Neutron-induced genetic effects: A review

Radiation Botany, 1970, Vol. 10, pp. 225 to 247. Pergamon Press. Printed in Great Britain.

N E U T R O N - I N D U C E D GENETIC EFFECTS: A REVIEW* MICHAEL A BENDER Department of Radiobiology, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

(Received 14 November 1969) A b s t r a c t - - E a r l y studies of the production of gene mutations and chromosomal aberrations by neutron irradiation of various plant and animal systems suffered from difficulties in establishing neutron dose. Gene mutation studies yielded estimates of relative biological effectiveness (RBE) of less than unity. More recent work incorporating more accurate dosimetry has shown that this value is always greater than one, and have established RBE values, dose-effect kinetics and dose rate effects for a variety of genetic end-points. In spite of a rather large variability among the results reported by various authors, it appears that several rather general conclusions can be drawn, at least regarding the genetic effects of fast neutron irradiation. 1. It seems clear that RBE values are always at least one and often much greater. 2. RBE values are much lower for point mutations than for chromosomal aberrations in animal cells. 3. RBE values are larger in plant than in animal ceils, and similar for mutation and for chromosome aberration production, supporting the idea that most plant mutations involve chromosomal rearrangements. 4. RBE is a function of neutron energy, increasing with increasing LET to a maximum at around 60 KeV/~ and then decreasing again at higher LET. 5. Neutrons induce multiple-hit events which have non-linear dose-effect kinetics for low LET radiations as a linear function of dose. 6. Neutron effects are in general much less subject to modification by other parameters, such as dose rate or oxygen tension, than are low LET radiations. Thermal neutrons deposit energy in tissue by capture rather than by production of a recoil proton, making determination of their relative effectiveness dependent on precise determinations of the energy deposited from proton and ~ particle production and from prompt and delayed [~particle and y ray production. Determination of thermal neutron RBE for chromosomal aberration production has yielded values about three times greater than would have been expected from the same doses of the various capture radiations given externally. The RBE for mutation production appears to be similar to that for fast neutrons. R~su~a&--Les premi6res 6tudes de la production de mutations de g6nes et d'aberrations chromosomiques par les neutrons dam divers syst~mes v6gdtaux et animaux ont souffert des difficult6s d'6tablissement de la dose. Des 6tudes des mutations gdniques ont donn6 des estimations de l'efficacit6 biologique relative (EBR) moindres que l'unit6. Des travaux pins r6cents qui comportent une dosimdtrie plus exacte ont montr6 que cette valeur est toujours sup6rieure ~t Iet ont 6tabli des valeurs d'EBR de cin6tique-dose-effets et des effets du d6bit de dose pour une vari6t6 de crit6res g6n6tiques. En d6pit d'une variabilit6 plut6t 61ev6epour quelque les r6sultats rapport6s par diff6rents auteurs, il apparalt que des conclusions g6ndrales peuvent atre formuIdes au moins en ce qui concerne les effets g6n6tiques des neutrons rapides : *Presented at the Symposium on Neutrons in Radiobiology, Oak Ridge, Tennessee, 11-14 November, 1969. Prepared at the Oak Ridge National Laboratory, operated by Union Carbide Corporation under contract with the United States Atomic Energy Commission. 225

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MICHAEL A BENDER 1. I1 paralt clair que les valeurs des EBR sont toujours au moins I lois et souvent supdrieurs. 2. Dans les cellules animales, les valeurs d'EBR sont beaucoup plus basses pour les mutations ponctuelles que pour les aberrations des chromosomes. 3. Les valeurs d'EBR sont sup6rieures pour les cellules v6g6tales que pour les cellules animales et semblables pour la production de mutations et d'aberrations chromosomiques, ce qui a donn6 l'id6e que la majorit6 des mutations des plantes implique des r6arrangements de chromosomes. 4. L'EBR est une fonction de l'dnergie des neutrons qui s'accrolt avec le TLE jusqu'a un maximum d'environ 60 KeV/~t et ensuite d6crolt pour les TLE plus 61ev6s. 5. Les neutrons produisent des effets multitopiques qui ne suivent pas une cindtique-doseeffet lindaire pour des radiations de TLE peu 6levds en fonction lineair de la dose. 6. Les effets des neutrons sont, en gdn6ral, par rapport aux radiations de TLE peu 6levds, moins sujet d'6tre modifids par d'autres param6tres, tels que le d6bit de dose ou la tension d'oxyg~ne. Les neutrons thermiques ddposent l'dnergie dam les tissus par capture plut6t que par production d'un proton de recul, ce qui rend la ddtermination de leur efficacit6 relative d6pendante de d6terminations pr6cises de l'6nergie d6pos6e ~ partir de la production de protons et de particules a e t de la production rapide ou retard6e de particules ~ et de rayons 3'. La d6termination de I'EBR des neutrons thermiques pour ]a production d'aberrations des chromosomes a donn6 des valeurs environ trois fois supdrieures h celles que l'on attendait /~ partir des m6mes doses de diverses radiations de captures diff6rents donndes de mani6re externe. L'EBR pour la production des mutations apparalt semblable ~ celle des neutrons rapides. Zusammet~fassung--Untersuchungen fiber die Induktion von Genmutationen und chromosomalen Aberrationen durch Bestrahlung verschiedener pflanzlicher oder tierischer Systeme mit Neutronen litten anfangs unter den Schwierigkeiten, die Neutronen-Dosis festzusetzen. Untersuchungen fiber Genmutation ergaben Sch~itzungen der relativen biologischen Wirksamkeit (R_BE) yon weniger als eins. Neuere Arbeiten mit genauerer Dosimetrie haben gezeigt, dass dieser Wert immer gr6sser als eins ist und sic haben RBE-Werte, DosisEffekt-Kurven und Wirkungen der Dosisleistung ftir verschiedene genetische Parameter bestimmt. Obwohl eine ziemlich grosse Variabilitiit bei den von versehiedenen Autoren vorgelegten Ergebnissen besteht, scheint es doch m6glich, einige ziemlich allgemeine Schli.isse zu ziehen, zumindest soweit es die genetischen Wirkungen der Bestrahlung mit schnellen Neutronen betrifft. 1. Es scheint klar zu sein, dass RBE-Werte zumindest eins betragen und oft viel gr6sser sind. 9. RBE-Werte sind viel geringer bei Punktmutationen als bei chromosomalen Aberrationen in tierischen ZeUen. 3. RBE-Werte sind bei pflanzlichen Zellen gr6sser als bei tlerischen Zellen und/ihnlich fiir die Bildung von Mutationen und Chromosomenaberrationen; dies unterstfitzt die Vorstellung, dass die meisten Mutationen bei Pflanzen chromosomale Rearrangements betreffen. 4. RBE ist eine Funktion der Neutronen-Energie; sie steigt an mit zunehmender LET bis zu einem Maximum bei etwa 60 KeV/tz und t'allt dann wieder ab bei h6herer LET. 5. Neutronen induzieren Vielfachtreffer-Ereignisse, die nicht lineare Dosis-Effekt-Kurven bei geringen LET-Bestrahlungen haben als lineare Funktion der Dosis. 6. Wirkungen von Neutronen sind im allgemeinen viel weniger Veriinderungen durch andere Parameter wie Dosisleistung oder Sauerstoffdruck ausgesetzt, als geringe LET-Bestrahlungen. Thermische Neutronen geben im Gewebe Energie eher durch Abfangen als durch Bildung yon Rtickstoss-Protonen ab, was die Bestimmung ihrer relativen Wirksamkeit abhiingig macht vonder genauen Bestimmung der Energie, die yon der Protonen- und a-Partikel-Bildung und v o n d e r sofortigen und verz6gerten B-Partikel- und y-Strahlen-Bildung abgegeben wird. Die Bestimmung der RBE thermischer Neutronen fiir die Bildung chromosomaler Aberrationen

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ergab Werte, die um das dreifache hbher waren als bei der gleichen Dosis der yon aussen gegebenen, verschiedenen Abfang-Strahlung zu erwarten ware. Die RBE ftir die Bildung yon Mutationen scheint der fiir schnelle Neutronen ~ihnlich zu sein.

