223

Mutation Research, 45 (1977) 223--231 © Elsevier/North-Holland Biomedical Press

THE RADIOSENSITIVITY OF CHROMOSOMES OF RABBIT LYMPHOCYTES

A. LI~ONARD, G.B. GERBER, G. DECAT and E.D. LI~ONARD Department of Radiobiology, C.E.N./S.C.K. B-2400 Mol (Belgium) (Received 22 April 1977) (Accepted 4 July 1977)

Summary Dicentrics were induced in human and rabbit lymphocytes in vitro by exposure to X-irradiation. The yields were compared with data reported in the literature. To elucidate the reasons for the different findings we have evaluated the parameters for the different models by maximum likelihood methods. From this analysis it can be concluded that the earlier claim of Bajerska and Liniecki [1] that both species have the same radiosensitivity must be discounted, and t h a t their similarity was due to the abnormally low values these investigators observed in irradiated human lymphocytes.

Introduction The t h e o r y of Brewen et al. [2] t h a t radiosensitivity of chromosomes is related to the number of chromosome arms has drawn much attention. Contradictory results, however, have been reported for a crucial experiment on two species with equal chromosome arm numbers, man and rabbit. Scott and Bigger [8], and somewhat later Muramatsu and Matsuoka [5], observed less than half the number of dicentrics in rabbit lymphocytes than in those from man, whereas Bajerska and Liniecki [ 1 ] claimed that both species have the same radiosensitivity. Sankaranarayanan [7] pointed out recently that the experimental design was not the same in the three investigations and t h a t this might explain, to a certain extent, the differences observed. Since important conclusions as to the validity of Brewen's t h e o r y are based on the outcome of this experiment we have studied the yield of dicentrics in man and rabbit under similar conditions and compared the data with those mentioned above.

224 Materials and methods Samples of heparinized venous blood from two young but sexually mature male rabbits were exposed to 0, 50, 100, 200, 300 or 400 rad of X-rays (250 kV, 15 mA, 0.5 mm Cu; dose rate 100 rad/min). Cultures were set up within one hour after sampling. From each sample, 0.5 ml was incubated at 37°C for 45.5 h in 5 ml Ham's F-10 medium supplemented with bovine serum, phytohaemagglutinin, streptomycin and penicillin. One ml of TC chromosome-arresting solution (Difco) was then added and the cells were incubated for an additional 2.5 h. After hypotonic treatment with KC1, the cells were fixed in methanol-acetic acid (3 : 1), spread on clean sides, left to dry and then stained with lacto~rcein. One hundred cells were analysed per animal per treatment. All abnormalities seen in the cells and distinguished unequivocally were scored. An estimation of the lymphocyte nuclear area, assumed to be proportional to nuclear volume, was obtained by measuring, for each donor, the diameters of 100 lymphocyte nuclei having regular spherical shape, in smears of fresh blood stained with May-Grtinwald, in an ocular micrometer. Results The frequency of chromosomal aberrations in rabbit lymphocytes exposed to different doses of X-rays and cultured for 48 h is presented in Table 1. From Table 2, where the yield of dicentrics in rabbits is compared with that in man TABLE YIELDS

1 OF

CHROMOSOMAL

ABERRATIONS

IN RABBIT

LYMPHOCYTES

AFTER

48 h OF

CULTI-

VATION

Dose

Animal

Cells examined

Abnormal cells

Aberrations Chromatid fragments

0

50

100

200

300

400

Chromofragments

.

Trans-

.

Dicentrics

1

100

0

.

2

100

1

--

1

--

--

--

200

1

--

1

--

--

--

1

100

1

.

2

100

3

--

2

--

--

1

200

4

--

2

--

--

2

1

100

6

--

--

--

3

2

100

4

.

200

10

--

5

--

--

7

1

100

14

--

12

--

--

9

2

100

27

--

29

1

--

14

200

41

--

41

1

--

23

1

100

40

--

23

--

--

34

2

100

47

--

34

1

1

47

200

87

--

57

1

1

81

1

100

62

1

34

--

3

70

2

100

62

2

63

2

--

200

124

3

97

2

3

.

.

.

Rings

.

5 .

.

.

1

.

