Chromosome aberrations and radiation-induced cell death

Mutation Research, 17 (I973) 355-366 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

355

CHROMOSOME A B E R R A T I O N S AND R A D I A T I O N - I N D U C E D CELL D E A T H II. P R E D I C T E D AND OBSERVED CELL SURVIVAL

A. V. CARRANO*

Laboratory of Radiobiology, University of California, San Francisco, California 94122 (U.S.A.) (Received August 3rd, 1972)

SUMMARY

The surviving fraction of synchronous V79 Chinese hamster cells was measured in two post-irradiation generations after a 3oo-rad X-ray dose in G1 by comparing the colony multiplicity in irradiated and control cultures. In addition, the ability of the irradiated population to form colonies was measured immediately after G1 irradiation or at 6, 32, 75 or 96 h after X-irradiation. Formulae were used in conjunction with previously observed transmission and survival parameters of chromosome aberrations 2 to predict the amount of cell death at any given time after irradiation. The results indicate that the survival pattern of these cells can indeed be predicted on the basis of cell loss from chromosome aberrations. It is likely that an asymmetrical chromosome exchange (dicentric, centric ring, or tricentric) and a chromosome deletion are equally capable of causing cell death, whereas translocations or inversions apparently do not lead to inviability. Furthermore, cell death is rapid: 45% of the total observed death occurs in the first two post-irradiation generations. The initial decrease in viability is caused predominantly by the formation of anaphase bridges, while cell death from fragment loss becomes increasingly important in later generations. In fact, it is probable that, on the average, a cell that loses a single acentric fragment will survive one generation.

INTRODUCTION

Several studies have established a relation between chromosomal aberrations and radiation-induced cell death1,6-9,14,18, ~1. Unfortunately, the chromosomal aberrations observed in most of these studies were classified on the basis of either total aberrations per cell or total number of breaks per cell, with no distinction made among the various types of aberrations. In other words, all chromosomal aberrations or breaks were presumed to possess the same killing efficiency. One m a y ask, for example, whether a cell with a single chromosome-type asymmetrical exchange (dicentric, centric ring, or tricentric) is as likely to survive as a cell with a single * Present address: Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439 (U.S.A.)

356

A.V. CARRANO

chromosome deletion (acentric fragment) and, if so, whether both cells are likely to survive for the same length of time. The answers to these questions would provide a basis for the prediction of the amount and time course of reproductive death if the initial frequency of each type of radiation-induced chromosomal aberration were known. Since transmission parameters for specific chromosome aberrations induced by X-rays in G1 for V79 Chinese hamster cells have been calculated 2, a basis exists for predicting cell death in the present study. To this end, then, the survival of V79 Chinese hamster cells was measured after X-irradiation in G1 by examining the colony multiplicity for consecutive 2-h periods up to 47 h after synchronization and determining the ability of the irradiated population to form macroscopic colonies as a function of time after irradiation. By using formulae incorporating the transmission and survival parameters previously calculated ~, a comparison could be made between the observed reproductive death and the loss of viability expected on the basis of chromosome loss. An X-ray dose of 300 tad was chosen to minimize the effects on macromolecular syntheses and to produce a sufficient yield of chromosome aberrations through three successive post-irradiation divisions 2. MATERIALS AND METHODS

