P R O C E E D I N G S S E R I E S 1 J

BIOLOGICAL EFFECTS OF N E U T R O N I R R A D I A T I O N

CZ.3 PROCEEDINGS OF T H l / s Y M P O S I U M

ON T H E E F F E C T S OF N E U T R O N I R R A D I A T I O N UPON C E L L F U N C T I O N

O R G A N I Z E D BY T H E I N T E R N A T I O N A L A T O M I C ENERGY AGENCY

A N D H E L D I N N E U H E R B E R G ( M U N I C H ) , 22-26 OCTOBER 1973

I N T E R N A T I O N A L A T O M I C ENERGY AGENCY V I E N N A , 1974

C O N T E N T S

N E U T R O N SOURCES, S P E C T R O M E T R Y A N D D O S I M E T R Y (Sessions 1 and I I )

N e u t r o n sources avai lable for r a d i o b i o l o g i c a l r e s e a r c h ( I A E A - S M - 1 7 9 / 3 5 , Inv i t ed paper) 3 G. B u r g e r , J . J . B r o e r s e

Fas t n e u t r o n spectra i n standard seed i r r a d i a t i o n f a c i l i t i e s f o r Τ RIGA r e a c t o r s ( I A E A - S M - 1 7 9 / 2 0 ) 21 J . K r a j n i k , M . N a j z e r , M . P a u k o , J . R a n t D i s c u s s i o n 28

Resonance energy neutron beams f r o m r e a c t o r s f or rad iob io l ogy studies ( I A E A - S M - 1 7 9 / 9 ) 31 K . R . B r y a n t , S. R. B u l l D i s c u s s i o n 47

Use of a c y c l o t r o n as a fast neutron source i n plant breeding and genetics ( I A E A - S M - 1 7 9 / 1 6 ) 49 J . H u c z k o w s k i , E. G a z e k , H . W o j c i e c h o w s k i D i s c u s s i o n 56

C a r a c t e r i s a t i o n d 'un faisceau de neutrons pour des etudes rad iob io log iques ( I A E A - S M - 1 7 9 / 2 1 ) 59 Nico le P a r m e n t i e r , A . B r i d i e r , M . C h e m t o b , P. F a c h e , R. L a g u e r r e , V . D . N g u y e n , A . R i c o u r t , R. S o u l i e , C. V e a u , A . D e b a u c h e , P. L e l e u x , J . P. M e u l d e r s , C. P i r a r t , A . W a m b e r s i e , M . W i n a n t D i s c u s s i o n 80

I n t e r m e d i a t e neutron- induced slow r e c o i l p a r t i c l e s applied i n rad iob io logy ( I A E A - S M - 1 7 9 / 4 ) 83 R. O. B e r g m a n Dis cuss i on 95

The b i o l o g i c a l effect of fast neutrons not i n c h a r g e d - p a r t i c l e e q u i l i b r i u m ( I A E A - S M - 1 7 9 / 5 ) 97 D . K . B e w l e y , N . J . M c N a l l y , B . C . P a g e D i s c u s s i o n 106

Neutron d o s i m e t r y and s p e c t r o m e t r y methods used i n c e l l m u t a t i o n exper iments ( I A E A - S M - 1 7 9 / 1 4 ) 109 P . D . H o l t , S .J . B o o t D i s c u s s i o n 118

Energy deposi t ion on a m i c r o s c o p i c scale , re levant to the b i o l o g i c a l effects of fast neutrons : Report on the F o u r t h E U R A T O M Symposium on M i c r o d o s i m e t r y ( I A E A - S M - 1 7 9 / 3 7 , I n v i t e d paper) 119 J . B o ο ζ

N E U T R O N E F F E C T S A T S U B C E L L U L A R AND C E L L U L A R L E V E L S (Sessions I I I and I V )

- Observations re l evant to the mechanism of R B E effects i n the k i l l i n g of ce l l s ( I A E A - S M - 1 7 9 / 2 ) 133 T i k v a h A l p e r Discuss ion 14.7

A t h e o r e t i c a l analysis of r a d i a t i o n sens i t i v i ty i n c e l l s f o l l owing neutron i r r a d i a t i o n ( I A E A - S M - 1 7 9 / 1 8 ) 151 H. P. L e e n h o u t s , Κ . Η. C h a d w i c k Discuss ion 161

D i f f e r e n t i a l effects of e lec trons and neutrons on c e l l k i l l i n g and ab i l i t y of D N A - m e m b r a n e complex to synthesize D N A i n E_. c o l i B / r and B s - 1 ( I A E A - S M - 1 7 9 / 1 1 ) 165 W. A . C r a m p , P . E . B r y a n t Discuss ion 175

Recovery and r e p a i r i n yeast ce l ls a f ter i r r a d i a t i o n w i t h densely i o n i z i n g p a r t i c l e s ( I A E A - S M - 1 7 9 / 2 3 ) 177 W . E . P o h l i t , M . S c h ä f e r Discuss ion 182

The influence of e x p e r i m e n t a l end po in ts , dose, dose r a t e , neutron energy, n i t rogen i o n s , hypoxia , c h r o m o s o m e vo lume and ploidy l e v e l on R B E i n Tradescant ia s tamen h a i r s and pol len ( I A E A - S M - 1 7 9 / 3 1 ) 185 A . G. U n d e r b r i n k , A . H. S p a r r o w Discuss ion 213

R B E of the 1 0 B ( n , a ) 7 L i capture reac t i on of t h e r m a l neutrons inside the green alga C h l o r e l l a pyrenoidosa ( I A E A - S M - 1 7 9 / 1 7 ) . . 215 H . - U . K n o o p , Μ. P a s c h k e

