons, and 250 kVp x rays were compared for their efficiency in the induction of dominant lethals following single exposures. RBE values were estimated to be about eight (later corrected to six) compared to x rays and the two neutron qualities were essentially of equal mutagenic efficiency. These studies employed single doses a t high dose rates. The neutron dose range included four doses between 20 and 100 "rep" (roentgen equivaIent physical or 93 ergs g-I). The report by Russell et al. (1954),and later studies of Pomerantseva (1964),Searle and Phillips (1964), Domshlak et al. (1970), and Grahn et al. (1979) all repeat the general finding of an RBE of four to six for this endpoint involving meiotic and postmeiotic male germ cell stages. Fission neutrons were used in all of these studies. Grahn (1983) has summarized comparisons of single and onceweekly 0.85 MeV fission neutron exposures from the JANUS reactor with single, once-weekly, and continuous 60Cogamma irradiation. Weekly and continuous exposures continued for 30 to 50 weeks and dominant lethal tests were performed a t intervals during this period. For postspermatogonial stages, the RBE is five following single fission neutron exposures, rises to about 10 when comparing weekly exposures and to 15 when comparing weekly neutron exposure with continuous low dose rate, approximately 0.02 to 0.04 mGy min-' gamma irradiation. No continuous neutron exposure data are available for direct comparison, so this RBE value assumes additivity for periodic or fractionated neutron exposures. The assumption of additivity is reasonable because the mutation rate following weekly neutron exposures does not decline as it does following weekly gamma ray exposures, in comparison with their single-dose values. In fact, the rate following weekly neutron doses may be as much as 1.5 fold greater than the single dose (Grahn, 1983),which accounts for part of the increase in the RBE value from 5 to 15. The assumption of neutron dose additivity also holds for comparisons involving spermatogonial irradiations, and the RBE values are more dependent upon the pattern of exposure to the low-LET radiation (Grahn et al. 1979,Grahn, 1983).RBE values are estimated from the ratios of dose-response regression coefficients (as they were for those noted above for mature germ cell stages) and are about four for single exposures, 15 for weekly exposures, and 30 for the comparison of weekly neutron exposures with continuous gamma irradiation. The shift from single to weekly to continuous gamma irradiation causes the mutation rate for lethals induced in spermato-

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4. HEREDr'ARY EFFECTS-I

gonia to drop significantly, in relative terms, from about 7 to 3 to 1. An important feature of these data on the dominant lethal mutation induced in stem cells is that they conform to a linear dose-response function so that estimated RBE values are valid over a wide range of doses. A study by Grahn et al. (1984) on the effects of extremely low single doses of neutrons involving levels of 0.01,0.025,0.05,0.1,0.2 and 0.4 Gy compared to a limited array of 'j°Co gamma ray doses down to 0.225 Gy, has confirmed the previous RBE value of five as prevailing over the full range of neutron exposures for dominant lethals induced in the postspermatogonial germ cells. There is one possible exception to this that may be of significance for considerations of occupational exposure standards. The response at 0.01 Gy and 0.025 Gy deviates from linearity by showing a mutation rate in excess of expectation based upon an extrapolation down from the linear dose response a t 0.05 Gy and above. This deviation is statistically significant, but does not exceed a factor of two above the expected value. The dose-response curve has a small "blister" above the linear trend a t the two lowest doses. It then joins the overall linear trend. At the lowest doses, the RBE is 10. It is five a t all levels above 0.05 Gy of neutrons. The data a t 0.01 and 0.025 Gy conform to the dose response regression describing the response to low weekly exposures to neutrons at 0.001 to 0.05 mGy wk-' (Grahn, 19831, so this phenomenon of a response in excess of expectation may be a more general one a t low doses and will influence RBE values in a significant way. Luning et al. (1975) reported on the occurrences of dominant lethals in mice following single exposures to 14 MeV neutrons. No comparison with a low-LET radiation was done. The mutation rate Gy-I for mature germ cells, which is not was 11.2 + 2.6 x significantly above that seen following 60Cogamma rays 10.1 + 0.5 X lo-' Gy - '. Pomerantseva (1964) compared the response to single doses of 660 MeV protons with fission neutrons, 190 kVp x rays, and 60Cogamma rays. The RBE value for neutrons versus gamma rays is four to five, for high energy protons versus gamma rays it is 0.6, and for x rays versus gamma rays it is 1.5. All of these data are based upon the induction of lethals in postspermatogonial cell stages by single doses.

Effects on Female Mice. The only cogent report on the induction of dominant lethals by neutron irradiation (0.7 MeV fission neutrons) of female mice is that of Searle and Beechey (1974b). Though the data were not analyzed statistically, the authors noted that the RBE value would be about one as compared to x rays for the

4.1.1.2

4.1

DOMINANT LETHAL MUTATIONS

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79

induction of all lethals (pre- and postimplantation) among litters conceived during the first week after exposure. However, a regression analysis of only the postimplantation fetal losses gives an RBE value of 4.8, derived from the ratio of slopes. Since the neutron series has an unusually high control level of fetal mortality, use of the x ray control for both radiation qualities gives an RBE value of 5.6. Thus, for single doses, both sexes would appear to show the same RBE for the induction of dominant lethals in postgonial germ cell stages. For protracted exposures, the RBE should increase by a factor of five, as this is the dose-rate factor for the induction of dominant lethals in the maturing oocyte, according to Baev et al. (1977). 4.1.2 A l p h Particle Irradiation

Effects on Male Mice. Three laboratories have studied the effects of gonadal burdens of 239pU on the induction of dominant lethal mutations (Luning et al., 1976; Searle et al., 1976; and Grahn et al., 1979). Luning et al. (1976)did not have a comparison group of animals exposed to low-LET radiations, Searle et al. (1976) used only a single-point comparison with a continuously gamma irradiated group, and Grahn et al. (1979) made comparisons with both neutron and gamma irradiated mice. In summary, Searle et al. (1976) derived an RBE of 22 for 5 MeV alpha particles when compared to W o gamma rays, Grahn et al. (1979) gave a value of 13 ? 3, while no value can be derived from Luning's data. The alpha particle induced mutation rate reported by Searle et al. (1976) was 190 x lo-' gamete-' Gy-' compared to 64 + 11 x lo-' gamete-' Gy-I given by Grahn et al. (1979), though both groups gave similar gamma ray rates (8.6 x lo-' versus 5 x lo-' gamete-' Gy-') for Searle and Grahn, respectively. The data of Luning et al. (1976) can be reanalyzed and converted to a rate per unit dose for the four injected dose levels of 1.85 x lo3, 3.7 x lo3 and 9.25 x lo3 Bq per mouse, by assuming that retention and distribution would have been the same in the CBA mouse he employed as has been seen for the two different hybrid mice used by Searle et al. (1976) and by Grahn et al. (1979). The regression of the dominant lethal mutation frequency on dose is estimated to be 87 ? 22 x lo-' lethals gamete-' Gy-' which is intermediate to the two rates noted above. It is likely, therefore, that the three independent studies present a consistent picture on the mutagenicity of gonadal burdens of plutonium. The variation among the studies may be more attributable to genetic differences in the sensitivity to both the induction and the scoring of lethals expressed as fetal deaths. All three studies derived most of their data from

4.1.2.1

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1

4. H E R E D M Y EFFECTS--I

males with initial budy burdens of 299Pu in the 1.85 x lo5to 3.7 x 106Bq kg-' range which produces an average dose to the gonad of 5 to 10mGy per week. As noted previously, the mutation rate in these circumstances is based upon the effects of continuous irradiation of all germ cell stages. Because the dominant lethal mutation rate from irradiated spermatogonia is one-tenth that in later stages, it is reasonable to presume that the majority of the lethals scored were induced in meitoic and postmeiotic cells. A more significant source of variation in the estimation of RBE values following the deposition of an alpha emitter in the gonads relates to the marked heterogeneity of distribution of the radionuclide in the organ. A number of studies with male and female mice, summarized by Searle (1979), have shown that the average dose to the whole gonad may be a poor estimate of the actual dose to the germ cells. In male mice, stem cells receive at least 2 to 2.5 times the average gonadal dose (Green et al., 19751,and possibly four times the average (Russell and Lindenbaum, 1979).No similar estimate is available for females. Taken at face value, the RBE values may be overestimated by a factor of two to four. An additional variable that influences the dose to the male germ cells is the histoarchitecture of the gonad (Brooks et al., 1979). Mice, for example, have a high ratio of tubular to interstitial tissue so that the burden in the interstitial tissue, which is the major place of deposition, is always close to the stem cell population in the spermatogenic tubules. The stem cell dose is accordingly higher than the average gonadal dose. In man, however, the situation is the opposite. About one-half the gonad is interstitial tissue as compared to only 10 percent in the mouse, therefore, the stem cell dose for man has been estimated by Brooks et al. (1979) to be less than the average organ dose. RBE values derived from laboratory animals cannot be applied to man without careful consideration of this histoarchitectural variance. A final variable concerns the tendency for the radionuclide to be scavenged by the macrophages and become somewhat more aggregated as the animal ages (see Searle, 1979, for a general discussion). This phenomenon may act to reduce the dose to the germline in both sexes, even though a conflicting effect of continuous alpha irradiation is the steady reduction of the gonadal mass thereby increasing the average dose to the whole gonad. In summary, the conservative approach would be to note that alpha particles and fission neutrons have about the same effectiveness for the induction of dominant lethal mutations, especially during the period soon &r deposition, and therefore the neutron RBE can be used safely in establishing the genetic hazard. Most human expo-

4.2 CHROMOSOME ABERRATIONS

1

81

sures are likely to be a t low rates over long periods, therefore this conservative approach could also be a better approximation of the RBE under conditions of long-term or repeated injections, an experimental condition that has not been tested.

Effects on Female Mice. There have been several studies addressing the effects of 239Puburdens in the ovary. The first compared 60Cogamma radiation a t two daily dose levels (24 hours of irradiation) of 0.1 and 0.2 Gy d-' with two dose levels of plutonium of 1.85 x lo5 Bq kg-' and 3.7 x lo5 Bq kg-' Searle et al.. (1980). There was no evidence of induced dominant lethals in either group, as the mean litter size was not reduced by the continuous gamma or alpha irradiation. Mean number of litters per female, the mean number of offspring per female, and the duration of fertility were all reduced. A crude estimate of 2.6 was given as the RBE value for oocyte killing. In a follow-up study, Searle et al. (1982)have reported the induction of dominant lethal mutations by =Pu, but only after 12 weeks of exposure and at an estimated dose accumulation of 5.91 Gy. At lower dose levels accrued over shorter periods (one to six weeks), the frequency of lethal mutations was not detectably different from that of the controls. The rate at 12 weeks, when compared to that induced by continuous gamma irradiation (Baev et al., 1977) gives an RBE (alphalgamma) of 3.6. As Searle has discussed, high LET radiations do not appear to have the same high level of mutagenic efficiency in females as in males; however, the complicating variables of age, cell stage, cell selection, type of genetic endpoint, total dose, and dose rate have not all been sorted out. 4.12.2

4% Chromosome Aberrations and Reciprocal Translocations Induced in Spermatogonia

Genetically transmissible balanced reciprocal chromo8ome translocations that are induced in spermatogonia can be scored readily a t the first meiotic metaphase by using the cytological procedures of Evans et al. (1964). Since that report was published, there has been an extraordinary increase in the amount of data on this genetic endpoint. Studies have examined the effeds of diverse radiation qualities, exposure patterns, and post exposure time intervals. The available data have been summarizedin considerable detail in recent UNSCEAR reports (UNSCEAR 1972, 1977, 1982, 1986, and 1988) and briefly, for low-LET radiations, in NCRP Report No. 64 (NCRP, 1980). Attention here will be focussed upon the effects of neutrons and alpha particles in comparison with the effects of single, weekly

82

/

4. HEREDITARY EFFECTS-I

and continuous gamma irradiation. Because data from irradiated females are sparse and only semiquantitative, discussions will be limited to effects on males. 46.1 Neutron Irradiation

The general appearance of the dose-response relationships for reciprocal chromosome translocations induced in spermatogonia has been essentially repeated or confhned in all studies (see Preston and Brewen, 1973). Single exposures induce a generally linear increase with dose up to about 6 Gy for low-LET radiations and to about 1 Gy for high-LET radiations. Beyond these dose levels, the response drops rather abruptly and does not conform to any interpretable regression function. This so called '?lumpedn curve, seen for a variety of genetic and somatic endpoints, is attributed to the competition between cell killing and mutation or aberration induction. Protraction andlor fractionation of the dose usually causes the linearity to continue to much higher levels of dose and response. The linear response seen a t the lower single-dose levels of low LET radiation is probably a result of the intrinsic heterogeneity in radiosensitivity of the spermatogonia. If the cells are scored a t or near the first major wave of meiosis after exposure, then a classical linear-quadratic response equation prevails up to 1.29 x lo7C kg-' of x rays (Preston and Brewen, 1973) and 3 Gy of gamma rays (Grahn et al., 1984). As most studies have used a nearly constant interval between irradiation and sampling, regardless of dose, they have scored a more heterogeneous cell population due to cells having passed through a number of mitotic divisionsbefore reaching the first meiotic metaphase. This appears to blur the cell stage distinctions needed to detect the non-linear component. Reducing the dose rate and the size of dose fractions act predictably to reduce the effect per unit dose for low-LET radiations because of the acknowledged 2-hit component of injury. This is especially notable for continuous low intensity gamma irradiation (Pomerantseva et al., 1972; 1975; Searle et al., 1976;Grahn et al., 1979,1983;Brewen et al., 1979).The dose rate effectiveness factor for gamma rays delivered a t approximately 1 Gy min-I compared to approximately 0.01 mGy min-') is between 10 and 15 (Brewen et al., 1979). In direct contrast, protraction of neutron irradiations augments the damage function, especially at higher doses (Searle et al., 1969; Dornshlak et al., 1970; Grahn, 1983; Grahn et al., 1979). At the very least, the dose response to protrticted neutron irradiation rises above the initial slope describing single dose effecta. For example, at 0.1 Gy or less,

4.2 CHROMOSOME ABERRATIONS

/

83

the response to protracted exposures is about 25 percent greater than the response to single doses. This difference steadily increases with dose to about 80 percent at 1Gy. RBE values for single doses of fission neutrons compared to *Co gamma rays are generally between three and six. The value could approach eight to ten at extremely low doses, due to two factors. First, the response to neutrons may be as high a s 8 to 11 x cell-' Gy - ' as seen by Searle et al. (1969) and Muramatsu et al. (1973) for doses up to 1Gy, and by Grahn et al., (1983) at extremely cell-' Gy- ' were noted low doses, <0.1 Gy. Values of 5 to 7 x by Domshlak et al. (1970), Grahn (1983), and Grahn et al., (1983) a t doses up to 1Gy. Second, the response to low-LET radiations may be distinctly linear-quadratic under the ideal experimental conditions noted previously, with an initial linear term at about 1 x cell-' Gy-' (Searle et al., 1971; Preston and Brewen, 1973; NCRP, 1980, Grahn et al., 1984). The RBE value of greatest interest is that relating to either continuous or protracted exposures. The best estimates are those derived from the ratio of dose-response coefficients, where, for statistical reasons, the individual regression is based upon five or more data points, a criterion that cannot be met consistently, however. The slope estimates for 60Cogamma rays delivered at rates below 0.1 mGy min-I to total doses up to 20 Gy, are: Gy-I (Brewen et al., 1979) 1.3 x 1.4 x Gy-I (Searle et al., 1976) Gy - ' (Pomerantseva et al., 1972; Grahn et al., 1979) 1.7 x As Searle et al. (1976) have noted, it is highly unlikely that the Gy-' and the value of 1.3 x lopsGy-' rate would go below 1 x from Brewen et al. (1979) may well be the lower asymptote for the relation between induction rate and dose rate. What then is the best estimate of the linear term for neutrons, especially a t doses below 0.5 Gy, before the response may become markedly nonlinear? The available estimates are as follows: 3.3 x Gy - ' (4.1 MeV neutrons, continuous exposure)Bajrakova et al. (1976) Gy - l (1.5 MeV neutrons, single exposure) Domshlak 4.9 x et al. (1976) 6.6 x Gy-I (0.85 MeV neutrons, once-weekly exposure) Grahn et al. (1984) Gy-I (0.7-0.85 MeV neutrons, single exposure) 9.0 x Searle et al. (1969), Grahn (1983) 10.5 x Gy-I (0.7 MeV neutrons, continuous exposure) Searle et al. (1969)

84

1

11.4 x

4. HEREDlTARY EFFECTS-I

Gy -

(2 MeV neutrons, single exposure) Muramatsu et al. (1973)

The best estimate of the linear term for the fission neutron (' 1 cell-' Gy-' for total MeV) is probably in the range of 5 to 9 x doses of 0.01 to 0.5 Gy. Data obtained following low once-weekly exposures of 5 to 50 mGy week-' for periods of 6 to 60 weeks and involving a total dose range of 0.075 to 1.6 Gy may best be described statistically by a linear-quadratic equation with a negative second degree term (Grahn, 1983).The linear term is 7.9 k 0.85 Gy-', rad- l) which does not differ significantly from (7.9 x 0.85 x the highest value for single exposures. The lowest and highest values for the nly RBE value would be 19 and 88 (11.4 x 10-4/1.3 x in a (3.3 x 10-4/1.7 x comparison of the above two arrays of data, but this mixes the results from separate studies, in time, place, radiation source, and mouse strain. For the fission neutron, one approach would be to take the midrange value for long-term weekly or continuous exposures to the for the neutron and 0.15 x two radiations. These are 7.5 x for60Cogamma rays. A nominal RBE value of 50 (7.510.15) with a range of 30 to 70 would accommodate the majority of the data. This nominal value might be considered a maximum value, as the RBE will fall to lower limits of 10 to 30 when comparisons involve large once-weekly fractions of gamma rays, for example, at levels above 0.4 Gy per week (Grahn, 1983). 4.2.2 A l p h Particle Zrradiation

Several studies have been reported that make comparisons between 239pUalpha emissions from an internal gonadal burden and external 60Cogamma radiation. These are the reports of Searle et d. (1976) and Grahn et al. (1979; 1983).The latter reports also included comparisons with single and weekly exposures to fission neutrons. Searle's data are based upon a single gamma ray dose point taken after 28 weeks of daily continuous irradiation a t 0.04 mGy min-'. Alpha-irradiated animals were sampled at 21,28 and 34 weeks after being exposed to a single injected dose of 239pUcitrate so as to deliver an average dose rate of 6 x mGy min-' or 0.88 mGy d-' to the whole gonad. Translocation frequencies were 1.7 percent and 0.74 percent for gamma rays and alpha particles, respectively. When reduced to rates per unit dose, the RBE estimate became 24. Grahn's data have a broader dose-response basis; however, they also identified a significant dose-response variable with regard to the internal emitter. Two dose levels of 239pUcitrate were employed,

4.3

EFFECTS ON CELIS IN MEIOSIS

1

85

1.85 x lo5 Bq kg - l and 3.7 x lo6 Bq kg-'.which delivered alpha doses of about 5 x loe4and 1 x mGy min-I or 7.5 x and 1.5 x lo-$ Gy d-l. Estimates of translocation frequencieswere made at a number of different intervals between 8 and 62 weeks after injection of 239pU.The response to 2s9pUdoes not continue to increase with increasing accumulated dose. The effect rises initially, plateaus, and then declines a t all sampling times beyond 45 weeks after exposure, although the average level of response is proportional to the injeded dose level. The initial rise in translocation frequency, during the first 20 weeks, occurs a t a rate that is only one-third to twothirds of the rate seen under weekly neutron exposurea (Grahn, 1983;Grahn et al., 1983). Though it was originally assumed that the neutron induced rate of response could be considered equal to that for alpha particles, which gave an RBE estimate of 38 + 5 (Grahn, et al., 19791, more complete data and interpretations (Grahn et al., 1983) suggested that the previous assumption that alpha particles and neutrons have the same mutagenic efficiencywas not warranted. Alpha particles may actually have only one-third to about one-half of the mutagenic efficiency of neutrons. It is difficult to recommend a specific RBE value for alpha parhcles.

4.3 Effects on Cells in Meiosis The resting and maturing oocyte and the sequence of spermatocyte stages provide the opportunity to evaluate damage in meiotic stages. Cells in meiotic stages are more sensitive to cell killing and apparently to the induction of chromosome and chromatid aberrations. The available data have been summarized by Sankaranarayanan (1982) and will not be detailed here as it can be noted that data involving LET variables are quite limited. In the male mouse, continuous irradiation will induce damage during the meiotic phases preceding the first meiotic metaphase, leading to the appearance of chromatid fragments and rearrangements at metaphase. The relevant dose is that accrued during the 13day interval from the establishment of the resting primary spermatocyte stage to the occurrence of first meiotic metaphase. In a com~ particles, Searle et al. parison of mCo gamma rays with 2 s 9 Palpha (1976) scored for fragments and estimated the RBE value to be 24. Grahn et al. (1979)developed data under similar exposure conditions leading to an RBE estimate of 33 + 5.Once-weekly fission neutron and "Co gamma ray exposures also induce a significant increase in fragments that can be detected if the mice are sampled immediately

86

1

4.

HEREDITARY EFFECTS-I

after exposure. The frequency of cells with fragments increases linearly with dose at the rate of 2 + 0.2 x Gy-I for gamma rays Gy-' for neutrons to give an RBE of about 18 + and 37 + 5 x 3 (Grahn et al., 1983). The estimated rate per Gy of alpha particles is 55 x to give an RBE of 28 +- 9. These induction rates are sensitive to age-related changes in the spontaneous frequency of fragments. Chromatid rearrangements or interchanges are seen at metaphase in spermatocytes exposed either continuously or a t selected times to low LET radiations during prophase (Adler, 1977; Walker, 1977). They are also seen following weekly exposure to neutrons and gamma rays (Grahn et al., 1983). In the latter report, the neutron/ gamma RBE was given as 7 + 1, significantly below the neutronefficiency noted for the induction of fragments. Alpha particles are less efficient than either neutrons or gamma rays. The one attempt to compare the effect of fission neutrons with x rays for the induction of aberrations in the maturing oocyte was inconclusive (Searle and Beechey, 1974b).

4.4 Abnormal Sperm Morphology

The screening of appropriate prepared samples for the frequency of sperm heads showing an abnormal morphology has been described by Wyrobek and Bruce (1978). Searle et d.(1976) did not see any influence of alpha irradiation on the frequency of abnormal sperm, though he did note an effect of continuous gamma irradiation. Grahn d al. (1979), however, did observe a significant effect of alpha particles, neutrons, and gamma rays on this measure of cellular damage. Compared to weekly gamma irradiations the RBE value for neutrons is about 10, but it is somewhat higher for alpha particles, reaching a value of 25 ? 8. This high value may be due to the necessity of making the comparison of continuous alpha irradiation with weekly gamma irradiations. Although the data are sparse, there is the suggestion that, for a given total weekly dose, continuous gamma irradiation may induce a greater response than that seen following onceweekly exposures. If so, then the alphdgamma RBE of 25 could drop back towards 10. For single doses of neutrons versus gamma rays, the RBE value for this endpoint is 5.5 + 0.5 (Grahn et al., 1984). The similarity of these RBE values to those seen for the more classical genetic endpoints is probably attributable to the fact that sperm abnormalities are due to the induction of complex chromosome aberrations in late spermatogonial and early primary spermatocyte

stages. In other words, this endpoint may be measuring some of the same information otherwise seen as translocations and dominant lethals.

4.5 Summary

There is the consistent finding that the RBE of fission neutrons, gamma followingdoses delivered a t high dose rates, compared to @'&I rays is five, with a range of three to six. In this instance, the endpoints are dominant lethals induced in the meiotic and postmeiotic male germ cell stages, reciprocal chromosome translocations and dominant lethals induced in spermatogonia, and the frequency of abnormal sperm. At the other extreme, that is, the comparison of protractedlfkactionated neutron exposures with continuous low dose rate gamma ray exposures, the RBE values are as follows: dominant lethals induced in mature germ cell stages, 15; dominant lethals induced in spermatogonia, 30; reciprocal translocations, up to 50. Alpha particles have the same RBE value as neutrons in comparisons involving dominant lethals or other genetic damage induced in mature cell stages, but for damage induced in spermatogonia, the RBE appears to be a factor of two or three lower. Much of the observed variation in RBE values for fission neutrons can be attributed to the fact that reduced dose rates and total doses of low-LET radiations clearly reduce the mutagenic effectiveness of those radiations, while the same does not occur for the high-LET radiations. One exception to this has been seen for once-weekly neutron exposures delivered over long periods. The mutagenic effectiveness for dominant lethal mutations actually increases by a factor of 1.5 to 2 over the effects of single neutron doses. This increaee in effectiveness may also .occur at extremely low single doses. It is not a universal finding. Nevertheless, taking it into consideration increases the RBE for certain comparisons against low-level continuous gamma irradiation. A brief tabular summary is presented in Table 4.1 to give an overview and range of the RBE values derived from the major genetic endpoints reviewed. These are all obtained from studies with male mice and are derived from comparisons of the responses to fission neutrons with those from @'Cogamma irradiations. The RBE values for alpha particles are summarized and discussed in detail in NCRP Report No. 89 (NCRP, 198713) but they are no greater than those seen for neutrons. They are o h n only one-half to two-thirds of the values for neutrons.

