Tritium and other radionuclide labeled organic compounds incorporated in genetic material: Recommendations of the National Council on Radiation Protection and Measurements (NCRP report ; no. 63)

NCRP REPORT No. 63

TRITIUM A N D OTHER RADIONUCLIDE LABELED ORGANIC COMPOUNDS INCORPORATED I N GENETIC MATERIAL Recommendations of the NATIONAL COLlNClL O N RADIA'TION PROTECTION AND MEASUREMENTS

Issued March 30, 1979 National Council on Radiation Protection and Measurements 7910 WOODMONT AVENUE / WASHINGTON, D.C. 20014

Copyright O National Council on Radiation Protection and Measurements 1979

All rights reserved. This publication is protected by copyright. No part 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 from the copyright owner, except for brief quotation in critical articles or reviews. Library of Congress Catalog Card Number 79-84486 International Standard Book Number 0-91339247-2

Preface In 1968 the NCRP Board of Directors, in an expansion of its internal emitter effort, assigned to Scientific Committee 24 the task of examining tritiated thymidine to determine whether the values for pennissible body burdens and concentrations of tritium as tritiated water given in NCRP Report No. 22 would be applicable. As the Committee studied the problem, they decided it would be appropriate to examine all radionuclides and additional compounds that have the potential for incorporation into genetic material. The Committee has made a sincere effort to review the world's literature on the subject and to arrive at conclusions which would make it possible to derive appropriate standards for radiation protection in the use of labeled organic materials. After a brief introduction, the report identifies those radionuclides of interest and the possible routes of exposure that may be encountered. There is a detailed review of the biological effects observed in man, animals, and plants. Approaches to calculations of dose and definition of a reference cell-nucleus are the subjects of the next section of the report. A separate section of the report is devoted to each of the radionuclides of particular concern. A summary chapter is included a t the end of the report to bring all of the ideas and conclusions together into a clear and concise form. The remainder of the report is devoted to appendices which gve background information on particular topics. The Council has noted the adoption by the 15th General Conference of Weights and Measures of special names for some units of the Systeme d' Unites International (SI) used in the field of ionizing radiation. The gray (symbol G y ) has been adopted as the special name for the SI Unit, of absorbed dose, absorbed dose index, kerma, and specific energy imparted. The becquerel (symbol Bq) has been adopted as the special name for the SI unit of activity (of a radionuclide). One gray equals one joule per kilogram and one becquerel is equal to one second to the power of minus one. Since the transition from the special units currently employed-rad and curie-to the new special names is expected to take some time, the Council has determined to continue, for the time being, the use of rad and curie. To convert from one set of ...

1u

iu

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PREFACE

units to the other, the following relationships pertain: 1 rad 1 curie

= 3.7

= 0.01

J kg-'

= 0.01

Gy

x .lOIOs-' = 3.7 x 10'' Bq (exactly)

Serving on the Committee for the preparation of this report were: EUGENEP . CRONKITE,Chairman Medical Department Brookhaven National Laboratory Upton, Long Island, New York EDWARD L. ALPEN Director Donner Laboratory and Pavilion University of California Berkeley, California

HORTONA. JOHNSON Professor and Chairman Department of Pathology School of Medicine Tulane University New Orleans, Louisiana

MICHAELA. BENDER Medical Department Brookhaven National Laboratory Upton, Long Island, New York

FRANKL. LOWMAN Deputy Director Environmental Research Lab. Environmental Protection Agency Naragansett, Rhode Island

JAMESE. CLEAVER University of California Medical Center Laboratory of Radiobiology San Francisco. California LUDWIGE. FEINENDEGEN Professor and Director Institute for Medicine Julich, West Germany MARYLOU INCRAM Director Institute for Cell Analysis University of Miami Hospital and Clinic National Children's Cardac Hospital Miami, Florida

EUGENEF. OAKBERG Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

F. PERSON STANLEY Department of Biophysics CoUege of Science The Pennsylvania State University University Park, Pennsylvania

EDWARD L. POWERS Professor Director of Zoology Laboratory of Radiation Biology The University of Texas at Austin Austin, Texas

NCRP Secretariat, James A. Spahn, Jr.

The Council wishes to express its appreciation to the members of the Committee for the time and effort devoted to the preparation of this report. Warren K. Sinclair President, NCRP Bethesda, Maryland December 15, 1978

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Radionuclides Incorporated into Nucleic Acid . . . . . . . . 2.1 Reason for Interest in Radionuclides Incorporated into Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Radionuclides Incorporated into Nucleic Acids . . . . . . . . 2.3 Radioactively-Labeled Nucleic Acid Precursors . . . . . . . . 2.4 Modes of Entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Incorporation of Nucleic Acid Precursors . . . . . . . . . . . . . . 2.6 Radioactively-Labeled Protein Precursors . . . . . . . . . . . . . 3 Biological Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Cells at Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Cell Renewal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Transmutation Effects . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Gene Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Chromosomal Aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Effects on the Fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Effects on Lifespan of the Individual . . . . . . . . . . . . . . . . . . 4 The Approach to Dose Calculations and the Reference Cell Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Tritium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Physical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Absorbed Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Dose Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Tritiated Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Tritiated Thymidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Other Tritium-Labeled DNA Precursors . . . . . . . . . 5.7 Tritium-Labeled RNA Precursors . . . . . . . . . . . . . . . . . . . . 5.8 Tritium-Labeled Amino Acids and Nonspecific Precursors 6 Carbon-14-Labeled Compounds . . . . . . . . . . . . . . . . . . . . . 7 Phosphorus-32 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Sulfur-35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Iodine-131 and Iodine-125-Labeled Precursors for DNA 10.Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTENTS

APPENDIX I APPENDIX II APPENDIX III APPENDIX IV APPENDIX V APPENDM VI APPENDIX VII

General Principles Underlying Establishment of Radiation Protection Standards . Environmental Contamination by Tritium Tritiated Water, and Metabolism The Quality Factor for Tritium Radiation The Toxicity of Tritiated Water (I-ITO) . . Metabolism of DNA, RNA, and Their Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem Cells, Somatic Mutations, and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Mutation, Chromosomal Aberrations, and Mammalian Cell Killing from Radionuclides Considered in This Report

41 47 56 64

69 83

97 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 TheNCRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 NCRP Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

1. Introduction This report addresses special hazards that might arise from the temporary or permanent incorporation of various radionuclides into the genetic material of the cell. The National Council on Radiation Protection and Measurements (NCRP) was originally concerned with the question: "Does incorporation of tritium labeled thymidine directly into the deoxyribonucleic acid (DNA) of synthesizing cells introduce complications that make the values of maximum permissible concentration (MPC) given in NCRP Report No. 22 (NCRP, 1959) inappropriate for this special case?" As the Council began to study this question, it became apparent that a broader consideration of various compounds labeled with several different radionuclides would be useful. As will be seen in the discussion that follows, the use of various types of labeled compounds does introduce complications that do indeed make the use of certain values given in NCRP Report No. 22 inappropriate. NCRP Report No. 22 recommends values for maximum permissible body burden (MPBB) of radionuclides and maximum permissible concentration (MPC) of these nuclides in air and in water. In general, these values are applicable to occupational exposure. The general principles involved in arriving at recommendations for these values are detailed in NCRP Report No. 39 (NCRP, 1971) and are summarized in Appendix I herein. The MPBB and MPC values given in Report No. 22 are primarily for occupational groups exposed to radioactive nuclides that are relatively widely, though not uniformly distributed throughout the body. For these radionuclides, the MPBB and MPC in air and in water are based on best estimates of the mean dose delivered to entire tissues under the various occupational exposure conditions considered. The MPC values were set to apply to radiation workers of age 18 or older on the assumption that no occupational exposures would be permitted before that age. Such values, based on the longest probable occupational exposure, would be conservative and would apply to radiation workers of any age greater than 18 years. The MPC's of radionuclides in air and in water in Report No. 22 are based on biological data and were calculated on the basis of the 1

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INTRODUCTION

maximum permissible dose equivalents established by the NCRP. These specify annual maximum permissible dose equivalents for occupational exposure of 15 rem for most individual organs of the body; and 5 rem when the critical organ is the gonads, the blood-forming organs, or the whole body (NCRP, 1971). A special concern in this report is the maximum permissible dose to the fetus resulting from the occupational exposure of the mother (NCRP, 1971). For this case, the MPD has been set at 0.5 rem (NCRP, 1971, 1977). Certain radionuclides, principally tritium and krypton, are released from nuclear power plants and fuel reprocessing facilities, and there is public concern about the possible hazards of the build-up of these radionuclides in the biosphere. Consquently, the Council has examined the significance of these releases in the context of radionuclide incorporation into cells and the consequences of this for tumor induction and genetic effects. Data on environmental contamination pertinent to this report are presented in Appendix 11.

2. Radionuclides Incorporated Into Nucleic Acid 2.1

Reason for Interest in Radionuclides Incorporated into Nucleic Acids

A situation of particular interest is posed by those radionuclides that are incorporated into the genetic material, deoxyribonucleic acid (DNA). Damage to DNA is the major cause of harmful effects of radiation and can result in cell killing, mutation, and other detrimental consequences. The DNA, located almost exclusively in the nucleus, directs the many specific cellular structures and functions, and must be replicated if daughter cells are to contain the full complement of genetic information. Incorporation of radionuclides exposes DNA to radiation and/or transmutation effects, and may thus interfere with DNA replication and affect the DNA structure in a manner that alters the genetic function. Ribonucleic acid (RNA) is similar in structure to DNA and is usually synthesized in the cell nucleus using DNA as a template. This transcription may occur throughout the cell cycle between two cell divisions. Much of the newly synthesized messenger RNA (mRNA) then leaves the nucleus for the cytoplasm, where the genetic message from the DNA is translated into protein, and the mRNA also may participate in the control of the genetic activity. The RNA that does not leave the nucleus rapidly usually turns over quickly so that there is little long-term retention of radioactive material in the nucleus. The important considerations with respect to potential radiation hazards from radionuclides incorporated into RNA are: (1) the close temporary contact of RNA and DNA in the cell nucleus during transcription; (2) the immediate metabolic conversion of some RNA precursors to DNA precursors; (3) the delayed metabolic conversion, upon RNA turnover, of some RNA building blocks to DNA precursors. The metabolism of DNA, RNA, and their precursors is discussed in Appendix V. 3

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2.

2.2

Radionuclides Incorporated into Nucleic Acids

RADIONUCLIDES IN NUCLEIC ACID

In examining injury to the genetic material of the cell by incorporated radionuclides, one must consider all radionuclides and radioactively labeled molecules that may be incorporated eventually into or be located close to DNA and RNA. The following nuclides are of particular interest: %, 14C,32P,%S,l3'I, and lZ5I.1251decays by electron capture and emits Auger electrons. The others emit beta particles and all are widely used. The energy from these incorporated nuclides is absorbed over a defined, relatively short path from the decay event. The radiation effects are primarily produced in close proximity to this path. Tritium, the only radionuclide of hydrogen, is used abundantly as a tracer in biology and medicine and frequently as a label for nucleic acid precursors. It may thus become incorporated into nucleic acids for the life span of a cell. Moreover, there is concern about environmental tritium in water that may be ingested over long periods of time and become incorporated into body constituents including DNA. Tritium-labeled nucleic acid precursors used in the laboratory and in clinical research may also accidentally enter the human body and pose a hazard. "S-labeled cysteine and methionine and 14C-and 3H-labeled amino acids are incorporated into proteins and distributed throughout the tissue and cells synthesizing proteins. They are not selectively incorporated into nucleic acids. They may, however, be brought into close proximity to these molecules by incorporation into nucleoproteins. 131 I and lz5I are mentioned because both are used as a label for 5iodo-2'-deoxyuridine (IUdR), an artificial analog of thymidine that is incorporated into DNA, instead of thymidine, through the thymidine "salvage" biochemical pathway, with less efficiency than thymidine (see Appendix V). 14 C-labeled nucleic acid precursors are frequently used for biochemical analysis of nucleic acid synthesis and turnover. 32Pphosphate may be incorporated into nucleotides and nucleic acids throughout the cells and may also enter body fluids with certain proteins and lipids. The hazards from these radionuclides are mainly due to the emitted electrons (beta particles or Auger electrons). As will be discussed, transmutations (chemical transmutation, nuclear recoil, and charge transfer) present much less hazard. (See also Section 3.4 and Appendix VIL) The site of immediate radiation effects is limited by the short range of the emitted electrons. In fact, for the case of very short range beta particles emitted by tritium and with the Auger electrons, the radiation is limited to the site of radionuclide incorporation within the cell and

2.2 RADIONUCLIDES IN NUCLEIC ACID

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may largely expose the nucleus. The injury to the organism from nucleic acid precursors labeled with short range electron emitters will therefore be determined by the number of cells synthesizing nucleic acids (cells at risk) at the time the labeled precursors are present. Transmutation effects are defined here as events that are localized to the immediate site of nuclear decay. They are thus limited to those molecules that have incorporated the radionuclides in question. They may involve effects from transmutation of the incorporated radionuclide into another element, concomitant excitation, nuclear recoil, and charge transfer processes resulting in molecular rearrangement of the labeled molecule. (See Section 3.4 and Appendix VII.)

2.3

Radioactively-Labeled Nucleic Acid Precursors

The radioactively-labeled compounds that may eventually be incorporated into DNA or RNA include the small molecular precursors, such as bicarbonates, formates, glycine, orotic acid, and the larger molecular precursors, such as purine derivatives, pyrimidine nucleosides, and halogenated analogs. The smaller precursors are used for the synthesis of the larger precursors that are ultimately linked into DNA or RNA. The four major precursors of DNA are the two deoxyribonucleosides, thymidine and deoxycytidine; and the purines, guanine and adenine. The corresponding precursors for RNA are two ribonucleosides, uridine and cytidine; and the two purines, guanine and adenine. These compounds and their roles in DNA and RNA synthesis are discussed further in Appendix V. Of all of the nucleic acid precursors, thymidine is especially important when radiation hazards are considered, because, like deoxycytidine, it is not only selectively incorporated into DNA but also widely used in biomedical research. The histones are proteins associated with the nucleic acids, are constituents of chromosomes, and have a slow turnover. Amino acids may be incorporated into histones and thus, if radioactively labeled, may constitute a hazard to genetic material. Tritiated water represents another possible pathway for the entrance of tritium into the nucleic acids (see Appendix 11).

2.4

Modes of Entry

Radionuclides such as 3H, 14C,and '"may enter the body in food, drinking water, and air and then be incorporated into genetic material.

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2. RADIONUCLIDES IN NUCLEIC ACID

Nucleic acid precursors labeled with radionuclides may enter the body with foodstuffs, or be ingested accidentally, or be injected into the bloodstream in the course of clinical studies. The efficiency of incorporation vmies with the route and rate of administration (see Appendix V). For example, in mice about 0.30 of thymidine rapidly injected intravenously is incorporated into DNA; in man, this value is about 0.50. After ingestion, however, only 0.5 to 0.10 of the thymidine enters the general circulation and may be incorporated into DNA.

2.5

Incorporation of Nucleic Acid Precursors

The nucleic acid precursors that have entered the bloodstream are rapidly distributed to the various organs and are incorporated by those cells engaged in the synthesis of DNA and RNA. The incorporation is usually completed within less than one hour after the precursor enters the bloodstream. RNA has a relatively rapid turnover rate and DNA is broken down upon cell death. Some degradation products are reutilized (see Appendix V). Two fundamentally important biological facts must be taken into consideration to arrive a t a realistic assessment of the toxicity of radionuclides incorporated into genetic material: first, radionuclide labeled nucleic acid precursors are concentrated in those cells that synthesize nucleic acids; and second, a large proportion of cells in any organ and in the whole body do not incorporate nucleic acid precursors into DNA and thus will not receive irradiation from DNA precursors (see Appendix VI). Most adult tissues consist of cells that divide rarely. Rapidly proliferating cells are found especially in the lining of the intestinal tract, in the bloodforming tissues (hematopoietic tissue), skin, and in the male germinal epithelium (sperm producing tissue). About 80 percent of the DNA synthesized in the adult at any one time is located in these organs. In the embryo, nearly all cells are dividing. All proliferating cells double their genetic material prior to division, but only a fraction of the proliferating cells synthesize DNA at any one time. In contrast to the limited number of proliferating cells in the whole body that synthesize DNA a t a given time between two cell divisions, aLl nucleated cells synthesize at least small amounts of RNA. The burden of radioactive material in cells containing labeled nucleic acids will depend upon the fate and function of the nucleic acids in these cells. Factors influencing the rate at which radioactive material is lost and perhaps redistributed from cells containing labeled nucleic

2.5 INCORPORATION OF NUCLEIC ACID PRECURSORS

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acids are: (1) rate of cell division, (2) metabolic turnover of labeled nucleic acids, (3) cell death, and (4) concomitant partial reutilization of the labeled components from metabolic turnover and dead cells. These factors vary with different types of cells and nucleic acids. Thus, in all cells the various molecular entities of RNA have defined and variable turnover rates. In contrast, the majority of DNA in a cell is generally considered metabolically stable in mammals and is distributed to daughter cells with each cell division. The rate of diminution of radioactive label bound to DNA per cell is a function of the time interval between consecutive cell divisions (generation time). Those. ceUs that divide only rarely will accumulate a higher radiation dose from radionuclides incorporated into DNA than cells with rapid renewal rates.

2.6

Radioactively-Labeled Protein Precursors

Although the present report is not directly concerned with the maximum permissible dose of radionuclides bound to protein precursors, radiation to the genetic material may occur if such compounds are incorporated into cells. Amino acids are built primarily into proteins, but may, in the course of their metabolism, also donate precursors for nucleic acid synthesis. Labeled proteins such as histones and protamines and chromatin-associated acid proteins have close contact with nuclear DNA. All labeled amino acids may have a t least temporary intimate contact with RNA.

3. Biological Effects

3.1 Cells at Risk When emitters of electrons are solely or predominantly localized in a small fraction of the cells in the body, the mean tissue dose may be negligible, but the dose to the labeled c e b may be large since nearly all energy emitted is deposited within the cell containing the radionuclide. These individually labeled cells and their progeny are at risk. I t is therefore appropriate to relate doses to the volumes that are actually exposed. The concept of average tissue dose is inadequate and inappropriate and, in fact, for defining maximum permissible body burdens under these conditions, would underestimate the hazard. The cells a t risk are those which, upon exposure, may give rise to acute or late radiation effects, such as cancer and genetic mutation. In the bloodforming tissues, the gastrointestinal tract, and the skin, for example, the cells at risk are stem cells. In the reproductive organs, the cells at risk are the spermatocytes, spermatogonia, and the oocytes (as long as they may become exposed) (see Appendices 11, 111, IV, VI, and VII).

3.2

Cell Renewal

Organs of the body have different rates of cell renewal. There is, for example, no replacement of lost nerve cells in the developed human brain. On the other hand, cells in liver, kidney, and connective tissue respond to injury by accelerating the rate of cell production, yet have a very low rate of turnover under normal conditions. The tissues with rapid cell renewal, such as bone marrow, lymphopoietic tissue, gut, and skin contain many cells that constantly proliferate and thus maintain a balance between cell loss and cell production. For example, there are more than 10'' red blood cells produced per day per reference man (ICRP, 1975).

3.2 CELLRENEWAL

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Cell production depends ultimately upon adequate numbers of undifferentiated progenitor cells. These are called stem cells. Stem cells have the potential for producing similar undifferentiated daughter cells as well as cells that will differentiate. The differentiated cells usually undergo further divisions and then mature into functioning cells, such as, for example, those that enter the peripheral blood from the bone marrow. Injury to and loss of bone marrow stem cells may cause a depletion of the number of mature cells, since the latter are constantly lost through cell aging and cell death. They must be replaced a t a rate that equals the rate of loss in order to maintain health. Renewal rates of cells in other proliferating tissues, such as the gut and skin, are not as well understood and correspondingly little information is available on these stem cells. Only a small fraction of cells are stem cells. Labeled stem cells are a t risk in proportion to the amount of incorporated radioactive material. However, stem cells containing lethal amounts of radionuclides may be killed before they can give rise to. a progeny of cells with malignant properties. Regarding late effects, all organ systems are at risk if radioactive material is incornorated into the DNA of their long-lived cells. Various organs and cell populations are known to be especially radiosensitive and to produce, when irradiated, particular symptoms relating to acute and late effects. The hematopoietic system is especially sensitive. Little is known about the renewal rates of the various cell populations in organs such as liver, kidney, and connective tissue so that one cannot make reasonable calculations of the number of cells a t risk. However, one can assume that the relative incorporation of radioactive material in all cells in the S-phase of the cycle will be similar. For calculation of radiation dose later in this report, it is assumed that the average time between successive divisions (generation time) of hematopoietic stem cells is approximately 30 days. The time between successive mitoses (generation time) in spermatogonial stem cells is 16-100 days. Additional details about stem cells are discussed in Appendices VI and VII.

3.3

Neoplastic Diseases

Tumors may be induced by chronic irradiation from incorporated radionuclides and the dose-effect relationships may differ for different types of tumors. Data principally demonstrating the relationship be-

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3. BIOLOGICAL EFFECTS

tween radiation exposure and development of tumors in man is well documented for leukemia, breast, and thyroid. The sum of the incidence rates of all possible tumors other than leukemia is now considered to be 5-10 times higher than that of leukemia, per unit dose (NCRP, 1971; UNSCEAR, Annex G, 1977)- see Appendix VI. In assessing the effects of radiation with respect to leukemia induction, one must consider the total dose, the dose rate, the volume of the body exposed (cells a t risk), and the distribution of absorbed dose. Previous studies have considered exposure of large volumes and have been conservative in that many accept for hazard evaluation the following assumptions: (1) Effects are independent of dose rate and there is no threshold dose of radiation below which leukemia is not induced; (2) increased incidence of leukemia above spontaneous incidence is linearly related to dose, and the risk, over a 20-year period, is about 20 cases per rad per million population a t risk. This number is derived from studies and data a t 100 rad or more and is probably an upper limit for the risk estimate at lower doses. It is assumed that injury solely to the hematopoietic stem cells by intranuclear irradiation may result in leukemia. After incorporation of radionuclides into the stem cell, its longer lifespan (time to next cell division) permits a higher number of decays and more accumulation of damage than occur in the equally labeled differentiated proliferating cells with relatively short generation times; the rate of accumulation of decays is halved with each cell division. Furthermore, differentiated proliferating cells have a finite lifespan and probably lack the capacity to establish a self-sustaining population of abnormal cells. Some cancers are monoclonal (arising from one cell) (Fialkow et al., 1967, 1977; Beutler et al., 1962)'-see Appendix VI. The probability of a cancerous event taking place in a single cell from one radiation event is a h c t i o n of the number of genes in the cell and the number of genes involved in induction of cancer. Thus, the probability of cancer is proportionate to the number of cells at risk times the dose of radiation to these cells. There will always be a large number of stem cells labeled from a single exposure to radioactively labeled nucleic acid precursors even though a specific radioactive DNA precursor, such as tritiated thymidine, is available only briefly. In addition, after even a brief period of labeling with nucleic acid precursors, reutilization of precursors from RNA turnover and from DNA of dead cells will increase the percentage of labeled stem cells. Tritium in water is distributed throughout the body water. Consequently, it is believed that it entails a risk of cancer induction that, per unit of absorbed dose, is similar to that associated with external radiation (Appendices 11, IV, and VI).

3.3 NEOPLASTIC DISEASES

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Transmutation Effects Effects from radiation, from chemical consequences of transmutation, from nuclear recoil and changes of charge of the daughter nuclide must be weighed and put into perspective when considering the effects of incorporated radionuclides. The hazards from transmutation have been studied in a variety of microorganisms, in Drosophila, and in mammalian cells in tissue culture (Appendix VII). Transmutation of tritium to 3He in the 6-position in DNA thymine produced on the average 0.3 single-strand and fewer than 0.01 double-strand breaks per tritium decay in frozen DNA that was sufficiently diluted to minimize intermolecular effects from the beta particles. For most types of cells that have been studied in vitro, single-strand breaks caused by various mechanisms can be repaired efficiently. The DNA double-strand breaks from tritium transmutation are expected to add insignificantly to the effects from the tritium beta particle, which may produce about 0.1 double-strand break per mammalian cell nucleus of average size. Decay of tritium in the 2-position of adenine has been shown to produce DNA strand-strand crosslinks with a n efficiency of about 0.5 per decay. The effect must be attributed to transmutation and probably does not occur for other positions on DNA bases. Data from bacteria show that, while mutations produced by tritium decays originating in DNA as 3H-methyl-thymidine, 3H-8-adenine, and 3H-8-guanine may be accounted for on the basis of ionizations from the beta particle, tritium decaying in the 5-position of cytosine, the 6-position of thymidine, and the 2-position of adenine yields significant increases in mutation production that must be attributed to transmutation. Confirmatory evidence for a transmutation effect for 3H-5-cytosine is available from Drosophila. The overall increase in mutation induction from transmutation from tritiated water is small, however, because hydrogen a t the 5-position of cytosine, 6-position of thymidine, and 2position of adenine constitutes a minor fraction, about 0.0005, of all nuclear hydrogen (Appendix 11). Decay of 14C may cause effects from chemical transmutation to nitrogen, and molecular bonds may be broken. These effects add to the radiation effects from the beta particle. Genetic data on the effectiveness of 14C decay in mutation induction and chromosomal aberration production are conflicting, but there are data suggesting a transmutation effect for I4C located in the 2-position of thymidine in DNA. Nevertheless, the contribution of such events to the total genetic effect of decay of 14Cin the cell nucleus is expected to be negligible in relation to the effects induced by ionizations from the beta particles because of the small fraction of nuclear carbon occupying this particular site. 3.4

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3. BIOLOGICAL EFFECTS

Each decay of 32P in isolated DNA produces by chemical consequences of transmutation and perhaps nuclear recoil a t least one single-strand break. There is also one double-strand break for about 20 single-strand breaks. By comparison, the rate of double-strand break production in isolated DNA from tritium decay in the 6-position of the pyrimidine ring is about one-fifth as great (appendix VII). When "P is distributed throughout the cell in all nucleotides and nucleic acids, the proportion of decays producing double-strand breaks due to a transmutation effect will be small. It may be pertinent that to date there is no well documented correlation between strand breaks and tumorigenesis. The radiation effects from the 32Pbeta particles will outweigh effects from 32P transmutation in DNA (IAEA, 1968). 32P transmutation effects in RNA are unlikely to have lasting biological consequences. No specific transmutation effect is known for decay of 1311. The situation is different for '"I. lZ51decays into 12Teby electron capture and internal conversion and produces an average of 8.6 Auger electrons having discrete energies from 0.5 to 34 keV. Hence, the daughter nuclide carries an average positive charge of 8.6 units. Charge transfer processes and molecular disruptions in consequence thereof are expected to add to the radiation effects from the low energy Auger electrons. '"I decay in the 5-position of the uracil ring in DNA was reported to produce double-strand breaks with an efficiency of 0.51.0. On the basis of radiation delivered to the cell nucleus from the Auger electrons, fewer than 0.5 double-strand breaks are expected. Therefore, in the case of lZ5Iit appears that the transmutation effect considerably exceeds the radiation effect (see Section 9 and Appendix VII). Since the effects due to radionuclides incorporated into a nucleic acid are generally taken to result entirely from ionization, except for the case of Auger electron emitters such as I2'I, the recommendations for maximum permissible burdens are based on the average beta energy absorbed per decay in the critical cells.

3.5

3.5.1

Gene Mutation

General

Radiation has been shown to induce gene mutations and this also applies to radiation from radionuclides incorporated directly into DNA, as well as from radionuclides not incorporated into DNA. It is

3.5

GENE MUTATION

/

13

estimated that the probability of gene mutation per rad is about to lop6 per gene (Searle, 1974). Transmutation effects have been demonstrated to be important for causing structural changes in DNA (see Section 3.4 and Appendix VII). As stated in Section 3.4, except for the special case of Iz5Itransmutation, it appears that the genetic effect of radioactive nucleic acid precursors may be estimated from the radiation doses delivered to the nuclei of the cells at risk.

3.5.2

In the Male Mammal

Radioactive DNA precursors are incorporated into the progenitor cells of the sperm (spermatogonia and resting primary spermatocytes). In man, the administration of a single dose of labeled nucleic acid precursor will result in the labeling of a cohort of spermatogonia that delivers radioactive sperm approximately 60-70 days later (Heller and Clermont, 1962, 19W. The most important cell in the mammalian testis, from the standpoint of genetic radiation effects, is the spermatogonial stem cell. The time between two consecutive divisions (i.e., generation time) of spermatogonial stem cells in man is estimated to lie somewhere between 16 and 100 days. Cell types in later stages of differentiation rapidly complete their development and disappear (see Appendices VI and VII).

3.5.3

In the Female Mammal

In the female, cells capable of differentiating into oocytes proliferate only during fetal life; in postnatal life there is no DNA synthesis connected with cell proliferation. Possibly repair replication may remove radionuclides .from DNA and/or permit their incorporation into DNA. This possibility has not been demonstrated as yet. However, oocytes continue to synthesize RNA, a process that thus permits incorporation of radionuclides into RNA in postnatal life. Low dose-rate irradiation of mouse oocytes has given a much lower induced mutation rate than in spermatogonia. A reliable spontaneous mutation rate for oocytes is not available. If radionuclides are present during fetal development, incorporation into the proliferating progenitor cells for oocytes could occur and mutations may be induced in these stages. Data for evaluation of this possibility are not available. but exposure to low dose-rate irradiation in utero has yielded very low mutation rates (see Appendices 111 and VII).

14

/

3. BIOLOGICAL EFFECTS

3.6 Chromosomal Aberrations

During mitosis the DNA in the nucleus is organized into microscopically visible chromosomes. Radiation induces breaks in chromosome continuity. If more than one is present at the same time, such breaks may recombine to form various classes of chromosomal aberrations. Either simple breaks or rearrangements may be lethal. If induced in germ line cells, aberrations may be transmitted to later generations and produce effects such as sterility or congenital malformation. If induced in somatic cells, aberrations may lead to cell death or altered function. It is suspected that chromosomal aberrations may be involved in the genesis of some malignancies. Chromosomal aberrations have been observed after incorporation of radionuclides into DNA and RNA. It appears that aberration frequencies may be predicted from consideration of absorbed dose to the nucleus, provided that allowance is made for the relatively low dose-rate and for its reduction in succeeding cell cycles. A more detailed discussion of chromosomal aberration production is provided in Appendix VII.