Ef f i cac i te r e l a t i v e de d i v e r s rayonnements m i x t e s g a m m a , neutrons pour l ' i n d u c t i o n i n v i t r o d 'anomal ies c h r o m o -somiques dans les lymphocytes humains ( I A E A - S M - 1 7 9 / 7 ) 221 M a r i e - T h e r e s e B i o l a , R. L e G o , G. V a c c a , Ginette D u c a t e ζ , Janine D a c h e r , Μ i r e i l l e B o u r g u i g n o n Discuss ion 2 36

The effect of d i f f e r i n g neutron energies on mutagenes is i n c u l t u r e d Chinese hamster ce l l s ( I A E A - S M - 1 7 9 / 1 5 ) 237 M a r g a r e t R i c h o l d , P . D . H o l t Discuss ion 243

L e t h a l effects of 14 -MeV fast neutrons of f r o g eggs and cu l tures m a m m a l i a n ce l l s ( I A E A - S M - 1 7 9 / 3 0 ) 245 K. T a k e s h i t a , S. S a w a d a Discuss ion 255

Mod i f i ca t i on of neutron effects upon ce l l s by r e p a i r , and by phys i ca l and c h e m i c a l means ( I A E A - S M - 1 7 9 / 3 9 , I n v i t e d p a p e r ) . . 257 R. J . B e r r y Discuss ion 2 70

N E U T R O N E F F E C T S ON M U L T I C E L L U L A R SYSTEMS (Sessions V and VI )

^^Response of mouse l e u k a e m i c ce l l s after i r r a d i a t i o n w i t h X - r a y s and fast neutrons ( I A E A - S M - 1 7 9 / 8 ) 275 J . J . B r o e r s e , P ia Μ . v a n O o s t e r o m D i s c u s s i o n 281

Ef fets hemato log iques compares des rayons y du cobalt et des rayonnements e m i s au cours d'une excurs i on c r i t i q u e ( I n s t a l l a t i o n CRAC) ( I A E A - S M - 1 7 9 / 1 9 ) 283 J . L . Μ a l a r b e t , R. L e G o , J . P r u d h o m m e , Lucet te G e n e s t D i s c u s s i o n 297

Ef fects of i n t r a c a v i t a r y i r r a d i a t i o n by c a l i f o r n i u m - 2 5 2 ( I A E A - S M - 1 7 9 / 2 9 ) 299 M . F . S u l l i v a n , J . L . B e a m e r , T . D . M a h o n e y D i s c u s s i o n 310

Regeneration of g a s t r i c g landular e p i t h e l i u m i n adult m i c e a f ter exposure to 1 -MeV fast neutrons (1AEA-SM-179 /12 ) 311 J . A . G. D a v i d s D i s c u s s i o n 323

S u r v i v a l of s p e r m a t o g o n i a l s t em ce l l s i n the mouse after exposure to 1 . 0 - M e V fast neutrons ( I A E A - S M - 1 7 9 / 2 5 ) 325 Anke L . de R u i t e r - B o o t s m a , M . F . K r a m e r , D . G. de R o o i j , J . A . G . D a v i d s D i s c u s s i o n 333

Use of the wound response to compare r e c o v e r y of neutron or X - i r r a d i a t e d i n t e r m i t o t i c ( G 0 ) c e l l s of the r a t lens e p i th e l iu m ( I A E A - S M - 1 7 9 / 2 4 ) 335 E. F . R i l e y , Β. T . A u s t i n , A l i c e L . L i n d g r e n , R. C. M i l l e r D i s c u s s i o n 346

RBE values of 4 0 0 - M e V and 1 4 - M e V neutrons us ing var ious b i o l og i ca l ef fects ( I A E A - S M - 1 7 9 / 6 ) 349 M a r i l e n a Β i a η c h i , J . B a a r l i , A . H . S u l l i v a n , M . D i P a o l a , M . Q u i η t i l i a n i D i s c u s s i o n 356

γ - Late effects of n e u t r o n o r gamma i r r a d i a t i o n i n mice ( I A E A - S M - 1 7 9 / 1 ) 359 E. J . A i n s w o r t h , R. J . M . F r y , D . G r a h n , F . S. W i l l i a m s o n , P a t r i c i a C. Β r e η η a η , S. P h y l l i s S t e a m e r , Α. V . C a r r a n o , J . H . R u s t

ι D i s c u s s i o n 378 < Neutron - induced m a m m a r y neoplasms ( I A E A - S M - 1 7 9 / 3 2 ) 381

Η. H . V o g e l , J r . D i s c u s s i o n 388

~ r Rat m a m m a r y carc inogenes i s f o l l o w i n g neutron or X - r a d i a t i o n ( I A E A - S M - 1 7 9 / 2 6 ) 391 C . J . S h e l l a b a r g e r , R. D . B r o w n , A . R. R a o , J . P. S h a n l e y , V . P. B o n d , A . M . K e l l e r e r , H . H . R o s s i , L . J . G o o d m a n , R. E. M i l l s D i s cuss i on 400

USE OF NEUTRONS I N P L A N T B R E E D I N G (Session V I I )

Re la t i on between m u t a t i o n y i e l d and c e l l l e t h a l i t y o v e r a wide range of X - r a y and f i s s i o n neutron doses i n m a i z e ( I A E A - S M - 1 7 9 / 2 8 ) 405 H . H . S m i t h , H . H . R o s s i , A . M . K e l l e r e r D i s c u s s i o n 415

The ef fectiveness of f i s s i o n neutrons , 14. 7 - M e V monoenerge t i c neutrons and 6 0 C o gamma r a d i a t i o n on seed l ing g r o w t h r e d u c t i o n and induct ion of c h l o r o p h y l l - d e f i c i e n t m u t a t i o n s i n bar ley ( I A E A - S M - 1 7 9 / 1 0 ) 417 Β. V . C o n g e r , M . J . C o n s t a n t i n D i s cuss i on 432