V79 Chinese hamster cells were grown in a modified Eagle's medium as previously described 2. Synchronization was performed by selecting the mitotic population after a brief (I h) colcemid arrest 2. The mitotic index was usually over 9o% (cells were discarded if the initial mitotic index was less than 85%). To measure colony multiplicity, approx. 5oo-2ooo mitotic cells (depending on the duration of culture) were added to 3o-ml Falcon plastic flasks with conditioned medium. After 1.25 h of incubation, the Gi cells were irradiated with 3oo rad by a G. E. Maxitron 3oo X-ray unit. The unit was operated at 3oo kVp (2o mA) with o.I m m A1 inherent and 2.o mra Cu (h. v. 1. 1.65 m m Cu) added filtration. The dose rate was 20o R/min. Beginning at 1.25 h and extending to 47 h after synchronization, control and irradiated cultures were removed from the incubator at 2-h intervals and examined with an inverted microscope for the number of viable cells per colony. Viability was determined by a modification of HANKS AND WALLACE'S eosin staining procedure 1°. A 0.3% solution of eosin Y in Saline A was added to the cell cultures at 37 ° to a final concentration of o.15%. After the mixture was incubated for 5 min, the flasks were examined for the number of cells per colony and the fraction of viable cells (the cells incorporating the dye being considered non-viable). The study was performed three times and approx. IOO colonies were counted for each time interval in each experiment. In one set of experiments the ability of irradiated cells to form macroscopic colonies was measured as a function of time after irradiation. After synchronization, mitotic cells were plated as single cells in 3o-ml Falcon plastic flasks and irradiated at 1.25 h. Some cultures were immediately trypsinized and replated for colony survival; others were allowed to grow for 6, 32, 75 or 96 h after synchronization and then trypsinized and replated for colony survival. At the time of replating, the numbers of control and irradiated cells inoculated in the flasks were adjusted for the dead-cell fraction as measured b y eosin staining.

II.

CHROMOSOME ABERRATIONS AND RADIATION-INDUCED CELL DEATH.

357

After IO days' incubation, the surviving fraction was compared with the controls. Normally, five flasks were seeded for each replating and the experiment was performed at least twice. Colonies containing more than 50 cells were fixed in lO% formalin, stained in lO% Giemsa solution, and then counted under a dissecting microscope. Plating efficiencies varied between 50 and 70%. RESULTS

Survival of cells in two post-irradiation generations The number of cells per colony, uncorrected for eosin staining, is shown in Fig. I. All the cells from the initial mitotic population entered G1 by the time of the first measurement at 1.5 h. Irradiation was performed at 1.25 h after synchronization (arrow in Fig. I). Neither control nor irradiated cultures showed a complete doubling immediately after synchronization (1.84 cells per colony), but in later divisions the control population was observed to double. There was a 2- to 3-h lag in the time required for the irradiated cells to attain equivalent cellular multiplicity to the unirradiated cells in the first post-irradiation division; this probably reflects the G~ block. It is remarkable that synchrony in doubling was maintained fairly well in the first two post-irradiation divisions and paralleled that of controls. However, loss of correspondence between the two populations occurred in the third post-irradiation division. The plateau seen at 41-43 h in the control cultures actually represented a mixed population of third- and later-division cells so that there was still a considerable fraction of third-division cells 2 dividing up to 47 h. The average fraction of surviving cells as a function of time after synchronization was calculated by dividing the number of cells per colony in the irradiated popula-

3°f 2O

>Z

£ o

io

1.00

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.80

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Q I 8

I 16

I 24

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TfME AFTER RELEASE (H)

J 40

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.60 i 48

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,~

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J0

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2~

TIME AFTER SYNCHRONIZATION (H)

Fig. I. N u m b e r of cells per colony in irradiated and control p o p u l a t i o n s as a function of time after release from the colcemid-induced synchrony. I r r a d i a t i o n was performed at 1.25 h (arrow). The cell n u m b e r in each colony is not corrected for dead cells as determined b y eosin staining. S t a n d a r d errors are indicated. ©, Control; 0, 3o0 rad. Fig. 2. Surviving fraction of cells after 300 tad at 1.25 h. The fractional survival was determined b y t a k i n g the ratio of the n u m b e r of cells per colony in irradiated cultures to control cultures. The ratio was t h e n corrected for tile n u m b e r of dead cells as determined b y eosin staining. The b r o k e n lines represent the collection intervals corresponding to first-, second- and third-division cells.

