Lack of adaptive response to low doses of ionizing radiation in human lymphocytes from five different donors

Mutation Research, 283 (1992) 137-144

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 0714

Lack of adaptive response to low doses of ionizing radiation in human lymphocytes from five different donors J. Hain, R. Jaussi and W. Burkart a Institute for Medical Radiobiology of the University of Ziirich and the Paul Scherrer Institute, Villigen, Switzerland and a Institut fiir Strahlenhygiene, Bundesamt fiir Strahlenschutz, Neuherberg, Germany

(Received 24 April 1992) (Revision received 5 June 1992) (Accepted 10 June 1992)

Keywords: Ionizing radiation; Adaptive response; Human lymphocytes

Summary Various investigators reported a reduced yield of chromosome and chromatid aberrations in short-term cultures of human lymphocytes if a 'challenge' exposure to ionizing radiation was preceded by an 'adaptive' exposure. In order to examine the cell cycle dependence of the 'adaptive response', chromosome and chromatid aberration yields were estimated after challenge doses in the G1, S or G 2 phase of lymphocytes which had been adapted in the early G 1 phase. On testing two donors no protective adaptive response was found. Blood samples of four donors were tested for their capability to evoke the adaptive response in a standard experiment with the adaptive dose in the S phase and the challenge dose in the G z phase. A synergistic response occurred in one out of two similar experiments performed with the same blood sample. The three other blood samples tested did not respond. Apparently these data indicate a high frequency of human lymphocyte cultures that do not display an adaptive response.

A p h e n o m e n o n similar to adaptive response was initially described in E. coli on recurring exposure of the cells to mutagens like N-methylN'-nitro-N-nitrosoguanidine (Samson and Cairns, 1977). The frequency of mutations was reduced and the survival of the bacteria was increased if the cells were preincubated with low concentra-

Correspondence: Jens Hain, Institute for Medical Radiobiology of the University of Ziirich and the Paul Scherrer Institute, CH-5232 Villigen, Switzerland.

tions of this chemical before it was applied in a highly mutagenic dose. The authors concluded that an adaptation to the mutagen had been elicited by the initial treatment. A similar adaptive response to chemical mutagens was found in experiments with Chinese hamster ovary and human skin fibroblast cell lines (Samson, 1980). An adaptive response of human cells to ionizing radiation was first reported in 1984 (Olivieri, 1984). Peripheral blood lymphocytes were cultivated in the presence of [3H]dThd, resulting in a low (adaptive) dose of radiation. Cells treated in

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this manner showed fewer chromatid aberrations after a challenge treatment with a high dose of X-rays than cells which had been cultivated in the absence of [3H]dThd. Essentially the same effect resulted when lymphocytes were adapted with low doses of X-rays instead of [3H]dThd (Shadley, 1987). This adaptation effect appeared to depend both on the dose and on the dose rate of the adaptive dose. With an adaptive dose of 0.01 Gy given at > 0.05 Gy/min or an adaptive dose of 0.5 Gy given at < 0.05 Gy/min significant reductions of chromatid aberrations were observed (Shadley and Wiencke, 1989). The adaptation needed at least 4 h to develop after the adaptive exposure and disappeared after three cell cycles (Shadley, 1987). Treatment of cells with clastogenic chemicals like bleomycin or mitomycin C led to cross-adaptation to ionizing radiation (Vijayalaxmi and Burkart, 1989, 1990). Adaptation of lymphocytes due to pretreatment with low doses of hydrogen peroxide suggested an involvement of free radicals in the adaptation process (CortEs et al., 1990). Incubation of blood cultures in the presence of cyclohexamide (an inhibitor of protein synthesis) (Youngblom et al., 1989), 3-aminobenzamide (an inhibitor of poly(ADP-ribose)polymerase) (Wiencke, 1986) or a deficiency of nicotinamide (Wiencke, 1987) apparently prevented the adaptive response. Thus lymphocytes may be able to induce a repair mechanism at the translational or pretranslational level. However, several investigators found donors whose cultured blood lymphocytes did not express an adaptive response or even expressed a synergistic response, i.e., a higher frequency of chromosome aberrations was observed in adapted cultures than in nonadapted ones (Bosi and Olivieri, 1989; Sankaranarayanan et al., 1989; Schmid et al., 1989; Bauchinger et al., 1989; Olivieri, 1990; Aghamohammadi and Savage, 1991). The knowledge of the prevalence of the adaptive response in individual donors and throughout the human population is sparse. Even less is known about the cell cycle dependence of the phenomenon. The adaptive response could not be induced by irradiating the lymphocytes in the G o phase (Shadley, 1987). It has been reported that the adaptive response in the G~ phase occurs only if the cells are challenged in the

interval between 5 and 9 h after the adaptive dose. This response was restricted to exchange aberrations (Wang et al., 1991). We intended to investigate in detail whether the adaptive response undergoes fluctuations during different phases of the cell cycle. For this purpose we gave an adaptive dose in the G 1 phase and the challenge doses in the late G 1, S or G 2 phase during the initial experiments. Because the two donors chosen did not show any adaptation we rescreened one of them and screened another three persons for adaptive response. We did not elicit a single adaptive response in any case tested. Material and methods