3.7

Effects o n the Fetus

Some data suggest that as little as 1 4 rad irradiating the fetus in pregnancy may increase the incidence of childhood leukemia. Some data show no effect. The fetus is most sensitive during the first trimester of pregnancy (see Appendices VI and VII). All fetal tissues proliferate. Hence, administration of radionuclide labeled precursors of the nucleic acids to the early fetus results in widespread labeling of proliferating cells. If 3H-thymidine, for example, is given throughout pregnancy, 100 percent of fetal cells become labeled. This is in contrast to the adult where extensive cell proliferation is restricted to only a few tissues and therefore a limited number of cells is labeled. Radionuclides that are not selectively incorporated into any tissue are distributed throughout the body in either fetus or adult. Such is the case, for example, for 3HOH or certain 14C-labeledmetabolites. However, a small percentage of such radionuclides may become linked metabolically to nucleic acids. Only a very small fraction of the tritium in body water exchanges with hydrogen of the tissue solids. In such cases, the biological effects in postnatal life will be determined by the metabolic turnover rate of the labeled molecules and the lifespan of the labeled cells (see Appendices I1 and V).

3.7 EFFECTS ON THE FETUS

/

15

3.8 Effects on Lifespan of the Individual Life shortening is an effect of long-term, low dose-rate radiation exposure that occurs in part through the induction of tumors. This effect has been observed from internally deposited radionuclides such as "Sr, 2 3 9 ~ and ~ ,2 2 6 ~ inaexperimental animals and for ''%a also in man. It is presumed that radionuclides incorporated into nucleic acids may induce tumors and hence shorten life although, to date, the data are equivocal from low doses of 3H-thymidine (Cronkite et al., 1973 and Mewissen and Rust, 1973). When life shortening due to tumors is subtracted out there appears to be no non-specific shortening at such low dose-rates.

4.

The Approach to Dose Calculation and the Reference Cell Nucleus

The foregoing discussion has emphasized that: 1) the radionuclides of concern are beta-emitters heterogeneously distributed within cells and subcellular structures predominantly or exclusively labeling the nuclear DNA during DNA synthesis and 2) it is appropriate to assume that the critical volume is the nucleus of stem cells since injury to nuclear DNA of stem cells is believed responsible for late somatic and genetic effects. Because of the heterogenous distribution of radionuclides incorporated into the genetic material, dose calculations may be performed either by averaging the energy absorbed per nucleus of labeled stem cells over all stem cell nuclei or by expressing the average energy absorbed per labeled cell nucleus. There will always be a large number of stem cell nuclei that become labeled when labeled nucleic acid precursors enter the body, even if only a small fraction are proliferating. Some cancers are monoclonal (originating from one cell). Whether all cancers are monoclonal or not is not known. As stated in Section 3.3, the probability of producing a tumor is proportionate to the number of cells exposed times the dose of radiation to these cells. Therefore, one must consider the dose to the labeled stem cell population. The average tissue dose or average dose to all stem cells is meaningless. In view of the preceding, it was decided to calculate permissible intakes of radionuclide labeled DNA precursors on the dose delivered to the labeled nuclei from the incorporation into the genetic material. I t is fully recognized that this approach is a departure from conventional dose calculations, but it realistically puts the labeled cell population into perspective and, moreover, is conservative since basing MPBB on average tissue dose would underestimate the hazard. In order to calculate dose to the labeled cell nuclei, it is necessary to formulate a reference nuclear volume. The reference nucleus is arbitrarily defined as a sphere with 8 pm diameter and a volume of 268 x 16

4. DOSE CALCULATION APPROACHES

/

17

10-l2 cm3, or 268 x 10-12 g (for simplicity 270 in all subsequent calculations) if unit density is assumed. The diameter selected is the average diameter of the cell nucleus that is measured in blood-forming tissue. Individual human cell nuclei, either in proliferating or resting cells, range in volume from about 30-900 x 10-l2 cm3, and the ratio of cytoplasmic volume to nuclear volume varies as much as tenfold. Ranges in nuclear size of the various cell types influence the individual doses absorbed per disintegration. The concept of absorbed dose (rad) is based on energy absorbed per unit mass of tissue. This approach may be used to calculate dose from beta particles to small volumes with dimensions of the reference nucleus. In the case where there is only a single disintegration in a reference nucleus, a large part of the nuclear volume is not affected. When there are several disintegrations a t random locations in a nucleus, all or almost all of the nuclear volume is affected. Some parts of the nucleus may have been affected by only one of the disintegrations and other parts by several. A relatively uniform dose to all of the DNA in reference nuclei is attained when there are sufficient disintegrations within the reference nucleus. Similarly, with internal or external radiation the estimated average tissue dose is a valid measure of the uniform distribution of dose throughout the tissue if the radiation absorption events pertain to a large number of randomly distributed nuclear volumes. If the average tissue dose is reduced there may, a t best, be in one nuclear volume a single or no absorption event. In different nuclear volumes, different portions of the DNA will be affected by single events. It can be shown that, after 300 mrem of approximately 80 kV x rays, there is on the average one Compton event of about 5.7 keV per reference nuclear volume. At lower doses, an increasing number of reference nuclear volumes receive no radiation. Accordingly, a t dose equivalents less than about 300 rnrem, an increasing fraction of tissue is not irradiated (fewer cells a t risk) and those nuclear volumes involved always receive a dose of about 300 mrem. A factor converting 3H beta disintegrations per volume of the reference nucleus (RN) to rads per RN can be derived as follows: 1. 1 erg = 6.25 x 10" eV 2. 100 ergs/g = 6.25 X loL3 eV/g = 1 rad 3. 6.25 x 10" eV RN volume (g) = eV/RN/rad 6.25 x 1013 270 x 10-l2 = 1.69 x lo4 eV/RN/rad Elr 3H 4. Dose to RN per 3H decay = eV/RN/rad 5.7 x 103 = 0.34 rad 1.69 X lo4

.

18

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4.

DOSE CALCULATION APPROACHES

Thus, in the case where 3H is randomly distributed throughout tissue, the dose to a RN is 0.34 rad. However, if the 3H is exclusively in the RN (none in cytoplasm or intracellular spaces) there is a loss of energy from the RN to the surrounding tissue. The amount of loss is i, ?tion of the nuclear volume. This loss reduces the average dose to the RN for a single 3H decay to 0.27 rad. The loss of energy from the reference nucleus depends upon the energy (or range) of the emitted particle. The factors to convert disintegration per volume of reference nucleus to average absorbed dose are listed in Table 1. In Figure 1 the relationship of dose per tritium disintegration to nuclear size is Illustrated. The values of absorbed dose to the reference nucleus from one radionuclide decay within that volume, listed in Table 1,are, of course, average values based on the mean energy of the emitted beta particles. Any absorbed dose calculated for uniform distribution in tissue (rad) that is lower than the average dose value per nuclear volume per decay simply indicates that some nuclear volumes are not irradiated. The preceding introduces at the microscopic level inhomogeneities of exposure with various fractions of nuclei receiving zero dose. The frequency of decays in the volume of the reference nucleus follows a Poisson distribution (see Appendix PI). The biological effect of inhomogeneities in absorbed dose and dose rate a t the cellular and subcellular level, where DNA may not be involved, is not a t all clear. Intuitively, one might expect less biological effect per unit dose as fewer cells are involved. There is no critical level of relative inhomogeneity associated with the average dose resulting from one decay per reference nucleus. At lower doses, the proportion of cells not involved increases and at larger doses the , , a

TABLE 1-Factors used to convert disintegrations to average absorbed dose in the reference nucleus -

Radionuclide

26

Average abaorbed dose (.ad) per decay in reference nucleus For random distribution in tissue

For random dintribution in nucleus"

" Corrected for loss of energy from the reference nucleus. The NCRP is indebted to M. J. Berger of the National Bureau of Standards, for the calculations needed to produce this table.

/

4. DOSE CALCULATION APPROACHES

19

- 10.0 8 9

4 >-

2 0 x

a : UI k

g

1.0

3

P

e B0 W

rn

0.1

IL:

0 V) m 4

NUCLEUS

YI

2 a: F

200

0

LL 2

400

600 800 1000 VOLUME (p3)

1200

1400

I

I

I

I

I

3

4

5

6

7

I

RADIUS (pl

Fig. 1. Relationship between absorbed dose per tritium disintegration and nuclear size.

proportion decreases. At sufficiently large doses, the proportion not involved becomes negligible, but inhomogeneity persists with a calculable proportion of cells receiving doses that are a portion of the average dose. Intuitively, again, one might expect less biologic effect per unit dose as fewer cells receive doses as little as half the average dose. These microdosimetric considerations are not unique to incorporated radionuclides but apply in principle to all external low level exposures to penetrating radiations.

5.

Tritium 5.1 Physical Considerations

Tritium has a half-life of 12.26 years. It decays to helium-3 by emitting beta particles with a mean energy of 5.7 keV' and a maximum energy of 18.6 keV. The mean range of the beta particle in water is 0.69 pm and the maximum range for an 18.6 keV beta particle approaches 6 pm (see Appendix 111). Experimental determinations of the Relative Biological Effectiveness (RBE) for beta particles from tritium have yielded values for different end points varying from 1 to more than 2. A quality factor (Q) of 1has been assigned by the NCRP (NCRP, 1971) and is used in this report. A discussion of and review of experiments on RBE of tritium are presented in Appendix 111. The daughter nuclide, helium, is a noble gas. I t will not form a stable chemical bond. When tritium is present as C-% before decay, the daughter nuclide will be lost from the carbon, where tritium was bound, to produce a reactive carbonium ion which may result in molecular alteration. The maximum nuclear recoil energy of 3 eV is too low to break most chemical bonds. These effects do not measurably contribute to early cell death, but may induce mutations (Appendix VII). As summarized in Section 3.4, present-day knowledge leads to the conclusion that, except for the special case of '251, energy deposition from beta radiation of nuclides into nucleic acid in man significantly outweighs the damaging contribution from transmutation effects.

5.2 Absorbed Dose

When tritium is randomly distributed throughout the cell, the average absorbed dose per disintegration occurring within the nucleus is 0.34 rad to the reference nucleus. When tritium is confined to the nucleus, the edge effect reduces the average absorbed dose from each

' The International Commission on Radiological Protection uses 10 keV for calculation (ICHP, 1959).

5.2

ABSORBED DOSE

/

21

intranuclear disintegration to 0.27 rad (see Figure 1). A maximum permissible dose of 5 rem per year to the reference nucleus is delivered by 28 tritium decays during a year. This represents about 1 decay every 20 days. For small numbers of decays per reference nucleus the fluctuation in this number for any nucleus is given by Poisson statistics (see Appendix IV).For the case where the radionuclide is incorporated into DNA in a non-exchangeable form, the absorbed dose will be inversely related to the rate of cell proliferation.

5.3

Dose Rate

One microcurie (pCi) of tritium distributed uniformly in one gram of material of unit density delivers a dose rate of 12.14 mrad per hour. At high levels of tritium incorporation into DNA of cell nuclei, where there are many disintegrations per hour, ordinary dose-rate considerations apply. Appropriate allowance for reduction in the amount of tritium a t each cell division must be made. Cells with a long lifespan may accumulate large doses. However, the energy from each individual disintegration is deposited in an instant. Little is known of the effect of dose-rate for infrequent decays. However, a t very low dose-rates, where only single disintegrations occur over long periods, the variation in energy of beta particles is a consideration. The energy of beta particles varies from nearly zero to about 3 times the average energy and hence the dose to any one reference nuclear volume varies from near zero to about 3 times the average of 0.27 rad. In other words, the average dose to the reference nucleus becomes a constant. Hence, as body burdens decrease, the average dose to reference nuclear volumes is constant with fewer cells being involved. Accordingly, average tissue doses are less than the average dose to the reference nucleus from beta emitters and this average tissue dose becomes increasingly meaningless. Similar considerations apply with external exposure to small doses of x or gamma rays (see Appendix IV).

5.4

Tritiated Water

About 30 percent of the total body hydrogen is in tissue solids. In animals continuously exposed to environmental tritium, tritium will equilibrate with the exchangeable hydrogen throughout the body. The rates of turnover of exchangeable hydrogen vary .considerably. With prolonged intake of tritiated water (3HOH) the specific activity of

22

/

5. TRITIUM

tissue-bound hydrogen has risen to as high as 35 percent of that of t,he body water. On the other hand, a single administration of 3HOH leads to an initial fixation in tissue solids of about 1-2 percent of the amount given. The rate a t which tritium enters and leaves tissue constituents may be influenced by isotopic mass effects of tritium. There is, however, no evidence in mammals that such isotopic effects on chemical reaction kinetics lead to a concentration of tritium in tissue solids above that of the tritiated water. Nevertheless, after cessation of tritium intake, tritium concentration in tissue solids decreases at a slower rate and thus finally becomes higher than that in body water. Additional details on metabolism of tritiated water are given in Appendix 11. A single intake of 1 mCi of tritiated water in reference man (60 percent water) results in a total concentration in body water of 0.023 pCi/rnl. However, tissue, exclusive of bone and fat, is about 70 percent water; thus the cell nucleus is assumed to contain 70 percent water. Hence, there will be 0.016 pCi/g of nucleus. One pCi of tritium per g uniformly distributed in tissue delivers a dose rate of 12.14 mrad per hour. Therefore, 0.016 pCi tritium per g will deliver an initial rate of 0.20 m a d per hour. Since the average biological half-life of tritiated water is 12 days (fractional turnover of 0.057 per day), the total dose will be 81 mrad or mrem since Q = 1. In the reference cell nucleus, 1 tritium disintegration corresponds on the average to 0.34 rad when tritium is randomly distributed. The dose to infinity of 81 mrem is thus equivalent to 1tritium decay in a volume corresponding to about 4 reference nuclei. In other words, one decay occurs in every fourth nuclear volume. A dose of 5 rem would therefore be delivered by a single intake of 60 mCi of tritiated water. This dose corresponds to 18 tritium decays per nuclear volume. With a chronic body burden of 1 mCi tritium in reference man (70 kg) the average concentration per g of tissue is 0.014 pCi/g. This amount of 3H gives a dose rate of 0.014 x 12.14 = 0.17 rnrad/hr. Tritium is distributed between body water and organic constituents of tissue. In reference man there is 7 kg of H, of which 4.7 kg is in body water and 2.3 kg is in the organic constituents of tissue. In the case where the specific activities of 3H in water and organic constituents are equal, the fraction of the dose from body water will be 4.666 + 7 = 0.67 and the fraction of the dose from organic constituents is 2.334 + 7 = 0.33. Therefore, the dose from 3H in water is multiplied by 1.5 to get the total tissue dose. In actuality, in one environmental study in which animals consumed water and food labeled with 3H, the specific activities of tissue water and inorganic constituents were nearly equal. As discussed in Appendix 11, the specific activities of 3H in urine and

5.4 TRITIATED WATER

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23

body water are nearly identical. Hence, in the case where food and water have equal concentrations of 3H, one can estimate tissue dose as follows: pCi 3H per ml urine x 12.14 x 1.5 = tissue dose in mad/h. When only tritiated water is consumed over long periods by rodents, the concentration of 3H in dry tissue becomes about 20 percent of that in body water (see Appendix 11). The body, exclusive of fat and calcified tissue, contains 70 percent water. The dose rate to tissue from 3H in water and organic constituents is estimated from 3H concentration in body water in pCi/ml. The absorbed dose-rate (D) to tissue is the sum of the absorbed dose-rates from the tritiated body water and the tritiated tissue organic constituents. As mentioned earlier, the concentration of 3H in body water and urine is the same, hence

0 D 0.7 0.2 0.3 12.14

= = =

= = =

(pCi/ml urine x 0.7 + pCi/ml urine x 0.20 x 0.3) 12.14 mrad h-' fraction of water in soft tissue fraction of 3H in organic constituents relative to water fraction of organic solids in soft tissue mrad (pCi g-')-' soft tissue

If the 3H concentration in urine is 1 pCi/ml

D = (1.0 x 0.7 + 1.0 x 0.2 x 0.3) 12.14 = 9.2 mrad h-'. With continuous concentration of 1 pCi/ml of urine the annual tissue dose will be 9.2

X

24

X

365 = 80.6 rad y-'.

Five rem per year (maximum permissible dose equivalent) to tissue will be delivered when the urinary concentration of 3H is constant a t 5 rem yr-' (80.6 rad yr-')-I (1pCi ml-') = 0.06 pCi d-'urine. The above concentration in urine means there is 2.52 mCi in body water and 0.5 mCi in organic constituents to give a total body burden of 3 mCi. The recommended total body burden of 2 mCi (NCRP, 1959) results in an accumulated annual tissue dose of 3.22 rad, and the 3H activity in the urine is 0.04 pCi/ml. Since the original body burdens were based on Q = 1.7 and for this report 1.0 is used, the body burden that will deliver 5 rem per year is 3 mCi and not 2 mCi. The fractional turnover of water per day is 0.057. Therefore, a maximum total body burden of 2 mCi (NCRP, 1959) will be maintained by intake of 0.11 mCi 3H/day and 0.005 mCi/hour. However, as noted above, using a Q = 1 the annual dose is 3 not 5 rem.

24

/

5. TRITIUM

5.5

Tritiated Thymidine

Tritiated thymidine is incorporated specifically into the DNA of proliferating cells. This DNA is nearly exclusively localized within the cell nucleus. The energy absorbed by the reference nucleus in this situation amounts to 0.27 rad per tritium decay (see Section 4).

5.5.1

Tritiated Thymidine Single Dose Injected Intravenously

Dose to Bone Marrow Stem Cells. The average turnover time of the bone marrow stem cell is considered to be 30 days. This value is used for the calculation of accumulated dose (see Appendix VI). Data from labeling of differentiated bone marrow cells in rodents and man indicate that after an intravenous injection of 1 pCi of tritiated thymidine per g body weight, there are 0.6-1.5 disintegrations per hour up to the first cell division. The value of 1.5 disintegrations per hour will be used in this report, with a range from 0.3 to 6 disintegrations per hour; i.e., from '/4 to 4 times the average dose (see Appendix V). It is assumed that the average radionuclide burden to the differentiated labeled bone marrow cell population applies to the labeled stem population. F'urthermore, the increase in nuclear volume during DNA synthesis, which decreases the dose per 3H disintegration, is ignored, since, as will be shown in Section 5.5.3, it involves a small fraction of cells. With these assumptions, the accumulated radiation dose for one year to the average nucleus, after a single intravenous injection of 1 pCi of tritiated thymidine per g body weight, can be calculated as follows: 1.5 decays per hour from the midpoint of DNA synthesis to the first cell division, which is an average time interval of 10 hours permitting an accumulation of 15 decays. Thereafter, during the next cell generation time the dose accumulation amounts to 0.75 decays x 24 hours x 30 days = 540 decays. After cell division and during the following 30 days, accumulated dose is calculated to be 0.38 x 24 x 30 = 270 decays. Continuing the dose accumulation for 1 year gives a total of 1078 decays with a range of about 270 for the lightly labeled cell nucleus to about 4312 for the heavily labeled cell nucleus. These decays will deliver an average 1078 x 0.27 = 291 rad to the average cell nucleus at risk, with a range of 73 - 1164 rad. It follows that, with Q = 1 and 1 rad = 1 rem, 5 rem per year would be delivered to the average labeled cell nucleus after a single intravenous injection of 0.02 pCi per g body weight, or 1.4 mCi per reference man. The 3HOH produced after injection of 3H-thyrnidine has a

comparatively short turnover time, and its contribution to dose can be neglected here. There is, in this instance also, practically no contribution from the thymidine reutilization. I t is to be noted that unlabeled cell nuclei will receive nearly zero doses. Dose to Male Germ Cells. The spermatogonial stem cell turnover time is quite uncertain, but estimated to lie somewhere between 16 and 100 days. Thirty days seems a reasonable value for the average. Labeled spermatogonial stem cells are thus assumed to have a turnover time of 30 days, the same as bone marrow stem cells. The size of their nuclei and the amount of 3H-thymidineincorporated per cell are also assumed to be comparable. Therefore, on the average, 1.5 disintegrations per hour per labeled cell would occur after an injection of 1&i of 3H-thyrnidine per g of body weight. Therefore, the accumulated dose that is listed for bone marrow stem cell nucleus in the discussion above also pertains to the spermatogonial stem cell nucleus, and 5 rern to the average labeled spermatogonial stem cell nucleus is expected to be delivered after an intravenous injection of 1.4 mCi per reference man. By taking into account the nuclear sizes and durations of the various stages of spermatogenesis, the accumulated average dose to the differentiating spermatocytes is estimated to be 70 rern after a single intravenous injection of 1pCi of tritiated thymidine per g body weight. If one assumes the range in labeling is from 1/4 the mean to 4 times this mean, as was done with bone marrow cells, the range of dose will be 17-280 rad. To assure that the average dose to sperm cell nucleus does not exceed 5 rem, the intravenous injection could be as large as & x 1 = approximately 0.071 pCi per g, or 5 mCi per reference man. This is more than 4 times the amount of 3H-thymidine that will deliver 5 rern to the average labeled spermatogonial stem cell nucleus. The introduction of 1.4 mCi of 3H-thymidine into the bloodstream of reference man will deliver a dose of 5 rern to the average labeled nuclei. It takes a single injection of 60 mCi of 3HOH to deliver 5 rern to the entire body. Thus, to deliver 5 rern to the stem cell nuclei at risk it takes 1.4/60 or 1/43 the amount of 3H-thymidine as it does 3HOH. However, in the case of 3H-thymidine perhaps only 10 percent of the stem cells are a t risk, whereas with tritiated water 100 percent are at risk. Thus, 3H-thymidine may only be 4.3 times as effective as tritiated water. The actual effect for any individual will depend upon the fraction of the stem cells that are in DNA synthesis and this is variable depending upon age and physiological state. To be conservative, therefore, it is recommended that the maximum amount of 3H-thymidine that may enter the bloodstream per annum from occupational exposure is 1.4 mCi.

26 5.5.2

/

5. TRITIUM

Tritiated Thymidine Ingested-Single

Dose

Only a part of thymidine ingested is absorbed through the intestinal tract. Calculation of dose from ingested 3H-thymidine is based on the following data obtained from rodents (see Appendix V). In rodents the ratio of incorporation of 3H-thymidine into DNA after oral administration and intravenous injection is 1:5. With 50 percent 3H-thymidine incorporation after i.v. injection, 10 percent of the ingested dose should be bound to DNA. Hence, the ratio of radiation doses in the cell nuclei after oral ingestion and intravenous injection of the same amount of 3H-thymidine is 1:5. Dose to Stem Cells a n d Bone Marrow. The radiation dose to the hematopoietic and gonadal stem cells after ingestion of 'H-thymidine will be 1/5 of that following injection (see Section 5.5.1). It is suggested that the maximum permissible intake by ingestion be 5 X 1.4 mCi = 7 mCi per reference man; i.e., 5 X 0.02 pCi = 0.1 pCi per g body weight. This recommendation means that 3H ingested in the form of thymidine is 60/7 = 8.6 times more hazardous than 3H ingested in water. This recommendation may require modification as more precise data on stem cells and on the distribution and rate of incorporation of ingested 3H-thymidine in man become available.

5.5.3

Continuous Intake of Tritiated Thymidine

When tritiated thyrnidine is continuously consumed or administered, it labels cells as they progressively come into DNA synthesis. Thus, with time, all cells in the dividing populations will become labeled; the faster the turnover, the more quickly they become labeled. In stem cell pools, where cells feed into differentiated pools and also feed back into the stem cell pool for self-perpetuation, the intensity of labeling will increase. The individual cells pass through the DNA synthesis phase where they double the DNA content from the diploid to tetraploid stage. At the end of this phase, the labeling intensity will depend on the amount of 3H-thymidine made available to the cell during this phase. It is assumed that the labeling intensity will be the same irrespective of whether the precursor is given as a single injection or whether the same amount is delivered over the entire length of the DNA synthesis period. The limit for continuous administration is expressed by the amount of 3H-thymidine given per unit time that will lead, during DNA synthesis, to the same intensity of labeling as is obtained from the maximum permissible amount for a single injection. Hence, this

5.5 TRITIATEDTHYMIDINE

/

27

will be, in yCi per hour, the allowed single injection in pCi divided by the time for DNA synthesis. During continuous administration of 3H-thymidine, the labeling intensity of the ceU is halved a t mitosis. If 3H-thymidine administration is continuous a t the same rate as that during the preceding cell cycle, the labeling intensity at the end of the second DNA-synthesis phase will be 1.5 times the preceding intensity. The next mitosis will again reduce the labeling intensity to one half, i.e., to 0.75 the value of the initial intensity, and the next DNA synthesis phase will increase labeling again to 1.75 the initial value. It is easily seen that, upon continuous exposure to the precursor a t a constant level, the average labeling intensity of the diploid cells will converge to unity with time and the average labeling intensity of tetraploid cells will converge to twice unity. In other words, the cells in S phase will have labeling intensities varying between 1 and 2 of the initial intensity after equilibrium is reached. During continuous exposure to 3H-thymidine, nearly all cells will become labeled as they pass through the DNA synthesis phase, and a t equilibrium the fraction of stem cells with tetraploid DNA will be

where k2= duration of G1-phase (3 h)2 t,,, = duration of mitosis (1h)2 T = turnover time (30 x 24 h = 720). Hence, with continuous consumption of 3H-thymidine, less than 1 percent of the cells will have twice the intensity of label. If human stem cells have the same DNA synthesis time (T,) of 12 hrs as cytologically identified human erythroid and granulocytic cells (Stryckmans et al., 1966)

will be the fraction of cells having labeling intensities between 1 and 2 times the value that is found for about 98 percent of the cells. The tetraploid cell nuclei (in the h1and t,,,), however, have about twice the volume of the diploid nuclei. Hence, the dose rate during tcz and t, will be nearly the same as the dose rate in diploid nuclei. Thus, Figure 1 shows that the dose per tritium disintegration decreases It is assumed that animal data apply to humans.

28

/

5.

TRITIUM

nearly by a factor of 2 as the volume increases from 270 x lo-'' ml to 540 x 10-l2ml. Since 1.4 mCi of 3H-thymidine to reference man is considered the maximum permissible amount to be allowed in a single intravenous injection (Di) per year, and since DNA synthesis time (t,) in the stem cell is assumed to be 12 hours,

will eventually lead to the same 3H content per labeled cell that is obtained from a single exposure to 1.4 mCi, but 10 times as many stem cells will be labeled. After a single administration of %-thymidine, there is a decreasing dose rate as labeled stem cells divide. With chronic exposure the dose rate will be constant at equilibrium. For the case of continuous exposure to 0.12 mCi/h, the dose rate per cell a t equilibrium level of labeling intensity may be calculated as follows: The single intravenous injection of 1pCi of tritiated thymidine per g body weight, or 70 mCi/reference man, results in an average of 1.5 decays/h in labeled cells (see Section 5.5.1). This corresponds, for the diploid cell, to 1.5 x 0.27 = 0.41 rad/h. The continuous intravenous administration of 0.12 mCi/h will, at equilibrium, result in the same number of decays as that produced by a single injection of 1.4 mCi. Therefore, t!le dose rate to labeled stem cells will be 1.4 x 0.41/70 = 0.008 rad/h and the accumulated dose/year is 70 rad. This is about 14 times higher than the dose limit of 5 rad. Hence, the correction factor 1/14 = 0.07 must be applied. Hence, it it is suggested that the limit for continuous exposure be

For the same amounts of 3H-thymidine injected or ingested, 5 times as much is incorporated into DNA after injection as after ingestion. Hence, the suggested limit is 5 x 8 = 40.0 pCi/h for chronic ingestion. 5.6

Other Tritium-Labeled DNA Precursors

Deoxycytidine, in addition to thymidine, is an immediate precursor that is also specifically incorporated into DNA. In order to calculate dose, it is assumed that the metabolism of thyrnidine and of deoxycytidine is similar. Consequently, it is suggested that the maximum permissible intake and dose values for deoxycytidine be the same as

5.6

OTHER TRITIUM-LABELED DNA PRECURSORS

/

'29

those for thymidine until more precise data are available. However, particular attention is drawn to the situation for 5-3H-deoxycytidine, (5-3HCdr)because of the 3H transmutation effect (see Section 3.4 and Appendix VII). Each decay of 3H in the five position of cytosine in DNA may possibly produce a mutation. Therefore, when 3H is in the 5 position of cytosine in DNA, there is a mutation from transmutation and another from radiation. Accordingly, it is suggested that the intake be limited to 1/2 of that for 'H thymidine. When 3H-deoxyuridine is used in biomedical research, after uptake by the cell it is methylated to thymidine (see Appendix V). If tritium is in the 6-position, it should be treated as thymidine; if tritium is in the Bposition, much will be lost during metabolic conversion to thymidine and little will .be incorporated into DNA. All other nucleic acid precursors are common to RNA and DNA. The rate of uptake of such precursors into DNA is governed by various factors such as rate of RNA synthesis, metabolic interconversion of nucleotides, and size of nucleotide pools (see Appendix V). The intake limits are listed in Section 5.7. Tritium-labeled halogenated nucleosides are incorporated less efficiently than thymidine by factors of 1/4 to 1/10 (see Appendix V). However, depending on the quantity administered, these halogenated nucleosides have been shown to be radiosensitizers when incorporated into DNA. Thus, in view of these offsetting factors, a conservative position would be to apply the limits for %-thymidine until more data on radiation effects of their labeled precursors are available.

5.7 Tritium-Labeled RNA Precursors

All nucleated cells synthesize at least small amounts of RNA, and will become labeled by precursors of these molecules. Since RNA, in contrast to DNA, is distributed throughout the cell, a large fraction of the beta particles from tritium bound to RNA is absorbed outside the nucleus. Tritium decays, originating at random in the cell, cause less damage to DNA than an equal number of decays of tritium localized in the nucleus only. Tritium decays in nuclear RNA may be as effective as in DNA or less effective, for example, when the 3H is located mainly in the nucleolus. Labeled RNA precursors are reutilized efficiently for new RNA and DNA synthesis. Essentially all cells will be labeled to some extent by RNA precursors, and prolonged availability of precursors from RNA turnover will increase the number of DNA labeled cells and the extent of DNA labeling. The objective is to keep the dose to

30

/

5. TRITIUM

the average labeled nuclei below 5 rem per year. Data generally indicate a lower efficiency of RNA precursor incorporation compared to thymidine, perhaps by a factor of 4 or more. Yet, through reutilization of RNA precursors, the ratio per individual labeled cell changes with time in favor of DNA labeling, but almost certainly does not reach the intensity of labeling from %-thymidine. Few data are available at the present time on the final extent of labeling of longlived cells after administration of labeled RNA precursors (see Appendix V). It is assumed for this report that the late hazard from incorporation of RNA precursors is certainly less than that for equally labeled thymidine. To be conservative, the limits for 3H-thymidine will be applied to tritiated RNA precursors as discussed in Section 5.5.1 for intravenous injection and in Section 5.5.2 for ingestion. For continuous exposure, it is suggested that the limits for tritiated thymidine be applied.