TABLE4.1--Summary ofRBE d u e s for fission neutrons compared to T o gamma mys Neutrons Gamma rays of D m range Dose rate Dose range Doae rate Genetic endpoint Exposure (GY) (mGy min-') (GY) m yn Single dosea 0.01-1.0 5-5.0 0.2-6.0 50300 Dominant lethal mutation: Postapermatogonial cell Spermatogonia Abnormal sperm Reciprocal chromosome translocation Once-weekly doses Dominant lethal mutation: (20-45 min) 0.005-0.15 0.25-1.5 Postapermatogonial cell 0.15-1.5 1.5-15 Spermatogonia Abnormal sperm 0.008-0.07 wk-I 0.08-0.8 wk-I Reciprocal chromosome translocation 0.1-1.5 1.5-20 Daily doses (not available, see below) Dominant lethal mutation: (22Wd) 0.50-2.0 Postspemtogonial cell 4.5-14 Spermatogonia Reciprocal chromosome translocation 1.0-20 "Comparisons made with re8pOmle8 to once-weekly neutron doeee.

w

+ Range of

RBE

I

5-10

2

Vduea

4

5-10 10-15 10-15 10

"

15-35

15-30.

30' 45-75.

-

5. Hereditary Effects-I1 5.1 Mammalian Germ Cell Mutagenesis 5.1.1 SpecificLocus Mutations-Spernatogonia The specific locus method entails screening for mutations at any of seven specific loci in mouse germ cells, primarily spermatogonia and oocytes. The extensive low LET radiation studies have been reviewed in a previous report (NCRP, 1980)and will not be recounted here except to provide baseline information for the discussion of high LET effeds. The specific locus endpoint, when induced by ionizing radiation, is thought to consist primarily of chromosome rearrangements, namely, small deletions, duplications, insertions etc; these are aberrations which include the specific locus under observation but frequently extend to adjacent loci. With neutrons, a larger proportion of the mutant events are recovered as multi-locus deletions than with x rays when induced in spermatogonia or oocytes (UNSCEAR, 1977, 1982). Therefore, it should not be surprising that the RBE values associated with the specific locus endpoint for a given germ cell stage are similar to that found for the endpoints discussed in the previous section, eg.,dominant lethality and translocation induction. Far spermatogonial studies, seven experiments have been reported (Table 5.1 taken from Searle, 1974 and Figure 5.1). Russell (1965a) has shown that doses of 0.52 to 0.55 Gy of fission neutrons produce essentially the same mutation rates when dose rates of 0.79, 0.008 or 0.002 Gy per minute are employed. Batchelor et al. (1967) found that the mutation rate of 0.62 Gy delivered at a dose rate of 1x Gy min-' was consistent with the data reported by Russell (1965a). Thus unlike low LET radiation there is no reduction in yield over an 80,000 fold dose rate change. In addition, the five experiments ranging between 0.52 to 0.88 Gy neutron exposures give essentially the same mutation rates per locus and unit absorbed dose, 1.6 to 1.9 x Gy -'indicating that over this narrow dose range there appears to be no evidence of a departure from linearity. At a higher acute dose, 1.88 Gy, a t 0.57 Gy min-' the mutation rate plummets such

i3

i2 TIUU~ 5.1-Fraguencien of specific locus rnutntions induced in spermatagonia q%r acute and chronic fision neutron eqmwea Doee (GY) Neutmn

0.52 0.52 0.55 0.88 1.88 0.62 2.14

Gamma

(Gy min-'1

Number of mp*

0.07 0.07 0.08 0.13 0.18 0.42 0.93

0.79 0,008 0.002 0.0001 0.57 0.000001 0.00002

16,758 17,041 18,194 19,506 39,028 39,083 41,875

h m rate

Numberof Mutatim

10 12 13 20 8 27 67

Raluenry per

! 4

-F 108 gametes

Itdexmm

8.5 10.1 10.2 14.6 2.9 9.9 22.9

Ruesell (1966a) Ruesell (1965a) Ruaaell (1965a) Rueaell(1965a) Batchelor et al. (1967) Batchelor et al. (1966) Batchelor et al. (1966)

m

B

a

5.1

MAMMALIAN GERM CELL MUTAGENESIS

1

91

High LET-/

L o w LET -----_----__-_----_--------

Fig. 6.1 Frequency of specific l m mutations induced in spermatogonia as a function of d m . Upper dashed line represents the best fit (weighted regression analysis) of all fast neutron doee points for specific locus mutations (excluding the 2.14 Gy chronic dose and the 1.88 Gy acute dose) induced in spermatogonia. The solid squarea are acute exposures, open circles are chronic exposures.The lower dashed line represents the low LET curve for comparison purposes (Adapted from Searle, 1974).

that the confidence limits overlap the spontaneous rate, in the same way as an acute x ray exposure of 2.58 x 10-I C kg- I. At a higher dose, delivered at low dose rate of 2.14 Gy, at 2 x Gy min-l, the mutation rate is significantly lower than predicted by linear extrapolation fiom the lower doses. Clearly, this means that at either high or low dose rates the mutant bearing germ cells from the stem cell population are selectively eliminated unlike the finding seen at lower total doses. Searle calculated the linear equation for all neu-

tron doses excluding the 1.88 Gy dose, at 0.67 Gy min-l, to be Y = 8.3 x + 1.25 x lo-' Gy. We calculate the regression coefficient to be 1.6 x Gy-' when the 2.1 Gy dose is also excluded. The RBE for fislsion neutrons compared with gamma rays delivered a t very low dose rates, such that a linear dose-effect relationship is expected, is between 17 and 23. 5.1.2 Specific Locus Mututwns in Oocytes

Mutation studies on mouse oocytes have shown a remarkable difference between oocytes recovered within the first six weeks after irradiation and those recovered at seven or more weeks for both low and high-LET radiations. The early dictyate oocytes manifest no mutational response to irradiation and, as discussed in NCRP Report No. 64 (NCRP, 1980) and more recently reported by Dobson et al. (1987)and Straume et al. (1987),it is likely that the immature mouse dictyate cells do not survive radiation damage to their hypervulnerable cell membranes and thus their lack of mutation response does not describe either a genetic insensitivity or remarkable repair capacity. Thus,the mouse immature oocytes response cannot be extrapolated to the human situation. However, Dobson et al. (1987) have shown that these immature oocytes were as mutable as maturing oocytes when 0.43 MeV neutrons are used. The short-ranged recoil protons, as a result of neutron irradiations, have a high probability of hitting the nucleus without also hitting the plasma membrane. Brewen and Payne (1979)and Lyon and Phillips (1975) have separated the maturing oocyte data into the f i s t week and the second thru the sixth week compartments which more clearly reflects differing sensitivities in the two distributions. For x rays, the oocytes, two to six weeks post irradiation, are about two times more sensitive than those recovered in the first week. The data for the neutron studies have not yet been reanalyzed in this way. When this is done the data should provide a more accurate estimate of RBE values for different oocyte stages. The results for all neutron studies in oocytes are shown in Table 5.2 (taken from Searle 1974). As can be seen from the data with acute doses (0.3, 0.6, and 1.2 Gy) the mutation rate increases with increasing doses but it is not clear a t 1.2 Gy exposure whether the curve has begun to bend over. Surprisingly the comparison of 0.6 Gy chronic, 1.5 mGy rnin-', with 0.6 Gy acute (0.79 Gy min- ') doses shows a dose rate effect of two (chronicis lower). While it is possible that such an effect is real, a radiobiological basis for this kind of dose rate effect with neutrons is not readily apparent. Russell (1965b3 points out that the caged females appeared to huddle

T~e~~.S.2-Frequencies of specifi locus mutations afir exposure of dicwte oocytes to fision neutrons (Taken from Table Xm,Searle, 1974) IrradiationT(~~ll~~ption Interval

< 7 weeks

Doet

Doee rate

(GYP

(Gy min-')

0.3b 0.6 0.6 1.2

0.08 0.0015 0.79 0.75

Number of

Offs-

5,870 46,301 43,000 6,058

Number of Mutations

1 22

37 7 * About one-eight of each dose conaista of gamma contamination (Russell, 196Sb).

Fres-per locm per 1@s5 gametea

References

2.43 6.79 12.29 16.51

Ruseell (1962) Russell (1965b);(1962) Russell (196Eib);(1962) Ruseell (196Eib);(1962)

.

@ 0

E

*

PB

with each other and may have provided an unexpected shielding factor that resulted in an overestimate of gonadal dose. Straume et al. (1987) suggest that the dose effect is ascribable to as much as a 35 percent gamma-ray contamination contribution; but if thie were the case, it would be indeed remarkable to see so large a dose rate effect with so low a gamma ray dose (approximately 0.2 Gy). The mouse oocyte data permit a wide range of RBE estimates to be derived. A weighted linear regression analysis of the three acute neutron doses in Table 5.2 suggest the a, coefficient is 2.0 x (when the dose is expressed in Gy). For low LET radiations the a, coefficient,depending on age of female and stage of maturing wcytes sampled (NCRP,1980), can range over a factor of five from 2.8 x to 1.4 x (when the dose is expressed in Gy), although unpublished data of Russell suggests the aging effect is not as great as the factor of five found in the published literature. RBE values can be estimated as follows:

The most recent x-ray data of Lyon et al. (1979) generates ax=6.0 x mutations per locus per Gy, and yields an RBE of 34, between the two estimates shown above.

5.1.3 Mammalian Germ Cell Summary and Conclusions (1) RBE values for spermatogonia (fission neutrons versus gamma radiation) are in the range of 20 for neutron doses of 0.5 to 1

GY. (2) Over the 0.5 to 1Gy neutron dose range, the mutation rate is 1.6 to 1.9 x lo-= events locus-I Gy-I and is independent of a wide range of dose rates. (3) Doses between 0.1 to 0.25 Gy should be studied to insure that the RBE, values have been determined. Should such studies be undertaken, they should include additional endpoints such as dominants and enzyme activity changes. (4) The immature mouse oocyte is assumed to be killed by radiation traversal across the cell's hypervulnerable membrane (Straume et al., 1987) and therefore is not an appropriate cell type h m which to determine RBE values. (5) In the maturing oocyte, there is a wide range of RBE values, 14 to 70, possibly reflecting differences in age of the females

5.2 NON-MAMMALIAN GERM CELL EXUDIES

I

95

studied and the differing sensitivities of the maturing oocyte stages. (6) The mutation rate for the maturing oocyte over the limited neutron dose range studied is 1.4 to 2.0 x lo-' eventa locus-' Gy -I. This is almost an order of magnitude greater sensitivity per unit dose than seen in spermatagonia and thus its adds to the concern of being able to establish an appropriate mammalian female mutation system for human extrapoliation purposes. (7) Ifthe mouse maturing oocyte is to serve as a sourcefor estimating the RBE value for genetic effects of various radiations for humans, then there is a need to obtain additional data at lower total doses and appropriate dose rates.

5.2 Non-Mammalian Germ Cell Studies 5.2.1 Dmsophila

The most extensive data on response versus dose for high LET radiations have been obtained with Dmsophila employing recessive lethal mutations, translocations, and dominant lethals as the primary endpoints of genetic damage over a wide spectrum of neutron energies. The major group of experiments, in a number of laboratories, (Table 5.3) have studied the effecta of neutrons on a variety of post-meiotic male germ cell stages, usually spermatozoaand spermatids. Many of the early studies with Drosophila (pre 1950s) suffered from a lack of accurate dosimetry and considerable germ cell heterogeneity. Thus, the interpretations of these early studies must be considered as uncertain (see review by Sankaranarayanan and Sobel, 1976). The dose-mutation response is linear for the three major endpoints with respect to neutrons. Gonzalez's (1972)data on post meiotic male germ cells are probably the most extensive on this point. Thus the conclusion reached in this report and those of other earlier workers is that single recoil proton tracks induced by neutrom are very efficient in producing two-break events (see Figures 5.2,5.3 and 5.4 taken h m Gomalez, 1972). Since for the low LET radiations these endpoints are the result of both one and two track events, the best representation of RBE a t low doses is derived from the ratio of U N 1 ax.Where possible, Table 5.3 employs this calculation. In Table 5.3, one observes that the RBE is, in general, a function of the neutron

TABLE 5.3-Neutron RBE Values from Drosophila Studies Mutation RBE and Neutron Energy

14-15 MeV

Germ cell atage Sperm Sperm spermatotids, late mixed sperm & spermatotids sperm spermatotida (hphila virilis)

sperm

Endpoint

Tranmlocation RBE -

Dominant lethal RBE

CD Q,

Reference

1 2

-

Fujikawa, & Inagaki, 1979 Sobel & Broeree, 1970

-

0.8-1.2 x-lethale

-

2.3 2-3

Edington & Randolph. 1958 Alexander, 1958

-

-

1-1.5

1.5 eat. x-lethals

f 2.7

-

0.5 sp. locue 0.8 x-lethal6 1.2 x-lethals

3 MeV

\

C" .

El E '

! 4

Gonzales, 1972 Munoz, 1971 I

2.5 MeV Spe-

2.3 x-lethale

5.7 (?)

Sperm late apematotids early spermatotids day 4 spermatotids

1.2 x-lethals 2.2 x-lethals

2.3 3.2

1.4 .

3.3

2.5

Nakao & Machida, 1970

2.0 MeV

1.3

1.7

1.6

-

1 MeV

mixed sperm mixed sperm

.

-

-

-

Dauch et d..1966

4.2

-

Edington, 1956 Edington & Randolph, 1958

5.2 NON-MAMMAUW GERM CELL WUJlIES

A W R B E D DOSE (Gy)

Fig. 6 2 The hquenciea of translocations between the -nd and third chromammes induced by 0.68 MeV neutrons and 250 kVp x rays in mature sperm ofDmsophila melanogastcr.Data points fmm different experiments are mpreeented by different symbols.The lines are the beat fitting regmion lines. (Adapted from Gonzales 1972). -

~

energy. The studies of Abrahamson et al. (1981)[see Figure 5.51 employed five neutron energies ranging from 0.43 MeV to 15 MeV for the induction of recessive lethals in oogonia for which an extensive dose-response curve for low LET radiation had been developed (NCRP, 1980).The highest RBE was found with the 0.43MeV monoenergetic neutron irradiation with the RBE decreasing with increasing neutron energies. Another important observation that emerges &om the data presented in Table 5.3 is that the RBE appears to be related to the type of endpoint studied in the same germ cell stage. Higher RBE values are generally found for dominant lethals, intermediate RBE values

1

5.2 NON-MAMMALIAN GERM CELL STUDIES

I

I

I

1

99

I

7-

6-

5U)

NEUTRONS

-

I

tW a

-

2

0

I

1

I

15 20 25 DOSE (GY) Fig. 6.3 The hquenciea of sex-linked receesive lethal mutations induced by 0.68 MeV neutrona and 250 kVp x r a y in mature sperm of D m p h i l a melanogasier. The linea are the beet fitting linear regressions. (Adapted from Gonzales 1972.) 5

10

for tranalocatione and the lowest RBE values are found for recessive lethal mutations. This could imply that there ia an increasing set of restrictions on these corresponding endpoints. For example, the breaks leading to dominant lethal damage may occur anywhere in the genome, those leading to translocation must be more spatially restricted and also still preserve both the integrity of chromosome organization and the viability of the zygotes derived from these

101

0

I

:

I 10

I

I

20

I

I

30

I

I

40

I

50

AB!X)RBED DOSE (Gy) Fig. 5.4 The frequenciesof surviving sperm after exposure tO 0.68 MeV neutrons or 250 kVp x rays. The lines are the beet fitting regreeeione.When two or more pointa were superimposed they were moved to one side. (Adapted from Gonzales 1972.)

gametes. Finally, the deletional events or aberration classes leading to recessive lethal damage must be even more spatially limited in order to permit survival of the zygote carrying the deleted segment of the chromosome; the larger the deletion of needed genetic material the greater the likelihood of an inviable zygote. Therefore, it appears that the changing RBE value reflects the changing capacity of the cell (or organism) to survive the type of genetic damage initiated. The data also suggest that the RBE value will also be germ cell stage specific (see for example Dauch et al., 1966where the same end point is measured in different stages of spermatogenesis). It are however the x-ray data that contribute the major variation to the RBE and may reflect different radiation sensitivities, due to, for example, oxygenation states ofthe sperm stages sampled. More data will, however, be needed to verify this.

Fig. 5.5 Dose response curves for Drosophih oogonia x-linked recedve lethal studies. Compares acute x rays, single d m (solid circles), fractionated (solid triangles) where x-ray responae is fitted by linear quadratic equation and neutron curves fitted by linear regression analyses. (Adapted from Abrahamson et al., 1981.)

If the packing and coiling of chromosomes is very tight, as in sperm, then a neutron track will potentially produce a much larger deletion than the same track traversing an extended chromosome, for example, in interphase (as in gonial cells). These larger deletions will often be inviable, reducing the yield of recessive lethals. The work of Sobels and Broerse (1970)on lethal and translocation production in sperm and spermatids with 15 MeV neutron energies may be an indication of this phenomenon. The RBE for translocations is one while it is less than one for recessive lethals. Sobels, on the other hand, suggests that the differences cited are due to different levels of oxygenation of the germ cell stages studied. 5.2.2 Silkworm Studies

The extensive work on silkworm germ cells has been primarily carried out at the National Institutes of Genetics in Japan by Tazima and colleagues. The end points involve "mutations" to pigment color changes in eggs and dominant lethality. The specific locus mutation studies have been carried out by irradiating different germ cell stages of the silkworm. It is important to note at the outset that

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5. HEREDITARY EFFECTS-Il

specific locus mutations recovered in eggs are not comparable to such mutations in Drosophila or the mouse, where these are scored in F, or F, adult progeny (see SchaIet and Sankaranarayanan, 1976,for a critique). Silkworm color mutants of the egg serosa membrane covering involve the pink gene (locus 0.0 chromosome 5) and the red gene (locus 31.7 on chromosome 5). The induced mutants recovered in the diapausing egg stage (the longest stage of development of the organism, lasting approximately one year) are, in the majority of cases, apparently not viable to the larval or adult stages andlor the experiment is rarely extended to determine this viability. Thus, in a sense, these specific locus events may well be a component of the much larger class of dominant lethals which prevent hatching of the fertilized egg. The dose response curve for the mutational events in mature sperm is linear for gamma rays in the dose range of 20 to 60 Gy and linear for 14 MeV neutrons in the dose range of 10 to 50 Gy. Thus,whatever the nature of these events, they appear to follow onehit kinetics in mature sperm. Non-linear, that is quadratic or linear quadratic type, kinetics is the typical response for both neutron (1.5 MeV and 14 MeV) and gamma ray irradiation in early and late spermatogonial stages, and oogonial stages and oocyte stages studied when the dose range was extensive. This non-linear response for neutrons is unusual. One possible explanation for these unusual results is that the pe and re mutations in these germ cell stages result primarily from large deletions or other chromosome rearrangements of a 2-break 2-track nature, which could permit survival of the germ cells through the subsequent mitotic and meiotic gametogenesis stages. (In the mature sperm studies, however, single chromosome breaks deleting the loci under study would suffice to give the required egg phenotype and subsequent dominant lethality.) The unique aspect of these results in gonia lies in the fact that the neutron radiation also requires that two independent tracks produce the event. With respect to RBE, 1.5 MeV neutrons are more efficient that 14 MeV neutrons which are, in most cases, more efficient than gamma rays (l3ICs) (see Table 5.4). Higher RBE values are obtained for dominant lethals in sperm (RBE = 10) than for the "mutational eventsn (RBE = 6). The RBE values in these papers are, however, underestimates because in all cases, they were compared with acutely delivered gamma radiation. However, a dose rate reduction effect for gamma irradiation ranging from two to four at the lowest dose studied, has been demonstrated in early gonial cells (Tazima and Kondo, 1963). For late gonial stages, a reverse dose rate effect has been found due to blockage of cells in G2 flazima, 1969).

/ 5. HEREDITARY EFFECTS-ll

6.2

NON-MAMMALIANGERM C E U STUDIES

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105

63.3 RBE Values For Interspecies Genetic-CytogeneticEndpoints The RBE value is a measure of the relative efficiency of two different forms of radiation inducing a particular type of genetic endpoint under equivalent biological conditions (cell type and cell stage, for example). While a range of RBE values can be noted for an endpoint aa a result of different physical and biological experimental conditions nonetheleas some generalities seem worthy of enumeration. (1) For many plant systems with either greater DNA content per cell or larger nuclear volumes than mammalian cells, the RBE values tend to be larger than those observed in mammalian test systems, by a factor of two or more. (2) Studies in mammalian cell or germ cell systemsreault in larger RBE values than those observed in insects, Dmsophilu and Bombyx, by factors of four or more. Again, this result probably reflects the different DNA contents, target size, and packing conditions of the target in the cell stages or types studied. (3) RBE values in general are higher for a centric-dicentric induction than for other genetic endpoints induced in the equivalent cell because little or no selection intervenes between the induction process and the observation of the induced event.

High-LET Radiation

6.1 Introduction

The determination of RBE values for the induction of cancer in various tissues depends on the determination of precise doseresponse curves for each tissue exposed to the specific radiation under study and to a reference radiation. The problem with obtaining adequate dose-response curves is that, on the one hand, experimental data are not sufficiently precise to choose among various models and on the other hand the understanding of the various mechanisms of carcinogenesis in different tissues is not sufficient to formulate appropriate models for fitting experimental data. Because of differences in sensitivities among tissues and also because of possible differences in mechanisms of carcinogenesis in different tissues, the alpha term, in equation 2.1, for low-LET radiation may vary considerably. In fact, data for at least two experimental tumor types, ovarian tumors and lung tumors in RFM mice, suggest an initial slope of zero, (i.e.,a threshold). The initial slopes of neutron dose-response curves also vary between tumor types but seemingly less than with low-LET radiation. Thus,it is not suprising that the estimates of RBE based on the experimental results vary markedly. This wide range of values underlines the problems of using experimentally derived RBE values for determining quality fadors. In the following sections, studies are reported of the comparative tumorigenic eff& of high- and low-LET radiations on various tissues and organs. It will be noted that it is only in a few studies that the initial slopes of the responses for both the high- and low-LET radiations have been determined and then, only with broad c o d dence limits. Thus, the estimated RBE values have considerable error.

6.2 LEWMIAINMICE

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6.2 Leukemia in Mice

6.2.1 Myeloid Leukemia Upton et al., (1970)carried out an extensive study of the life shortening and tumorigenic effects in both female and male RFM/ Un mice, using protracted neutron irradiation with a broad range of dose rates. The induction of myeloid leukemia was studied in male mice with single doses of fission neutrons delivered a t 0.85Gy min-I to over the range of 1.3 to 3.32 Gy and at dose rates of 1.2 x 2.43 x Gy min-I over the range of 0.17 to 1.2 Gy (see Figure 6.1).Other groups were exposed to x rays at 0.8 Gy min-I over the range of 0.25 to 1.5 Gy and to low dose-rate gamma radiation at 3.7 x to 5.7 x Gy min-I over the range of 1.48 to 58.13 Gy. The aim of the experiment was to investigate the effects of dose rate. It was found that the effect of neutrons was not diminished by daily fractionation or by lowering the dose rates, whereas, with gamma rays a marked reduction occurred with such exposure regimens (Figure 6.1).Since the effects of single doses below 1 Gy were not studied,

Fig. a1 Incidence of myeloid leukemias in RFM male mice as a function of dose. Single neutron expoeurea M. Single gamma exposures M. Daily neutron exposures -. Daily gamma irradiation -. (Adapted from Upton et al., 1970.)

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the RBE for single low doses cannot be estimated directly. It was possible from these studies, however, to derive an RBE estimate for low dose rate exposures since both the neutron and gamma ray responses appeared linear following low dose irradiation. Upton et d., (1970) estimated the RBE value to be 16 for myeloid leukemia when the effects of daily chronic neutron and gamma irradiation were compared. In a later study, Ullrich et al. (1976) also used the R F W n mouse and with lower dose levels (0.048 to 0.47 Gy at 0.05 Gy min-' and 0.24 to 1.88 Gy at 0.01 and 0.25 Gy min-I). However, they employed "barrier protected" animal quarters. The incidence of myeloid leukemia was too low to allow analysis. Despite the use of the same mouse strain as in Upton's study it appears that the change in microenvironment resulting from the different housing altered the susceptibility to both naturally occuning and induced myeloid leukemia. This dramatic effect of a change in the environment underlines the importance of host factors in carcinogenesis. Because other major neutron studies a t Harwell, Oak Ridge National Laboratory, Argonne National Laboratory and the I.T.A.L. reactor in the Netherlands either were not designed to study the induction of myeloid leukemia at low doses, or used mouse strains with low susceptibility, there has been a lack of data suitable for the determination of the initial slope of the dose-response curve for myeloid leukemia. Attempts to c o r d the gap in the information for the induction of myeloid leukemia with low doses of fission neutron radiation have been made recently. Mole and Davids (1982) have reported on the induction of tumors in general and acute myeloid leukemia, in particular, after exposure of CBAIH male mice to fission neutrons (0.02,0.05,0.1,0.2,0.50,1 and 2 Gy)The best fit to the data was I = clDe-AD,where I is the incidence, D is the dose and -ADrepresents the probability that an initiated cell will retain the productive capacity and a is the coefficient of the linear component, with a = 4.05 2 1.25 x lo-' cases of myeloid leukemia per Gy-l. In a separate study of the induction of myeloid leukemia by x rays, the incidence was thought to depend on the square of the dose; and the dose-response curve could be adequately described by a dose-squared response adjusted for cell killing (Figure6.2);I = a D2e-)rD(Major and Mole, 1978;Mole et d., 1983). Based on these reported dose-response relationships, the RBE for the induction of myeloid leukemia by fission neutrons would be a function of D,-* where D, is the neutron dose. It is obvious that this conclusion depends on whether or not the response to the reference radiation is proportional to D2. An analysis of these data by Ullrich

6.2 LEUKEMIAINMICE

1

109

rnyeloid

4

2

X ray dose (Gy) Fig. 6.2 Incidence of acute myeloid leukemia in CBAIH male mice as a h c t i o n of fission neutron dose. The bars represent 2 80 percent Poisson confidence limits. (Adapted from Mole et al., 1983).

and Preston (1987) assuming linear responses suggested an RBE value of 2.8. The induction of myeloid leukemia has been studied by Ullrich and Preston (1987) in RFM male mice exposed to 0.5 to 0.8 Gy of fission neutrons at the ORNL Health Physia Research Reactor (HPRR). It can be seen in Figure 6.3 that the neutron curve is consistent with a linear response (y = 0.89 k 0.16D), with no evidence of bending over. The slope of the curve is about 2.5 greater than the slope for the response to gamma rays when the data are fitted by a weighted linear regression. Based on the ratios of the slopes for the two radiation qualities, an RBE value of 2.5 was obtained. While both studies (Mole and Davids, 1982; Ullrich and Preston, 1987) provided data for neutron response of low doses, the RBE estimates were for high dose rate exposures. It is not known whether higher RBE values would have been observed following low dose rate exposures. Specific types of leukemia appear to be associated with specific chromosome aberrations (Rowley, 1984). In RFM mice, aberrations, especially involving chromosome two, appear to be associated with myeloid leukemia (Hayata et al, 1983). The shapes of dose-response curves of tumor incidence as a function of dose are influenced by a

. 0

Neutmns

y rays

Fig. 8.3 The percent incidence of myeloid leukemia in male RFM mice as afunction of dose. Single neutron expoeuree W. Single lS7Cegamma ray expoeure M. (Adapted from Ullrich and Preston, 1987.)

number of factors. Therefore, the shapes of such curves cannot be necessarily deduced from the shapes of curves for the induction of the initial events, for example, from dose-response curves for the induction of chromosome aberrations. However, it may be noted that neither a D2 nor a linear response are consistent with the observed induction curves for deletions following low-LET radiation. In view of the above, despite the considerable amount of data obtained a t low doses, especially for fission neutrons, any estimate of RBE for the induction of myeloid leukemia must still be considered tentative.

