On the biological effectiveness of intermediate neutrons: Induction of chromosomal aberrations

Radiation Botany, 1969, Vol. 9, pp. 449 to 458. Pergamon Press. Printed in Great Britain.

ON T H E B I O L O G I C A L EFFECTIVENESS OF I N T E R M E D I A T E N E U T R O N S : I N D U C T I O N OF C H R O M O S O M A L ABERRATIONS G. AHNSTROM, L. EHRENBERG, A. T. NATARAJAN and C.-G. ROS~N Department of Biochemistry, University of Stockhobn, Sweden

J. and M. MOUTSCHEN-DAHMEN Laboratoire G~ndtique, University of Liege, Belgium

(Received 19 February 1969) A b s t r a c t - - S e e d s of aVigella damascena and excised embryos of barley were irradiated with neutrons of maximum energies 507, 250 and 76 keV produced by (p,n) reaction with a thick Li target, and with somewhat moderated fission neutrons in the R-1 reactor, Stockholm. Neutrons of the lower energy were found to induce chromosome aberrations (minutes, dicentrics and rings, observed in the first metaphase in roots after onset of germination) with a reduced effectiveness compared to 507 keV and fission neutrons. This reduced RBE at lower neutron energy is suggested to be due to a shorter track length in the sense of NEARV et al. rather than to a changed LET. In Wigella simple chromosome breaks were induced at an increasing effectiveness with decreasing neutron energy. This relatively rare aberration type (not determinable in barley) is possibly a consequence of incomplete exchanges. Elastic nuclear collisions are not expected to play a role in the effects observed. Further approaches to study the action of neutrons at the still lower energies where this mechanism of energy dissipation predominates are discussed. R ~ s m a a ~ - D e s graines de aVigella damascena ainsi que des embryons d'orge excis6s ont dt~ irradi6s par des neutrons d'dnergies maximales 507, 250 et 76 keV produits par une rdaetion (p,n) avec une cible dpaisse de Li ainsi que par des neutrons de fission assez ralentis du r6acteur R-1 de Stockholm. Les neutrons d'~nergie la plus basse induisent des aberrations des chromosomes (microfragments, dieentriques et anneaux observ6s au tours de la premi6re m6taphase consdcutive au d6but de la germination) avec une efficacit6 rdduite par rapport aux neutrons de 507 keV ainsi qu'aux neutrons de fission. On sugg~re que la r~duction de cet EBR des neutrons d'6nergie plus basse dolt 6tre due h des traces plus courtes au sens de NEARY et al. plut6t qu'~t un changement de LET. Chez aVigella, des cassures chromosomiques simples sont induites avee une efficacitd croissante en fonction de la r6duction d'~nergie des neutrons. Ce type d'aberrations relativement rare (et non d~terminable chez l'orge) peut ~tre la consdquence d'dehanges incomplets. On ne s'attend pas ~t ce que les collisions nucl6aires 6lastiques jouent un r61e darts les effets observds. On discute une nouvelle voie d'dtude de l'action des neutrons ~t des 6nergies encore plus basses o~ ce m6canlsme de dissipation prddomine. Z u s n m m e n f a s s u n g - - S a m e n yon Wigella damascena trod herausgetrennte Embryonen yon Gerste wurden mit Neutronen einer maximalen Energie yon 507, 250 und 76 keV bestrahlt, die durch eine (p,n) Reakfion mit einem dicken Li-Target und durch etwas abgeschw~ichte SpaltneutronenimR-1 Reaktor, Stockholm, hergestellt wurden. Neutronenmit der geringeren Energie induzierten Chromosomenaberrationen (minutes, dicentrics und rings, die in der ersten Wurzelmetaphase nach dem Einsetzen der Keimung beobachtet wurden); ihre Effektivit~it ist im Vergleich zu der Energie yon 507 keV und Spaltneutronen reduziert. 449

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G. AHNSTROM et al.