INTRODUCTION

THEM exists a substantial literature on the genetic effects, including chromosomal aberration production, of neutrons. These effects have been studied in a variety of organisms, from fungi to mammals. No attempt will be made here to review the work that has been done on quantitative variation, mainly with thermal neutrons, as this has been done by BROCK.(11) Early work was doubtless prompted in part by curiosityabout the newly-discoverednuclear particle. But another motive was the insight it was hoped would be given by studies with high linear energy transfer (LET) into the mechanism of mutation production by ionizing radiation. The advent of the nuclear era in 1945 created new and powerful practical motives for studies of the effects of neutrons. Not only possible future use of nuclear weapons, but the development and lately widespread installation of nuclear reactors makes accurate assessment of neutron radiation hazards manditory. And this, of course, requires determination of doseeffect kinetics, relative biological effectiveness (RBE), and of the influence of variations in neutron energy, dose protraction, and other modifying factors. An interest also developed in possible medical use of neutrons in radiation therapy, both because neutron-induced damage is m u c h less influenced by modifying factors such as oxygen tension than is damage induced by X or y rays, and because of the possibility of differentially affecting the target tissue by exploiting, for example, differences in recovery rates. Also, there has been investigation of the use of neutrons for mutagenesis in plant breeding and crop improvement. Accurate determination of RBE requires accurate dosimetry, which in turn requires a knowledge of energy deposition on a microscopic scale in the target tissue. T h e difficulty of such "micro-dosimetry" often comes as a surprise to the biologist, though doubtless not to the physicist. Unfortunately, m a n y of the

early studies of biological effects of neutrons were confounded by gross dosimetric errors. And the lack of adequate knowledge of microscopic dose distribution is a continuing problem for RBE determinations. Part of the difficulty lies in the different mechanisms of energy loss of neutrons of various energies. This is not a particularly serious problem for mono-energetic beams of high energy fast neutrons, which lose most of their energy through the production of recoil protons in elastic collisions with hydrogen nuclei. It becomes serious, though, for the broad spectra of fast neutron energies produced by nuclear weapons or reactors, and is an almost impossible problem for thermal neutrons which lose their energy through capture reactions with various elements. O f particular importance is the micro-distribution of high cross-section trace elements, especially boron. EARLY STUDIES

Studies of the biological effects of neutrons were started soon after their discovery in 1932. WmTINO040) was the first to report data on mutagenesis. H e irradiated Habrobracon males with fast neutrons from E. O. Lawrence's cyclotron, and observed dominant lethality among their progeny. Shortly thereafter, NAOAI and LOCHER(94,95) reported the production of sex-linked recessive lethals in Drosophila mdanogaster by fast neutrons generated by y-irradiation of beryllium. T h e y also reported an apparent difference from X or y ray mutagenesis, a clustering of the neutron-induced mutants. At about the same time, SNELL (128) and SNELL and AEBERSOLD(129) reported dominant lethality and translocation production in male mice subjected to testicular irradiation with neutrons from the Berkeley cyclotron. SNELL and AEBERSOLD(1~9) noted, furthermore, that the neutrons appeared to be 5-6 times as effective as X rays (based on dose measurements with-a Bakelite R-meter chamber) in producing dominant lethals. Snell also looked for, and failed to find, any recessive mutations among the F 1 mice,

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but the number of animals was very small. TIMOFEEFF-P~ESSOVSKY and ZIMMER,(13s,139,147) however, studying the production of sex-linked recessive lethals in Drosophila melanogaster, reported that although fast neutrons did induce recessives, they were only about two-thirds as effective as X or y rays, based on ionization chamber measurements. There followed a period during which a number of Drosophila experiments done at various laboratories appeared to confirm Timofeeff-Ressovsky and Zimmer's low RBE values for fast neutrons. Thus DEMPSTER(2s) found that the RBE for sex-linked recessive lethals was about 0.75. He also found an RBE of only about 1-5 for dominant lethals and of about 1.25 for translocation production. KAUFMANN and collaborators,(~L el) also reported an RBE of less than one for sex-linked recessive lethals, and an approximately equal effectiveness of X rays and fast neutrons for translocation production as determined by examination of F 1 larval salivary glands. A little later FANO and D~.MEREC (~e,45,46) reported an RBE of about two for dominant lethal production and of less than one for sex-linked recessive lethals. FANO also tested NAGAI and LOCHER'S(95) finding of a nonrandom distribution of the neutron-induced sex-linked recessive lethals, which had apparently been confirmed in experiments by NXSmNA and MORIWAKI(99) but failed to find any significant difference in distribution between those induced by fast neutrons and those induced by X rays. In a limited test of the effectiveness of fast neutrons in inducing sex-linked recessive lethals in Drosophila, GILES(49)found the neutrons less effective than X rays. EBERHARDTO1)studied the production of cubitus interuptus mutants (thought to be single chromosome breaks) by fast neutrons in Drosophila and reported an RBE of less than one as compared with X rays. CATSCH, PETER and WELT0S) also reported an RBE of less than one for the induction of translocations between chromosomes two and three. By the end of World W a r II, the Drosophila data all seemed to confirm RBE values for fast neutrons of no more than two for dominant lethal production, of one or less for translocations, and of less than one for sex-linked recessive lethals. The sex-linked recessive lethal results

were in fact frequently taken as evidence that point mutations were caused by single ionizations, the lesser effectiveness of neutrons being explained as caused by wastage of ionizations because of their high density along recoil proton tracks.(Sg) During the same period, however, a rather different picture was emerging from direct cytological study of chromosomal aberration production by fast neutron irradiation. In an early series of papers, MARSHAK and collaborators(73,74,76,78) reported chromosomal aberration production in both mitotic and meiotic plant cells irradiated with fast neutrons. The cells were scored for the presence of anaphase bridges and fragments at various times after treatment, and RBE values ranging from 2-5 to almost 7 were found, depending on both the post-irradiation interval and the particular experimental material. Similarly high RBE values for fast neutrons were also reported by NISHINA, SINOTO and SATO(100) from less extensive studies of anaphase abnormalities in Vicia faba root tips. Somewhat later, GILES(4°,50) and THODAY(lsS) conducted more detailed investigations of fastneutron-induced metaphase chromosomal aberrations in Tradescantia microspores. Both authors noted that although the aberrations produced were of the same types, there were several important differences between the effects of fast neutrons and those of X or y rays: the neutrons were m u c h more efficient on the basis of doses measured with small Bakelite R-meter chambers (even when corrected for a 2-2-5 fold greater ionization in tissue than in air), and the doseresponse curves for two-break aberrations were linear, in contrast to the approximately dosesquare curves obtained with low L E T radiations. Giles also noted that there was no intensity factor, such as had been demonstrated for X rays, for the induction of two-break aberrations by neutrons. In studies with neutron beams of different mean energies, GXLES(5°) found those with the lower energy to be significantly more efficient. H e also demonstrated that neutron irradiation resulted in a lower ratio of inter- to intrachanges than did X rays. These results had obvious bearing on the mechanisms ofchromosome breakage by ionizing

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW radiation and of the rejoining of the broken chromosome ends to form exchange aberrations. In fact, as was pointed out by both Giles and Thoday, they would be expected from consideration of the high L E T of the recoil protons from fast neutron irradiation, provided that chromosomal breaks were induced by multiple rather than single ionizations, and provided that most multiple-break aberrations induced by low L E T radiation resulted from statistically independent ionizing events. These early studies of the relation of chromosomal aberrations production to L E T were thus very important in the development of the still-accepted theories of chromosomal aberration. Nevertheless, the cytological studies of aberration production in Tradescantia were difficult to reconcile with the then existing Drosophila results, especially those on translocation production, which appeared to show an RBE no greater than one for this type of chromosomal aberration. It is interesting that MULLER and VALI~NGIA(91) in a brief abstract some years later called attention to the meaning of the linear curves observed for translocation production in Drosophila, and also noted that there was, as Muller liked to put it, concatenation of visible and recessive lethal mutations with neutrons but not with X rays. This, of course, suggested that the mutations caused by neutrons were frequently really chromosomal aberrations, and thus that higher RBE values might be expected than had actually been reported. W E A P O N S ' TESTS