66.2 Thymic Lymphoma In the study of Ullrich et al. (1976), there was a greater increase in the incidence of thymic lymphoma after single exposures to fission neutrons a t the Health Physics Research Reador (HPRR)compared to 13'Cs gamma radiation up to 2 Gy. Because the lowest dose level of exposure was 0.24 Gy and the dose-effect curve tended to bend over and reach a plateau a t 1 Gy, it was impossible to estimate the initial slope (Figure 6.4). To investigate the lower dose levels, exposures to HPRR neutrons at 0.05 Gy min-I were used (Figure 6.5). A linear relationship described the curve: I = 5.2 + 0.5 D. Where I = percent incidence of thymic lymphoma, 5.2 is the percent incidence in the unirradiated population and D the dose in Gy of fission neutrons. A potential complexity is the ratio of gamma ray

6.2 LEUKEMIA IN MICE

1

111

DOSE (Gy) Fig. 6.4 Excess incidence of thymic lymphoma in RFM female mice as a b e t i o n Chronic neutron exposures W.Single gamma of dose. Single neutronexpoeure U. radiation exposures M.(Adapted from Ullrich et al., 1976.)

dose to neutron dose in the HPRR beam. Although for the 0.048 to 0.47 Gy portion of the dose response, the gamma radiation component of the dose is considered to be of minor importance. About 0.07 Gy of the total 0.47 Gy dose was gamma radiation, a dose that has no measurable effect on thymic lymphoma incidence. Also, the f a d that there is little difference in the initial slope, irrespective of the neutron dose rate, suggests that the contaminating gamma radiation had little effect. By combiningthe data obtained from different series exposed a t somewhat different dose rates, it appeared that the initial response was linear followed by a plateau. The dose response following x ray irradiation was more complex. Over the 0 to 0.5 Gy dose range the response was adequately described by a dose squared relationship. Since, over this dose range, the gamma ray dose response varied with the square of the dose and the neutron response varied linearly, it followed that the RBE value for neutrons varied with the square root of the neutron dose in Gy according to the equation: RBE = 2 D$6 (6.1) While the dose response was markedly reduced following low dose-

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Fig. 6.6 Excess incidence of thymic lymphoma in RFM female mice as a function of dose. Single neutron exposure O-O. Single gamma radiation exposure M. (Adapted from Ullrich et al., 1976.)

rate gamma ray irradiation, Ullrich et al., (1976) did not observe an effect of dose rate over 0 to 1 Gy dose range following low doserate neutron irradiation. A comparison of the slopes of linear dose response relationships for low dose rate gamma rays and neutrons results in an RBE estimate of approximately 27. Maisin et al. (1983) found that with BALBIc mice, single doses of 23 MeV (modal energy) neutrons were not more effective in the induction of thymic lymphoma than 13Tsgamma radiation over the dose ranges studied of 0.02, 0.06, 0.18, 0.55 and 3 Gy neutrons and 0.25, 0.5 1, 2, 4 and 6 Gy gamma rays. The data from Argonne National Laboratory comparing fission neutrons and gamma rays also suggest induction of thymic lymphoma (Grahn et al., 1986). It should be noted that the optimal regimen of irradiation for the induction of thymic lymphoma with neutrons has not been determined as it has for low-LET radiation, and thus no comparison has been made using the optimal regimens for both radiation qualities. Radiation-induced damage to hematopoetic tissue has long been considered an essential feature in the induction of thymic lymphoma. If this is so, then RBE values that are consistent with RBE values for cell killing should be expected.

6.2 LEUKEMIA IN MICE

1

113

66.3 Other Lymphomas Neither the studies of Upton et al. (1970)nor those of Ullrich et al. (1976; 1977) reveal the precise nature of the dose-response relationships of the induction of non-thymic lymphomas by neutron irradiation. Mewissen and Rust (1976) reported enhancement of the incidence of reticulum cell sarcoma aRer neutron exposures. Since the data are presented as age-specific rates, the dose response is not immediately apparent but the age-specific rates aRer exposure to 0.032,O. 045,0.063,0.088 and 0.123 Gy are above control levels over the age-span studied. The authors noted that the type of lymphoma seemed to change from the histiocytic (Type A Dunn's nomenclature) in control mice to the Hodgkin's like lymphoma (Type B), in irradiated mice (gamma and neutron). Neither Covelli et al. (1976) nor Ullrich et al. (1976) found any increase in reticulum cell sarcomas &r exposure to neutrons. Covelli et al. (1976), in a study of the comparative effects of 250 kVp x rays and 400 MeV neutrons found no effect on the incidence of nonthymic lymphomas of doses ranging from 0.11 to 0.82 Gy with the direct beam or 0.07 to 0.58 Gy of an attenuated beam. The 4 Gy x ray group was the only group to show an effect and this was interpreted to be an advancement in time of appearance of lymphomas rather than a change in incidence. Covelli et al. (1984) have compared the decrease in reticulum cell sarcoma observed &r exposure to x rays and neutrons. The effect was greater with neutrons. The dose-effect pattern was influenced by the age at exposure with x rays but not with neutrons.

6.2.4

Dose-Rate Effects

Data from RFM male mice obtained by Upton et al. (1970) are insufficient to define precisely the effect of neutron dose rate on the induction of myeloid leukemia. However, the evidence does not suggest that exposures a t very low neutron dose rates (1.7 to 34 mGy d-'1 influence the leukemogenic effect over total doses below 1Gy. In the case of thymic lymphoma, a decrease in the dose rate does not change the leukemogeniceffeds significantly in the range 0 to 1Gy. But a total dose of 1.88 Gy fission spectrum neutrons from 2S2Cfat 0.01 Gy d-l was significantly more tumorigenic than 1.88 Gy of fission spectrum neutrons h m the HPRR reador a t 0.25 Gy min-'. A similar effect has been found for life shortening and for some epithelial tissue tumors to be discussed later.

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6.3 Epithelial Cell Tumora 6.3.1 Ovariun Tumors

In the RFM mouse the incidence of ovarian tumors was found by Ullrich et al. (1976)to rise rapidly as a function of dose with single exposures to fission neutrons up to about 1 Gy and then plateau at an incidence of 40 to 50 percent (see Figure 6.6).In BALBIc mice a similar effed was found (Ullrich et al., 1977) although the effect reaches a plateau a t about 0.5 Gy (see Figure 6.7). In further studies with the BALBIc mouse (Ullrich, 19831, the shapes of the dose-response curves have been determined after exposures to fission neutrons at six dose levels of 0.025 to 2 Gy or 0.1 to 2 Gy of 13'Cs gamma rays. The overall similarity of the responses to the two radiation qualities can be seen in Figure 6.8. A possible difference between the two curves is limited to doses below about 0.20Gy.Both sets of data suggest a low RBE for ovarian tumors. An

Fig. 6.6 Excegs incidence of ovarian tumor~in RFM mice aa a function of doee. HPRR neutrone at 0.25 Gy min-I w.ss3Cf neutrana at 10 mGy d-I M. ImCa gamma raye, 0.45 Gy min-I A-A. (Adapted fmm Ullrich et al., 1976.)

6.3 EPITHELIAL CELL TUMORS

1

115

801OVARIAN TUMORS

zui

/

0

// &o/o-

0

o A

acute neutron chronic neutron acute gamma my

I

Fig. 6.7 Incidence of ovarian tumors in BALB/c mice as a function of dose. HPRR neutrons, 0.25 Gy min-I M. W!f neutrons, 10 mGy d-I m.lacs gamma rays, 0.45 Gy min-I A-A. (Adapted h m Ullrich et al., 1977.)

important finding is that a linear neutron dose response could be rejected and that the response to gamma radiation is consistent with a threshold type of response which, in turn, is consistent with the accepted mechanism of radiation-induced ovarian tumors. It is thought that ovarian tumors arise when the oocytes are killed and the perturbed pituitary-gonad axis results in elevated levels of gonadotrophins. Such a mechanism would entail a threshold dose, and the extent of the threshold for a particular radiation quality would depend on the effediveness of the radiation to inactivate oocytes. In the experiment shown in Figure 6.8 the threshold, for gamma radiation, is about 0.1 Gy. This complex tissue response is not observed for other tissues and may be unique to the mouse ovary. 6.3.2 Dose-Rate Effects

In RFM and BALBlc mice Ullrich et al. (1976; 1977) and Ullrich (1984) found that the tumorigenic effect of 262Cfneutron radiation was reduced by reducing the dose-rate (Figures 6.6,6.7, 6.9). This

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Fig. 6.8 Age adjusted incidence of ovarian tumors in BALBJc mice as a function of dose. Fission neutrons M. '3'Cs gamma radiation M. (Adapted h m Ullrich, 1983.)

finding was surprising. The reduction in the tumorigenic effect that results from lowering the dose rate of low-LET radiation has been explained by the reduced killing of oocytes. However, lowering the dose rate of neutron radiation should not decrease the efficiencyof cell inactivation or, therefore, decrease the tumorigenic effect. Either the accepted explanation of the mechanism of induction of ovarian tumors in mice is incorrect, or, as seems likely, the gamma contamination of 25%f radiation is important. It was pointed out in Section 6.2.2 that i t was very unlikely that a small gamma radiation contamination would make a significant contribution to the resulta in the case of thymic lymphoma. However, in the case of the ovary, which is known to be exquisitely sensitive, the gamma radiation may be important, although the difference in the effects of exposures at the different dose rates is greater than is expected from a low-LET dose-rate effect. Another fador of importance could be the reduction in susceptibility for radiation-induced ovarian tumors with age (Yuhas, 1974), particularly with longer

6.4 LUNGTUMORS

1

117

='1

Fig. 6.9 Incidence of ovarian tumors in mice as a function of neutron dose and dose rate. HPRR neutrons 0.25 Gy min-I M. Data pooled h m 10 mGy d-I and 0.1 Gy d-' experiments O-O. (Adapted from Ullrich, 1984.)

protraction and higher total doses. The explanation of the dose-rate effect will have to be resolved using a high-LET radiation beam with minimal gamma ray contamination. Because of these unexplained results the RBE value estimates for ovarian tumors may not be useful for estimates of RBE in other species.

6.4 Lung Tumors

6.4.1

Types of Lung Tumors

If tumors arise from different cells a difference in their doseresponse reIationships would seem possible. Equally important is whether or not the type of tumor occurring naturally in the particular mouse strain under study is the same type of tumor induced by the radiation. Murine lung tumors arise from two cell types, namely, Type 2 alveolar cells and Clamcells. While identificationby electron micros-

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EXPERIMENTAL CARCINOGENESIS

copy is the method of choice for determining the cell of origin of lung tumors, this is neither practical, nor economically poseible, in large scale radiation studies. Based on light microscopicexamination, lung tumors are frequently classified into alveolar and bronchiolar. The former are thought to arise from Type 2 cells and the latter from CZum cells. The proportion of the two types of tumors occurring naturally, and after exposure to carcinogenicagents is strain dependent and is influenced by the type of inducing agent. It has been suggested that the adenocarcinomas arise from Clam cells (Kaufmann et al., 1979).Others believe a clear distinction between pulmonary adenomas and adenocarcinoma is not possible (Stewart et al., 1977),although it is apparent that murine lung tumors can differ markedly in their malignancy.

6.4.2

Lung Ademma

In female RFM mice, single doses of whole-body gamma radiation, below about 0.10 Gy, appeared to reduce the lifetime incidence of lung tumors (Ullrich et al.. 1976). The incidence was increased with doses between 0.1 and 1Gy. Following neutron irradiation, the incidence of adenomas was increased at all doses tested. This increase in incidence was independent of neutron dose rate. With the lower dose rate of 0.01 Gy d-I, the data suggest that the tumorigenic effect may saturate at about 0.25 Gy (see Figure 6.10). Maisin et al., (1983) have reported a significant decrease in lung carcinomas after exposure to 0.02 Gy of 23 MeV neutrons. The effects of irradiation by fission neutrons and 300 kVp x rays on the production of lung tumors in RFM female mice has been studied by Ullrich et al. (1979). The average number of tumors per mouse was determined as a function of neutron dose, up to 1.5 Gy, a t nine months after irradiation (see Figure 6.11). The number of tumors per tumor-bearing mouse rose in a linear fashion, in the range of 0.05 to 0.25 Gy, while from 0.25 to 1Gy there was a less marked increase. The maximum number of tumors per mouse was about three times the control value. At 1.5 Gy the average number of lung tumors decreased to about twice the control value (see Figure 6.11). A similar pattern is seen when the prevalence of lung tumors (the percent of tumor-bearing mice) is plotted as a function of dose (Figure 6.12). The relationship between tumors/mowe and x-ray dose could be adequately described by either a linear quadratic or a threshold model (see Figure 6.11), while the data for neutrons could be described by either a linear or a threshold model (see Figure 6.11). Questions about the biological significance of these tumors in

Fig. 6.10 A g e - a d j W incidence of lung adenomas in RFM female mice an a 262Cfneutrons, 10 mGy d-' function of d m . HPRR neutrons, 0.25 Gy min-' M. M.'"Ce gamma rays, 0.45 Gy m i n - I D-LI. (Adapted from Ullrich et al., 1976.)

relation to human cancer have been discussed and debated by many authors. Whether radiation induces these tumors or rather accelerates their time of appearance is also an unresolved question. Because of these biological uncertainties and the uncertainty about the form of the dose response, the application of these data for derivation of generally applicable RBE estimates may not be appropriate. In BALBIc mice, a decreased incidence was found for neutrons in the range between about 0.05 to 0.2 Gy dose levels and even in the 0.24 to 1.88Gy dose levels the tumor incidence did not exceed control levels. With exposures a t 0.01 Gy d- the incidence decreased from 28 percent a t 0.24 Gy to 13 percent a t 1.88 Gy commpared to a control level of about 25 percent. Exposure to 0.5 or 2 Gy of gamma radiation had no effect on the lung tumor incidence. 6.4.3

Lung Adenocarcinoma

In the case of pulmonary adenocarcinomas, Ullrich et al. (1977) found an equally unexpected neutron dose response in their initial

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0.6 0.8 1.0 NEUTRON DOSE (Gy)

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1

1.5

4 5 6 7 8 9 X-RAY DOSE (Gy) Fig. 6.11 Average number of lung turnore per mouse (RFMfemale) ae a function of dose. A: 300 kVp x rays at 4 Gy min-I,and B: HPRR neutrone at 0.05 to 0.25 Gy min-I. (Adapted from Ullrich et al., 1979.)

0

1

2

3

1

6.4 LUNGTUMORS

80

I

0 1 0

121

LUNG TUMORS

r

I

I

0.2

0.4

I

0.6

I

0.8

I

1.0

2

.

d

1.5

DOSE (GY) Fig. 6.12 Percent of tumor-bearing mice (RFMfemale) at nine months as a function of dose. HPRR neutrone at 0.05 to 0.25 Gy min-I. (Adapted from Ullrich d al., 1979.)

studies. There was a steep initial rise in incidence reaching a maximum incidence that was about three times the control incidence of pulmonary adenocarcinoma, followed by an equally abrupt fall to the level of control incidenceswith no subsequent increase with dose. With an exposure rate of 0.01 Gy d-l, the incidence after 0.47 Gy was about the same as control levels and less than after exposures at 0.04 Gy min- but with increasing total doses the incidences rose above those after single exposures. These unusual dose-response curves remain unexplained. In a more recent and more detailed study, Ullrich (1982) found the initial part of the neutron dose-response curves rose rapidly between 0.01 to 0.20 Gy and started to plateau between 0.2 to 0.4 Gy (Figure 6.13). It was not possible to determine statistically whether the response should be represented by a continuously bending curve or an initial linear component with an additional component influenced by one or more factors other than induction. However, based on the results of fractionation and protraction regimens, it is considered that the initial slope is linear (Ullrich, 1982,1984).The dose response following gamma ray exposures is best described by a linear-quadratic model. A comparison of the initial linear slopes results in an RBE estimate of 18.5.

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DOSE (GY) Fig. 6.13 Incidence of pulmonary adenocarcinomas in female BALBIc mice as a function of dose. Fission neutron u. 137Csgamma rays M. (Adapted from Ullrich, 1983.)

6.4.4 Fractionation and Dose-Rate Effects on Lung Tumors

The results of the study by Ullrich (1982) of the influence of splitting the neutron dose into two equal fractions separated by 24 hours or 30 days on the dose response are shown in Figure 6.14. When the interval between the two doses was 24 hours the response was not significantly different from that after a single exposure. In contrast, the response to two fractions separated by 30 days was different from the response to a single dose when the total dose was 0.5 Gy. The curve for the total dose range (0 to 0.5 Gy) was fitted by a linear regression (Y = 14.8 + 72 D) where D is the neutron dose in Gy and the slope coefficientwas not significantly different from that obtained for the 0 to 0.2 Gy dose range. Protraction of neutron exposures of 0.01 to 0.1 Gy d-I gave results similar to those found with the 30-day split doses. The dose-response curve for protracted irradiation appears to bend over less than the single dose-response curve (Ullrich, 1984). Since dose rate had little influence on the initial slope of the neutron dose response, the RBE (f$y) for low dose rate exposure when estimated from the ratio of the linear slope coefficients is 18.5. Ainsworth et al. (1977) analyzed the data from studies a t Argonne based on whether the lung tumor was the cause of death or found

6.5 WMMARY TUMORS

1

123

FlSSlON NEURON DOSE (Gy)

Fig. 6.14 Excess incidence of pulmonary adenocarcinomas as a function of total neutron dose. Single doses o-e. Two equal doses separated by 24 hours M.Two equal doses separated by 30 days w.(Adapted from Ullrich, 1982.)

incidentally a t autopsy. The change in age-specific mortality rates as a result of exposures of female B6CF,/Anl mice to single or fractionated exposures to neutrons suggest an earlier time of appearance rather than an induced increase in incidence (see Figure 6.15). The shortening in time of appearance was greater when 0.8 Gy was given in 24 fractions than when given in a single exposure. Preliminary analyses from the Argonne National Laboratory study with JANUS reactor fission neutrons suggest that the RBE for mortality due to lung tumors after single doses for both sexes combined is 15 to 20 and may be somewhat higher with either 24 or 60 weekly fractions.

6.5 Mammary Tumors 6.5.1 Mammary Adenocarcinomas

Earlier studies in BALBIc mice, which have a natural incidence of mammary tumors of about eight percent (Ullrich et al., 1977), indicated a complex response to single doses of fission neutron radiation. With exposures a t low-dose rates, the incidence increases with

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.

AGE, days

Fig. 6.15 Age-specific mortality rates for lethal lung tumors in female BGCF,/Anl mice exposed to JANUS reactor fission spectrum neutrons. Fission neutrons of 2.4 Gy in 24 weekly fractions M, fission neutrons of 0.8 Gy in 24 weekly fractions w, fission neutrons of 0.8 Gy in a single dose X--X; and controls W. (Adapted from Ainsworth et al., 1977.)

dose but much less steeply than with single high dose-rate exposures. At about 1Gy the effect is similar irrespective of dose rate, but after a total dose of about 2 Gy, the tumor incidence is over three times greater in the low dose-rate exposure group. More recently, Ullrich (1983)has examined effects on mammary tumor induction in more detail. These studies have determined the dose-response relationships for the induction of mammary cancer in BALBIc mice exposed

6.5

MAMMARY TUMORS

1

125

to single doses of 0.025 to 2 Gy h i o n neutrons and 0.1 to 2 Gy 13'Cs gamma rays (see Figure 6.16). After exposure to 0.025 and 0.05 Gy of fission neutrons the incidence rises steeply but starts to bend between 0.05 and 0.1 Gy. The data for the 0 to 0.1 Gy doses of h i o n neutron were fitted by a linear regression (Y = 7.0 + 114 D) where D is the dose in Gy. Over the range of 0 to 0.5 Gy either a linear or a square root of the neutron dose response describe the data. Similarly, the doses of importance for determining the initial slope of the dose response to gamma radiation are below 0.25 Gy. These details underline the requirement for data for very low doses in order to determine, experimentally, the shapes of the dose-response curves for the increase of cancers after exposure to either fission neutron or gamma radiation. The appropriate models for the responses to gamma and neutron irradiation are equally difficult to select from current data. However, if the slope of the response after exposure to low dose-rate gamma radiation of 3.5 excess cases Gy-I (Ullrich et al., 1977) is assumed to represent the initial slope of the response, an RBE of about 33 is obtained from the ratio of the initial slopes of the responses of the two radiation qualities.

Fig. a16 Incidence of mammaqj adenoeareinomas in female BALBlc mice as a (Adapted h m function of d m . Fiseion neutrous u.ImCs gamma radiation u. Ullrich, 1983.)

In a study of C3H mice (that have a high natural incidence of mammary tumors) exposed to 14 MeV cyclotron neutrons or x rays, Hornsey (1982) found the appearance of tumors was advanced in time at each dose level down to 0.02 Gy. An RBE value of 11 was obtained when the time a t which 50 percent of the mice had developed tumors was used as the endpoint. 6.5.2 Dose-Rate and Fractionatton Effects on Mammary Tumors

Low-dose rate exposure to ==Cf neutrons appeared to induce a higher incidence of mammary adenocarcinomas than a t low dose (0 to 0.2 Gy), high dose-rate exposures. However, split doses with a 30day interval did not increase significantly the incidence (Ullrich, 1984). Because of the marked increased incidence of mammary tumors at low doses following chronic exposures, the RBE estimate for low dose rate neutrons versus gamma rays increases from 33 to 77.

6.6 Harderian Gland

In RFM female mice (Ullrich et al., 1976), after exposure to single doses of fission neutrons, the incidence of Harderian gland tumors increased with dose, from the very low natural incidence, linearly up to 0.5 Gy; the response was somewhat greater a t 1 Gy but after 2 Gy the effect was minimal. Reduction of the dose rate resulted in a similar dose response but with a somewhat lower incidence, especially a t 1 Gy (see Figure 6.17). In BGCF, mice (with or without pituitary isografts) exposed to fission neutrons, the incidence rises steeply, but possibly with a complex response even over the 0 to 0.16 Gy dose range; above 0.16 Gy the response curve bends over (Fry, 1981a)see Figure 6.18. When the data were analyzed by the method of Kaplan and Meier-(1958) and the excess incidence (at 850 days) is plotted as a function of dose, an equally complex curve was obtained between 0 and 0.32 Gy and the curve bent over between 0.32 and 0.64 Gy. The ratio of Harderian gland tumors that caused death to the number of Harderian gland tumors found incidentally was higher in neutron irradiated animals than those exposed to gamma radiation, suggesting that the degree of malignancy could be associated with radiation quality. With total doses of 0.8 Gy, or greater, of fission neutrons, Fry et al. (1976) found that fractionation induced a higher incidence than with a single dose. However, with total doses below 0.4 Gy and

DOSE (Gy) Fig 6.17 Excess age-a4ueted incidence of Harderian gland tumors as a b c t i o n of dose of RFM female mice. HPRR neutrons, 0.25 Gy min-I M.26aCfneutrons, 1 mGy d-' M.lS7Csgamma 0.45 Gy min-1 M.(Adapted from Ullrich et al., 1976.)

fractions of 0.025 Gy per fraction, the dose-response curves for single and fractionated exposures were similar (Fry, 1981b).

6.7 Tumorigenesis in Rats

6.7.1

Mammary Tumors

The rat mammary gland, particularly of Sprague-Dawleyrats, has been used extensively by a small number of research groups to study mammary tumors. Tumor data have been obtained for neutrons of different energies: 0.43 MeV (Shellabarger et al., 1974,1980,1982); 0.5 MeV (van Bekkurn et al., 1979; Broerse et al., 1978,1982,1983); 14.5 MeV (van Bekkum et al., 1979; Broerse et al., 1978,1982,1983); 35 MeV neutrons (Montour et al., 1977) and fission neutrons (Vogel, 1969, 1978; Vogel and Dickson, 1982).