Source of lymphocytes and culture conditions The methods of blood cultivation and analysis of chromosome aberrations were essentially the same as described in other papers dealing with adaptive response in human lymphocytes (for examples, see Olivieri, 1984; Wiencke, 1986).

Irradiation The cultures were irradiated at 37°C with 250 kV X-rays and 10 mA current (Philips MCN 321 X-ray tube). The adaptive dose was given at a dose rate of 200 mGy/min and the challenge dose at a dose rate of 1 Gy/min. For filtering 1 mm A1 and 1 mm Cu were used. Doses were determined with a Farmer ionization chamber (NE 2571). In the first experiment the adaptive dose (0.01 Gy) was applied 5 h after PHA stimulation. The challenge dose was given at different time points corresponding to the desired cell cycle phases of the cells finally scored. The total culture time for donor 1 was 52 h and that for donor 2 was 54 h. For the second experiment comparing donors 2, 3, 4 and 5 the adaptive dose (0.01 or 0.02 Gy) was given 32 h after PHA stimulation followed by the challenge dose (0.5, 1 or 1.5 Gy) at 48 h.

Metaphase preparation In experiments with a challenge dose of 0.5 Gy, colcemid (0.2 I~M final concentration) was added immediately following irradiation. After challenge doses of 1 and 1.5 Gy colcemid was

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added 4 h later to allow cells to escape the induced Gz delay and to reach metaphase. Cells incubated for 2 h with colcemid were fixed and washed three times in methanol/acetic acid (3 : 1). Metaphases were spread on cold wet slides and stained with Giemsa (Merck) according to the protocol obtained from the supplier.

[3H]thymidine (0.02 izCi/ml) was added to the cultures immediately after irradiation (Mindek, 1992). Metaphases from cells with incorporated [3H]thymidine (more than 5 grains per metaphase) were considered to have been irradiated in S phase. The number of these metaphases was recorded but they were not analyzed further.

Labeling of metaphases

Aberration analysis

In order to recognize metaphases of cells that had been irradiated in the second or third cell cycle or that had been challenged in the S phase of the first cell cycle, two differential staining methods were used. (1) Hoechst 33258 plus Giemsa staining was applied in order to distinguish metaphases of the first division from metaphases of cells having already cycled twice or more during the incubation time (Perry and Wolff, 1974). Only metaphases from cells in the first cell cycle were scanned for aberrations. (2) For the blood samples from donor 5 with the challenge dose (1.5 Gy) given in the G z phase,

Only well-spread metaphase plates displaying a complete chromosome set were used to acquire data. For each data point 100-200 metaphases were scored on double coded slides. Description of the different aberrations listed in the tables: gaps are achromatic lesions smaller than the width of a chromatid and are not included in the evaluation of aberrations per 100 cells; exchanges are translocation figures (number of aberrations = number of involved chromosomes) and rings or dicentric chromosomes (counted as two aberrations); deletions include acentric fragments, chromatid breaks and isochromatid breaks.

TABLE 1 CHALLENGE DOSES IN DIFFERENT PHASES OF THE CELL CYCLE Irradiation (time after PHA)

Gaps

Exchanges

Deletions

Number of cells

Damaged cells (%)

Aberrations in 100 cells

3 5 7 1 3 3 8 7

65 96 69 63 8 12

5 5 119 93 106 129 40 57

200 200 200 200 200 200 200 200

2.5 2,5 62 60 56 62 21 31

3 3 125 143 122 128 28 41"

6 5 37 16 55 44 60 89

58 72 6 11 1 5

2 166 102 69 67 70 65

100 100 200 200 200 200 200 175

2 66 60 34 33 34 34

2 141 123 41 45 36 43

Cell cycle phase

Second division metaphases (%)

Donor 1 (male, age 32) None 0.01 Gy (5 h) 1.5 Gy (22 h) 0.01 Gy (5 h) + 1.5 Gy (22 h) 1.5 Gy (26 h) 0.01 Gy (5 h) + 1.5 Gy (26 h) 1.5 Gy (48 h) 0.01 Gy (5 h)+ 1.5 Gy (48 h)