Rad iosens i t i v i ty of plant seeds to neutrons and i t s m o d i f i c a t i o n ( I A E A - S M - 1 7 9 / 2 7 ) 433 A . R. G o p a l - A y e n g a r , N . S. R a o , Β. B . S i n g h D i s cuss i on 43 8

C o m p a r a i s o n des effets physiologiques et genetiques provoques par des i r r a d i a t i o n s aux neutrons et aux rayons g a m m a de boutures d ' o i e l l e t Dianthus caryophy l lus L . ( I A E A - S M - 1 7 9 / 2 2 ) 441 P. P e r e a u - L e r o y D is cuss i on 449

O T H E R P R A C T I C A L I M P L I C A T I O N S OF N E U T R O N R A D I A T I O N BIOLOGY (Session V I I I )

Fundamenta l and p r a c t i c a l aspects of the a p p l i c a t i o n of fast neutrons i n c l i n i c a l rad i o therapy ( I A E A - S M - 1 7 9 / 3 ) 453 G. W. B a r e n d s e n , J . J . B r o e r s e D i s cuss i on 458

Genetic effects of neutrons i n m a m m a l s and t h e i r i m p l i c a t i o n s f o r r i s k assessment i n man ( I A E A - S M - 1 7 9 / 3 8 , I n v i t e d paper) . . . 461 A . G. Se a r l e D i s cuss i on 471

C h a i r m e n of Sessions and Secre tar ia t 473 L i s t of P a r t i c i p a n t s 475 A u t h o r Index 483

IAEA-SM-179/28

R E L A T I O N B E T W E E N M U T A T I O N Y I E L D AND C E L L L E T H A L I T Y O V E R A W I D E R A N G E O F X - R A Y A N D F I S S I O N N E U T R O N D O S E S I N M A I Z E *

H . H . S M I T H Department of Biology, Brookhaven Nat ional Laboratory, Upton, N . Y .

H . H . ROSSI , A . M . K E L L E R E R Department of Radiology, C o l u m b i a Univers i ty , New Y o r k , N . Y . , United States of A m e r i c a

Abstract

RELATION BETWEEN MUTATION YIELD AND C E L L LETHALITY OVER A WIDE RANGE OF X-RAY AND FISSION NEUTRON DOSES IN MAIZE.

Dry maize seeds, of a genetic stock heterozygous for the yellow-green locus (Yg^ Λ ^ ) ι were irradi­ated with fission neutron doses up to 2700 rads and with 250 kVp X-rays to 40 000 rads. The frequency of yellow-green (yg) sectors in seedling leaves 4 and 5 is largely a measure of the frequency of breakage (or incomplete exchange) and loss of the part of the short arm of chromosome I X carrying Yj>. Plots of dose versus yj* sector frequency per leaf show a linear increase with neutrons which reaches a maximum (leaf 4 = S. 5, leaf 5 = 2. 5) followed by a rapid decline; and a curvilinear increase with X-rays reaching a higher maximum (leaf 4 = 8. 5, leaf 5 = 3.6) followed by a decrease. The observed rise and subsequent fall of numbers of yg_ mutations can be accounted for by a model in which the decline of the curves is due to cell killing which may also be due to chromosome breakage and deletion. The interpretation is based on the principles of the dual action theory, which explains radiobiological action in terms of micro-dosimetry. One of the observations accounted for is the higher maximum mutation rate for X-rays as compared to neutrons.

INTRODUCTION

The aim of t h i s paper i s to explore the e f f e c t s of neutron and χ r a d i a t i o n , over a wide range of doses, on a p a r t i c u l a r genetic e f f e c t and on l e t h a l i t y i n maize; and to relate the observations t o microdosimetric data f o r χ rays and neutrons.

MATERIALS AND METHODS

Seeds of a genetic stock of maize (Zea mays L.) heterozygous at the yellow-green locus, Y%Jl$2> w e r e u s e d i n a H experiments. The dominant Yg? produces f u l l green color, so that the heterozygote has normal green leaves. The Yg£ locus i s located near the end of the short arm of chromo­some 9. Loss of the Yg 2 a l l e l e (deletion) or change i n i t s function (mutation) i n heterozygotes gives a yellowish-green phenotype i n leaf

Research carried out at Brookhaven National Laboratory under the auspices of the US Atomic Energy Commission.

405

406 SMITH et a l .

c e l l s and c e l l lineages of the altered genotypes. The frequency of yellow-green streaks or sectors i n leaves of seedlings grown from i r r a d i a t e d seeds was used as a measure of the frequency of r a d i a t i o n induced genetic change or damage [ 1 ] . This was considered to be due mainly, i f not exclusively, to breaks i n chromosome 9 between the centromere and the Yj* locus, w i t h loss (following involvement i n chromosomal aberrations) of the Y g o ~ containing segment.

Prior to exposure to r a d i a t i o n , the seeds were equ i l i b r a t e d over a saturated aqueous solution of Cr0~ i n an atmosphere of 35% r e l a t i v e humidity. The water content of Y g ^ / y g n e m b r y o s , excised from maize seeds so stored, was determined to be 6.7%>.

Seeds were i r r a d i a t e d w i t h χ rays under the following conditions: 30 ma, 45 cm from source, 1.0 mm Al f i l t e r , 4.4 mm HVL A l , dose rate 1658-1845 rad/min. Other seeds were exposed to f i s s i o n neutrons generated by thermal neutron bombardment of a 235y plate i n the thermal column of a Brookhaven National Laboratory reactor. The computed dose rate was from 27.5 to 198 rad/min. During the i r r a d i a t i o n the seeds were maintained i n the ambient atmosphere, i . e . w i t h oxygen a v a i l a b i l i t y of about 21%. Immediately a f t e r i r r a d i a t i o n , w i t h i n 4 sec, the seeds were immersed i n d i s t i l l e d deionized water (22° C) which was determined to have the same content of dissolved 0 2 (about 21%) as the a i r . The oxygen a v a i l ­a b i l i t y during and a f t e r i r r a d i a t i o n was such as to enhance r a d i a t i o n response w i t h low LET i r r a d i a t i o n .