5.8 Tritium-Labeled Amino Acids a n d Nonspecific Precursors Although the present report is not directly concerned with the maximum permissible intake of radionuclides bound to protein precursors, radiation from labeled protein precursors may be delivered to the genetic material. There may be labeled nuclear proteins such as histones that have close contact to DNA. Protein is distributed throughout the cell, and has defined turnover rates. Radiation damage to the genetic material produced by 3H decay in labeled cellular proteins is assumed to be comparable to that from 3H distributed randomly in the cell. In addition, proteins are distributed throughout the body, as in connective tissue, blood, lymph, and interstitial fluid. The effect on the cell nucleus of 3H incorporated into such proteins may be neglected. Tritiated amino acids, after administration, are widely distributed throughout the body and incorporated into protein. In addition, amino acids may be incorporated into other constituents because of multiple metabolic pathways; e.g., glycine into the purine ring. Besides amino acids, other substances, such as orotic acid and formate, may enter nucleic acids through multiple metabolic pathways. These substances have large natural pools in which labeled materials are diluted. These labeled materials have variable biological half-lives, as do the tissue constituents into which they are incorporated. Comparatively small amounts become incorporated into DNA. It is rec-

5.8 LABELED AMINO ACIDS AND NONSPECIFIC PRECURSORS

/

31

ornmended that the maximum permissible amounts of 3H-aminoacids should be determined by the usual criteria of calculating tissue dose on the basis of distribution and turnover rates of radionuclides in tissue and not in single cells.

Compounds The half-life of '*C is 5730 years and the emitted beta particle has an average energy of 0.049 MeV. This energy is 8.6 times greater than the mean energy of the 3H beta particle. Correspondingly, the I4Cbeta tracks have a mean range of 33 pm, exceeding the diameter of the typical mammalian cell by a factor of three. Transmutation effects resulting from I4C decaying within the DNA are known and have been discussed in Section 3.4 and Appendix VII. They are outweighed by radiation effects. The dose to the labeled standard nucleus from the beta particles is the limiting factor for defining maximum permissible intake. Experiments have shown that comparable activities of 3H and I4C uniformly incorporated into DNA of mammalian cells produce damage that is related to the radiation absorbed by the nucleus. The greater ranges of the I4C beta particles in comparison with 3H beta particles lead to a larger fraction of energy absorbed outside the cell nucleus. This loss of energy from the nucleus where decay occurs reduces the total energy absorbed by that nucleus to a value of 0.19 of that obtained if all of the energy were absorbed within the nucleus (see Table l ) , or 0.68 of the energy that is deposited from a 3H decay under comparable conditions. In most tissues, the ratio of cell diameter to nuclear diameter, and the space between cells, are sufficiently large that the radiation effect on a nucleus from a I4Cbeta particle originating in the nucleus of an adjacent cell is negligibly small. In some tissues, containing densely packed small cells such as lymph node and spleen, neglecting this effect may not be fully justified. This effect is expected to be small, relative to the effect on the beta-emitting nucleus and therefore, in this report, it is neglected until micromorphometric data are obtained on human tissues that will determine the contribution of dose to adjacent cells. Since the absorbed dose to mammahan cell nuclei determines the magnitude of effect, it is recommended that the intake by injection of I4C-labeledthymidine and other I4CDNA precursors be limited to %.ss or 1.47 that of 3H-thyrnidine and should not exceed 2.0 mCi per reference man. The maximum permissible intake by ingestion would 32

6. CARBON-14-LABELED COMPOUNDS

/

33

be 10 mCi per reference man. For chronic exposure, the suggested limit 2.0 is - x 0.07 = 11 pCi/h for injection and 55 pCi/h for ingestion. 12 Because of the relative randomness of RNA distribution in cells, the 14 C toxicity from 14C-labeledRNA precursors is to be compared with that of tritium on the basis of the ratio of their average beta energies of 8.6 (see Section 4). Thus, with random 14Cand 3H in tissue, it is recommended that the intake for 14C-labeledRNA precursors be % of that for tritiated RNA precursors (see Section 5.7). For I4C-labeled amino acids and for nonspecific precursors, the intake for both injection and ingestion should be limited to Ye of that for the corresponding tritium-labeled precursors and the same principles apply as stated for tissue dose in Section 5.8.

Because phosphorylated nucleic acid precursors are considered to be mainly dephosphorylated prior to incorporation into nucleic acids, this section will consider the hazard of administration of 32Pphosphate. 32P is distributed throughout the body and incorporated into the nucleic acids and nucleotides throughout the cell. Thus, cells, including those synthesizing nucleic acids, will be irradiated with the 32Pbeta particles having a mean energy of 0.695 MeV and mean range of about 1.53 mm, exceeding by more than 100-fold the diameter of most mammalian cell nuclei. The half-life of 32Pis 14.3 days. When 32Pis present within tissue and cells, one 32Pdecay will deliver a dose which is proportional to the beta energy. This is approximately 122 times the dose absorbed for one tritium beta particle of average energy (see Section 4). Each transmutation of 32P to 32S affects the molecule in which transmutation occurs. Since most decays of incorporated 32Pdo not occur in DNA but throughout the cell, effects from 32Ptransmutation (see Section 3.4 and Appendix VII) are far outweighed by radiation effects. Therefore, it is recommended that the maximum permissible body burden, 6 pCi in the critical organ, bone, and 30 pCi for the total body, given in NCRP Report No. 22 be observed (NCRP, 1959).

Sulfur-35-labeled cysteine and methionine are distributed throughout the tissue and cells synthesizing proteins. They are not incorporated into nucleic acids. They may, however, be brought into close proximity to these molecules. 35Shas a half-life of 87.9 days. The mean beta particle energy is 0.049 MeV and the maximum energy is 0.167 MeV. The mean range of the beta is 32.6 pm and thus exceeds by about fourfold the diameter of the standard nucleus. For 35S distributed in tissue, one 35Sdecay delivers a dose that is proportional to the beta energy, or eight times the dose absorbed per tritium decay (Section 4). It is recommended that the maximum permissible body burden, 90 pCi in the critical organ, testis, and 400 pCi for the total body, given in NCRP Report No. 22 be observed (NCRP, 1959).

9. Iodine-131 and Iodine-125-

Labeled Precursors for DNA 131

I and "51 are discussed because both are used as labels for 5-iodo2'-deoxyuridine (IUdR) that is incorporated into DNA in the place of thymidine. The distribution of the IUdR to the tissues and cells is practically identical to that of thyrnidine. IUdR, however, enters DNA less effectively than thymidine. In mice about 5-10 percent of the intravenously given quantity of IUdR is incorporated into DNA and about 1-2 percent of the ingested quantity (see Appendix V). More than 90 percent of the iodine in IUdR is thus made labile and enters the inorganic iodine pool. Unless the iodine uptake in the thyroid is blocked, approximately ?hof the radioiodine is finally concentrated in the thyroid gland. 131 I has a complex decay scheme with beta and gamma radiation being emitted. The half-life is 8.1 days. The major (87 percent) beta energy is 0.61 MeV and the major (80 percent) gamma energy is 0.36 MeV. The average range of beta particles from I3'I is 450 pm and exceeds by 50-fold the diameter of the reference nucleus. Radiation absorption will be relatively uniform to labeled and unlabeled cells in those tissues where cell proliferation occurs and where I3'IUdR is incorporated, such as in bone marrow, intestinal tract, skin, and male reproductive tissue. The maximum permissible dose to the thyroid is 15 rem (NCRP, 1971). Since 90 percent of the iodine in IUdR is released into the inorganic iodine pool and 1 pCi of iodine given to the adult human delivers approximately 2 rad to the thyroid, the maximum amount of I3'IUdR that can be given when the thyroid is not blocked by stable iodine is about 8 pCi I3'IUdR. On the assumption that 10 percent enters the DNA of the body and one-fifth of that goes into the bone marrow (1.3 x lo3 g per reference man), the concentration of I3'I will be about 1.2 x lop4pCi/g of bone marrow. This delivers an initial dose of about 0.16 rnrad/hour. The decay of Iz5I,on the average, leads to the emission of about E orbital electrons (Auger electrons) that have discrete energies ranging from about 0.5-34 keV. When '251is uniformly distributed in the cell nucleus, for example, after incorporation of lSIUdR into DNA, thc 36

9.

IODINE-131 AND IODINE-125

/

37

radiation dose to the reference nucleus will be 0.71 rad per decay (see Section 4, Table 1); this is approximately 2.6 times greater than that calculated for the beta particle from tritium decay in the nucleus (see Section 4). Experimental evidence indicates that Iz5Idisintegrations in DNA of cultured cells are very efficient in producing lethality, perhaps up to 40 times as efficient as 3H decays in DNA. Furthermore, survival curves of mammalian cells labeled with Iz5IUdRshow the reduction of the shoulder region characteristic of high-LET curves. In whole mice, decays of Iz5I in DNA following labeling with lZ51UdRare about 10 times more efficient in altering turnover rates of labeled DNA than are decays from 3H incorporated into DNA with 3H-IUdR (see Appendix VII). The great efficiency of Iz5Icould be due to a high ionization density from the Auger electrons, or to charge transfer in the DNA molecule caused by the Auger process itself. The latter interpretation is supported by the high efficiency of 1251decay in DNA in producing DNA double-strand breaks (see Appendix VII). lZ51UdRis incorporated into DNA with an efficiency about ?kof that of thymidine. Since in mammals there is a 25-fold higher toxicity of L251decay compared to 3H decay in DNA, until further data are available it is recommended that the maximum intake by intravenous injection be 6/25 = 0.24 that of 3H-thymidine, or about 300 pCi in a reference man. The effect of the relative half lives of Iz5Iand 3H would increase the maximum intake of Iz5IUdR. However, in view of the uncertainty of the toxicity of lz51as compared to 3H, the half lives have been ignored in order to err on the conservative side. Since the amount of precursor incorporated after ingestion is about 20 percent of that after intravenous injection for both thymidine and IUDR (see Appendix V), it is recommended that the maximum intake by ingestion again be 6/25 = 0.24 that of 3H-thyrnidine, i.e., about 1.5 mCi in a reference man. In making this recommendation, it is assumed that sufficient stable iodine will be administered to minimize uptake of 1251by the thyroid. For the case of continuous accidental exposure, it is considered that there is no blockage of the iodine uptake into the thyroid gland. Hence the suggested limit is that for inorganic radioiodine (about 8 pCi).

10. Summary The incorporation of nucleic acid precursors labeled by short range beta emitting radionuclides (3H, 14C, 32P,lZ51)leads to a microscopic heterogeneous dose distribution where cell nuclei are exposed to much higher doses of radiation than the adjacent cytoplasm. The degree of radiation effect on a single cell that has incorporated these radionuclide-labeled DNA precursors depends upon the congruity among the site of absorption of the radiation, the radiosensitive volume, and the number of radiosensitive volumes, in addition to the total dose absorbed by the nucleus. In the case of single 3~ decays a t infrequent intervals, critical volumes of DNA leading to carcinogenic or genetic mutation may not be involved or repair may take place. The critical cells for acute and/or late radiation effects are those cells which may later produce cancer or may carry a genetic mutation that can be transmitted upon conception. To produce an effect, the damaged cells must be self replicating and produce viable progeny. Self replicating cells are by definition the stem cells. This report deals with the hematopoietic and spermatic stem cells along with the special case of the oocyte that is non-replicating after birth. Stem cells of skin, gut, and other tissues are not considered because of inadequate knowledge about their behavior. For this report the nucleus of the hematopoietic stem cell is selected as the critical microvolume and termed the reference nucleus with a mass of 270 x lopL2g. This mass is also used in calculating the dose to the spermatogonia (stem cells for sperm). The incorporation of %, 14C,32P,35S,13'1, and lZ5Iinto or close to the genetic material, i.e., the DNA in the stem cell nuclei, has been considered. Labeled nucleic acid precursors are initially incorporated into the cell nucleus when DNA and RNA are synthesized. Whereas nearly all nucleated cells synthesize at least some RNA almost continually, DNA is synthesized during a finite time period between two cell divisions. When nucleic acid precursors are administered to the body, they are incorporated within hours, and the half time of disappearance of nucleic acid precursors from the peripheral blood is in the order of minutes. If not incorporated, these precursors are rapidly catabolized and excreted. About 10 percent of the hematopoietic stem cells are in DNA synthesis and incorporate specific precursors for DNA, such as 38

lo. SUMMARY

/

39

thymidine. Turnover of cellular RNA and breakdown of the DNA of dead cells provide nucleic acid precursors for reutilization in DNA synthesis. If the degree of labeling of hematopoietic stem cell nuclei from various labeled nucleic acid precursors at various times after precursor administration is known, the dose to the stem cell nucleus can be calculated from the defined volume of the nucleus and the rate of division of stem cells, i.e., generation time or cycle time. This approach to calculating dose is a departure from the conventional expression of average dose to tissue volumes. In the case of the extreme heterogeneity of dose distribution specifically to radiosensitive sites at the cellular level, as observed after administration of radionuclide-labeled nucleic acid precursors emitting short range particles, average tissue dose will underestimate the dose to the reference nuclei and thus underestimate the potential hazard. In this report dose calculations are based on the dose delivered to the labeled reference nuclei by the labeled nucleic acid precursors, rather than being based on the average tissue dose. This dosimetric approach should not be considered as analogous to the "hot particle" approach where the congruity between the site of radiation absorption and the radiosensitive site is entirely a matter of chance distribution of the "hot particle" and the stem cell nucleus. The basic objective in this report is to keep the average radiation dose to stem cell nuclei below 5 rem per annum even though only a fraction of the stem cells will be labeled after a single intravenous injection or ingestion of radionuclide DNA precursors. If there is continued intake by injection or ingestion, almost all of the stem cells will become labeled with time depending on the kinetics of their turnover. When, as described earlier, a sufficient amount of %-thymidine is given continuously so that almost all stem cells will be labeled and receiving an annual dose of 5 rem, there is still a wide divergence from average tissue dose. For example, the average cell volume is roughly 1 x lo-' g and the reference nuclear volume is 0.270 x lo-' g. Thus, if all bone marrow cell nuclei were equally labeled and receiving 5 rem per year the average bone marrow dose would be 5 x 0.270 = 1.35 rem. Since it is believed that the dose to the DNA determines carcinogenic potential, the hazard would have been underestimated substantially. For dose calculations, a reference nuclear volume of 270 x 10-l2 ml (sphere of 8 pm diameter) was used; the average generation time of the hematopoietic stem cell was taken to be 30 days. Identical assumptions were made for the spermatogonial stem cell. Experimental data on rate of incorporation and turnover of labeled

40

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10. SUMMARY

nucleic acid precursors in the average labeled bone marrow cells were taken to apply to the average stem cell. The amounts of labeled nucleic acid precursor administered at one time or continuously by different routes that would deliver 5 rem per year to the average labeled hematopoietic and spermatogonial stem cell nucleus were calculated and the results are listed in Table 2, the Summary Table. TABLE 2-Summary table Amount of labeled compounds that delivers 5 rad per year to hemopoietic and spermatogonial stem cell nuclei. Calculations based on reference man Haute a n d % v t i o n

.-

Source

single i.v. injection

'H-water (p. 22.23) "H-TdR (p. 24-26.28) 5-'H-CdR Ip. 29. 30) AU other 'H-DNA precurnors (P. 29. 30) 'H-HNA precursors (p. 30. 31) " C T d R (p. 33) and dJ "CDNA precursors "C-RNA precursors (p. 34) 'fil-UdR ( p 37) "'I-UdR ( p 38) VP

.as l a b . Amino Acids

"Provided thyroid ia; blocked.

60 mCi 1.4 mCi 07mO 1.4

rnC~

1.4 mCi 2.0 mCi

continuous ingestion

60 rnCi 7.0 mCi 3.5 mCi 7.0 rnC~ 7.0 ~ C I 10 mCi

i.v, injection

ingestion

0.11 mCi/d 0.005 mCi/h 0.008 mCi/h 0.004 mCi/h 0.008 m C i h

0.11 mCi/d 0.005 mCi/h 0.04 mCi/h 0.02 m C i 0.04 m C i h

0.008 mCi/h 0.012 mCi/h

0.04 mCi/h 0.06 mCi/h

0.175 mCi 0.875 mCi 0.001 mCi/h 0.336 mCi 1.680 ~ C I ' see page 30 limited by dose M tissue, see page 38 limited by dose to tissue. see page 35 limited by dose to cissue, see page 36 limlted by dose to tissue. see pages 32. 34, 36

0.005 mCi/h

APPENDIX I

General Principles Underlying Establishment of Radiation Protection Standards A. Basic Criteria Basic radiation protection criteria have been established by the NCRP and are set out in NCRP Report No. 39 (NCRP, 1971). The current dose-limiting recommendations are summarized in Table 3 (which is taken from Table 6 in NCRP Report No. 39 (NCRP, 1971)). Table 3 is provided for convenience, but the reader should keep in mind that the application of the limits given is substantially conditioned by the qualifications and comments to be found in NCRP Report No. 39 (NCRP, 1971).

B. Internal Exposure The Maximum Permissible Body Burden (MPBB) and the Maximum Permissible Concentration (MPC) of individual radionuclides must be based upon the basic criteria outlined above and are more fully presented in NCRP Report No. 39. The main recommendations found in NCRP Report No. 39 which deal with internal emitters are contained in the following paragraphs.3 (149) "When radioactive materials enter the body they are absorbed, metabolized, and distributed to the tissues according to the chemical properties of the elements and compounds in which they are contained. This is, of course, the basis for the widespread use of radionuclides as tracers in biological, biochemical, and medical research. Since most radionuclides of importance in radiation protection evaluations localize Paragraphs 149, 150, 151, 152,153, 154, 155, 156,and 157 of NCRP Report No. 39

(NCRP, 1971). 41

42

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APPENDIX I

TABLE 3-Dose- limiting recommendations Maximum Permissible Dose Equivalent for Occupational Exposure Combined whole body occupational exposure Prospective annual limit Retrospective annual limit Long term accumulation to age N years Skin Hands Forearms Other organs, tissues and organ systems Fertile women (with respect to fetus) Dose Limits for the Public, or Occasionally Exposed Individuals Individual or Occasional Students Population Dose Limits Genetic Somatic

5 rems in any one year 10-15 rems in any one year ( N -18) X 5 rems 15 rerns in any one year 75 rems in any one year (25/qtr) 30 rems in any one year (10/qtr) 15 rems in any one year (5/qtr) 0.5 rem in gestation period

0.5 rem in any one year 0.1 rem in any one year

1

0.17 rem average per year 0.17 rem average ~ e vear r

to a greater or lesser degree in certain tissues or organs, the internal emitters produce typically the phenomenon of partial body irradiation. There are important contrasts, however, with irradiation of portions of the body by external sources. (150) "The biological effects of ionizing radiation are essentially the same whether the source is external to or within the body, provided the absorbed dose and its distribution in the site of interest are the same. Nevertheless, there are material differences in concept and emphasis. For example, alpha particles, easily shielded as an external source, assume considerable importance within the body because of the high LET. Likewise, low energy beta emitters are relatively innocuous externally and potentially damaging internally. The size of an organ also plays an important role, since penetrating radiation may or may not be essentially totally absorbed in the organ of interest. In rare instances (e.g., natural uranium), chemical toxicity may compound the radiation problem, or even predominate over radiation effects. (151) "Internal sources delivering absorbed doses rather uniformly distributed throughout the body, and exceeding a few hundred rad in a short time, produce effects closely resembling those of a single moderately large dose of external radiation. Many aspects of the early or acute radiation syndrome may appear. This is particularly true of radioactive nuclides of hydrogen, sodium, and potassium which are distributed throughout the body fluids. With nuclides which localize

B. INTERNAL EXPOSURE

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43

markedly, the course of events is understandably quite different and does not resemble in any detail the acute whole body radiation syndrome. (152) "Acute or early effects from internal sources occur only following relatively large doses. They tend to develop more slowly than with a comparable dose of whole or even partial body external irradiation. This is due in part to the time required for absorption, distribution, and localization, as well as to the non-uniform distribution of the radiation dose within tissues and organs, and the protracted dose. The time required for normal metabolic processes and radioactive decay frequently results in a protracted radiation dose, even if the intake per se is a single short event. All of these factors result in a different, usually more delayed and more prolonged, time course of events. (153) "It is apparent from the above observations that the effects of internal sources are not likely to correspond to the effects of comparable doses of partial body external irradiation. Even if there were no complexities, such as subsequent transfer of the material, the localization is by tissue or organ system rather than by area of the body. Thus, an element which localizes in lymphoid tissue may be present wherever such tissue occurs unless it is sequestered by other processes. Further, the localization may sometimes be by individual cell type or process such as incorporation of radio-strontium in or on the crystalline structure of bone, especially growing bone. On the other hand, internal sources may require special attention, as when radionuclides are incorporated into the molecular structure of DNA. (154) "A special consideration pertains to those radionuclides that emit radiations of high LET. Most studies have been carried out with the alpha-emitting bone seekers, such as plutonium and radium, and soft-tissue seekers, such as radon and polonium. The high relative biological effectiveness of these nuclides in producing various types of damage has been clearly demonstrated and has resulted in the adoption of a quality factor (QF)4 of 10 for alpha particles. On deeper analysis, it is clear that the RBE for alpha emitters in tissues such as bone, from which estimates of QF are derived, is most difficult to determine. In practice, the spatial distribution of some of the alphaemitting nuclides such as radium and its progeny is different from that for representative beta-emitters (low LET). In addition, the rnicrodosimetry for alpha tracks necessarily differs from that for beta tracks. Also, the gross pattern of energy deposition is quite different for deposited nuclides from that of external radiations, which are the usual

' The currently accepted symbol for quality factor is Q (ICRU, 1971).

44

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APPENDIX I

reference radiation. Thus, it is very difficult, if not impossible, to isolate differences in effect attributable to LET from those attributable to differences in deposition of absorbed energy in space and in time. Apparent RBE values (and the derived QF values) vary from as low as one up to ten or more depending on the conditions of the measurement and the process under study. From studies in simpler systems it is known that the passage of a single alpha particle is more likely to kill a cell through which it passes than is radiation of lower LET. Put another way, the process can be described as having 'single-event' kinetics. There is little or no effect of dose rate with alpha particles. As also indicated in Chapter 3 there is little ability of cells (as studied in cell culture) to repair damage from high-LET radiation. (155) "With a nonspecific endpoint such as lifespan shortening as the criterion of damage, differences in effectiveness as measured in animals may appear even among nuclides with roughly comparable radiation characteristics. This may be related to the kinetics of distribution of the nuclide. For example, radium, which deposits directly and primarily in bone mineral volume, appears to be less damaging per unit of absorbed dose, for both early and late effects, than plutonium. The latter deposits primarily on bone surfaces rather than throughout the bone volume and also irradiates soft tissue transiently during the initial phases of its absorption and translocation to bone surfaces. The difference is of the order of five fold. The same extraeffectiveness, compared with radium, is seen with the soft tissue-seeker polonium, which does not deposit at aLl in the mineral phase of bone. The effectiveness of a given nuclide is frequently dependent on the effect studied and the duration of the exposure. For example, radon can be given in a dosage pattern that more or less resembles the radiation treatments with external sources. When doses from radon large enough to produce acute lethality are given to animals, the relative effectiveness of the radiations is only slightly greater than one when compared to x or gamma irradiation. Yet, when longer term effects, such as lifespan shortening in animals, are measured a t less acutely toxic doses, there is no doubt that radon is considerably more effective per unit absorbed dose than emitters of lower LET radiation, although it does not quite reach the effectiveness of plutonium or polonium. (156) "A possible phenomenon, mainly peculiar to internal emitters, is the 'transmutation effect'. Since many radionuclides such as sulphur, carbon, and hydrogen may be incorporated in essential biological molecules, there is the possibility of the following effects when they decay radioactively:

B. INTERNAL EMITTERS

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45

1. energy deposition from the ionizing process in the conventional

way. 2. molecular disruption due to possible recoil effects. 3. molecular disorientation as a result of nuclear transmutation. As an example of the third case, a complex molecule may be equally satisfied with a 12C atom or 14Catom a t a regular carbon position. If the radioactive carbon 14C then decays to nitrogen, the molecular structure is affected. If the molecule were DNA, the result might be equivalent to gene mutation. Several calculations and a few experiments with lower organisms have indicated this potentiality. While it cannot be dismissed as a possible problem with the internal emitters (or as one sequel of neutron irradiation), the effect does not seem to be large enough to be of ~ o n c e r n . ~ (157) "In summary of this relatively brief consideration of irradiation by internal emitters, compared with external irradiation, it may be concluded that the complications are primarily associated with quantitative aspects. There are many residual difficulties in constructing, a t the detail level, a common frame of reference for internal and external irradiation. Radiation protection practice succeeds because it is always possible to include conservative round-offs in attempting to set a common scale in terms of dose equivalents. The principal features to be kept in mind for the internal sources case are: 1. difficulties in estimating absorbed dose 2. possible chemical toxicity 3. possible transmutation and recoil effects. With these reservations, the attempt to develop permissible limits for internal emitters based on the broader background experience with external radiation is appropriate. Partial exceptions include consideration of the bone-seekers, where the long experience with radium in man is relevant, and the few instances where chemical toxicity rather than radiation controls the permissible intake." (End of quotation) Maximum permissible body burdens, maximum permissible concentrations, and maximum permissible intakes for internal emitters should be consistent, as far as possible, with the principles outlined in NCRP Report No. 39 (NCRP, 1971). Control of the internal dose will be achieved by limiting the body burden of radionuclides. Generally, this can be accomplished by control of intakes via ingestion, inhalation, or ' injection or by control of the average concentration of radioactive materials in the air or water. Since it would be impractical to set intake or concentration values for radiation workers as a function of age, See Krische and Zelle (1969) and Appendix VII for further discussion.

46

/

APPENDIX I

values are selected in such a manner that they conform to the above stated limits when applied to the most restrictive case; they are set to be applicable to radiation workers of age 18. Thus, the values are conservative and are applicable to radiation workers of any age .(assuming there is no occupational exposure to radiation permitted at age less than 18). NCRP Report No. 22 (NCRP, 1959) gives concentration values for numerous nuclides generally independent of the chemical compounds in which they may be administered. This report is concerned with intake values for 3H, 14C,32P,35S,lZ5I,or 1311that may be incorporated into the cell nucleus, and to which the dosimetric concepts set out in Section 4 may be applied.

APPENDIX 11

Environmental Contamination by Tritium and Tritiated Water, and Metabolism Introduction Natural Occurrence of Tritium Tritium (3H),an isotope of hydrogen, has an extremely small natural abundance in comparison to H and 2H. It is produced naturally in the upper atmosphere by the interaction of cosmic radiation with oxygen and nitrogen with a production rate of 10,000 to 20,000 curies per day (Jacobs, 1968).The 3H in the upper atmosphere'is oxidized to tritiated water (3HOH) and mixes with the hydrosphere generally through movements of air masses and by rainfall. There are also several man-made sources of tritium. The most important of these are nuclear explosions and power reactors, the latter of which may produce tritium by several different types of reactions that depend upon the structure and operation of the reactor. In 1969, the world's 3H burden had been estimated to be about 1700 megacuries (MCi), primarily from weapons, and almost all in the ocean. The equilibrium contribution from natural 3H was around 70 MCi (Jacobs, 1968; Cowser et al., 1966; Logsdon and Hickey, 1971; Terpilak et al., 1971; Peterson et al., 1969).The present 3H concentration in water thus amounts to about 3.2 x lop9pCi per cm3. The amounts of tritium being contributed to the hydrosphere from reactors and fuel processing plants in general have been considered in detail by Peterson et al. (1969). The Radioactivity Concentration Guide (RCG) for tritium in water is pCi/rnl (Jacobs, 1968). All experience today indicates that reactors and fuel processing plants will be able to stay well below the RCG in effluent water without difficulty under normal operating conditions. It is not likely that power reactor production will equal the sum of natural and weapons produced tritium

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until the year 2000. Bogen et al. (1973) estimated reactor tritium production to be 0.01 MCi in 1970,0.7 MCi in 1980, and 4 MCi in 2000 compared to about 5 MCi per year from natural sources.

Behavior of Tritium in the Environment The spatial and temporal distributions of any radionuclide, including tritium, released into the environment are governed by the same processes that control the transport and distribution of the corresponding natural stable element occurring in the same chemical-physical form. In addition, the distribution of the radionuclide is influenced by the degrees of isotope dilution that occur in the atmosphere, lithosphere, hydrosphere, and biosphere. Tritiated water follows the pathways of natural water in the environment, in plants, and in animals, except for some cases that are subject to processes influenced by difference in vapor pressure. Thus, tritium introduced into the waters of the earth enters into the hydrologic cycle and into food webs, some of which lead to man. Significant fractionation does not occur when a mixture of natural and tritiated water passes across a liquid-liquid boundary either in the environment or in organisms. However, fractionation may occur when tritiated water and natural water cross a liquid-gas phase boundary. Because of the difference in mass between Hz0 and tritiated water, the vapor pressure of tritiated water is only 90 to 92 percent that of normal water a t environmental temperatures (Avinur and Nir, 1960; Sepall and Mason, 1960; Jones, 1963; Smith and Fitch, 1963). I t has been shown that the evaporation of 86 percent of a volume of water (containing tritiated water) by dry air resulted in an increase of specific activity of 1.2 in the remaining 14 percent of the water (Horton et al., 1971). However, evaporation from natural bodies of water usually does not occur a t the very low relative humidities that were used in this experiment. Although the lower vapor pressure of tritiated water causes discrimination in favor of the evaporation of natural water over that of tritiated water from lakes and ponds, rainout and direct condensation of atmospheric water vapor back to these relatively shallow bodies of water result in preferential overall transport of tritiated water from a contaminated body of water to the atmosphere. Under environmental conditions, with relative humidities greater than 8 percent, the net loss of tritiated water from a shallow basin to the atmosphere exceeds that of natural water because the atmospheric water vapor is essentially free of tritiated water and thus provides a

ENVlRONMENTAL TRITIUM BEHAVIOR

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49

reservoir for isotope dilution before back transport to the basin by condensation. Tritiated water, introduced into the surface waters of the sea, disperses in the upper mixed layer. Between 50°N and 45"s the oceans usually consist of an upper "warm-water sphere" separated by a vertical transition zone of rapid temperature and density change, and a deeper "cold-water sphere" that reaches to the bottom (Dietrich, 1963). The warm upper waters vary in depth, but are often 50 to 100 meters thick and are rapidly mixed by winds because the density and salinity are nearly uniform. A shear zone often exists just below the mixed water in the layer of rapid change in density. This layer, the pycnocline, constitutes a barrier to the downward movement of tritiated and normal water from the upper mixed layer and to the upward movement of water from the depths. Tritiated water, added to the surface of the sea, rapidly mixes with the water of the mixed layer above the pycnocline. Because this layer is usually 50 to 100 meters in depth, the added tritiated water is subjected to appreciable dilution by the normal sea water. Evidence that tritium in fresh water is introduced into the ocean at the surface has been presented by Ostlund et al. (1969).These authors demonstrated an inverse relationship between tritium content and salinity in Atlantic samples collected at depths, a finding suggesting a dilution of tritium-rich fresh water with tritium-poor sea water. The known tritium content of rain water at that time was only about onehalf that required to account for the observed tritium levels in the sea water. The authors proposed that direct molecular exchange of water vapor a t the sea surface contributed the other half of the net tritium supply in the sea water of the region of their study. On the basis of salinity and temperature, the upper mixed layer in this area was about 50 meters thick. The average tritium content in the upper mixed layer was about 30 times that in the deeper layers that constituted the thermocline, a situation indicating that tritiated water did not penetrate below the thermocline. Broecker et al. (1966) observed a cut-off for "Sr content in Pacific Ocean water at the upper edge of the thermocline with the values in the upper water being about 10 times those of the deeper waters. Thus, both mixed water and soluble radionuclides are effectively retained in the upper mixed layer. The seas are relatively large and repeated cycles of evaporation and condensation usually occur between the water of the mixed layer and the atmosphere. The water in the sea and atmosphere thus tends to equilibrate in tritium content at a lower specific activity than the rainfall (Bainbridge, 1963). In contrast, rainfall on land is subjected to

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less dilution, and rainfall over land usually contains greater amounts of tritiated water than rainfall over oceans (Brown, 1967; Ericksson, 1965; Israel et al., 1963). In summary, although the difference in vapor pressure between tritiated water and normal water results in differential evaporation of tritiated and normal water from lakes, rivers, ponds, and seas, these differences are masked by other physical processes and are not significant in shifting the amount of tritium available to the biota.