The explosion of the atomic bombs at Hiroshima and Nagasaki in 1945 naturally created a great practical interest in possible novel effects of the radiation from such weapons. At first, much of the interest was focused on somatic, primarily medical, effects, but even in the very early post-war testing period some genetic work was done. Seeds of corn, barley, wheat and oats were included among biological specimens exposed to a nuclear detonation at Bikini Atoll in 1946 as part of Operation Crossroads. The exposed seed, as well as parallel seed samples exposed to known doses of X rays, were used to study pollen abortion, seedling leaf mottling and chromosomal aberrations in microspore

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mitosis. The results of these studies were reported in a series of papers by ANDERSON,(2,8) RANDOLPH(I04,105) and SMITH.(127) No striking differences were observed between the X-rayed samples and those exposed to the b o m b detonation for any of the biological endpoints. These early tests of the effects of weapons radiation were followed a few years later by a much more extensive and detailed series of investigations of a variety of biological specimens exposed during nuclear detonations. A primary interest was to establish more carefully whether the various biological endpoints responded quantitatively in the same manner to the weapons radiations as to the various radiations administered in the laboratory; that is, to calibrate and test the biological systems and determine whether they could be used as dosimeters.(1~) While the T ray experiments did not present any real problems, the difficulties of neutron dosimctry were formidable. This led to extensive laboratory calibrations with neutrons, and contributed to important developments in neutron physical dosimctry. T h e result was that much more accurate R_BE values were established for genetic effects, and m a n y of those reported earlier were shown to be too low. The results of these investigations were reported in a series of papers in 1954. Included among the genetic endpoints were recessive lethal mutation in Neurospora,(4) dominant lethal, sex-linked recessive lethal and visible mutation in Drosophila,(6,6°,se, s~) visible mutations in the wasp Mormoniella,(s2) translocations and other chromosomal rearrangements in Drosophila(7°, 134) dominant lethal mutation in mice,(ll7) pollen abortion and chromosomal aberrations in microspores in Datura(132,x45) and chromosomal aberrations in Tradescantia.(l~,65) SHEPPARD et al.(x26) later revised the dose estimates for some of the laboratory neutron exposures done in O a k Ridge. For more detailed discussion of the results of these experiments, as well as more recent ones, it is convenient to consider the various genetic endpoints separately. However, the later weapon tests and the laboratory neutron exposures done in connection with them m a y be summarized in a very general way. T h e results with the

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various genetic systems established that there is no difference between the effects of neutrons from the detonation of nuclear weapons and those from laboratory sources producing neutrons of comparable energy distributions. Nor are there any special effects caused by the extremely high dose rates from weapons. T h e y also established that the RBE for genetic effects induced by fast neutrons is always greater than unity, and that the RBE is greater for chromosomal aberrations than for gene or point mutations. These points will be more thoroughly discussed in the succeeding sections.

FAST NEUTRONS

The importance of L E T in determining the biological effects of ionizing radiation has, of course, long been recognized.049) Early studies with neutrons demonstrated that biological effects which gave sigmoidal or power-function curves with radiations of low L E T tended to yield linear curves with neutrons.(49, is°) RBE determinations present no problem when the dose-effect curves for the two radiations being compared are linear. RBE is simply the ratio of the two slopes. Biological dose-effect curves are rarely strictly linear, however, and even curves for genetic endpoints usually thought of as having linear kinetics often have a higherorder component. RBE values are thus somewhat dependent on dose for virtually all genetic effects, and are, of course, completely so for effects having nearly pure multiple-hit kinetics with low L E T radiations.

A. Mutation Microorganisms. Although there have been a number of investigations of the efficiency of killing of microorganisms by neutrons, as part of general investigations of the influence of LET, there is very little information on neutron mutagenesis in microorganisms. ATWOOD and Mug~I(4) determined the frequencies of recessive lethals in a Weurospora heterokaryon after exposure to neutrons from two nuclear detonations and from a cyclotron and compared them to results from 6°Co y rays and from X rays. T h e y found the effectiveness of the different treatments to be roughly comparable, but

precise RBE estimation was not possible. Data on RBE for neutron mutagenesis in microorganisms would be of interest because, as pointed out by KONDO(eT) and by SPARROW,(13°) it appears from comparative survival data and from data on mutagenesis by heavy ions and alpha particles that microorganism m a y show much less L E T dependence than higher organisms, and that the RBE for neutrons of very high L E T might go below unity. Insects. As noted already, early work on neutron mutagenesis in Drosophila seemed to show that RBE values for recessive lethal and visible mutation were less than unity. Later work has however shown this conclusion to be in error. MICKEY and YANDERS(86,87) found that neutrons from a nuclear detonation and from the Oak Ridge 86 in. cyclotron were more efficient than X rays in inducing sex-linked recessive lethals in D. melanogaster sperm, with an RBE of about 2. IvEs, LEVINE and YOST(6°) also found an RBE of about 2 for simple sexlinked recessive lethals (those not associated with detectable chromosomal rearrangements) in sperm exposed to a nuclear detonation. It appears, however, that the neutron dose estimates for at least some of Mickey and Yanders' experiments were too low, and that the RBE value should consequently be somewhat lower.(35,~2s) EDINGTON(35) determined an RBE of 1.62 for fast neutrons from the O a k Ridge 86 in. cyclotron in relation to 6°Co Y rays for the induction of sex-linked recessive lethals in D. melanogaster sperm. He also studied the effect of X rays, but because of a significant nonlinearity in his X ray curve, as compared with the linear dose-effect curves for the neutrons and g rays, a precise RBE for neutrons vs. X rays could not be determined; it varied from about 1.1 at low doses up to a m a x i m u m of 1.6. Later experiments compared the induction of sex-linked recessive lethals in D. melanogaster sperm irradiated with 14.1 M e V neutrons from the D, T reaction and with e°Co y rays. An RBE of only 1.23 was foundOS) as compared with the 1.62 found for the cyclotron-generated neutrons (estimated average of 1 MeV) used earlier. As cited by DUBININ,(30) Glembotskii, Abeleva and Lapkin found an RBE of 1"5-2 for sex-linked

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW recessive lethal mutations in D. melanogaster males irradiated with fission neutrons. ZIMMERING, OSTER and MULLER(14s) also reported briefly that they had obtained an RBE of about 2"5 for sex-linked recessive lethals in D. melanogaster sperm irradiated with fission-spectrum neutrons. The frequencies of sex-linked mutations in D. melanogaster males treated with X rays or neutrons from deuteron bombardment of a lithium target were studied by D A U C H et al.t24) at various times (broods) probably corresponding to irradiation of stages from spermatogonia through mature sperm. They found RBE values ranging from 1.16 for the most mature cells to a high of 2.21 on the second day after irradiation and back down to 1-25 by the fourth day. The average RBE for the whole testing period was 1"52. The RBE for second chromosome recessive lethals induced in D. melanogaster males irradiated with X rays or with neutrons from a reactor (the average energy of the neutrons was estimated to be 0.7 MeV) was measured by LAMB, M c S H E E H Y and PURDOM.(69) Two germ cell stages, pre-meiotic and post-meiotic, were studied. For the pre-meiotic gonia the RBE was 2"05, while for spermatids the RBE was 2.24. The difference is not statistically significant. MICKEY(se) also studied the production of recessive visible mutations in the third chromosome of D. melanogaster by nuclear detonation and cyclotron-generated neutrons. He found RBE values for eight specific loci ranging from 1"0 to 16"9 when compared with X ray results. The differences were not significant, however, and the averages for the cyclotron and the nuclear detonations were respectively 4.0 and 4.5. Presumably these values, like those for the sex-linked lethals should be revised downward because of the under-estimation of the neutron doses noted by SHEPPARD et al.(1~5) In their tests of male D. melanogaster exposed during the nuclear weapons tests, IVES, LEVlNE and YOST(e°) also measured the frequency of autosomal recessive visible mutations in a specific locus test. Again, they found an RBE of approximately 4. The induction of visible mutations by neutron irradiation of another insect, MormonieUa vitripennis, was also studied during the nuclear

