125

Mutation Research, 37 (1976) 125--136 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

X-RAY-INDUCED MUTATION TO 6-THIOGUANINE RESISTANCE IN CULTURED HUMAN DIPLOID FIBROBLASTS

ROGER COX and W.K. MASSON

M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon. 0 X l l ORD (England)

(Received February 12th, 1976) (Revision received May 4th, 1976) (Accepted May 13th, 1976)

Summary

X-ray induced mutation to 6-thioguanine (6TG)-resistance was studied in early passage cultures of human diploid fibroblasts.

The appearance of phenotypic induced mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings and to the duration of the growth period prior to mutant selection; the maximum yield of X-ray induced mutants was observed when cells surviving radiation had com- pleted 3--4 doublings (6--7 days growth) in non-selective medium.

The maximum induced mutation frequency was linearly related to X-ray dose and the mutation rate was estimated to be 3.1 • 10 -7 mutations per viable cell per tad.

The data obtained for X-ray induced mutations in cultured human diploid fibroblasts were compared with (a) similar experimental data obtained with established cell cultures and (b) with theoretical predictions of X-ray mutation rates in human germ cells.

Introduction

The induction of mutations in cultured human diploid fibroblasts with phys- ical and chemical mutagenic agents may give insights into the mechanisms of mutagenesis in normal human cells and also provide useful quantitative in- formation about genetic risks to man. Cultured human somatic cells having mutations affecting the activity of the purine salvage enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT, EC 2.4.2.8) may be selected by their resistance to the cytotoxic purine analogues 8-azaguanine (8AG) [2] or 6-thioguanine (6TG) [7] and mutation to 8AG resistance in human cells has been used to quantitate the mutagenicity of X-rays and chemical agents [2, 12,13]. However, if quantitative experiments are to be useful, even in terms of

126

simple comparative risk estimates, then it is essential to recover the maximum number of mutant clones from the treated cell population. Purine analogue concentration, cell plating density and antagonism of the analogue by serum components have been examined as factors influencing the recovery of HGPRT- deficient mutant clones [7]. However, the duration of post-treatment growth in non-selective medium before mutant selection, which is of paramount im- portance in the detection of the maximum number of induced mutants, has not been investigated in cultured human cells. The use of a post-treatment growth period before mutant selection is based on the assumption that a muta- gen-treated cell population requires a period of growth in non-selective medium to allow induced mutant genotypes in that population to be recognised by means of mutant assay techniques that rely upon phenotypic differences be- tween mutant and wild type cells. In previous studies various post-treatment growth periods of 3 days, 3 generations and 7 days were used before the selec- tion of induced 8AG-resistant mutants of human cells [2,12,13]. However, in none of these studies were data presented to show that the chosen post- t reatment growth period was optimal for the recovery of induced mutants and as a consequence the results cannot be reliably assessed in quantitative terms.

In this report we describe the effects of post-irradiation growth on the detec- tion of X-ray induced mutations to 6TG-resistance (HGPRT-deficiency) in cul- tured human fibroblasts and show that (a) the appearance of induced pheno- typic mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings completed and (b) the X-ray induction of mu- tation was linearly related to dose.

Materials and methods

Cell cultures HF19 human diploid fibroblasts were initiated from 9 foetal lung tissue and

the cells maintained in monolayer culture using methods previously described [8]. Exponentially growing early passage (4--15 in vitro population doublings) cultures were used in all experiments. The cells were removed from the growth surface of culture vessels with 0.1% trypsin plus 0.4 mg per ml ethylene di- amine tetra acetate (EDTA), resuspended in normal growth medium and the cells counted using a haemocytometer .

Media The normal growth medium was Eagle's Minimal Essential Medium (MEM)

plus 10% foetal calf serum (Gibco Biocult). Medium for the selection of HGPRT-deficient mutants was normal growth medium supplemented with 2 pg per ml 6TG (Sigma). Four different batches of serum were used in these ex- periments, and before use each was shown to be suitable for the selection of HGPRT-deficient mutants [ 7 ].

Irradiation o f cells Cells were X-irradiated 20 h after at tachment to the growth surface of Petri

dishes (Sterilin). X-ray (250 kV) doses of 0--200 rad were used and dose rates varied between 50 and 200 rad per min. For assay of induced mutat ion 2 • 104

127

cells were irradiated at each X-ray dose. Cells for feeder layers were irradiated in suspension with 3 krad X-rays.