Metabolism of Tritium The difference in vapor pressure between tritiated and normal water may also be important in connection with the limited concentration of tritiated water that has been observed in some plants and animals. Tritiated water may be retained in excess of environmental levels when water contaminated with 3HOH is transferred across a liquidgas phase boundary. This process occurs in transpiring plants and, to a lesser degree, in air breathing animals. The increase of the specific activity of tritiated water in plants from preferential transpiration of non-tritiated water may result in enhanced incorporation of tritium into food webs, especially in those food webs that include plants with high rates of photosynthesis. Koranda and his associates (Koranda and Martin, 1969) studied the distribution of tritium in organic matter, tissue water, and transpired water of the desert shrub Artiplex canescens in an area contaminated by fallout. The specific activity of tritium in the tissue water and organic matter was about the same. The specific activity of tritium in the transpired water vapor, however, was only about 22 percent of that in the tissue water and organic matter. The average specific activity in the soil water, at root-depths, was only about % the specific activity in the leaf tissue, but was 1-$5 times the specific activity of the transpired vapor. On the basis of these data it appears that the specific activity of tissue water and organic matter in plants may be increased by a factor of a t least 3 over the specific activity of the environmental soil water as a result of preferential transpiration of non-tritiated water from the plants into the air. Those plants with more rapid rates of transpiration would be expected to exhibit greater discrimination against loss of tritiated water. The fixation of tritium is rapid in the metabolic pathways of photosynthesis. Amino acids tagged with tritium have been observed to appear as early as 3 minutes after exposure of Chlorella to tritiated water (Moses and Calvin, 1959). In addition, tritium is incorporated

TRITIUM METABOLISM

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51

into plant carbohydrates and fats. Once this labeled food is broken down to small molec'ules in the gastrointestinal tracts of animals, the tritium enters the general body pool of carbohydrates, fatty acids, amino acids, and other organic precursors of larger molecules. These precursors, after entrance into the body, are either incorporated into newly synthesized molecules, or are degraded through the ordinary pathways, a t rates dependent upon the species, and are excreted. I t is impossible to imagine a situation in which the specific activity of tritium in the organic material of an animal would greatly exceed the specific activity of the tritium in the body water under the conditions of constant intake and in which most of the source-tritium was in the water. However, an herbivore receiving most of its food and water from plants containing tritium at levels 2 to 3 times those of environmental water might be expected to attain specific activities of tritium in its organic components that exceed the values in environmental water. Siri and Evers (1962) investigated tritium exchange from tritiated water administered intravenously or by mouth to humans, rats, guinea pigs, pigeons, and rabbits. In humans, the tritium of the expired water was about 88 percent of that in the urine and blood. In pigeons, the specific activity of the expired water was 35 percent to 55 percent of that in the blood. In these animals, the specific activity of the tritium in the body water and the organic material could be increased significantly by the preferential expiration of non-tritiated water vapor. In desert animals that obtain most of their water from tritium-containing food, the specific activity of tritium in the body water and organic constituents might well exceed the values in the environmental soil water and environmental water vapor. However, there appears to be no mechanism whereby tritium can become significantly concentrated in the organic constituents of the bodies of manunals a t levels much greater than that of intake water. With very long-term exposure, under conditions where an animal has received essentially all of its water from sources with a given concentration of tritium, the tritium bound to tissue organic constituents has ranged from 1 percent to as much as 35 percent of the concentration in the intake water (Seelentag, 1973; Woodard, 1970; Smith and Taylor, 1969; Khan and Wilson, 1965). In one study carried out over 5 months, in which the tritium concentration of 11 separate tissues was studied during constant intake of tritiated water, the concentration in the organic constituents varied from 0.1 to 10 percent of that in the body water (Pinson and Langham, 1957). Evans made observations on 52 deer that were exposed to tritium from a tritium-producing installation (Evans, 1969). In this situation

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the water and organic components of the food were tritiated. The concentration of tritium in the body water of these deer ranged from 4.2 to 54.5 picocurie per rnl. Assays of heart, liver, kidney, spleen, muscle, and fat for organically bound tritium suggested to Evans that about 0.85 of equilibrium specific activity had been reached. He further assumed that eventually all of the whole body hydrogen would become uniformly labeled if there was constant intake of tritiated water. The tritium-hydrogen ratios in both body water and the water of combustion of dried tissues were the same. However, if only 3HOH is consumed the specific activity of organic constituents is about 25 percent of the water (Laskey et al., 1973). The incorporation of tritium in the organic component of deer tissue results in whole-body radiation doses 1.4 to 1.5 times higher than estimated if only tritium in body water were to be considered. Evans further extrapolated to man, estimating that with reference man containing 7 kg of body hydrogen and that with a sustained tritium concentration of 1 pCi/l in body water the ultimate body burden would be 63 pCi, a sum of the tritium in body water (43 pCi) and that incorporated into tritiated organic compounds. This value will pertain to man when the total biosphere and all constituents consumed by man have the same specific activity of tritium. Koranda and Martin (1973) found that the lyophilized residues of six organs of kangaroo rats living for several generations in the tritium contaminated environment around a nuclear test site exhibited an average tritium activity 1.5 times that in body water. Robertson (1973) noted that the data of Evans and that of Koranda and Martin were not consistent with the data of Thompson and Ballou (1956). The latter investigators exposed mature female rats to constant levels of tritiated water for 6 weeks, mated them, and continued the offspring on the same level of tritiated water for 6 months. The specific activity of the tissue organic components was 20 to 30 percent of the activity in the body water during the exposure. This was interpreted to mean that only 20 to 30 percent of the hydrogen of organic components in the body comes from the water. One can, therefore, postulate that the higher amounts found in deer and kangaroo rats indicate that these animals obtained some of their ingested tritium from food as well as from water (Hatch and Mazrimas, 1972). It has been shown that specific activity in expired tritiated water vapor relative to tissue water is 0.44 in kangaroo rats and 0.64 in mice (Koranda and Martin, 1973), a result indicating that the lungs discriminate against the excretion of tritium. The effect of this isotopic discrimination in the lung on the specific activity of body water clearly depends upon the fraction of water excretion that is handled by the lungs. Robertson (1973) has shown that this isotopic discrimination in

TRITIUM METABOLISM

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53

the lungs might lead to an increased specific activity in the body of the order of 15 percent above that in the ingested water. Tritium passes equally well in both directions through liquid interfaces, but is discriminated against, to some extent, in passing from a liquid to gaseous phase as in respiration through lungs or transpiration by leaves of plants. Metabolism of Tritiated Water in Human Beings Tritium may enter the human body by absorption from the respiratory tract, either as tritiated water or tritium gas, through the intestinal tract, and by diffusion through the skin (Pinson and Langham, 1957; Pinson, 1952a; Delong et al., 1954; Osborne, 1966). The uptake from tritiated water and the air is about equal for respiration and diffusion through the skin. In general, 98 to 99 percent of inhaled tritiated water is absorbed through the lungs. Woodard (1970) has carefully studied data on the metabolism of tritiated water and reviewed this information analytically. Tritiated water can be absorbed through skin, the lungs, or the gut. In each case the tritium is rapidly distributed throughout the body via the blood. When tritium enters through the lungs or gut, maximum blood concentrations are reached within a few minutes. When absorption is through the skin, maximum blood concentrations are reached within two hours. Tritiated water in blood equilibrates with extracellular fluid (lymph, interstitial and intestinal fluids, or cerebrospinal fluid) in about 12 minutes. However, in poorly vascularized tissues, such as bone and fat, equilibrium with plasma water may take days to weeks. Tritiated water is excreted in the urine, sweat, breath, and stool. It has been shown that tritiated water in urine has the same specific activity as that of blood (Feinendegen, 1967; Seelentag, 1973; Pinson and Langham, 1957). Reasonably detailed studies have been made on 300 individuals who have been accidentally contaminated with tritiated water (Wylie et al., 1963; Butler and LeRoy, 1965). The range in the biological half-time has been between 2.4 and 18 days. The shortest half-time was observed when fluids were forced, to produce diuresis. Generally, the biological half-time of water decreased as the ambient temperature increased. Under tropical conditions, the biological half-time of water has been studied in human beings by Sadarangani et al., (1973) and it was found to range from 4 to 9 days. After a single administration of tritiated water to man, the excretion rate can be represented as the sum of three exponentials with half-times of disappearance of approximately 6, 23, and 345 days (Robertson, 1973; Butler, 1962). The ICRP and

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NCRP (ICRP, 1959; NCRP, 1959) have adopted, for practical purposes, a "biological half-time" of excretion of 12 days. The slower components of turnover of tritium listed above after the administration of tritiated water, represent the entrance of tritium into slowly exchanging pools of water, exchange with labile hydrogen, incorporation into organic constituents, and their subsequent turnover. Smith and Taylor (1969) have analyzed the methods by which tritium may enter organic constituents of the body. Tritium may enter organic compounds by exchanging with hydrogen at any of the labile sites in the molecule. In addition, tritium may be incorporated into stable configurations. In a general sense, the more rapidly a molecule i s turning over, the more tritium will be incorporated into it per unit of time and the more rapidly will the label be removed from tl$s ~nolecular species. Long-lived molecules will incorporate smaller amounts of tritium per unit time, but will turn over much more slowly, and will thus retain the radionuclide longer if the tritium is in a stable position within the molecule. Smith and Taylor (1969) have shown that the diverse routes by which tritium can enter the various body constituents, including proteins, carbohydrates, lipids, purine, and pyrimidine bases of DNA, represent a catalog of aLl known enzymatic reactions in which hydrogen is exchanged. There have been extensive studies in animals that have also been confirmed in a single study in man in which skin and fat were biopsied from a man who had been exposed to tritiated water 8 months earlier (Pinson et al., 1952). The tritium detected in the nonvolatile part of the tissue showed that it was firmly incorporated. The specific activity of the tritium was lower in fat than in skin. In a general sense, hydrogen bonded to oxygen, nitrogen, sulfur, or phosphorus will readily exchange with tritium in water, whereas hydrogen bonded to carbon is u s u d y not exchangeable except during some enzyme-mediated reactions. The only mechanism by which tritium can bond to carbon is apparently by de novo biosynthesis. There are extensive studies on other animals that show. that between 1 and 3 percent of a single administration of tritium in water is incorporated into organic constituents of the body. The rate at which those turn over is dependent upon the half-life of the molecules into which they are incorporated. It is true that some amino acids, purines, and pyrimidines are reutilized in new synthesis after macromolecular degradation. However, a large fraction of amino acids, purines, and pyrimidines enter the general body pool and are degraded to water, carbon dioxide, urea, and other metabolites and thus, by their entrance into the general body pools, may be recycled into macromolecules, degraded, and excreted.

HUMAN TRITIUM METABOLISM

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Tritium in 5-Position of Cytosine in DNA There is some special concern regarding tritium in the 5-position of cytosine in DNA, since a t this site tritium decays were found to produce more mutations than in other positions in the DNA. Only 0.02 of the DNA hydrogen is located in the 5-position of the cytosine ring. The average mammalian cell nucleus contains about 0.3 organic molecules and 0.7 water, and, in d,about 0.02 DNA. From these fractions it can be calculated that about 0.00015 of the total hydrogen of the cell nucleus is in the 5-position of the cytosine ring. At the present time there is a tritium concentration of about 3.2 x pCi per cm%f terrestrial water in the United States. If one assumes that tissue will contain the same concentration a t equilibrium, then one can calculate the number of tritium decays per cm3 of tissue per year. 3.2 x pCi per cm3 x 2.2 x lo6 decays per minute per pCi x 5.3 x lo5minutes per year = 37.3 x lo2 decays per cm3per year. Since the reference nucleus (see Section 4) has a volume of 270 x 10-l2 cm3. there will be 37.3 x lo2 x 270 x 10-l2 = 1.0 x lop6decays per reference nucleus per year. With 0.00015 of the total nuclear hydrogen being in the 5 position of the cytosine ring, there will be 1 x x 1.5 x lop4 = 1.5 x lo-'' decays and probably mutations per year a t this position. This is a comparatively negligible risk in view of the number of mutations produced per reference nucleus per year from background radiation. per nucleus per year This is estimated to amount to about from a whole body exposure to 150 rnrem. In summary, the plants and animals that may develop tritium specific activities greater than those in environmental water derive most of their water across liquid-liquid boundaries and transpire a significant amount of water back into the atmosphere. In most organisms, however, the kinetics of water and food intake, metabolism, and excretion result in little, if any, enhancement of tritium specific activities over those in the environment. In mammals, including man, the specific activities of tritium in the body water or organic constituents are slightly lower than those in the environment, including drinking water and food. For a more complete discussion of the topic of environmental tritium, see NCRP Report No. 62 Tritium in the Environment (NCRP, 1979).

APPENDIX 111

The Quality Factor for Tritium Radiation The International Commission on Radiological Protection originally recommended a value of 1.7 for the quality factor (Q) of electrons with maximum energy (E,,,) less than 30 keV (ICRP, 1966). Today, ICRP Report 26 (ICRP, 1977) recommends Q = 1. The earlier Q of 1.7 was based upon the linear energy transfer (LET) and the experimental relative biological effectiveness (RBE) values of tritium, the only important nuclide providing beta emissions in this energy range. Later, in view of the uncertainties inherent in the RBE values and in the interest of simplicity, it was suggested that Q be reduced to unity (Bond and Feinendegen, 1966; Vennart, 1968; Dunster, 1969; NCRP, 1971).Recent data confirm this Q value, yet emphasize the importance of dose rate when different photon energies are used (Berry et al., 1973; Bond et al., 1978a). It is instructive to review the experimental evidence for the RBE of the tritium beta particle with emphasis on the causes of uncertainties involved.

LET of Tritium Beta Radiation The values of Q recommended by the ICRP (1966) are related to the LET or stopping power. The initial LET of an average 3H beta particle (E., = 5.7 keV) is about 3.5 keV per micron in water. The LET increases thereafter as the electron loses energy so that an average LET over the whole track has been estimated to be about 5.5 keV per micron. This estimate does not consider any possible redundancy in the track, however. The effective track width will depend upon the sizes of biological targets. Each time an electron track doubles back upon itself and takes a second pass through a biological target, it increases the LET. In the extreme case the track will be entirely redundant and the effective track length will be equal to the range of the electron. The mean range of a 5.7 keV electron in water is about 0.68 pm. If the energy were distributed evenly over this distance, the LET would be about 8.25 keV per pm in water. What has been said here for the % beta particle is equally applicable to any electron of 56

LET OF TRITIUM

the energy of the "-beta, of its production.

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irrespective of its origin or the mechanism

Uncertainties in RBE Calculations The experimental RBE is calculated as the ratio of doses of radiations of different qualities that are required to produce the same biological effect. Dose of reference radiation RBE = Dose of radiation from tritium I t is to be expected that the value of RBE will vary somewhat from one biological endpoint to another, but what is far more troublesome is the fact that for any given endpoint there are many uncertainties inherent in the calculation of doses. Some of these are outlined below. Reference Dose: Tritium Dose:

(1)Gamma rays vs. x rays (2) Dose rate (1)Mean energy (2) Effective half-life (3) Distribution of 3H

Reference Dose (1) Most experimental values of the RBE for *Co gamma rays relative to 250 kVp x rays are close to 0.85 when the dose rate is more than 50 rad per minute (Sinclair, 1962). With low dose-rate gamma radiation a t 46 rad per hour, the RBE relative to 220-250 kVp x rays a t 50-65 rad per minute was reported to range between 0.63 and 0.70, whereas a t still lower dose rates of 1.5 rad per hour, the RBE ranged from 0.29 to 0.61 (Dewey et al., 1965).Thus, the RBE of beta radiation from tritium can be changed by a factor of approximately 2 depending upon whether gamma rays or x rays are used for the reference radiation a t higher dose rates. The reason for these differences Lies in the different energies of the Compton electrons produced by x and gamma rays. In fact, a careful analysis of the energy deposition per critical volume of 1pm diameter, showed that the 3H-beta particle carries the same quality as about 6080 kVp x rays (Booz, 1978) and the energy imparted per critical volume became less as the photon energy was increased (Bond, 1978a). Since, in the low dose region, single events per critical volume occur, it is the ratio of imparted energy from single events per critical volume from the 3H decay and from the reference radiation that is likely to determine the RBE (Bond, 1978b). Thus, it is to be expected that at low dose and at low dose-rates the RBE of the 3Hbeta particle changes

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with the energy of the reference radiation and with the size of the critical volume. The higher the dose-rate, the higher are the multiple events per critical volume per unit time, and the more equal become the effects from different energy x and gamma rays. Also, the larger the critical volume (i.e., from 1-5 pn in diameter) the more reduced become the difference between energies imparted to that volume from different photon energies and from the 3H beta (Bond, 1978b). It can then be stated that the RBE of the 3H beta particle tends to be 1with the reference being that energy of x rays that imparts to the reference nucleus the same energy per single absorption event as that absorbed from the 3H beta. (2) Dose rate. For proper determination of RBE, the dose rates of % and reference radiation should be comparable. Ideally, the reference radiation should be given a t an exponentially decreasing dose rate corresponding to the turnover of tritiated water, if the latter is used as the 3H carrier. Even if both 3H and reference radiation are given a t the same rate, the experimental RBE can vary depending upon the dose rates of both radiations. Berry et al. (1973) have investigated in Vicia faba the effects of dose rate on RBE of 3H relative to x rays and to gamma radiation and found that the reproducibility for the two reference radiations appeared to be considerably less in low dose-rate versus high dose-rate exposures. Hall et al. (1967) have shown a 2-fold increase in RBE of tritium relative to gamma rays in growth suppression and killing of HeLa cells when the dose rate was decreased from about 32 rad per hour to 1rad per hour. Yet more detailed information from Berry et al. (1973) on the RBE of gamma versus x rays showed a change from high to low dose-rates by a factor of more than 2. Tritium Dose (1) Mean energy. The generally accepted E,, for tritium beta particles is 5.7 keV (Lederer et al., 1968), although values of 5.5 and 6.0 keV have been used for dosimetric calculations. This range can cause up to a 5 percent variation in the calculated RBE. (2) Effective half-life. In experiments where the dose is accumulated from a single injection of tritiated water, the effective half-life of tritiated water is an important determinant. This situation is complicated by the fact that in toxicity studies the water balance is altered by the radiation sickness of the animals, the half-time of 3H increasing with increasing dose (Furchner, 1957). Estimates of effective half-lives of 'H administered to the mouse as tritiated water and measured by different techniques give values in the range of 1.75 to 2.3 days for the fast component (Furchner, 1957; Brues et al., 1952; Lambert and Clifton, 1967).

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(3) Distribution of aH. Distribution factors are vague enough when 3H is distributed in water, but they become far more complex for tritium bound to other metabolites. In the case of tritiated thymidine, for example, tritium is incorporated only into nuclei of cells in the Sphase of the cell cycle, and then is diluted by cell division at different rates in different tissues (Bond and Feinendegen, 1966; Johnson, 1970). Because of their much greater simplicity, only estimates of RBE based upon tritiated water will be considered here. The central problem in calculating the dose from tritiated water usually amounts to the following: Given a certain average whole-body dose or given a certain level of radioactivity in body water, what is the dose absorbed by critical cells and their nuclei? Dose calculations are customarily based upon the water content of soft tissues, which is taken to be 0.70, 0.75, or 0.80. This, in itself, gives an additional arbitrary variation in the calculated RBE, but in principle it is not correct to use the average tissue water content for calculation of dose to cell nuclei. Since the range of one-half of tritium beta particles is less than 0.68 pm (Berger, 1971),the dose to cells is due largely to that from tritium in intracellular water. The important factor, then, is not the concentration of water in tisslles, but the concentration within the cells of interest, or, perhaps more important, the concentration within their nuclei. The concentration of water within cells may be quite different from that in extracellular tissue. A considerable part of tissue water is interstitial so that one might expect the concentration of water in the cell to be less than that of the whole tissue. This would tend to lower the calculated dose to the cell and would tend to raise the calculated RBE above those based upon tissue water. The water content of the cell is difficult to measure and possibly differs from one cell type to another. By comparing the wet weight of isolated single neurons with the dry mass as measured by x-ray microradiography, Hyden estimates that the neuron is about 75 percent water (Hyden, 1960). Using a different technique and a different cell type, Harrison (1953) finds a smaller water content. From desiccation and analysis of bulk tissue he estimates the liver cell of the rat to be only 60 percent water. It is not hard to imagine sources of error in either of these methods, so that these figures are only approximate. As for the distribution of water between nucleus and cytoplasm, x-ray absorbance studies show no significant difference in dry mass per volume in neurons that are simply fresh frozen and dried (Hyden, 1960). The data of Maurer and Prirnbsch (1964), which show a lesser dry mass per volume in the nucleus than in cytoplasm, are based upon cells that have been thoroughly leached out by fixation and embedding of the tissue. In the calculations that follow, it seems realistic to assume that nucleus and cytoplasm contain somewhere between 0.60

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APPENDIX I11

and 0.75 water and that the radioactivity per mass everywhere within the cell is 0.60 to 0.75 that of body water depending upon tissues and cells involved. It is important t o recognize that these limits are based upon only two cell types. It is conceivable that the nucleus of the small lymphocyte, for example, which is densely stained and has only 2/3 to 3/4 the volume of the hepatocyte nucleus, might have a water content of less than 60 percent. In experiments in which the radiation dose from tritium is estimated by measuring the radioactivity of body water, a significant error will result from not including the radiation dose from organically bound tritium. After administration of tritiated water, initially about 0.03 of the total body activity is tissue bound, and after 30 days when the tritium water has largely been excreted, 0.50-0.70 of the tritium remaining in the body is bound to exchangeable and non-exchangeable positions of tissue solids (Lambert and Clifton, 1967) (see also Appendix 11).

A Review o f RBE Experiments in Mammals (1)Mammalian cells in vitro. Hall et al. (1967) compared tritiated water and gamma radiation with respect t o the suppression of HeLa cell growth rate. The radiation was administered a t low dose rates, 0.5-3 rad per h, comparable to the dose rates in in vivo toxicity studies. Cells were considered to be 0.80 water, and the dose to the cell was thus considered to be 0.80 of the dose to the medium. This gave an RBE of about 2. If the water content of the cells is somewhere between 0.60 and 0.75, the calculated RBE should have been in the range of 2.1-2.7. Yet this value carries the uncertainty of dose rate effects, considering the relatively low effect of gamma rays at low dose rates compared to x rays. T h e RBE tends toward 1if x rays and not gamma rays were the reference radiation (Bond et al., 1978). ( 2 ) The Work of Brues et al. T h e first toxicity study on whole animals from which an RBE for tritiated water could be calculated was carried out by Brues et al. (1952). They measured the LD50~30of tritiated water for CF1 female mice and found it to be very nearly 1 mCi/g of body weight when given in a single injection. They also measured the water turnover in these animals, and determined a rate constant of 0.4 per day for the first 4 days (Tlp = 1.7 days) and 0.64 per day thereafter = 1.1 days). T h e accumulated dose was calculated to be 678 rad. This is the average whole-body dose from a single injection of 1 mCi per g body weight of tritiated water. Yet,

MAMMALIAN R B E EXPERIMENTS

/

61

there is considerable inhomogeneity of water distribution in fat and bone on one hand and in parenchymal soft tissue organs on the other. This tends to increase the dose to the critical tissues such as bone marrow and the gastrointestinal tract. If one assumes, as the authors did, that their mice were on the average 0.75 water, then the cells that were 0.75 water received the same dose, or 678 rad. For cells with 0.60 water, the total dose would be 543 rad. At that time, Brues had no reference value for the LDs/m of x or gamma radiation delivered at an exponentially declining rate. Furchner (1957) determined 1350 rad as the LD%/mfor @"'o radiation given a t an exponentially decreasing rate. This results in a n RBE based on gamma radiation of somewhere between 2 and 2.5. Inhomogeneous distribution of tritiated water in the body would lower the RBE by a factor of perhaps 0.7, and the lower effectiveness of gamma radiation a t low dose rates further decreases the RBE toward 1.0 for the reference x radiation. (3) The Work of Storer et al. In the experiments of Storer et al. (1957), nearly constant levels of tritiated water were maintained in body water for 5 days. Three endpoints were used: Atrophy of mouse spleen and thymus and depression of 59Feuptake in red cells of rats. Initial body water concentrations were established by intraperitoneal injection of tritiated water. Urine samples, considered representative of body water, were monitored daily. The dose to soft tissues was calculated by assuming these tissues to be 0.75 water, so that the activity measured in body water was multiplied by 0.75 to obtain the activity in the tissues. The authors used E,, = 6 keV, which tends to increase the calculated dose. Also, their measurements of radioactivity in body water did not record some 5 percent of organically bound tritium. In the mouse experiments the RBE of tritium relative to radium gamma radiation was calculated to be: Splenic atrophy: Thymic atrophy:

RBE = 1.32 RBE = 1.52

The 0.05 corrections for average energy and organically bound tritium more or less cancel out. If the uncertainty in intracellular water (0.600.75) is considered, the RBE values vary as follows: Splenic atrophy: Thymic atrophy:

RBE RBE

= =

1.32 - 1.63 1.52 - 1.90

The RBE of 250 kVp x rays and O ' Co gamma rays relative to radium gamma rays was unity at high doses in both of these systems. For depression of 59Feuptake by red cells in rats, Storer et al. (1957)

62

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APPENDIX 111

obtained an RBE of 1.64 relative to '%o gamma rays. Allowing for the uncertainty in cell water, the RBE is in the range of 1.64 - 2.05. The lower effectiveness of gamma rays a t low dose-rates tends to reduce the above RBE values toward 1 for the reference x radiation. (4) The work of Furchner (1957) determined the RBE of tritium beta radiation by using the 30-day mortality of CFI mice after a single intraperitoneal injection of tritiated water. The reference radiation was 60Co radiation given a t a n exponentially decreasing rate. The effective half-life of tritium, in animals receiving an LDmpo dose, was about 50 hours. Here the E,, was taken to be 6 keV. Also, the dose rates were calculated from the activity of body water (blood in this case) and did not take into account organically bound tritium. This calculation leads to an underestimation of dose that may be compensated for by the overestimation due to the use of 6 keV as the mean energy. The whole-body doses for 0.50 mortality were 804 rad from tritium and 1350 rad from gamma radiation. The 804 rad represents a whole-body dose. The local dose depends again upon the local concentration of tritium in tissue water as compared with the whole-body water content. If the whole body is 0.75 water, then cells containing 0.75 water wdl also absorb 804 rad. Cells containing only 0.60 water will receive only 640 rad. This yields an RBE range, depending upon intracellular water, of 1.7-2.1 relative to gamma rays. However, considering inhomogeneity of tritium distribution in total water with increased radiation to soft parenchymal tissue sites, and the lower effectiveness of gamma rays a t low dose rates, the RBE tends toward 1 for the reference x radiation. (5) The work of Lambert (1969) has compared the effectiveness of tritium and x rays with regard to the killing or injuring of spermatogonia in the mouse testis. Lambert found that, 72 hours after the intraperitoneal injection of 20 pCi/g of body weight of tritiated water, the population of resting primary spermatocytes was reduced by 0.27, equivalent to the effect of 30 rad of 200 kVp x rays. The latter was given at an exponentially decreasing dose rate over a 72-hour period. The tissue activity of the testis was measured directly and the integrated dose was calculated. This method accounted for bound tritium as well as for that in water. The radiation dose to the tissue from tritium was calculated to be 12.2 rad, resulting in an RBE of 30/12.2 = 2.4. Because of the uncertainty of tritium distribution in the intracellular and extracellular space, however, the calculated tissue dose may be too low compared to the dose to the cell nuclei. Moreover, 250 kVp x rays have a lower effectiveness at low dose-rate compared to lower energy x rays; thus, the RBE tends to be substantially reduced when the reference radiation is 80 kVp x rays.

MAMMALIAN RBE EXPERIMENTS

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63

A similar experiment was carried out using tritiated thymidine (Lambert, 1969). The dosimetry was based on autoradiographic grain counting. This probably gives a less accurate evaluation of the nuclear dose than does the dosimetry of tritiated water. This experiment gave an RBE of 1.6. Again, the dose-rate effects lead to a reduction of RBE for the reference 80 kVp x radiation. Dobson and Cooper (1974) and Dobson and Kwan (1976) exposed adult female mice to tritium water throughout the period of pregnancy and lactation. The female offspring were sacrificed at day 14 after delivery and the oocytes were counted and compared to control. Doses to the oocytes from the tritium decays were calculated on the basis of the radioassay of the urine, and the urine activity was taken to be representative of tritium in body water. Cobalt-60 gamma rays were used as reference with a dose rate comparatively low as was that from tritium. At the very low dose region below 30 rad there was an apparent increase in the 3H beta RBE from about 1.7 at about 30 rad to about 2 or more, depending on the linear extrapolation of the non-linear portion of the dose-response curve, from the gamma irradiation. Yet an analysis of the dose-response curve from 3H and from gamma rays indicated that the increase of RBE of 3H in this experiment can be ascribed to a reduced effectiveness of the gamma rays in the low dose region (Bond, 1978a). This is in concurrence with the observations cited previously. A Quality Factor for Tritium

The ranges of uncertainty for the various experimental RBE values are obvious. However, taking into account the necessity of carefully choosing the reference radiation, it may be concluded that there is ample evidence to ascribe to the 3H beta an RBE of 1 provided the reference radiation is in the order of 60-80 kVp x rays.