The i r r a d i a t e d seeds, together with unirradiated controls, were sown immediately i n moist s o i l , one per 4-inch pot, coded, and arranged i n a completely randomized experimental design i n a controlled environment growth chamber. The conditions i n the chamber were 18 hr of l i g h t at 1700 to 2000 foot candles i n t e n s i t y i n a temperature of 21° C (dark) and 24° C ( l i g h t ) .

When f u l l y mature, the fou r t h seedling leaf (leaf 4) was harvested and scored, under coded number, for frequency of yellow-green ( y g o ) sectors. The f i f t h seedling leaf (leaf 5) matured somewhat l a t e r and was then scored i n the same way. The size of the v j * 2 sectors i s a function of the number of c e l l divisions and the amount of c e l l expansion that takes place subse­quent to i r r a d i a t i o n . Consequently, the sectors are larger i n leaf 5. The r e l a t i v e frequency of v_g2 sectors i n the two leaves i s a function of the number of primordial leaf c e l l s i n the seed embryo at the time of i r r a d i a t i o n and the target size [ 2 ] . I n these stocks the leaf 5 primordium appeared i n the dormant seed as a four-layered structure with s l i g h t l y more than 500 c e l l s . However, since the epidermal layers of the l e a f are devoid of chloroplasts and therefore not involved i n v_g2 expression, leaf 5 at the time of i r r a d i a t i o n presented a relevant target of about 250 c e l l s . The number of c e l l s i n embryonic leaf 4 was more d i f f i c u l t to establish but was estimated by d i f f e r e n t techniques to be about 2900. I t was concluded that the p o t e n t i a l number of relevant leaf 4 target c e l l s was about 1500. This estimate i s probably too high, however, since many (possibly about h a l f ) of the c e l l s i n leaf 4 are d i f f e r e n t i a t e d and may not be capable of co n t r i b u t i n g v_g2 sectors.

To determine the volume of the nucleus at the time of i r r a d i a t i o n , primordia were dissected from the seed embryo, placed i n a 0.1% solution of the surface-active compound Tween 20 for 5 min, then fi x e d and stained w i t h acetocarmine. The diameter of the nucleus (or length and width i f c y l i n d r i c a l ) was measured, and the volume was calculated i n cubic microns. The mean nuclear volumes were 159 μ^ f o r leaf 4 and 122 μ^ f o r l e a f 5.

IAEA-SM-179/28 407

RESULTS AND COMPUTATIONS

The dose-response data f o r frequency of jg^ sectors per leaf are presented i n Table I . The range i n doses f o r χ rays i s from 0.50 to 40.0 krad and f o r neutrons from 0.03 to 2.69 krad. These results are shown p l o t t e d a r i t h m e t i c a l l y i n Fig. 1 f o r leaf 4 and i n Fig. 2 for leaf 5. The neutron and x-ray curves have i n common that a f t e r an i n i t i a l r i s e they reach a peak, saturate, then decline [ 3 ] .

I n Fig. 3 the experimental results f o r leaf 4 are shown i n a logarithmic p l o t of the frequency of ygp sectors per leaf as a function of absorbed dose. I n the logarithmic p l o t s t r a i g h t lines of slope 1 correspond to p r o p o r t i o n a l i t y between observed y i e l d and absorbed dose; li n e s of slope 2 correspond to a quadratic dependence between y i e l d and absorbed dose. I n these experiments no spontaneous incidence of ys^ sectors was observed. The curves d i f f e r insofar as: 1) neutrons are much more e f f e c t i v e per u n i t of absorbed dose; 2) the neutron-induced y i e l d of mutations peaks at a somewhat lower frequency; 3) the slope of the r i s i n g part of the neutron curve i s approximately 1 which corresponds to p r o p o r t i o n a l i t y between e f f e c t and absorbed dose. The x-ray curve, on the other hand, i s consistent w i t h a l i n e a r increase at smallest doses which turns i n t o a quadratic dependence wi t h increasing dose.

T A B L E I . DOSE-RESPONSE D A T A FOR NUMBER OF yg 2 SECTORS PER L E A F F O L L O W I N G I R R A D I A T I O N OF SEEDS W I T H X - R A Y S OR NEUTRONS

X rays Neutrons

Absorbed Dose

k r a d

No.

leaves

2 £ 2 s e c t o r / l e a f Absorbed

Dose

krad

No.

l eaves

yg^ s e c t o r / l e a f Absorbed Dose

k r a d

No.

leaves

L e a f 4 L e a f 5 Absorbed

Dose

krad

No.

l eaves

L e a f 4 L e a f 5 Absorbed Dose

k r a d

No.

leaves Mean SE Mean SE

Absorbed

Dose

krad

No.

l eaves Mean SE Mean SE

0.50 343 0.08 .014 .002 .007 0.03 60 0.92 .131 0.50 .090

0.75 477 0.09 .014 0.04 .009 0 .07 60 1.33 .150 0.83 .106 1.00 165 0.05 .017 0.15 148 3.80 .194 0,87 .077 1.80 87 0.30 .052 0.12 .034 0.27 119 5.08 .340 0.98 .089

3.95 87 0.92 .117 0.30 .064 0.39 118 5.30 .332 1.69 .115 5.92 60 2.08 .216 0.85 .111 0.65 116 3.46 .221 1.40 .101 8.10 146 3.97 .321 1.30 .121 0 .95 131 4.91 .253 2.41 .118 9.17 87 5.21 .425 1.93 .177 1.16 83 5.43 .259 2.54 .123