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weapons tests.(62) Females were irradiated, and the progeny scored for eye color mutants. Since the dose-effect curves for X rays were not completely linear, an exact RBE value cannot be calculated; at various doses the values appear to range between about 2 and about 20. An "average" value of 4-5 does not seem unreasonable on inspection of the curves, however. Since the eye color mutants were not analysed further, it is not known what fraction were associated with chromosomal rearrangements. The RBE for recessive visible mutation at two specific loci in spermatogonia and oogonia of silkworms exposed to 14.1 MeV or to fissionspectrum (average of about 1 MeV) neutrons as compared with 137Cs y rays was determined by MURAKAMI and collaborators.C92,93) The RBE for the 14.1 MeV neutrons varied from 1 to 3 depending on the developmental stages of the gonia at the time of irradiation. For the fission-spectrum neutrons the RBE varied from about 2 to about 4, again depending on the developmental stage. Curiously, the dose-effect curves for both the neutron energies as well as for the y rays were distinctly non-linear, and the RBE values are calculated from the dose needed for a given level of effect, rather than from the ratio of slopes. Dominant lethal mutations, thought to be caused largely by chromosomal rearrangements have also been studied extensively. As already pointed out, the early neutron work showed that the RBE for this class of mutations was higher than for recessive lethals or visibles. In connection with the nuclear weapons effects tests, BAKER and VON HALLE(5) determined dominant lethal RBE values in D. melanogaster sperm irradiated with neutrons from either the detonations or from the Oak Ridge 86 in. cyclotron. Because of non-linearity of the X ray dominant lethal dose-effect curve, exact RBE values could not be established; they ranged from about 7.3 for low doses to about 4.8 at high doses. Because of the dosimetric errors reported by SHEPPARD el al.~l~s) these values were revised downward to about 4.5 at low doses and to 2"9 at higher doses. These authors also present additional data on dominant lethality obtained by Edington with the same cyclotron facility. Edington's curve is in good

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agreement with the corrected curve of Baker and von Halle. EDINOTON and RANDOLPH(36) determined RBE values for D. melanogaster dominant lethals induced by 14.1 M e V D, T neutrons. Again, because of the non-linearity of the X or g ray dose-effect curves, a single RBE cannot be given. Edington and Randolph found the RBE for 50 per cent effect, and in relation to their g ray curve, to be 2-27. They also give the RBE in the same terms for the earlier cyclotron studies cited by Sheppard et al. as 4.23. ALEXANDER(a) studied the induction by fissionspectrum neutrons of dominant lethals in a different species of Drosophila, D. virilis, in relation to the stage of germ cell development at the time of irradiation. She found RBE values (calculated for 50 per cent effect because of the non-linearity of the X ray curves) ranging from 6-6 for mature sperm down to a low 1-6 for meiotic ceils. Another class of dominant mutations in Drosophila that are thought to consist mainly of small chromosomal rearrangements are the Minutes. These mutations display linear X-ray dose-effect curves, and have been used by several groups for RBE determinations. MICKEY(ae) found RBE values of from 4.6 to 9.8 in his tests of the effects of nuclear detonation and cyclotron neutrons. IVES, LEVINE and Yosw(e0) also reported that the RBE for Minutes induced by fission-spectrum neutrons was of the order 3.5-5 relative to X rays. Mice. RUSSELL and KELLy(ala,alS) studied the RBE for the induction of recessive visible mutations in the spermatogonia of mice irradiated at various dose rates with fission-spectrum neutrons. Because there appeared to be no difference in mutation yields from different dose rates for neutron irradiation (unlike the case for low L E T radiation in mouse spermatogonia) they calculated the RBE from the combined data for all dose rates. The value, compared with acute X rays, was 5.8. RUSSELL(lla) calculated that the RBE for chronic exposures would be about 19, since the difference between acute and chronic X ray mutation rates had been found to be about 3.3. Using data from an additional fission neutron experiment at low dose rate he found an RBE of

18.1 in good agreement with the calculated value. In a series of papers BATCHELOR, PHILLIPS and SEA~LE(s-8,122-124) reported the results of similar specific locus mutation studies with mouse spermatogonia irradiated at both low and high dose rates with fission-spectrum neutrons. For chronic neutron exposures they found RBE values of about 23 when compared with chronic X ray exposures and of about 5 when compared with acute X ray exposures. These values are in reasonable agreement with those reported by Russell. Although the chronic neutron exposures in the experiments of both the Oak Ridge and the Harwell groups gave essentially linear doseeffect curves for recessive visible mutation, both groups noted that mutation yields were lower for high doses of neutrons delivered at high dose rates than for lower doses, giving rise to a "reverse dose rate effect" for ~eutrons, possibly caused by germinal selection against more sensitive cells among the spermatogonia given high acute doses of neutrons.(a,xla,n4,124) Batchelor et al. also measured the yields of dominant visible mutations induced by chronic fission-spectrum neutron irradiation of spermatogonia.(~, a~4) An RBE of about 20 was calculated in comparison with chronic y irradiation. Searle and Phillips also reported a relative ineffectiveness of large, acute doses of neutrons for inducing dominant visibles similar to that already described for recessive visibles.(TM) T h e induction of dominant lethals in the mouse was one of the first examples of neutron mutagenesis reported. As already mentioned, SNELL and ASBERSOLDCI2g) found an RBE for fast neutrons of about 5. RUSSELL and collaboratorsOa6,uT) studied the induction of dominant lethals in mouse sperm by neutrons from nuclear detonations and from the Oak Ridge 86 in. cyclotron. T h e y found no difference between the b o m b and the cyclotron neutron effectiveness, and an RBE value when compared with X rays of about 8. This value was, however, revised downward to about 6 following the dose corrections reported by SHEPPARD et al.(1~5) POMERAm'SEVA and RAMADA(a03) also reported data from fission-spectrum neutron irradiation of mouse sperm, as well as data on neutron

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW irradiation of spermatids. T h e y found an RBE value of only about 3"7 for the irradiated sperm and about 2-5 for spermatids. SEARLE and PmLLIPS,~122) however, also studying dominant lethal frequencies in sperm irradiated with fission-spectrum neutrons at low dose rates, found an RBE of about 6, in agreement with Russell's earlier results. RUSSELL et al.O12) compared the effectiveness of 14.1 M e V D , T neutrons to that of the neutrons in their earlier experiments on dominant lethality in mouse sperm.OXs,lXT) T h e y found that the 14.1 M e V neutrons were only about one-third as effective as the b o m b and cyclotron neutrons, that is, the RBE for these higher energy neutrons is only about 2. Plants. All of the investigations that have been made of fast neutron mutagenesis in plants have involved somatic mutation, virtually all with seed irradiation. Most work has been done with barley and wheat. The early weapons test studies already mentioned with corn and several cereal species established that fast neutrons were much more efficient than X or ,( rays, and that RBE values for somatic mutation in seeds were considerably higher than those for mutation in animal ceils. GUSTAFSSON and MACKEYOS,~I,7*) irradiated barley seeds, either dry or pre-soaked, with fast neutrons from a cyclotron deuteron beam on a beryllium target. They used a neutron activation [*TAl(n,~)*4Na] method for dosimetry, arriving at a unit called dis, which according to M a c K e y was equal to 1/42 dV unit or about 1/17 R units. It appears from the RBE values obtained in these early barley experiments in comparison with those from later investigations that these dose calculations were fairly accurate. For chlorophyll mutants detected in the second generation (M,), they found an RBE of approximately 70. Continuing the work on barley, EHRENBERO and collaborators(s7-41) further refined the relationship of the dis unit to the Roentgen, and concluded that the RBE for chlorophyll mutations was " a t least 20". Fast neutron mutagenesis was studied by MATSUMURA(s0-s2) using wheat seeds. T h e neutrons used were from the D , T reaction, having an energy of 14.1 MeV. In diploid

