Accumulative effect of two low doses of irradiation in inducing an adaptive response in human lymphocytes

Mutation Research, 302 (1993) 191-196

191

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00

MUTLET 00809

Accumulative effect of two low doses of irradiation in inducing an adaptive response in human lymphocytes Y o n g l i Bai a n d D e q i n g C h e n Laboratory of Industrial Hygiene, Ministry of Public Health, Beijing 100088, China (Received 19 June 1992) (Revision received 17 March 1993) (Accepted 2 April 1993)

Keywords: Adaptive response; Chromatid breaks; Adaptive dose; Co-60 y-rays; Human lymphocytes

Summary A chromosomal adaptive response to Co-60 y-rays in human lymphocytes was observed over a range of 1-20 cGy pre-exposure doses, with 1 cGy giving optimal reduction in chromatid breaks. A 0.5 cGy dose, which in itself did not induce an adaptive response, did so when given twice within the same cell cycle, and the magnitude of the accumulative effect was strongest when there was an interval of 6 h between the two adaptive doses and between the second adaptive dose and a challenge dose. Reductions equivalent in effect to a single 1 cGy dose were seen when a 0.5 cGy dose was given twice. Delivering two 1 cGy doses had no greater effect than did a single 1 cGy dose.

Olivieri et al. (1984) first reported that pre-exposure of human lymphocytes to low levels of radioactive thymidine led to a significant reduction in the chromosome damage induced by a subsequent challenge with a high dose of X-rays. This has been termed an adaptive response to ionizing radiation, and has been observed by others using X-rays (Wiencke et al., 1986; Wolff et al., 1988; Sankaranarayanan et al., 1989; Vijayalaxmi and Burkart, 1989; Cai and Liu, 1990; Wang et al., 1991; Shadley and Dai, 1992; Shadley

Correspondence: Prof. Deqing Chen, Laboratory of Industrial Hygiene, Ministry of Public Health, P.O. Box 8018, Beijing 100088, China.

and Wiencke, 1989) or y-rays (Osmak et al., 1992) to induce the adaptation. It has been hypothesized that the low dose exposure induces a chromosomal repair mechanism that is responsible for the reduction in aberration (Olivieri et al., 1984). Lymphocytes exposed to an adaptive dose of 1 cGy X-rays were observed to express some novel proteins which could be considered potential candidates for DNA repair enzymes (Wolff and Wiencke, 1989). It might be possible that exposure of lymphocytes to two adaptive doses, instead of one, could induce higher levels of repair enzymes or preventive substances resulting in a greater reduction of the chromosome damage induced by the challenge dose, but results by Fan et al. (1990) showed

192 the incidence of c h r o m o s o m e d a m a g e to be similar in the lymphocytes pre-exposed to a single adaptive dose of 1 cGy X-rays or two adaptive doses of 1 cGy, suggesting that, u n d e r the conditions tested, the second adaptive dose did not offer any additional protection against the chrom o s o m e d a m a g e induced by the challenge dose. We a d o p t e d a lower dose to explore w h e t h e r the second adaptive dose would offer any additional protective effect.

Experiment 2 Lymphocytes from d o n o r B were exposed to one or two adaptive doses of 1 c G y Co-60 y-rays (1 c G y / m i n ) at 6 a n d / o r 42 h as well as at 36 a n d / o r 42 h after P H A stimulation, and one or two 0.5 cGy doses of Co-60 y-rays at 6 a n d / o r 12 h, 36 a n d / o r 42 h or at 6 a n d / o r 42 h, and a dose of 150 cGy at 48 h (95 c G y / m i n ) . All of the cultures were simultaneously incubated.

Experiment 3 Materials

and methods

Preparation of chromosomes V e n o u s blood samples were collected from two healthy male donors aged 26 years ( d o n o r B and d o n o r S). W h o l e blood (0.5 ml) was a d d e d to 4 ml of R P M I 1640 m e d i u m including 20% fetal calf serum, 100 u n i t s / m l penicillin, and 100 / z g / m l streptomycin, 0.1 ml P H A . At the beginning of the culture, colchicine was a d d e d to cultures so as to gain pure populations of lymphocytes at the first mitotic division (M1) in vitro (Chen and Zhang, 1992). T h e cultures were kept at 37°C for 54 h, and then lymphocytes were harvested, treated with 0.075 M KCI hypotonic solution and fixed in m e t h a n o l : a c e t i c acid mixture (3 : 1, v/v). T h e fixed cells were spread onto clean glass slides, air-dried and stained with Giemsa.