APPENDIX IV

The Toxicity of Tritiated Water ( 3 ~ 0 ~ ) The Problem There has been considerable apprehension that tritium constitutes a unique hazard because a small amount of tritium introduced as tritiated water will eventually, in part, find its way through metabolic pathways into newly synthesized DNA. Accordingly, it is necessary to review the knowledge on the toxicity of tritium to put the entire picture into perspective and to 'see if there is any unique hazard from the incorporation of tritium into DNA. For the metabolic pathways and degree of incorporation of tritium into tissue, see Appendix 11. Several studies have been performed in which deleterious effects of radiation from tritium were observed. Brues et al. (1952) have shown that the LD50/30 dose of %OH administered to mice in a single injection was approximately 1 mCi/g body weight. This dose delivered about 700 rad total body radiation. Furchner (1957) also determined the toxicity of tritium in mice and obtained comparable results. TrujiUo et al. (1955) studied the toxicity of tritium as gas in mice. Again, the toxicity was comparable to that observed for "OH. Cahill and Yuile (1970) maintained pregnant rats throughout pregnancy at constant ~ from 1-100 pCi/ml body water, body activities of 3 H O ranging delivering 0.3-40 rad/day. At 1 pC/ml there were small but statistically significant effects. As the tritium concentration was increased, the incidence of effects on the fetus also increased. Hatch et al. (1970) studied kangaroo rats living in a tritiated environment around the crater of a nuclear detonation in Nevada. The tritium activity in body water ranged from 0.1-0.4 pCi/g body weight. No effects were observed over the period of time the animals were studied. Larnbert (1969) studied the effect of injection of 3HOH upon spermatogonia in the mouse. Twenty pCi/g body weight of aHOH approximated the effect of 30 rad of external 200 keV x rays. There have been two cases of death by destruction of bone marrow

cells in human beings after incorporation of tritium. The details of these cases have been presented by Seelentag (1973). In this instance, tritium was introduced into the bodies as a result of working with tritiated luminous compounds in the watch industry. In each instance, in addition to the tritium, the individuals had also incorporated small amounts of radium, thorium, and strontium-90 from earlier work in the watch industry. The estimated doses over several years of work were many thousands of rem. There are other ample examples of the harmful effect of tritium from 3HOH and other tritiated compounds. These have been reviewed extensively by Bond and Feinendegen (1966) and Cronkite et al. (1973). Many studies with animals that had been given tritium over long periods revealed no deleterious effects. When rats were given 86 pCi/ g body weight in a single injection, no harmful effects were observed during 9 months (Thompson and B d o u , 1954). With tritium given to young rats a t a concentration of 4-5 pCi/ml in drinking water, beginning in early life and continuing for 6 months, no harmful effects were seen. No visible effects were produced in rats exposed to similar levels from conception throughout life also produced (Pinson, 1952b; Thompson and B d o u , 1956). The concentration of tritium in body water was approximately 3 pCi/ml. Since 1 pCi/ml delivers 0.3 rad/day, this dose delivered 0.9 rad/day plus the dose from the amount of tritium that was incorporated into the organic constituents of tissue. Baserga and Lisco (1966) followed mice for 2 years after giving newborn animals 15 pCi 3HOH/g body weight and observed no harmful effects. The amount of tritium incorporated into the DNA of mice chronically exposed to tritium in their drinking water was measured for seven different organs. The specific activity of tritium permanently incorporated into DNA averaged about 0.73 that of tritium in tissue water for spleen, kidney, thymus, liver, and bone marrow and was only slightly higher in testes (0.85) and ovaries (1.07). Too little DNA was isolated from ovaries to establish the specific activity accurately enough to determine whether it is r e d y higher than that of tissue water. Therefore, these results give no evidence for significant isotope discrimination in favor of tritium incorporation into DNA in any tissue and suggest in fact significant isotope discrimination against tritium in most tissues (Commerford et al., 1977). Calculations based on the measured tritium content of DNA, histone, and other chromosomal components indicate that during chronic exposure to tritiated water the radiation dose due to tritium incorporated into the chromosomes is trivial in comparison to the dose due to tritium in the water in the cell nucleus. Chromosomal tritium makes

66

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APPENDIX IV

a significant contribution to total radiation dose after acute exposure in those cells with a long life span which proliferated at the time of exposure. The tritium content of hemoglobin and glycogen isolated from these mice was also measured in order to determine the tritium content of metabolic precursors of DNA. These values, taken together with known species differences in patterns of DNA synthesis, suggest that humans similarly exposed to tritiated water would incorporate less tritium into their DNA than mice. These results have been published in detail (Commerford et al., 1977). The preceding observations deserve some discussion in the context of the concept of a reference nucleus (see Sections 4 and 5.4). The studies of Brues et al. (1952) on LDmfrom 3HOH pose a situation in which the tritium would be randomly distributed in tissue. Under this condition, the dose per 'H decay in the reference nucleus is 0.34 rad. Thus, an LDS0of 700 rad in the mouse corresponds to 700/0.34 = 2059 3H disintegrations per volume of reference nucleus during the 30-day period of observation. Cahill and Yuile (1970) detected small but statistically significant biological effects in the fetus when the plasma concentration of the mother was maintained a t 1 pCi/ml. A concentration of 1 pCi/ml corresponds to 0.86 disintegration/day/reference nuclear volume. Hatch et al. (1970) detected no biological effects in rats living in a tritiated environment with a concentration in body water up to 0.4 pCi/g body weight. This yields about 0.32 3H decays per reference nuclear volume per day. The failure to detect biological effects when the number of disintegrations per reference nuclear volume is between an average of 0.32 disintegration/day and 0.86 decays/day is probably statistical and does not prove a threshold. The existence of inhomogeneities of dose a t the cellular level is compounded by stochastic considerations. When the concentration of 3H is sufficiently low so that the reference nuclear volume receives on an average only a few disintegrations over several hours or days, the frequency of decays in a reference nuclear volume follows the Poisson distribution:

P.

rnnevm n!

=-

(1)

where n = number of disintegrations m = average number of disintegrations/day in a nuclear volume P, = fraction of nuclear volumes receiving n number of disintegrations. Table 4 lists the number of disintegrations for the two situations

HTO TOXICITY

/

67

TABLE4-The bwlogic response in rat fetuses to infrequent tritium disintegrationPh No Effect m

n o 1 2 3 4

0.32/day

Pn 0.726 0.232 0.037 0.004 0.0003

Detectable Effect

m = O.RG/dsy

P. 0.42 0.364 0.155 0.045 0.009

" Number of disintegrations per nuclear volume for the situation where no biologic effect has been detected (average of 0.32 disintegration per day) and where a barely detectable effect was observed (average of 0.86 disintegration per day) in rat fetuses whose mothers were maintained on =HOH(see Cahill and Yuile, 1970). See Equation (1)

described above where the average disintegrations wefe 0.32 and 0.86 per day, respectively. Perusal of Table 4 shows that there is no detectable effect so far as the particular endpoint is concerned when 23.2 percent of the reference nuclear volumes receive 1decay/day and 3.7 percent receive 2 decays/day. However, when 15.5 percent are receiving 2 decays/day and 4.5 percent are receiving 3 decays/day, biological effects are observed. This suggests, but does not prove, that the biological effects are either nonexistent or only become detectable when there are multiple disintegrations in a respectable fraction of cells a t risk per day. I t is believed that when the number cf decays is spread out in time the probability of repair is increased. Single decays wdl produce mainly single strand DNA breaks for which probability of repair is great. The spatial distribution of decays at low decay rates is such that the probability of the same volume of DNA being hit twice in a short period of time is close to zero. It has been shown in various biological systems that lowering dose rates to only one decay or radiation absorption event per nuclear volume substantially reduces the biological effect per unit dose absorbed (see Appendix VII).

Carcinogenesis of Tritiated Water No evidence is yet available on carcinogenesis from tritiated water. Yet, Mewissen and Rust (1973) observed an increase in the incidence of tumors after 0.3-1.5 yCi/g of body weight of 3H-thymidine was administered to newborn male and female mice. Johnson and Cronkite (1967) failed to find an effect of 3H-thymidine on tumor incidence or longevity when it was administered to other strains of weanling animals and in similar and different amounts. It is likely that the carcinogenesis

68

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APPENDIX IV

observed in the studies of Mewissen and Rust (1973) was brought about by the selective exposure to relatively high doses of critical cell nuclei having incorporated 3H-thymidine. The average dose rates to the labeled cells may have initially ranged between 0.2-1 decay/h and for most heavily labeled cells to 4 decay& i.e., to 25 rad per day (see Appendix V and Section 4).

Conclusion It appears unlikely that tritium in the form of water in the environment from natural sources and from nuclear installations at presently projected levels would ever produce deleterious, detectable effects. This is not to imply that tritium beta irradiation in sufficient amounts would not be carcinogenic or mutagenic.

APPENDIX V

METABOLISM OF DNA, RNA, AND THEIR PRECURSORS The genetic material (deoxyribonucleic acid, DNA) of any living cell determines the cell's specific functions in tissues and the characteristics of that cell's descendants. If the cells are part of the germ line (e.g., spermatocytes, oogonia), then the DNA determines the characteristics of the offspring. Except for specific situations, DNA is considered to be metabolically stable. Radionuclides in tissues will irradiate the DNA within range of the emitted radiation and, if the nuclide is in DNA itself, the transmutation associated with decay may also have some effects (see Appendix VII). Transmutation effects in DNA may produce changes in molecular structure at or near the site of decay and add to the molecular alterations produced by the radiation effects. In establishing permissible levels of radionuclides that may be incorporated into DNA, the factors to be considered include the chemical form in which the radionuclide enters the DNA, i.e., the type of isotopically labeled nucleic acid precursor, its biological role and half-life, pathways of ingestion and metabolism, sites of incorporation into cells and tissues, proportion of cells that will incorporate the labeled compound, and the biological roles of the affected cells and tissues. In the metabolism of nucleic acid precursors there may be unique radioactive metabolites excreted (e.g., beta-amino-isobutyric acid from thymidine). In principle, a useful approach might be to establish the source of the tritium by investigating the labeled metabolites in the urine. It might be possible by this method to also recognize specific metabolic pathways of the tritiated precursor. This problem is, however, beyond the scope of this report.

RNA and DNA Distribution in Cells Most nucleic acid precursors contribute to both DNA and ribonucleic acid (RNA) (Davidson, 1965). DNA in mammalian cells is almost 69

70

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APPENDIX V

entirely stable and located in the cell nucleus, where it controls the many specific cellular structures and functions. In contrast, RNA, in the form of various molecular species, is distributed throughout the cell as ribosomal RNA, transfer RNA, and messenger RNA. The various RNA fractions have defined turnover rates. Nearly all RNA is synthesized in cell nucleus by use of DNA as template (Du Praw, 1970). The nucleolus is particularly rich in RNA. A large fraction of the newly synthesized RNA then leaves the nucleus for the cytoplasm where it translates the genetic message from DNA into proteins and finally into all cellular specific structures and functions (Watson, 1965), and part of the RNA may remain in the nucleus. RNA turnover supplies precursors again for DNA synthesis. Cells Synthesizing RNA and DNA

Any cell that divides duplicates its genetic material in order to deliver a complete set of genetic information to the daughter cells. The limited time interval between two consecutive divisions, during which the cell synthesizes DNA prior to cell divisions, is called S-phase (for DNA synthetic phase) of the cell cycle. The time from cell division to the beginning of S-phase is called GI-phase,and between the end of Sphase and cell division lies Gz-phase (see Figure 2) (Howard and Pelc, 1953). RNA is synthesized throughout most of the cell cycle. However, little or no precursors are incorporated into RNA during cell division.

C E L L C Y C L E PHASES

lrr terpliase

GI

- Phdse

S - Phase G 2 - Phase

M~tosls

Fig. 2. T h e cell cycle indicates that only during a particular limited period of time the genetic material is duplicated prior to the next cell division. This period is called the S-phase. Between the S-phase and the next cell division lies the Gn-phase. The GI-phase denotes the time interval between the fmt division and the S-phase. NormaUy, cells are distributed at random throughout the various phases of the cell cycle so that the cells of a particular population do not pass through the cycle in synchrony.

CELLS SYNTHESIZING RNA

& DNA

/

71

TABLE 5-Distribulion of labeled 5-iodo-2-deoxyuridine(IUDR) in the whole body of the mouse (in percent of whole body DNA labeling) Organ

Percent Label~ng

Gastrointestinal tract bone m m o w skin spleen lymph nodes thymus liver muscle kidney lungs testes pancreas brain Because IUDR is specifically incorporated into DNA-synthesizing cells, the precursor distribution signals the distribution of DNA-synthesizing cells between the organs, in percent of all DNA-synthesizing cells in the whole body of the mouse. Adapted from: Hughes et al. (1964) Heiniger el al. (1970)

The rate of RNA synthesis is highest during Gz-phase (Mitchison, 1971). Nearly all cells in the body synthesize at least some RNA, whereas DNA is synthesized nearly exclusively in cells preparing for division. Application of a precursor for RNA leads, therefore, to the distribution of this precursor to nearly all cells including DNA-synthesizing cells, whereas specific precursors for DNA are incorporated only into those cells that are in DNA synthesis. RNA and DNA precursors that are not taken up by cells within about 1 hour after entering the blood circulation are almost totally degraded. Most degradation products other than water are excreted within hours, mainly with the urine. The DNA-synthesizing cells are located mainly in tissues with relatively rapid cell renewal. The gastrointestinal tract contains approximately 0.50 of all the DNA-synthesizing cells in the mammalian body, and about 0.10-0.20 are located in the bone marrow. Other proliferating ceUs are found in the lymphopoietic tissues, skin, male gonads, and parenchymal organs (see Table 5). Adult man has a total bone marrow cell mass of 1.3 kg-1.5 kg (ICRP, 1975), i.e., about 1.3 x 1012 cells. About 0.35 of bone marrow cells are differentiated. If the average generation time is 20 hours, .DNA of an individual proliferating cell will be synthesized approximately once a day (Stryckmans et al., 1966). Therefore, 1.3 x 0.35 x 1 0 ' ~= about 0.5 x 10" ceUs in the bone marrow are estimated to synthesize DNA once per day. The diploid

72

/

APPENDIX V

cell contains about 6 x lo-" g DNA which is doubled prior to cell division (Davidson, 1965). Hence, 0.5 x loL2x 6 X lo-" = 3 g of DNA are synthesized per day in the bone marrow, and close to 20 g of DNA may be synthesized per day in the entire body.

RNA and DNA Precursors There are many compounds that may eventually be linked into RNA and DNA. If molecules are incorporated into nucleic acids after a few biochemical steps, they are considered immediate nucleic acid precursors. They may be taken up by cells and accepted into the metabolic chains leading to DNA and/or RNA. Of these compounds, the pyrimidine ribosides and deoxyribosides (also called pyrimidine nucleosides) cytidine, uridine, deoxycytidine, deoxyuridine and thymidine easily cross the cellular membrane, in contrast to phosphorylated compounds. The purine bases, adenine and guanine and also purine ribosides, adenosine and guanosine are accepted as precursors from the extracellular space. T h e structural formulas of these immediate nucleic acid precursors are shown in Figure 3. These compounds have a relatively small intracellular pool compared to other compounds that may potentially enter many different pathways and hence may be metabolized extensively. T o this latter group of compounds belong, for example, formate, certain amino acids, and molecules donating methyl groups. These molecules have large pools and are distributed with relatively little or no specificity throughout the cells and the body. This report considers the immediate nucleic acid precursors that may be incorporated by cells and are not diluted by more nonspecific metabolites, such as formate or phosphates, and thus constitute, if they are labeled by radionuclides, a greater potential hazard.

Metabolic Pathways of RNA and DNA Precursors The metabolic pathways of the immediate nucleic acid precursors incorporated from the extracellular space are schematically summarized in Figure 4 (Davidson, 1965). The pyrimidine nucleosides are cytidine (C), uridine (U), deoxycytidine (dC), deoxyuridine (dU), and thymidine (dT). Prior to incorporation into RNA or DNA, they are phosphorylated inside the cell to mono-, di-, and triphosphates. Phosphorylated nucleosides are called nucleotides. It is noteworthy that dT, dU, and dC are specifically incorporated into DNA and not into RNA, whereas C and U are both finally linked to RNA and DNA. The

PRECURSOR METABOLIC PATHWAYS

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73

0

NH2

Orotic Acid I

PYRIMIDINE RIBOSIDES A N D DEOXYRIBOSIDES

Deoxvcvtidine

I1

Adenine

Deoxvuridine

PURINE BASES

Thymidine

Ill

Guanine

PURINE RlBOSlDES

Admmine

Gunnosins

Fig. 3. The immediate nucleic acid precursors are listed. Orotic acid is included, because it is an intermediate product in intracellular pyrimidine synthesis. Decarboxylation of orotic acid produces uracil, which is the pyrimidine base of uridine.

purine bases adenine (A) and guanine (G) enter, inside the cell, linkages to ribose phosphates to form purine nucleotides. Both purine nucleotides are finally incorporated into RNA and DNA as well. Reduction of the RNA precursors, i.e., conversion of the ribose moiety to deoxyribose, and hence of RNA precursors to DNA precursors, occurs at the diphosphate level, for both purine and pyrimidine nucleotides. For RNA synthesis there are finally available cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and adenosine triphosphate (ATP). The four corresponding nucleotides for

74

/

APPENDIX V

CMP

UMP

4I

C

CDP

I I

II

UDP-dUDP*

C

T

li

P UTP

dUTP

dTTP

Lp J J i GTP

ATP

dATP

dGTP

dGDP

JI

GMP 1 - - - - - - - AMP

T

GUANOSINE5:; h

Fig. 4.

T

ADENOSINE h

Metabolic interconversions in the nucleic acid precursor pool.

DNA are deoxycytidine triphosphate (dCTP), thymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), and deoxyguanosine triphosphate (dGTP). Pyrophosphates are formed as byproducts of the polymerization of these nucleotides to nucleic acids. The structural formula of a single chain of RNA is shown schematically in Figure 5. I t is recognized that two individual neighboring nucleotides are linked together by one atom of phosphorus between position 3 and 5 of adjacent ribose moieties; correspondingly, in DNA the phosphorus is bound between position 3 and 5 of two neighboring deoxyribose moieties. Two complementary strands of RNA and DNA are held together by hydrogen bridges between the bases; uracil opposing adenine, and cytosine opposing guanine in RNA. In DNA, the bridges are formed between thymine and adenine, and between cytosine and guanine, and form a helical structure, shown schematically in Figure 6.

/

PRECURSOR METABOLIC PATHWAYS

75

Thymidine Thymidine (see Figures 3 and 7) is specifically incorporated into DNA. It may be labeled with 3H in the nonexchangeable H positions a t carbon-6, or in the methyl group attached to carbon-5. I t may be also labeled with 14C,and it is commercially supplied with I4Cin the 2 position. It is most widely used to specifically measure the kinetics of cellular proliferation (Cleaver, 1967). In the adult organism, about 0.10-0.20 of the total thymidine incorporated is located in the bone marrow, about 0.50 in the gastrointestinal tract, and the rest is distributed mainly among lymphopoietic tissue, skin, and DNA-synthesizing cells in the parenchymal organs and muscle (see Table 5). In the embryo, depending on the state of development, thymidine may be incorporated into all cells of the body (Leblond and Walker, 1956; Fliedner et al., 1971). With DNA synthesis of about 20 g per day in the adult, there is a requirement of about 4 g of thymidine per day. The body provides this amount by de novo synthesis and by reutilization of thymidine from dead cells. In mice, about 0.30 of intravenously injected W-thymidine is incorporated into the DNA of the entire body (Feinendegen et al., 1973). There may be species-specific differences. In rats, this value was

O-P-O-

I 0 RlBONUCLElC ACID (RNA) (Partial Formula)

I

~

o

p

-

H II

O -

I 00

Fig. 5. Structural formula of a portion of one strand of RNA.

-

76

/

APPENDIX V

S-A== =T-S P \ S-Ga=z==;C-

'

P = phosphorus S = deoxyribose A = Adenine T =Thymine G = Guanine C Cytosine

-

Fig. 6.

Model of a double-stranded DNA.

' C H =~ z.oA

'I= 2 . 1 5 A

THYMIDINE IODODEOXYURIDINE Fig. 7. The structural formulas of thymidine and iodoeoxyuridine indicate thc analogy of the two molecules.

THYMIDINE

/

77

reported to be approximately 0.15 (Lambert and Clifton, 1968). In the human, about 0.50 incorporation and 0.50 catabolism to tritiated water has been estimated (Rubini et al., 1960). Thymidine may also enter the body by mouth. Thus, if 3H-thymidine is ingested by rats, approximately 0.13 of the activity appears to be absorbed by the gastrointestinal tract (Vennart, 1969) and about 0.02-0.08 percent may be incorporated into whole body DNA in rats and mice (Vennart, 1969; Wade and Shaw, 1970; Friedrich, 1974).Thus, ingestion of labeled thymidine leads to DNA labeling that is about 0.20 of that observed following i.v. injection (see Table 6). In mice, as much as 0.60 of the ingested 3Hthymidine is catabolized to yield tritiated water (Wade and Shaw, 1970). After intravenous injection of 1yCi of 3H-thymidine per gram body weight in rats, about 0.30 of the bone marrow cells become labeled and it was calculated that the average labeled bone marrow cell had 1.2 (range from 0.3 to 4.8) tritium disintegrations per hour (Bond and Feinendegen, 1966).A value of 0.6 tritium disintegrations per hour per yCi injected intravenously was obtained from mouse bone marrow (Feinendegen and Cronkite, 1977) and an average value of 1.5-2.0 was calculated from human bone marrow cell data (Cronkite et al., 1962). ad., 1962). Thymidine is rapidly cleared from the blood and appears in DNA (Cleaver, 1967). As early as 30 seconds after intravenous injection of 3 H-thymidine in mice, a few labeled bone marrow cell nuclei were observed (Feinendegen and Bond, 1962). The half-time of disappearance of trace amounts of thymidine from the peripheral circulation was estimated to be 2 - 3 minutes (Hughes et al., 1964). A single intravenous injection of 3H-thymidine, therefore, leads to a short period of availability of the precursor, and the incorporation into DNA-synthesizing cells in the mammalian body is practically complete within less than an hour. Since the period of DNA synthesis is limited to a fraction of the time a cell needs to pass from one division to the next, labeled thymidine is incorporated only into those selected cells TABLE 6-Ratw of the amount of DNA.precursor incorporated into listed organs after ingestion and intravenous injection of the same amouni ofprecursor. Organ

intestine bone marrow spleen lymph node thymus average

.lH~,,hymidine

5-iodo-2'-deoxyundine

l'r'Il

0.21 f 0.04 0.17 f 0.03 0.17 + 0.03 0.20 0.05 0.24 0.06 0.20

+ +

0.15 0.02 0.12 0.04 0.29 -1- 0.03 0.17 0.06 0.38 0.13 0.22

+ + +

From: G. Friedrich; Thesis, University Duesseldorf, FRG,1974.

78

/

AF'PENDIX V

that happen to be in or nearly in DNA synthesis at the time of thymidine administration. Depending on the parameters of proliferation of different cell populations, the fraction of the cells labeled varies. Thus, in mice, about 0.80 of the nucleated red cell precursors, yet only approximately 0.10 of the stem cells in bone marrow, are labeled from one intravenous injection of thyrnidine (Vassort et al., 1973). The situation of the stem cells is discussed in Appendix VI.

Iododeoxyuridine Five-iodo-2'-deoxyuridine (IUdR),a thymidine analog, is specific for DNA synthesis (Hughes et al., 1964) and has found wide application in biomedical research. The structural formula is shown in Figure 7. The methyl group of thyrnidine, in the 5 position of the pyrimidine ring, is replaced by iodine, the atomic radius of which is similar to the radius of the methyl group. IUdR is distributed between cells in the body in a manner practically identical to thymidine (Feinendegen et al., 1973). Thus, as indicated in Table 5, about 0.10-0.20 of the activity is found in the bone marrow, about 0.50 in the digestive tract, and the rest is distributed mainly between lymphopoietic tissue, skin, and parenchymal organs. Little IUdR is incorporated into tissues such as muscle, liver, kidney, pancreas, lungs, and brain (Hughes et al., 1964). It was shown in mouse tissue in vivo and in vitro that the efficiency of incorporation of IUDR is about % that of thymidine (Feinendegen et al., 1973). Only about 0.05-0.10 of the amount of IUdR injected intravenously in mice is incorporated in the whole body. If IUdR is ingested in mice, approximately 0.01-0.02 of the precursor is absorbed by the digestive tract and enters the whole body DNA. Thus, the ratio of incorporation of precursor injected to ingested is about 5:l for IUdR, and the same ratio was found for thymidine (see Table 6).

Thym.idineReutilization and Turnover of Labeled DNA Incorporated thymidine and iododeoxyuridine remain bound to cellular DNA until the cell dies. Dead cells are digested. The degradation of DNA leads to the liberation of DNA subunits, nucleotides and nucleosides, which are partially reutilized for DNA synthesis in other cells. The majority of the precursors appear to be reincorporated in the form of nucleosides. Thus, thymidine appears as a physiological intermediate in the salvage pathway. Between 0.40 and 0.60 of the thymidine liberated from tissue DNA is reutilized (Feinendegen et al.,

THYMIDINE REUTILIZATION

/

79

1973). In the bone marrow of rats and mice, the thymidine reutilization amounts to about 0.40. Reincorporation of DNA breakdown products into DNA leads not only to a redistribution of label to such cells that did not incorporate the tracer during its initial availability, but also it causes a rate of turnover of DNA-bound tracer in the entire tissue that is slower than the rate of DNA turnover. In mice and rats, 3H-thymidine bound to DNA shows the first component of exponential turnover for a period of 6 - 9 days. In young rats, the initial turnover rates per day, after 3H-thymidine labeling, were 0.86 for the jejunum, 0.38 for the colon, 0.23 in thymus, and 0.46 in bone marrow (Steel and Lamerton, 1965). In adult mice, the initial turnover rates per day were found to be about 0.5 for the lining of the digestive tract, about 0.23 for thymus, about 0.14 for lymph nodes, and about 0.25 for bone marrow. Yet turnover rates of DNA with near exclusion of thymidine reutilization were calculated from parallel experiments with labeled IUdR, and amounted to about 0.44 per day for bone marrow and 0.71 for thymus (Feinendegen et al., 1973). Later than day 9 and up to at least 6 weeks after labeling, when the initially labeled differentiating cells had matured and disappeared, the turnover of whole body DNA as well as DNA in whole femora of adult mice labeled with trace amounts of lZ51UdRwas considerably slower with a rate of only about 0.03 per day (Kronenberger et al., 1976);yet the bone marrow flushed out of the mouse femur had a faster corresponding late turnover rate of DNA of about 0.09 per day, whereas the selective assay of stem cell turnover in the mouse yielded a turnover rate of 0.15 per day. If no toxic effect of the tracer dose of lZ51UdRis assumed, these data indicate that the average half-time of all cells a t steady state in bone marrow of the mouse is about 8 days and 4.5 days for the hematopoietic stem cell (Siegers et al., 1978).

Other Specific DNA Precursors Detailed data on the efficiency of incorporation in and on distribution between cells and tissue are not available for the specific DNA precursors, deoxycytidine and deoxyuridine. Circumstantial evidence suggests that these precursors are distributed throughout the body in a manner similar to that of thymidine and iododeoxyuridine. The relative amount of 3H-deoxycytidine incorporated into the long lived cell population in bone marrow, gastrointestinal tract, skin, thymus, spleen, and mesenteric lymph node varied from 0.29 (thymus) to 0.65 (lymph node) that of 3H-thymidineadministered in equirnolar amounts (see Table 7b).

80

/

APPENDIX V

RNA Precursors The immediate precursors for RNA that may enter the cell art cytidine, uridine, adenine, and guanine, and to some degree, adenosini and guanosine. They may be incorporated also into DNA (Davidson 1965). The metabolic pathways in the nucleotide pool of the cell an presented in Table 7a. These indicate the conversion of RNA-nucleo tides to DNA-nucleotides but not the reverse. RNA breakdown alsc supplies precursors for DNA and, therefore, causes a delayed labelin1 of DNA after a single administration of labeled RNA precursor (Fei nendegen et al., 1964). The extent of the delayed DNA labeling fron labeled RNA exceeds that from thymidine reutilization but does no cause a transfer of all RNA labeling to DNA. Because of delayed DNI labeling, tracer is finally made available to, and incorporated by, al cells that eventually synthesize DNA during turnover of labeled RNA including the stem cells. Since nearly all cells synthesize some RNA, labeled RNA precw sors are incorporated into nearly all cells in the body. Differentiatin; cells, such as in bone marrow, intestinal tract, and lymphoid tissuc and cells particularly engaged in protein synthesis, such as ceUs o pancreas, the parotid gland, etc., synthesize larger amounts of RNf than do mature, non-dividing cells; for example, the peripheral blooc leukocytes. TABLE 7a-Incorporation of nucleic acidprecursors into whole tissue of the mouse, normalized to the data obtained from 3H-thymidine

I I

'H-thymidine

Bone I00

I I

TfJCt I00

Skin

1

Thymus SpIeen I

I

1

100

100

lE~~:,"d~l ( .

K ~ d n e y Liver

.

M~F

I

LM)

LOO

I 5-'H deoxycytidine 5 - ' H cytidine 6:'H uridine 2-'H adenine 8-'H adenine

28-'H adenosine 8-'H guanosine

B ' H deoxyeuanosine 'H-deoxyadenosine

75.0

32.3

8.9

The compounds were i . v . administered in a n amount of lo-*mM/rnouse: 2 hours after in~ecuonsamples wer counted. The dam are averages of3 mice *standard deviations. From Feinendegen (1976).