233

wheat he found an RBE for chlorophyll mutants detected in the M s generation of about 10, while, as expected, there were very few chlorophyll mutants from species of higher ploidy, and no RBE could be determined. Fuju~ 47) also studied fast neutron mutagenesis in wheat seeds. Using tiller striping in the M 1 plants grown from irradiated seeds heterozygous for a chlorophyll mutant of the chlorina type, he found the RBE for 14.1 MeV D , T neutrons to be at least 20 as compared with e°Co g rays. The RBE values for mutation were measured by Fuju(4s) at a specific locus in Arabidopsis thaliana seeds irradiated with either 14.1 MeV D , T or fission-spectrum fast neutrons in comparison with "t" rays. For both neutron sources he obtained values of about 15-16 at the level of 1 per cent mutants. A single RBE figure could not be given, however, because the g ray dose-effect curves were of a clearly different shape than the neutron curves. Using somatic mutation in the stamen hairs of Tradescantia flowers DAVIES and BATEMAN(25) determined the relative effectiveness of 0-65 M e V fast neutrons from the 3H (p,n)3He reaction as compared with X rays. Again, since the X ray curve showed a large dose-square component, while the neutron curve did not, RBE was dependent on dose. At the arbitrary level of 15 mutations per flower, the RBE was about 17.5. Davies and Bateman calculated the m a x i m u m RBE to be about 40 (for chronic exposures, where the low L E T dose-square component would be minimized). Mutation spectrum. One of the obvious questions about the genetic effect of neutrons is whether the proportions of various types of mutants is the same when they are induced by neutrons as when they are induced by X or -( rays. Since the RBE for the induction of chromosomal aberrations in animal cells is higher than that for gene mutations (see succeeding section), one might anticipate that " m u t a n t s " in such categories as sex-linked recessive lethals in Drosophila might more frequently be deletions or other chromosomal rearrangements if induced by neutrons. This, in fact, appears to be the case.(e°, sg) Similarly, the proportion of dominant lethals, for example, among all mutants will be greater following neutron

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irradiation than following y irradiation. Aside from this sort of effect, however, there seems to be little evidence for a different spectrum for fast-neutron-induced mutations. B. Chromosome aberration Plant cells• Unlike the early work on fast-

neutron-induced mutations, the first work on chromosomal aberrations elucidated the important aspects of their induction by neutrons. As already mentioned, in a series of papers starting in 1939 MARSHAK and collaborators(~) established that the RBE for anaphase chromosomal aberration production in plant and animal cells ranged from 2.5 to about 7 depending, in part, on the stage of the cell cycle irradiated. GILES(49,s°) and THODAY(Iz~) then established a number of essential points about the neutron induction of metaphase chromosome aberrations in Tradescantia. These included high RBE, lack of an intensity factor, linear dose-effect kinetics for two-break aberrations, and dependence of RBE on neutron energy. The later weapons test studies of cytogenetic effects of neutrons did little but confirm some of these facts. In particular, the experiments of CONGER(17)and KIRBY-SMITHand SWANSON(65) with Tradescantia microspores and those of SPENCER and BLAKESLEE(lz2) and YOST, CUMMINOS and BLAKESLEE(145) confirmed high RBE values for fast neutrons. GILES and CONOER(51) extended Giles' earlier observations in experiments with Tradescantia microspores exposed to fast neutrons in the Oak Ridge Graphite Reactor. They estimated the average neutron energy at about 1.0 MeV, lower than the averages for the two neutron. sources used by GILES(5°) in the experiments that established the dependence of RBE on neutron energy. They observed that the RBE was even higher for the lowest energy neutrons and also that the ratio of inter- to intrachanges was even lower. KIRBY-SMITH, SHEPPARD and CRAIG (66) irradiated both dry pollen and inflorescences of Tradescantia with neutrons from the Oak Ridge 86 in. cyclotron. T h e y found RBE values of 8.0 for the pollen grains and I0.0 for microspore divisions in the influorescences, but these values should probably be revised downward

somewhat because of the dosimetric problems discussed by SHEPPARD et al.(125) A more extensive investigation of the relation between RBE and L E T for 1.3, 2"5 (D,D) and 14.1 (D,T) M e V fast neutron-induced chromosomal aberrations in Tradescantia microspores was conducted by CONOER et al.( TM Careful calculations were made of the relative contributions to dose, L E T and RBE of the heavy particles generated by the various reactions of the D , T neutrons other than elastic collisions with hydrogen.(l°s, l°s) The relation between L E T and RBE was calculated for both chromatid and chromosome type aberrations, using both track average and energy average L E T values for the various radiations• 049) Because of the difference in doseeffect kinetics between the low L E T X and y rays and the neutrons used in the experiments, RBE values were calculated at the 50 per cent effect point for each radiation. It was found that the RBE's for chromatid and chromosome type aberrations agreed, ranging from about 3.5 for the 14.1 MeV neutrons, through about 6.5 for the 2"5 M e V neutrons, to about 7 for the 1.3 M e V average energy cyclotron-generated neutrons. When plotted against track average L E T the RBE values generated a smooth curve, increasing with increasing LET. When plotted against energy average L E T , however, the RBE for the 14.1 MeV neutrons was much lower than that for the lower energy neutrons. This resulted from the great contribution of the heavy particle component to the energy average LET. Conger et al. concluded that the track average method of L E T calculation is more realistic, biologically, in cases where an appreciable fraction of the dose is delivered by extremely densely ionizing particles. T h e y also concluded that for neutrons of energies above about 5 M e V the contribution to L E T of heavy particles is great enough so that this component should be considered separately from the hydrogen proton component• When Conger et al. did consider the RBE for this heavy component separately, and also considered data on aberrations induced in Tradescantia microspores by Rn ~ particles previously reported by KOTVAL and GRAY,(6s) they found that the RBE versus L E T curve rose smoothly from unity at a track average of 0.27 •

.

,

•

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW KeV[~ (e°Co7 rays) to a peak (extrapolated) of about 10 in the range between 50 and 70 KeV/[x, and then falls off to a value of about 5 at around 120 KeV/lx remaining at that value up to over 200 K e V / ~ (the L E T calculated for the 14.1 M e V neutron irradiation heavy particle component). Such a curve shape might be predicted of course, as it is the sort of curve generated when the probability of producing an aberration increases faster than the number of particle traversals of the nucleus up to some point, but increases less rapidly beyond that point (ionization "wastage"). SMITH et al.O~6) further investigated the influence of fast neutron energy on a cytogenetic effect in corn. T h e endpoint used, leaf sectoring, is believed to reflect chromosomal deletion, and exhibits essentially linear dose-effect kinetics even with low L E T radiation. Corn seeds were irradiated with essentially monoenergetic neutrons with energies of 0-43, 0.65, 1.00, 1.50 and 1.80 MeV by taking advantage of the angular distribution of neutron energies around a tritium target bombarded with 2"8 MeV protons. The slopes of the dose-effect curves were compared with those obtained with X rays. The RBE values thus derived ranged from 42 to 135, the lower neutron energies giving the higher RBE values. Animal cells. During the testing of the biological effects of neutrons from nuclear weapons detonations a n u m b e r of groups determined RBE values and dose-effect kinetics for the production of chromosome aberrations in Drosophila. STONE et al.Oa4) measured translocation production in D. virilis following exposure to neutrons in the weapons tests. In addition to confirming the essentially linear dose-effect kinetics noted by CATSGH, PETER and WELT,(15) they found the neutrons considerably more efficient than X rays, with RBE values ranging from about 7.5 at low doses to about 3 at high doses. Chromosomal rearrangement production in D. melanogaster was measured by LEWIS,(7°) using the " b i t h o r a x " method. H e compared the effects of fast neutrons from the weapons tests and from a nuclear reactor with those of X and y rays. Again, he found the neutrons to be much more efficient, with an RBE of the order of 9. MULLER(90) reported the results of a series of