450

Es wird angenommen, dass diese reduzierte RBE bei geringerer Neutronenenergie eher einer kiirzeren Traek-L/inge im Sinne yon NEARV et al. als einer ver~tnderten LET zuzusehreiben ist. In Arigdla wurden einfache Chromosomenbrtiehe bei zunehmender Effektivit~it mit abnehmender Neutronenenergie induziert. Dieser relativ seltene Aberrationstyp (bei Gerste nicht zu bestimmen) ist m6glieherweise eine Folge unvollst/indigen Austausehes. Man nimmt an, dass elastische Kemkollisionen bei den beobaehteten Wirkungen keine Rolle spieler,. Weitere etwaige Methoden zur Untersuchung der Wirkung yon Neutronen noeh geringerer Energien, bei denen dieser Mechanismus der Energieverschwendung tiberwiegt, werden diskutiert. 1. I N T R O D U C T I O N

IN R.ADIOBIOLOOICAL experiments with neutrons of different energies and energy distributions certain variations in quality of the observed effects as well as in effectiveness of the radiations are discerned. Such variations m a y partly at least be related to the L E T distribution of the recoil protons, which carry the major fraction of the dose. It is very unsatisfactory, however, that an evaluation cannot be done of a possibly deviating biological effectiveness, eventually also action spectrum, of the heavier ions (mainly of C, O and N) which carry about 10 per cent of the dose in fast neutron irradiation. One property of these ions as well as of low energy protons, which is of importance especially in work with partly moderated fission neutrons, e.g. in a reactor, is their low velocity. When the latter falls below that of the outer shell electrons in tissue, the probability of ionization decreases, and with a further decrease of velocity an increasing fraction of the energy dissipation occurs by a different mechanism, v i z . , elastic nuclear collisions.(20,s, xs) Such collisions might have the effect that whole atoms leave their molecules, with an immediate and irreversible chemical change leading possibly to other biological effects in consequence than those induced by ionization. The velocity of L electrons of oxygen and carbon will be reached by ca. 20 keV protons, ca. 2 5 0 k e V carbon ions, and ca. 300 keV oxygen ions. It is probable, however, that a measurable or dominating effect of elastic collisions will require particles of still lower velocities. (is) Neutrons of a few keV kinetic energy would give rise predominantly to slow particles since in this energy range the contribution from inelastic collisions is negligible. (Assuming the

embryonal tissue of seeds to contain 10 per cent hydrogen and 5 per cent nitrogentS) the contribution to the total high L E T dose of the particles--mainly a 0.56 M e V p r o t o n - - f o r m e d in X4N(n,p)14C will amount to 2 per cent at 1 keV and then increases rapidly with decreasing neutron energy; at 0.1 keV this contribution amounts to 40 per cent of the dose.) I f these have special biological effects, or if they are highly effective, they would, in principle, present a safety problem for thepersonnel in reactors or atomic plants: Most safety monitors are based on ionization or excitation, and these neutrons will therefore escape monitoring if they are not thermalized in the monitoring device.t24,O Slow charged particles are easily produced in accelerators. Their ranges are, however, very short, as demonstrated in the following table for protons (data from PERSON et al.09) and JUNO, Ca) assuming the absorber to be tissue of unit density) :

Proton energy (keV) 1000 30 5 1 "2

range (nm) (1 nm in unit density material = 10 Izg em -2) 23,000 300 100 13

Heavier particles of corresponding velocities will loose kinetic energy faster because of greater charge; therefore their ranges will not be very m u c h longer than those given for protons. A cell diameter is of the order of 10,000 nm. Irradiation of cells with 20 keV protons or other particles of similar velocities is therefore