358

A . V . CARRANO

tion by cells per colony in the control population (Fig. 2). The ratio at each interval was corrected for the viability of the cells observed with eosing staining. The broken lines in the figure indicate the separations between divisions as determined in the preceding analysis by the labeling pattern on the chromosomes ~. The data points for the surviving cell fraction were arbitrarily fitted with the illustrated curve. Though the data suggest a possible step function, the errors are sufficiently large not to warrant this analysis. The curve indicates that essentially no death occurs before the first mitosis (begimfing at 9 h). The surviving fraction is approx, o.87 through the first post-irradiation division (9-21 h), o.79 through the second post-irradiation division (23-31 h), and o.73 through the third postdrradiation division (33-47 h). In order to compare cell death in each generation with the loss of chromosomal aberrations, the comparison must be made for the same time interval. Since the chromosome aberrations were scored in successive metaphasesL a generation, as defined here, is the time required for a ceil to proceed from one metaphase to the next. Consequently, survival must be measured on this same basis. Since there is no a priori reason to assume that death occurs only in mitosis or only in interphase, a cell dividing for the first time after irradiation might die as the result of the anaphase behavior of an aberration either in anaphase or at any time thereafter. In order to conform to the time of the first post-irradiation generation, then, the loss of viability is measured as that occurring before the initiation of the third post-irradiation division. Similarly, death in the second post-irradiation generation might occur any time from the beginning of the second post-irradiation division up to the beginning of the fourth post-irradiation division. The cell loss in the first post-irradiation generation must therefore be computed by averaging the surviving fraction from 9 to 31 h and in the second post-irradiation generation by averaging the surviving fraction from 23 to 47 tl (Table I). TABLE I SURVIVAL

OF

V79

CELLS AFTER

EACH POST-IRRADIATION

Post-irradiation generation

Time interval (h) Average survival

o I 2

1,5-7 9-~3I 23-47

GENERATION

0-99 0.82 0.75

A measure of the rate of cell death can be derived from this average fraction of cells surviving after each generation. Thus, there is approximately I7°io cell death in the first post-irradiation generation and about 7% in the second post-irradiation generation. The death after two generations, about 25%, represents 45% of M1 the death that will occur.

Calculation of expected survival The transmission and survival parameters of chromosome aberrations calculated in the preceding study 2 can be used to predict the aberration frequencies and survival in successive generations. Since a cell containing both asymmetrical exchanges and independently induced acentric fragments is presumed to die from only one of these two types of aberrations, cells in the preceding study were classified

CHROMOSOME ABERRATIONS AND RADIATION-INDUCED CELL DEATH.

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359

as containing either two-hit aberrations (dicentrics, centric rings, or tricentrics) or only acentric fragments. The total surviving fraction is then the product of the surviving fractions of each independent classification. Consequently, the surviving fraction for each of the two groups of cells must be calculated. The relative frequency of cells containing I, 2, 3 .... n two-hit aberrations at the ith + i division can be calculated from the frequencies at the ith division by the following formula: - 2F. w"

(1)

where F~ is the relative frequency of cells in the ith + I generation possessing n two-hit aberrations; Fn is the relative frequency of cells in the ith generation with n two-hit aberrations; and W n is the probability of transmission of n two-hit aberrations. The factor 2 is incorporated since two daughter cells are produced in a fall-free situation, each with n two-hit aberrations. The relative frequency of cells in the zero class Fo (cells without two-hit aberrations) is given at the ith + I division by

Fo = 2F0+

2rn S

(2)

where S is the probability that a bridged two-hit aberration will rupture and both daughter cells will survive and ( I - W n) is the probability that a two-hit aberration witl form an anaphase bridge. For example, in the preceding study* the two-hit frequency in the first postirradiation division was o.41 per cell and the aberrations were distributed as indicated in Table II. W, the probability that a two-hit aberration falls free, was found to be 0.56. Thus, considering the frequency of cells with two aberrations per cell, equation (I) becomes F~ = 2(0.056)(0.56) 2 = 0.035 For S = o the number of cells in the zero class becomes F'o = 2(0.663) = 1.328 TABLE II EXPECTED

T R A N S M I S S I O N OF T~VO-HIT A B E R R A T I O N S

Distribution o f aberrations a Relative Two-hit aberrations per cell

Total number o f cells expected after cell death ( s u m o f classes)

Total number of Expected cells expected survival with x o o % S = o survival

frequency o f cells in each generation

o

z

2

3

4

Generation o (observed)

0.664

0.272

0.056

0.007

o.ooi

i.oo

I

--

Generation I (expected) S=o

1.328

0.305

0.035

0.003

--

1.671

2

0.834

Generation 2 (expected) S=o

2.656

0.342

0.022

o.ooi

--

3.o2~

4

0.755

S = o

a P r o b a b i l i t y of a fall-free a b e r r a t i o n , W, is 0.56, D i s t r i b u t i o n of a b e r r a t i o n s is s h o w n o n l y for t h e c a s e w h e n t h e p r o b a b i l i t y of b r i d g e r u p t u r e a n d p r o g e n y s u r v i v a l , S, is zero.