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60

W

50 -

0

z

40U

z

fi 3 0 -

6. EXPERIMENTAL CARCINOGENESIS

-

Pituitary Isoqraftfn Pitu~taryIsoqraft

0 fn

A fn

I-

m 3

$ 20-

Fig. 6.18 The age-adjusted increase of Harderian gland tumors as a function of and , without (MI dose of JANUS reador fission neutrons in BGCF, mice with (H) pituitary isografts. The incidence of tumors in mice exposed to 0.64 Gy fission neutrons (fn) before receiving pituitary isograRs (Adapted from Fry, 1981b.)

There are a number of types of mammary tumors that occur naturally and after irradiation. The probability that benign fibroadenomas will occur in Sprague-Dawley rats, even without irradiation, is high (virtually 100 percent) and almost certainly they occur earlier after irradiation. The data can be analyzed in terms of the advancement in time of tumor appearance, which in turn can be used to estimate RBE. Shellabarger et al. (1980) used this approach to estimate the RBE of 0.43 MeV neutrons compared to x rays (see Figure 6.19). As can be seen, the response to x rays appears to be linear, but there are no data below 0.28 Gy. The response for neutrons bends over at doses below 4 mGy or as the authors state is "sublinear" (Figure 6.19). Figure 6.20 gives the mean number of tumors as a function of neutron dose. This neutron curve also bends over between 4 and 16 mGy. The authors drew four conclusions from these studies: (1) That single doses as low as 1 mGy of neutrons (0.43 MeV) increased the total mammary tumor rate significantly. (2) The dose-response relationship for neutrons is consistent with a slope of about 0.5 on a log-log plot, and of one for x rays.

6.7 TUhIORIGENESIS IN RATS

129

1

Dn(cV) 0 1

1

I

I

.05 I

.1 1

1

I

1

NEUTRONS

I

Fig. 6.19 Advancement in time of occurrence of mammary fibroadenomas as a The scales for the neutron dose and function of neutron dose (D,,) and x-ray dose 0,). x-ray dose differ by a fador of 10 (Adapted from Shellabarger et al., 1982).

(3) The comparative responses to neutrons and x rays suggest that RBE values for neutrons may reach a t least 100 a t low doses. (4) The relationship of RBE to dose suggests that RBE is inversely proportional to the square root of the neutron dose. This relationship was formulated by Rossi and Kellerer (1972). The finding of a dose exponent of about 0.5 by Rossi and Kellerer (1972) was interpreted as evidence that deposition of energy in more than one cell must be involved in tumorigenesis in the mammary gland. The great interest in the experimental data obtained from Sprague-Dawley rats is, in part, due to the fact that effects have been determined directly a t very low doses. When the total tumor rate in the Sprague-Dawley rats was examined the contribution of carcinomas was not more than 14 percent, and therefore the conclusions are based primarily on the appearance of benign tumors. It is not known why benign tumors arise more rapidly and a t a greater frequency than their malignant counterparts. In addition, i t is not known whether different targets are involved in the two types of tumors nor whether the time required for the expression of malignant tumors is

1

130

EXPERIMENTAL CARCINOGENESIS

6.

1

I

1

I

1

I

I

4

-

1

1

-

X - RAYS

-

Fi.620 Mean number neoplasms (benign and malignant) per animal as a funcand x-ray dose (03. The scales for the neutron doee and x-ray tion neutron dose (D,) differ by a factor of 10 (Adapted from Shellabarger et al., 1982).

greatly in excess of that required for benign tumors. The forms of the dose-response curves for induction and the advancement of the time of appearance may well be different if the nature or.number of the targets involved in the mechanisms are themselves different. Shellabarger et al. (1983)have investigated whether the high RBE values obtained from the Sprague-Dawley data are a characteristic of rat strains with a high natural incidence (of which the SpragueDawley rat is one), by investigating RBE values in rats with a low natural incidence. In AC1 female rats the natural incidence of mammary tumors and the susceptibility to induction of tumors by irradiation is lower than in Sprague-Dawley rats. For a number of reasons it was possible to only estimate RBE values for higher doses than in the Sprague-Dawley rat, but the RBE values a t the higher doses were in agreement with those found in the Sprague-Dawley rats.

6.7 'lWtf0RIGENESISIN RATS

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131

The results obtained with high dose-rate fission neutron irradiation by Vogel(1969,1978)and Vogel and Dickson (19821,with Sprague-Dawley rats, are consistent with those of Shellabarger and coworkers. Montour et al., (1977)exposed Sprague-Dawleyrats to 0.025 to 0.2 Gy with 14.5 MeV cyclotron neutrons. The incidence of mammary neoplasms (about 10 percent were adenocarcinomas) at 11 months after irradiation, as a function of neutron dose could be fitted by a linear plot. These workers compared their results for the number of tumors per rat at 11 months with those of Shellabarger and his coworkers and contended that both sets of data could be represented by linear responses. On the assumption that the two sets of data could be compared on the basis of linear dose-response relationships, the response to 0.43 MeV neutrons was four to five times greater than to 14.5 MeV neutrons. The RBE values for 14.5 MeV neutrons were estimated to range h m five at 0.4 Gy to about 14 at 0.025 Gy. Broerse et al. (1983) have studied mammary tumorigenesis in three strains of rat:4 Sprague-Dawley, Wistar derived rats (WAG1 Rij), and Brown Norway (BNIBiRij) exposed to 0.5, 4 and 15 MeV monoenergetic neutrons and to x rays. Sprague-Dawley rats have been exposed to 0.5 MeV neutrons (0.025, 0.08 and 0.32 Gy), 4 MeV neutrons (0.04, 0.12 and 0.4 Gy), and 15,MeV neutrons (0.05,0.15 and 0.5 Gy) and x rays (0.1,0.3, 1 and 2 Gy). The other two strains were exposed to somewhat higher doses of all radiation qualities, for example 0.05, 0.2 and 0.8 Gy of 0.5 MeV neutrons and 0.25, 1 and 4 Gy x rays. Broerse et al. (1982, 1983)examined incidencesof benign and malignant tumors in WAG/ Rij rats separately. They found the dose-response curves for the induction of carcinoma in WAGIRU rats after exposure to 0.5 MeV neutrons or x rays to be linear. Thus, over the dose ranges examined, the RBE was constant and had a value of 10. In the case of benign tumors, the dose-response curves for the 0.5 MeV and 15 MeV neutrons bent over whereas the response to x rays was linear (see Figure 6.21). Thus, RBE values apparently increased with decrease in dose (Table 6.1). The RBE values determined at 0.05 Gy of 0.5 MeV TABLE6.1-Estimates of RBE values (0.5 MeV neutronslx mys) for mcrmmcuy tumors in different strains of rats (from Broerse et al., 1983) Neutron Dose (GY)

Mailgant tumors WAGlRij

WAGRij

Benign turnom Sprague-Dawley

BN/BiRij

Vrhe term strain is used for convenience but is not accurate because the SpragueDawley rats used in these and other experiments are not an inbred pure strain.

132 / 6. EXPERIMENTAL CARCINOGENESIS neutrons versus xrays for the induction of benign tumors was strain dependent varying from 5 to 25 (Table 6.1). The RBE values estimated from the data for Sprague-Dawley rats appear to be lower than those reported by Shellabarger et al. (1980). However, a comparison of the results for Sprague-Dawley rats with those obtained by Shellabarger et a2. (1980)is difficult because of the differences in the range of dose and the analyses that have been used. Furthermore, Broerse and coworkers (1982)have noted that the times of appearance of mammary tumors in unirradiated rats from their colony of Sprague-Dawley are considerably later in life than those found by Shellabarger and coworkers. It is clear that the 0.5MeV neutrons are more tumorigenic than the 15 MeV neutrons (see Figure 6.21).The studies of Yokoro et al.

0.5 MeV neutrons

absorbed d o s e (Gy )

Fig. 6.21 Incidence of benign mammary tumors in WAGIRij rats exposed to x rays neutrons A-A and 0.5 MeV neutrons -(Adapted from Broerse et al.,

M,15 MeV

1982).

6.8 STlJ'DIES IN OTHER SPECIES

1

133

(1980) and Fry (1981a) have also demonstrated that lower neutron energies are more effective. 6.7.2 Fmctwnation and Protraction The fractionation study of Broerse et al. (1982) revealed no difference in the induction of carcinomas in WAGIRij rats exposed to 2 Gy of 0.5 MeV neutrons in a single dose or 10 fkactions. Vogel and Dickson (1982) found that neutron radiation protracted over 1month with exposure for 13.5 hours d-I to total air kerma of 0.02, 0.06 and 0.50 Gy a t air kerma rates of 8.79 x 2.62 X and 2.15 x Gy h-l, and 2.15 x 10-l rad-l h-I), respectively, was more effective than single, acute exposures of 0.5 Gy air kerma. The assessment of the radiation effect was based on the percentage of tumor bearing rats at 10 months after irradiation. 6.7.3 Skin Burns et al. (1968) investigated the comparative effects of alpha particles (37 MeV) from a cyclotron and monoenergetic electrons (0.7 MeV) on skin turmorigenesis. From the responses to the series of doses for both radiation qualities, it is clear that the increase in tumorslrat occurs at lower surface dose levels for the alpha particles, reaching a peak effect a t 10 Gy compared to about 30 Gy for the electrons. However, the data are not adequate for precise RBE estimates.

6.8 Studies in Other Species There are few quantitative studies of radiation carcinogenesis in species other than Mus and Rattus and fewer that address the question of RBE and external high LET-radiation. Bradley and coworkers (Bradley et al., 1981and Zook et al., 19851, studied the effects of partial body fractionated irradiation with 15 MeV neutrons and cobalt-gamma rays on male beagles at high dose rates. Large total doses were used. Tumors appear to have a shorter latent period and to be multiple in the neutron irradiated dogs compared to those exposed to photons. The authors suggest that the data, although not suitable for estimating maximum RBE values, indicate an RBE value for tumors (types pooled) of six to seven. Bradley and coworkers (1977, 1979) have also studied the effects of partial-body

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EXPERIMENTAL CARCINOGENESIS

irradiation in rabbits exposed to 12 fractions of either 15 MeV cyclotron neutrons or 4 MeV photons from a linear accelerator. Of interest was the appearance of osteosarcomas within one year of exposure to neutron irradiation. The RBE values for reador fission neutrons for the induction of tumors in rabbits was also studied by Hulse (1980) using 2.5 MeV gamma rays as the reference radiation. The neutron doses ranged from 1.80 to 5.5 Gy and gamma ray doses from 4.4 to 14.1 Gy. Basal cell carcinomas, fibrosarcomas, testicular tumors and osteosarcomas were found. The doses in this study, while lower than in those of Bradley et al. (1977,1979) are high for the determination of RBEs of most interest. Hulse estimated an RBE of about 2.5 for osteosarcomas and 3.0 to 3.5 for other tumors. Broerse et al. (1981) have reported on a study of the effects of x rays and fission neutrons on Rhesus monkeys that were observed for more than 16 years after irradiation. Based on 79, 195 and 341 monkey-years for the fission neutrons, x rays, and unirradiated y-I Gy-I for x rays and 2.23 x groups, risk values of 5.7 x y-' Gy-' for neutrons were estimated. These risk estimates are based on the assumption of linearity of the dose-response relationship for both radiation qualities. The RBE based on these estimates is about four. 6.9 Dose R a t . and Fractionation It is clear that with the high-LET radiations there is a greater degree of additivity and dose-rate independence than with low-LET radiations. However, when doses are protracted, the effect for high LET radiation may be significantly greater than single exposures. This was seen to be the case for some but not all tumors in RFM and BALBIc mice (see Table 6.2). TABLE6.%Tumorigenic Tieaue

effect of neutrons in BALBlc mice (Adapted from Ullrich 1983, 1984) Single expures Slope coefficient

?

S.E.a

Lung Mammary gland ovary Protracted exposures

Lug 0.86 2 0.11 Mammary gland 2.7 2 0.94 ovary 0.27 2 0.09 The initial slopes of the dose-reapnee curves have been estimated from weighted linear regreasiona using the data for dosea from 0 to 0.2 Gy except in the case of the mammary gland for which data for 0 to 0.1 Gy single exposurea and 0 to 0.05 G y protracted exposum were used.

6.10 THE RELATIONSHIP OF LET AND RBE

1

135

Also, fractionation or protraction of fission neutron exposures has been found to increase the effectiveness somewhat in some tumor systems (Ainsworth et al., 1977; Vogel and Dickson, 1982; U'llrich, 1982,1983).It also has been reported that no increase in effect occurs with fractionation (Broerse et al., 1982). It appears that the dose per fraction and the total dose are important factors in the enhancement found with fractionation. For example, in one tissue, enhancement of fractionation has been reported with higher total doses but not with total doses below 0.4 Gy and small doses per fraction (Fry, 1981b).While it is clear that the timedose relationships for tumorigenesis are influenced markedly by the radiation quality, there is little understanding of the underlying reasons for the differences. One explanation of a greater effect of fractionation compared to single exposures, than is predicted by additivity, would be an increase in the number of cells at risk. In cycling cells, fractionated or protracted exposures could affect more cells than single exposures. Also, an increase might occur in proliferative tissues with doses that caused some cell killing. Ifcells are killed, say by the initial fractions, proliferation involved in replacing the lost cells may result in an increased number of susceptible cells being at risk. If this hypothesis is correct, and the data of Ullrich (1982) suggest it is, tissues that are either nonproliferative or have a very slow cell renewal should not show the enhancement effect of fractionation. Both the dose per fraction and fraction interval would be important factors influencing cell repopulation. Alternatively, cell killing may alter the cell-cell interactions and influence expression. The data are inadequate to test the possible explanations but it is of interest that systems that do exhibit increased cell proliferation in response to radiation-induced cell killing also show an enhanced response. No enhancement effect of fractionation or protraction would be predicted for ovarian tumors since no cell renewal can be involved if oocyte killing is the dominant factor in radiation induction of this type of tumor because oocytes do not proliferate. No enhancement has been found with protracted neutron exposure (U11rich, 1984, see Table 6.2).

6.10 The Relationehip of LET and RBE

Most of the data for tumors induced by external high-LET radiation are for neutrons and particularly fast neutrons. Studies have been, or are being carried out, with neutrons of different energies so that in the h t u r e it will be possible to draw some general conclusions

136

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6. EXPERIMENTAL CARCINOGENESIS

about the relationship of neutron energy to carcinogenic effect. Studies of the relationship between the spatial distribution of energy and carcinogenic potency that can be carried out with radiations of different LET and track structure should provide useful information but it is only recently that such experiments could be performed, and the available data remain limited. It is a general radiobiological finding that the degree of radiationinduced damage depends not only on the absorbed dose and the system irradiated but also on the spatial distribution of the energy deposited at the subcellular level. For example, the RBE of radiations increase for cell killing (Todd, 1976; Barendsen, 1968; Cox et al., 1977b; Blakely, et al., 1979) and mutagenesis (Cox et al., 1977a; 197713)with increasing LET up to a peak of 100to 200 keV pm-' and then decreases. The decrease is considered to be due to a saturation of events in the targets and an overkill effect. KraR et al. (1984),in studies that involved greater ranges of LET (from 10 to 15,000 keV pm-l) and energies (from 0.5 and 960 MeV a m - ' ) than any previous study, found that for LET values 200 keV pm- ' it was total energy transfer and not track structure that is important for cell killing. The inactivation cross section is independent of the atomic number of the particles in this lower LET value range. In the case of particles with LET values > 200 keV pm-' the picture is different; with increasing atomic number the inactivation cross section increases. Although there is a saturation effect, as indicated by the earlier work, the saturation cross section does not correspond to the geometrical cross section of the cell nucleus as implied by the suggested overkill effect. The rate of induction of chromosome aberrations is greater with the greater track diameters and not the higher LET values when different energies of the same heavy ion are compared. The investigations of the relationship of track structure and energy transfer to carcinogenesis have not progressed as far as the cell survival studies. However, some studies have provided some information about the relationship of LET to RBE. A systematic investigation of the LET-RBE relationship has begun using helium, carbon, neon, argon, and iron ion beams generated by the BEVALAC and with the mouse Harderian gland as the test system (Fry et al., 1985).In the completed experiments, the effect of heavy ions, with the exception of iron, were studied with the Bragg peaks of heavy ion beams, spread by a ridge filter. The dosimetry and estimate of an average LET of such beams is complicated by fragmentation and the inherent problems of LET determinations. However, the comparative tumorigenic effect of the various heavy

1

137 ion beams is of interest. It can be seen in Figure 6.22 that the curves for prevalence as a function of dose show an increasing effect from Wo, argon and iron, respectively. The estimated effect of the heavy ions, argon and iron, appears to be comparable to h i o n neutrons. The estimates of RBE given in Table 6.3 are based on the slopes of the initial part of the dose-response curves for fission neutrons 0.25 Gy rnin-I and the slope of the dose response for lS7Csgamma radiation 0.083 Gy d - I (Ullrichet al., 1976;Ullrich and Storer, 1979; Ullrich. 1984).The error associated with the RBE values have been approximated, i.e., S.E.= RBE [ ( s ~ +~ ) ' where fn and 6.10 THE RELATIONSHIP OF LET AND RBE

'*])I-

Y

HISTOLOGICAL DATA

0

0.4

Q0

1.2

1.6

2.0

2.4

2.8

3.2

Eigure 632 Prevalence of Harderian gland tumow as a function of dose of heavy ions and 60Co Y rays: 'He, 228 MeVIamu; 12C,400 MeVIamu;T e , 425 MeVlamu;T e , 600 MeVIamu; and 'Oh,570 MeVIarnu. Irradiation of the mice was in spread Bragg peaks for all ions except q e , for which the plateau region of the beam was used. Precise LET valuee are diflicultto determine for the spread Bragg peak m u r e 8 but the doae-average LET values were estimated to be as follows: "He, 1 - 2 keV '2C, 80 keV p,m-l; mNe, 160 keV pm-I; q e , 190 keV pm-I; and *Ar, 650 keV pm-l. (Adapted from Fry et al., 1985).

TABLE 6 . 3 - ~ s t i m t e s of RBEM b m initid sbpea

Mow Strain

RFM

Sex

P

'l'ieeue-tumor

Thymic lymphoma Ovarian tumor Pituitary Harderian gland Lung tumor

BALB/c

9

of f i s s h neutron and gamma-ray response c u w r for difiervnt tissue-tumor t y p s fn 0.56 2 0.004 0.52 -C 0.04 0.41 2 0.21 0.54 + 0.03 1.7 -c 0.15

Initial elope Y

RBEM

0.021 +. 0.02 0.0 0.007 0.005 0.015 r 0.004 0.29 + 0.151

27

+

a

59 36

6

Approximate S.E.of RBEy 2

26

+-52

+

*

10 3

4

I3 L?

g

.n

Lung adenocarcinoma

0.76 2 0.19 0.041 2 0.009 2 6 19 Mammary carcinoma 1.14 0.27 0.035 2 0.01 33b 2 12 'RBE, values are not given for ovarian tumors because it is not clear that RBE, is appropriate for these tumors. For example, it can be seen that infinity is the arithmetical value for the RBE of ovarian tumors. This illuetrates the problem of the use of RBE for tumom that demonstrate a threshold responee. bHigher values (approximately 70) have been estimated for low dose rate neutron expmures.

8

6.11 CONCLUSIONS

1

139

y = the coefficients for the initial slopes for the responses to fission neutron and gamma radiation, respectively.

6.11 Conclusions Concerning the Muence of Radiation Quality on Carcinogenesis The following conclusions seem to be justified concerning the influence of radiation quality on radiation carcinogenesis. (1) The LET-dependent differences in the tumorigenic effeds of different radiation qualities are assumed to be quantitative and not qualitative. That is, there is little evidence that different radiation qualities influence the biological characteristics (such as malignancy) of the tumors induced. The RBE of a particular radiation is not only LET dependent but also dose dependent, tissue dependent, dose rate and dose fractionation dependent. In the case of mice, the risk of death from tumors in females is about twice that in males for both gamma and fission neutron radiation, and therefore, RBE values for comparable tissues should be independent of sex. The RBE value may vary with age. The excess incidence of tumors induced per unit dose of radiation varies between tissues. At low doses the tissuedependent differences are greater with low LET radiation than with neutrons. As a result of these differences the RBE values have a very wide range. (2) Dose-response curves for tumor induction by some high-LET radiations, such as fission neutrons, that include doses greater than about 0.2 Gy are not linear, although the initial part of the curve almost certainly is linear. Thus, linear interpolation cannot be used to estimate the effects of low doses from data obtained a t higher doses. The dose range over which the response appears to be linear varies with different tissues. A number of factors influence the shapes of the dose-response curves for tumor induction, and as yet no acceptable model has been derived that takes into account all the physical factors, such as the distribution of dose in the tissue and cell, and all of the biological factors that include cell killing. Doseresponse curves (tumor incidence as a function of dose) reflect the sum of the effeds of all the factors that influence the induction of the initial events and their expression. There are endogenous and exogenous factors that determine whether or not the induced tramformation in initiated cells is expressed and that cells progress to overt cancers. Therefore, the inci-

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EXPERIMENTAL CARCINOGENESIS

dence of tumors is a poor measure of the number of initiated cells capable of forming tumors (and vice versa). (3) The response to high-LET radiation is generally less dependent on dose rate than with low-LET radiation. However, the induction of rodent breast cancers following protracted exposure to fission neutrons has been found to be greater than single exposures at a high dose rate. (4) Although, there is limited information for tumorigenesis both in relation to total doses and tumor types, fractionation of the total doses of fast neutrons does not usually result in a reduction in the tumorigenic effeds in contrast to low-LET radiation. The results suggest that fractionation, protraction and lowering the dose rate of fission neutrons may increase the tumorigenic effect compared to single exposures in some tissues and for some fractionation regimens. However, the total dose, the dose per fraction, and in the case of split doses, the time interval between doses, all appear to be important in determining the effect of fractionation. (5) The RBE value of neutron radiation for tumorigenesis, in general, varies inversely with the neutron energy down to about 0.4 MeV. The RBE increases with LET,probably up to 100 to 200 keV pm-l, but the precise relationship of RBE to LET has not been delineated. It is of interest that other highLET radiations, such as some heavy ions, may be as effective as fission neutrons, but not more so. (6) Currently, there is an insflcient amount of suitable data for validation of possible models of the dose-response relationships for cancer induction &om external exposure to highLET radiations. The reasons for the inadequacy include: (a) a t very low doses of high-LET radiation the absorbed dose is an inadequate descriptor for energy deposition within the biologically significant target(s), (b)there are insuf%cientdata on the induction of cancer with very low neutron doses < 0.1 Gy in different tissues and in different strains and species, (c) most of the data from the mouse have been obtained from females and the high sensitivity of the ovary results in an altered hormonal balance (tumor incidences in hormonedependent tissues cannot be considered independent under such conditions), (d) some of the published conclusions about the dose-effect and dose-RBE relationships are based on data from experiments that determined changes in the times of appearance of tumors and not changes in tumor induction rates. Different mechanisms may be involved in the induction of a higher cumulative incidence than in the advancement of

6.11 CONCLUSIONS

1

141

time of appearance of tumors and it is not clear what generalizations can be made based on one or the other. (7) The range of values of RBEs is broad (from1to approximately 80, depending on tissue, dose rate, method of analysis, etc.), largely, but not entirely, because of marked differences in the responses to low-LET radiation. This makes RBE an unsatisfactory, but a t this time a necessary guide, in the choice of Q values for different radiation qualities. For tumors in which the dose response characteristics are sufficiently defined to derive RBE,the range in values is not as wide but substantial variability still remains (60 to 60).

Internal Emitters Extensive investigations have been made on the toxicity of internallydeposited alpha emitters in humans and laboratory animals (NAS/NRC, 1988). These studies have been very valuable in establishing the risks of cancer and other effects from radioactivity within the body. However, many of these results are of limited applicability in evaluating the RBE of alpha particles relative to low LET radiation in terms of dose to the target cells. This is because the alpha emitters usually deposit energy nonuniformly within tissue; the alpha-particle range in soft tissue is short (about 24 pm at 4 MeV for 232Thto 82 pm at 8.78 MeV for 212Po);and the precise locations of the target cells relative to the alpha emitters are often uncertain. As the distance from an alpha-particle source increases, the dose progressively decreases and is zero beyond the alpha-particle's range. The few studies in which these complications are minimal are the following: (1) Bone sarcoma RBE of alpha versus beta particles, (2) Bone sarcoma RBE of fission fragments versus alpha particles, (3) Liver chromosome aberrations of alpha particles versus beta particles or gamma rays. Discussion here of RBE values derivable from internal emitters is limited to the above three examples. In addition, the toxicity ratios (the ratio of averaged organ doses a t equal levels of effect) will be given for lung cancers produced by alpha versus beta particles and for bone sarcomas produced by selected radionuclides versus 226Ra.