INTERMEDIATE NEUTRONS AND CHROMOSOMAL ABERRATIONS

451

excluded because of the poor penetration. A magnesium walls and argon gas measuring only homogeneous irradiation of objects up to about the y-component, the other with the wall and 1 m m thickness m a y be attained, however, gas composed of C H measuring the y + n e u t r o n with neutrons of corresponding energies. dose rate. T h e preliminary measurement exWith accelerators available in Sweden neu- hibited that the y-radiation component origitron doses up to a few rad per hour could be nated mainly from Bremsstrahlung, since it produced in (p,n) reactions with medium increased very slowly with the proton energy. weight nuclei (63Cu, 51V, 45Sc), but with a con- Above the energy threshold for the nuclear siderable y-radiation contamination. Since such reaction the neutron dose rate rose rapidly with dose rates were insufficient with respect to the increasing proton energy (Ep); see T a b l e 1. sensitivity of available biological test reactions, For the more accurate dosimetry in the bioa few exploratory experiments to be described logical experiments a CH-ion chamber of the in the present communication m a d e use of (p,n) • same shape as the sample holder was conneutrons from a thick Li target, with the structed. Using this chamber, the y-dose rate at higher neutron energies obtainable with this different Ep was determined by extrapolation light nucleus.~) from values obtained below the threshold In the investigation seeds of Nigella damascena energy, i.e. where no neutrons were generated. were used, in addition to excised embryos of This y-dose determination was performed for barley. Both these materials are sufficiently each set of experimental conditions. small and Nigella has a low chromosome It was demonstrated through measurements n u m b e r with large chromosomes suited for with the ion chamber at different analyses cytological analysis.03) around the irradiation position that the neutron dose received in different parts of the disc-shaped sample would not vary more than 2. E X P ~ . ~ J ~ 10 per cent. Field homogeneity was also verified at the lowest neutron energy used, which 2.1 Irradiation and dosimetry Neutrons of different energies were pro- is most critical in this respect, by a cytological duced through the 7Li(p,n)~Be (~)reaction, the analysis of 20 cells per seed in all seeds of a protons being accelerated to different kinetic sample and comparing the distribution of the energies above the reaction threshold in the frequencies of aberrant cells per seed with the 5 M e V V a n de Graaff accelerator of AB binomial distribution expected in case of homoAtomenergi, Studsvik (Sweden). T h e bio- geneous irradiation. Za = 13.2 was obtained for logical samples, which were circular with 1 cm equality of the two distributions, which, with dia. and ca. 0.1 g/cm 2 thickness, were mounted 20 d.f., corresponds to 0 . 9 0 > P > 0 " 8 0 . Dosiat distances 1.5-4 cm from a rotating, thick Li m e t r y as well as biological irradiation were done at a proton current of ca. 20 ~tamp. T h e target, cooled by air under pressure. A preliminary determination of the y- neutron dose rates were determined at a radiation and neutron dose rates was done known and stable ion current, and the doses with a pair of ionization chambers, one with given were then obtained by integrating the Table I. y-radiation and neutron dose rate in CH at 1.5 erafrom the Li target

Proton energy, Proton MeV keV above current, threshold ~amp 1.852 1.942 2.25

17 77 385

19.2 19.5 22.5

Dose rate neutrons, -f-radiation, rad/hr rad/hr 40 160 1010

27 27 32

Neutron energy, keV

76 175 507

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G. AHNSTROM et al.

current over the duration of the irradiation, using a ~tC meter. The neutron doses obtained from the ionization chamber were recalculated for the atomic compositionCS) of the seed embryos from cross sections for elastic collisions with H, O, C and N. In parallel experiments irradiation with moderated fission neutrons was done in the core of the Stockholm reactor R-1. In an additional study aVigella seeds were irradiated with y-rays in a e°Co source. 2.2 Biological materials and cytological technique Barley embryos (diploid, Hordeum vulgare v. Bonus) were prepared by cutting dry kernels transversely into two parts of unequal size (2/3 and 1/3), the smaller part always containing the embryo. The embryos were then mounted on the sample holder disc with the flat endosperm side down and the embryo facing the Li target. In addition seeds of Nigella damascena (var. Miss Jekyll from Weibull Plant Breeding Institute, Landskrona) were irradiated mixed with the barley embryos, i.e., in each exposure the two materials were given the same dose. Between irradiation and the initiation of germination, the seeds and embryos, respectively, were stored in plastic containers at q-4°C. Germination was initiated by soaking in water for 2 hr followed by plating on moist filter paper in petri dishes at 20°C in the dark. Twenty-four hours after initiation of germination, the barley embryos were transferred to 0.1% colchicine for 3 hr and fixed in acetic alcohol (1:3). aVigella embryos were dissected out 36 hr after initiation of germination. They were pretreated for 3 hr in 0.1% colchicine before fixing in a modified Carnoy's fluid (3:1:1 of alcohol, chloroform and acetic acid). The slides were prepared as Feulgen squashes and brightly stained meristematic portions of the radicle of each embryo were squashed on a slide. The slides were made permanent by the dry ice method. Twenty or thirty ceils from each slide were scored for aberrations of different types (dlcentrics, rings, minutes, fragments; el. Section 3.1). The cells were found to be in the first mitotic division at the time of fixation since (a) there were no micro-nuclei present in any of

the preparations, and (b) the two-hit aberrations such as dicentrics or rings were always accompanied by acentric fragments. 3. RESULTS

