Theoretical and Applied Genetics 44, 167-172 (1974) 9 by Springer-Verlag 1974

Radiation Induced DNA Double Strand Breaks and Chromosome Aberrations H. P. LEENHOUTS and K. H. CHADWICK Association E u r a t o m -- ITAL, Wageningen (The Netherlands)

Summary. This paper presents arguments which favour an alternative approach to the interpretation of radiation induced chromosome aberrations. Starting from the modern concept that a chromosome has a DNA double stranded helix backbone and that the induction of DNA double strand breaks has a quadratic dose relationship, it is concluded that most chromosome aberrations arise from only one chromosome break. The direct correlation between chromosome aberrations and cell death derived from the model is demonstrated by the analysis of experimental results. The effect of dose rate, LET and the occurrence of chromatid aberrations after irradiation in G1 are all logically explained by the ' theoretical model.

Introduction In a recent publication (Chadwick and Leenhouts, 1973) we have developed a theory, which is based on the induction of double strand breaks in the DNA helix, to explain radiation induced cell killing. The application of this theory to the dependence of cell survival on radiation dose rate and the variation of radiation sensitivity in the cell cycle gave analyses which were both logical and could be explained b y the behaviour and metabolism of the DNA molecules in the cell. If this theory is considered in the light of the modern proposals of chromosome structure (DuPraw, t965; Callan, 1967; Whitehouse, t967; DuPraw, t970; Prescott, 1970; Crick, 1971; Laird, t 9 7 1 ) certain arguments can be developed which question the validity of the basic assumptions of the current theories on radiation induced chromosome aberrations. These arguments and their consequences are presented here in the form of an alternative theoretical approach. D N A D o u b l e Strand Breaks, Cell Killing and C h r o m o s o m e Aberrations The DNA helix forms a structured molecular target for radiation which induces double and single strand breaks in the sugar-phosphate chains of the helix. In aqueous solution the induction of double strand breaks in DNA molecules has been found to have a quadratic relation with radiation dose (Hagen, t 967 ; Freifelder and Trumbo, t969). A double strand break can be induced in one radiation event when both strands of the helix are broken simultaneously (proportional to dose), or b y the combination of two unrepaired single strand breaks which are induced in two independent radiation events (proportional to the square of the dose).

Thus if allowance is made for repair processes the total number of DNA double strand breaks after a dose D can be calculated to be N=aD

+flD

2.

The basic tenet of the new molecular theory is t h a t the induction of double strand breaks in DNA in the cell also has a quadratic dose relation and t h a t this molecular damage forms the critical biological lesion. In the molecular theory of cell survival we have assumed t h a t each double strand break in a cell has a probability p of leading to cell death so t h a t the probability for cell survival is given b y the equation S

~

e -pN

--=- e

P(~

.

Recent experimental work on chromosome structure (DuPraw, t965; Laird, t97t) supports the view t h a t the chromosome has a single long DNA double helix backbone (Callan, t967; Whitehouse, t967; DuPraw, 1970; Prescott, t970; Crick, t971). Thus, on the basis of this concept a break in the DNA double strand helix is in fact a break in the chromosome backbone. This means t h a t the induction of chromosome backbone breaks will also have a quadratic dose relation given b y N =aD

+flD

29

This straight forward conclusion is of fundamental importance as it contradicts the basic assumption of the current theories of chromosome aberrations (Sax, t939, 1940, t941 ; Lea, 1955 ; Revell, t955, t959, 1963, 1966). These theories, which were developed prior to the recent considerations on chromosome structure, assume t h a t the p r i m a r y break or lesion in the chromosome is linearly related to radiation dose. The conclusion that the induction of chromosome backbone breaks is quadratic forms the starting point of the new proposal t h a t a chromosome back-

168

H. ]). Leenbouts et al. : Radiation Induced I)NA Double Strand Breaks and Chromosome Aberrations

Fraction unbroken D N A molecules

1.C

Survival of Chinese Hamster cell

1.0[~ Ref:F,.e~lder (a)tin)

05

,

Chromosome aberrations per cells

b)' 9 ' Ref:Ptettlm

1.5

Ref: Fex =nclNImp('1970)

'

C ~'l

e t 11 11972)

0.5 1.0 0.3

0.1 0.5

ol

lObO

2obo

o.ol

soo

Dose (rad)

looo Dose (rad)

2o0

~o

q u a d r a t i c dose relationship. Fig. t b shows the s u r v i v a l of chinese h a m s t e r cells following i r r a d i a t i o n (Fox a n d Nias, 1970) a n a lysed according to the molecular t h e o r y a n d Fig. I c shows the i n d u c t i o n of dieentrics plus centric rings plus acentric f r a g m e n t s in chinese h a m s t e r cells (Preston et al., t972) a n a l y s e d according to the q u a d r a t i c relationship.