Cell survival experiments X-ray survival curves of HF19 cells were obtained using the feeder cell tech-

nique previously described [5]. Cells surviving radiation were cloned in 9 cm diameter polystyrene petri dishes (Sterilin) containing 10 ml of normal growth medium. The dishes were incubated in 95% air plus 5% CO2 at 37°C for 14 days; after this time clones were stained with 0.25% Azur A dye and counted using a low power microscope. The fraction of cells surviving each dose of radiation was calculated with respect to the cloning efficiency of unirradiated cells.

Post-irradiation growth o f cells to allow the expression o f induced mutations Following irradiation, 2 • 106 cells were plated into 14 cm diameter poly-

styrene Petri dishes (Sterilin) containing 30 ml normal growth medium and incubated in 95% air plus 5% CO2 at 37°C. At 3 day intervals 2 . 1 0 6 cells were subcultured into fresh medium to maintain exponential growth. Post- irradiation growth periods of 1 to 22 days were used in these experiments and the viability of control and irradiated cultures was estimated throughout the growth period using the feeder cell technique [5]. The average number of cell doublings during each growth period was calculated as: N/No = 2 n where N is the number of viable cells in the final culture, No is the number of surviving viable cells in the original inoculum and n is the number of population doubl- ings. For post-irradiation growth periods > 3 days the value of N was corrected to account for cell doublings before subculture.

Selection o f induced HGPRT-deficient mutants Following growth in non-selective medium cells were transferred to selective

medium to permit the detection of mutant phenotypes. Previous experiments [7] showed that 6TG resistant, HGPRT-deficient mutants of HF19 human diploid fibroblasts were efficiently selected by plating cells at densities < 1 0 a cells per cm 2 into normal growth medium containing 1--5 pg per ml 6TG. In experiments reported here HGPRT-deficient cells were selected by their ability to form clones in growth medium containing 2 pg per ml 6TG. Two cell plating methods were used. In early experiments 5 • 104 cells were plated into each of 34--68 Petri dishes of 9 cm diameter containing 10 ml of selective medium. In later experiments mutants were also cloned on the growth surface of bulk cell culture vessels (Sterilin) using a method previously described [9]. Petri dishes were incubated in 95% air plus 5% CO2 ; selective medium in cell culture vessels was equilibrated in 95% air plus 5% at the beginning of the incubation period and the vessels required no further gassing. Incubation of both Petri dishes and cell culture vessels was at 37°C for 21 days and no medium changes were neces- sary for op t imum recovery of mutants. Clones arising in 6TG medium at the end of the incubation period were stained with 0.25% Azur A dye for 1 h and counted by eye against a white background. Small clones were examined with a low power microscope to distinguish them from aggregates of stained cell debris.

128

Determination o f induced mutation frequency Mutation frequencies in control cell populations were calculated as:

Nov M o - So (1)

Where, N O is the number of mutants in the unirradiated population, So is the number of viable cells in the same unirradiated population at the time of mu- tant selection, c is a correction factor of 2 to allow for the loss of mutants un- der the selective conditions used [7] and Mo is the mutat ion frequency per viable cell in the unirradiated population.

Similarly, mutat ion frequencies in X-irradiated cell populations were calcu- lated as:

Nxc Mx = Sx (2)

Where, Nx is the number of mutants in the X-irradiated population, Sx is the number of viable cells in the same X-irradiated population at the time of mu- tant selection, c is the same correction factor used in eqn. (1) and Mx is the mutat ion frequency per viable cell in the irradiated population. The frequency of mutants induced by the radiation is then given by:

Mx - -Mo (3)

Results

Dose-response for cell inactivation Pooled data from five different experiments showed that the X-ray dose-re-

sponse for cell inactivation was exponential throughout with a Do value of 126.2 + 2.5 tad {Fig. 1). Similar unshouldered X-ray survival curves have been published for other cultured human diploid fibroblasts of foetal origin [ 5,6].

Post-irradiation growth o f cells The growth curve of Fig. 2 illustrates the doubling of surviving cells during a

13 day growth period after X-ray doses of 0, 50, 100, 150 and 200 rad. Growth was exponential with no indication of radiation-induced division delay. The doubling time of viable cells in irradiated and control populations was 32 h.