3.1 Chromosome aberration types The aberrations encountered in this study were all of chromosome type. This indicates that during irradiation the cells in the resting seeds had been in a G 1 or pre-G 1 stage with regard to their DNA synthesis. The types of aberrations scored included dicentrics and centric rings accompanied by acentric fragments, as well as minutes. The minutes, usually occurring in pairs, were the most common type of aberration. Though an interstitial origin of the minutes cannot be ruled out, there was strong evidence for a terminal origin. In some cases one fragment out of a pair of minutes was still found attached to the terminal end of the chromosome. The last mentioned type of minutes may originate from single hit events. The exponential shape of the dose-effect curve for induction of minutes with y-rays strongly indicates their origin as two-hit events, however (ef. Table 4). The origin of the acentric fragments is less clear. In barley the distribution of breaks in the eentromeric region (17 out of 19 breaks scored) was not at random among the slides studied. Thus, 8 of these fragments were found in three cells of one and the same root. This aberration seems, consequently, to have several traits in common with the centromeric fragments frequently found in materials treated with irradiated media,~4.12) and to have a physiological mechanism rather th.an being a direct radiation effect. In contrast, the acentric fragments in Wigella were indicated to be binomially distributed over ceils and roots, and no tendency for centromeric localization was observed (2 out of 48 breaks had occurred in the centromerit region). T h e y may or may not be true one-hit events (see MOUTSCHEN et al.~14) and Table 4). In so far as the mechanism of formation of chromatid fragments according to Revell's exchange theory has a counterpart on the chromosome level, one plausible interpretation of this aberration type would be semi-

INTERMEDIATE NEUTRONS AND CHROMOSOMAL ABERRATIONS incomplete exchanges especially of the X-shaped symmetrical type.~ s,ls) 3.2 Influence of neutron energy on aberration frequencies In the following presentation and discussion the neutron energy spectra with m a x i m u m energies (Emax) = 507, 125 and 76 keV will be referred to as high, medium and low energy, respectively. In Tables 2 and 3, the frequencies of the different aberration types are given for the two species as number of aberrations (or cells with aberrations, respectively) per 100 cells and per rad. I n barley (Table 2) the frequencies of dicentrics + rings or of minutes obtained at high energy agreed acceptably with the values in material irradiated with moderated fission neutrons. A lowering of the neutron energy provoked a drop of the frequencies. For

453

minutes this drop which exceeds a factor 2, is highly significant (a t-test of the mean values at the highest and lowest neutron energies gives P < 0 . 0 0 1 ) . The drop in dicentrics + rings is not significant ( 0 . 1 0 > P > 0 . 0 5 ) , but m a y be real in view of the fact that the corresponding effect is significant in Nigella. In this species (Table 3) the frequencies of minutes at high energy agree acceptably with the effect obtained in the reactor. Here both dicentries + rings and minutes are induced at a lower effectiveness at the lower energy, compared to the medium and high energies. (The lower frequency of dicentries + rings at high compared to medium energy is not significant. T h e value at high energy m a y be fortuitously low especially in view of the fact that it is significantly l o w e r - - P ~ 0 - 0 1 - - t h a n the reactor value for dicentrics + rings.) It m a y be concluded that, in both species,

Table 2. Chromosome aberrations in barley at different neutron energies Energy dose

No. of % cells with analyzed aberrations cells per rad

Aberr. (100 cells)-Xrad-X Total

Minutes

Die. + rings Fragments*

Low (76 keV) 37 rad 77 rad Mean value

140 140

0.306 0.269 0-288

0.290 0"288 0.289

0.174 0.111 0.142

0.116 0.167 0.142

0 0.01 0.005

Medium (175 keV) 54 tad 106 rad Mean value

140 140

0.332 0.310 0.321

0.344 0.351 0.348

0.185 0.236 0-210

0.159 0.108 0.134

0 0.007 0.004

140 140 140 140

0.615 0.367 0.318 0.296

0.765 0.436 0.382 0"449

0.542 0.242 0.227 0.316

0-209 0" 193 0.155 0.129

0.014 0 0 0.004

0.491 0.399

0.601 0.508

0.392 0.332

0.201 0.172

0.007 0.005

0.484 0.443 0.464

0-501 0.579 0.540

0'272 0.372 0"322

0.200 0.207 0.204

0.029 0 0.015

High (507 keV) 58 tad 115 rad 148 tad 188 tad Mean values a. 58 q- 115 rad b. All doses Reactor 50 rad I00 rad Mean value

140 140

* Centromeric fragments excluded (cf. text).