Gamma-ray irradiation Blood samples were exposed to Co-60 y-rays; adaptive dose and challenge dose were from the s a m e Co-60 irradiation source. R a d i a n t intensity at different distances from the irradiation source were m e a s u r e d by using an ionizing c h a m b e r (model N E 2 5 0 5 / N E , m a d e in England) so as to d e t e r m i n e the place of the blood samples in t h e irradiation field.

Experiment 1 Lymphocytes from d o n o r B were exposed to a single adaptive dose of 0.5, 1, 5, 10, 20 or 30 cGy from Co-60 y-rays (dose rate of 1 c G y / m i n ) at 6 h after P H A stimulation, and a challenge dose of 100 cGy at 48 h (dose rate of 95 c G y / m i n ) .

Lymphocytes from d o n o r B and from d o n o r S were exposed to one or two adaptive doses of 0.5 or 1 c G y at 36 a n d / o r 42 h after P H A stimulation respectively, and to a challenge dose of 150 cGy at 48 h. All of the cultures were simultaneously incubated.

TABLE 1 CHROMATID BREAKS INDUCED BY 100 cGy Co-60 y-RAYS AT 48 h IN HUMAN LYMPHOCYTES FROM DONOR B PRETREATED WITH A SINGLE ADAPTIVE DOSE OF Co-60 y-RAYS AT 6 h AFTER PHA STIMULATION Adaptive

Challenge

Chromatid breaks

dose (cGy)

dose (cGy)

Observed a

-

2

100

67

0.5

2

1

2

2 5 10 20 30

2 4 4 7 8

0.5 1 2 5 10 20 30

100 100 100 100 100 100 100

62 (0.21 +0.03) Ms 31 (0.10+0.02) ** 38 (0.13 _+0.02) * 44 (0.15+_0.02) * 46(0.15-+0.02)* 53 (0.18_+0.02) * 63 (0.21 _+0.03) Ms

Expected

a

67 (0.22 + 0.03) 67(0.22_+0.03) 67 (0.22 -+0.03) 69 (0.23+_0.03) 69(0.23-+0.03) 72 (0.24-+0.03) 73 (0.24 + 0.03)

Data are shown as mean _+1 standard error per cell. Expected values are the sum of the two individual treatments minus the control. a Chromatid breaks per 300 M1 cells. * p < 0.05, ** p < 0.01, Ns not significantly different from expected value (p > 0.05), t-test.

193 TABLE 2 C H R O M A T I D B R E A K S I N D U C E D BY 150 cGy Co-60 y - R A Y S A T 48 h A F T E R P H A S T I M U L A T I O N IN H U M A N L Y M P H O C Y T E S F R O M D O N O R B P R E T R E A T E D W I T H A SINGLE A N D / O R T W O DOSES O F 1 cGy Co-60 y-RAYS Adaptive dose 1 (cGy)

Adaptive dose 2 (cGy)

Challenge dose (cGy)

Adaptive dose

-

-

-

2

2

-

-

150

97

97

1

-

-

3

2

-

1

-

2

2

1

1

-

1

3

1

-

150

-

1

150

At 6 and 42 h Obs.

1

Exp. a

a

55 * (0.18+0.02) 50 * (0.16_+0.02) 53 * (0.17 _+0.02)

150

1

At 36 and 42 h Obs.

Exp.

98 (0.32_+0.03)

43 * (0.14_+0.02)

97

50

(0.32_+0.03)

(0.16_+0.02)

100

46

(0.33 _+0.03)

*

*

(0.15 _+0.02)

97 (0.32 -+ 0.03) 97 (0.32 _+0.03) 99 (0.33 -+ 0.03)

Data are shown as m e a n + 1 standard error per cell. a Chromatid breaks per 300 M1 cells. The expected values are the sum of 2 - 3 individual treatments minus the control. * p < 0.01, t-test.

Statistical analysis

Results

300 cells were scored for each point. Very few chromatid exchanges were found; they were recorded but not included in the analysis. Only chromatid breaks were subjected to statistical analysis (t-test).

Optimal adaptive dose Table 1 shows that the lymphocytes pre-exposed to a single adaptive dose of 1, 2, 5, 10 or 20 cGy became significantly less susceptible to in-

TABLE 3 C H R O M A T I D B R E A K S I N D U C E D BY 150 cGy Co-60 y - R A Y S A T 48 h A F T E R P H A S T I M U L A T I O N IN H U M A N L Y M P H O C Y T E S F R O M D O N O R B P R E T R E A T E D W I T H A S I N G L E A N D / O R T W O D O S E S O F 0.5 cGy Co-60 y - R A Y S Adaptive dose 1 (cGy)

Adaptive dose 2 (cGy)

Challenge dose (cGy)

Adaptive dose At 6 and 12 h

-

-

-

2

2

2

97

97

97

Obs. a

At 6 and 42 h Exp.

a

Obs.