7.1 RBE of Alpha-Particles Versus Beta-Particles for Inducing Bone Sarcoma

A preliminary analysis was published on the RBE of alpha-particles versus beta-particles for bone sarcoma induction in beagles and mice (Mays and Finkel, 1980).Their analysis has been updated here to reflect the following changes: (a) all of the beagles have died, (b) three additional bone cancers are included, (c)dogs dying before their minimal bone sarcoma appearance time of 500 days are excluded, (dl the assumed skeletal weight for young adult beagles is changed from

7.1 RBE OF ALPHA-PARTICLES

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143

7.5 percent of body weight at injection to a more realistic 10 percent, (el skeletal retention equations are slightly revised (Miller and Buster, 1986) and (0the minor dose from 210Pband progeny is included in the 226Radecay series (Lloyd et a1. 1986b). No changes were required for the mice. These studies are of a particular importance because of the large amount of data on the effects of radium in man collected by Evans (Evans, 1974). 226Raand 90Srwere injeded intravenously into young adult beagles a t 17 months of age at the University of Utah (Miller and Buster 1986) and into young adult CF1 female mice at 70 days of age at Argonne National Laboratory (Finkel et al., 1959 & 1969a). Bone sarcomas, mostly osteosarcomas, were the main radiation-induced cancer in beagles (see Table 7.1 and Figure 7.1) and in mice (see Table 7.2 and Figure 7.2). 2asRais an alpha emitter and @OSris a beta emitter. Both are bone volume seekers so that the mean endosteal dose, 0-10 pm from bone surface, is roughly equal to the skeletal dose averaged over bone and marrow (Beddoe and Spiers, 1979; Mays and Lloyd, 1972). The average skeletal dose was computed at the assumed start of tumor growth, which was taken as 1 year before death in the beagles (Thurman et al., 1971), 140 days before death with bone sarcoma in the mice injected with and 100 days before first radiogmphic

AVERAGE SKELETAL DOSE IN GY AT I YEAR BEFORE DEATH F'ig. 7.1 Bone sarcoma incidence

2

1 S.D. in beagles iqjected with %or %r.

AVERAGE SKELETAL DOSE IN GY 100 DAYS BEFORE TUMOR APPEARANCE OR 140 DAYS BEFORE DEATH Fig. 7.2 Bone sarcoma incidence +- 1 S.D.in female CFI mice injeded with 226Ra or gOSr.

appearance of the tumors in the mice injected with 226Ra,to allow for the typical 40 days between first radiographic appearance and death with tumor (Mays and Finkel, 1980).Because the slopes of the retention curves for nGRa and 90Srwere similar, assumptions on the time span of the "wasted" radiation had little influence on the calculated RBE (Mays et al., 1969). The RBE of alpha particles versus beta particles in producing bone sarcomas was taken as the ratio of 90Srd ~ s e / ~ ~dose ~ Raat a given level of incidence (Table 7.3 and Figure 7.3).The RBE increased as the incidence decreased, reaching an RBE value of 25 at 7.7percent incidence in mice and a value of 26 a t 8 percent incidence in the beagles at Utah. Similar results were obtained for beagles at Davis, California, that received eight injections of 226Ra a t two week intervals beginning at 435 days of age or that received 90Srin food from mid-gestation until 540 days of age (Raabe et al., 1983).The basic tabular data for these beagles (Book et al., 1983)lists 125 dogs with bone sarcoma following 226Rainjection and 36 dogs with bone sarcoma following 90Srfeeding. When these data were analyzed in the same way as for the beagles a t Utah, the RBE of 226Rarelative to 90Srprogressively increased

hid

Nuclide

kw k p - I

TABLE7.1-Bone Time Or) From iqi. To death

sarcomus in beagles injected with 22sRaor Number of Dogs Number of Dogs With bone at 500 days Sarcoma Post injection

Incidence (percent)

Skeleton dose (Gy)one year Before death

TABLE7.2-Bone sammaa in female CFl mice iqjected with PsRa or soSr betted

=%a

%r

bBq b-' 4440 2960 1480 740 370 185 92.5 46.3 37.0 27.8 18.5 9.25 3.70 1.85 0

Number of mice at 150 days Post injection

Number of mice with Bone eareoma

Incidence or Mortality (percent)

45 44 45 44 43

14 31 33 38 34 28 45 22 56 94 80 19 5 11 6

31.1 70.5 73.3 86.4 79.1 62.2 43.3 21.2 23.4 18.7 11.7 7.7 2.0 4.3 1.2

45 104 104 239 504 683 247 252 254 521

81400 26 19 32600 45 41 16300 42 34 7400 59 8 3260 74 2 83 3 1630 329 104 0 166 119 2 48 148 2 0 149 2 Dose for 329 kBq kg-' level calculated at 460 days (600 days

-

73.1 91.1 81.0 13.6 2.7 3.6 0 1.7 1.4 1.3 140 days)

Bone sarcoma mice Av. days iqi. Skeltal dose (Gy) To appear (Ra) 100 days before appearance (Ra), Or death (Sr) Or 140 days before death (Sr)

328 359 394 428 484 544 639 657 643 686 655 580 853 710 730

289 213 118 64.2 36.4 20.4 11.9 6.14 4.80 3.83 2.44 1.09 0.62 0.26 0

z

Q)

, 9

aiz

C

%

7.1 RBE OF ALPHA-PARTICLES

TABLE7.3--Bone

I

147

samoma RBE ofaasRaversus 90Sr pe6Ra

Incidence percent

&particla (Gy)

Beagles

66.7 41.7 16.7 8.7 8.0

71.4 65.4' 59.3 23.2 21.1'

Mice

86.4 81.0 79.1 62.2 43.3 21.2 23.4 18.7 13.6 11.7 7.7

65'

Species

63 62a 55' 46' 37" 38" 35' 33 3 1. 27' 'Interpolated from curve8 on Figurea 7.1 and 7.2

a-particlea

(GY) 13.5' 8.77 3.37" 1.66 0.80

RBE

5 7 18 14 26

64.2 44.P 36.4 20.4 11.9 6.14 4.80 3.83 2.8P 2.44 1.09

1 1.4

2 3 4 6 8 9 12 13 25

AVERAGE SKELETAL DOSE IN GY FROM =RA a-PARTICLES Fig.7.3 Relativebiological effectivenessofalpha particlee from %and progeny, relative to beta particles from BOSr and progeny.

from an RBE value of nine a t 56 percent bone sarcoma incidence, to an RBE value of 20 at 12 percent incidence and RBE value of 35 at three percent incidence. Below three percent incidence, it is unknown whether the RBE of alpha particles, relative to beta particles, continues to increase or reaches some constant value. The high RBE at low incidence is due mainly to the low effediveness per Gy of beta particles for bone sarcoma induction at low doses and low dose rates. If the target cells for bone sarcoma production were deeper than the assumed 0 to 10 pm from bone surfaces (ICRP, 19771, then the doses to the target cells would be decreased proportionally more for the short-range alpha particles than for the long-range beta particles. This would increase the RBE values shown in Figure 7.3 by a constant multiplication factor, but the shape of the curves would remain unchanged. 7.2 RBE of Fission Fragments Versus Alpha-Particles for Inducing Bone Sarcoma When a heavy atom fissions, the two fission fragments recoil in opposite directions, producing tracks of ultra dense ionization averaging about 4,800 keV pm-' in soft tissue. To evaluate the effectiveness of fission fragments in bone sarcoma induction, beagles and mice were injected with 252Cfor 249Cf(Tayloret al., 1983).The skeletal dose from 252Cfis about one-half from fission fragments and one-half from alpha particles. The comparison isotope was 2*Cf, which emits alpha particles in 100 percent of its disintegrations and is identical chemically and metabolically to 252Cf.From combined results in beagles and mice, the average RBE ? S.D.for bone sarcoma induction by fission fragments relative to alpha particles was 0.1 2 0.1 (Mays et al. 1989). The low RBE of fission fragments for cancer induction agrees with the findings of Brooks et al. (1972), that fission fragments from 262Cf were very much less effective per Gy than alpha particles from "'Am in the induction of chromosome aberrations in the livers of Chinese hamsters. For equal doses, from %*Cffission fragments and alpha particles, the summed length of all the alpha tracks in tissue is about 34 times longer than that of all the fission tracks, as can be seen from the ratio of their average LETS in water (about 4,800 keV pm-' for fission fragments to 140 keV pm-I for alpha particles). Thus, about 34 times more cells were traversed by alpha particles than by h i o n fragments.

7.3 RBE OF ALPHA PARTICLES

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149

7.3 RBE of Alpha Particles Versus Beta Particles or Gamma Rays for Inducing Liver Chromosome Aberrations

Injected monomeric 239Pu,241Amand lUCe, in citrate solution, deposit uniformly throughout the livers of Chinese hamsters. Brooks et al. (1972)and Brooks (1975)found that low LET radiation from the beta particles of internallydeposited lUCe and protracted external gamma rays from 60Cowere equally effective per Gy in producing chromosome aberrations in the liver. However, the alpha particles from 239Puand 241Amwere about 15 to 20 times as effective as the protracted low LET radiation, Their results are plotted in Figure 2.9.

7.4 Lung Cancer Toxicity Ratio from Alpha Versus Beta

Particles A detailed analysis of world-wide data on radionuclide-induced lung cancer concluded that, in terms of the calculated dose averaged over the entire lung, the alpha radiation from inhaled radionuclides such as 239Puwas roughly 30 times as effective as the beta radiation from intratracheal intubation of radionuclides such as 14Ce in producing a given incidence of lung cancer (ICRP, 1980). Throughout the lungs of these laboratory animals, mainly rats, the local distribution of dose was nonuniform, especially for the alpha emitters. Results from inhaled =Rn progeny were not included in the analysis. Preliminary results have recently appeared from the Inhalation Toxicology Research Institute at Albuquerque on beagles that inhaled the beta emitters OOY,91Y,'We, or 90Srin fused aluminosiliin monodisperse parcate particulates, or the alpha emitter 239h02 ticulates. Within the lung, these particulates are retained mainly within the alveolar region. In terms of the averaged lung dose, the alpha dose from 239Puappeared to be about 10to 18 times as effective as the beta dose from 9'Y in producing a given risk from lung cancer (Boecker et d.,1988).However, many of the beagles exposed to received high dose rates exceeding 1 Gy d-l. Using the same data base, Griffith et al. (1987)found that the lung cancer risk per Gy from the beta emitters decreased with decreasing dose rate, and below 1 Gy per day appeared to be a constant one-third of that for the combined 91Yexposures. Thus, Griffith et al. (1987)suggest that it might require 33 to 58 times the average lung dose from a beta emitter, at low dose rate, to produce the same lung cancer risk as

the alpha dose from 239Pu. These conclusions are preliminary because about 30 percent of the lowdose 239pUdogs were still alive in 1987.

7.5 Toxicity of Selected Radionuclides Relative to aaeRa Bone-seeking radionuclides are classified as either bone-surface seekers or bone-volume seekers, depending on the skeletal location of their early deposition. Complications to this simple classification are that some of the volume seekers have a transient deposition on bone surfaces, whereas long-lived surface seekers become partially buried under the apposition of newly formed bone. A further complication is that a single intake of either a surface seeker or a volume seeker usually deposits nonuniformly. For example, radium concentrates in regions of bone accretion, whereas plutonium concentrates on endosteal surfaces. While attempts have been made to predict the toxicity of internallydeposited emitters by simplified mathematical models (ICRP, 1979), it may be more reliable to determine relative toxicities by direct experimental observation in laboratory animals. Radium-226 is often used as the comparison radionuclide, based on extensive knowledge of radiation effects in the U.S.radium dial workers (Aub et al., 1952; Evans, 1974;Finkel et al., 196913, Rowland et al., 1978 and 1983; Mays et al., 1985 and 1986). Table 7.4 shows the relative toxicity of selected bone-seeking radionuclides compared to 22% in terms of the average skeletal dose in GY. The alpha emitting surface seekers (%%f, wpWm, "'Am, =Pu, and 228Th)were all more toxic per unit of average skeletal dose than the bone volume seekers (n8Ra and 226Ra,both of which have alpha emitters in their decay series). Plutonium-239 was about 16 times TABLE7.4-Taxicity of selected bone-seekingmdionlrclides relative to =Ra in bone-samma induction in beagles and C57BLIDo mice (Mayset al., 1986, Taylor et al.. 1983 and Jones d al.. 1985) Toxieitv ratio

Radionuclide

mCf

a*zwcm %'Am

Beaglea

-

5.4 2 1.6 =TJll 16.6 2 4.5 % 8.5 2 2.3 228Re 2.0 0.5 ZL8Ra 1 .Based on average akelatal dose (in Gy)

.o

2

S.D..

Mice 5.0 4.4 4.9 15.3

2 1.4 2

1.8

2 1.4

2 3.9

-

1.0

7.6 SUMMARY OF INTERNAL E m R S

1

151

more toxic than 226Ra,both in beagles and in mice. Furthermore, 239PUwas about three times more toxic than the trivalent transplutonium radionuclides P41Am, 2430244Cm, and 249CfJ,suggesting that 239Pupreferentially deposits in high concentration in regions that are richly populated with cells that can be transformed into bone sarcomas. Among the bone volume seekers, the higher toxicity of relative to 226Ra, may result from, (a) the longer average range of the alpha particles in the 22sRadecay series (50 pm in soft tissue) compared to 40 pm for 226Raand its retained progeny, (b) the tendency of 22Thformed from the decay of 22eRain bone volume to redeposit on bone surfaces when the bone volume is resorbed, and (c) the redeposition on bone surfaces of some of the short-lived 3.62-day 2uRa produced by the decay of nsTh. In human radium dial workers, the effectivenessfor bone sarcoma induction, per Gy of average skeletal dose, for 228Rawas about 1.5 times that for (Rowland et al., 19781, in good agreement with the relative effectiveness of 2.0 & 0.5 derived for beagles (Mays et d., 1986).

7.6 Summary of Internal Emittera

The effectiveness of alpha emitters is high, relative to beta emitters,being in the range of 15to 50 times as effectivefor the induction of bone sarcomas, liver chromosome aberrations, and lung cancers. The RBE of alpha emitters tends to increase as the dose decreases, probably mainly due to the decreased effectiveness per Gy of low LET radiation a t low doses and low dose rates. Alpha emitters, like 2SgpUthat deposit on bone surfaces, are more toxic than alpha emitters like 226Rathat deposit within bone volume, and thus irradiate fewer target cells. The low RBE of fission fragments, relative to alpha particles, may be because mainly, at equal doses, many more cells are traversed by alpha particles than by fission fragments.

8. Life Shortening in Mice-

RBE 8.1 Introduction

Many end points of radiation injury that have been established quantitatively with low-LET (x or y) irradiation have been also measured with high-LET radiations, particularly neutrons, yielding RBE estimates that may vary over two orders of magnitude, depending on the biological system under study and the radiation doses and dose-rates employed. This section is a review of most of the large-scale rodent studies carried out during the past 30 years in which life shortening from all causes of death was the end point of radiation iqjury. In practically all of these studies, the experimental animals were well-characterized strains (or hybrids) of the house mouse (Mus musculus), and the high-LET radiations were neutrons. However, it needs to be recognized that one of the major differences among mouse strains is their different susceptibilities, both spontaneous and radiationinduced; and, furthermore, within a given strain, there are frequently sex differences in the incidence and time of onset of specific tumor types. Nevertheless, in all mouse strains examined, regardless of the spontaneous incidence of various tumor types, the radiation-specific excess mortality is almost entirely attributable to tumors, regardless of radiation quality, total dose, or dose rate (Grahn et al., 1978).This statement holds true for other species that have been examined, including the white-footed mouse (Peromyscus leucopus), a rodent that is taxonomically closer to the hamster than to the house mouse (Thomson et al., 1985a)],the beagle (T. E. Fritz, personal communication), and man (Beebe et al., 1978). Section 6 of this Report shows that each tumor type has an essentially unique RBE value, and that the extremes of these values may vary by a fador of five or more. This range is considerably greater than that observed for the RBE values for life shortening from all causes in different mouse strains, where the extremes differ by a factor of no more than two (Storer and Mitchell, 1984).

8.1 INTRODUCTION

I

153

Therefore, the use of life shortening from all causes, including deaths from infectious and degenerative diseases as well as tumors, has certain advantages, not the least of which is the objective and unequivocal nature of the end point. (There is often a degree of subjectivity in the assignment of a specific cause of death.) Of equal importance is the modulatory effect of this end point; the effect of an unusually high or low RBE value of a specific tumor type, particularly one of low incidence, will be diminished, and a more realistic RBE value for the high-LET radiation may be obtained. Earlier attempts to measure the RBE of neutrons for life shortening aRer single exposures at relatively high instantaneous dose rates and total doses gave estimates of about three, roughly the same as In these the RBE value for acute radiation toxicity (30day LD50). experiments, the investigators generally concluded that the doseresponse curves for both neutrons and gamma rays, at least over the appropriate dose ranges, were linear, so that the RBE value was essentially the ratio of the slopes of the two curves. Under some conditions, straightforward relationships between neutron RBE values and neutron dose can be derived; under other conditions, particularly in the lowdose range, the relationship may not be at all simple. The problem is that the establishment of reliable RBE estimates requires the establishment of dose-response curves for two radiations, the shapes of which may be difficult to determine satisfactorily (Land, 1980). More recent data, however, have shown that after small single doses of neutrons, the dose-effect curve for life shortening rises rapidly, then tends toward a plateau at higher doses. The proper description of the curve thus becomes the matter of primary concern, i.e., whether or not there is a linear initial segment, and which of a variety of curvilinear functions best describes the nonlinear portions of the curve. There are several problems associated with the measurement of RBE values for small doses of radiation. One of these relates to the fact that the effects of the low LET radiation-generally 60Coor 13'Cs gamma rays-are markedly dependent on dose rate: the more a given total dose is protracted or fractionated, the less effective it becomes, and, consequently the higher the RBE value for the highLET radiation becomes. In the case of high-LET radiation, however, there is strong evidence that a t least under some circumstances, fractionation of a given total dose may be more damaging than the same dose given in one exposure. In the case of life shortening in BGCF, mice (Thomsond al., 1981a),this augmentation phenomenon L dose), is most pronounced a t high doses (- 75 percent of the,D but there is no evidence of augmentation at a total dose of 0.2 Gy of

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/

8. LIFE SHORTENING LN MICE-RBE

0.8 MeV fission neutrons (Ainsworth et al., 1974, 1976; Thomson et al., 1981a). It follows then that any estimate of the RBE value must precisely specify the exposure conditions. The extreme values would be represented by: (1)a comparison of the effects of single doses of neutrons and gamma rays delivered a t instantaneous dose rates that are sufficiently high to obviate problems of dose-rate dependence (low RBE); and (2)a comparison of the effects of fractionated or protracted neutron doses with those of continuous low dose rate gamma radiation, where the differences in magnitude and direction of dose-rate dependence would be maximal (high RBE). Intercomparison of results from different laboratories is often difficult for several reasons: radiation sources, dosimetric procedures, and experimental animal strains are generally different. Although the differences in physical parameters can often be adjusted, the biological variables-genetic makeup, age at exposure, diet, housing conditions, bacterial and viral disease incidence, etc.-are not easily reconciled. There are, however, some radiation responses that seem to be essentially strain-independent (Grahn et al., 1978). A more important problem, however, is the difficulty in establishing the shape of the dose effect curves for both high and low-LET radiations in the low dose range, i.e., doses one to two orders of magnitude or so above background. Thus, the approach customarily employed is to use data obtained a t high doses as the basis of a model for extrapolation to low doses. As will be pointed out, however, the mathematical function that best describes the neutron dose-response curve for single doses above 0.1 to 0.2 Gy does not satisfactorily predict the observed results for life shortening after lower doses. A survey of data published before 1981suggests that the relationship between neutron dose (D)and the RBE value for life shortening, regardless of the mode of neutron exposure (single, fractionated, or continuous) could be expressed as: RBE value

=

ADB

(8.1)

where the value of B was approximately a negative 0.5 and that of A (the RBE value a t 0.01 Gy) ranged from 10 to 80, depending on a number of factors, the most important of which was the instantaneous dose rate of the reference low-LET radiations (Thomson et al., 1981a, 1981b). This analysis employed the radiation-specific excess mortality rate [approximated by the method of Sacher (1976); see Thomson et al. (1981a) for the complete derivation], and led to a conclusion compatible with that advanced by Rossi (1977a1, i.e., that the neutron RBE value varied inversely with the square root of the neutron dose

8.2 SINGLEEXPOSURES

1

155

However, almost all of the data then available on life shortening after neutron exposures involved total doses greater than 0.2 Gy the exception being the experiments of Storer et al. (1979). More recent data from the Argonne and the Oak Ridge National Laboratories on both single and fractionated neutron exposures at doses as low as 0.01 Gy have cast doubt on the usefulness of Equation 8.1, and suggest that: (1)the initial segment of the neutron dose-response curve, like that of the gamma-ray curve, is indistinguishable from linear, and (2) there may be a limiting value for the RBE (Storer and Mitchell, 1984; Thomson et al., 1985a). Dennis (1987)has suggested that Equation 8.1 is a special form of a complex equation, and is not valid at low neutron doses. The more general form of his equation is: RBE value

=

A (K + D)B

(8.2)

The constant K is about 0.04 Gy for single exposures, and about 0.2 Gy for fractionated exposures. Clearly, when D is large relative to K, Equation 8.2 approaches Equation 8.1. Conversely, when D is small relative to K, the RBE reduces to a constant value.

8 2 Single Exposures

The results of studies of the effects of single exposures to 0.85 MeV fission neutrons (from the JANUS reactor) and 60Cogamma rays on life shortening in BGCF, mice (C57BLI6 x BALBIc hybrids) have been published by Thomson et ul. (1981a, 1983,1985a, 1985b).In the experiments reported in the first two papers, the dose ranges were 0.05 to 2.4 Gy for neutrons, and 0.9 to 7.88 Gy for gamma rays. In general, exposure times were 20 minutes, and the instantaneous dose rates were varied from 0.0025 to 0.12 Gy min-I for neutrons and 0.045 to 0.39 Gy min-I for gamma rays. It was clear that the life shortening per Gy of gamma radiation was relatively constant over the entire dose range studied, so that the data could be fitted with a single linear equation with a slope of about 40 days of life lost per Gy. However, for neutron exposures the life shortening per Gy varied inversely with neutron dose, and the data could be best fitted by a power function [see Thornson et ul. (1983) for the parameters]. If one examines the lowest end of the dose range (0.05 to 0.2 Gy) it is evident that a linear dose-response curve cannot be rejected (Thomson et al., 19831, and, therefore, a limiting value for the neutron RBE is possible, about 12 for males and 18 for females. An

156

1

8. LIFE SHORTENJNG M MICE-RBE

independent analysis by Storer and Mitchell (1984) of these data as well as those from Oak Ridge National Laboratory (Storer et al., 1979; Storer and Ullrich, 1983) suggested a range of RBE values from 13 to 23, depending on sex and strain (and possibly, differences between the laboratories in physical factors, such as neutron energies and dosimetry). An experiment a t Argonne National Laboratory (Thornson et d., 198%) was specifically designed to examine directly the shape of the dose-response curves for female mice at low doses of neutrons (0.01, 0.025,0.05,0.1,0.2 and 0.4 Gy) and gamma rays (0.225,0.45 and 0.9 Gy). The results show that the prediction equations presented by Thomson et al. (1983) seriously overestimate the life shortening a t the lowest doses, although the agreement between observed and predicted values is reasonably good at doses above 0.05 Gy. Over the range of 0.01 to 0.1 Gy a linear dose response is satisfactory; inclusion of the data at 0.2 Gy and above cause the linear fit to be rejected. The slopes of the neutron (0 to 0.1 Gy) and gamma ray dose-response curves are 561. and 38. days of life lost per Gy, respectively, and the RBE value, given by the ratio of the slopes, is 15.0 + 5.1. The entire range of neutron doses 0.01 to 0.4 Gy can also be fitted reasonably well by a linear-quadratic equation of the form: Y = a + b,x + b2x2,where x is the dose and Y is the mean after survival. The limiting RBE value will then be the ratio of the fist power coefficient, b,, to the slope for the gamma dose-responsecurve, or 14. In 1979,Storer et al. published considerabledata on life shortening in female RFM and BALBIc mice after gamma (13'Cs) or neutron radiation from either the HPRR reactor or a 252Cfsource, depending on the dose rate desired. In a subsequent publication, Storer and Ullrich (1983) presented additional data on the BALBIc strain. The radiation responses of the two strains were appreciably different. Over the dose ranges of 0.048 to 0.47 Gy for neutrons and 0.1 to 0.5 Gy for gamma rays, Storer et al. (1979) concluded that the neutron dose-response curve for the RFM strain was linear, whereas that for gamma rays was parabolic (i.e., life shortening was a function of the square of the dose). Their estimate of the RBE was: RBE = 0.95 D,-0.5

(8.3)

where D, is the dose in Gy i.e., the same form as Equation 8.1. In the case of the BALBIc mice, the picture more closely resembles that seen with the BGCF, mice. The most recent analysis ofthese data (Storer and Mitchell, 1984)suggested linear dose-responsecurves for both neutrons and gamma rays, with a limiting RBE value of 13.3, similar to the value of 15.0 derived for BGCF, females.