10.00 85 5.93 .453 1.86 .151 1.36 102 4.98 .193 2.52 .098 11.52 108 5.17 .338 1.98 .164 1.63 68 4.21 .167 2.44 .112 13.68 81 6.37 .379 3.04 .166 1.98 20 2.30 .231 2.30 .231 16.96 83 7.25 .454 3.21 .168 2.37 109 3.23 .117 2.47 .102 20.00 55 7.56 .436 3.15 .190 2.69 25 2.73 .219 1.62 .198 22.79 60 8.18 .495 3.62 .230

30.00 30 4.39 .310 2.30 .187

40.00 9 3.44 .709 2.22 .364

408 SMITH et a l .

10 15 20 25 30 35 40 ABSORBED DOSE (krod)

FIG. 1. Frequency of y j 2 sectors induced in maize leaf 4 by a wide range of neutron and X-ray doses; linear representation.

τ 1 1 1 1 1 r

L i I I I 1 I I L _

5 10 15 20 25 30 35 ABSORBED DOSE (krod)

FIG. 2. Frequency of yg 9sectors induced in maize leaf 5 by a wide range of neutron and X-ray doses; linear representation.

IAEA-SM-179/28 409

10 ι 1 1 Γ

ο.οι Ι 1—— 1 I —1 10 I0 2 I0 3 10* io5

ABSORBED DOSE (rod)

FIG. 3. Mean numbers of sectors in leaf 4 as a function of X-ray and neutron dose (log-log plot). The vertical bars indicate standard deviations due to the Poissonian fluctuations of the numbers of observed events. The curves represent the least squares fit according to Eqs (4) and (5) with the parameters given in Table I I .

The corresponding r e l a t i o n s f o r l e a f 5 are shown i n Fig. 4. One finds the same c h a r a c t e r i s t i c s , although the i n i t i a l shape of the neutron curve i s doubtful and seems to be subject to considerable s t a t i s t i c a l u n c e r t a i n t i e s .

The results are i n agreement w i t h the assumption that the e f f e c t p r o b a b i l i t y i s proportional to the square of the energy concentration i n sensitive sites of the c e l l nucleus. I t has been shown [ 4 ] that the dependence of the e f f e c t on the square of the sp e c i f i c energy, z, leads to a linear-quadratic dependence of the observed y i e l d on dose:

y - k ( C D + D2) (1)

where ζ i s the energy average of z, produced by any one single charged p a r t i c l e i n the s i t e . The value of ζ f o r neutrons i s generally so high that the quadratic component can be neglected. For χ rays, on the other hand, ζ i s so small that the quadratic component dominates except at smallest doses, k i s a p r o p o r t i o n a l i t y constant and can be understood i n terms of the d i f f e r e n t number of c e l l s i n the le a f primordia.

Since, i n the present experiments w i t h χ rays, both the i n i t i a l l i n e a r component and the subsequent quadratic increase of the y i e l d can be observed, one i s able to determine the values of k and ζ. For t h i s purpose a least square f i t of the data to the linear-quadratic model:

y = a D + b D wi t h a = k£ and b = k (2)

410 SMITH et a l .

T A B L E I L E S T I M A T E S OF T H E P A R A M E T E R S I N R E L A T I O N TO T H E Y I E L D OF y g 2 SECTORS

A«, Estimates of the Parameters i n Relation to the Yield of Sectors Produced by Neutrons

- a l D y = a D e ^ η η η

Leaf 4 Leaf 5

a n (rad" 1) .024 .006

a' (rad" 1) .0013 .0012 η '

Β. Estimates of the Parameters i n Relation to the Yield of Sectors Produced by X Rays

• 2 y - (a D + b D2 ) e" b D *

X X X

Leaf 4 Leaf 5

a (rad" 1) 9.5 χ 1θ" 5 3.3 χ 1θ" 5

χ b (rad" 2) 4.5 χ 1θ" 8 1.7 χ 1θ" 8

b' (rad" 2) 5.0 χ 1θ" 9 4.5 χ 1θ" 9

has been performed. Only data up to 10 rad have been included i n t h i s analysis i n order to avoid complexities due to c e l l k i l l i n g and to the corresponding decline of the observed y i e l d of mutations at higher doses.

Together with the estimate of the two c o e f f i c i e n t s i n Eq. (2) the j o i n t 95%-confidence region of these parameters has been derived. As pointed out i n a separate discussion of the s t a t i s t i c a l analysis [ 5 ^ t h i s confidence region i s an e l l i p s e . Figure 5 depicts the results for leaf 4 and 5. From the estimates of the parameters a and b l i s t e d i n Table I I one obtains values, ζ = a/b, for χ rays of 2100 rad for leaf 4 and 1900 rad f o r leaf 5. The close s i m i l a r i t y i n these two essentially independently computed values of the average specific energy produced by one absorption event implies that the same primary biophysical phenomena are involved i n c e l l s producing sectors i n each leaf. These values belong to diameters of the sensitive sites of approximately 0.2 to 0.3 μπι.

IAEA-SM-179/28 411

10 ΙΟ2 ΙΟ3 ΙΟ4 ΙΟ5

ABSORBED DOSE (rod)

F I G . 4. Mean numbers of y g 9 sectors in leaf 5 as a function of X-ray and neutron dose. Same remarks as for Fig. 3.

F I G . 5. Least squares estimates and 95% confidence regions for the parameters a and b in Eq. (2) for X-rays. Only dose values up to 10 4rads are included in the analysis. The joint regions of standard deviation are represented by broken lines.