4

54

124

225 TABLE

2

YIELD

OF

DICENTRICS

Dose (rad)

IN MAN

(3)

AND

RABBIT

Yield of dicentrics per 1 0 0 cells Man

Rabbit

0

0

50

6.5

-+ 1 . 3

100

12.5

-+ 1 . 8

3.5

+ 1.3

200

31.5

+- 2 . 8

11.5

+ 2.4

300

85.0

-+ 4 . 6

40.5

-+ 4 . 5

400

138.0

+ 5.9

62.0

_+ 5 . 6

a Mean

a

0

-+ 0 . 5

1.0

+- 0 . 7

Poisson standard error.

-+

obtained earlier [3], it can be seen that, in agreement with the observations o f Scott and Bigger [8] and of Muramatsu and Matsuoka [5] but in contradiction to those of Bajerska and Liniecki [1], human lymphocytes were more radiosensitive than rabbit lymphocytes. To elucidate the reasons for the different finding we assembled the original data from these investigators for culture periods of 48 h {Tables 3 and 4) and evaluated the parameters for the different models by maximum likelihood methods [6]. Tables 5 and 6 present the means and standard errors of the parameters, the

TABLE YIELDS

3 OF

DICENTRICS

OBSERVED

IN

RABBIT

LYMPHOCYTES

BY

DIFFERENT

INVESTIGA-

TORS

X-ray dose (tad)

Present results

0

0/200

50

2/200

53.2 96 100

--7/200

S c o t t and Bigger [8]

Muramatsu and M a t s u o k a [5]

0/220

0/200

--

--

--

--

-.

--2/230

2/100 .

.

Bajerska and Lin~ecki [ 1 ]

-.

106.4

--

--

--

153

--

16/120

--

10/260 --

172.8

--

--

--

27/230

192

--

--

12/100

--

196.8

--

--

--

29/180

2 0 0

2 3 / 2 0 0

--

--

--

2 0 3

--

2 1 / 1 0 0

--

--

43/290

249.3

--

--

--

288

--

--

20/100

300

81/200

--

--

305

--

60/120

--

--

353.5

--

--

--

56/170 --

364 400

-124/200

-122/470

--

36/100

--

--

--

--

--

66/165

406

--

407

--

35/51

--

--

458

--

III/158

--

--

230/178

--

--

102/63

--

--

610 763

-

-

--

226 TABLE

4

YIELDS

OF

DICENTRICS

X-ray dose (rad)

OBSERVED

L~onard

IN MAN

Scott

and

Bigger

0

1/400

BY DIFFERENT

INVESTIGATORS

Muramatsu

[8]

Matsuoka

1/1 O0

and

Linieeki

0/3000

1/3000

49

--

--

--

50

26/400

--

--

--

54

--

--

--

3/70

75

--

24/140

--

--

89

--

--

--

10/100

6/70

96

5/100

--

--

44/400

100

50/400

--

--

--

101

--

--

--

9/100

150

--

22/100

--

--

151

--

--

--

15/100

192

--

--

193

--

--

126/400

2 0 0

2 2 5

--

143/400

201100 28/150

--

--

9 1 / 1 4 0

--

--

--

--

2 5 0

--

--

288

--

--

298

--

--

--

52/I00

300

340/400

--

1 7 / 5 0

143/200

--

98/100

--

56/I00

355

--

--

--

50/75

384

--

--

400

554/400

319/200

--

--

et al [4]

[5]

-48/60

402

--

--

--

39/50

449

--

--

--

48/60

residual mean squares and P the probability for the goodness of fit. The standard error was multiplied with a heterogeneity factor when P was less than 5%. It can be seen that all experiments performed on the rabbit had a reasonably good fit regardless of whether the power law or quadratic law model was applied.

-

200 ~

~

_u

/

160"

120

g u

=

80

o

260

~

4;0

6;0

8;0 D o s e

Fig. 1. Plot tained from [5];

of dicentrics, in rabbit all the data combined.

e Bajerska

and

Liniecki

[1].

(rad)

lymphocytes, against X-ray dose according to the quadratic law obX Present results; • Scott and Bigger [8]; + Muramatsu and Matsuoka

227

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