8.3 FRACTIONED AND PROTRACTED EXPOSURES

TABLE8.1-Summary Neutron Doee Range

157

of projected RBE values at 0.05 Gy

Projected

Mouse

(Gy) Strain 0.28-2.5 C57L x M e male C57L x M e female CP-5 reador 0.36-2.75 CF No. 1 female Cyclotron, 1 MeV 1.3 -3.32 RF male Neutron Source

Weapons

1

B E value at 0.05 Gy

Reference

16

Upton et al. (1960)

12 10

Vogel et al. (1961) Upton et al. (1967)

Other large scale studies include those of Upton et al., (19601, Vogel et al. (1961), and Upton et al. (1967). In each case, the range of neutron doses was well above the level (0.1 or 0.2 Gy) where departure from linearity becomes obvious. The data conformed reasonably well to Equation 8.1; the parameters are given by Thomson et al. (1981a). From this equation, we have estimated the RBE value at 0.05 Gy of fission neutrons assuming that below this dose the RBE value will be constant, with the summary given in Table 8.1. This analysis is by no means precise, but it at least provides answers that are consistent with the results obtained by Storer and Mitchell (1984) and Thomson et al. (1985a) in the low-dose range. 8.3 Short-Term Fractionated and Protracted Exposures

This section examines some of the experiments in which mice were given either periodic exposures to radiation up to a predetermined total dose, or continuous exposure over an extended period of time ranging from a few days up to a few months (20 to 25 percent of the life span of the animal). In general, only those protraction or fractionation protocols in which fewer than 10 to 20 percent of the animals died during the exposure period have been considered. Concurrent with the single exposure series (see Section 8.2) using 0.85 MeV neutrons at Argonne, animals were exposed to similar total doses given in 24 weekly fractions (Ainsworth et al., 1974, 1976). From a comparison of the results of single and fractionated exposures (Thomson et al., 1981a), it is clear that the effectiveness of neutron radiation is greater (augmentation) when a given total dose is divided into 24 fractions. In the case of male mice, the life shortening per Gy is consistently 50 to 60 percent higher at all fractionated dose levels over the range of 0.2 to 2.4 Gy. In the case of females, however, the augmentation is less pronounced at low doses and is not obsewed a t 0.1 Gy total dose (Thomson et al., 1983). Thomson et al. (1981a) used the same analytical approach to these data on fractionated neutron exposures as they had used with single

158

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8. LIFE SHORTENING IN MICE-RBE

exposures, and concluded that the coefficient of Equation 8.1 (i.e., the RBE a t 0.01 Gy) was 60 to 80, three-fold greater than seen for single exposures. Much of this difference could be attributed to the decreased effectiveness of the total gamma dose when divided into 24 weekly fractions; the days of life lost per Gy dropped from about 40 for single exposures to about 20 for fractionated exposures. A reevaluation of these data (Thomson et al., 1983)showed that if linearity is assumed in the lowdose range for neutrons, the RBE value approaches a limiting value of 20 for males and 25 for females. Storer and Mitchell (1984) have reached essentially the same conclusions. The conditions under which the augmentation phenomenon occurs for neutrons have not been fully defined. There were no significant differences in life shortening following 2.4 Gy of fission neutrons given: (1)in 24 weekly fractions of 0.1 Gy; (2) 6 fractions of 0.4 Gy per fraction given at 4-week intervals; or (3) 72 fractions of 33.3 mGy given three times per week for 24 weeks (Ainsworth et al., 1974, 1976). However, an exposure series completed a t Argonne in which mice received neutron doses of 2.4 Gy in 1,2, 4, or 6 fractions with a one week interval between fractions did show a difference (Thomson et al., 1985a). Splitting the dose into two fractions of 1.2 Gy resulted in significantly greater life shortening than a single exposure; division into four fractions of 0.6 Gy each was even more effective. Six fractions of 0.4 Gy produced no greater effect than four fractions of 0.6 Gy; both regimens were as effective aa those employed earlier involving 24 weekly fractions. The augmentation of neutron-induced life shortening by fractionation or protraction of a given total dose is not universally observed. Storer et al. (1988) studied it in BALB/c mice exposed to 1.88 Gy delivered both continuously (0.01 Gy per day for 188 days) and in eight fractions (0.235 Gy per fraction) given seven weeks apart in RFM mice: the augmentation was observed only after continuous exposure. No differences in life shortening were seen in either strain following exposure to a total dose of 0.47 Gy, regardless of the mode of exposure. Considering all the data now available, it seemsunlikely that the neutron RBE a t total neutron doses below about 0.4 Gy will be significantly augmented by fractionation. In an experiment involving the white footed mouse (Peromyscus leucopus),the animals received total doses of 0.4 and 1.6 Gy delivered in either 24 weekly fractions or as single doses. The augmentation of life shortening was only 10 percent, rather than the 60 percent seen with the BGCF, Mus musculus (Thomson et al., 1986). Storer et al. (1979) exposed RFM and BALBIc female mice to 262Cf neutrons a t a dose rate of 0.01 Gy d-I for 24,47,94, and 188 days,

8.3 FRACTIONED AND PROTRACTED EXPOSURES

1

159

and to gamma rays at a dose rate of 6.9 x Gy min- I, 0.083 Gy per 20 hour exposure dayfor6,12,24,and48 days(0.5,1,2 and4 Gy). At high total neutron doses, as mentioned above, the augmentation effect was clearly discernible in the BALBIc strains; at low doses, the dose-response curves tended to merge. Supplementary data on fractionated and protracted neutron exposures to BALB/c mice at lower total doses were presented by Storer and Ullrich (1983). From all these data, Storer and Mitchell (1984) concluded that the RBE value is about 13. As was the case for exposures to single doses of neutrons, the response of the RFM mice was different from that of the BALBIc strain. As mentioned above, the augmentation phenomenon was observed only a t the highest total dose (1.88 Gy). At the lowest dose (0.24 Gy), the life shortening following lowdose-rate exposure was only half of that observed after exposure at a rate of 0.25 Gy min-'. Another complication is that the effectiveness of both neutrons and gamma rays, as measured by days of life lost per Gy, diminished with increasing doses. for BALBIc mice, it can be estimated that the limiting value for days of life lost per Gy of protracted neutron exposure will be about 260 (not significantly different from the value of 290 for single exposures), while that for protracted gamma radiation will be about 35, significantly less than 105 observed for single exposures. Thus,for continuous short-term neutron exposures (24 to 188days, 3 to 20 percent of the life span) in BALBIc mice, the limiting value for the RBE is eight. In addition to the studies on the effects of single exposures, Upton et al. (1967) studied the effects (for the most part in RF female mice) of different rates, ranging from 0.003 to 8.3 x Gy rnin-' of neutrons, with total doses ranging from 0.016 to 9.3 Gy; and from 0.4 to 49.3 x lop6Gy min-I of gamma rays, with total doses from 1.01 to 98.75 Gy. Exposure times varied between 3 and 560 days. Unfortunately, no one dose rate was employed at a sufficient number of data points, spread over a broad enough dose range, to warrant a detailed examination of the dose-response curves for different dose rates. Therefore, the results obtained were pooled a t the same total accumulated dose, regardless of instantaneous dose rate, omitting from the analysis those data points at which more than 20 percent of the animals died during exposure. The data show that the life shortening per unit dose of neutron irradiation did not vary inversely with the dose but rather (within limits of error) remained constant, whereas the life shortening per unit dose of gamma ray irradiation increased rather markedly with increasing dose, at least between 0 and 6.1 Gy. Inspection of the data suggested that polynomial fits might be attempted; for days of life shortening (LS) the most satisfac-

160

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8.

LIFE SHORTENING IN MICE-RBE

tory fits were a linear equation for the neutron data and a quadratic with a zero coefficient for the first-power dose for the gamma-ray data: LS (n) = 86.1 ( 2 12.1) D, (8.4) where D, is the dose of neutrons in Gy and Dy is the gamma dose in GY. The RBE equation derived from these equations becomes: (8.6) RBE = 6.63 D;0.5 Although the dose-response curves for RF female mice exposed to protracted gamma and neutron radiation do not resemble those seen with other strains, the relationship between RBE and neutron dose seems to be consistent. The coefficient of 6.63 may be compared with the value of 0.95 reported by Storer et al. (1979)(Equation 8.2),using the same analytical approach, for RFM female mice exposed to single doses of gamma rays and neutrons of high dose rates. The difference is largely attributable to the greatly reduced effectiveness of gamma radiation delivered to RFM or RF mice at low instantaneous rates. Neary et al. (1957) and Mole and Thomas (1961)presented results of experiments in which female CBA mice received either continuous or once-a-week exposures to neutrons at weekly dose rates of 0.022, 0.064,0.15, or 0.17 Gy (air doses) for varying periods of time. All of these data have been combined here because there were too few doses a t any given dose rate to treat separately. Further, the doses have been multiplied by the fador of 0.75 given by Neary et al. (1957) to convert air doses to absorbed doses. Over the range of 0.5 to 1.12 Gy, the dose-response curve is indistinguishable from linear, and the days of life lost per Gy of neutron exposure is given by: LS(n) = 278 ( + 50) (8.7) The RBE value will then be given by 278 b,-l, where b, is the days of life lost per Gy of gamma irradiation. Mole and Thomas (1961) provided data for continuous gamma irradiation at three weekly dose rates, 1 , 2 and 3.25 Gy, for which values of b, can be determined over a total dose range of 0 to 30 Gy. These are, respectively, 14.2, 16.6, and 28.3, and the RBE values become, respectively, 20,17, and 10. Although the absolute accuracy of these numbers is questionable, their relative values are less open to question, and emphasize the point made earlier that the neutron RBE values will depend on the conditions under which the dose-response curve for gamma radiation is established.

8.4 DURATION-OF-LIFE & OTHER LONGTERM EXPOSURES

1

161

It is instructive to compare these dose rates with those used by Sacher and Grahn (1964) in their duration-of-life gamma-ray studies. The lowest daily dose rate used by Mole and Thomas (1961)falls in the range of doses (below approximately 0.2 Gy d-'1 where the survival of the animal is dependent only on total dose, and is doserate independent (Sacher, 1976).The highest dose rate used by Mole and Thomas (1961) is in the dose-rate dependent portion of the response, and their intermediate rate is near the transition point. By analogy, it is possible that the RBE value of 20 will, in fad, be the limiting value, and would not be increased if still lower gamma rates had been studied.

8.4 Duration-of-Lifeand Other Long-Term Fractionated or Protracted Exposures

This section considers a number of experiments in which mice were exposed continuously or in weekly fractions for the duration of their lives. Also included are some experiments in which mice were exposed continuously or repeatedly for sufllciently long periods of time so that it may be presumed that any additional exposure would not have influenced their survival. The criteria for selection were (I) irradiation over longer than half the expected mean life span of the animal; andlor (2) death of more than 50 percent of the animals during the course of exposure. The use of total or mean accumulated doses in duration-of-life experiments may be misleading. Therefore, these sets of data have been analyzed in terms of dose rate, expressed as Gy per week or Gy per weekly fraction. The experiments described in this section (Thornson et al., 1981b)involved once-weeklyexposures, so that dose rate is defined as dose per weekly fraction. In the analysis of other data in which long-term continuous exposures were involved, the given daily dose rates were converted to weekly dose rates for the sake of conformity. Experiments have been described by Thomson et al. (198:Lb) in which mice were exposed once a week to small doses of neutrons (6.7 to 26.7 mGy per weekly fraction) and gamma rays (0.07 to 0.32 Gy per weekly fraction) either for 59 weeks (60 fractions) or for the duration of their lives. In the 60-fraction series, the total neutron doses were 0.4 to 1.6 Gy, the life shortening was the same as that observed after the same doses given in 24 fractions, and about 60 percent greater than that seen after single exposures. The total garnma-ray doses were 4.17 to 19.18 Gy and the life shortening was

162

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8. LIFE SHORTENING IN MICE-FU3E

less than that produced by 24 weekly fractions, which in turn was less than that produced by single exposures. The only difference in life shortening between the two protocols was observed in male mice receiving the lowest gamma-ray dose, 0.07 Gy per weekly fraction. The difference was not statistically significant and was largely attributable to a slightly higher proportion of deaths during the first 60 weeks of exposure in the durationof-life series. Therefore it was concluded that the radiation received after the 60th exposure did not contribute measurably to life shortening (i.e., the lethal process had been unalterably established by the time that 60 exposures were completed), and accordingly the data were treated as a single experiment. As in the other Argonne experiments reported a t the same time (Thomson et al., 1981a), a n inverse relationship between neutron dose and RBE value was observed readily. However, the range of total neutron doses (in the 60-week series) did not extend into the range where linearity might be expected. It could be established, however, that the RBE value would be a t least 21. Another series employed 60 once-weekly exposures to considerably Gy per weekly fraction lower dose rates, 3.3 x to 6.7 x for neutrons, 0.016 to 0.1 Gy per week for gamma rays (Thornson and Grahn, 1988). The dose-response curves for gamma radiation (both sexes) are linear throughout the total dose range of 1to 6 Gy, with slope coefficients of 18days of life lost per Gy per weekly fraction for males and 22 for females. It is equally clear that the neutron dose-response curves are linear over a range of total doses from 0.02 to 0.3 Gy, with slopes of 330 days of life lost per Gy (males) and 388 (females). Consequently, the RBE value for life shortening from longterm fractionated radiation is about 18 for both sexes. This number is lower than the limit suggested from the earlier study (Thomson, 1981b), principally because of a much greater sensitivity to gamma radiation shown by the mice in the more recent study, 1,100 to 1,300 days lost per rad per weekly fraction versus 700 to 900 in the earlier experiments. There are relatively few other experimental data with which these can be compared. Neary et al. (1957,1962) exposed CBA mice of both sexes to 0.7 MeV neutrons on a nearly continuous basis (average 140 hours per week) for the duration of life. Neutron doses ranged from to 0.157 Gy per week (air doses), with considerable 8.5 x gamma contamination; average total accumulated neutron doses ranged h m 0.087 to 8.75 Gy. Only two gamma-ray exposures, 0.158 and 1.11Gy per week with cumulative exposures of 16.26 to 72.52 Gy were carried out. Their data have been reexamined by Thornson et ad. (1981b). As in most other experiments, the life shortening per

8.5 DISCUSSION OF LIFE SHORTENING IN MICE

1

163

unit dose for neutron exposures decreased with increasing neutron dose rates, and when the data were fitted to a power function, the parameters for the CBA animals were very close (within 5 to 20 percent) to those for the B6CF1 mice. However, the RBE values differed by a fador of over two (45 for the CBA mice, 18 for the B6CF1's at the presumed limiting value). This difference reflects the fact that weekly exposure to gamma irradiation given in a 45 minute period is twice as effective as the same total weekly dose protracted over 8 to 12 hours per day, seven days per week (Sacher and Grahn, 1964). Consequently, the RBE value for continuous exposures to a given neutron dose rate will be about twice that observed for once-a-week fractionated exposures to an equivalent weekly dose rate, provided that the reference (gamma) radiation is delivered in the same mode as the neutron radiation.

8.5 Discussion of Life Shortening in Mice

In the absence of direct observations of life shortening following low doses of radiation, one approach is to search for an adequate mathematical description of data from higher doses. In many cases, the modified power function equations (Thornsonet al., 198la, 198Ib) provide the most satisfactmy fit for high doses but are poor predictors of the results at low doses (Storer and Mitchell, 1984). It is certainly true that in the case of the gamma ray dose-response curves, equally good fits can often be obtained with linear, linear quadratic, or quadratic h c t i o n s with a zero coefficient for the linear term regression equations. In the case of the neutron data, however, there are only a few cases where a linear fit is applicable except over a rather limited dose range; and although the neutron dose-response data can sometimes be fitted with a linear-quadratic equation (LS = a D + hD2), the coefficient of the dose-squared term is invariably negative. The results of the analyses can be summarized as follows. For single exposures to neutrons at relatively high doses P 0 . 2 Gy), data from several laboratories show a rather consistent pattern of an inverse relationship between RBE and the square mot of the neutron dose over substantial dose ranges, a pattern seen with many other test ol?jecta (Rossi, 1977a, 1982). It appears, however, that the pattern breaks down in the lowdose range; below 0.1 to 0.2 Gy for single exposures, the dose-response curve for neutrons is linear, or a t least indistinguishable from linear,

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8. LIFE SHOIZTENINGIN MICE-RBE

up to 0.05 to 0.1 Gy; and that the RBE value should be constant. Present estimates of the values range from 10 to 16. For short-term fractionated or protracted exposures, a similar relationship is observed. The initial slope of the neutron dose-response curve is about the same as for single exposures; the augmentation phenomenon associated with fractionation of aneutron dose is rather dramatic, a t least in some strains of mice, but it is negligible at low doses and does not appear to affect the initial slope. The slope for fractionated gamma radiation is appreciably flatter, so that the neutron RBE value is higher, lying between 11and 30 depending on sex and strain. For duration-of-lifeand other long-term protraction studies, a generally similar dose-response curve for neutrons is seen in the data from three laboratories. The dose-response curves for gamma radiation show considerable variation, depending on whether the expoures were given continuously or in weekly fractions. The RBE values consequently vary from 15 to 20 when based on weekly fractionated gamma-ray exposures to about 40 when based on continuous exposures. In conclusion, for single neutron exposures the best analyses to date suggest a limiting neutron RBE value of somewhere between 10 and 15, with a preponderance of data favoring the higher number (see Table 8.2).For continuous or long-term fractionated exposures, the answers are less definitive, but it seems probable that the RBE value will be as high as 40 to 50, when compared to a low LET radiation delivered at low dose rates and total doses. These numbers apply to RBE values for life shortening from all causes of death; RBE values for specific causes may vary by fact.org of two to four (Thomson, 1982;this report, Section 6). One final caveat: almost all of the above-mentioned data have been obtained on mice, specifically various strains of Mus musculus. The applicability of these numbers to man is, of course, uncertain, although much thought has been given to interspecies comparisons [see, e.g., Grahn et al. 1978, and NCRP, 19801.

8.5 DISCUSSION OF LIFE SHORTENING IN MICE

/

165

Neutron radiation &erange (Gy)

Low LET radiation range (Gy) Doae rate

Mouse strain and sex I. Single e q + m BALBic

W f

0.24-1.88

0.01 Gy d-I

Ia7Cey

0.54

0.083 Gy d-I

RFM

"Wf

0.24-1.88

0.01 Gy d-I

lB7Csy

0.50-4

0.083 Gy d - I

RF CF No. 1

PeBe. 5MeV CP-5 reactor

0.15-2.91 0.29-9.10

BOCo y %y

0.25-40.9 0.56-27

CBA

GLEEP reactor, 0.7 MeV

Variable 0.01-0.05 Gy min- 1 0.022-0.17 Gy wk-I

Woy

1-30

Variable 0.01-0.13 Gy min - 1 1-30 Gy wk-I

Source

0.5-3.75

Dose rate

Souree

Range of RBE values for 6nctionated and shortterm protracted expomm Ill. Duration-of-life and long-termfractionated exposurea BGCF,, d JANUS reador, 0.02-2.44 0.00033-0.0267 MCo y 0.85 MeV Gy ark-' BGCF,

P

CBA, 8 CBA, 2'

RF

JANUS reactor, 0.85 MeV

0.02-2.1 1

GLEEP reactor, 0.09-8.75 0.7 MeV GLEEP reactor, 0.09-8.76 0.7 MeV Po-Be, 5 MeV 0.016-9.3

C

Q, Q,

RBE

Reference

13b Starer et al., 1979, 1983, 1984 11-1C S-r el d.,1979, lW. 1984 3 4 Upton et d.,1967 12' Vogel el al., 1959

-

10-2P

Mole and Thomas, 1961

0.00033-0.0267 Gy wk-I

%y

1-27.50

0.000860.157 Gy wk-I 0.00085-0.157 Gy wk-I Variable

7

16.26-72.52

MCo 7

16.26-72.52

%.V

2.4-98.76

0.0167-0.319 Gy wk-I ? 3.6 0.0167-0.319 Gywk-'2 2.3 0.161.11 Gy wk-I 0.161.11 Gy wk-1 Variable

nn

thp . e m

mf

+ha u -1..me

rh-m

'

8 M

X

0

!

18.'P

E! Thomaon et al., l98lb, 1988

17Ab Thomson et al.. 1981b. 1988

- 16'

- 44.

- 2%

Neary et d., 1957, 1962 Neary et al.. 1957, 1962 Upton et a1.. 1967

Range of RBE value8 for duration of life and Long-term protracted expoeuree 17.In eases where a linear doserespome m e wuld not be established, the limiting value of the RBE was estir@d from the extrapolated neutron dose m n s e at 0.05 Gy (single and shortterm protracted exposures) or 0.005 Gy per week (duration of life exposures). bAuthors estimate. These value8 are highly dependent on the dose rate of the low-LFT radiation (see text). dPUE As-nAm

-

*

0 11-34

1-28.88

' r

i5

d

m

I

9. Discussion and Conclusions As emphasized in the introductory section 1.1, the scope of this report is limited to the influence of radiation quality on dose response functions as evidenced by experimentally determined values of RBE. Thus RBE, even though it plays a large role in determining Q, is sharply distinguished from Q. This is because several variables in addition to RBE, such as those discussed in Section 1.1, have to be taken into account in arriving a t a value for Q for a particular radiation. Accordingly, this report does not present values of Q. Any RBE value obtained experimentally is specific to the endpoint studied and to the physical factors, such as the dose, dose rate, and the protraction schedule used. It is also specific to the applicable biological and environmental conditions, and time elapsed between exposure and the observation of the endpoint concerned. For this reason, it is difficult to generalize RBE data from the various systems. In order to see if any groupings or patterns might emerge that would be useful in estimating a range of values within which those for radiation carcinogenesis in man might be expected to lie, RBE values for fisson neutrons obtained from data for low doses and dose rates6, for a number of end points including carcinogenesis in animals, are compared. This was done for the same end point across a limited number of species, and for several different end points within a species. Results from the various systems evaluated are listed in summary Table 9.1, derived from larger tables in the text. The values given are the closest available to the maximum value for the relative biological effectiveness, RBE,, taken as the ratio of the estimated slopes of the initial "linear" portions of the relevant experimentally determined dose-response curves. It has not been possible to determine by direct observation, for either the human or other mammalian species, the slopes for the 'More properly termed "temporal distributionof d m " because the effect is the result of increased time for intracellular repair with low doses spread out over time, or for restitution of cell populations in "renewal systems," and not to the physical dose rate per se.

168

/

9. DISCUSSION AND CONCLUSIONS

dose-response curves for cancer induction that have sufficiently narrow confidence limits for low doses of radiations. This is because of severe statistical limitations with small exposures. Therefore, most values for low doses must be derived either from lower systems, or by "extrapolation" (interpolation)from the higher, but still relatively low dose ranges. Statistical limitations often preclude obtaining an ;ga term when data for doses lower than 0.25 to 1Gy are available, due to the contribution of a higher order term at low dose. Such estimates of the initial slope of the linear term may systematically overestimate the slope of the linear component. If no significant higher order term is found and none can reasonably be assumed to exist, then an estimate of the initial slope derived from linear interpolation may be accepted as unbiased. An additional method, often used to estimate the slope of the initial linear term for low doses delivered a t any dose rate involves determination of the slope of the response curve following low dose rate exposures a t total doses sufficient to result in observable responses. Based on predictions of the linear quadratic model, this slope should estimate the slope of the initial linear term for low doses delivered at any dose rate. However, the "low dose rates" used experimentally, as emphasized in the earlier report on "dose rate" (NCRP, 19801, are frequently well above those commensurate with average occupational exposure limits or background radiation. The reason is that such extremely low exposure rates do not allow a total dose to be accumulated, even over most of the life span of a rodent, that is large enough to cause statistically significant or even observable increase in the mutation rate or tumor incidence. An.apparently linear initial portion of a dose-response curve does not necessarily rule out the existence of a dose rate effect. In view of the large range of RBE values obtained for different species and endpoints, three endpoints were selected for additional discussion based on relevance to radiation protection. The three selected are carcinogenesis, life shortening and chromosome abnormalities. Results for animal carcinogenesis are listed in Tables 6.1, 6.2 and 6.3, and in Table 9.1. It is seen that the dose response curves for both the low-LET and high-LET radiations differ considerably. Although the variation in the value of slope of the dose response curve may be somewhat less for the high-LET radiation, a range of 10 or more is seen for each radiation type. Accordingly, the RBE;zM values also vary widely, from close to unity to multiples of 10. For those tumors in which no excess can be found in the lower dose ranges of low LET radiation, (and where there are grounds for expecting a threshold), an RBE value cannot be determined. Life shortening in mice exposed to low doses or low dose rates is due almost exclusively to death from cancer. This end point has been

9. DISCUSSION AND CONCLUSIONS

TABLE9.1-Summary

of estimated RBEp values

1

169

for fission neutrons

versus gamma rays End point

Range of valuesa

Cytogenetic studies, human lymphocytes in culture 3 4 53 Transformation 3- 80b 5- 7W Genetic endpoints in mammalian systems 2-100 Genetic endpoints in plant system Life shortening, mouse 10- 46 Tumor induction 16- 59 a Values taken from larger tables or data given in earlier sections of this report. This value of 80 was derived h m one set of experiments only. ' The value of 70; derived h m data on specific locus mutations in mice, is not necessarily an RBE,,,.

frequently used, as the data available are more extensive than for other end points associated with carcinogenesis. Fission neutron RBE values for life shortening obtained from the low dose range lie in the range of 10 to 25 or 30 when high energy gamma radiation is used as the standard. Very low dose rates may yield somewhathigher values of RBE. The range of RBE values for cytogentic studies at low dose and dose rates is the smallest of all the end points studied. This is in contrast to RBE values for genetic end points in plant systems which have a range of 2 to 100. (Table 9.1). Results in chromosome abnormalities in human cells, some of which have been implicated in the etiology of human cancers (Rowley, 1984; Sandberg, 1980), are summarized in Section 2, see Table 2.12. The variation in RBE values for neutrons of a given energy appears to be relatively small. However, it is seen from Table 2.12 that the RBE values for neutrons, using x rays or gamma rays as the comparison radiation can differ by as much as factors of 1.5 to 3 or more. The RBE values are in the 1.1to 1.2 range for high doses delivered at low dose rates. The RBE values for induction of human chromosome aberrations for fission neutrons are in the range of 10 to 19 when 250 kVp x rays are used as the reference radiation and 34 to 53 when 60Cogamma rays (1.17 and 1.33 MeV) are used as the reference radiation. The above observations, also identified in other systems (Underbrink et al., 1976; Bond et al., 1978) suggests that a specification of the reference radiation finer than the present range that includes high energy gammas and relatively low energy x rays may be in order. The question merits attention particularly because a principal source of risk coefficients for low LET radiations is the Japanese experience, in which the radiation exposure was overwhelmingly to high-energy gamma rays. However, as noted in Section 1.2, such

170

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9.