412 SMITH et a l .

These distances are smaller than those observed i n the effects on most other eukaryotic c e l l s [ 4 ] , The difference i s , however, not unexpected since i n the present case one i s dealing with a dry system. As a conse­quence of the large values f o r ζ and small s i t e diameters, the dry seed system shows a l i n e a r dose-effect r e l a t i o n f o r χ rays up to about 2500 rad, i n contrast to only about 5 rad f o r a sensitive wet higher plant system [ 6 ] . For doses which i n most b i o l o g i c a l systems would be considered high, a c h a r a c t e r i s t i c low dose response i s obtained i n t h i s dry system.

Since, as yet, no precise microdosimetric data are available f o r sites as small as 0.2 μπι, one cannot predict the value of ζ f o r neutrons. The values would be expected, however, to be roughly 50 times larger than the values f o r χ rays. Accordingly, the observed l i n e a r component f o r neutrons should exceed the l i n e a r component f o r χ rays by about the same factor of 50. The actu a l l y observed values of the lin e a r c o e f f i c i e n t f o r neutrons are also given i n Table I I . One finds that for leaf 4 the value i s considerably larger than expected. The same observation holds for l e a f 5, although i n t h i s case the value i s at best te n t a t i v e due to considerable s t a t i s t i c a l f l u c t u a t i o n s i n the i n i t i a l part of the dose-effect r e l a t i o n . I t should i n t h i s context be noted that the standard deviations given i n Table I and Figs. 3 and 4 represent only the Poissonian flu c t u a t i o n s i n the number of observed sectors; other systematic variations may we l l occur and are not represented by these standard deviations. The data for l e a f 5 i n the range of low doses indicate p r o p o r t i o n a l i t y to a power of dose less than 1. I f substantiated t h i s would be a find i n g of considerable i n t e r e s t . The question must, however, remain open as long as no systematic analysis of the experimental uncertainties has been performed. For the present discussion only a l i n e a r r e l a t i o n at small doses w i l l be considered although t h i s may lead to conservative estimates of the neutron RBE.

The essential observation i s that the l i m i t i n g RBE of neutrons vs. χ rays at lowest doses appears to exceed the expected values of roughly 50 by a wide margin. The value f o r leaf 4 appears to exceed 200. Although there i s at present no explanation f o r t h i s r e s u l t , i t should be pointed out that values of neutron RBE of as high as 135 have been reported i n t h i s material [1,2] and RBE values exceeding 100 have also been found i n studies of the o p a c i f i c a t i o n of the murine lens [ 7 ] , i n experiments w i t h the induction of mammary tumors i n the Sprague-Dawley r a t by neutrons [ 8 ] , and i n results w i t h somatic aberrations i n Tradescantia [ 9 ] . There are some indications that these very high values of the neutron RBE at low doses are due to an increased production of sublesions i n the densely ionizing tracks of heavy charged p a r t i c l e s . Such an increase could be represented i n Eq. (1) by a larger value of the c o e f f i c i e n t k for neutrons than for χ rays. Since there have been no systematic microdosimetric studies w i t h the necessary s p a t i a l resolution of several nanometer one cannot, at present, attempt a quantita t i v e evaluation of the e f f e c t .

I t remains to study the plateau of the curves and t h e i r subse­quent decline. The simplest explanation of the observed curves i s that induction of mutations and c e l l k i l l i n g go on simultaneously. I n a f i r s t approximation one may consider the case that these processes are inde­pendent, although t h i s i s probably an over s i m p l i f i c a t i o n . The simplest assumption i s then that c e l l s u r v i v a l declines exponentially w i t h c e l l u l a r damage that follows the same linear-quadratic k i n e t i c s that are observed for other e f f e c t s . The r e s u l t i n g r e l a t i o n f o r the observed y i e l d over the f u l l dose range i s then the product of the survival p r o b a b i l i t y and the p r o b a b i l i t y for mutation induction:

y = (a D + b D2) e" 3' D " b ' D * (3)

IAEA-SM-179/28 413

For neutrons, the quadratic terms can be neglected i n both parts of the equation. For χ rays, on the other hand, the l i n e a r component plays no role i n the term for s u r v i v a l since that term i s s i g n i f i c a n t only i n the range of high doses. One, therefore, deals w i t h the two d i f f e r e n t equations for neutrons and χ rays:

y = a D e" a^ D n (4) J η η v '

y = (a D + b D 2 ) e U x (5) J v χ χ x' v ' The s o l i d curves i n Figs. 3 and 4 follow these r e l a t i o n s w i t h the values of the c o e f f i c i e n t s given i n Table I I .

Although the parameters l i s t e d i n Table I I r e s u l t i n a s a t i s f a c t o r y f i t we consider i t premature to re l a t e them to microdosimetric quantities which would necessarily have to pe r t a i n to the d i f f e r e n t sensitive volumes involved i n mutation and k i l l i n g . However, i t may be noted that due to the s i n g l e - h i t k i n e t i c s of neutrons one must expect events i n which mutation and k i l l i n g occur simultaneously r e s u l t i n g i n a smaller maximum observable mutation frequency, which i s consistent w i t h our r e s u l t s .

DISCUSSION

Loss of the Yg^ locus to give V J * sectors i s considered to be due to chromosome breaks, one occurring between t h i s locus and the centro­mere, followed by rearranged j o i n i n g so that i n subsequent somatic c e l l divisions the part of chromosome 9 containing dominant Yg„ i s eliminated. I n the early cytogenetic l i t e r a t u r e [10] i t was shown th a t : 1) simple deletions are rare; 2) x-ray induced aberrations involving two chromosomes are dependent on two independent breaks l i m i t e d i n both time and space; and 3) the frequency of these two-break aberrations increases more than l i n e a r l y w i t h increasing dose. I t was then shown [11] that a f t e r neutron i r r a d i a t i o n (7.5 and 15 MeV) there was a l i n e a r r e l a t i o n of two-break aberrations to dose. This was a t t r i b u t e d to both breaks being produced by single proton i o n i z a t i o n paths, i n contrast to t h e i r production w i t h x rays by two independent electron i o n i z a t i o n paths. Further extensions of these considerations, r e l a t i n g a dependence of the number of chromosome exchanges on the square of the dose for χ rays and a l i n e a r r e l a t i o n f o r neutrons, were developed by, e.g. Lea [ 1 2 ] , Wolff et al. [13] and Neary [ 1 4 ] .