The relationship between post-irradiation growth and induced mutation fre- quency

The relationship between post-irradiation growth and observed mutat ion fre- quency is shown in Fig. 3. Mutation frequencies after 1 and 2 days growth were generally indistinguishable from those observed in unirradiated cultures. The mutat ion frequency detected increased progressively in all irradiated cell popu- lations when growth was prolonged to 3, 4 or 5 days; the extent of the increase was approximately proportional to the initial radiation dose. With post-irradia- tion growth periods greater than 6 days no further increase in induced muta- tion frequency per viable cell was observed and, for all radiation doses, muta- tion frequencies after growth for 6 or 7 days were indistinguishable from those after growth for 10--13 days. The extent of this plateau region on the curves of

129

1.0

Q5

~d

Q2

0.1

X-roy dose (rod)

0 50 100 150 I I I

\ \

200 L

6

~ 3

2

±

F

/ /

J I l I I I

0 2 4 G 8 10 12

Duration of post-irradiation growth(doys)

Fig. 1. Survival of h u m a n diploid f ibroblas ts a f t e r X-irradiat ion. Bars r ep r e sen t one s t anda rd dev ia t i on on d a t a c o m p i l e d fo r five sepa ra t e expe r imen t s . The curve was f i t ted by l inear regress ion analysis (by var iance ra t io t e s t P = 0.31) .

Fig. 2. The pos to i r radia t ion g r o w t h of c on t ro l and i r r ad ia ted h u m a n diploid f ibroblas ts in n o r m a l g r o w t h m e d i u m . Each p o i n t includes d a t a fo r un i r r ad ia t ed cells and for cells surviving doses of 50, 100, 150 an d 200 rad and d i f f e r en t doses are n o t dis t inguished. Bars r e p r e sen t one s t anda rd dev ia t ion on the doubl ing of viable cells. A l t h o u g h the curve is c lear ly l inear up to seven days of pos t - i r rad ia t ion g r o w t h the re is some dev ia t i on wi th longer g r o w t h per iods (by var iance r a t io tes t P = 0 .015) .

Fig. 3 was not fully examined, but in one experiment on cells given 150 rad the mutat ion frequencies were indistinguishable after growth for 11 and 13 days and for 22 days. This result suggests that, once fully expressed, HGPRT-defi- cient phenotypes induced by low doses of X-rays were not at any selective dis- advantage in the mixed cell population growing exponentially in non-selective medium.

The data from two mutat ion experiments using post-irradiation growth for 7 days and one experiment using post-irradiation growth for 10 days are shown in Tables I and II. In addition to illustrating the similarity of induced mutat ion frequencies detected after post-irradiation growth for 7 and 10 days the data also show good agreement between the two methods of mutant cloning i.e. Petri dishes and bulk cell culture vessels [9].

The data of Figs. 2 and 3 were also used to estimate the rate of appearance of radiation-induced, HGPRT-deficient phenotypes during post-irradiation growth. Each induced mutat ion frequency can be expressed as a fraction of the mean of the induced mutat ion frequencies obtained for the same X-ray dose after post-irradiation growth periods of 7--13 days (5--9 populat ion doubl- ings) i.e. as a fraction of the maximum. This fraction, for each mutant frequen- cy found after 1--6 days growth (0--4 population doublings), was then plot ted

T A B L E I

M U T A T I O N F R E Q U E N C I E S IN C O N T R O L A N D X - I R R A D I A T E D C E L L P O P U L A T I O N S : M U T A N T S S E L E C T E D IN P E T R I D I S H E S A F T E R 7 D A Y S

E X P R E S S I O N

Dose D i s h e s Cel l s C l o n i n g V i a b l e T o t a l M u t a t i o n I n d u c e d C o m p a r a b l e

( rad) s c o r e d a p l a t e d e f f i c i e n c y b ce l l s m u t a n t f r e q u e n c y p e r m u t a t i o n i n d u c e d m u t a t i o n

(%) p l a t e d c l o n e s p e r v i a b l e f r e q u e n c y p e r f r e q u e n c i e s

ce l l e v i a b l e ce l l o b t a i n e d u s i n g b u l k

ce l l c u l t u r e ves se l s d

0 66 3 . 3 " 1 0 6 67 .1 2 . 2 - 1 0 6 1 0 9 . 0 " 1 0 - 6 0 0

50 6 4 3 . 2 - 1 0 6 6 5 . 0 2 . 1 - 1 0 6 21 2 . 0 . 1 0 -5 1 . 1 - 1 0 -5 1 . 6 - 1 0 -5