DISCUSSION AND CONCLUSIONS

a change would interact strongly with other factors that must be considered in setting exposure limits. The substantial variation in the slope of the dose-response curves and thus RBE, noted above for both low and high-LET radiations, is for those endpoints considered most relevant to carcinogenesis and therefore to radiation protection. The higher values obtained at low doses, or higher doses a t low-dose rates, are the result largely, but not entirely, of changes in the slope of the dose response curve for the low-LET reference radiation. In addition, as noted above, the RBE value for a given high-LET radiation can differ by a factor of as much as three, depending on the reference radiation used for comparison. Average values, perhaps weighted on the basis of frequency of tumor types or other bases could be provided. However, these would be of limited use because of the larger variation in individual values. Because of the large range of RBE values for all endpoints reviewed, it must be a matter of judgement as to which values are to be used for selecting Q values for use in radiation protection.

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hairs and pollen," page 186 in Biological Effects of Neutron Irradiation, IAEA/STJ./F'UB/352 (International Atomic Energy Agency, Vienna). R. C., SPARROW, A. H. AND Ross& H. H. (1970). UNDERBRINK, A. G., SPARROW, "RBE of x rays and 0.43 MeV monoenergetic neutrons on somatic mutations and loss of reproductive integrity in Tmdescantia stamen hairs," page 373 in Symposium on Neutrons in Radiobiology,(Oak Ridge National Laboratory), Oak Ridge, Tennessee 1969, COW-691106. (U.S. Technical Information Service, Springfield, Virginia) Also Radiat. Res. 44, 187. R. C., SPARROW, A. H. AND ROSSI, H. H. (1971) UNDERBRINK, A. G., SPARROW, "Relative biological effectiveness of 0.43 MeV and low energy neutrons for somatic aberrations and hair length in Tradescantia stamen hairs," Int. J. Radiat. Biol. 19, 215. UNSCEAR (1977). United Nations Scientific Committee on the Effects of Atomic Radiation, Sources and Effects oflonizing Radiation. Report to the General Assembly with Annexes, Pub. E.77.M.1, (United Nations, New York). UNSCEAR (1982). United Nations Scientific Committee on the Effects of Atomic Radiation. Ionizing Radiation: Sources and Biological Effects (United Nations, New York). UNSCEAR (1988). United Nations Scientific Committee on the Weds of Atomic Radiation. Sources,Effects andRisks of ionizing Radiation, United Nations Scien-c Committee on the Effects of Atomic Radiation 1988 Report to the General Assembly, with annexes (United Nations, New York). A. W., FURTH, J.,CHRISTENBERRY, K. W.,ANDBENEDICT, UPTON,A. C.,KIMBALL, W. H. (1960). "Some delayed effects of atomic-bomb radiations in mice," Cancer Res. 20, 1. UPTON,A. C., RANDOLPH,M. L., AND CONKLIN, J. W. (1967). "Late effects of fast neutrons and gamma rays in mice a s influenced by the dose rate of irradiation: Life shortening," Radiat. Res. 32,493. UPTON.A. C., RANDOLPH,M. L., AND CONKLIN,J. W. (1970). "Late effeds of fast neutrons and gamma-rays in mice as influenced by the dose rate of irradiation: Induction of neoplasia," Radiat. Res. 41,467. VAN BEKKUM, D. W..BROERSE.J. J., ZWIETEN,M.J. VAN,HOLLANDER, C. F.. AND BLANKENSTEIN, M. 1. (1979). "Radiation induced mammary cancer in the rat," page 743 in Radiation Research, Okada, S., Imamura, M., Terasima, T., and Yamaguchi, H., Eds. D. (1980). "Recovery kinetics of radiation-induced VIRSIK,R. P. AND HARDER, chromosome aberrations in human Go lymphocytesn, Radiat. Environ, Biophys, 18, 221. VOGEL,H. H., JR.(1969). "Mammary gland neoplasms after fission neutron irradiation," Nature 222, 1279. VOGEL,H. H., JR. (1978). "High LET irradiation of Sprague-Dawley female rats and mammary neoplasm induction," page 147 in Late Biological Effects of Ionizing Radiation, Vol. 11, STIIPUB/489 (International Atomic Energy Agency, Vienna.) H. W. (1982). "Mammary neoplasia in SpragVOGEL,H. H.,JR.AND DICKSON, ue-Dawley rats following acute and protracted irradiation," page 135 in

Neutron Carcinogenesis, Broerse J. J. and Gerber, G. B., Eds. EUR Report 8084 EN. (Office of Official Publications of the European Communities, Luxembourg). VOGEL, H. H.,JR.,FRIGERIO, N. A., AM) JORDAN, D. L. (1961). "Life shortening in mice irradiated with either fission neutrons or cobalt40 gamma rays," Radiology, 77, 608. VULPIS,N. (1973). "Chromosome aberrations induced in human peripheral blood lymphocytes using heavy particles from 1°B(n,a)' Li reaction," Mutat. Res. 18, 103. VULPIS,N. AND SCARPA, G. (1986). "Induction of chromasome aberrations by 90Srp-particles in cultured human Golymphocytes," Mutat. Res. 163,277. V W I S ,N., PANEITA, G., AM)TAGNACCI. L. (1976). "Radiation-induced chromosome aberrations in radiological protection. Dose-response curves a t low dose-levels," Int. J. Radiat. Biol. 29,595. VULPIS,N., TAGNACCI L.. SCARPA, G. (1978). "Chromosome aberrations as a dosimetric technique for fission neutrons over the dose range 0.2-50 rad," Int. J. Radiat. Biol., 33. WAGNER, R., SCHMID, E.,AND BAUCIUNGER, M. (1983)."Application of conventional and FPG staining for the analysis of chromosome aberrations induced by low levels of dose in human lymphocytes," Mutat. Res., 109, 65. WALDREN, C., CORREU,L., SOGENIER, M. A. AM) PUCK,T. T. (1986) "Measurements of low levels of x-ray mutagenesis in relation to human disease." Proceed. Nat. Acad. Sci. 83,4839. WALKER, H. C. (1977). "Comparative sensitivities of meiotic prophase stages in male mice to chromosome damage by acute X- and chronic garnmairradiation," Mutat. Res. 44,427. WYROBEK, A. J. AND BRUCE.W. R. (1978). "The induction of sperm-shape abnormalities in mice and humans," page 257 in Vol. 5. Chemical Mutagens, Principles and Methods for their Detection, Hollaender A. and de Serres, F. J., Eds. (Plenum Press, New York). YANDEU,D. W., DRYJA,T.,AM) Lmm,J. B. (1986). "Somatic mutations at a heterozygous autosomal locus in human cells occur more frequently by allele loss than by intragenetic structural alterations." Somatic Cell and Molecular Genet. 12,255. YANG, T. C. H. AND TOBIAS, C. A., (1984). "Mechanisms of radiation-induced neoplastic cell transformation," presented at the Neyman Kiefer Conference, report LBG16793 Lawrence Berkeley Laboratory, (Berkeley, California). YANG,T. C. H., TOBIAS,C. A.. BWCELEY, E. A., CRAISE,L. M., W m s , I. S., PEREZ,C., AND HOWARD, J., (1980). "Enhancement effects of high energy neon particles on the viral transformation of mouse C3WlOT% cells in vitro," Radiat. Res. 81, 208. YOKORO, K.,m,C.. ITO, A., -A, K..KANDA,K.,AND KOBAYASHI, T. (1980). "Mammary carcinogenic effect of lowdose fission radiation in Wistar/Furth rats and its dependency on prolactin," J. Nat. Cancer Inst. 64, 1459.

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YUHAS,J. M. (1974). "Recovery from radiation-carcinogenic iqjury to the mouse ovary," Radiat. Res. 60, 321. ZHANGX-Z, JINC-Z,LENGRP, QIAOF., AND YANG,J. (1982). "Chromosome aberrations induced in human lymphocytes after irradiation with neutron sources ?Wf and %lAm-Be,"Page 255 in Proceedings of the First Academic Session of the Chinese Society of Radiation Protection, (Atomic Energy Press, Beijing). ZAIDER.M. AND BREEINER, D. J . (1985). "Microdosimetricdefinition of quality factors," Radiat. Res. 103,302. ZHOU,H., QIAD, F.. GUO.Z., ZHANG.X.. LENG,R.. ZHENG,S., ZHU,Y., YAM, J., AND ZHAO, X. (1979). "14 MeV neutrons induced chromosomal aberrations in human peripheral blood lymphocytesirradiated in vitro," Acta Cenetica Sinica 6, 363. E., ROSIEK,0.. AND SABLINSKI, J. (1980). ZIEMBA-ZOKTOWAKA, B., BOCIAN, "Chromosome aberrations induced by low doses of X-rays in human lymphocytes in vitro," Int. J . Radiat. Biol. 37,231. J., AND BARENDSEN, G. W. (1983). "Dose-effect relationships for ZOETELIEF, induction of cell inactivation and asymmetrical chromosome exchanges in three cell lines by photons and neutrons of different energy," Int. J. Radiat. Biol. 43,349. E. W., C A S ~G. W., , AND ROGERS, C. (1985). "NeoZOOK,B. C., BBADLEY, plastic and other pathologic effects of fractionated fast neutrons or photons on the thorax and anterior abdomen of beagles," p. 193 in Proceedings of the 22nd Hanford Life Sciences Symposium. Life-Span Radiation Effects in Animals-What Can They Tell Us? Thompson, R. C. and Mahaffey, J. A., Eds., CONF-830951 (National Technical Infonnation Service, Springfield, Virginia)

The NCRP The National Council on Radiation Protection and Measurements is a nonprofit corporation chartered by Congress in 1964 to: 1. Collect, analyze, develop, and disseminate in the public interest information and recommendations about (a) protection against radiation and (b) radiation measurements, quantities, and units, particularly those concerned with radiation protection; 2. Provide a means by which organizations concerned with the scientific and related aspects of radiation protection and of radiation quantities, units, and measurements may cooperate for effective utilization of their combined resources, and to stimulate the work of such organizations; 3. Develop basic concepts about radiation quantities, units, and measurements, about the application of these concepts, and about radiation protection; 4. Cooperate with the International Commission on Radiological Protection, the International Commission on Radiation Units and Measurements, and other national and international organizations, governmental and private, concerned with radiation quantities, units, and measurements and with radiation protection. The Council is the successor to the unincorporated association of scientists known as the National Committee on Radiation Protection and Measurements and was formed to carry on the work begun by the Committee. The Council is made up of the members and the participants who serve on the over sixty scientific committees of the Council. The scientific committees, composed of experts having detailed knowledge and competence in the particular area of the committee's interest draft proposed recommendations. These are then submitted to the full membership of the Council for careful review and approval before being published. The following comprise the current officers and membership of the Council: officers President WARREN K . SINCLAIR Vice President Secretary and

Treasurer Assistant Secretary Assistant Trwsurer

S. JAMES A D E ~ I N

W.ROGERNEY

CARLD. HOBELMAN

JAMESF . BERG

THENCRP Members SEYMOUR ABRAHAMSON JACOB I. FABRMANT s.JAMES ADEU~~EIN R J. MICHAELFRY PEI%XR. ALMOND THOMASF.GESELL EDWARDL. ALPEN ETHELS. GILBERT LYNNR. ANSPAUGH ROBERTA. GOEPP JOHN A. AUXIER JOEL E. GRAY WILLIAMJ. BAIB ARTHUB W. G w MICHAELA. BENDER ERICJ. HALL B GORDONBLAYLOCK NAOMIH. HARLEY BRUCEB. BOECKER WILLUM R. HENDEE JOHN D. BOICE,JR DONALDG. JACOBS ROBERTL. BRENT A EVEREITEJ m ,JR. ANTONEBROOKS BERNDKAHN KENNETHR KASE PAULL. CARSON MELVINW. CARTER HAROLD L.KUNDEL RANDALL S. C m L L CHARLESE. LAND JAMES E. CLEAVER RAYD.LLOYD FRED T. CRO89 HARRY R. WON STANLEY B. CURTIS ROGER0.MCCLELLAN GERALDD. DODD BARBARAJ. MCNEIL PATRICIAW. DURBIN CHARLESB. MEINHOLD CARLH. DURNEY L.MENDEMHN MORTIMER KEm F.ECKERMAN FRED A METIZER CHARLESEBENHAUER WILLIAM A. MILLS THOMAS S. ELY DADEW. MOELLER

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A. ALAN MOGHISSI JOHN W. P ~ N ANDREWK.POZNANSKI NORMANC. RASMUSSEN C H E R ~RICHMOND GENEVI~VE ROESSLER MARVINRO~ENSTEIN LAWRENCE N. F~YIRENBERG LEONARDA. SAGAN KEm J. SCHIAGER ROBERTA SCHLENKER ROY E. SHORE WARRENK.S ~ C L A I R PAULSLOVIC RICHARD A TELL W ~ L I AL.MTEMPLETON THOMASS. TENWRDE J. W. T H I E ~ ~ E N JoHN E. TILL ROBERTL. ULLRICH ARTHUBC. U ~ N GEORGEL. VOELZ GEORGEM. WILKENING MARVINZ w m

Honorary Members L T R I ~ ~S.~ TAYLOR, N Honorniy

ROBERT0.GORSON JOHN H. HARLEY JOHN W. HEALY L o r n H.

HEMPELMANN. JR PAULC. HODGES GEORGEV. LEROY WILFRID B. MANN KARLZ. MORGAN ROBERTJ. NELSEN

Presided

WESLEYL NWORC HARAI,DH.ROSSI WILLIAM L RUEWELL JOHN H. R u m EUGENEL. SAENGER J. S c m WILLIAM J. NEWELLSTANNARD JOHN B. S ~ O R E R ROY C. THOMPSON EDWARDW. WEBSTER HAROLD 0.WYCKOW

Currently, the following subgroups are actively engaged in formulating recommendations: SC 1

SC 16

Basic Radiation Protection Criteria SC 1-1 Probability of Causation for Genetic and Developmental Effects SC 1-2 The Aese8ament of Risk for Radiation Protection Purposes SC 1-3 Collective Dose X-Ray Protection in Dental Offices

THE NCRF' Biological Aspects of Radiation Protection Criteria SC 40-1 Atomic Bomb Survivor Dosimetry Operational Radiation Safety SC 46-2 Uranium Mining and Milling-Radiation Safety Programs SC 46-3 ALARA for Occupationally E x p d Individuals in Clinical Radiology SC 46-4 Calibration of Survey Instrumentation SC 46-5 Maintaining Radiation Protection Records SC 46-7 Emergency Planning SC 46-8 Radiation Protection Design Guidelines for Particle Accelerator Facilities SC 46-9 ALARA a t Nuclear Plants SC 46-10 ABsessment of Occupational Doee8 from Internal Emitters SC 46-11 Radiation Protection During Special Medical P d u r e e Conceptual Basis of Calculations of Dose Distributions Internal Emitter Standards SC 57-2 h p i r a t o r y Tract Model SC 57-6 Bone Problems SC 57-9 Lung Cancer Risk SC 57-10 Liver Cancer Risk SC 57-12 Strontium SC 57-14 Placental Transfer SC 57-15 Uranium Human Population Exposure Experience Radiation Exposure Control in a Nuclear Emergency SC 63-1 Public Knowledge About Radiation SC 63-2 Criteria for Radiation Instruments for the Public Environmental Radioactivity and Waste Management SC 64-6 Screening Models SC 64-7 Contaminated Soil as a Source of Radiation Exposure SC 64-8 Ocean Diaposal of Radioactive Waste SC 64-9 Effects of Radiation on Aquatic Organism SC 64-10 Xenon SC 64-11 Disposal of Low Level Waste Quality Assurance and Accuracy in Radiation Protection Measurements Biological Effects and Exposure Criteria for Ultrasound Biological Effects of Magnetic Fields Efficacy of Radiographic Procedures Radiation Exposure and Potentially Related Iqjury Guidance on Radiation Received in Space Activities Effects of Radiation on the Embryo-Fetus Guidance on Occupational and Public Exposure Resulting from Diagnostic Nuclear Medicine Procedures Practical Guidance on the Evaluation of Human Exposures to Radiohquency Radiation Extremely Low-Frequency Electric and Magnetic Fields Radiation Biology of the Skin (Beta-Ray Dosimetry) Identification of Research Needs Contamination of Materials, Objects and Soils Risk of Lung Cancer from Radon Hot Particles in Eyes, Ears and Lunge

THENCRP

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Ad Hoc Committee on Comparison of Radiation Exposures Ad Hoc Group on Plutonium Ad Hoc Group on Radon Ad Hoc Group on Video Display lbrminals Study Group on Comparative Risk %k Force on Occupational Exposure Levele

I n recognition of its responsibility to facilitate and stimulate cooperation among organizations concerned with the scientific and related aspects of radiation protection and measurement, the Council has created a category of NCRP Collaborating Organizations. Organizations or groups of organizations that are national or international in scope and are concerned with scientific problems involving radiation quantities, units, measurements, and effects, or radiation protection may be admitted to collaborating status by the Council. The present Collaborating Organizations with which the NCRP maintains liaison are as follows: American Academy of Dermatology American Association of Physicists in Medicine American College of ~ e d i c a Physics l American Collem of Nuclear Physicians American college of Radiology American Dental A~eociation American Industrial Hygiene Agsociation American Institute of Ultrasound in Medicine American Insurance Services Group American Medical Association American Nuclear Society American Occupational Medical Association American Pediatric Medical b c i a t i o n American Public Health Amciation American Radium Society American Roentgen Ray Society American Society of Radiologic Technologists American Society for Therapeutic Radiology and Oncology Association of University Radiologists Bioelectromagnetics Society College of American Pathologists Conference of Radiation Control Program Directors Electric Power Research h t i t u t e Federal Communicatione Cornmimion Federal Emergency Management Agency Genetics Society of America Health Effects Research Foundation (Japan) Health Physics Society Institute of Nuclear Power Operations National Electrical Manufacturers k i a t i o n National Institute of Standards and Technology Nuclear Management and Resources Council Radiation Research Society '

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Radiological Society of North America Society of Nuclear Medicine United States Air Force United States Army United States Department of Energy United States Department of Housing and Urban Development United States Department of Labor United States Environmental Protection Agency United States Navy United States Nuclear Regulatory Commission United States Public Health Servica

The NCRP has found its relationship with these organizations to be extremely valuable to continued progress in its program. Another aspect of the cooperative efforts of the N O relates to the special liaison relationships established with various governmental organizations that have an interest in radiation protection and measurements. This liaison relationship provides: (1)an opportunity for participating organizations to designate an individual to provide liaison between the organization and the NCRP, (2) that the individual designated will receive copies of draft NCRP reports (at the time that these are submitted to the members of the Council) with an invitation to comment, but not vote; and (3) that new NCRP efforts might be discussed with liaison individuals as appropriate, so that they might have an opportunity to make suggestions on new studies and related matters. The following organizations participate in the special liaison program: Auetralian Radiation Laboratory Commiesariat a 1'Energie Atomique (France) Commission of the European Communities Defense Nuclear Agency Federal Emergency Management Agency Japan Radiation Council National Institute of Standards and Technology National Radiological Protection Board (UnitedKingdom) National Reeearch Council (Canada) Otlice of Science and Technology Policy Otlice of Technology Assessment Ultrasonics Institute of Australia United States Air Force united states Anmy United States Coast Guard United States Department of Energy United States Department of Health and Human Services United States Department of Labor United States Department of Tramportation United States Environmental Proteetion Agency United States Navy United States Nuclear Regulatory Commission

THENCRP

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The NCRP values highly the participation of these organizations in the liaison program. The Council's activities are made possible by the voluntary contribution of time and effort by its members and participants and the generous support of the following organizatiom: A h d P. Sloan Foundation Alliance of American Ineurera American Academy of Dental Radiolugy American Academy of Dermatology American Aaeociation of Physicists in Medicine American College of Medical Physics American College of Nuclear Physicians American College of Radiology American College of Radiology Foundation American Dental h i a t i o n American Hospital Radiology Administratom American Industrial Hygiene h i a t i o n American Insurance Services Group American Medical A m i a t i o n American Nuclear Society American Occu~ationalMedical Aeeociation American osteopathic College of Radiology American Pediatric Medical b i a t i o n American Public Health Amciation American Radium Society American Roentgen Ray Society American Society of Radiologic Technologiete American Society for Therapeutic Radiology and Oncology American Veterinary Medical h i a t i o n American Veterinary Radiology Society Association of University Radiologists Battelle Memorial Institute Center for Devices and Radiological Health College of American Pathologiets Committee on Radiation Research and Policy Coordination Commonwealth of Pennsylvania Defense Nuclear Agency Eastman Kodak Company Edison Electric Institute Edward M a l l i n c M t , Jr. Foundation EGLG Idaho, Inc. Electric Power Reeearch h t i t u t e Federal Emergency Management Agency Florida Institute of Phoe~hateR d Genetics Society of Amehca Health Effects Research Foundation (Japan) Health fiysics Society Institute of Nuclear Power Operatione James Picker Foundation Martin Marietta Corporation National Aeronautics and Space Administration

National Asstxiation of Photographic Manufacturers National Cancer Institute National Electrical Manufacturere Association National Institute of Standards and Technology Nuclear Management and Resources Council Radiation Research Society Radiological Society of North America Richard Lounsbery Foundation Sandia National Laboratory Society of Nuclear Medicine united States Department of Energy United States Dmartment of Labor United States ~'-nmental Protection Agency United S t a b Navy United States Nuclear Regulatory Commission Victoreen, Incorporated

To all of these organizations the Council expresses its profound appreciation for their support. Initial funds for publication of NCRP reports were provided by a grant from the James Picker Foundation and for this the Council wishes to express its deep appreciation. The NCRP seeks to promulgate information and recommendations based on leading scientific judgment on matters of radiation protection and measurement and to foster cooperation among organizations concerned with these matters. These efforts are intended to serve the public interest and the Council welcomes comments and suggestions on its reports or activities from those interested in its work.

NCRP Publications NCRP publications are distributed by the NCRP Publications' office. Information on prices and how to order may be obtained by directing a n inquiry to: NCRP Publications 7910 Woodmont Ave., Suite 800 Bethesda, Md 20814 The currently available publications are listed below. Proceedings of the Annual Meeting No. 1

Title Perceptions of Risk, Proceedings of the Fifteenth Annual Meeting, Held on March 14-15,1979 (Including Taylor Ledure No. 3) (1980) Quantitative Risk in Standurds Setting, Proceedings of the Sixteenth Annual Meeting, Held on April 2-3, 1980 (Including Taylor Lecture No. 4) (1981) Critical Issues in Setting Radiation Dose Limits, Proceedings of the Seventeenth Annual Meeting, Held on April 8-9, 1981 (Including Taylor Lecture No. 5) (1982) Radiation Protection and New Medical Diagnostic Procedures, Proceedings of the Eighteenth Annual Meeting, Held on April 6-7,1982 (Including Taylor Lecture No. . 6) (1983) Environmental Radioactivity, Proceedings of the Nineteenth Annual Meeting, Held on April 6-7, 1983 (Including Taylor Lecture No. 7) (1984) Some Issues Important in Developing Basic Radiation Protection Recommendations, Proceedings of the Twentieth Annual Meeting, Held on April 4-45, 1984 (Including Taylor Ledure No. 8) (1985) Radioactive Waste, Proceedings of the Twenty-first Annual Meeting, Held on April 3-4, 1985 (Including Taylor Lecture No. 9) (1986)

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NCRP PUBLICATIONS

Nonionizing ElectromugneticRadiation and Ultnrsound, Proceedings of the Twenty-second Annual Meeting, Held on April 23,1986 (IncludingTaylor Lecture No. 10) (1988) New Dosimetry at Hiroshima and Nagasaki and Its Implications for Risk Estimates, Proceedings of the Twenty-third Annual Meeting, Held on April 5-6, 1987 (Including Taylor Lecture No. 11)(1988). Rcrdon, Proceedings of the Twenty-fourth Annual Meeting, Held on March 30-31,1988 (IncludingTaylor Lecture No. 12) (1989). Radiution Protection Today-The NCRP at Sixty Years, Proceedings of the Twenty-fifth Annual Meeting of the National Council on Radiation Protection and Measurements, Held on April 5-6,1989 (Including Taylor Lecture No. 13) (1990). Symposium Proceedings

The Control of Exposure of the Public to Ionizing Radidbn in the Event of Accident or Attack, Proceedings of a Symposium held April 27-29,1981 (1982) Lauriston S. Taylor Lectures No. 1 2 3 4

5

6

Title and Author The Squares of the Natural Numbers in Radiation Protection by Herbert M. Parker (1977) Why be QuantitativeAbout Radiution Risk Estimates? by Sir Edward Pochin (1978) Radiation Protection-Concepts and Trade O f f . by Hymer L. Friedell (1979)[Availablea h in Perceptions of Risk, see above] From "Quantity of Radiation" and 'Dose" to "Exposure" and "AbsorbedDose''-An Historical Review by Harold 0.Wyckoff (1980)[Availablealso in QuantitcrtiveRisks in Standards Setting, see above] How Well Can We Assess Genetic R i s k Not Very by James F. Crow (1981) [Available also in Critical Issues in Setting Radiution Dose Limits,see above] Ethics, Td-off' and Medical Radicrtion by Eugene L. Saenger (1982) [Available also in Radiation Protection and New Medical Diagnostic Approaches, see abovel

NCRPPUBLICATIONS

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The Human Environment-Past, Present and Future by Merril Eisenbud (1983) [Available also in Environmental Radioactivity, see abovel Limitation and Assessment in Radiation Protection by Harald H . Rossi (1984) [Available also in Some Issues Important in Developing Basic Radiution Protection Recommendrrtions, see abovel Truth (and Beauty) in Radiation Measurement by John H. Harley (1985:)[Available also in Radioactive Waste, see above] Nonionizing Radiation Bioeffects: Cellular Properties and Intentions by Herman P. Schwan (1986) [Available also in Nonionizing Electromagnetic Radiations and Ultrasound, aee abovel How to be Quantitative about Radiation Risk Estimates by Seymour Jablon (1987) [Available also in New Dosimetry at Hiroshima and Nagasaki and its Implidations fir Risk Estimates, see abovel How Safe is Safe Enough? by Bo Lindell(1988) [Available also in Radon, see abovel Radiobiology and Radiation Protection: The Past Century and Prospects for the Future by Arthur C. Upton (1989) [Available also in Radiatwn Protection Today, see above] Radiation Protection and the Internal Emitter Saga by J . Newel1 Stannard (1990).