The data on production of χ% sectors are consistent w i t h those on chromosome exchanges and amenable to the same explanation. The chromo­somes i n leaf primordia of the dormant seed embryo i n maize would be expected to be mainly i n Gl of interphase [ 1 5 ] .

Recently i n s i g h t i n t o the u l t r a s t r u c t u r e of interphase chromosomes has been gained through electron microscopy. From observations on certain animal chromosomes, which may apply generally, i t appears that each un-replicated interphase chromosome consists of a continuous folded f i b e r [ 1 6 ] . This f i b e r i s comprised of a single DNA double h e l i x t i g h t l y packed by supercoiling and coated by p r o t e i n . DuPraw states, "For example, a length of Watson-Crick double h e l i x 20 Ä i n diameter and 56 μ long would be wound into a supercoiled f i b e r 80 to 100 Ä i n diameter and 7 or 8 μ long; the l a t t e r could then be supercoiled again i n t o i t s f u l l y packed form, 230 X i n diameter and only 1 μ long" [ 1 6 ] ,

The following t e n t a t i v e i n t e r p r e t a t i o n of the elementary processes leading to the r a d i a t i o n response described i n t h i s paper i s consistent

414 SMITH et al .

w i t h the Information available at t h i s time. Radiation-induced lesions i n two of the continuous folded chromatin f i b e r s , on the same or d i f f e r e n t chromosomes, j o i n i n rearranged p o s i t i o n so that two-break exchange aberrations ( d i c e n t r i c s , centric rings, i n t e r s t i t i a l deletions), as commonly seen i n the l i g h t microscope, are formed. Certain of these, involving the short arm of chromosome 9, can lead to subsequent loss of a chromosome segment containing the Yg^ l°cus and eventual appearance of y g o sectors i n the l e a f .

The diameters computed from microdosimetric considerations of the maize data, namely 2000 to 3000 Ä, can be formally interpreted as the diameter of sensitive sites which are subject to dual damage, or as the e f f e c t i v e diameter of the sphere of pote n t i a l i n t e r a c t i o n around a break produced by i o n i z a t i o n . The i n t e r a c t i o n , scored eventually as a yg^ sector, i s due p r i m a r i l y to the i l l e g i t i m a t e j o i n i n g of two breaks produced by two independent charged p a r t i c l e s i n the s i t e (as with χ rays) or the i l l e g i t i m a t e j o i n i n g of two breaks produced by the same charged p a r t i c l e i n a s i t e (as w i t h neutrons and low doses of χ rays) . The amount of energy produced w i t h i n a s i t e by a charged p a r t i c l e to y i e l d the observed cytogenetic e f f e c t i s computed as about 2000 rad.

ACKNOWLEDGEMENT

We acknowledge w i t h thanks the excellent technical assistance of Mr. Nicholas Combatti.

REFERENCES

[1 ] SMITH, Η. Η., BATEMAN, J. L e, QUASTLER, Η., ROSSI, Η β Η., RBE of mono-energetic fast neutrons: cytogenetic effects i n maize. B i o l o g i c a l Effects of Neutron and Proton I r r a d i a t i o n s (Proc. Symp. Upton, 1963) 2, IAEA, Vienna (1964) 233.

[2 ] SMITH, Η. H., ROSSI, Η. Η., Energy requirements and r e l a t i v e b i o l o g i c a l effectiveness f o r producing a cytogenetic phenomenon i n maize by i r r a d i a t i n g seeds w i t h x-rays and monoenergetic neutrons. Radiat. Res. 28 (1966) 302.

[ 3 ] SMITH, Η. H., Radiation probes of maize meristems. The Dynamics of Meristem Cell Populations ( M i l l e r , M. W., Kuehnert, C. C, eds.), Plenum Press, New York (1972) 271.

[ 4 ] KELLERER, Α. Μ., ROSSI, Η. Η., The theory of dual radiation a c t i o n . Current Topics Radiat. Res. Quart. 8 (1972) 85.

[ 5 ] KELLERER, A. M,, BRENOT, J., On the s t a t i s t i c a l evaluation of dose-response functions. Biophysik, i n press.

[ 6 ] SPARROW, A. H., UNDERBRINK, A. G., ROSSI, Η. H., Mutations induced i n Tradescantia by small doses of x-rays and neutrons: analysis of dose-response curves. Science 176 (1972) 916.

[ 7 ] BATEMAN, J. L., ROSSI, Η β Η., KELLERER, Α, Μ β, ROBINSON, C. V., BOND, V. P., Dose-dependence of fast neutron RBE for lens o p a c i f i c a t i o n i n mice. Radiat. Res. 51 (1972) 381.

[ 8 ] SHELLABARGER, C. J,, KELLERER, Α. Μ., ROSSI, Η. Η., GOODMAN, L. J., BROWN, R. D., MILLS, R. E., RAO, A. R., SHANLEY, J. P., BOND, V. P., Rat mammary carcinogenesis following neutron- or x - i r r a d i a t i o n . These proceedings.

[ 9 ] UNDERBRINK, A. G., SPARROW, R. C, SPARROW, A. H., ROSSI, Η β H., Relative b i o l o g i c a l effectiveness of 0.43 MeV and lower energy neutrons on somatic aberrations and hair-length i n Tradescantia stamen h a i r s . I n t . J. Radiat. B i o l . 19 (1971) 215.