G. AHNSTROM et al.

454

Table 3. Chromosome aberrations in Nigella at different neutron energies Energy dose

Low (76 keV) 37 rad 77 rad Mean value

No. of % cells with analyzed aberrations cells per rad

330 330

0.327 0.193 0.260

Aberr. (100 cells)-arad-t Total

Minutes

0.361 0.217 0.289

0.156 0.099 0.128

Die. +rlngs

Fragments

0.115 0.059 0.087

0.090 0.059 0.075

~r.___d

k_____y_____)

0.215

Medium (175 keV) 54 rad 106 rad Mean value

150 150

0.310 0.358

0"351 0.434

0.196 0.164

0.334

0.393

0.180 k

0.162 0.127 0.226 0.177 ~

"y

250 250 150 150

b. all doses

0.034 ~r"__.__d

•

0.357

High (507 keV) 58 rad 115 rad 14Brad 188 rad Mean values a. 5 8 + 1 1 5 rad

0.023 0.044

0.211

0.296 0.292 0.262 0.191

0.296 0.418 0.329 0.245

0.152 0.264 0.162 0.088

0.117 0.144 0.162 0.139

0.028 0.010 0.005 0.018

0.294

0,357

0.208 0-131 0.019 ~ w ~ ~ - - - - w-'----~ 0.339 0.151

0.260

0.322

0.167

0.141

k..._ _.__.,,(. _ _ )

0.015

k.__~

0.308

)

0.157

Rgac,lor

50 100 200 4O0 Mean values a. 50 -4- I00 rad b. all doses

I00 100 100 100

0.30 0.35 0.29 0.21

0"32 0.53 0.52 0.64

0.12 0.33 0.21 0.39

0.20 0.18 0-30 0.24

0.00 0.02 0.00 0.02

0.325 0.288

0.425 0.500

0.225 0.262

0.19 0.23

0.01 0.01

Table 4. Chromosome aberrations in Nigella following y-irradiation Dose, krad

5 10 20 40

No. of analyzed cells

Total

Min

200 200 100 100

0-0063 0.0043 0'0100 0.0152

0.0034 0.0011 0-0039 0.0094

Aberr. (I00 cells)-lrad-1 Dic + rings Fragments 0.0026 0.0026 0.0040 0.0050

0.00030 0.00055 0"00210 0.00085

INTERMEDIATE NEUTRONS AND CHROMOSOMAL ABERRATIONS the neutrons become less effective with decreasing energy as regards induction of minutes and exchanges. The decrease is about a factor of 2 when going from 507 to 76 keV maximum neutron energy. More extensive data would be needed for the evaluation of differences between the two species with respect to their response to neutron energy. In aVigella the acentric fragments were sufficiently common to permit an analysis of the dependence of the frequency on the neutron energy (Table 3). This aberration type exhibits very low frequencies (I-2 per cent per rad) at high energy and in the reactor treated material. The fragments become considerably more common with a decrease of neutron energy, i.e., they show an opposite energy dependence compared to the other aberration types studied. Thus the following ratios of fragments to exchanges were found at the different neutron energies:

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4. DISCUSSION

maximum units.* In the case of monoenergetic neutrons of energy En that major part, ca. 90 per cent, of the dose which is dissipated to protons gives with equal probability protons of all energies between 0 and En. T h e fractions of the total dose carried by protons of different energies are thus proportional to the proton energy. The relative biological effectiveness (RBE) of ionizing radiations is a function of LET.(~,16, ~x) In most systems, especially higher organisms, RBE exhibits a maximum at an L E T which, depending on the biological material, assumes a value in the range lO0-200keV/~t.(2~ For protons, a fairly broad maximum L E T of 100 keV/~t is obtained at kinetic energies around 75 keV.(as, 2~) In the studies of SMXrH eta/.( ~x} of Yg2 sectors in maize the increasing RBE with decreasing energy of (monoenergetic) neutrons down to the lowest energy studied, 430 keV, is therefore certainly due to the increasing LET, and the remarkably high value for chromosome aberrations induced in AUium root tips, in the studies of TRorrsraI et al.,(~8) by intermediate neutrons (of average energy 200 keV) is probably close to the maximum RBE. In the present study with aVigella R B E values in the range 50-90 are obtained, with the lower value at the lowest neutron energy, in comparison with the effect of the lower yradiation doses (cf. Tables 3 and 4). Careful calculations would be needed to evaluate the role of L E T in the decrease of RBE found to accompany a decrease of neutron energy down to ca. 50 keV. This role is however expected to be small or negligible, e.g. in view of the fact that the surface dose in a mouse phantom of 500 and 100 keV neutrons were calculated to have rather similar L E T distributions, os) A more likely explanation to the lower biological effectiveness of low energy neutrons might be related to track-length. NEARY et al.(xT) have demonstrated that soft X-rays with electron ranges below 0.25 ~ induce isochromatid aberrations in Tradescantia pollen