RNA PRECURSORS

81

/

TABLE 7b-Relative uptake of tritiated nucleic acidprecursors in long-lived cell populations of various tissues of the mouse, compared to the labeling intensity observed in these ceUs after administration of equimolar amounts of 3 H - t h y m ~ i n e (Feinendegen, 1978) Precursor

Gi

Bone marrow

tract

Skin

Thymus

1.OO 0.38

1.00

0.35

1.00 0.33

1.00 0.29

1.00 0.68

1.00 0.65

0.16

0.16 0.23 0.65 0.65 0.35 0.03 0.05

0.42 0.54 0.70 0.70 0.28 0.09 0.10

'&'Ieen

Mesenteric Lymphnode

DNA precursors: "H-thymidine 'H-deoxycytidine RNA-DNA p r e c u ~ o r s : 'H-cytidine 'H-uridie 2-"H-adenine 8-'H-adenine 'H-adenosine '%-panosine "Hdeoxyguanosine

0.41 0.72 0.85 0.85

0.30 0.06 0.12

0.43 0.73 0.62 0.62 0.28 0.07 0.03

0.45 0.6i 0.47 0.26

0.20 0.01

0.07

0.42 0.46

0.46 0.21 0.03 0.05

In rats, after intravenous injection of tracer amounts of 3H-~ytidine, nearly all of the proliferating cells in the bone marrow were found to be labeled in autoradiograms. The labeling intensity diminished with increasing stages of cellular differentiation. The 3H activity initially incorporated into the average labeled bone marrow cell amounted to about 0.25 of that observed per average labeled bone marrow cell after intravenous injection of an equivalent amount of 3Hthymidine (Feinendegen et al., 1964). The initial rate of incorporation of various % -nucleosides and %-purines into whole mouse tissue in relation to the incorporation rate of %-thymidine is given in Table 7a. The incorporation of various 3H-nucleic-acid precursors into the population of long lived cells in various tissues in relation to the incorporation following administration of equimolar amounts of 3H-thymidine into these tissues is shown in Table 7b. Approximately one day after 3H-cytidine injection, the ratio of 3H bound to RNA and to DNA in whole bone marrow was found to be about 4:l and it changed over a period of 9 days to about 2.5:l. From day 5 to 9 after labeling, the turnover rates were about 0.35 per day for RNA, and about 0.25 to 0.3 for DNA (Feinendegen et al., 1964). It was shown by autoradiography that with progression of cellular differentiation the ratio of 3H-RNA to 3H-DNA per single cell decreased to less than 1in the segmented granulocytes. Thus, in individual cells the distribution of radionuclides between the various RNA fractions on the one hand and the metabolically stable DNA on the other changes in favor of DNA as a function of time after application of labeled RNA precursors. The tritium bound to DNA obviously constitutes the longterm hazard in cells with long lifespans. Because 3H-nucleosides in the rat and mouse are less efficiently incorporated than 3H-thymidine into the population of long lived cells, one may expect a lesser hazard from

82

/

APPENDIX V

the labeled nucleic acid precursors other than labeled thymidine. Yet many more cells are eventually a t risk from labeled RNA precursors and long-term effects to the whole animal may perhaps be not much different from those caused by the correspondingly labeled thymidine. More experimental evidence is needed.

Conclusion The hazard from radionuclide-labeled thymidine stably bound to DNA is determined by the dose rate to the long-lived labeled cells and the generation time of these cells. However, the situation is more complicated in the case of labeled precursors common to RNA and DNA. Turnover of radionuclide label bound to RNA and DNA of tissue is determined by various factors, such as renewal rates of different RNA entities in the different cell types, reutilization of RNA breakdown products also causing delayed DNA labeling, salvage of labeled DNA breakdown products, and the rates of cellular proliferation. Because DNA is metabolically stable, in contrast to RNA, label bound to RNA precursor will be distributed, as a function of time, with increasing preference to DNA. Hence, for cells with a long lifespan, it is finally the rate of cellular proliferation that defines the biological half-life of the radionuclide incorporated into the cell nucleus, irrespective of whether it was initially bound to RNA or DNA. On this basis, the dose to the stem cell nucleus from 3H-thyrnidine is about 50 times higher than from the same amount administered as "HOH. In the case of application of precursors for both RNA and DNA the initial intensity of total labeling (RNA and DNA) of single cells is smaller perhaps by a factor of 4 or more compared to that from labeled thymidine, yet the number of labeled cells in critical tissue, such as bone marrow, is larger by a factor of 2 and more. In addition, RNA renewal and partial reutilization of labeled RNA breakdown products for DNA synthesis lead to an increasing labeling intensity of DNA of long-lived cells and this labeling is not expected to exceed the labeling intensity observed after application of the same amount of labeled thymidine. For the purpose of this report, it appears justified to estimate the long-term hazard from the labeled RNA precursors as specified in Figure 4, to be not larger than that from the same amount of equally labeled thymidine. Until more experimental evidence is available, late hazards from labeled RNA precursors should be considered similar to those from equally labeled thymidine.

APPENDIX VI

Stem Cells, Somatic Mutations, and Carcinogenesis Introduction The term, stem cells, refers to relatively undifferentiated progenitor cells that have the capability of producing daughter cells, some of which undergo differentiation into the mature functional cells of the various tissues during subsequent divisions, and some of which remain undifferentiated. In this way, a population of progenitor cells is maintained to meet the demand for new mature, functional cells to replace the mature cells lost through cell senescence, utilization, or loss from the body. Stem cells continue to reproduce themselves throughout the life of the individual, in contrast to differentiated end cells, which lose the capacity for self-renewal. In studies of cell proliferation kinetics in the post-embryonic human, it is customary to assume that significant numbers of stem cells are found only among such rapidly proliferating cell lines as in intestinal epithelium, the male gonads, and hematopoietic tissues (i.e., bone marrow and lymphoid tissue). This concept of "significance", however, reflects a strong bias introduced by the specific requirements of experimental programs in which "significance" can be taken to mean that the concentration of actively proliferating stem cells is great enough to allow the investigator to measure cell proliferation rates with a minimum of tedium and with good statistical accuracy. In considering the relationship between radiation and an increased incidence of cancer, it is necessary that such risk estimates take into consideration the stem cells in all tissues, not merely in those that are rapidly proliferating. In this context, the cells a t risk from radionuclides that are incorporated in DNA are those stem cells in any tissue that are synthesizing DNA during the time the radionuclide precursors are available. As tissues, the gastrointestinal epithelium, male germinal epithelium, and the hematopoietic tissue are a t especially great risk, not only because of the high radiosensitivity of their stem cells, but also the

/

84

APPENDIX VI

high probability that they will undergo division during the life of the individual. If one thinks not in terms of tissues, however, but in terms of individual stem cells, then all stem cells that have incorporated a given amount of radioactive precursor into DNA are considered to be at equal risk. The carcinogenic risk to the individual, however, will depend on the probabilities that: (1) the labeled cell will undergo a potentially carcinogenic change in its genetic apparatus; (2) this change or other changes will neither be lethal to the cell nor eliminate its capability to divide. The rate of stem cell proliferation is not known. However, estimates may be made regarding structure and turnover of bone marrow cell populations. The presently accepted structure of hematopoietic cell proliferation is shown in Figure 8. There are cogent reasons to believe that there is a primitive pluripotent stem cell pool that feeds cells into committed stem lines which will give rise to erythropoiesis, rnegakaryocytopoiesis, and granulo-monocytopoiesis. The pluripotent and committed stem cell pools differ in their cell kinetic properties. Since most quantitative data are derived from studies of murine hematopoiesis, data from these animals will be summarized. DIFFERENTIATED POOL COMMITTED STEM CELL POOL

PLURIPOTENT STEM CELL

N

-

z

- 1140

5 3 6 x l o 7 / kg

I 0 xl0'1kg

NS

14 x 10l/kg

N

- 4 0 x 10'1kg

Kg

i

0 015 x 101/k~/hr

Kg: 0 0 3 x 1 0 ~ / k ~ / h r :

0.015rlo7/kglhr

F R I C T I O N I N S 0.10

A

2.32 x 10~/kg/hr

FRACTION IN 5 0 . 3 5 Is : 12 h r r

Is

x 10'1 k ~

= 1676 X lo7/ hQ

NE +

NS:O.lOrlO'lh~

KO,

N€ NG

12 hrs

c

1 0 . 0 rl0'/kplhr

1.17 x 1o7/hplhr • NS

225 X IO'/~O (CALCULATED1 139, OF NE t NG NS FROM I, :

* 179

X

lo7/ kp

11% OF N ~ I N '

K0 -. % % 2 IS

A M P L I F I C A T I O N x 16 K8:2(KwT-K,NI

K

0

.% Is

Fig. 8. A concept of stem cell and differentiated cell pools in man (Cronkite and Feinendegen, 1975).

The terms used in Figure 8 are defined as follows:

N N, t,

= = =

total number in a pool of cells/kg body weight number in DNA synthesis/kg body weight time for DNA synthesis is 12 hours

INTRODUCTION

Kg Idut Kin

/

85

birth rate (cells produced per 107/kg/h) number leaving a pool per 107/kg/h = number entering a pool and is equal to number leaving previous pool (Kout) NE = number of nucleated erythrocytic precursors x 107/kg NG = number of granulocytic precursors x 107/kg IL = index of labeling with tritiated thymidine = =

The pluripotent stem cell is called the colony forming unit-spleen (CFU-S). It has the following characteristics: 1. Abundance in the bone marrow about 1 per 1000 nucleated bone marrow cells (Siminovitch et al., 1963). 2. The fraction in DNA synthesis varies according to: a. Strain of mouse (0-20 percent) (Vassort et al., 1973). b. Hematopoietic stresses from hypoxia, endotoxin, and infection (Boggs et al., 1972, 1973; Eaves and Bruce, 1972; Quesenberry et al., 1973; Murphy and Lord, 1973). 3. It migrates and circulates in the blood presumably with a "steady state" exchange with bone marrow and perhaps other tissues (Brecher and Cronkite, 1951; Gidali et al., 1974). 4. The DNA synthesis time of murine stem cells is about 5 hours (Siegers et al., 1978). 5. The average turnover time is of the order of 6 days under "steady state" conditions (Siegers et al., 1978). There is no known way to study human pluripotent stem cells a t the present time. The committed stem cells for erythropoiesis and granulopoiesis are believed to be capable of measurement by in uitro culture techniques. The committed stem cell (CSC) has accordingly been named the colony forming unit culture (CFU-C) for erythroid a n d granulocytic cell lines. In man, the abundance of the granulocytic CFU-C is 0.1-1 per lo3 bone marrow cells (Senn and McCulloch, 1970) with a fraction in S-phase (thymidine succinate technique) of 0.35 (Moore and Williams, 1973). The abundance of CFU-E for the erythrocytic cell line is also of the order of 1 per 1000 bone marrow cells (Iscove et al., 1973) with a comparable fraction in DNA synthesis. These estimates should be considered minimal since there is a tendency for an increased yield of clones in the assay system with the development of more potent stimulatory substances. Some insight into the probable relationship between the stem cell pool and the differentiated cell pools of the human bone marrow can be obtained by estimating the fraction of human marrow cells that are

86

/

APPENDIX VI

stem cells. In making this estimate, the following values are accepted: (1) Mean lifespan of red cell = 120 days with loss by senescence. (2) Granulocytes are lost from the blood randomly with a half-time of 6.7 hours (mean lifespan of 9.7 hours). (3) Granulocyte turnover rate (GTR) is 6.8 x lo7kg-' h-'. (4) Red cell turnover rate (RTR) is 12 x lo7 kg-' h-'. (5) Total number of erythroid marrow cells is 536 x 107/kg (Donohue et al., 1958). (6) Total number of granulocytic bone marrow cells is 1140 x lo7/ kg (Donohue et al., 1958). (7) Total number of bone marrow cells is 1800 x 107/kg (Donohue et al., 1958). (8) DNA synthesis time of differentiated human marrow cells (red and white) is about 12 hours (Stryckmans et al., 1966). (9) The average amplification from the immediate precursor of the differentiated cell lines to non-dividing granulocytic cells and erythropoietic cells is 16 (Cronkite and Vincent, 1969; Bond et al., 1959). A minimal influx (Ki.) of stem cells into the erythropoietic and granulopoietic pathways can be estimated by dividing the respective turnover rates by the amplification factor of 16.

~ f =' p R T R + 16 = 12 x lo7 kg-' h-' t- 16 = 0.75 x lo7kg-' h-' K& = GTR +- 16 = 6.8 x lo7kg-' h-' + 16 = 0.42 x lo7kg-' h-' Total influx (Kz) = 1.17 X lo7 kg-' h-'. Stem cell pools are self sustaining and in addition supply cells for is half of the the differentiated pool. In other words, the flux out (Lut) birthrate (KB) of the pluripotent stem cell pool under steady conditions. In the committed stem cell pools there is self-reduplication and KB thus the Kmr = -+ Kin. See Figure 8 for details. 2 In the system established in Figure 8, the pluripotent stem cell pool contains 1.8 X lo7 cells/kg (N) on the assumption that mouse and man have the same ratio of pluripotent stem cells to total marrow cells of 1to 1000. In this model it is assumed that 0.10 of the human pluripotent stem cells are in DNA synthesis (N,): A total of 0.18 x lo7 cells/kg. If the measured DNA synthesis time for the differentiated pool of 12 hours applies, the birthrate is

INTRODUCTION

/

87

and the K0ut is 0.015 x lo7 kg-' h-'. Again referring to Figure 8, the Kg in the committed stem cell pool is

KB = 2

(Idut

= 2.31

- Kin)

x lo7kg-' h-'

and the number in DNA synthesis (N,) is

the total number of committed stem cells (N) is

N=

Ns - 14 X lo7 = 40 x 107/kg. fraction in S 0.35

I n this estimate it is assumed that the fraction of cells in S phase for in vitro colony forming cells that produce erythroid and granulocytic colonies applies. T h e ratio of committed stem cells to total marrow cellularity is 40 X 10' = 0.02 or 1 in 50 bone marrow cells are CSC. The ratio of 1800 X l o 7 40 X lo7 CSC to differentiated erythroid and granulocytic cells is 1676 X lo7 = 0.024 or 1 in 42 erythroid and granulocytic cells are CSC. If it is assumed that the cells leave the CSC randomly, the average turnover time (T) is

T=-

N

x 1.44 =

Idut

40 x lo7 1.44 = 49 hours. 1.17 x lo7

-

Similarly, under the conditions established for the human pluripotent stem cell pool and assuming a random exit irrespective of whether cells are out of cycle (Go)or in cycle the average turnover time (T) in the pluripotent stem cell pool is

T

N

= -X

Idut

1.44 =

1.8 X lo7 - 1.44 = 173 hours 0.015 X lo7

=

7.2 days

In the mouse the measured stem cell turnover time is 6 days (Siegers et al., 1978). To be conservative, it is arbitrarily assumed that the human stem cell pool WLU have a turnover time of 30 days rather than the calculated 7.2 days above. Under this condition one desires to know the number of stem cells a t risk (in DNA synthesis)

88

/

APPENDIX VI

= .0025

x lo7cells kg-' h-'a 1.44

= 0.0036 x 10' cells kg-' h-'

hence Ke = KUt x 2 = 0.0072 x lo-' cells kg-' h-'

since

and fraction in DNA synthesis is

From studies of Fialkow et al. (1967, 1977) and Beutler et al. (1962) it is clear that some cancer is monoclonal arising from one cell. It is therefore reasonable to assume that the probability of growing out a harmful clone of cells after exposure to radiation is a function of the number of cells at risk and the dose to these cells. With uniform external exposure, all cells receive nearly the same dose. Accordingly, the hazard per rad to the pluripotent hematopoietic stem cell (PHSC) from 3H-thyrnidine should be about 0.024 that of external radiation delivering the same dose to all cells. The major determinants of risk are summarized diagrammatically in Figure 9. The turnover times of cell populations in several tissues are presented in Table 8. Although not necessarily related to carcinogenesis, one additional aspect of radiation-induced stem cell injury must be considered, namely, that organs and tissues represent complex organizations of several cell types and cell products. If a portion of a tissue such as liver, for example, is damaged and cells divide to replace the damaged portions, it is not only liver cells per se that must be produced but other components of liver tissue as well, e.g., biliary epithelium, blood and lymph vessels, connective tissue, and so forth. Furthermore, the various components must be produced and organized in keeping with

INTRODUCTION

Make radioactive DNA precursor available

-w

/

A l l stern cells

Is cell

I

No Carcindgenic Risk

Will mutation?

No Carcinogenic Risk

No Carcinogenic Risk

Maximum Carcinogenic Risk Associated with this Cell

No Carcinopnic Risk

Obviously. other decisions c w l d be included. Pertinent questions might.include the following. Is one maximum risk all enough to produce cancer? Will cancer beginning at age x years develop fast enough t o be clinically significant before the individual reaches the end of a normal lifespan? Will regulatory factors such as immune response destroy a malignant cell line before it becomes clinically significant?

Fig. 9.

Decision p a t t e r n for carcinogenesis.

89

TARLE 8-Some estimated turnover times of epithelium in man Technique male germinal epithelium (dul.ation of spermatogenesis) corneal cpitheliwn small intestine epithelium colon epithelium rectum epithelium skin. forearm skin, thorax skin, abdomen utsrine cervix bone marrow, erythroid bone marrow, mycloid

"HTdR "HTdR colchicine ;'HTdR "HTdR mitotic index :'HTdR mitotic tnder 'HTdR various methods "HTdR

tci","zy;",:,

Reference

Heller and Clermont (1962) Hnnna el oL (1961) Bertalanfy and Nagy (1961) Cole and McKalen (1963) Cole and MeKalen (1961) Johnson el a1 (1960) .Johnson rf at. (1960) Katzberg (1952) Richart (1903) Lajtha and Oliver (1960) Cronkite el a/.(1960) or Cronkite and Vincent (1969)

'"HTdR-tritiated thymidine

the genetically controlled architectural plan for liver tissue. Each cellular line has its own stem cells, hence the coordinated proliferation of the several cell lines that make up tissues represents a level of complexity far greater than that associated with any single cell line. A wide spectrum of cancer types has been reported to follow high doses (hundreds to thousands of rad) to localized areas of the body. Indeed, after exposure of man to whole-body or large volume exposure the incidence of leukemia (acute lyrnphocytic, acute and chronic myeloid, but not chronic lymphocytic), cancer of thyroid, female breast, and lung has been well documented in human populations. Semiquantitative dose-effect relationships are available in the survivors of the atomic bombing in Japan and in the patients with ankylosing spondylitis treated by irradiation of a large volume of the spine (UNSCEAR, 1972). Other neoplasms have been induced by whole- or partial-body irradiation in man and animals. Per unit dose absorbed, the incidence of all other neoplasms is quoted to be four to six times higher than that of leukemia (UNSCEAR, 1977). With the caveats set forth above kept in mind, it is appropriate to pay particular attention to those tissues in which radiation-induced stem cell injury has definitely been shown to result in an increased incidence of cancer.

Leukemogenesis The stem cells that may give rise to radiation-induced leukemia reside primarily in the bone marrow. In this tissue, the various hematopoietic cell lines are intermingled, occurring as clusters or islands of cells representing all cell lines and stages of differentiation. These islands are supported and enclosed by a fairly loose, delicate reticulum

LEUKEMOGENESIS

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91

and by the bony spicules of the marrow cavity. Blood flows or percolates slowly and intermittently through sinusoids that are defined by the supporting structure. Marrow sinusoids contain mature blood cells of the circulating blood. The extra-sinusoidal hematopoietic islands contain all of the maturation stages of the various cell lines. Conceptually, the bone marrow is divided into three pools (a pool of stem cells, a pool of proliferating cells, and a storage pool of nonproliferating cells). Among the three marrow pools, all maturation stages of each cell line are represented. Since each mitosis yields two cells for one, the later stages of differentiation are present in greater numbers than are the earlier stages. For this reason, mitoses in the proliferating pool of differentiating cell lines are referred to as "amplifying" divisions. The various morphologically recognizable stages of dividing and differentiating cells in a pool may be considered as separate "compartments" of each cell line. After the last cell division has been completed, bone marrow cells mature. Cells in the final stage of maturation also are considered as a separate pool and each morphologically distinct type may be considered as a separate compartment. These cells are destined to leave the marrow and ultimately die. A fluctuating "steady state" between birth rates and death rates is regulated by as yet obscure mechanisms. Injury to the stem cell pool is a serious threat to hematopoiesis, for depletion of the stem cell compartment could shut off the production of mature cells by the hematopoietic tissue. Since blood cells are constantly being lost through cell senescence or utilization, they must be replaced a t a rate that equals the rate of loss. When blood cell production fails, the failure is manifested, relatively promptly and dramatically, as a deficiency of circulating cells and all the serious clinical problems associated with such deficiency. Somatic mutations can result from irradiation of the stem cells in cell renewal systems. However, one must ask whether low dose rate irradiation of DNA of long-lived non-proliferating cells has any biological significance. It is noteworthy that fetuses can be 100 percent labeled in utero by 3HTdR.4If the doses of 3HTdR to the mother are not too high, the litters are normal in size and weight, grow normally, and are fertile (Fliedner et al., 1968). The females may be sterile depending upon the dose administered to the mother. Rats, so labeled, that have been kdled up to one year after birth, appear normal and have a very high fraction of labeled neurones, as well as labeled skeletal and cardiac muscle and other somatic tissue. Studies on the long-term effects of such labeling are incomplete. WTdR-tritiated thymidine.

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In tritium-labeled proliferating tissue, cells divide and deliver 3HDNA to both daughter cells. In the absence of further label, this results in dose reduction until the cell reaches the mature, functional stage. Actually, the dose-rate to the cell nucleus falls step-wise and may be taken to approach an exponential decrease when averaged to the population until the progeny of a labeled cell exceeds the number of chromosomes of that cell which, for human cells, would be produced by the seventh post-labeling division (Cronkite et al., 1961). The reasoning behind this statement is as follows: 3H-thymidine is either incorporated into DNA of dividing cells or rapidly catabolized. The radiation exposure from 3HTdR of a cell population will depend on the percentage of cells in the population synthesizing DNA at the time of a single injection of 3HTdR and on the subsequent division of labeled cells. At each subsequent division, duplication of chromosomal material proceeds from unlabeled material and, on the average, the number of labeled chromosomes is halved at each subsequent division. Random distribution of labeled and unlabeled chromosomes to daughter cells a t each division and sister chromatid exchange will result in deviation from the theoretical even distribution of the labeled material between daughter cells, but these deviations will be random and will not affect the overall consideration of halving of the labeled chromosomes at successive divisions. Eventually a single labeled chromatid must go to only one of the two daughter cells and thus completely unlabeled cells will arise (Cronkite et ai., 1961). As the progeny of a labeled cell increases, a greater percentage of the cells will be unlabeled because of the discrete nature of the chromosomes. Sister chromatid exchange will result in partial labeling of chromatids but this cannot alter the ultimate consequences of the discrete nature of the chromatid material and will only delay the time a t which, after a somewhat larger number of divisions, unlabeled cells arise. Two additional properties of renewal populations must be considered. In some of the renewal population, there is a more or less efficient reutilization of nucleotide or nucleoside material that may come from DNA of dead cells or from nuclei that are lost from cells during the production of c e h devoid of DNA. Such a cell is the red blood cell. The mature red blood cell is always produced by extrusion of the nucleus after the last division of a dividing red cell precursor. Similarly, the end product of a megakaryocyte is the blood platelet which does not contain DNA. Mature megakaryocytes contain a multiple of the diploid set of chromosomes and apparently can continue for many days to produce platelets by shedding their cytoplasm. Finally, the nucleus remains and is subject to degradation. Extruded nuclei or nuclear debris are rapidly phagocytized and deliver labeled nucleotides

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or nucleosides which are available to be reutilized for DNA synthesis by any cell in the bone marrow. This reutilization has been amply demonstrated, but even if such reutilization should attain a level of 50 percent, this would merely result in doubling the time until disappearance of the label from the cells. From the foregoing it may be estimated that, on the assumption that each labeled cell will divide again, the rate of disappearance of the label from the renewal population becomes a function of the interval between mitoses (generation time) of the population. In human cells with 46 chromosomes, without sister chromatid exchange or reutilization of the label from the extruded nuclei or dying cells, only half of the cells would be labeled after the sixth division, and thereafter the number would drop off to %, %, etc., with each lapse of an additional generation time. Chromatid exchange and reutilization can merely delay the time of appearance of unlabeled cells and disappearance of labeled cells (Cronkite et al., 1961). With a population that turns over slowly it is statistically conceivable that a cell might divide only once in an individual's lifetime. The hazard from such cells is probably negligible because they will accumulate very high total exposures and are likely to be killed before they can divide again. At the other end of the scale, very rapidly dividing populations will dilute the label rather quickly and, consequently, fewer and fewer cells will be at risk. Between these extremes, there are cells that do receive a-significant dose of radiation and may give rise to mutation or a defect in DNA that, on duplication, may result in an abnormal progeny before a sufficient dose of radiation to the cell prevents duplication or kills it. The lack of direct observations of stem cell proliferation kinetics in man and the sparsity of data on the dilution of label precludes prediction of the number of cells in a given population likely to become a potential source of mutant cells (cancer, etc.). One must rely on experimental data to gauge the magnitude of the hazard.

Epidemiology of Radiation Leukemogenesis in M a n The incidence in all types of leukemia except chronic lymphocytic leukemia has been increased by whole-body irradiation under various conditions. In considering the potential hazard from radiation exposure it is necessary to consider the total dose, the dose rate, the volume of the body exposed, the distribution of absorbed dose, and the number of pluripotential hematopoietic stem cells a t risk. In the case of radiation of sufficiently low penetration so that it is all absorbed in the

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first few millimeters of soft tissues, one would not expect an increased incidence of leukemia. Evidence suggests that a substantial volume of bone marrow must be irradiated before an increased incidence of leukemia will be observed in man or animals. To evaluate the possible influence of radiation upon the induction of any tumor or to evaluate the probability that a given case of leukemia may have been due to some prior exposure to radiation, the age-specific incidence must be considered. For leukemia, the agespecific incidence is high in the first years of life, falls progressively during the first ten years of life, remains roughly constant until about age 35, and then again begins to climb. In the patients given largevolume spinal irradiation for treatment of ankylosing spondylitis an increased incidence of leukemia was observed within 20 years thereafter and with increasing age the incidence rose at a rate that was about the same as that observed among unirradiated subjects (CourtBrown and Doll, 1957; UNSCEAR, 1972). The incidence was about five times what would have been expected due to natural causes at any given age. However, in the Japanese and the patients with spondylitis, a detectably increased incidence of leukemia was observed in those individuals who had received doses of radiation in excess of about 100 rad. Although linearity in the dose-effect curve is assumed at doses from low-LET radiation to the bone marrow of less than 100 rad, an increased incidence in man at such low doses has not yet been observed with certainty. With high LET radiation, for example the neutron component a t Hiroshima, an increased incidence of leukemia was observed in the 10-49 rad exposure group (UNSCEAR, 1977). The excess incidence of leukemia in a population rises to a peali after the radiation exposure of the population, and then falls. In the case of the Japanese, the peak in leukemia mortality occurred about 6 years after exposure and thereafter declined. Even so, thirty years after exposure, the incidence is still higher than one would predict in a normal population. It is probable that the peak for acute myelocytic leukemia was earlier than that for chronic myelocytic leukemia. The hazard of developing leukemia as a result of radiation exposure has been estimated on the basis of the following assumptions: (a) Linearity between dose received and incidence of leukemia in the Japanese and in patients with spondylitis treated with largevolume irradiation. (b) No threshold dose below which the incidence is not increased. With these assumptions one predicts that there will be twenty more cases of leukemia than expected per rad exposure per million population a t risk over a 20-year period (UNSCEAR, 1972). The probability of leukemia induction from the incorporation of

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radionuclides into hematopoietic stem cell nuclei is believed proportionate to the number of stem cell nuclei labeled times the dose to the labeled stem cells times the risk coefficient per rad exposure. The average risk coefficient for leukemia (derived from exposure of human beings to external radiation) is 20 cases over the period of 20 years per lo6 persons at risk per rad (UNSCEAR, 1977). With the above considerations it is of no consequence whether the hematopoietic stem cells are exposed as a result of total or partial body external exposure, or by incorporation of radionuclides into the DNA of hematopoietic stem cells. The only considerations are the number of hematopoietic stem cells and the average dose to their nuclei.

Susceptibility of the Fetus Data suggest that exposure of the fetus in utero, particularly during the first trimester of pregnancy, to doses of the order of 1-4 rad wiU increase the incidence of leukemia (UNSCEAR, 1972). Other data discussed in the UNSCEAR report do not support this contention. An increased incidence of other childhood malignancies is also reported following whole-body irradiation of the fetus in the course of prenatal diagnostic examination, but this is of questionable significance.

Chronic Exposure and Small- Volume Exposure In the situation of chronic exposure to small tissue volumes of the body, the dosimetry is so poor that adequate estimates of hazard cannot be made. An increased incidence of leukemia was observed in various occupational groups such as the American radiologists (UNSCEAR, 1972). With better protection of radiologists, this excess incidence of leukemia has disappeared. For small-volume exposure, such as occurs after radiotherapy of tumors of the breast, lungs, and other parts of the body where only a small fraction of the bone marrow is irradiated, an increased incidence of leukemia has not been observed. Therapy of carcinoma of the thyroid by radioactive iodine, however, in some cases has been followed by leukemia. In this case, the whole bone marrow is irradiated during the time period when the radioiodine is in the circulation prior to incorporation into thyroid hormone in the thyroid gland or its excretion. In the case of small volume exposure, absence of an observed increased incidence of leukemia is most likely statistical with a very low probability of induction because of the small number of stem cells irradiated. In the case of high therapeutic doses of radiation to small marrow volumes, the stem cells in the irradiated

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area are probably killed so that there are no surviving mutant cells to produce clinical leukemia. Whereas there are insufficient data on human populations to determine the influence of lower dose rates upon the incidence of leukemia, there are sufficient animal data to indicate that one would expect a smaller yield of harmful effects when the dose rate is reduced to low levels of chronic exposure. The lower incidence is presumably due to repair mechanisms reducing the net injury to the DNA.

Experimental Observations on Carcinogenic Effect of 3H Thymidine I t is to be expected that 3HTdR in sufficiently large doses will cause a measurable increase in the number of neoplasms in a population. The important question is whether this compound, because of its incorporation into DNA, has a unique oncogenic potential beyond that which would be expected according to the dosimetric considerations that are usually applied to other internal emitters. Only a few large-scale studies of the late effects of "TdR have been reported thus far: (1) Baserga et al. (1965) reported an increased incidence of tumors of various kinds in CAF, mice injected with 1pCi of 3HTdR per gram of body weight at the ages of 2, 6, and 12 months; (2) Cottier et al. (1963) found no increase in lymphomas in C57B mice injected with 10 pCi/g of 3HTdR at the age of 6 weeks; (3) Johnson and Cronkite (1967) found no increase in tumors in Swiss albino mice injected with 5 pCi/g a t 6-12 weeks of age; (4) Mewissen and Rust (1973) observed an increase in the incidence of tumors after 0.3-1.5 pCi per g of body weight of "TdR when given to newborn male and female mice. If these data are valid and if the differences in results are due to differences in strain susceptibility, then tumor incidence for the dose range of 1 to 10 pCi per g of body weight is detectable in mouse experiments. I t is of interest to compare this dose with an estimate based upon radiation dosimetry. The injected amount of 3HTdR can be related approximately to the absorbed radiation dose (see Chapter 4 and Appendix V). In nuclei primarily labeled after an injection of 1 pCi/g body weight, the absorbed dose rate is estimated to be about 4 rad per day in mice. This agrees well with observations on dose-effects in mouse spermatogonia (Johnson and Cronkite, 1959) and other systems (Bond and Feinendegen, 1966). Nuclei labeled to this extent in slowly proliferating tissues could be irradiated at this dose rate over a long period of time and would lead to a dose accumulation of several hundred rad (see also Appendix IV).