[10*] SAX, K., An analysis of x-ray induced chromosomal aberrations i n Tradescantia. Genetics 25 (1940) 41.

IAEA-SM-179/28 415

[11] GILES, Ν. Η., Comparative studies of the cytogenetical effects of neutrons and x-rays. Genetics 28 (1943) 398.

[12] LEA, D. E., Actions of Radiation on Living Cells. Cambridge Univ. Press, Cambridge (1947).

[13] WOLFF, S#, ATWOOD, K. C., RANDOLPH, M. L., LUIPPOLD, H. E„, Factors l i m i t i n g the number of radiation-induced chromosome exchanges. I . Distance: Evidence from non-interaction of x-ray- and neutron-induced breaks. J. Biophys. Biochem. Cytol. 4 (1958) 365.

[14] NEARY, G. J . , Chromosome aberrations and the theory of RBE. I . General considerations. I n t . J. Radiat. B i o l . 9 (1965) 477.

[15] VAN'T HOF, J . , The regulation of c e l l d i v i s i o n i n higher plants. Brookhaven Symp. B i o l . 25 (1973) i n press.

[16] DuPRAW, E. J., DNA and Chromosomes. Holt, Rinehart and Winston, New York (1970).

D I S C U S S I O N

Β. V . CONGER: The R B E values you obtain are v e r y h igh , but since the condit ions under which the seeds were t reated were such that some oxygen-enhancement would be expected, they are probably not as high as they could be. By c o n t r o l l i n g seed water content and oxygen a v a i l a b i l i t y so as to p r o ­v ide m a x i m u m r a d i o r e s i s t a n c e , you could possibly obtain R B E values 2 to 3 t i m e s h igher , i . e. i n the range of 600 to 700. I f you l owered the water content of your seeds s t i l l f u r t h e r and soaked them i n oxygenated water af ter i r r a d i ­a t i on , I suspect that you would have even greater oxygen enhancement, w h i c h would l o w e r the R B E values .

H . H . S M I T H : Yes , I agree, we could have mod i f i ed the condit ions to obtain s t i l l higher R B E values .

Β. V . CONGER: What were the approximate t i m e s after sowing when counts were taken of the frequency of y e l l o w - g r e e n sectors i n leaves 4 and 5?

H . H . S M I T H : T h i s i s done as soon as the leaves r e a c h m a t u r i t y , w h i c h i s approx imate ly 4 weeks after sowing i n the case of leaf and about 5 weeks i n that of leaf 5 under our growing condit ions .

Κ. H . CHADWICK: As I have already mentioned that the peak heights f o r mutat ions should be the same regard less of r a d i a t i o n type, I should l i k e to comment on the apparent c o n t r a d i c t i o n betweenyour r e s u l t s and our proposa ls . I do not believe that there i s i n fact a cont rad i c t i on . I would be i n c l i n e d to draw a double peak curve through the neutron data i n your F i g . 3. I t w i l l then be found that the f i r s t peak i s matched by a s m a l l peak at the edge of the X - r a y curve at the same height . I would say that you have two d i f f e rent s e n s i t i v i t i e s i n your c e l l populat ion. The f i r s t peaks agree quite w e l l ; the second peak for the neutrons and the m a i n peak for the X - r a y s are d i f f e rent because of the add i t i v i ty of the mutat ions f r o m the m o r e sensi t ive c e l l s . A t the second neutron peak I suspect that the m o r e sensit ive ce l l s are no longer c o n t r i b u ­t i n g , whereas i n the case of the X - r a y s some sensi t ive ce l l s are s t i l l p r o ­ducing mutat ions and pushing up the m a i n X - r a y peak.

Y o u r u n i n e m e proposals for the chromosome are most welcome; i n t h i s connection we would r e f e r you to the paper c i ted as Ref.[ 6] i n paper I A E A -SM-179/18, where we discuss the associat ion between DNA d o u b l e - s t r a n d breaks , the unineme concept and chromosome a b e r r a t i o n produc t i on . A m o r e detai led paper on th i s subject w i l l be appearing i n the j o u r n a l M T h e o r e t i c a l and Appl ied Genetics" s h o r t l y .

416 SMITH et a l .

Η. Η. S M I T H : I l ook f o r w a r d to r ead ing your papers . W i t h r e g a r d to the suggestion that the peak areas of our X - r a y and neutron curves may con­s i s t of two peaks each, the confidence l i m i t s around these points are not su f f i c i ent ly n a r r o w at p resent , i n m y op in ion , to w a r r a n t such an i n t e r p r e t a t i o n .

P. D. H O L T : You have assumed i n your paper that mutat i on and c e l l k i l l i n g are independent, but have indicated that th is assuption i s probably only an a p p r o x i m a t i o n . Have you any idea what e r r o r i s involved?

Η. H . S M I T H : We have made some computations on the basis of the assumption that i n the produc t i on of a Y g 2 sector the targe t i s p r i m a r i l y the distance of the Y g 2 l ocus f r o m the c e n t r o m e r e i n chromosome I X , that i n the case of c e l l k i l l i n g the t a r g e t i s the whole length of the genome, and that both phenomena invo lve two chromosome breaks and i l l e g i t i m a t e r e j o i n i n g w i t h a loss of c h r o m o s o m a l m a t e r i a l — c e l l k i l l i n g i n v o l v i n g m u l t i p l e events of t h i s same type . Our computat ions indicated tentat ive ly that two such dual events would on average cause c e l l death, but we do not believe that our neutron data are s u f f i c i e n t l y accurate at present to c o n f i r m an i n t e r p r e ­t a t i o n of th i s k i n d .