360

A . V . CARRANO

The equations may similarly be applied to the first-to-second post-irradiation generation. Predicted cell survival is then calculated by summing the relative frequencies of cells within each generation and dividing by the relative frequency expected on the basis of lOO% survival. The mechanics are shown in Table II for the case when S = o, and the results are listed for other values of S in Table IV. A formula for determining the distribution of fragments in later generations from cells containing only acentric fragments has also been derived~, 4 and is given simply as (3)

F' = Z F P~ ~ 4 e all e

where F ' is the frequency of cells containing o, 2, 4.-.Je~'e~ acentric fragments in the ith + I or later generations. (Only even fragment multiples are expected to be observed in subsequent generations, since the fragment is transrnitted as a paired structure ~) ; F is the frequency of cells in generation zero containing o, I, 2 . . . f acentric fra~nents; ps is the probability that a daughter cell will survive if it loses f fragments. P is equal to I.O for one generation and to 0.70 for two generations~; ne is the number of daughter cells produced that contain o, 2, 4 ~ . . f ~ n fragments as the result of a transmission event, e, and is ascertained for each class of cells in generation o; q~ is the probability associated with the occurrence of each transmission event, e, and is a function of the transmission probability, 2T, or o.57 (refs. 2,3). The application of this formula has been demonstrated elsewhere a and only the results are shown here. The expected distribution of acentric fragments in two successive generations based on an acentfic fragment frequency of o.556 in generation 0 ~ is given in Table III. With the values of P given above, the expected fractional survival resulting only from fragment loss after one generation is Lo but after two generations is o.89. The total expected surviving fraction, then, is the product of the expected survival of cells with two-hit aberrations and cells with acentric fragments. This is shown in Table IV for various values of S (probability of bridge rupture and progeny survival). These calculated values are to be compared with the surviving fraction

TABLE

11I

EXPECTED TRANSMISSION OF FRAGMENTS IN CELLS WITHOUT T~VO-HIT ABE.RRATIONS

Distributio~a ~[ aberrations ~ Relative Fragments per cel~

Totag number of cells expected alter celt death (s,~m of classes)

Total number of Expected cells expected survival with z o o % s~trvivcd

frequency of cells in each generat:ion

o

•

2

3

4 or more

Generation o

o.574

o.3I 9

0.089

o.o16

o.oo2

t.oo

t

......

Generation I (expected) P=I,O

1,7I

~,,,-

0.270

_4

0.020

2,oo

2

I,ooo

Generation 2 (expected) P=-o, 7

3.o3

---

o.481

.....

o.o37

3,551

4

o.888

(observed)

P r o b a b i l i t y t h a t a f r a g m e n t will b e t r a n s m i t t e d a s a p a i r e d s t r u c t u r e , 2 T , is 0.57. P r o b a b i l i t y t h a t a d a u g h t e r ceil will s u r v i v e t h e loss of a s i n g l e f r a g m e n t , P , is I.O o v e r o n e g e n e r a t i o n b u t 0. 7 o v e r t w o g e n e r a t i o n s ,

CHROMOSOME ABERRATIONS AND RADIATION-INDUCED CELL DEATH.

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TABLE IV EXPECTED

Generation

I 2

SURVIVAL

OF CELLS a

Surviving fraction From two-hit aberrations ( S ) o o.25 o.5 0.75

x.o

From acentric fragments

0.834 0.755

I.OO I.OO

I.O 0.888

0.877 o.817

o.918 0.878

0.96o o.94 °

Total survival ( S ) o 0.25 0.5

0.75

I.O

0.83 0.67

0.96 0.84

I.O 0.89

0.88 0.73

0.92 0.78

a E x p e c t e d values were calculated a s s u m i n g t h a t the probability of a fall-free aberration, W, is 0.56 and the p r o b a b i l i t y of acentric f r a g m e n t transmission, 2T, is o.57. Observed survival: generation I. o.82; generation 2, o.75.