1 0 0 50 2 . 5 - 1 0 6 6 5 . 5 1 . 6 " 1 0 6 3 3 4 . 1 - 1 0 - s 3 . 2 - 1 0 -5 2 . 8 - 1 0 -5

1 5 0 68 3 . 4 - 1 0 6 6 9 . 2 2 . 3 - 1 0 6 7 6 6 . 5 " 1 0 -5 5 .6 " 1 0 - 5 3 . 8 - 1 0 -5

2 0 0 48 2 . 4 . 106 5 2 . 6 1 . 3 - 1 0 6 4 6 7 . 3 " 1 0 -5 6 . 4 . 1 0 -5 6 . 5 - 1 0 -5

a Pe t r i d i shes o f 9 c m d i a m e t e r e a c h c o n t a i n i n g 5 • 1 0 4 cel ls .

b D e t e r m i n e d a t t h e t i m e o f m u t a n t s e l e c t i o n .

c V a l u e s c o r r e c t e d f o r e f f i c i e n c y o f m u t a n t r e c o v e r y (see t e x t ) .

d D a t a f r o m T a b l e II , see a lso ref . [ 9 ] .

T A B L E II

M U T A T I O N F R E Q U E N C I E S I N C O N T R O L A N D

A F T E R 7 A N D 1 0 D A Y E X P R E S S I O N X - I R R A D I A T E D C E L L P O P U L A T I O N S : M U T A N T S S E L E C T E D I N B U L K C E L L C U L T U R E V E S S E L S

Dose E x p r e s s i o n Ce l l s

( rad) t i m e ( d a y s ) p l a t e d C l o n i n g V i a b l e ce l l s T o t a l m u t a n t M u t a t i o n f r e q u e n c y I n d u c e d m u t a t i o n e f f i c i e n c y a p l a t e d c l o n e s p e r v i a b l e ce l l b f r e q u e n c y p e r

(%) v i a b l e ce l l

0 7

0 1 0

50 7

50 10

1 0 0 7

1 0 0 10

1 5 0 7

1 5 0 1 0

2 0 0 7

2 0 0 1 0

7 1 0 6

7 1 0 6

7 1 0 6

7 1 0 6

7 1 0 ° 7 1 0 6

7 1 0 6

7 • 1 0 6

7 - 1 0 6

7 • 1 0 6

8 6 . 5 6 .0 • 106 53 1 .7

6 3 . 2 4 .4 • 106 3 3 1 .5

5 6 . 4 3 .9 • 106 6 6 3 .3

6 2 . 8 4 . 4 • 106 6 5 2 .9

5 8 . 0 4.1 • 106 9 3 4 .6

5 9 . 0 4.1 • 106 8 4 4 .1

6 1 . 2 4 .3 • 106 1 1 8 5 .5

6 7 . 0 4 .7 • 106 1 2 7 5 .4

6 1 . 2 4 .3 • 106 1 7 6 8 .2

6 2 . 2 4 .3 • 106 1 7 2 7 .9

• 1 0 - 5 0

1 0 -5 0

1 0 -5 1 .6 • 1 0 -5

1 0 -5 1 .5 • 1 0 -5

1 0 -5 2 .8 - 1 0 -5

1 0 -5 2 .6 • 1 0 -5

1 0 -5 3 .8 • 1 0 -5

10 -5 3 .9 • 1 0 -5

1 0 -5 6 .5 • 1 0 -5

1 0 -5 6 .4 - 1 0 -5

a D e t e r m i n e d i n P e t r i d i s h e s a t t h e t i m e o f m u t a n t s e l e c t i o n .

b V a l u e s c o r r e c t e d f o r e f f i c i e n c y o f m u t a n t r e c o v e r y (see t e x t ) .