At 36 and 42 h

Exp.

Obs.

-

-

150

0.5

-

-

3

3

2

-

0.5

-

2

4

4

0.5 0.5

0.5 -

150

-

0.5

150

0.5

0.5

150

5 96 MS (0.32_+0.03) 93 MS (0.31 _+0.03) 50 * (0.16 + 0.02)

98 (0.32+0.03) 97 (0.32 _+0.03) 100 (0.33 + 0.03)

Data are shown as mean_+ 1 standard error per cell. a Chromatid breaks per 300 M1 cells. * p < 0.01, NS p > 0.05, t-test.

5 96 NS (0.32_+ 0.03) 95 MS (0.32 _+0.03) 51 * (0.17 + 0.02)

98 (0.32_+ 0.03) 99 (0.33 + 0.03) 102 (0.34 _+0.03)

2 87 MS (0.29+0.03) 95 MS (0.32 ___0.03) 26 * (0.09 + 0.02)

Exp.

97 (0.32 _+0.03) 99 (0.33 _+0.03) 101 (0.34 _+0.03)

194 TABLE 4 C H R O M A T I D B R E A K S I N D U C E D BY 150 cGy Co-60 y-RAYS A T 48 h IN H U M A N L Y M P H O C Y T E S F R O M D O N O R S B A N D S P R E T R E A T E D W I T H A S I N G L E A N D / O R T W O DOSES O F 0.5 cGy Co-60 y - R A Y S A T 36 A N D / O R 42 h A F T E R PHA STIMULATION Adaptive

Adaptive

Challenge

Donor B

dose 1 (cGy)

dose 2 (cGy)

dose (cGy)

Obs. a

-

150 0.5 0.5 0.5

0.5

150

0.5

150

0.5

150

Obs.

1

2

91

99

1

3

-

0.5 0.5

Donor S Exp. a

2 2 86 NS (0.29 _+0.04) 84 NS (0.28 + 0.03) 25 * (0.08 + 0.03)

91 (0.30 _+0.03) 92 (0.31 ± 0.03) 93 (0.31 ± 0.03)

2 2 89 NS (0.30 _+0.03) 92 NS (0.31 ± 0.03) 40 * (0.13 _+0.02)

Exp.

100 (0.33 ± 0.03) 99 (0.33 ± 0.03) 102 (0.34 + 0.03)

Data are shown as mean_+ 1 standard error per cell. Chromatid breaks per 300 M1 cells. * p < 0.01, NS p > 0.05, t-test.

duct±on of chromatid breaks by 100 cGy, but pretreatment with a single dose of 0.5 or 30 cGy did not affect the subsequent response to 100 cGy. The most significant effect was found after pretreatment with 1 cGy: the chromatid breaks were reduced by 54%.

Accumulative effect of adaptive dose Table 2 shows that the lymphocytes treated with one or two adaptive dose(s) of 1 cGy prior to 150 cGy had fewer than the expected number of chromatid breaks, but the results were similar whether the adaptive doses were given at 6 and

TABLE 5 C H R O M A T I D B R E A K S I N D U C E D BY 150 cGy Co-60 y-RAYS A T 48 h IN H U M A N L Y M P H O C Y T E S F R O M D O N O R S B A N D S P R E T R E A T E D W I T H A SINGLE A N D / O R T W O DOSES O F 1 cGy Co-60 y - R A Y S A T 36 A N D / O R 42 h A F T E R PHA STIMULATION Adaptive dose 1 (cGy)

Adaptive dose 2 (cGy)

Challenge dose (cGy)

1

Exp. a

1 150

Donor S Obs.

99

-

1

1

-

2

2

2

4

1

1

-

150

1

150

1

150

48 * (0.16 ± 0.02) 51 * (0.17 _+0.02) 47 * (0.16 _+0.02)

Data are shown as mean_+ 1 standard error per cell. a Chromatid breaks per 300 M1 cells. * p < 0.01, t-test.

Exp.