observed in each generation, shown at the bottom of Table IV. Note that the closest agreement of the observed and expected values for both generations occurs when S < o.5. The expected values in Table IV were calculated on the assumption that W , the probability of obtaining a fall-free, two-hit aberration, was o.56 (true only if S = o). Other values of S can be similarly applied. For example, values of W can be obtained for any value of S by reworking equations (I) and (2) with the observed aberration frequencies. Of course, for each new value of W , the probability of cell survival due to fragment loss, P, will also change 4 and consequently the expected survivals in generations I and 2 will be altered. The effect of the variation of S on these parameters is illustrated in Table V. It is evident from the agreement with the observed survival that the probability of bridge rupture and progeny survival, S, most likely varies between o and o.5. Therefore, the probability that a two-hit aberration will fall free, W , lies between o.56 and o.59, and the probability that a cell survives one generation after a fragment loss is i.o but survival over two generations is about 0.66-0.70. Since the distribution of aberrations can be predicted for cells in successive generations, the mean aberration frequency can readily be calculated. In the case of acentric fragments, it has been shown that the fragment frequency in any succeeding generation is equal to the product of the transmission frequency of acentric fragments, 2T, and the mean frequency in the previous generationL so that the loss of fragments through cell death does not affect the observed frequency in the succeeding generations. For the two-hit aberrations, the mean frequency is obtained simply by taking the mean of the expected distribution of aberrations calculated from equations (I) and (2), The decreases in aberration frequencies for each class of aberrations are shown for eight generations in Table VI. TABLE V RANGES

OF

W, P

AND

E(S)

S

W

P1-2

P~-a

E ( S ) 1a

E ( S ) 2a

o 0.25 o.5o 0.75 I.O

0.56 0.57 0.59 o.61 o.64

I.O i.o I.O I.O 0.93

o.7o 0.68 0.66 0.64 o.62

0.83 0.87 o.91 o.96 0.93

0.67 0.70 0.75 0.78 0.78

a E ( S ) 1 and E ( S ) 2 are the expected survivals in the first and second post-irradiation generations respectively. Observed survival: first generation, 0.82; second generation, o.75.

362

A.V. CARRANO

T A B L E VI EXPECTED

ABERRATION

FREQUENCIES

AND LIMITING

S U R V I V A L OF S U C C E S S I V E G E N E R A T I O N S

Postirradiation generation

Two-hit aberrations Frequency Fraction of Sa = o population in zero class

Fragments b Frequency

o I 2

o.41o 0.230 o.129

0.663 0.795 0.879

0.556 o.316 o.18o

0.574 o.855 0.854

o.381 o.68o o.745

3 4 5

0.072 0.040 0.022

0-931 o.961 0.978

O.lO3 0.059 0.034

0.902 0.943 0.967

0.840 0.906 0.946

6 7 8

o.oi2 °.°°7 0.004

0.988 o.993 0.996

o.o19 o.oi I 0.006

o.981 0.989 0.994

0.969 0.982 0.990

Fraction of population in zero class

Expected survival after to days' growth

a S is t h e p r o b a b i l i t y of b r i d g e r u p t u r e a n d p r o g e n y s u r v i v a l . b I n cells w i t h o u t t w o - h i t a b e r r a t i o n s .

The table was derived for S = o and for the aberration frequencies obtained by analyzing metaphase cells in the first generation after G1 irradiation 2. The fraction of the population without two-hit aberrations is based on the Poisson distribution except that, in the case of the acentric fragments, the zero class is taken from the expected distribution for two generations (Table III) since it is not Poisson. After three generations the Poisson distribution is found to be a good approximation for the frequency of cells without aberrations. If a population of cells with any of the aberration frequencies given in Table VI were plated for a survival analysis, the surviving fraction expected after io days' growth could then be obtained by multiplying the two zero class fractions.

Replating experiments A test was performed on the formulae's ability to predict cell suvival for various aberration frequencies by replating the cell population. Cells irradiated initially in G1 of generation zero (1.25 h after synchronization) were trypsinized and plated after various periods of cell growth (and hence loss of cells and aberrations). The fraction of colonies surviving after IO days' growth was then compared to the predicted survival from Table VI. The results are indicated in Table VII. As observed in the analysis of cellular multiplicity (Fig. 2), no cell death occurred before the first TABLE VII REPLATING

OF S Y N C H R O N O U S

CELLS

Time of replating (h after synchronization)

Approximate post-irradiation generation

Observed survival a ( ± S.E.)