Doselrad)

I n T a b l e I we p r e s e n t the details of the coefficients which have been derived from the analyses of the curves shown in Fig. t. I n the last c o l u m n of the T a b l e the coefficients for double s t r a n d breakage per n u c l e o t i d e pair have been calculated using an estim a t e of the n u m b e r of n u c l e o t i d e pairs per m a m m a lian cell g i v e n b y Swanson, Merz a n d Y o u n g (1967). I t has been a s s u m e d t h a t the p r o b a b i l i t y p for cell d e a t h per double s t r a n d b r e a k is u n i t y a n d t h a t each double s t r a n d b r e a k causes a chromosome a b e r r a t i o n . These a s s u m p t i o n s form an o v e r s i m p l i f i c a t i o n which does n o t however d e t r a c t from the c o n v i n c i n g agreem e n t b e t w e e n the coefficients f o u n d in all three cases. This a g r e e m e n t b e t w e e n the coefficients is a s t r o n g i n d i c a t i o n t h a t the same r a d i a t i o n i n d u c e d process, i . e . D N A double s t r a n d breakage, is i n v o l v e d directly as the basic m e c h a n i s m leading to r a d i a t i o n i n d u c e d cell d e a t h a n d chromosome a b e r r a t i o n s .

Fig. 1. Comparison of dose kinetics for DNA double strand breaks, cell survival and chromosome aberrations b o n e b r e a k m a y lead to a chromosome a b e r r a t i o n . T h u s , the yield, Y, of a chromosome a b e r r a t i o n can be w r i t t e n as

Y=

k N = k (a D + fl D 2)

where k is a c o n s t a n t which is d e p e n d e n t u p o n the a b e r r a t i o n t y p e b e i n g scored a n d the scoring efficiency. The i n t e r p r e t a t i o n of e x p e r i m e n t a l results should be m a d e on the basis of the p a r a m e t e r s which form the coefficients a a n d fl a n d which have been g i v e n special radiobiological significance in reference 4 where the complete d e r i v a t i o n of the q u a d r a t i c e q u a t i o n has been presented. I n Fig. t we p r e s e n t some evidence which s u p p o r t s the t h e o r y which has j u s t been developed. Fig. i a shows the dose kinetics f o u n d for the i n d u c t i o n of D N A double s t r a n d breaks in D N A o b t a i n e d from the phage B 3 a n d i r r a d i a t e d in buffer solution (Freifelder a n d T r u m b o , 1969), a n a l y s e d according to the

The Correlation between Cell Killing and Chromosome Aberrations A direct e x t e n s i o n of these a r g u m e n t s is t h a t we p r e d i c t t h a t for m e a s u r e m e n t s carried out on the

Table I. Comparison of the induction of D N A double strand breaks, cell survival and chromosome aberrations after irra-

diation Nucleotide pairs per molecule or cell

Coefficient for d. s. b. per nucleotide pair

DNA double Fraction of unbroken a = 2.3 • 10-4 rad-1 strand breaks DNA molecules fl = 2.2 X 10 .7 rad -~" in buffer F = e-~D--, qD2

t.4 • 108

a 1 = t.6 • 10-~2 rad-1 fit = 1.6 • 10 -15 rad -2

Freifelder and Trumbo (1969) Fig. I a

Survival of Survival of cells chinese S = e P~D--P#D2 hamster cells

p a = t.6 • to .3 rad -1 p f l = 2.8• 10-~rad -2

~ 3 • 100

a t = 5.3 • 10 -13 rad-Z fll ~ 9.3• t0-1~rad -2

Fox and Nias(1970) Fig. 1 b

dicentrics + centric k a = 6 • 10 -4 tad -1 rings + acentric kfi = 6.7 • 10-~ rad -2 fragments per cell Y= kaD +kflD 2

~ 3 • 109

ae * = 2.0 X 10 -la rad -1 fil = 2.2 • lo-ts rad -2

Preston et al. (1972) Fig. 1 c

Process

Chromosome aberrations in chinese hamster cells

Analysis

Numerical values

* It is assumed that the probability p for cell death per double strand break = t. ** It is assumed that each double strand break causes a chromosome aberration.

Ref.

H. P. I,eenhouts et al. : Radiation Induced DNA Double Strand Breaks and Chromosome Aberrations

100 200 300 400 500 600

01

ration we can now consider how this mechanism is related to the various chromosome configurations found at mitosis and what consequences arise from this mechanism. The Terminal Deletion The simplest explanation for the occurrence of this chromosome aberration is that proposed in the classical theory (Sax, t939, 1940, 194t; Lea, t955) t h a t the terminal deletion arises from a single break in the chromosome. The classical theory predicted t h a t the terminal deletions would exhibit a linear dose relationship. We would expect t h a t an aberration which arises from a single break in the chromosome backbone would have a quadratic dose relationship in general. Fig. 3 shows the curve for terminal deletions found in Wallabia bicolor b y Brewen and Brock (1968) fitted with a quadratic equation which is in agreement with our theoretical expectations.