APPENDIX VII

Genetic Mutation, Chromosomal Aberrations, and Mammalian Cell Killing from Radionuclides Considered in This Report Introduction Genetic and chromosomal effects of radioisotopes incorporated in or near the genetic material have attracted a good deal of research attention, mainly because of the possibility that effects of atomic transmutation might be observed that would give some insight into the organization and functioning of the genetic apparatus. Most information is available for effects of the radionuclides that can be substituted directly into normal DNA: 3H, 14C,and 32P. Unfortunately, from the point of view of human hazards evaluation, much of the mutation information comes from experiments with bacteria and bacteriophage. Nevertheless, it appears possible to answer, a t least tentatively, the question of whether there are any special hazards associated with several of the radionuclides that may be incorporated into nucleic acids (i.e., hazards not predictable from ordinary considerations of the dose of ionizing radiation to the target). Although the mechanisms by which ionizing radiation causes the death of cells are not well understood, it is clear that the primary damage site is nuclear, quite possibly the DNA itself. Cell killing is thus considered here along with mutation and chromosomal aberration production.

Effects From Radiation Versus Transmutation The production of point mutations by incorporated radionuclides has been studied in a variety of prokaryote and eukaryote systems. 97

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The existence of atomic transmutation effects has been firmly established for radionuclides located in some of the stable positions of several of the DNA bases. For purposes of this discussion, transmutation effects will be considered to include those resulting from recoil and excitation energy as well as from the actual change of the parent atom to one of different atomic number. I t was pointed out early (Strauss, 1958) that while the biological effects of such transmutations in important biomolecules such as DNA, RNA, and protein might seem easy to detect, in fact it requires a rather special set of circumstances to allow separation of the effects of the ionizations produced by the beta particle from those of transmutation. Where the energy of the emitted beta particle is large, as for example in 32Pdecay, it is possible to arrange for most of its energy to be deposited outside of the cells of interest and thus to separate any ionization effects from those of transmutation. But for radionuclides emitting a beta particle of such low energy that many of its ionizations are produced within a few nm of the parent atom, it becomes operationally almost impossible to separate transmutation and radiation effects; any separation must depend on exceedingly accurate microdosirnetry. Indeed, the effech of dissipation of recoil and excitation energy of the parent atom are expected to be similar to those ordinarily produced by ionizing radiation; so when the beta energy is dissipated in a very small volume there is really no distinction possible.

Mutation The mutagenic effects of incorporated beta-emitting radionuclides have been reviewed by Krisch and Zelle (1969) and more recently by Person et al. (1976), and were also the subject of a symposium sponsored by the International Atomic Energy Agency (IAEA, 1968) in which more detailed discussions of much of the work summarized below may be found. Tritium.The mutagenic effects of incorporated tritium have been studied extensively in microorganisms, especially in Escherichia coli by Person and his collaborators (Person, 1968; Person et al., 1976). Experiments with tritiated DNA, RNA, and protein precursors showed that while the mutagenic effects of decay of tritium located in RNA, protein, and some of the stable hydrogen positions of DNA can be accounted for entirely on the basis of the beta particle dose absorbed within the bacterial "nucleus" (the central one-fourth volume), decay at three positions in DNA bases produces an additional transmutation effect. The most notable exception occurs when the bacteria incorpo-

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rate 3H-uridine or "H-uracil. I t was shown that the labeled uridine or uracil was incorporated into DNA as cytosine, and that decays of tritium attached to the number five ring carbon were about seven times as effective in producing mutations at a specific locus than decays of tritium attached to the number six ring carbon. Person's group was also able to demonstrate, using an amber suppressor system, that the excess mutations induced by 5-3H-cytosine were produced almost entirely by a cytosine to thymine coding change. I t appears that decay of tritium in the five position leads to deamination (Krasin et al., 1976b), and that the deaminated cytosine is "mistaken" for thymine a t the next DNA replication, thus leading to the substitution of a thymine-adenine (TA) pair for the original cytosine-guanine (CG) pair. In any case, the efficiency with which decay of 5-3H-cytosine in DNA leads to the CG + TA transition is certainly very high, possibly approaching 100 percent. In a recent comparative study of the nature of the mutations produced by gamma radiation and decay of tritium incorporated as methyl-3H-thymidine, 6-3H-uracil, or 'H-histidine, Phillips et al. (1972) found no specificity such as that found for 5-3Huridine or uracil. Sands et al. (1972) also determined the relative efficiencies of 3H decays originating from growth and storage in tritiated water for the production of both lethality and mutations in E. coli. The efficiency in terms of radiation dose delivered to the bacterial "nucleus" was about the same as that per tritium decay in protein. Sands et al. (1972) also conducted experiments in which the bacteria were grown, in chemostat cultures, in medium containing tritiated nucleic acid precursors in order to compare the mutagenic and lethal efficiencies of tritium decays in metabolically active cells with efficiencies previously determined for decays occurring while the cells were in the frozen state. As in the earlier experiments, 3H-uracilwas more mutagenic when labeled in the 5-position than when labeled in the 6-position. The absolute numbers of mutations per decay in the metabolically active cells were, furthermore, very similar in the chemostat experiments to the numbers obtained in the earlier frozen state experiments for 3 ~ - u r a c iin l either the 5- or 6-position and for 3H-thymidine labeled in the methyl group. More recently it has been shown that tritium located in two other stable DNA positions also produces a local transmutation effect (Person et al., 1976) in bacteria. These are the 6-position of thymidine and the 2-position of the purine adenine. It was estimated that about 2.6 percent of 3H-6-thymidine decays produce reversion in an argininerequiring strain of E. coli in addition to those caused by the beta particle radiation. Thus the transmutation effect for this position in DNA appears to be considerably smaller than for 3H-5-cytosine. The 2-position of adenine and the 8-positions of both adenine and

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guanine are stable ones, but in the bacterium used by Person, Snipes, and Krasin there is interconversion between 3H-8-adenine and 3Hguanine. Thus the mutagenic effectiveness of 3H-2-adenine was compared with the effectiveness of % a t the 8-position of both purines. Decays at the 2-position were considerably more effective. It was estimated that about 8 percent of the 2-position decays cause mutation in the E. coli system by a transmutation effect. The mechanisms by which decays in both 3H-6-thymidine and 3H2-adenine produce their transmutation effects are clearly different from those involved in the case of %-5-cytosine (Person et al., 1976). Those involved for 3H-6-thymidine remain obscure, but those for 3H2-adenine appear to result from the production of a specific DNA lesion. Krasin et al., (1976a) have shown that decays in the 2-position of adenine in DNA result in strand-strand crosslinks, presumably at the site of the AT pair a t which the decay occurs. They were able to determine that about half of such decays result in a crosslink. Just how the crosslinks produce mutations is unknown, but presumably an error-prone repair mechanism is involved. The production of mutations by tritiated nucleic acid precursors has also been investigated extensively in the fruit fly, Drosophila. Much of this work has been reviewed by Kieft (1968), by Oftedal and Kaplan (1968), and by Krisch and Zelle (1969). While it is now clear that all types of mutations are indeed induced by tritiated nucleic acid precursors, there remains in this system some controversy over whether there are any "special" effects, i.e., effects different from those that would be anticipated from the beta particle-dose alone. For example, Kaplan and co-workers (Kaplan et al., 1964; Kaplan et al., 1965) found that the distributions of "-thymidine- and 3~-cytidine-inducedsexlinked recessive lethals, along the length of the x chromosome, differed at the 4 percent probability level and also that the distributions were significantly non-random. This result was taken as evidence for a local, or transmutation, effect. However, Rudkin (1965) questioned the statistical basis for the non-random distribution, claimed by Kaplan et al. for tritium labeled thymidine, and concluded that the relative frequencies of lethals induced by the label were not different from those expected on the basis of the DNA content of the region. A greater mutagenic effect of tritiated uridine than tritiated thymidine has been reported for Drosophila spermatogonia (Olivieri and Olivieri, 1965), but when this was further investigated by Kieft (1968), 6-%-uridine and "-thymidine were found to be equally effective. In the same study, Kieft also tested mutagenic efficiency of 3H-uridine labeled in the 5-position and found it several times more effective than the other tritiated precursors. I t seems likely that in Drosophila, as in

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the E. coli strain used by person, a fraction of the 5-3H-uridine is converted to DNA-bound 5-3H-deoxycytidine,and thus that the effect noted by Kieft in Drosophila is the same as that seen in Person's studies (Person, 1968). Understandably, there are only few data available on mutagenesis by incorporated tritiated compounds in mammals. Greulich (1961) and Bateman and Chandley (1962) established that tritiated thymidine is indeed mutagenic in the mouse. No useful estimates of efficiency or comparisons with the effects of other labeled nucleosides are available upon which to make any judgment as to possible transmutatioli effects. Recently Russell has reported preliminary results from a program designed to determine specific-locus recessive visible mutation rates, dominant mutation rates, and translocation production in mice incorporating tritium either as tritiated water or in the form of tritiated thymidine (Russell et al., 1971). More detailed data were reported by Carsten and Commerford (1976) who observed a significant increase in dominant lethal mutations in mice kept throughout life on drinking water containing 3 pCi ' H per ml. In summary, there is clearcut evidence in prokaryotes of a transmutation effect for cytosine tritiated in the 5-position and incorporated into DNA, as well as suggestive evidence for such an effect in Drosophila; the decay of 'H in the 5-position of cytosine appears to cause a specific coding change from cytosine to thymidine with high efficiency, and the 3H-5-cytosinewas more effective than other tritiated DNA precursors by a factor of 6 or 7. In prokaryotes, decays in 3H-6thymidine and in 3H-2-adenine also produce transmutation effects, although with lower efficiency than in the case of 'H-5-cytosine. No transmutation effect has been detected for decays in any of the other stable hydrogen positions in DNA (the 6-position of cytosine, the methyl group of thymidine, and the 8-positions of adenine and guanine), nor for decays originating in RNA or protein. Since the relative abundance of hydrogen atoms attached to the three DNA positions for which transmutation effects have been demonstrated among all DNA hydrogens is relatively low, it appears reasonably conservative to assume, for the purpose of practical hazards considerations, that there is no significant transmutation effect for tritium incorporated in DNA, and that one may estimate hazards solely on the basis of absorbed beta dose; yet, administration of tritiated precursors that specifically introduce %I-5-cytosine, 3H-6-thymidine, or 3H-2-adenine into DNA represents a special case, of course, and the associated hazards must be treated separately. Carbon-14.Very few data are available upon which to base estimates of genetic hazards associated with 14C-labelednucleic acid precursors.

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Carbon-14 is difficult to use for studies on mutation induction by radiation and transmutation effects, partly because of the relatively high energy of its beta particle, but mainly because of its very long half-life, which requires high specific activities to be achieved in order to obtain reasonable decay rates. Suomalainen et al. (1956) showed that '"-labeled sugars were mutagenic in Drosophila when fed to larvae throughout their development. Some of the I4Cmust have been incorporated into the flies' DNA. The levels of sex-linked lethal production observed appeared consistent with crude estimates of the beta particle dose absorbed. Stroemnaes (1962) also trested the mutagenic effectiveness of 14Clabeled compounds in Drosophila. He injected 8-'"-adenine into male larvae and was able to demonstrate an increase in the frequency of sex-linked lethal mutations, although no increase in dominant lethal frequency could be established. Further experiments were carried out with Drosophila by Purdom (1965) who raised both males and females on media containing 14C-labeled glucose. Both autosomal and sexlinked lethal frequencies were measured and compared with those induced by 60 Co gamma rays administered a t dose rates approximating those calculated to result from the flies' 14C-burdens.No evidence for the induction of mosaic mutations was found, and the 14C-induced mutation rates could be accounted for by the beta dose absorbed. Purdom calculated that the maximum transmutation contribution to the I4Cmutagenesis consistent with his experimental data was 0.01 in the males, and even less in the females. Lee et al. (1972) have reported an extensive investigation of possible 14 C transmutation effects on mutation in Drosophila. Uniformly (generally) labeled 14C-thymidine was fed to male larvae. When they became adults they were mated to females which were either allowed to lay their eggs immediately or forced to delay sperm utilization for three weeks. Because of the Drosophila sperm geometry only about 4 X lop4 of the total beta energy from 14C decays is absorbed in the sperm head, so storage in the females allows accumulation of decays with very little accumulation of ionizing radiation dose. Sex-linked lethal production was compared in the stored and unstored sperm, and was followed to the Fa generation in order to detect possible mutant mosaics. The results were completely negative. Furthermore, a concomitant Y chromosome loss test was carried out to test the possibility that transmutations might generally lead to chromosome loss and thus fail to produce detectable mutation frequencies mainly because of the mutations' failure to be transmitted. This test was also negative. Lee et al. concluded that a transmutation effect, if present a t all, must be rather small.

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Kuzin et al. (1964), on the other hand, have reported a positive effect of I4Ctransmutation on mutagenesis in Drosophila. Larvae were fed '4C-glucose and I4C-glycine. The production of sex-lined lethal mutations was compared with that from chronic gamma irradiation. The authors report a somewhat greater mutagenic efficiency of the 14C labeling than could be accounted for on the basis of the ionizing radiation doses alone. They estimate the increase in the range of 2.3 to 2.9 times when compared to the chronic gamma rays, and attribute it partly to the different LET of the '"Co gamma rays and the 14Cbeta particles and partly to transmutation effects. Furthermore, Anderson and Person (1972) have reported evidence. for a transmutation effect in E. Coli produced by incorporated 14C-thymidinelabeled specifically in the 2-position of the pyrimidine ring. For both lethality and mutation, the effects of I4C decays in the thymidine methyl group could be accounted for by the beta radiation dose alone, but when the 14Cwas in the thymidine 2-position the effect was greater, a result suggesting a transmutation effect. For the mutation endpoint, it was shown by genetic analyses that both transitions and transversions were produced by decays at the 2-position, and that a fairly high percentage involved an AT pair, again suggesting a transmutation effect (Anderson and Person, 1972; Person et al., 1970). The results reported by Kuzin et al. (1964) and by Anderson and Person (1972) are in apparent conflict with those of earlier workers. However, as pointed out by Strauss (1958), 14C is an unfavorable radionuclide with which to demonstrate transmutation effects because of its physical characteristics. Totter et al. (1958), considering the excitation and recoil energy of the 14Cdecay as well as the possible chemical consequences of the conversion of a carbon atom into a nitrogen atom in DNA, assumed that the transmutation efficiency for the production of mutation might be as high as unity. While there is no experimental evidence to support this assumption, it is a conservative one. Certainly, it would be surprising if decay of I4Cin DNA did not at least occasionally produce mutation by a transmutation mechanism as well as through its beta irradiation. Phosphorous-32. The biological effects of 32Pincorporated into nucleic acids are much better known than those of other radionuclides. The classic experiments of Hershey et al. (1951) and of Stent and Fuerst (1955) very clearly showed that lethality in bacteriophages resulted from the transmutation of 32Pin their DNA and further that essentially every decay in single-stranded DNA produced a break in the continuity of the molecule while only about one in ten decays in double-stranded DNA produced breakage and lethality. Phosphorus-32, because of the high energy of its beta particle, deposits most of its ionization energy

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outside of volumes of the order of those of bacteria and even of eukaryote germ cells, so transmutation effects are clearly separable from those of the beta particle dose. The existence of such transmutation effects has been extensively studied in prokaryote systems (Krisch and Zelle, 1969). Early tests for transmutation effects in mutation induction by incorporated 32P in Drosophila were, however, essentially negative (Oftedal and Kaplan, 1968).In more recent, and somewhat more elaborate, experiments Lee and co-workers (Lee et al., 1966; 1967) were unable to demonstrate any production of full sex-linked lethals by 32Ptransmutation. It was calculated that if there were transmutation mutagenesis the efficiency was likely to be below one lethal per 11,000decays. Hence the efficiency for mutation induction by decay of 3ZPin Drosophila DNA must be orders of magnitude lower than in prokaryotes. I t seems likely that the difference is the result of a more complex organization of eukaryote chromosomes. While the results of Lee et al. (1967) for F2 "fuIl" lethals were negative, they did obtain evidence for the production of Fg mosaic lethals through 32P transmutation. From the relative frequencies of lethals detected in the F2and the F3 generation and their 95 percent confidence limits Lee et al. were able to estimate that less than 25 percent of the tissue of the F1flies could have been mutant. This result may be interpreted as evidence for a multistranded structure of Drosophila chromosomes; but as pointed out by Lee et al., the possibility that the low proportion of mutant tissue results from some peculiarity of the mutational process in a chromosome with a single DNA double helix cannot be ruled out. In summary, as is also the case for 14C,it has been very difficult to establish the existence of a transmutation effect for mutagenesis by 32P decay in eukaryotic chromosomes, although it is readily demonstrated in prokaryotic chromosomes. Although the demonstration of mosaic mutation induction by "P is of great interest, the very low efficiency of 32Pin producing mutations in Drosophila by transmutation suggests that one may estimate with confidence the genetic hazards of =P incorporated into human DNA from consideration of the beta particle dose alone. Sulfur-35.The decay of incorporated 35Sto 35C1might be expected to produce transmutation effects. Only two studies of possible mutagenesis by this mechanism have been reported, and both have produced equivocal results. Hungate and Mannell (1952) studied mutation production by "S in Neurospora, but were unable to produce convincing evidence for a transmutation effect. In view of the lack of any evidence for incorporation of 35Sinto DNA, any genetic effect would in any case

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have to be indirect. It appears, therefore, safe to estimate genetic hazards for this radionuclide on the basis of the ionizing radiation dose to the germ cells. Iodine-125.There is reason to suspect that the multiple ionizations resulting from the Auger effect might cause the decay of lZ5I to be particularly effective in producing mutation and other biological effects and experimental evidence for this has begun to accumulate. Krisch (1972) has recently reported studies of efficiency of 12" incorporated as tracer of the thymidine analog, 5-iodo-2'-deoxyuridine (IUdR), into the DNA of various strains of E. coli with differing repair capabilities, and into TI coliphage, in producing lethality. While killing of these organisms is not strictly a genetic effect, the end point has often been studied in parallel with experiments to determine the mutagenic efficiencies of other incorporated radionuclides. Krisch (1972) found that the beta radiation dose could only account for about 33 percent of the killing of the TI phage, and only about 5 percent of the killing of the bacteria. He concluded that most of the effect is due to direct disruption of the DNA molecule through the Auger effect. By analogy to the effects of other radionuclides incorporated into DNA, it thus seems prudent to assume for the present that 12"UdR may constitute a greater genetic hazard than would be expected on the sole basis of beta particle radiation dose to the nucleus. Similar experiments, with '"IUdR incorporated into the DNA of mammalian tissue culture cells, that lead to similar conclusions, will be discussed in a later section of this appendix.

Genetic Hazards in Mammals Dynamics of Germ Cell Production. The peculiarities of the population dynamics of germ cell production in mammalian gonads must be taken into account in evaluating mammalian genetic hazards from radiation and transmutation effects. This is particularly the case when considering the incorporation of labeled radionuclides from brief exposures ("pulse" labeling). These considerations are in fact similar to those arising in evaluating the consequences of single, acute exposures to radiation from external sources. The primordial germ cells in the early embryo can first be recognized in the region of the root of the allantois. They then migrate to the germinal ridge and give rise to the definitive germ cells. During the period of migration and mitotic activity, radiation response is similar in male and female; but this is the only time in gametogenesis when

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radiation response of the two sexes is comparable. During embryogenesis (13.5 days post-conception in the mouse), primordial germ cells in the female enter meiosis, and mitotic stages no longer are present. Although there is some variation between species, most oocytes are in meiotic prophase a t birth. In the human female, mitosis ceases a t about 7 months gestation, and all oocytes have reached the diplotene stage a t birth (Baker, 1963). Thus, in the female, there is no cell comparable to the stem spermatogonium of the male; the young female begins life with a finite supply of germ cells in the diplotene stage of meiosis, and they remain in this stage until they enter diakinesis just prior to ovulation. All DNA synthesis occurs in utero, or within two weeks after birth, and should radionuclides enter DNA a t this time, the resulting radioactivity would not be diluted by cell division until cleavage of a n embryo which could be as much as 20-40 years later in the human female. Therefore, in women, considerable dose could accumulate in the 20-40 years between incorporation and ovulation. T h e hazard from exposure after the oocytes have reached the arrested stage would appear small, for it would be limited to contact with tritiated water, labeled protein, and RNA precursors not specifically entering DNA. In the male, in contrast, the possibility of incorporation of labeled DNA precursors exists throughout the lifespan, for both primordial germ cells and spermatogonia are mitotically active. They are characterized by a long but variable cell cycle (Huckins, 1971a; 1971b), and a high resistance to radiation. Since the time required for development of all cellular stages later than the spermatogonial stem cell type G is short in relation to the reproductive lifespan in mammals, the type A, spermatogonium is the most important cell in terms of both fertility and genetic effects in irradiated males. T h e general radiation response of the human testis has repeatedly been demonstrated to be the same as for other animals, and the most radiation resistant type A spermatogonia closely resemble A, spermatogonia of mouse and rat. I n making comparisons with experimental animals, corrections must be made for differences in the duration of spermatogenesis. These adjustments can be made with relative ease for spermatocyte and spermatid stages, but become increasingly difficult a s one progresses backward in the spermatogonial sequence. For example, duration of spermatogenesis from A, spermatogonia to mature spermatids is 35 days in the mouse and 72-74 days from Ad spermatogonia to mature spermatids in man. Yet, cell cycle length for A2-B spermatogonia is 26-28 hours in the mouse and 16 days for Ad spermatogonia in man (Heller and Clermont, 1964). Wherever they can be measured, the time for development of specific cell types bears

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a constant relationship to the total duration of spermatogenesis, i.e., even though the total time varies, the relative time required for a specific stage remains the same. Although not yet proved, there is strong evidence for similarity of stem cell behavior in all mammals. Thus, whereas a cell cycle length of 26-28 hours has been observed for differentiating spermatogonia of the mouse, 41-42.5 hours for the rat, and possibly 16 days for man, cycle lengths for some stem cells may be as long as 8.5 days in the mouse (Oakberg, 1971), 13 days in the rat (Huckins, 1971b), and may be as long as 100 days in man (Rowley et al., 1974; Chowdhury and Steinberger, 1976). Therefore, the magnitude of the effect of stem cell kinetics on both depletion and recovery may be much greater than anticipated on the basis of present estimates of the duration of spermatogenesis, for these estimates ignore the time required for the stem cell divisions that give rise to the differentiating spermatogonia. T h e above set of relationships is important in estimating the dose received from radionuclides incorporated into the DNA of stem spermatogonia. T h e long generation times of these cells (for a few cells in man possibly as much as 100 days) will result in a very slow dilution of label by cell division, and accumulation of high doses by some cells. However, this irradiation should occur at a dose rate giving significantly lower mutation frequencies than that observed a t acute dose rates. Furthermore, the average dose to the entire population of stem cells will be proportional to the number of stem cells that actually incorporate the radionuclide, that is, the fraction of the population in the S phase during exposure. If, for example, 10 percent of the A, spermatogonia are labeled by a "H-thymidine exposure, and the average dose to the labeled nuclei is 100 rad, the "genetically significant dose" would only be 10 rad because of the 90 percent of the spermatogonial stem cells receiving no dose. Uncertainty regarding the duration of the cell cycle for the human spermatogonial stem cell is somewhat mitigated by this factor, for if the average cycle time is longer, so that there is a greater dose accumulation from radionuclides incorporated into DNA, then presumably the fraction of stem cells in the S-phase of the cell cycle that is capable of being labeled by a pulse of radioactive DNA precursor is lower. Johnson and Cronkite (1959) and Lambert (1969) have reported direct observations on the effectiveness of tritiated thymidine for killing of mouse spermatogonia compared with the effect of both tritiated water and externally administered x rays. The endpoint used was depletion of primary spermatocytes that presumably resulted from killing of type B spermatogonia. Although it was difficult to estimate doses to the target cell nuclei accurately, Lambert concluded that his

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results were compatible with tritium RBE values in the range of 1.32.4 (see Appendix 111).

DNA Strand Breakage While breakage of DNA, in either one or both chains, is not a genetic effect in the strict sense, it is certainly closely related. Misrepair by cellular repair mechanisms is postulated to lead to mutation, and failure to rejoin strand breaks can lead to chromosomal aberrations. The question of DNA strand breakage by the transmutation effect of incorporated radionuclides has been examined directly only relatively recently. Rosenthal and Fox (1970) determined the efficiencies of both incorporated "P and incorporated 3H for producing single and double strand breaks in frozen, dilute solutions of DipZococcus pneumoniae transforming DNA. Both measurements of DNA breaks and of biological transforming efficiency were made. It was found that disintegration of "'P caused single strand breaks with unit efficiency and double strand breaks with an efficiency of only about 0.05. This ratio agrees reasonably well with earlier biological results for bacteriophage. The decay of tritium incorporated into DNA from 3H-uridine or 3H-uracil, labeled in the 6-ring-position, produced single strand breaks with an efficiency of 0.30 and double strand breaks with less than 1 percent efficiency. In the case of =P decay, virtually all of the single strand breaks must have been from transmutation. For tritium decays the 0.30 efficiency must represent an upper limit for transmutation. More recently, Cleaver et al., (1972) determined the efficiency of incorporated methyl-3H-thymidine for producing DNA strand breaks in frozen Chinese hamster V79 tissue culture cells. After the cells were allowed to incorporate the label for one complete generation time, they were stored frozen for various times to accumulate decays. Upon thawing, the DNA was extracted and strand breaks determined by alkaline sucrose gradient centrifugation. I t was calculated that one tritium decay induced about 2.1 single strand breaks. Control x-ray experiments and calculations of tritium beta dose based on nuclear volume determinations yielded an RBE of about 1-1.4. Krasin et al., (1973) have compared the relative efficiencies of decays of tritium incorporated as either 5-3H- or 6-3H-cytosine in DNA isolated from E. coli for producing DNA strand breaks. Decays from tritium in the 5-ring position produced no more breaks than could be accounted for on the basis of the beta radiation dose, but decays originating from label in the 6-position were much more efficient, a result suggesting a transmutation effect. It is interesting that this

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situation is precisely the reverse of that for mutation induction, where decays of tritium a t the 5-position have the greater efficiency. Krasin et al. (1976a) have recently determined the efficiency of decays in %-2-adenine, and in "-8-adenine and 3H-8-guanine in producing single strand breaks in E. coli and bacteriophage T4 DNA in uitro. Decays in the 2-position produced single strand breaks with an efficiency estimated to lie between 0.08 and 0.5 per decay, while those in the 8-position produced breaks with an efficiency of not more than 0.08 per decay. It thus may be that decay of tritium in the 2position of adenine is particularly efficient in producing single strand breaks, as it also is in producing strand-strand crosslinks.

Chromosomal Aberration Production There are only scant direct data upon which to base comparisons of chromosomal aberration production from incorporated radioactive nucleic acid precursors with those from other radiation sources. The general questions involved are much the same for chromosomal aberration production as for other biological endpoints such as cell killing or mutagenesis. These involve the RBE for the radiation produced and possible transmutation and recoil effects. The evidence has been reviewed for the particular case of tritiated thymidine by Bond and Feinendegen (1966) and more recently by Vennart (1968). These authors agreed in concluding that the RBE for tritium particles is probably about one for chromosomal aberration production and the former authors concluded that there was no real evidence for transmutation or recoil effects. No contrary evidence has appeared since. In the absence of experimental data, only theoretical conclusions or extrapolations from other endpoints can be given for other isotopically labeled nucleic acid precursors. Tritium. The production of chromosomal aberrations by incorporated tritiated thymidine has been studied in plant material for a number of years, beginning with the work of Taylor (1958). More recently, such studies have included animal cells as well. However, there are few data on yields of aberrations in relation to the radiation dose. Only one report (Dewey et al., 1965) has compared yields from tritiated thyrnidine with those from external radiation. Similarly, there is only one report (Bender et al., 1962) on aberration production by a tritiated nucleic acid precursor other than thymidine. Few studies are available on the production of chromosomal aberrations by nucleic acid precursors labeled with radionuclides other than tritium; because of the possibility of visualizing the chromosomal location of incorporated

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tritiated thymidine, aberrations may be examined as to their localization near the position of tritium labeling. Localization ofAberrations. Bender et al. (1962) studied the induction of chromosomal aberrations in human leukocytes in uitro by incorporated tritiated thymidine and found no correlation between the site of labeling and the site of breakage. Hsu and Zenges (1965), on the other hand, found that the late-labeling X and Y chromosomes were broken more frequently after "terminal" labeling than expected on the basis of length. I t is difficult to attribute this increase to selective labeling of these chromosomes, however, since other late-labeling chromosomes had fewer breaks than expected, and early-labeling chromosomes had almost as many as expected on the basis of length. Dewey et al. (1965) also examined their Chinese hamster chromosome aberration data for evidence of selective breakage of particular chromosomes, but were unable to demonstrate such an effect. In a later study, however, Ockey (1967) found that the frequency with which the late-labeling X chromosome was broken in XX and XXY human leukocytes, labeled in uitro with tritiated thymidine during the end of the S-period, was much greater than that of the X chromosome labeled early in the Speriod. The same difference was observed when sex chromosome breakage was compared in XX and XY leukocytes labeled late in the S-period. As Ockey points out, part of the former difference may have simply reflected a dose-rate effect, but this does not explain the difference in the XX-XY experiments. The question of selective breakage of specific chromosomes thus remains unanswered. As Bond and Feinendegen (1966) have pointed out, however, even if the late-labeling human X chromosome is more subject to breakage in cells labeled late in the S-period, this appears unlikely to present a serious hazard, since only a small proportion of the cells in an exposed individual are at any time at the end of their Speriod. Furthermore, the excess X breakage in such cells would probably be counterbalanced by a relative diminution in X breakage in cells labeled early in the S-period. Aberration Types Produced. Bender et al. (1962) reported that both tritiated thymidine and tritiated uridine induced "double gaps" or "iso achromatic lesions" not usually observed in x-irradiated human leukocytes. They also found that the ratio of single gaps or achromatic lesions to other classes of aberrations was higher than that seen in xirradiated cells. The other aberration types observed were the same as those induced by conventional radiation sources. None of the other studies with incorporated tritiated thymidine have reported unusual aberration types. The significance of the observations on achromatic lesions from tritiated compounds thus is unclear; in any case such

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lesions are probably not true chromosomal breaks, such as are involved in the major aberration types. Apart from the case of achromatic lesions or gaps already mentioned, the data of Bender et al. (1962), although scanty, do not show any notable deviation from the expected ratios of aberration types. I t should be noted, however, that since multiple-break aberrations are markedly dose-rate dependent, while single-break aberrations are not, it must be anticipated that aberration yields from incorporated radioactive nucleic acid precursors will reflect the relatively low dose-rate at which these compounds irradiate the labeled nucleus. The question of possible transmutation or recoil effects on chromosomal aberrations resulting from the decay of the 3H atom to stable 3He is, naturally, intimately related to the questions of RBE and aberration types. Both the RBE of 1 or less in the experiments of Dewey et al. (1965) and the apparent lack of any correlation between site of labeling and site of breakage in the experiments of Bender et al. (1962) argue against any transmutation or recoil effects for 3H decay. This might not be the case with '4C- or "P-labeled nucleic acid precursors. Dose-Effect Relationship. Dewey et al. (1965, 1967) found an essentially linear dose-effect curve for chromosomal aberration production by tritium incorporated into the DNA of Chinese hamster tissue culture cells as tritiated thymidine. More recently, however, Hori and Nakai (1978) have reported two-component curves for exposures of human peripheral lymphocytes in culture to either tritiated water or tritiated thymidine. The exposure-effect curves were linear for the higher exposures, but were less than linear for the lower exposure levels. Since the curves of Dewey et al. are for cells in which incorporation of a pulse label was actually measured, it seems safe to assume, at least provisionally, that aberration production by tritium incorporated into DNA is an essentially linear function of the number of decays occurring in the labeled nuclei. Sister Chromatid Exchange. The initial method of detecting the phenomenon of sister chromatid exchange was by means of tritiated thyrnidine labeling of DNA and autoradiography. Almost immediately there arose the question of whether or not sister chromatid exchanges were induced by the beta particle radiation from the tritium (Wolff, 1964) and, by inference, whether there might be a transmutation effect involved. Unfortunately, although Gibson and Prescott (1972) have clearly established that sister chromatid exchanges are induced by endogenous radiation, and furthermore that the process saturates a t low dose levels, no data are available to answer the question of possible transmutation effects. The hazard imposed by a n increase in sister

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chromatid exchange frequency is difficult to assess. Sister chromatids are identical in such mitotic cells in which sister strand exchange is observed, hence, exchange cannot result in any genetic recombination. However, if such a phenomenon also occurs in meiotic prophase between synapsed homologs, it would result in an increase of genetic recombination. Whether such an effect could be considered a hazard, however, is unknown. The induction of mutations in DNA-labeled germ cells appears to be more of a genetic hazard than the induction of recombinations. Carbon-14.Only two investigations of the induction of chromosomal aberrations by 14Cincorporation have been reported, but both suggest the existence of a transmutation effect. McQuade and co-workers studied the effectiveness of "C-labeled thymidine in producing chromosomal aberrations in onion root tip cells (McQuade et al., 1956a; 1956b; McQuade and Friedkin, 1960). Comparison of the aberration frequencies from 14C-2-thymidinewith those from 14C-methyl-thymidine showed that the I4C in the methyl group was twice as effective as when it was in the 2-position of the ring. This variation in effectiveness with location within the thymidine base is suggestive of an effect of transmutation. Interestingly, the suggested transmutation effect would be for 14Cin the methyl group, but not for the 2-position of the base, just the opposite of the situation reported by Person et al. (1970) and Anderson and Person (1972) for mutation induction in bacteria. Kuzin et al. (1961) (cited by Kuzin et al., 1964) reported experiments in which chromosomal aberration production was measured in Vicia faba meristems containing a general I4C label, derived from exposure to 14C02,and compared the effect to that produced by chronic '%o gamma-irradiation. The 14C was reported to induce aberrations with at least an order of magnitude greater efficiency, again suggesting a transmutation effect.