1 3 1

8

x

u

~ G

>

# . 4

o

.G

E

0

Average cell doublings

2 .4 6 8 1.4 n i I i / / - - - - ' q

O . - f ~ - O O . . . . . . . . . . 200rod O /

, , '+Orod

O ~ ° • I

I I i i I 1 I / ~ - - J

2 4 6 8 10 12 1.4 22

Duration of post-irradiation growth (days)

-100 rad

50rod

0

1.0

0.8

u~

E "6 0.4

0.2 i

ol I

0

Duration of post-irradiation growth (days)

2 .4 6

+

2

i I _1 I 2 3 a

Average celt doubhngs

Fig. 3. T he re lat ionship b e t w e e n durat ion o f post - irradiat ion g r o w t h and induced m u t a t i o n f r e q u e n c y observed in cu l tured h u m a n diploid f ibroblasts af ter X-ray do se s o f 5 0 tad ( e ) ; 1 0 0 tad ( a ) ; 1 5 0 tad (m); and 2 0 0 zad (0 ) . M u t a t i o n frequenc ies in unirradiated contro l s varied b e t w e e n 0 . 5 and 1 .8 • 1 0 -5 m u t a - t ions per viable cell .

Fig. 4. T he re lat ionship b e t w e e n n u m b e r o f post - irradiat ion po pu la t io ns doubl ings and frac t ion o f to ta l m u t a n t s d e t e c t e d in X-irradiated cu l tures o f h u m a n diploid f ibroblasts . The s y m b o l s referring to X-ray dose are the s a m e as in Fig. 3. The curve was f i t t ed by l inear regress ion analysis ( see t ex t ) .

against the number of cell doublings occurring in the same culture during the same post-irradiation growth periods (Fig. 4). The number of phenotypic mu- tants detected increased linearly with both the number of cell doublings and the duration of post-irradiation growth, independent of radiation dose. The line of best fit was calculated for y = a + b x where x was the average number of cell doublings, y was the fraction of mutants detected, a was the intercept and b was the slope. From the data of Fig. 4 a was 0.196 -+ 0.068 and b was 0.320 + 0.035. In empirical terms the data show that 50% of radiation induced mutants were detectable by their resistance to 6TG approx. 2 cell dOublings (3 days post-irradiation growth) after irradiation. The line of best fit in Fig. 4 did not extrapolate to zero but intercepted the X axis at 0.6. This result is evidence that one post-irradiation cell doubling was probably required before any radia- tion-induced genotypic mutants were detected by means of their resistance to 6TG and is also evidence that cell doubling may be a more important factor than chronological time in the expression of radiation induced mutation.

D o s e - r e s p o n s e f o r cel l m u t a t i o n If the induced mutation frequencies obtained after post-irradiation growth

periods of 6--13 days can be regarded as representing the maximum X-ray in-

132

10

× 8

o

c

c 4 o

2

12

I I I I I

0 50 100 150 200 X- ray d o s e ( t a d )

Fig. 5. ' D e p e n d e n c e o n X-ray dose of m a x i m u m de t ec t ed f r e q u e n c y of i n d u ced m u t a t i o n in h u m a n di- ploid f ibroblasts . Bars r e p r e s e n t one s t anda rd dev ia t i on on da ta c o m p i l e d fo r b e t w e e n th ree and seven separa te d e t e r m i n a t i o n s at each dose poin t . The curve was f i t ted by l inear regress ion analysis (by var iance ra t io tes t P = 0 .51) .

10

%

.Q

6

C

0

, Z • Z

y÷ I

10 0 5 02 Surv i v ing f ract ion

I

01

Fig. 6. The relationship between surviving fraction and induced mutation frequency in X-irradiated hu-

man diploid fibroblasts. Bars represent one standard deviation for data taken from Figs. 1 and 5 and the

curve was fitted by linear regression analysis (by variance ratio test P = 0.31).

133

duced mutat ion frequencies that can be obtained using HGPRT-deficiency as a genetic marker in cultured human cells, then the dose-response for mutat ion to HGPRT<leficiency in human diploid fibroblasts is as shown in Fig. 5. Induced mutat ion frequency increased linearly with dose up to at least 200 rad and the induced mutat ion rate was 3.10 + 0.08 • 10 -7 mutations per viable cell per rad. As predicted by the shapes of the dose-response curves for inactivation (Fig. 2) and mutat ion (Fig. 5), the plot of induced mutat ion frequency versus log sur- viving fraction (Fig. 6) was also linear, indicating a constant relationship be- tween radiation induced mutat ion and inactivation in cultured human diploid fibroblasts [14].