2

91

-

1

1

Donor B Obs. a

3

91 (0.30 ± 0.03) 92 (0.31 ± 0.03) 93 (0.31 ± 0.03)

63 * (0.21 +_0.03) 61 * (0.20 ± 0.03) 65 * (0.22 _+0.03)

100 (0.33 ± 0.03) 99 (0.33 _+0.03) 102 (0.34 ± 0.03)

195 42 or 36 and 42 h after PHA stimulation. A single dose at 36 h may be more effective than a single dose at 6 h. It was very interesting that pre-exposure of lymphocytes to a single adaptive dose of 0.5 cGy did not lead to a reduction of chromatid breaks by a subsequent challenge dose of 150 cGy, but two adaptive doses of 0.5 cGy did (Table 3). This indicated that the second adaptive dose of 0.5 cGy, which was given to the lymphocytes at 12, 36 or 42 h after PHA during the same cell cycle as the fist adaptive dose (Chen et al., 1992), offered additional protection against the chromosome damage induced by the challenge dose of 150 cGy. The adaptive response induced by two 0.5 cGy doses was similar to or in excess of that of a single 1 cGy dose, especially, at 36 and 42 h after PHA stimulation; by using two 0.5 cGy doses the chromatid breaks were reduced by 74% (see Table 3).

Accumulative adaptive response reproducibility Tables 4 and 5 show the results from repeated experiments in blood from donor B and blood from donor S, in which the above results were further confirmed and the observations in both donors were consistent. Interindividual and interexperimental variability was found as reported by Bosi et al. (1987). Discussion

Human lymphocytes exposed to doses of X-rays as low as 0.5 cGy (20 cGy/min) or 1 cGy administered at 32 h after PHA became adapted so that less cytogenetic damage in the form of chromatid breakage is induced by 150 cGy at 48 h (Shadley and Wolff, 1987), but doses of Co-60 y-rays as low as 0.5 cGy (1 cGy/min) cannot reduce the induction of chromatid breaks produced by 100 or 150 cGy Co-60 y-rays given at 48 h in our experiments. Optimal dose ranges for the adaptive response to ionizing radiation have been shown to be 0.5-20 cGy for X-rays (Shadley and Wolff, 1987) and 1-10 cGy for 3H-TdR (Ikushima, 1989). The observations presented here suggest that an optimal dose range for Co-60 y-rays is estimated to be 1-20 cGy for adapting human lymphocytes to ionizing radiation. 1 cGy is con-

sidered to be the optimal dose or saturating dose, which induces maximum activity of repair enzymes. 3-Aminobenzamide (3-AB), a potent inhibitor of poly(ADP-ribose)polymerase, which was given before the challenge dose, abolished the adaptive response induced by a low dose of radiation (Ikushima, 1989; Shadley and Wolff, 1987). Cycloheximide has also been shown to eliminate the adaptive response (Youngblom et al., 1989). These results indicate that proteins, poly(ADP-ribose)polymerase and some unknown substances are involved in the adaptive response. It was proposed that the induced repair system became saturated between 0.01 and 1 Gy for radon 222 (Pohl-Riiling and Fisher, 1979); this appears to happen at 1 cGy for Co-60 y-rays in our study, and could explain why the second adaptive dose of 1 cGy Co-60 y-rays cannot offer an additional preventive effect against induction of chromatid breaks caused by a challenge dose as reported by Fan et al. (1990) and confirmed here. When two very low doses of 0.5 cGy were administered at 6 and 12 h or at 6 and 42 h, the second 0.5 cGy dose appears to add to the repair induced by the first dose, and to attain saturation equivalent to that caused by 1 cGy; thus, the second 0.5 cGy can afford an additional preventive effect against the induction of chromatid breaks by 150 cGy. Maximal expression of the adaptive response was found by Shadley et al. (1987) when an interval of 5 or 6 h between the adaptive dose and the challenge doses was allowed, whereas similar resuits were not observed when an adaptive dose of 1 cGy was given at 42 h, followed by a challenge dose of 150 cGy at 48 h. But our results showed maximal adaptation when two adaptive doses were given at 36 and 42 h, followed by a challenge dose of 150 cGy at 48 h. In this study colchicine was added to the cultures at the beginning of culture to be sure of having all scored ceils in the first metaphase (Chen et al., 1992) so that the results become comparable with others. In spite of the fact that colcemid inhibits transition of G 0 / G a in mitogen-stimulated lymphocytes (Kenter et al., 1986), no effect on chromosome aberrations of the continuous presence of colchicine has been observed. Chen and Zhang (1985) reported that levels of

196

aberrations induced by 1 Gy of Co-60 y-rays were similar whether colchicine was added to the cultures at 0 or at 46 h after P H A stimulation.

Acknowledgement We are very grateful to technician-in-charge Chaoyang Zhang for her help and technical instruction during the experiment.

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