Summary In spite of the inconclusive evidence thus far available, chromosome aberration yields from radioactive nucleic acid precursors, with the possible exception of 14C-label,appear to be predictable from the dose absorbed in the nucleus, from a consideration of dose rate, sensitivity of cell stage, and other known effects. Transmutation or recoil effects are most probably not important in chromosome breakage by tritiated precursors and asynchronous labeling of chromosomeq does not likely cause preferential breakage of particular chromosomes or chromosome regions. There is, at present, no reason to consider the RBE for chromosome aberration production by beta rays from incorporated

tritium to be different from one. It appears that the RBE for 14C,at least in the methyl group of thymidine, may be greater than unity; but since there would be only one in thirty-nine carbon atoms in DNA labeled in this particular position in case of random labeling, damage from the beta particle dose to the chromosome is expected to outweigh the transmutation effects.

Mammalian Cell Survival There has been a number of studies of the killing of mammalian cells in tissue culture by incorporated tritiated nucleosides and by tritiated water. Unfortunately, there is little information available on the killing of mammalian cells by incorporated radionuclides other than tritium. Much of the available information for tritium was reviewed by Bond and Feinendegen (1966), who concluded that the effects of intranuclear tritium in thymidine are predictable from the absorbed dose, that there is no reason to assume any transmutation effect, and that the RBE for tritium beta particles is approximately 1.0. More recently Vennart (1968) drew essentially the same conclusion regarding the RBE of the tritium beta particle (see,however, Appendix 111). Tritiated Uridine and Amino A c d s . Marin and Bender (1963) studied the survival of tissue culture cells treated with tritiated uridine, which essentially labels the RNA. Since RNA is largely located in the cytoplasm, this precursor was much less effective in preventing colony formation than was tritiated thymidine incorporated into DNA. The killing by tritiated uridine could be accounted for entirely on the basis of the doses which the labeled RNA delivered to the nucleus, again supporting the idea that the RBE for tritium beta particles is 1.0. Burki and Okada (1968) also compared the efficiency of cell killing resulting from tritium labeled RNA and from tritium labeled protein of tissue culture cells with that resulting from tritium labeled DNA. In order to meassure effects from doses delivered while the labeled RNA was stiU restricted to the nucleus they allowed the radionuclide to irradiate the cells while frozen in liquid nitrogen for various times after either a pulse label alone or a pulse label followed by growth during a generation time in the absence of labeled uridine. They found the tritium label in cytoplasmic RNA only somewhat less efficient than the tritium label in nuclear RNA. This, in turn, was approximately only one-fifth as efficient as tritium in DNA, at least for cases where only one DNA strand was labeled. The efficiency of the tritiated protein for killing was similar to that of cytoplasmic RNA. Puck and

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Yamada (1962) reported similar qualitative results for tritium incorporated into mammalian tissue culture cells as labeled thymidine, uridine, or leucine, but no quantitative results were given. Effect of Tritium Location Within DNA. Burki and Okada (1970) have compared the killing effects of tritiated thyrnidine that was incorporated either in one or both strands of DNA of L5178Y tissue culture cells. The labeled cells were stored for various times in liquid nitrogen to allow the nuclei to accumulate various doses. They found the survival curves for cells labeled in only one strand of their DNA to be approximately exponential, whereas labeling in both DNA strands caused a sigmoid survival curve as well as a higher D37 value. Furthermore, they found a higher D37 for gamma rays than for the nuclear doses from 3H beta particles (i-e., a high RBE for 3~ decays). Burki and Okada concluded that these findings suggest an important effect of transmutation and/or recoil from 3H decay. There are, however, several technical difficulties with these experiments, and recently Bedford et al. (1975) have repeated and extended them in an attempt to resolve the questions of the RBE for 3Hthyrnidine and of whether there is really a difference between the survival curves for unifilar and bifilar labeling. The L5178Y mouse tissue culture cell used by Burki and Okada had previously been found to exhibit essentially a simple exponential survival curve for acute doses of gamma rays (Caldwell et ad., 1965), unlike most mammalian tissue culture cell lines. Bedford et al. (1975) found such curves for gamma rays, tritiated water, and both unifilar and bifilar label with 3H-thymidine (labeled on the methyl group). No difference was seen between the slopes of the curves for unifilar and bifilar labeling, nor between those for the tritiated water and the gamma ray curves. There was, though, a difference in slope between the two pairs of curves amounting to an apparent RBE for tritiated thymidine of 4.4. However, in parallel experiments with the Chinese hamster V79 cell line, in which the cells were exposed either in the frozen state (as were the L5178Y cells) or unfrozen but held at 5OC to accumulate decays, it was found that the high RBE was peculiar to the exposure of cells in the frozen state. When the V79 cells were not frozen, both the divergence between the curves for tritiated water and tritiated thymidine and the high RBE disappeared. An RBE for tritium of 1.7 to 1.9 relative to 60 Co gamma rays was estimated for unfrozen cells. The observed increased relative effectiveness of tritium beta particles in frozen cells may be caused by changes in nuclear volume and by changes in the distribution of DNA and of water, and lack of rapid repair of lesions induced in the frozen cells may also contribute. It is of interest that Cleaver et al. (1972) found that sensitivity to x rays as measured by

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DNA single strand breakage was decreased in frozen V79 cells. On the other hand, Sands et al. (1972) reported tritium decays from DNA labeling somewhat more effective for producing mutations in frozen than in unfrozen E. coli. The evidence so far available lets one assume that transmutation effects can be ignored in assessing the cell-killing hazard associated with tritiated thymidine. Killing by 32PIncorporation. Ragni and Szybalski (1962) and Leach (1964) have measured mammalian cell survival after incorporation of 32P.Both reports agree that 32P incorporated by mammalian cells is much less effective for cell killing than is "9in bacteriophage or bacteria. The reason for this is probably the relatively low contribution in mammalian cells from 32Ptransmutation effect to overall damage mainly caused by the beta radiation. Ragni and Szybalski compared the efficiency of "2P in only one of the DNA strands with the efficiency of label in both strands and found the latter more effective, a finding giving support to the contribution of transmutation of 32P to its effectiveness in cell killing. None of the experiments allows a calculation of ionization dose from the 32Pdecay. Thus, the question of RBE and of the relative importance of transmutation and recoil cannot be quantitatively answered. It does seem likely, though, that transmutation is relatively much less important in mammalian cells than it is in phage and bacteria. and 13'1 (Incorporated as 5-Iododeoxyuridine). Iodine Killing by lZ5I may be incorporated into DNA as a tracer of the thymidine analog 5iodo-2'-deoxyuridine (IUdR).Depending on the quantity incorporated, this analog may be a radiosensitizer. The decay of lZ5Iin DNA is, however, especially effective, quite apart from any radiosensitization of IUdR, because of the multiple ionizations produced by the Auger effect in consequence of K-capture decay of this nuclide. Hofer and Hughes (1971) have compared the efficiencies with which cells of a transplantable murine leukemia are killed by '?UdR, 13'IUdR, and 3H-thymidine. They found that 3H and 13'1 decays were equally effective, based on the beta radiation delivered to the cell nuclei, and that the killing could be accounted for on the basis of the doses delivered. Furthermore, the shapes of the survival curves for these radionuclides were typical of those obtained with low-LET radiations. Decays of lZ5I, on the other hand, were 4-5 times more effective, when the expected RBE of the lZ5Ibeta particles is allowed for, and the survival curves obtained resembled those obtained with high-LET radiation in having a near single exponential shape with very little shoulder. The comparative radiotoxicity of 12"UdR and %-IUdR incorporated into the whole-body DNA of the normal mouse bone marrow cells was investigated by Ertl et al. (1970) and Feinendegen et al. (1971),by use

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of a direct measure of the whole-body DNA turnover. They found the 125 I decays 10 times and more as effective as tritium decays. Roots et al. (1971) have compared the effectiveness for cell killing, as measured by failure of colony formation, of 3H-IUdR and Iz5IUdR incorporated into the DNA of L5178Y cells in culture. In this case, the efficiency of Iz5I decays was about 17 times higher than could be accounted for on the basis of beta particle dose alone. More recently Burki et al. (1975) compared the killing efficiencies of %-thymidine and IzIUdR incorporated into DNA of V79 cells and L5178Y cells in culture. The cells were frozen to allow for dose accumulation. I n spite of considerable differences in radiosensitivity of the two cell strains, the killing efficiencies of Iz5I in both cell types were very similar, approximately 0.025. By comparison, the killing efficiencies from incorporated %-IUdR or "-thymidine were 4-7 times lower for the L5178Y cells and 25 times lower for the V-79 cells. It seems clear, then, that the Auger process does actually contribute to cell killing, probably through extensive excitation and charge transfer in the DNA molecule. The "quality factor" for 1 2 5 ~when supplied as IUdR must thus be taken to be much higher than that for 1251in forms that do not lead to incorporation into DNA.

Summary The available experimental data on cell killing by tritium incorporated into nucleic acids suggest that the dose of ionizing radiation delivered t o the nucleus accounts for all of the effect, and that transmutation and recoil effects, if they exist, are negligible. Iodine-125, when incorporated into DNA, presents a special hazard. Lack of experimental data precludes any assessment of possible special hazards from the incorporation of other radionuclides.

References ANDERSON,F. A. A N D PERSON,L. (1972). "Incorporation of I4C labelled precursors in the Escherichia coli: the lethal and mutagenic effects," page 10 in Abstracts of the Third Annual Meeting of the Environmental Mutagen Society. AVINUR, P. AND NIR, A. (1960)."Separation factor for tritiated water fractional in distillation," Nature 188, 652. BAINBRIDGE, A. E. (1963). "Tritium in surface waters of the North Pacific," page 129 in Nuclear Geophysics (National Academy of Sciences-National Research Council, Washington). BAKER,T. G. (1963). "A quantitative and cytological study of germ cells in human ovaries," Proc. R. Soc. London Ser. B. 158,417. R. AND LISCO,H. (1966). "Tumor induction in mice by radioactive BASERGA, thymidine," Radiat. Res. 29, 583. BASERGA,R., LISCO,H. A N D KISIELESKI,W. E. (1965). Tumor Induction in Mice by Radioactive Thymidine. TID-22292 (U.S. Atomic Energy Cornmission, Washington). BATEMAN, A. J. AND CHANDLY, A. (1962). "Mutations induced in the mouse with tritiated thymidine," Nature 193, 105. BEDFORD, J. S., MITCHELL,J. B., GRIGGS,H. G. A N D BENDER,M. A. (1975). "Cell killing by gamma rays and beta particles from tritiated water and incorporated tritiated thymidine," Radiat. Res. 63, 531. BENDER,M. A., GOOCH,P. C. A N D PRESCOTT,D. M. (1962). "Aberrations induced in human leukocyte chromosomes by 3H-labeled nucleosides," Cytogenetics, 1,65. BERGER,M. J. (1971). "Distribution of absorbed dose around point sources of electrons and beta particles in water and other media." J. Nucl. Med. 12, Suppl. No. 5. BERRY,R. J., CAVANAGH, J., OLIVER,R. AND WINSTON,B. M. (1973). " V h ation of relative biological effectiveness with dose rate for different radiation qualities," Health Phys. 24, 369. BERTALANFLY, F. D. A N D NAGY,K. P. (1961). "Mitotic activity and renewal rate of the epithelial cells of human duodenum," Acta Anat. 45, 362. BEUTLER,E., YEH, M. AND FAIRBANKS, V. F. (1962). "The normal human female as a mosaic of X-chromosome activity: studies using the gene for G6-PD deficiency as a marker," Proc. Natl. Acad. Sci. U.S.A. 48,9. BOGEN,D. C., HENKEL,C. A., WHITE,C. G. C. AND WELFORD, G. A. (1973). "Tritium intake in New York City," page 639 in Tritium, Report No CONF710809, Moghissi, A. A., and Carter, M. W., Eds. (Messenger Graphics, Phoenix, Arizona). P. A. A N D BOGGS,D. R. (1972). "The effect of BOGGS,S. S., CHERVENICK,

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Control and Removal of Radioactive Contamination in Laboratories (19.51) Recommendations for Waste Disposal of Phosphorus-32 and Iodine-131 for Medical Users (1951) Recommendations for the Disposal of Carbon-I4 Wastes (1953) Radioactive Waste Disposal in the Ocean (1954) Maximum Permissible Body Burdens and Maximum Permissible Concentrations of Radionuclides in Air and in 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 of Absorbed Dose of Neutrons and of Mixtures o f Neutrons and Gamma Rays (1961)

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Stopping Powers for Use with Cavity Chambers (1961) Safe Handling of Radioactive Materials (1964) Radiation Protection in Educational Institutions (1966) Medical X-Ray and Gamma-Ray Protection for Energies Up to 10 MeV-Equipment Design and Use (1968) Dental X-ray Protection (1970) Radiation Protection in Veterinary Medicine (1970) Precautions in the Management of Patients Who Haug Received Therapeutic Amounts of Radionuclides (1970) Protection Against Neutron Radiation (1971) Basic Radiation Protection Criteria (1971) Protection Against Radiation From Brachytherapy Sources (1972)

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A Handbook of Radioactivity Measurements Procedures (1978) 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)

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NCRP NCRP NCRP NCRP NCRP NCRP NCRP

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8,9, 12, 16, 22 23,25, 27,30 32, 33, 35, 36, 37 38,39,40,41 42, 4 3 , 4 4 , 4 5 , 4 6 47, 48, 49,50, 51 52, 53, 54, 55, 56, 57

(Titles of the individual reports contained in each volume are given above.) The following NCRP reports are now superseded and/or out of print: No. 1 2 3 4

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X-Ray Protection (1931). [Superseded by NCRP Report 31 Radium Protection (1934). [Superseded by NCRP Report 41 X-Ray Protection (1936). [Superseded by NCRP Report 61 Radium Protection (1938). [Superseded by NCRP Report 131

No. No. NO. No.

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Safe Handling of Radioactive Luminous Compounds (1941). [Out of print]

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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 Protection Against Radiations from Radium, Cobalt-60 and Cesium-137 (1954). [Superseded by NCRP Report No. 241 Protection Against Betatron-Synchrotron Radiations Up to 100 Million Electron Volts (1954). [Superseded by NCRP Report No. 511

Safe Handling of Cadavers Containing Radioactive Isotopes (1953). [Superseded by NCRP Report No. 211

Permissible Dose from External Sources of Ionizing Radiation (1954) including Maximum Permissible Exposure to Man, Addendum to National Bureau of Standards Handbook 59 (1958). [Superseded by NCRP Report No. 391

X-Ray Protection (1955). [Superseded by NCRP Report No. 261

Regulation of Radiation Exposure by Legislative Means (1955). [Out of print]

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 Reports Nos. 33, 34, and 401

Medical X-Ray Protection Up to Three Million Volts (1961). [Superseded by NCRP Reports 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-Structural Shielding Design and Evaluation (1970). [Superseded by NCRP Report No. 491

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Statements

The following statements of the NCRP were published outside of the NCRP Report series: "Blood Counts, Statement of the National Committee on Radiation Protection," Radiology 63,428 (1954) "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) X-Ray Protection Standards for Home Television Receivers, Interum Statement of the National Council on Radiation Protection a n d Measurements (National Council on Radiation Protection and Measurements, Washington, 1968) Specification of Units of Natural Uranium a n d Natural Thorium (National Council on Radiation Protection and Measurements, Washington, 1973)

Copies of the statements published in journals may be consulted in libraries. A Limited number of copies of the Iast two statements listed above are available for distribution by NCRP Publications.

Index Absorbed dose, 20,21 Reference nucleus, 20 Tritium, 20 Approach to dose calculation, 16 Average energy absorbed per labeled cell nucleus, 16 Biological effects, 8, 9, 11, 14, 15 Chromosomal aberrations, 14 Effects on the fetus, 14 Effects of the life span of the individual, 15 Gene mutation, 12 Life shortening, 15 Neoplastic diseases, 9 Transmutation effects, 11 Bone marrow, 24 Carbon-14, 101-103, 112 2-" C-thymidine, 103 Genetic mutations, 102 Carbon-14 labeled nucleic acid precursors, 4 Carbon-14 labeled compounds, 32, 33 Chronic exposure, 33 Energy absorbed reference nucleus. 32 MPD, 33 MPD-injection, 32 RNA precursors, 33 Transmutation, 32 Carbon-14- and aH-labeled amino acids,.4 Carcinogenesis, 88, 89, 90, 96 Cellular decision pattern, 89 Determinants of risk, 88 From 'H-thymidine, 96 Risk analysis, 90 Cells a t risk, 8. 83 Mutations and carcinogenesis, 83 Oocytes, 8 Somatic, 83 Spermatocytes, 8 Spermatogonia, 8 Stem cells, 8, 83 Cell renewal, 8, 9 Differentiated cells, 9 Generation time, 9

Renewal rates, 9 Stem cells, 9 Chromosomal aberrations, 14,97,109-112 Aberration types, 110, 111 Breaks in, 14 Carbon-14, 112 Chromosome, 14 Effects on the fetus, 14 Dose effect relationship, 111 Localization of aberrations, 110 Methyl - "C-thymidine, 112 Rearrangements, 14 Sister chromatid exchange, 111,112 Tritium, 109, 110 Chromosomal aberration production, 109 Comparison of dose-tritiated thymidine to tritiated water. 25 Conversion factors, 17, 18 Dose calculation approaches, 17 Deoxycytidine, 28, 29 MPD, 28, 29 DNA precursors, 5 Adenine, 5 Deoxycytidine, 5 Guanine, 5 Thymidine, 5 DNA strand breakage, 108, 109 32P,108 5-'H-cytosine, 108 6-3H-cytosine, 108 6-3H uridine, 108 'H-thymidine, 108 Dose calculation approaches, 17, 18, 19 Absorbed dose, 17 Average tissue dose. 17 Conversion factor, 17, 18 Inhomogeneities, 18, 19 Microdosimetric considerations, 19 Dose to male germ cell, 25 Tritiated thymidine, 25 Accumulated dose. 25 Effects on the fetus, 14 Chromosomal aberrations, 14 Incidence of childhood leukemia, 14 143

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INDEX

Environmental contamination, 47, 48, 50 Behavior of tritium, 48 Man-made sources of tritium, 47 Metabolism of tritium, 50 Occurrence of tritium, 47 Environmental tritium behavior, 49 Fresh water, 49 Ocean, 49 Rain water. 49 Tritiated water, 49 Female mammal, 13 Oocytes, 13 Gene mutation, 11-13.97-108 Biological effects, 12 Coding change, 99 Deamination, 99 Diplotene, 106 Dominant lethal, 101 Effects from radiation versus transmutation, 11, 98 Efficiency. 99 Eukaryote, 97 Generation time, 13 Immature oocyte, 106 Increase in the frequency, 102 In the female mammal, 13 In the male mammal, 13 Iodine-125, 105 Mammals, 101 Mosaic, 104 Mutation, 97 Oogenesis, 106 Phosphorus-32,103, 104 Probability, 13 Prokaryote, 97 Relative efficiencies, 99 Resulting from recoil and excitation energy, 98 Specific-locus recessive visible mutagen rates, 101 Spermatogenesis, 106, 107 Spennatogonia, I07 Strand-strand crosslinks, 100 Spermatogonial stem cells, 13 Sulfur-35, 104, 105 Translocation production, 101 Tritium, 98-101 2-'H-adenine, 99, 1Ob 5-3H-cytosine,99 6-3H thymidine, 99, 100 'H-uracil, 99 3H-uridine,99 ,

Hazards of nucleic acid precursors, 82 Intensity of total labeling, 82 Lifespan, 82 Rates of cell proliferation, 82 Renewal rates. 82 Salvage of, 82 3H-thyrnidme.82 Tritiated water (3HOH,HTO),82 Turnover, 82 Histones, 5 Iodine-125, 4, 36, 37, 115, 116 Auger electrons, 36, 37 Characteristics, 36,37 Charge transfer, 37 High ionization density, 37 MPD, 37 Radiation dose, 37 Toxicity, 37 Iodine-131, 4,36, 115, 116 Characteristics. 36 MPD, 36 5-iodo-2'-deoxyuridine (IUdR), 4 Incorporation of nucleic acid precursors, 6 7 Cell deaths, 7 Concomitant partial reutilization, 7 Metabolic turnover of labeled nuclei1 acids, 7 Proliferating cells, 6 Rate of cell division, 7 Synthesis, 6 Leukemia, 94.95 Chronic exposure and small volume ex posure, 95 Fetus susceptibility, 95 From radioiodine, 95 Incidence, 94 Risk estimates, 95 Risk for incorporated radionuclide, 95 Specific incidence, 94 Leukemia induction, 10 Assumptions, 10 Dose rate, 10 Cells at risk, 10 Intranuclear irradiation, 10 Risk, 10 Total dose, 10 Leukemogenesis, 90-94 Cells a t risk, 93 Determinants of absorbed dose distribu tions, 93 Determinants of body volume, 93

INDEX Determinants of dose rate, 93 Determinants of pluripotent hematopoetic stem cells, 93 Determinants of total dose, 93 Incidence, 94 In man, 93 Risk estimate, 94 i f e shortening, 15 Biological effects, 15 damrnalian cell killing, 97 damrnalian cell survival, 113-116 Auger effect, 115 lZ6I,115 K-capture decay. 115 1311, 115 32 P incorporation, 115 Tritiated amino acids, 113 Tritium-RBE, 114 Tritiated thymidine, 114 Tritiated uridine, 113 detabolism of DNA, RNA and their Precursors, 69-82 Cells synthesizing RNA and DNA, 70 Degradation products, 71 Efficiency of incorporation, 78 Iododeoxyuridine. 78 Long-lived cell populations. 81 Metabolic interconversions. 74 Metabolic pathways of RNA and DNA precursors, 72 Other specific DNA Precursors, 79 Rate of DNA synthesis, 71 RNA and DNA Distribution in Cells, 69 Relative uptake, 81 RNA precursors, 72,80 RNA and DNA precursors. 72 Thymidine, 75 Thymidine bound to DNA, 79 Thymidine cleared from blood, 77 Thymidine incorporation, 77 Thymidine utilization and turnover of labeled DNA, 78 Turnover rates, 79 kfodes of entry, 5 , 6 air, 5 drinking water, 5 food, 5 ingested accidentally, 6 injected, 6 kfonoclonal, 16 \leoplastic diseases, 9, 10 Biological effects, 9

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145

Breast, 10 Cells a t risk, 10 Distribution of absorbed dose, 10 Leukemia, 10 Monoclonal, 10 Probability of cancer, 10 Thyroid, 10 Total dose, 10 Oocytes, 13 In the female mammal, 13 Repair replication, 13 Other tritium-labeled DNA Precursors, 28 Phosphorus-32, 35 MPD, 35 Phosphate, 35 Physical characteristics, 35 Radiation effects, 35 Transmutation, 35 Phosphorus-32 incorporation, 115 Phosphorus-32 phosphate, 4 Quality factor for tritium, 56-63 Atrophy of mouse spleen and thymus, 61 Depression of "FE uptake, 61 Distribution of 'H, 59 Dose rate, 58 Effective half-life, 58 Inhomogenous distribution, 61 Killing or injuring of spermatogonia, 62 LD 50/30 of tritiated water, 60 LD 50/30 dose, 62 Let of tritium, 56 Mammalian cells in vitro, 60 Mean energy, 58 Mortality, 30-day, 62 Oocytes, 63 Organically bound tritium, 60 Whole animals, 60 RBE, 57-59,6143 RBE experiments in mammals, 60 Reference dose, 57 Reference nucleus, 58 Reference radiation, 58, 63 n i t i u m dose, 57,58 Water in cells of interest, 59 Water in tissues, 59 Whole animals, 60 Radiation protection standards, 41-46 Basic criteria, 41 Energy deposition, Internal exposure, 41 Maximum permissible concentrations, 45 Maximum permissible intakes, 45

146

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INDEX

Molecular disorientation, 45 Molecular disruption, 45 Transmutation effect, 44 Radioactive decay, 4 Auger electrons, 4 Beta particles, 4 Electron capture, 4 Reference nuclear volume, 17, 18 RNA precursors, 5, 29, 30, 80, 81 Adenine, 5,80 Adenosine and guanosine, 80 Cytidine, 5, 80 Guanine, 5, 80 Incorporation of nucleic acid precursors. 80 Long-lived cell populations, 81 Metabolic pathways, 80 MPD, 30 Relative uptake, 81 Tritium, 29 Uridine, 5,80 Sister chromatid exchange, 111, 112 Spermatogonial stem cells, 13 Gene mutation, 13 Somatic mutations, 83 Stem cells, 83-88 Birthrate, 86 Cells a t risk, 83, 87 Concept, 84 Flux, 86 Gastrointestinal epithelium, & Hematopoietic tissue, 84 Male germinal epithelium, 84 Monoclonal, 88 Proliferation, 84 Turnover time, 85 Sulfur-35, 35 MPD, 35 Physical characteristics, 35 35 S-labeled cysteine and methionine, 4 Summary, 38 Thymidine, 78 Salvage pathway, 78 Toxicity of tritiated water, 64-68 Carcinogenesis, 67 Effects in the fetus, 65 Hemoglobin and glycogen, 66 Histone and other chromosomal components, 65 Human beings, 65 LDm, 66 LDWNdose, 64

Ovaries, 65 Pregnant rats, 64 Reference nuclear volume, 66 Spermatogonia, 64 Stochastic considerations, 66 Strand DNA breaks, 67 Testes, 65 Tritium content to DNA, 65 Tritium in tissue water for spleen, kidnej thymus, liver and ovaries, 65 Transmutations, 5 Charge transfer processes, 5 Excitation, 5 Molecular rearrangement, 5 Nuclear recoil, 5 Transmutation effects, 4. 11, 12 Auger electrons, 12 Bacteria, 11 Biological effects, 11 Changes of charge, 11 Chemical, 11 Decay, 12 Decay of 14C,11 '''1 decay, 11 Decay of "'I, 11 Decay of tritium, 11 DNA strand-strand crosslinks, 11 Drosophila, 11 Nuclear recoil, 11 Tissue culture, I 1 Tritiated thymidine, 23-28, 39,40 Accumulated, 25 Accumulated radiation dose, 24 Carcinogenic potential, 39 Chronic ingestion limit, 28, 39 Continuous exposure, 27,% Continuous exposure limit, 28 Continuous intake, 26 Dose, 24,25 Dose to male germ cell, 25 Dose rate, 28 Ingested single dose, 26 Labeling intensity, 27 Maximum permissible amount, 28 Recommended maximum dose, 26 Single administration, 28 Tritium, 24 Tritiated water, 5,21-23,64-68 Biological half life, 22 Chronic body burden, 22 Concentration in tissue, 22 Fractional turnover, 23

INDEX Prolonged intake, 21 Single administration, 22 Tissue concentration, 23 Tritium, 23 Urinary concentration, 23 Tritium, 4,20-31,40,98-101, 113-115 Absorbed dose,20 2-adenine, 100 Chronic body burden. 22 5-cytosine, 99 Dose rate, 21 Fractional turnover, 23 Labeled amino acids and non specific precursors, 30 MPD, 23, 25, 28, 29.30, 31,40 Physical considerations, 20 Quality factors, 20 Relative biological effectiveness, 20 Tissue dose, 23 Thymidine, 24, 114 6-thymidine, 100 Tritiated uridine, 113 Tritiated water, 21, 23 Urinary concentration, 23 Tritium-labeled amino acids and nonspecific precursors, 30 Tritium, 30

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147

Tritium labeled nucleic acid precursors, 4 Tritium metabolism, 50-55 Absorbed through skin, lungs or gut, 53 Absorption from respiratory tract, 53 Animals, 51 Body water, 52 Biological half-time, 53, 54 Carbohydrates, 51 Constant intake, 51 Dose for organic bound, 52 Environmental contamination, 50 Excretion, 53, 54 Fatty acids, 51 Guinea pigs, 51 Humans, 51 Isotropic discrimination, 52 Mutations, 55 Organic matter, 50 Pigeons, 51 Plants,50 Rabbits, 51 Rats,51 Specific activity, 50 Tissue concentrate, 51 Tritium in 5 position of cytosine in DNA, 55 Tritiated water in human beings, 53