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experiments on translocation production by fast neutrons in D. melanogaster sperm. He found that uranium fission neutrons and the neutrons from the O R N L 86 in. cyclotron were of comparable efficiency, and much more efficient than X rays. Because of the linear dose-effect kinetics for neutrons no overall RBE could be calculated, but at the 10 per cent effect level the neutrons were approximately twice as effective as X rays. ALEXANDER(1) studied the production of translocations in D. virilis by uranium fission neutrons. Males were irradiated, and premeiotic, meiotic and postmeiotic cells sampled. Linear dose-effect kinetics were observed for all stages, and the RBE value for mature sperm appears comparable to that observed by Muller. A distinct stage sensitivity was observed, the least mature cells being the least sensitive. More recently DAUCH et al.(~4) reported experiments on translocation induction by fast neutron irradiation of D. melanogaster males. Again, linear dose-effect kinetics were observed, and a distinct stage sensitivity demonstrated. RBE values relative to X rays ranged from a high of 3"27 to a low of only 1"65 when calculated on the assumption of linear dose-effect kinetics for the X rays. Interestingly, much of the RBE variation in successive broods was caused by changes in the X ray sensitivity rather than the neutron sensitivity. Dauch et al. suggested that the stage sensitivity changes, rather than dosimetric errors, might have been responsible for the low relative efficiencies of neutrons reported by early Drosophila workers. SEARLE, EVANS and WEST(121) have reported studies of the induction of translocations in mouse spermatogonia by neutrons. Spermatocytes were examined cytologically following either chronic or acute exposures to fission neutrons. They found that while the yield of translocations increased in approximately linear fashion up to about 100 rad of acute exposure, the yields actually became lower with higher doses. In contrast, with 12 week chronic exposures there was no evidence for such an effect. For acute exposures below 100 rad of neutrons, Searle et al. calculate an RBE of about 3-7. For chronic exposures they estimate the RBE to be 20-25. Anaphase chromosome aberrations were

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studied by CURTIS and coworkersOg,~2.2s) in the regenerating livers of mice previously irradiated with fission neutrons. They found not only that neutrons were much more efficient than X rays in producing these abnormalities, but also that their frequency was relatively independent of the time intervening between irradiation and examination, unlike the case with X irradiation, following which aberration yields decreased with increasing time. They also observed that chronic or fractionated neutron exposures, again unlike the case for X irradiation, were just as effective as acute ones. NO'C/ELLel al.OOa) on the other hand, did find an apparent dose rate effect for neutron-induced chromosomal aberrations in mouse liver, lower dose rates giving lower aberration frequencies. However, CONGER and CURTIS(19) have pointed out that this apparent effect might have been caused instead by the different average energies of the neutrons used for the acute and the chronic exposures. The induction of chromosome aberrations in human peripheral leukocytes irradiated in vitro with acute doses of fast neutrons of various energies was studied by GOOGH, BENDER and RANDOLPH.(54) For single-break deletions, which display essentially linear dose-effect curves for both low and high L E T radiations, they obtained RBE values relative to X rays of 2.6 for 14.1 MeV (D,T) neutrons and about 4.5 for 2"5 MeV (D,D) neutrons. They found the expected linear dose-effect relationship for the induction of two-break ring and dicentric chromosomes by 2"5 M e V neutrons, but the data for the induction of this class of aberrations by 14.1 MeV neutrons could be equally well fitted by either a linear or a dose-square model. Assuming the dose-square kinetics for 14.1 MeV neutrons, the calculated RBE for rings and dicentrics was only 1.4. Preliminary results for fission-spectrum neutrons(9) in the same h u m a n leukocyte system yield an RBE of about 4.5 for deletions. The ring and dicentric data fit a linear model with a slope somewhat greater than that for their induction by 2-5 M e V neutrons. GOOCH, BENDER and RANDOLPH(54) also made estimates of fission neutron RBE for peripheral leukocytes irradiated in vivo, using the available data on chromosome aberrations in three men exposed in the Recuplex criticality

accident (BENDER and GOOCH(9)). These estimates ranged from 2.5 to 10.2 for the three subjects, leading the authors to conclude that the true RBE for in vivo irradiation is probably of the order of 5-6. SCOTT et al.01,,120) have also reported experiments in which h u m a n peripheral leukocytes were irradiated with both acute and chronic (6"75 or 3.41 rad/hr) doses of fission neutrons. An unusual feature of the work of these authors with the h u m a n leukocyte system is that, in contrast to the results of others, they obtain virtually linear dose-effect kinetics for twobreak aberrations induced by low L E T radiations. The reason for this peculiarity is unknown, but it does allow straight-forward calculation of RBE from the ratio of slopes. Scott et al. found no dose rate effect with neutrons for twobreak aberrations (although, curiously, they do for low L E T radiation). The RBE value was approximately 3.5. They did not calculate an RBE for one-break aberration production, but rough estimation from their tabular data yields a value of approximately 4.5 similar to that found by BENDER and GoocH.~ 9) C. Modifying factors Neutron energy. The work of GILES,C~0) GILES and CONGER,(5x) CONGER et al.,(~x) GOOCH, BENDER and RANDOLPH,~~4) SMXTHet al.OZe) and BENDER and GOOCHO) with neutron fluxes of different average energy clearly established a relationship between fast neutron energy (more properly average LET) and efficiency for chromosomal aberration production. The studies Of EDINOTON and RANDOLPH,(s~) Of MURAKAm and KONDO(92) and MURAKAMI, KONDO and TAZIMA,(9s) and of RUSSELL et al.Cne) and RUSSELL, RUSSELL and KIMBALL(I17) established the same sort ofenerg'y dependence for mutations of various sorts. In addition, the work of CONGER et al.~21) called attention to the increasing contribution to average L E T of the heavy particles generated by inelastic collisions with C, N and O nuclei at neutron energies above about 5 MeV. In view of this demonstrated energy dependence, which Conger et al. estimated to be as much as 2.5-fold over the L E T range they studied, and of the great variation in the neutron energy spectra of the neutron fluxes used

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW in the various studies of fast neutron mutagenesis, it is perhaps not surprising that the RBE values reported in the literature show substantial variation for the same biological endpoint. Dose rate andfractionation. Dose rate is expected to affect the yield of biological effects that depend on multiple ionizing events, except in unusual systems (such as Drosophila sperm, in which chromosome breaks remain open until fertilization). I t is well known, for example, that while acute and chronic (or fractionated) doses of low L E T X or "~ rays give essentially the same yields of single-break aberrations, they do not give the same yields of two-break aberrations.(43) This is because the "two-hit" component of the two-break aberration yield gets smaller with decreasing dose rate as a result of there being a time limit on rejoining of separately induced chromosome breaks. I f the dose rate is decreased enough, one expects to get no "two-hit" twobreak aberrations at all, and to be left with a linear dose-effect curve of very small slope, representing only those very few aberrations in which both breaks were fortuitously caused by a single ionizing event. Because the breaks involved in multiple-break aberration production are not statistically independent with high L E T radiations such as are produced by fast neutron irradiation, however, no such dose rate dependence is expected. GILES(49,5°) and THODAVOSS) first noted the lack of a dose rate dependence for two-break chromosome aberration production by neutrons. GILES, DESERRES and BEAa~rY¢~3) carried out extensive observations on the effect of dose fractionation in Tradescantia microspores. As expected, they found no decrease in exchange yields with fast neutron dose fractions separated by time intervals ranging from zero to 12 hr. SEARLE, EVANS and WEST(121) have reported a similar lack of dose rate dependence for translocation induction in mouse spermatocytes by fast neutrons, but this is complicated by the falloff in yield they observed with large, acute doses. NOWELL et al.OOl) reported an apparent dose rate effect for chromosomal aberrations in mouse liver, but this was not confirmed by CONOER and CURTIS.tl°) Lack of effect of dose rate has also been confirmed recently for h u m a n leukocytes by SCOTT et al.tX19,l~o)