Expected survival ~

1.25 6 32 75 96

o o 1-2 4-5 6 7

0.44 0.45 0.63 0.85 0.97

0.38 0.38 o.68-o.75 o.91-o.95 o.97 0.98

a Aftei IO d a y s ' g ro wth. b F r o m T a b l e VI.

-~ 4± ± ±

0.03 0.06 o.o5 0.09 0.05

CHROMOSOME ABERRATIONS AND RADIATION-INDUCED

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post-irradiation division, i.e., the colony-surviving fraction after replating was the same at 1.25 and 6 h. Also, as the irradiated cells divided, their survival increased with time in a manner consistent with that predicted on the basis of aberration loss. DISCUSSION

The method used to compute the surviving fraction in the first and second post-irradiation generation gives only an approximation, since cells from two generations always overlapped. Thus, it is difficult to ascertain, for example, whether the cell death observed at 25 h results from first generation cells dying in interphase or second generation cells dying in mitosis. Consequently, the survival data had to be averaged over two generations. The problem arises, then, that not all of the deaths will have occurred within the time intervals selected (9-31 h for first-generation cells and 23-47 h for second-generation cells). If this were the case, the colony multiplicity observed for the irradiated population would be higher than predicted. This might explain the death in the second generation, where the observed surviving fraction was 0.75 and the predicted was 0.67 (Table IV). There is another possible explanation for this discrepancy: the time interval for the second generation deaths (23-47 h) might not have been representative of all third-division cells since more secondgeneration deaths probably occurred after 47 h, which would tend to lower the observed survival. GREENBLATT8 has obtained a very similar survival pattern based on total cell counts after irradiation of asynchronous V79 cells. The replating experiments, however, suggest that only a small amount of the cell death, if any, produced by the aberrant anaphase behavior of broken chromosomes, might not occur within one generation. The evidence for this lies in the fact that the observed survival after replating agrees well with survival predicted on the basis of cell death within one generation. The formulae predicted that death within the first generation would be caused primarily by the anaphase behavior of asymmetrical exchanges, i.e., the probability, P, that a cell would survive after fragment loss was I.O. The predicted surviving fraction, 0.83, agrees well with the observed, 0.82, when the probability of bridge rupture and progeny survival, S, is zero. This, then, suggests that the bridging of chromosomes accounts for almost all of the death observed. This first-generation death, 3o% of all the death that occurs, can be attributed either to the anaphase bridge's failure to rupture or, if the bridge ruptures, to failure of the progeny cells to survive one generation (S ~ o for both possibilities). Fusion of daughter cells (many of which ultimately died) has been observed in irradiated populations by time lapse cinemicrography for other cell lines~5,16. The lethal effect of asymmetrical exchanges has also been observed before in vivo~7, 2°. The quantitative estimation of the parameters T, P and W is restricted by the assumptions used in their derivation 4. In the case of fragments it was assumed that a cell was likely to die from deficiencies rather than from duplications of genetic material. In tissue culture systems it has not been ascertained which is the more injurious, although it is known that stability is often conferred on a primary culture by altering its karyotype to a higher modal chromosome number ~a. Further, considering the diploid (pseudodiploid) character of the V79 cell line, it is difficult to