Discussion

The data of Fig. 3 on the influence of the duration of post-irradiation growth on mutat ion detection, illustrate the importance of controlling this factor in quantitative experiments on mutagenesis in cultured mammalian cells. Maxi- mum detect ion of X-ray induced mutations to HGPRT-deficiency was not reached until cells surviving irradiation had completed between 3 and 4 doubl- ings (6--7 days) and, if a single post-irradiation growth period of less than 6 days had been used, the dose-dependent increase in mutat ion frequency ob- served would have been underestimated. X-ray mutagenesis provides a relatively simple tool for examining factors affecting mutat ion detect ion in cultured human cells because the dosimetry of the mutagenic t reatment is relatively straightforward and also because at low doses of radiation there is no signifi- cant division delay in cells surviving irradiation. With many toxic and mutagen- ic chemical agents dosimetry is a major problem and treated cells often show considerable division delay [4], probably because toxic agents cannot be wholly removed from the intracellular environment by changing the extracellu- lar medium. Thus, the rate of division of cells surviving chemical t reatment may be expected to be dose dependent and, if so, the use of multiple post-treatment growth periods before mutant selection is of paramount importance if it is de- sired to measure the maximum mutat ion response of a cell population treated with a toxic chemical agent.

In experiments reported here the maximum induced mutat ion frequencies were obtained after post-irradiation growth for between 6 and 13 days and ap- pear to be unchanged thereafter for up to 22 days of growth. This result is evidence that radiation-induced HGPRT-deficient mutants of human cells in which mutat ion has been stabilised are not at a nkajor selective disadvantage when growing in a normal growth medium (Eagle's MEM) with non-mutant cells. If correct, this observation has two important implications. First, if there was strong selection against "early" expressed HGPRT-deficient mutants during the growth period before maximum mutant detection becomes possible, the maximum detected mutat ion frequency would be lower than the true value. The apparent lack of a selective effect against mutants induced by low doses of X-rays may mean that the maximum mutat ion frequencies obtained may ap- proximate to the true values. Secondly, the stability of the frequency of muta- tion detect ion with increasing duration of post-irradiation growth means that the duration of post-irradiation growth is no longer a critical factor in obtaining

134

meaningful data, providing of course that maximum mutat ion detect ion has been obtained. Strong selection against induced mutants during post-irradiation growth of mixed populations would tend to produce humped mutat ion ex- pression curves [11] and in such cases an analysis of the variation of mutat ion frequency with duration o f post-irradiation growth would seem to be impera- tive if deductions are to be made about the true frequency of mutat ion induc- tion.

The influence of analogue concentration on the recovery of induced 6TG- resistant mutants and the maximum induced mutat ion frequencies observed was not fully examined but, in one experiment not included in this report, in- duced mutat ion frequencies obtained for 150 rad X-rays after post-irradiation growth for 7 and 10 days were not significantly different when 6TG at 2 and 4 pg ml -~ was used to select HGPRT-deficient mutants.

The appearance of X-ray induced mutat ion to HGPRT-deficiency in cultured human cells was linearly related to the average number of post-irradiation cell doublings and to the duration of the post-irradiation growth period before selection (Fig. 4). The half t ime tl/2 for complete phenotypic expression of mu- tants was 2 doublings, a figure in good agreement with the data of Caboche [4] on the detection of chemically induced mutations to bromodeoxyuridine-resis- tance in a subline of BHK21 cells. A dependence of mutat ion expression on cell division would suggest that segregation of DNA damage and/or the dilution of normal gene products may be the most important factors in the expression of induced mutat ions to purine and pyrimidine analogue resistance in cultured mammalian cells. If so expression of induced mutat ion to analogue resistance may be species dependent and also influenced by factors such as the mutagen used, t he rate of decay of normal gene products and the concentration of the analogue used for mutant selection. General statements about the chronological time or number of cell doublings necessary for expression of induced mutat ion are therefore not yet possible and, if meaningful quantitative mutat ion data are to be obtained, it seems necessary to examine the influence of post- treatment growth for each cell type, mutat ion system and mutagen studied until general statements become possible.