4.1 The three neutron energy levels used in the study, and referred to as 507, 175 and 76 keV, or high, medium and low, respectively, represent distributions of energies with the given

*A computation, performed by R. Bergman, of the energy distributions under the irradiation conditions for low energy gives the average energy 53 keV.

76 0.88

175 0.19

507 keV 0.11

The significance of the ca. four-fold increase (per rad) or eight-fold increase (relatively to exchanges) was estimated in the following Z2 tests, comparing high and low energy: (a) Frequencies of fragments vs. exchanges, P < 0-001 ; (b) Frequencies of cells with fragments (i.e. some allowance for an eventual clustering) vs. cells with exchanges, P < 0.001 ; (c, d) Same as (a) and (b) but fragments vs. exchanges + minutes, P < 0.001 ; (e) Frequencies of roots with and without fragments (all roots with 20 analyzed cells); this analysis gives full allowance for any clustering. P < 0-01. It may thus be concluded that at least in Nigella a shift of the spectrum of aberration types occurs when the (maximum) neutron energy is decreased from ca. 500 to ca. 70 keV.

456

G. AHNSTROM et al.

grains with a decreasing effectiveness. With due regard to the fact that the present study is concerned with other species and cells in other stages, it might be inferred that particle range would play a similar role in the present case. A range of 0.25 ~ in water, i.e. 25 ~ g c m -2, would be displayed by ca. 20 keV protons.119) With monoenergetic 76 keV neutrons (20/76) 2 ----- 7 per cent of the dose dissipated to protons (in addition to the dose dissipated to heavier particles) would be carried by particles with ranges below 25 [~g cm -2, and since in fact a spectrum of lower neutron energies was used, this fraction is certainly appreciably higher.* At high energy on the other hand, the fraction of the dose dissipated to protons with ranges <25 ~ g c m -2 is certainly of the order of 1 per cent or less, the short range particles being thus mainly restricted to C, N and O ions. The investigation indicates that protons in the energy range below 20 keV, and other particles of corresponding range do not exhibit effects of specially high RBE. The increasing effectiveness, with lowered neutron energy, of single fragments which appear in N i g e l l a is, however, of considerable interest. In this context, it is interesting to note that Tgoizsmi et al.('~S)have demonstrated a very high R_BE for intermediate neutrons (in the range of 200 keV) for chromosome aberrations in onions. The higher ratio of breaks to exchanges in N i g e l l a at lower neutron energies m a y be related to the high value to this ratio following y-irradiation of seeds, as compared of the effect of densely ionizing particles. It might be assumed that the y-radiation effect is mainly produced by ion clusters or 8-rays of relatively short range. 4.2 The particle energies used in the present study are by an order of magnitude too high to permit an elucidation of the relative biological effectiveness of nuclear collisions. Proton energies as low as ca. 1.5 keV would be required for the energy loss through nuclear collision and through ionization and excitation to be of equal magnitude (el. calculations by NF.UF~.LD *At the computed average energy, 53 keV, ca. 14 per cent of the dose would bc carried by protons < 20 keV (cf. footnote, p. ¢55).