364

A.V. CARRANO

understand why the loss of a fragment will lead to cell death if there is homologous genetic material elsewhere in the genome. The present analysis implies that loss of all copies of homologous material might not be necessary to produce cell death but that loss of substantial regions of the genome will suffice. As the results indicate, a cell might be capable of completing one generation after fragment loss, but the probability of undergoing further generations decreases. It is expected that if the third post-irradiation generation could have been measured, P would have been very much less than o.7. Thus, the cell might require at least one generation to deplete itself of any accumulated intracellular material that would have been manufactured by the genetic material in the lost fragment. Microtus agrestis cells, irradiated with 35o R of X-rays in vivo, were found to be capable of surviving up to 3 ° days in vivo or in primary cultures with deletions of heterochromatic segments of the sex chromosomes 5. Unfortunately, the number of cell divisions through which these cells passed over the 3o-day period is not known with certainty. In any case, these results are not inconsistent with the data presented here, for there could be a finite probability, P, that a cell will survive the loss of certain genetic material even after m a n y generations. The classification of aberrations into asymmetrical exchanges (two-hit aberlations) and acentric fragments disregards both translocations and inversions of genetic material. The data presented here suggest that all the observed death can be accounted for if translocations and inversions did not lead to loss of viability. HEDDLE 11 has evidence that symmetrical exchanges are as frequent as asymmetrical exchanges in Vicia faba. If symmetrical exchanges do indeed cause a cell to die, the rate and amount of death is extremely difficult to reconcile with the observed fractional survivals. Consequently, it appears that a cell capable of preserving its genetic information in either the original or a rearranged state will survive. This agrees with the results of GROTE9, who found that in a diploid Syrian hamster cell line, the only cells that did not survive were those forming micronuclei. From her analysis of micronuclei in diploid Syrian hamster cells, GROTE9 has further suggested that chromosome fragment loss can account for all the observed death after X-irradiation in G1. For the V79 Chinese hamster cells this does not seem to be the ease. The amount of death that can be attributed to fragment loss depends on the value of S, the probability that anaphase bridges rupture and both progeny cells survive. Thus, when S = 1.o, death can not be caused by anaphase bridges and must be attributed entirely to fragment loss. The data best fit a range of S between o and o.5 (Table IV). In Tradescantia it has been shown that the probability of bridge rupture is proportional to the exchange frequency 1~. At the asymmetrical exchange frequency observed here (o.41 aberrations/cell) the probability of bridge rupture should be about 0.5, using the Tradescantia correlation. Since the parameter S also incorporates the probability of progeny survival, the observed range is reasonable. At the upper limit on the range of S (o.5) it is estimated that the m a x i m u m amount of death that might be attributed to fragment loss is about 5o% in the first postirradiation generation and about 4o% in the second. The remainder of the cell death must be due to asymmetrical exchanges. The analysis of cell death in successive generations indicates that if the transmission and survival parameters for specific aberrations are known, formulae can

CHROMOSOME ABERRATIONS AND RADIATION-INDUCED CELL DEATH. i i .