NCRP Commentaries No. 1

Title

Ktypton-85 in the Atmosphere-- With Specific Reference to the Public Health Significance of the Proposed Controlled Release at Three Mik Island (1980) Preliminary Evaluation of Criteria fir the Disposal of Tmnsumnic Contuminated Waste (1982) Screening Techniques for Determining Compliance with Environmental Standards (19861, Rev. (1989) Guidelines for the Release of Waste Water fiom Nuclear Facilities with Special Reference to the Public Health Signifiance of the Proposed Release of Treated Waste Waters at Three Mile Island (1987) A Review of the Publication, Living Without Landfills (1989)

/

NCRP PUBLICATIONS

NCRP Reports

No. 8

Title Control and Removal of Radioactive Contamination i n Laboratories (1951) Maximum Permissible Body Burdens and Maximum Permissible Concentrations of Radionuclides in Air and i n Water for Occupational Exposure (1959)[Includes Addendum 1 issued in August 19631 Measurement of Neutron Flux and Spectra for Physical and Biological Applications (1960) Measurement o f A bsorbed Dose ofNeutrons and Mixtures of Neutrons and Gamma Rays (1961) Stopping Powers for Use with Cavity Chambers (1961) Safe Handling of Radioactive Materials (1964) Radiation Protection in Educational Institutions (1966) Dental X-Ray Protection (1970) Radiation Protection in Veterinary Medicine (1970) Precautions in the Management of Patients Who Have Received Therapeutic Amounts of Radionuclides (1970) Protection Against Neutron Radiation (1971) Protection Against Radiation from Brachytherapy Sources (1972) Specifications of Gamma-Ray Brachytherapy Sources (1974) Radiological Factors Affecting Decision-Making i n a Nuclear Attack (1974) Krypton-85 i n the Atmosphere-Accumulation, Biological Significance, and Control Technology (1975) Alpha-Emitting Particles in Lungs (1975) Tritium Measurement Techniques (1976) Structural Shielding Design and Evaluation for Medical Use of X Rays and Gamma Rays of Energies Up to 10 MeV (1976) Envimnmentul Radiation Mecrsurements (1976) Radiation Protection Design Guidelines for 0.1-100 MeV Particle Accelemtor Facilities (1977) Cesium-137 fi-om the Environment to Man: Metabolism and Dose (1977) Review of NCRP Radiation Dose Limit for Embryo and Fetus in Occupationally Exposed Women (1977) Medical Radiation Exposure of Pregnant and Potentially Pregnant Women (1977)

NCRP PUBLICATIONS

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209

Protection of the Thyroid Gland in the Event of Releases of Radioiodine (1977) Instrumentation and Monitoring Methods for Radiation Protection (1978) A Handbook of Radioactivity Measurements Procedures, 2nd ed. (1985) Operational Radiation Safety Program (1978) Physical, Chemical, and Biological Properties of Radiocerium Relevant to Radiation Protection Guidelines (1978) Radiation Safety Training Criteria for Industrial Radiography (1978) Tritium in the Environment (1979) Tritium and Other Radionuclide Labeled Organic Compounds Incorporated in Genetic Material (1979) Influence of Dose and Its Distribution in Time on DoseResponse Relationships for Low-LET Radiations (1980) Management of Persons Accidentally Contaminated with Radionuclides (1980) Mammography (1980) Radiofreqency Electromagnetic Fields-Properties, Quantities and Units, Biophysical Interaction, and ' Measurements (1981) Radiation Protection in Pediatric Radiology (1981) Dosimetry of X-Ray and Gamma-Ray Beams for Radiation Therapy in the Energy Range 10 keV to 50 MeV (1981) Nuclear Medicine-Factors Influencing the Choice and Use of Radionuclides i n Diagnosis and Therapy (1982) Operational Radiation Safety-Training (1983) Radiation Protection and Measurement for Low Voltage Neutron Generators (1983) Protection in Nuclear Medicine and Ultrasound Diagmstic Procedures in Children (1983) Biological Effects of Ultrasound: Mechanisms and Clinical Implications (1983) Iodine-129: Evaluation of Releases from Nuclear Power Generation (1983) Radiological Assessment: Predicting the ~ r a n s ' ~ o r t , Bioaccumulation, and Uptake by Man ofRadionuclides Released to the Environment (1984) Exposures from the Uranium Series with Emphasis on Radon and its Daughters (1984)

NCRP PUBLICATIONS

Evaluation of Occupational and Environmental Exposures to Radon and Radon Daughters in the United States (1984) Neutron Contamination from Medical Electron Accelerators (1984) Induction of ThyroidCancer by IonizingRadiztion (1985) Carbon-14 in the Environment (1985) SI Units i n Radiation Protection and Measurements (1985) The Experimental Basis for Absorbed-Dose CalcuLations in Medical Uses of Radionuclides (1985) General Comepk for the Dosimetry of Internally Deposited Radionlcclides (1985) Mammography-A User's Guide (1986) Biological Effects and Exposure Criteria for Radiofrequency Ebctromagnetic Fields (1986) Use of Bioassay Procedures for Assessment of Internal Radionuclide Deposition (1987) Radiation Alarms and Access Control Systems (1987) Genetic Effects of Internally Deposited Radionuclides (1987) Neptunium: Radiation Protection Guidelines (1987) Recommendations on Limits for Exposure to Ionizing Rarliation (1987) Public Radiation Exposure fkom Nuclear Power Generation in the United States (1987) Ionizing Radiation Exposure of the Population of the United States (1987) Exposure of the Population in the United States and Canadu from Natuml Background Radiation (1987) Radiation Exposure of the U.S. Population fi.om Consumer Products and Miscellaneous Sources (1987) Compamtive Caminogenesis of Ionizing Radiation and Chemicals (1989) Measurement of Radon and Radon Daughters in Air (1988) Guidance on Radiation Received i n Space Activities (1989) Quality Assumme for Diagnostic Imaging Equipment (1988) Exposure of the U.S. Population jiom Diagnostic Medical Radiation (1989) Exposure of the U.S. Population From Occupational Radiation (1989)

NCRP PUBLICATIONS

102 103 104

105

106

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Medical X-Ray, Electron Beam and Gamma-RayPmtactwn For Energies Up To 50 MeV (Equipment Design, Perfbrmance and Use) (1989) Control of Radon in Houses (1989) TheRelative Biological EffectivenessofRadiahnsofDifferent Quality (1990) Radiation Protection for Medical and AUied Health Personnel (1989) Limits of Exposure to "Hot Particles" on thiskin (1989)

Binders for NCRP Reports are available. Rvosizes make it possible to collect into small binders the "old series" of reports (NCRPReports Nos. 8-30) and into large binders the more recent publications (NCW Reports Nos. 32-106). Each binder will accommodate from five to seven reports. The binders cany the identification "NCRP Reports" and come with label holders which permit the user to attach labels showing the reports contained in each binder. The following bound sets of NCRP Reports are also available: Volume I. NCRP Reports Nos. 8, 22 Volume TI. NCRP Reporta Nos. 23,25,27,30 Volume III. NCRP Reports Nos. 32,35,36,37 Volume IV. NCRP Reporta Nos. 38,40,41 Volume V. NCRP Reports Nos. 42,44,46 Volume VI. NCRP Reports Nos. 47,49,50,51 Volume VII. NCRP Reports Nos. 52,53,54,55,57 Volume VIII. NCRP Reports No. 58 Volume IX. NCRP Reports Nos. 59, 60, 61, 62, 63 Volume X. NCRP Reports Nos. 64,65,66,67 Volume XI. NCRP Reports Nos. 68,69, 70, 71,72 Volume Xn. NCRP Reporta Nos. 73,74,75,76 Volume XIII. NCRP Reports Nos. 77,78,79,80 Volume XTV. NCRP Reports Nos. 81,82,83,84,85 Volume XV. N C W Reports Nos. 86,87,88,89 Volume XVI. NCRP Reports Nos. 90,91,92,93 Volume XVII. NCRP Reports Nos.94,95,96,97 Volume XVIII. NCRP Reporta Nos. 98,99,100 (Titles ofthe individual reports contained in each volume are given above). The following NCRP Reports are now superseded and/or out of print:

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Title X-Ray Protection (1931).[Superseded by NCRP Report No. 31 Radium Protection (1934).[Supersededby NCRP Report No. 41 X-Ray Protection (1936).[Superseded by NCRP Report No. 61 Radium Protection (1938).[Superseded by NCRP Report No. 131 Safe Handling of Radioactive Luminous Compounds (1941).[Out of Print] Medical X-Ray Protection Up to TwoMillion Volts (1949). [Superseded by NCRP Report No. 181 Safe Handling of Radioactive Isotopes (1949).[Superseded by NCRP Report No. 301 Recommendations for Waste Disposal of Phosphorus32 and Iodine-131 for Medical Users (1951).[Out of Printl Radiological Monitoring Methods and Instruments (1952).[Superseded by NCRP Report No. 571 Maximum Permissible Amounts of Radioisotopes in the Human Body and Maximum Permissible Concentrations in Air and Water (1953).[Superseded by NCRP Report No.221 Recommendations for the Disposal of Carbon-14 Wastes (1953).[Superseded by NCRP Report No. 811 Protection Against Radiations t o m Radium, Cobalt-60 and Cesium-137 (1954).[Supersededby NCRP Report No. 241 Protection Against Betatron-Synchrotron Radiations Up to 100 Million Electron Volts (1954).[Superseded by NCRP Report No. 53.1 Safe Handling of Cadavers Containing Radioactive Zsotopes (1953).[Superseded by NCRP Report No. 211 Radioactive Waste Disposal in the Ocean (1954).[Out of Print] Permissible Dose from External Sources oflonizing Radiation (1954)including Maximum Permissible Exposure to Man, Addendum to National Bureau of Standards Handbook 59 (1958).[Supersededby NCRP Report No. 391 X-Ray Protection (1955).[Superseded by NCRP Report No. 261 Regulation of Radiation Exposure by Legislative Means (1955).[Out o f Print]

NCRP PUBLICATIONS

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Protection Against Neutron Radiation Up to 30 Million Electron Volts (1957). [Superseded by NCRP Report No. 381 Safe Handling of Bodies Containing Radioactive Isotopes (1958). [Superseded by NCRP Report No. 371 Protection Against Radiations from Sealed Gamma Sources (1960). [Superseded by NCRP Report Nos. 33, 34, and 401 Medical X-Ray Protection Up to Three Million Volts (1961). [Superseded by NCRP Report Nos. 33, 34, 35, and 361 A Manual of Radioactivity Procedures (1961). [Superseded by NCRP Report No. 581 Exposure to Radiation in an Emergency (1962). [Superseded by NCRP Report No. 421 Shielding for High Energy Electron Accelerator Installations (1964).[Superseded by NCRP Report No. 511 Medical X-Ray and Gamma-Ray Protection for Energies up to 10 MeV-Equipment Design and Use (1968). [Superseded by NCRP Report No. 1021 Medical X-Ray and Gamma-Ray Protection for Energies Up to 10 MeV-Structural Shielding Design and Evaluation (1970). [Superseded by NCRP Report No. 491 Basic Radiation Protection Criteria (1971). [Superseded by NCRP Report No. 911 Review of the Current State of Radiation Protection Philosophy (1975). [Superseded by NCRP Report No. 911 Natural Background Radiation in the United States (1975). [Superseded by NCRP Report No. 941 Radiation Protection for Medical and Allied Health Personnel [Superseded by NCRP Report No. 1051 Radiation Exposure from ConsumerProducts and Miscellaneous Sources (1977). [Superseded by NCRP Report No. 951 A Handbook on Radioactivity Measurement Procedures. [Superseded by NCRP Report No. 58,2nd ed.] Other Documents The following documents of the NCRP were published outside of the NCRP Reports and Commentaries series:

"Blood Counts, Statement of the National Committee on Radiation Protection," Radiology 63,428 (1954)

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"Statements on Maximum Permissible Dose from Television Receivers and Maximum Permissible Dose to the Skin of the Whole Body," Am. J. Roentgenol., Radium Ther. and Nucl. Med. 84,152 (1960) and Radiology 75,122 (1960) Dose Effect Modifiing Factors In Radiation Protection, Report of Subcommittee M-4 (Relative Biological Effectiveness) of the National Council on Radiation Protection and Measurements, Report BNL 50073 (T-471) (1967) Brookhaven National Laboratory (National Technical Information Service, Springfield, Virginia). X-Ray Protection Standards for Home Television Receivers, Interim Statement of the National Council on Radiation Protection and Measurements (National Council on Radiation Protection and Measurements, Washington, 1968) Specification of Units of Natuml Umnium and Natuml Thorium (National Council on Radiation Protection and Measurements, Washington, 1973) NCRP Statement on Dose Limit for Neutrons (National Council on Radiation Protection and Measurements, Washington, 1980) Control ofAirEmissions ofRadionuclides (National Council on Radiation Protection and Measurements, Bethesda, Maryland, 1984) Copies of the statements published in journals may be consulted in libraries. A limited number of copies of the remaining documents listed above are available for distribution by NCRP Publications.

Index Alpha particles 84-87.143 Effecta on apem morphology, 86 Hereditary effects, 84 Mutagenic efficiency, 85 RBE values, 87,143 Arabidopsis (plant), 15,23,24 RBE Values, 23,24 Assay system, 4 9 , m BALBI3T3 cells, 50 C3W10T1I2cells, 50 Syrian hameter embrgos, 49 Auger electrons, 58 RBE Values, 58 Augmentation, 153, 158 Neutron irradiation, 163, 158 BALBl3T3 cells, 50 Transformation assay system, 50 Basal cell carcinomas, 134 Bone sarcoma, 142,148 RBE of h i o n fragments, 148 C3HI1(YT1/2cells, 5 0 , 5 2 4 5 Transformation assays, 60, 5 2 , M Transformation frequency, 53 Carcinogenesis, 139 Influence of radiation quality. 139 Cataract formation, 11 Cell killing, 66 Californium 252 neutrons, 119,127 Induction of harderian gland tumors, 127 Induction of lung adenomas, 119 Chromosome aberrations, 29,30, 3336,37-44,46,81, 140, 149 Alpha particle induced, 39,41 Beta particle induced, 39 Chinese hamster cells, 39 Chromitid exchanges, 38 Deletions, 29 Dicentrics, 29,33,41 Dose-response relationship, 30 Exchange type, 29 Fisaion fragment induced, 39 Gamma ray induced, 37,39,42,43 Heavy ion induced, 38 High LET radiation induced, 44 Human cells, 46 Human lymphocytes, 40.42

Internal emitter induced, 140 Lymphocytes, 30,41 Neutron induced, 35,37,42,43 Roton induced, 41 RBEy, 33 RBE valuee, 149 Single track, 40 Versue lineal energy, 34 x ray induced, 38,43 Chromoeome volume. 23 Chinese hamster V79 cells, 39,65,66 Chromosome aberrations, 39 Mutation studies, 66 RBE-LET relationship, 66 Cesium 137 gamma rays, 122,127 Induced harderian gland tumors, 127 Induced lung adenocarcinomas, 122 Cyclotron neutrons, 77 Induced dominant lethal mutations, 77 Cytogenetic effecta, 14,27,28,40 In mammalian cells, 28,40 In planta, 14.27 RBE values, 40 Dominant lethal mutations, 75 Dose equivalent, 10 Dose-rate dependencies, 55 Dose-rate effects, 72, 113, 122, 126 Lung tumors, 122 Mammary tumors, 126 Myeloid leukemia, 113 thymic lymphoma, 113 Dose-response relationehip, 29 Low-LET radiatiom, 29 Dose-response curve. 7 Linear quadratic, 7 Drosophila, 95, 96, 99 Dominant lethal mutations. 95 Neutron induced mutations, 99 RBE values ('lbble), 96 Recessive mutations, 95 location mutations, 95 x-ray induced mutations, 99 Effective doae equivalent, 10 Environmental decta. 15 Response of plants to irradiation, 15

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Epithelial cell tumors, 114 Ovarian tumors, 114 Fibrmarmmas, 134 Fieeion neutrons, 21,24,43,83,87, 88,90,93,94,116,122,124,127, 128 Dominant lethal mutations, 77 Harderian gland tumore, 128 Lung adenocarcinomas, 122 mamma^^ tumors, 127 Ovarian tumors, 116 Pulmonary tumors, 124 RBE, 21,24,83.87,94 RBEM, 43 RBE ('hble), 88 Specific locus mutations, 90,93 Fractionation, 122,126, 134 Enhancement effecte, 134 Lung tumors, 122 Mammary tumors, 126 Gamma rays, 84,86 Effects on sperm morphology, 86 Translocation frequenciee, 84 Harderian gland tumors, 126-128, 137 Cesium 137 gamma ray induction, 128 Fission neutron induction, 128 Heavy ion induction, 137 HPRR neutron induction, 127 Neutron induction, 127 RBE values, 126 Heavy ions, 24 RBE values, 24 Hereditary effects, 75,82,84,89 Alpha particles, 84 Dominant lethal mutations, 76 Mammalian germ cells, 89 Neutron induced, 82 HGPRT assay system, 64.69 HPRR neutrons, 119,127 Harderian gland tumore, 127 Lung adenomas. 119 Hodgbin like lymphoma, 113 H u m cells, 46,66 Chromosome aberrations, 46 Mutation studies, 66 Internal emitters, 142 LET, 136 Relationship to RBE,136 LET-RBE, 10 Relationship in water, 10

Leukemia, 107.108 Dose-response curve, 108 Incidence vs. type of radiation, 107 Single gamma ray expowre. 107 Life shortening, 152,155,157, 162, 165 Neutron irradiation, 155 Protracted exposures, 157 FU3E value, 152,162 FU3E values (nble), 165 Lineal energy (y),5,34 Dicentric induction, 34 Linear collision stopping power, 4.5 Restricted stopping power, 5 Lung adenocarcinorna, 117,119,122 Californium 252 neutrons, 119 Cesium 137 gamma rays, 119 Fission neutrons, 122 HPRR neutrons, 117 Lung cancer risk, 149 Lung tumors, 117, 123 RBE values, 123 Lycopersicon esculentum (tomato), 23, 24 Lymphocytes, 30 Chromosome aberrations, 30 Lymphomas, 113 Dm-rate effect, 113 Mammalian cells, 49,64,68 In vitro transformation, 49 Mutations Mammary tumors, 123,125,127,129, 131,132 Adenouminoma, 123,125 Dose-rate effecte, 125 Fractionation effects, 125 Fiesion neutron induced, 127 Neutron radiation induced, 129, 132 Neutron radiation induced 35 Mev, 127 RBE values, 129 X-ray radiation induced, 129,132 Muons, 26 RBE, 26 Mutagenesie, 49.71.72, 78-81, 85, 87,99,102 Alpha particle efficiency, 88 Mammalian germ cells, 99 I n vivo,49 Dominant lethal mutations, 78-80 Neutron radiation induced, 74,85, 87

INDEX Plutonium 239 induced, 79,81 RBE, 71.89 Mutation studies, 64,65, 70, 71 AL cell system, 70, 71 Chinese hamster V79 cells, 65 Human fibroblasts, 65 Mammalian cells, 61 Myeloid leukemia in mice, 109, 110 RBE values. 109. 110 Neutron radiation, 27.34.51.53, 78, 82,85,87, 134, 153, 155, 158 Augmentation effect. 153, 158 Hereditary effects, 82 Induced transformations, 51 Induction of dominant lethal mutations, 78 Life shortening effects, 155 Maximum effects, 34 Mutagenic efficiency, 85, 87 RBE, 27 Transformation fi-equency, 53 Tumorigenicity in mice, 134 Neoplastic transformation, 61 Nonstochastic effeda, 11 Cataract formation, 11 Sterility, 11 Nigella damascena (black caraway), 15, 23, 24 RBE values, 23,24 Ooeytee, 92 Specific loam mutations. 92 oryza sativa (rice), 15 RBE values, 15 Oncogenic transformation, 49,56,57 CgH/10TY2cells, 57 LET dependency, 56 Radiation induced, 49 ~steosarcomas,134,143 Internal emitters, 143 Ovarian tumors, 114-117 Cesium 137 gamma ray induction, 116 Fission neutron induction, 116 Incidence in mice, 115 RBE valuee, 114 Oxygen effeds in plants, 21 Partial body radiation (PBR). 9 Penetration factor (PF), 10 Proton radiation, 41 Chromosome aberration induction, 41 Plutonium 239, 79,81,85

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Translocation mutation induction, 85 Mutagenicity, 79 Ovarian burdens, 81 Pulmomq tumors, 124 Fission neutron induction, 124 Quality factor, 6 Definition, 6 Radiation quality. 4 Radiation toxicity, 153 RBE values, 153 Radiosenaitivity, 82 Spermatogonia, 82 Recessive lethal mutations, 95 Droeophila, 95 Reciprocal transloeations, 81,82 Neutron induced, 82 Spermatogonia, 81 Repair of potential lethal damage, 59 Restricted stopping power, 5 Reticulum cell sarcoma, 113 RBE, 1, 3,6, 13, 15, 16, 21-24, 25-28,40, 52,54, 58, 61, 63, 67, 71, 72, 77, 78,83,87-89, 94, 96, 109, 112,123,126, 129,130, 134,135, 138, 140,142, 144, 148, 152,154, 162,165,169 Alpha particles, 87, 143, 144 Alternatives, 13 Arabidopsis, 23,24 Auger electrons, 58 Bone sarcoma,142 Cell survival, 72 Chromoeome volume (plants).,23 Cytogenetic effect, 40 Definition, 3, 6 Dose and dose rate, 21 Environment effects, 15 Fission fragments, 148 Fission neutrons, 21, 24,83, 87,88, 94, 134 Heavy ions, 24,63 Hereditary effects, 83 Incorporated radionuclides, 58 LET relationship, 22, 67, 135 Life shortening, 152, 162, 165 Lung turnore, 123 Lycopersicon eeculentum (tomato), 23 Maize, 21 Mammalian cells. 40 Mammary tumom, 126,129

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Moisture effect, 21 Muons, 26 Mutagenesis, 16, 71, 72, 89 Myeloid leukemia, 109 Neutrons, 27,52.54,96,134,140 Nigella damaeeena (black caraway), 23 Oxygen effects, 21 Planta, 21 Postimplantation fetal loee, 78 Postepermatogonial stages, 77 Radiation quality, 3 Thymic lymphoma, 112 Tradescantia, 21, 27 !hamformation, 61 b o r s , ('hble), 138 Versus neutron energy, 28 Variation with d m , 154 WEM, 1-3,33,4244,46,54,69,67, 84,169 Chromosome aberrations ('lhble), 44 Chromosome aberrations, 42 Fieaion neutrons. 43 LET effects, 42 Neutmm, 59 Reference radiation, 46 'hble, 169 X rays, 33 RBE-LET relationship. 66 Chinese hamster V79 cells, 66 Radium dial workers, 150 Reference radiation, 5,46 W E M ,46 Risk coeficienta, 11, 149 Lung cancer, 149 Silkworm, 103 RBE values CIgble), 103 Spermatid sensitivity, 76 Spermatogonia. 75.82.69.91 Radimnsitivity, 75-82 Specific locus mutations. 89,91 Sperm morphology. 86 RBE value for neutrons, 86 Sperm survival curve. 100 Neutron irradiation, 100 X-ray irradiation, 100 Sterility, 11 Syrian hameter cells, 50,51 Tramformation assay system, 50 Specific locus mutations, 89,90,92, 93, 102 Oocutes, 92

Spermatogonia, 89,90 'Igble, 102 Stochastic effecta, 11 Carcinogeneaie, 11 Mutagenesis. 11 Thymic lymphoma, 110-112 Incidence with neutrons. 110 Incidence with gamma rays. 111 RBE values, 112 ~ l o c a t i o n s84,8Ei, , 95,98 Droeophila, 95 Frequencies, 98 Gamma ray induced, 84 Plutonium 239 induced, 85 Triticum genus (wheat), 15 Tradeacantia, 15,23,26-28 W E , 27 WE, 28 'Ibsticular tumors, 134 'Ibxicity ratio, 149 Lung cancer, 149 Thioguanine CrCf) resistant mutante, 64 Thymidine kinase locus. 72 b o r i g e n e s i s in rate, 127 Transformation frequency, 49-61, 53-57,59,60,62 Alpha particles, 60 C,H/lOTlW cells, 55 Fieeion neutrons, 53,55 Gamma rays, 55 Gamma rays, hctionated, 56 Gamma rays, low dose, 56 In v i h , 49 Mammalian cells, 49 Neutrons, kactionated, 56 Neutron irradiation, 51,53,57 Per surviving cell, 62 X ray irradiation, 51,63,54,57,60 k f o r m a t i o n assay system, 49-64 BALBI3T3 cells. 60 C3H/1OT1/2cells, 50,52,54 RBE, 63 RBEM,54 Syrian hamster cells, 49-52 V i l transformations, 62 Radiation enhanced, 62 Weighting factors (W,), 2, 11 X rays, 51,54,77 Dominant lethal mutations, 77 Transformation frequency, 51, 54 Zen mays (maize), 15, 24 RBE, 24