~ ' ~ ~ 2

Survival

X

169

0"3

o

Terminal deletions

// 100

///2

50% y=1.7.10-2D+

.

~ j . Y/ o = ( 4 / ~/3 M:t~ 4h~+C~ : [ :~!}i~

A

40 O00ii

30

2O _

_

100 200 300 400 500

Dose( rad )

Fig. 2. Correlation between cell killing and total chromosome aberrations in synchronised chinese hamster cell (Dewey et al., 197t) same cell under the same conditions the coefficients derived for cell survival and chromosome aberrations will be related to each other b y a constant factor. One such correlation is illustrated in Fig. 2 for synchronized chinese hamster cells irradiated at different stages of tile cell cycle (Dewey et al., t97t). The coefficients p c~ and p/5 derived for the cell survival curves, multiplied by a constant 'K', give curves which fit in each case the total production of chromosome aberrations. I t is interesting to note that the correlation is found in different phases of the cell cycle, this means t h a t the changing shape of the cell survival curve through the cell cycle is mimicked b y the chromosome aberration curve. This variation in radiation sensitivity through the cell cycle has been analysed to provide strong support for the association between DNA double strand breaks and cell killing (Chadwick and Leenhouts, t973, 1973a). Starting from the basic mechanism of a DNA double strand break leading to a chromosome aberTheoret. Appl. Genetics, Vol. 44, No. 4

100

200

L

30(7

I

400

I

500 Dose (rad)

Fig. 3. Terminal deletions in Wallabia bicolor fitted to Y -- k (aD + //D ~) (Brewen and Brock, 1968) It is worth noting, at this point, t h a t under certain experimental conditions the general quadratic relationship m a y be reduced to a linear dose relationship. This occurs when the coefficient fl is close or equal to zero. This is i m p o r t a n t because the actual dose relationship for terminal deletions, linear or quadratic, has been a topic of some controversy between different schools of radiation cytologists.

The Exchange Aberrations The dose relationship for exchange aberrations has often been fitted using a quadratic dose relationship and as a result these aberrations have been interpreted in the classical and exchange theories as arising from two chromosome breaks.

170

H.P. Leenhouts et a!. : Radiation Induced DNA Double Strand Breaks and Chromosome Aberrations Dicentrics.rings per ceff

Aberrationtype] G1

1-4- t.5MeV X y=6,B7.1026D 2 2.0

Terminal deletions +

2-+-60Co ~ yo2.2'1(j4D+6.7.106D 2

IRR. Joining Metaphase

t

~

minutes

3--A- 200 kVpXy, S.5.104D+10.4"10"6D 2 4-.0- 14MeVn y,2.18.1(53Doh.4.1(36D 2

H

t

11

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l

11

1.5

Acentric rings t

~)l(~_~

~C_~

1 1.0

Pericentric inversion

I

Dicentricand fragment

~ 6~ l r ~ , L ~ I ~ : ~

Symmetrical interchange

0.5

1co

2oo

3oo

4oo

5oo Dose(red) Fig. 4. The effect of different radiation qualities on the production of dicentrics and rings in whole blood (Unscear, 1969) The consequence of the arguments presented in this paper is that an exchange aberration which arises from only one break in the chromosome backbone would have a quadratic dose relationship and would therefore fit tile experimentally determined dose relationships. Dicentric and ring aberrations form one group of easily recognisable and accurately scorable exchange aberrations. Fig. 4 presents the fitting of a quadratic equation to the production of rings and dicentrics in blood for different qualities of radiation (Unscear, t969). The theory predicts, in analogy with the theory for cell killing (Chadwick and Leenhouts, 1973), that as the LET of the incident radiation increases the ~ coefficient will increase and dominate and the relation will eventually become linear with dose. The value of the ~ coefficient (Fig. 4) is increasing consistently with increasing radiation quality and the limiting RBE of 4.0 found for the t4 MeV neutrons against the 200 kVp X-rays is in agreement with that found for cell killing in the Tt-g kidney cells (Chadwick and Leenhouts, t973). If we accept that the basic mechanism involved in a chromosome aberration is a radiation induced DNA double strand break and that an exchange aberration must be developed from one chromosome break it is necessary to find an explanation for the considerable variety of exchange aberration configur-

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Fig. 5. Diagrammatic representation of the formation of chromosome type aberrations at mitosis on the basis of rejoining between a broken and normal chromosome end

Aberrationtype G2

IRR. Joining Anaphase

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