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p r e d i c t t h e f r a c t i o n of t h e p o p u l a t i o n s u r v i v i n g a t a n y g i v e n t i m e . T h e d a t a , in f a c t , s u g g e s t t h a t i t is l i k e l y t h a t e v e r y a s y m m e t r i c a l a b e r r a t i o n , w h e t h e r a t w o - h i t a b e r r a t i o n o r a n a c e n t r i c f r a g m e n t , is c a p a b l e of c a u s i n g t h e cell t o die. T h e r a t e a t w h i c h e a c h i n d u c e s loss of V i a b i l i t y differs. T w o - h i t a b e r r a t i o n s a p p e a r t o i n d u c e cell d e a t h r a p i d l y , p r o b a b l y b e c a u s e of t h e f o r m a t i o n of a n a p h a s e b r i d g e s , w h e r e a s f r a g m e n t s a r e d e l a y e d i n t h e e x p r e s s i o n of t h e i r m a x i m u m l e t h a l effect ( p r o b a b l y for o n e or m o r e g e n e r a t i o n s a f t e r t h e i r loss f r o m t h e cell). T h e t o t a l a b e r r a t i o n f r e q u e n c y ( t w o - h i t p l u s f r a g m e n t s i n cells w i t h o u t t w o - h i t a b e r r a t i o n s ) o b s e r v e d a f t e r 300 R in G1 w a s a b o u t I.O p e r cell a n d p r o d u c e d a c o l o n y - s u r v i v i n g f r a c t i o n of 0.44. ACKNOWLEDGMENTS I a m i n d e b t e d t o SHELDON WOLFF for h i s c r i t i c i s m s a n d s u p p o r t t h r o u g h o u t t h e c o u r s e of t h i s w o r k . T h i s w o r k w a s s u p p o r t e d i n p a r t b y N . I . H . B i o p h y s i c s T r a i n i n g G r a n t No. 5 - T O I - G M 0082 9 a n d w a s p e r f o r m e d u n d e r t h e a u s p i c e s of t h e U.S. A t o m i c E n e r g y C o m m i s s i o n . REFERENCES I BENDER, M. A, AND S. WOLFF, X-ray-induced chromosome aberrations and reproductive death in mammalian cells, Am. Naturalist, 95 (1961) 39-52 . 2 CARRANO, A. V., Chromosome aberrations and radiation-induced cell death, I. Transmission and survival parameters of aberrations, Mutation Res., 17 (1973) 341-353 • 3 CARRANO,A. V., A formula to predict the transmission frequency of acentric fragments, Genetics, in the press. 4 CARRANO,A. V., AND J. A. HEDDLE, The fate of chromosome aberrations, J. Theoret. Biol., in the press. 5 COOPER, J. E. K., AND T. C. HSU, Radiation-induced deletions and translocations of Microtus agrestis sex chromosomes in FIFO, Exptl. Cell Res., 67 (1971) 343-351. 6 DEWEY, W. C., S. C. FURMAN AND H. H. MILLER, Comparison of lethality and chromosomal damage induced by X-rays in synchronized Chinese hamster cells in vitro, Radiation Res., 43 (197 ° ) 561-581. 7 DEWEY, W. C., L. E. STONE, H. H. MILLER AND R. E. GIBLAK, Radiosensitization with 5bromodeoxyuridine of Chinese hamster cells X-irradiated during different phases of the cell cycle, Radiation Res., 47 (1971) 672-688. 8 GREENBLATT, C. L., The evaluation of X-ray-induced chromosome aberrations in cell cultures of the Chinese hamster, Intern. J. Radiation Biol., 4 (1961) 185-21o. 9 GROTE, S. J., Radiation-induced lethality and genetic damage in mammalian cells, Ph. D. Dissertation, Univ. of London, 1972 . io HANKS, J. H., AND H. WALLACE, Determination of cell viability, Proc. Soc. Exptl. Biol. Med., 98 (1958) 188-192. i I HEDDLE, J. A., Randomness in the formation of radiation-induced chromosome aberrations, Genetics, 52 (1965) 1329-1334 . 12 HEDDLE, J. A., AND D. SCOTT, The rupture of radiation-induced anaphase bridges, Radiation Botany, io (197 o) 11-17. 13 Hsu, T. C., D. BILLEN AND A. LEVAN, Mammalian chromosomes in vitro, XV. Patterns of transformation, J. Natl. Cancer Inst., 27 (1961) 515-541. 14 Hsu, T. C., W. C. DEWEY AND R. M. HUMPHREY, Radiosensitivity of cells of Chinese hamster in vitro in relation to the cell cycle, Exptl. Cell Res., 27 (1962) 441-452. 15 HURWlTZ, C., AND L. J. TOLMACH, Time lapse cinemicrographic studies of X-irradiated HeLa $3 cells, I. Cell progression and cell disintegration, Biophys. J., 9 (1969) 6o7-633. 16 MARIN, G., AND M. A. BENDER, Radiation-induced mammalian cell death; Lapse time cinemicrographic observations, Exptl. Cell Res., 43 (1966) 413-423 . 17 PONTECORVO, G., AND H. J. MULLER, The lethality of dicentric chromosomes in Drosophila, Genetics, 26 (1941) 165. 18 PUCK, T. T., Action of radiation on mammalian cells, III. Relationship between reproductive death and induction of chromosome anomalies by X-irradiation of euploid h u m a n cells in vitro, Proc. Natl. Acad. Sci. (U.S.), 44 (1958) 772-780.

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19 SASAKI,M. S., AND A. NORMAN, Selection against c h r o m o s o m e aberrations in h u m a n l y m p h ocytes, Nature, 214 (1967) 502-503 . 20 SAX, K., The behavior of X-ray-induced c h r o m o s o m a l aberrations in Allium root tip cells, Genetics, 26 (1941 ) 418-425 . 21 Yu, C. K., AND W. I~. SINCLAIR,Mitotic delay and c h r o m o s o m a l aberrations induced by Xrays in synchronized Chinese h a m s t e r cells in vitro, J. Natl. Cancer Inst., 39 (1967) 619-631.