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As already mentioned, multiple-break aberration yields from low L E T radiation become small at low dose rates, and the dose-effect curves become linear. Thus at very low dose rates the RBE values for neutrons compared to X or "~ rays should become independent of dose and should eventually reach some limiting, very large value. NEARY and collaboratorsOT,gs) have studied the effect of dose rate on fast neutron RBE from this point of view, and have determined the "ultimate" m a x i m u m value of fast neutron RBE for various classes of aberrations induced in Tradescantia microspores. The various RBE values were all high, of the order of 100. T h e y also point out that since the dosesquare component for two-break aberrations induced by low L E T radiation is minimal at very low doses, the RBE should also increase with decreasing dose, possibly approaching the same limiting value as with dose protraction. Although not really expected tbr gene mutations, there is a dose rate effect for mutation production when mouse spermatogonia are irradiated with X or y rays. Both RUSSELL and KELLY,(lls,ll5) and BATGHELOR, PHILLIPS and SEARLE(8-S,I**,1~3) reported that with fast neutron irradiation, on the contrary, there was no such dose rate effect. This difference between low and high L E T radiation response in the mouse leads to RBE values increasing from 5 to 6 for acute exposures to 20 or more from chronic exposures. Oxygen effect. The existence of an oxygen effect for both gene mutation and chromosomal aberration production by low L E T radiation is well known. GILES, BEATY and RILEY(52) investigated the influence of oxygen on the production of chromosomal aberrations by fast neutrons in Tradeseantia microspores. They found that the oxygen effect was still present with neutrons, though it was not as great as with X-rays. By comparison with data of THODAY and READOSe,137) on the effect of oxygen with X rays and with ~ particles, they concluded that the magnitude of the oxygen effect is inversely related to average LET. EHRENBERG et al.,(ag) on the other hand, were unable to demonstrate any effect of oxygen concentration on mutations induced by fast neutron irradiation of barley seeds, though they

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did find the usual oxygen effect when the seeds were irradiated with X rays. Using an indirect indicator of chromosomal aberration production, the frequency of micronuclei, EVANS,NEARYand WILLIAMSON (44) also were able to demonstrate an oxygen effect in Vicia faba root tips irradiated with fast neutrons, though it was smaller than that observed in the same system when irradiated with 6°Co y rays. They pointed out that this differential oxygen effect would have the effect of increasing RBE for neutrons for irradiation under anoxic conditions. CONGER (ls) investigated the effect of oxygen on chromosomal aberration production in an Ehrlich mouse ascites tumor irradiated in vitro with fast neutrons. In agreement with Giles, Beatty and Riley's results, he found the oxygen effect with neutrons, and also that it was much smaller than with X rays. HORNSEY, HOWARDFLANDERS and MOORE(59) later reinvestigated the oxygen effect for chromosome aberration production in Ehrlich mouse ascites tumor cells by fast neutrons and by X rays. Their results were similar to those of CONOER.(ls) At the 50 per cent level they found the RBE in oxygen to be 3.2, while in nitrogen it was increased to 6"5. Other effects in seeds. In addition to the dose rate and oxygen effects already mentioned, the effects of other possible factors have been investigated in neutron-irradiated seeds. These include the effects of polyploidy,(s4) water content,(39) and various pre- and post-treatments.(96) With the exception of seed water content, however, these effects appear to be the same for neutrons as for low L E T radiation. It seems possible that the seed water content effect is actually an oxygen effect.

D. Interaction, site size and saturation In experiments with soaked Vicia faba seeds WOLFF et al.(~42) observed that there was no interaction between fast-neutron- (14.1 MeV) and X-ray-induced chromosome breaks. The yields of two-break exchange aberrations from consecutive doses of the two types of radiation were simply the sum of the yields from either dose alone, rather than the higher yield expected if neutron-induced breaks were free to recombine with those induced by the X rays. This,

plus the linear dose-effect kinetics with neutrons, enabled them to calculate the distance that could separate two breaks and still allow recombination between them (i.e. the site size). They reasoned that since interaction between X ray and neutron breaks was negligible, either the site number must be very large or the site volume must be very small. T h e sites must further be small enough so that they can be fitted into the nucleus in the required number without overlapping; otherwise they would not be expected to be independent. Wolff et al. calculated that for these conditions to be met, the site diameter could not be greater than 0-3~. in Vicia root tip cells. NATARAJANand NARAYANAN(96)carried out a similar experiment with X ray and fast neutron (average energy about 5 MeV) irradiations of barley seeds. T h e y reported significant interaction for exchanges. This suggested that the rejoining distance must be g r e a t e r ' f o r barley than for Vicia, and posed the possibility that the size of the site in barley was different from that in Vicia. The possibly non-linear dose-effect kinetics for 14.1 MeV-neutron-induced exchanges in h u m a n leukocytes reported by GOOCH, BENDER and RANDOLPH(54) suggested a similar difference between species. An attempt was made by HEDDLE(ss) to confirm the interaction observed in barley seeds by Natarajan and Narayanan. Contrary to the prediction from the observation of neutronX ray interaction, he obtained a linear doseeffect curve with 14.1 MeV neutrons. Also, he failed to obtain any evidence for interaction in his repetitions of Natarajan and Narayanan's experiment. It appears likely, therefore, that in barley, as in Vicia, there is no interaction between neutron- and X-ray-induced breaks, and thus no compelling evidence for a site size difference between species. Using preliminary data of REDDIOll) that suggested a significant interaction between X rays and neutrons for the induction of translocations, HEDDLE and WOLFF(5~) calculated that the rejoining distance in Drosophila was similar to that in Vida and barley (0.1tz). Later, however, some question as to the reality of the reported interaction arose.(ss) As pointed out by WOLFF,(TM) the concept

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW of interacdon sites of limited size and number leads to the prediction that two-break exchange dose-effect curves should saturate at high doses. Such a saturation is difficult to demonstrate for low L E T radiation because of the non-linear dose-effect kinetics for exchanges. Recently SAVAGEOZS) has investigated the saturation question using fast-neutron-induced exchanges in Tradescantia microspores, thus taking advantage of the linear dose-effect kinetics expected. He found clearcut evidence for saturation and calculated that the site number for these cells was about 2.7. The calculated site number for X rays, on the other hand, was much larger, leading Savage to suggest the possibility that X ray and neutron sites are somehow different and perhaps independent of each other, thus raising some question as to the validity of site size calculations from non-interaction data.

THERMAL NEUTRONS

Because the effects of fast neutrons (at least up to about 5 MeV) are largely caused by protons from elastic collisions with hydrogen nuclei, dosimetry is relatively uncomplicated (in spite of early difficulties), and the concept of RBE easily understood. This is not true for neutrons of thermal energies, however, because of the different mechanisms by which they deposit energy in tissue. Thermal neutrons react with elements of biological importance largely by nuclear capture reactions. The most important of these are with isotopes of the elements hydrogen, nitrogen and boron. Each forms an unstable isotope which emits a different type of radiation; hydrogen a y ray, boron an particle, and nitrogen a proton (plus a [3 particle later). In addition, of course, the recoil nuclei, such as the lithium nucleus from the l°B (n, 0c) Li reaction, also deposit some energy in the tissue. Finally, there exists the possibility of transmutation effects. The relative amount of the energy deposited in the tissue depends, then, on its atomic composition. Since very high L E T ~ particles are produced through neutron capture by Z°B which has a very high capture cross section, the exact boron concentration in the tissue must be known precisely in order to calculate dose; in addition, because of the