and SNYDER (18) and experiments of JuNo.(8) At I0 k e V protons the former mechanism is still some 30 times less important than the interaction with atomic electrons. Possible biological effects with nuclear collisions as a primary mechanism would therefore require work at considerably lower proton energies than used in the present study, unless these effects had an extraordinarily high RBE. Very few radiation-chemical and no radiobiological, investigationshave yet been carried out with the purpose of measuring the relative effectiveness of neutrons in the k c V range. O u r investigationO) of the discoloration of plcxiglas by 5 k e V protons showed an effectiveness to induce this non-specificchemical change about equal to that of fission neutrons. Using still lower energies, JuNo and ZIMM~.R(10)reveal, however, spectral changes indicating a specific action of protons of energies around 1 keV. Further, measuring enzyme inactivation by slow protons in thin layers of ribonuclease the same authors(S, x°) find the inactivation cross section to increase ca. 5 times with decreasing proton energy below ca. l ' 3 k e V . At the latter energy the cross section was found to exhibit a m i n i m u m attained from a plateau around 50-60 keV. In the work of JuNG and ZIMMER the dose was expressed, however, in n u m b e r of incident protons per cm z. Available data indicate that the L E T of protons rises below 1.3 keV to about the same extent as the inactivation cross section. It seems therefore that the enzyme inactivation per unit dose and, hence, the ' R B E ' is rather independent of the particle energy in the range 0-2.5 keV. t In similar studies on the inactivation of infective D N A of bacteriophage ~X 174, the energy dependence of the effectiveness of the slow protons was found to be less than in the case of RNase. (g,n) T h e problem is, however, interesting enough to motivate efforts to study the biological action in cellular materials of protons of energies d o w n to ca. I kcV, and of other particles of I" In thiscalculationpar ticlcrangesgiven by Jingo(s) and PZRSON(19)wcrc used. Espcclallyat low energies the ranges arc very short; they correspond to onc--a few ribonuclcasc molecule diarnctcrs and their determination is thereforedifficult.

I N T E R M E D I A T E NEUTRONS AND C H R O M O S O M A L ABERRATIONS corresponding velocities. Since the short ranges do not permit irradiation of cells directly with accelerated particles, possibilities of producing these particles in the tissue via neutrons have to be utilized. T h e following ways of doing this m a y be envisaged: (a) Production of neutrons in nuclear reactions with atoms heavier than Li, such as eSCu, 51V, 45Sc, with incident protons just over the threshold energy. (With p r o t o n beams of 1 m a m p or more sufficient dose rates of 5 keV neutrons might be obtained.) (b) Moderation of Li(p,n) neutrons or neutrons p r o d u c e d in (p,n) reactions with heavier targets according to (a), with comparison of different energy spectra. (c) Resonance neutrons, with 23Na or 5sCo. A calculation shows that very high epithermal fluxes would be needed to reach dose rates useful in biological experiments. For the BR2 reactor (Mol, Belgium) exposure of a piece of N a at the exit of a radial canal would give ca. 0-2 r a d / d a y of 2.8 keV neutrons. Presumably the V e r y H i g h Flux R e a c t o r at Brookhaven m i g h t permit a r o u n d 5 r a d / d a y to be achieved at a suitable position in the 'epithermal canal'. I n view of the y-contamination, this m e t h o d is not feasible at present. (d) 1.3 keV deuterons from H ( n y ) D , or 4 0 k e V c a r b o n ions from X4N(n,p)14C. This is only possible in very small objects, to avoid the d o m i n a t i n g influence of the reaction products carrying the m a i n fraction of the energy. Since in most cases the attainable dose rates are very low, suitable, more sensitive biological test reactions should be developed. Acknowledgements--We owe thanks to Dr. M. N#.vE DE MI~VERGNIS, Mol, for calculating resonance neutron yields and to Dr. R. BEROS~a~, Gustaf Werner Institute, Uppsala University, for computation of neutron energy distributions. The investigation was supported by the Swedish Atomic Research Council and the Knut and Alice Wallenberg Foundation.

REFERENCES 1. AHNSTR6M G. and EHRENBERG L. (1961) Dosimetry of mixed neutron-gamma radiations in the Stockholm Reactor, R-l, pp. 603-607. In