In the experiments reported here the induced mutat ion frequency in human cells increased linearly with dose and there was no evidence of any cumulative effect of radiation on the rate of induced mutation. This result contrasts with experiments reported by Albertini and DeMars [2] on X-ray induced mutat ion to 8AG-resistance in cultured human diploid cells where a cumulative mutat ion dose-response was observed. However, in the Albertini and DeMars study the analogue (8AG) was applied to micro-colonies formed by the growth of cells surviving radiation (the in situ method) whilst in the experiments reported here cells were introduced to the selective agent (6TG) after the appropriate post- irradiation growth period so that the response of individual cells could be as- sessed without complications due to intercellular influences (the replating method). The difference in the shape of the dose-response curves for mutat ion between Albertini and DeMars and ourselves may be explained by the result of preliminary experiments, so far unpublished, suggesting that the cumulative dose-response for mutat ion obtained using in situ mutant selection may be an artefact of the methodology. The linear X-ray induction of mutat ion obtained

135

here using the replating method of selection seems more likely to represent the true mutat ion dose-response of X-irradiated cultured human diploid cells.

It has also been possible to compare our data obtained for radiation inac- tivation and mutat ion to HGPRT-deficiency in human diploid cells with results of similar experiments with cultures of established mammalian cells [14]. The plot of induced mutant frequency against log surviving fraction (Fig. 6) gave a straight line with the value for the slope m, the ratio between the probabilities of mutat ion and inactivation, of 4.0 + 0.1 • 10 -s for human cells. The value of m for X-ray inactivation and mutat ion to HGPRT-deficiency of established mouse lymphoma cells [11] and of V79 Chinese hamster cells is 3 . 4 . 1 0 -s [15]. The similarity of these m values suggests that, for any one locus in cul- tured mammalian cells, the probabilities of radiation induced mutat ion and in- activation remain in a fixed ratio that may be relatively species independent. Confirmation of these observations and their biological interpretation must await further cell mutat ion studies, preferably with diploid cell cultures of mammalian species rather than the atypical established cell lines more frequent- ly used in such studies.

The unshouldered exponential survival curve (Fig. 1), the linear mutat ion induction curve (Fig. 5) obtained after X-irradiation and the results of frac- t ionated dose experiments [5] are evidence that inactivation and mutat ion of cultured human cells result from the interaction of single electron tracks with cellular DNA. A "single t rack" mechanism for the radiation-induction of initial DNA lesions has also been postulated to explain the relationship between in- activation and mutat ion in established mammalian cell lines [15]. If correct, these hypotheses may have important implications for basic theory on the mode of action of low LET radiations on mammalian cells.

Using X-ray mutat ion data from organisms ranging from the bacterium Escherichia coli to the mouse, Abrahamson et al. [1] suggested that the mean induced mutat ion rate for a variety of loci in a given species is proportional to DNA content per cell (the ABCW relationship). No reliable human mutat ion data were available and on the basis of DNA content alone it was proposed that the average forward mutat ion rate for acute X-irradiation of human germ cells was 2.6 • 10 -7 mutations per locus per tad. Our observations on forward muta- tion to HGPRT deficiency in cultured human somatic cells after acute X-irrad- iation give a value for the mutat ion rate of 3.1 • 10 -7 mutations per viable cell per rad, in good agreement with the ABCW prediction. Using the experimental techniques described here it should also be possible to test the ethyl methane- sulphonate (EMS) induced mutat ion rate for human cells of 10 -~ mutations per locus per mol of EMS and the "radequiv" [3] value of 4 • 10 ~ M for EMS pre- dicted by Heddle and Athanasiou (HA) using the ABCW method of analysis [10]. However, whilst an experimental confirmation of ABCW and HA predic- tions for cultured human cells would add weight to both the practical value of such predictions and the concept of radequiv, it seems unlikely to contr ibute to our understanding of the biological basis of the rather unexpected relation- ship between induced mutat ion rate and cellular DNA content.

The experiments described here show that quantitative mutat ion data can be obtained with diploid human cells only if the culture conditions and meth- odology employed allow maximum detect ion of induced mutant genotypes.

1 3 6

Although the mutation rate obtained here for X-ray induction of HGPRT-de- ficiency in freshly isolated human somatic cells with a fibroblastic morphology was very similar to theoretical predictions of average mutation rates in human germ cells, the true predictive value of human somatic cell mutation systems may be realised only when mutation data are available for other gene loci and when culture techniques are developed for the assay of induced mutations in "differentiated" human diploid cells.

Acknowledgements

We thank the John Radcliffe Hospital, Oxford, for samples of foetal tissue, D.G. Papworth for assistance with statistical analyses and Drs. R.J. Munson and R.H. Mole for their advice on the preparation of the manuscript.

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