239

extremely short range of the ~ particle in tissue, the distribution of boron in the tissue is critical. CONGER and GILESC~0)studied the production of both chromosome and chromatid aberrations by thermal neutron irradiation of Tradescantia microspores. From an analysis of the elemental composition of Tradescantia anthers, they were able to calculate that the reaction of hydrogen, nitrogen and boron accounted for 99 per cent of all of the ionization in the tissue. Boron, though present at a concentration of only 2.9 p.p.m, contributed some 29 per cent of the dose. T h e y calculated R_BE values for thermal neutrons based on the combined doses from the nitrogen protons and the boron ~ particles relative to X rays. These ranged from 8.8 to 19.9 (using the 50 per cent effect level for cells without aberrations) with a "best" value of 15. They noted that this RBE was reasonable, since although low L E T radiation actually contributed a large portion of all the tissue ionization, the dose-effect curves for exchanges were linear, as had been shown for protons from fast neutron irradiation and for 0cparticles. They noted that this R_BE was some three times higher than would be expected from the earlier determinations of the RBE values for external and fast neutron irradiation. This may be explained at least in part, however, by differences in dosimetry in early experiments. Chromosomal aberrations in root tip anaphases and in meioses of seedlings grown from thermal-neutron-irradiated barley seeds were studied by CALDECOTT and collaborators.CZ~,z4) They noted linear dose-effect kinetics for exchanges, and also that the neutrons produced more chromosomal damage at the same seedling survival level than did X rays. No elemental analyses were made, and no ionization dose estimates attempted. Similarly, SPENCER, SINGLETON and BLAKESLEE(z88) studied pollen lethals, and YOST, SINGLETON and BLAKESLEE(14s) chromosomal aberrations in plants grown from thermal-neutron-irradiated Datura seed. T h e y were unable to detect any difference between the thermal neutrons and X rays in the types of pollen lethals induced ("gene" or "chromosomal") or in the chromosomal aberration types.

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MICHAEL A BENDER

KL~O and WOODteS,64) studied the induction of sex-linked recessive lethals in male and female Drosophila melanogaster by thermal neutrons. From elemental analyses of flies and from feeding experiments which increased their X°B content, King concluded that the contribution of boron a particles to lethal production in Drosophila is negligible, and that the nitrogen protons were primarily responsible for the mutations observed. He found that these protons were approximately 1.5 times as effective as X rays. He also observed that thermal neutrons were only about threequarters as effective in the females as in the males, and suggested that this might reflect a difference in the nitrogen content or distribution in male and female gonads. KAYHARTCe~) irradiated Mormoniella females with thermal neutrons, and observed that the dose-effect curve for visible eye color mutations was linear, but no ionization dose estimates were made. The effects of thermal neutron irradiation on chromosome rejoining capacity were compared with those of X rays in Trillium microsporogenesis by DESCI-INER and SPARROW(29) They found that although similar stage sensitivity changes occurred with the two radiations when given at various times in the cell cycle, the neutrons produced a more uniform breakage effect, regardless of stage in the cell cycle, than did the X rays. Rejoining of broken ends, however, appeared to be similar with the two radiations. Y A G Y U and MORRIS(14s) irradiated tomato seeds with thermal neutrons and obscrvcd anaphase aberrations and seedling mutations. N o ionization dose cstimatcs werc m a d c and the authors did not observe any diffcrcnccs between the effects of the two treatments except for the cxpcctcd linear dose-cffcct kinctics for anaphasc bridgcs induced by thc neutrons as opposed to non-linear kinetics for X rays in root tips from sccds germinated shortly after irradiation. Diploid wheat sccds were exposed by MATSUMURA(8°) to thermal ncutrons. H c thcn studicd both chromosomal aberrations in root tips and seedling mutations. H e observcd linear dosc--cffcctkinetics, but no elcmcntal analyses wcrc attempted, and no ionization dosc calculationscould be made.

BROCKtl0) irradiated

seeds of the

clover

Trifolium subterraneum with thermal neutron and determined seedling mutation frequencies. From elemental analyses of the seeds he was able to estimate RBE for the nitrogen protons and boron a particles in comparison with X rays and obtained a value of about 15. Mice were exposed by CURTIS,TILLEY and CROWLEY¢~n) to either acute or chronic doses of thermal neutrons. T h e y observed that dose rate made little if any difference in the yields of anaphase chromosomal aberrations in regenerating liver cells, as was also the case for fast neutrons. A n u m b e r of authors have treated rice seeds with thermal neutrons and observed chromosome aberrations and mutations.(~9,s6,x°9,n°, 1~4) In no case were ionization dose estimates made. No important differences between the effects of thermal neutrons and low L E T rad~tions were noted. RAO and GOPAL-AYENGAR(l°g)reported a decrease in mutation frequency in seeds stored for 7 days after neutron irradiation before further treatment with diethyl sulphate and subsequent germination. RAo et al.01o) conducted further experiments with a 10-day storage period, during which they measured the -( and ~ ray decay of isotopes created by neutron capture by various elements. T h e effect of the storage on mutation frequency was determined in rice and on chromosomal aberrations in barley. Some classes of mutations showed an increase with post-irradiation storage, but others showed a decrease. The frequency of chromosomal aberrations was increased by storage. The authors concluded that the decay and ~ radiation caused at least part of this effect, largely because of the absence of such an effect of storage following X or y irradiation. Nitrogen protons. One way in which the RBE for nitrogen protons from the X4N (n,p)14C reaction can be studied is to enrich the organism in the isotope XSN which has a low neutron capture cross section, thus depleting the X4N abundance. PITTENOERand ATWOOD(10~)briefly reported such experiments with survival in Neurospora crassa, and ATWOOD and Mur~Aa{*) used the technique, as well as boron enrichment (see below), for the study of mutation production in Neurospora. As expected, the neutron

NEUTRON-INDUCED GENETIC EFFECTS: A REVIEW effectiveness was reduced in the aSN enriched organisms. Boron ¢¢particles. The importance of l°B, which has a much higher neutron capture cross section than the more abundant ~aB isotope has been studied in a number of enrichment experiments. CONGER (18) used X°B enrichment in experiments with thermal neutrons and Tradescantia microspores. The yield of chromatid aberrations was increased by X°B enrichment about 5 times per unit of thermal neutron fluence. From boron analyses of the anthers he was able to calculate that the increase in ionization from the increased I°B content was about sixfold. Using KOTVAL and GRAY'S(6s) RBE values of 14"1 for R a particles he calculated that the biological effect should have increased some 18-fold. He ascribed the difference to anther sterility caused by the higher boron in some buds, a factor which would have made the boron content analyses yield a higher average value for the pooled samples than for the anthers actually used for cytological analysis. MATSUMURA, KONDO and MABUCHI(ss) also used the boron enrichment technique to study the RBE for the boron = particles in experiments with diploid Einkorn wheat seeds soaked in solutions of boron of natural isotopic abundance. They studied both chromosomal aberrations in root tips and pollen mother cells and seedling chlorophyll mutations. They calculated boron ~¢particle RBE values in comparison with lS6Cs y rays of 23 for chromosomal aberrations and 29 for chlorophyll mutations. Enrichment with the l°B isotope was used by MOUTSCHEN et al.(ss) to determine boron c¢ particle RBE values for chromosome aberration production in seeds ofNigella damaseena irradiated with thermal neutrons. Boron analyses of the seeds were carried out and RBE values for both normal and enriched seeds calculated. Although the enrichment did increase the aberration yield per unit neutron influence, they found a RBE relative to ls~Cs y rays of 54 for the unenriched seeds, which fell to about 23 for the enriched seeds. The authors emphasize the difficulty of enriching NigeUa seeds, and suggest that the decrease in RBE with boron enrichment may be due to the normally high boron content of Nigella seeds (about 57 p.p.m., vs. 2"9 for

241

Tradescantia anthers) and probable distribution of much of the boron in the seed coat, or at least far enough away from the nuclei to make its dose contribution ineffective for chromosome breakage. ECOGHARD and collaboratorsO2-s4) have also studied the effect of I°B enrichment in both Vicia and tomato plants irradiated with thermal neutrons. I n Vicia chromosomal aberrations in meiotic divisions were studied; in the tomato experiments mature pollen was irradiated and seedling mutations scored. In both sets of experiments an increase in yield per unit neutron fluence was observed in the l°B enriched cells, the increase amounting to about 20 per cent.

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