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Selected Topics in Radiation Dosimetry, IAEA, Vienna. 2. BARENDSEN G. W. (1967) Relative biological effectiveness as a function of linear energy transfer, pp. 249-267, In Proc. Conf. Microdosimetry, Ispra. 3. Bm~WENJ. G. and BROCK R. D. (1968) The exchange hypothesis and chromosome type of aberrations. Mutation Res. 6, 245-255. 4. CHOPRA V. L., NATARAJANA. T. and SWAMINATHAN M. S. (1962) Cytological effects observed in plant material grown on irradiated fruit juices. Radiation Botany 3, 1-6. 5. EHRENB~.ROL. and SAELANDE. (1954) Effects of pile radiation on barley seeds. 07. Nucl. Energy 1, 150-169. 6. FANO U. (1954) Principles of radiologieal physics, pp. 1-144. In A. HOLLAENDER (ed.), Radiation Biology Vol. I, part 1. McGraw Hill, New York. 7. GmBONSJ. H. and NEWSON H. W. (1960) The Li'(p,n)BC reaction, pp. 133-176. In J. B. MARXON and J. L. FOWLER (eds.), Fast Neutron Physics, part 1. Interscience, New York. 8. JuNo H. (1965) Zur biologischen Wirksamkeit elastischer Kernst6sse, I. Inaktivierung yon Ribonuclease durch langsame Protonen. Z. Naturforsch. 20b, 764-772. 9. JuNo H. and KURZINOERK. (1969) Zur biologischen Wirksamkeit elastischer Kernst6sse III. Einwirkung von langsamen Protonen auf infekti6se DNS des Bakteriophagen ~X!74. Z. Naturforsc. 24b, 328-332. 10. JUNO H. and ZIMM~RK. G. (1966) Some chemical and biological effects of elastic nuclear reactions, pp. 69-128. In M. EBERT and A. HOWARD (eds.), Current Topics in Radiation Research, Vol. 2. North Holland, Amsterdam. 11. KURZINOER K. and JUNO H. (1969) Action of elastic nuclear collisions on infectious DNA of bacteriophage ~X174. Intern..7. Radiation Biol. 14, 493-495. 12. MOUTSCHENJ. and MATAONE R. (1965) Cytological effects of irradiated glucose. Radiation Botany 5, 23-28. 13. Mou'rSCHENJ., MotrrscHEN-DAH~rEN M., GmOT J. and Raz~zm~a~s M. (1966) Caryotype et mdiose chez Nigella damascena L. La Cellule 66, 83-94. 14. MOUTSCHENJ., MOUTS(IHEN-DAHMEN M., WOODLEY R. and A R C ~ E A U J. (1968) The RBE of heavy particles from the reaction l°B(n, oc)'Li for chromosome aberrations in aVigella damascena L. Radiation Res. 34, 488--500.

458

G. A H N S T R O M et al.

15. MOUTSCHEN-DAHMAN M., MOUTSCHEN J. and EHRENBERG L. (1966) On post irradiation modification of biological effects of neutrons. I. Effect of myleran on chromosomal aberrations in neutron irradiated seeds. Radiation Botany 6, 251-264. 16. NSARY C. J. (1965) Chromosome aberrations and the theory of R.BE. I. General considerations. Intern. 07. Radiation Biol. 9, 477-502. 17. NEARY C.J., PRESTONR . J . and SAVACEJ. R. K. (1967) Chromosome aberrations and the theory of RBE. III. Evidence from experiments with soft X-rays, and a consideration of the effects of hard X-rays. Intern. 07. Radiation Biol. 12, 317-345. 18. N E ~ L D J. and SNYDER W. S. (1961) Estimates of energy dissipation by heavy charged particles in tissue, pp. 35-44. In Selected Topics in Radiation Dosimeter, IAEA, Vienna. 19. PE~ON S., HUTCHINSON F. and MARVIN D. (1963) Range of low-energy protons in proteins. Radiation Res. 18, 397--406.

20. PLATZMAN R. L. (1952) On the primary processes in radiation chemistry and biology, Chap. 7. In J. J. NmKSON (ed.), Symposium on Radiobiology. John Wiley, New York. 21. SMITH H. H., BATEMANJ. L., QUASTLERH. and Rosst H. H. (1964) Role of monoenergetic neutrons: cytogenetic effects in maize, pp. 233-248. In Biological effects of neutron and proton irradiations Vol. 2. IAEA, Vienna. 22. SNYD~R W. S. (1963) The LET distribution of dose in some tissue cylinders, pp. 3-19. In Biological effects of neutron and proton irradiation. IAEA, Vienna. 23. TRorrsKII N. A., BYLINSKIIN. F. and PHILIPPOVXCHA. S. (1966) The mutagerfic effects of intermediate neutrons, pp. 175-178. In Z. LANDA (ed.), Symposium on mechanisms of mutation and inducing factors, Prague, 1965. Academic Publishing House, Prague. 24. ZIMMER K. G. (1956) Probleme der Neutronendosimetrie. Strahlentherapie 101, 143-151.