G1 G1 G2

Donor 2 (female, age 29) None 0.01 Gy (5 h) 1.5 Gy (18 h) 0.01 Gy (5 h) + 1.5 Gy (18 h) 1.5 Gy (40 h) 0.01 Gy (5 h) + 1.5 Gy (40 h) 1.5 Gy (46 h) 0.01 Gy (5 h) + 1.5 Gy (46 h)

G1 late S G2

17 17 11 11 <1 <1 <1 <1

Blood cultures of two donors were tested for the influence of the challenge treatment during different stages of the cell cycle after an adaptive dose at 5 h after PHA stimulation. All cultures from donor 2 were stained differentially in order to detect cells that had undergone two divisions (second division metaphases). * Synergistic, p < 0.05 (Student's t-test).

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Stat&tical analysis Student's t-test was applied to determine whether the observed number of chromosome or chromatid aberrations was significantly lower than the expected number. The values for expected numbers of aberrations were calculated as follows: the aberrations per 100 cells resulting from the two individual treatments (adaptive dose and challenge dose) were added and the control value for unirradiated cells was subtracted. Results

The first experiment (Table 1) aimed at investigating possible alterations of the adaptive response during the first cell cycle after PHA stimulation. The adaptive dose of 0.01 Gy was given in the G~ phase (5 h after PHA stimulation). The challenge dose of 1.5 Gy was given in the Gt, S or G 2 phase. A control which did not receive the adaptive dose was scored for each time point. Blood samples of two donors were divided into aliquots and used to investigate three time points

and controls. The samples of donor 1 received the challenge dose in the mid Gt phase (22 h), in the late G~ phase (26 h) or in the G 2 phase (Table 1). The samples of donor 2 received the challenge dose in the G 1 phase (18 h), in the late S phase (40 h) or in the G 2 phase (Table 1). For determining the percentage of second division metaphases in blood cultures of donor 1 two extra samples were incubated in the presence of BUdR. One sample was left unirradiated, the other was irradiated with 1.5 Gy at 48 h after PHA stimulation. Both of them contained 35% metaphases from the second division. To determine the percentage of metaphases from the second division in blood cultures of donor 2, BUdR was added 8 h after stimulation to each culture. As shown in Table 1 both donors did not express a significant protective adaptive response at any of the cell cycle time points tested. A challenge in the G 2 phase apparently resulted in a synergistic effect in the lymphocyte culture of donor 1. In order to search for donors showing a protective adaptive response, a second experiment

TABLE 2 TEST F O R A D A P T I V E R E S P O N S E , S T A N D A R D C O N D I T I O N S Blood sample (sex, age) and tr eatment

Gaps

Exchanges

Deletions

N u m b e r of cells

Damaged cells (%)

Aberrations in 100 cells

Second division metaphases

1 6 12 5

3

2 2 22 22 36

100 100 100 100 100 100

2 2 21 21 33

2 2 22 22 42 *

n.d. n.d. n.d. 29 26 28

1 15 11

7 5

1 1 55 57

100 100 191 200

1 1 27 30

1 1 36 34

n.d. n.d. n.d. n.d.

21 22

1 1

1 28 28

100 100 100 100

1 27 26

1 30 30

n.d. n.d. 16 13

Donor 2 (f, 29) None 0.01 Gy 0.02 Gy 1.00 Gy 0.01 Gy + 1.00 Gy 0.02 Gy + 1.00 Gy

Donor 3 (f, 28) None 0.01 Gy 1.00 Gy 0.01 G y + 1.00 Gy

Donor 4 (m, 26) None 0.01 Gy 1.50 Gy 0.01 G y + 1.50 Gy

The adaptive dose was given 32 h after P H A stimulation and the challenge dose was given 48 h after P H A stimulation. Cells were fixed at 54 h. n.d., not determined. * Synergistic, p < 0.05 (Student's t-test).

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was performed. An irradiation scheme which had been used successfully in earlier experiments of other investigators was applied: the adaptive dose of 0.01 Gy or 0.02 Gy was given 32 h after PHA stimulation and the challenge dose 48 h after PHA stimulation. Three donors were tested with this experimental set-up. The results are displayed in Table 2. None of the three donors showed an adaptive response. The blood culture of donor 2 adapted with 0.02 Gy even displayed a synergistic response. Donor 5 was tested for possible effects of the fetal calf serum in the culture medium on the frequency of chromosome aberrations (Ghosh et al., 1991; Wolff, 1984). Aliquots of a blood sample were cultivated with or without 10% FCS. The lymphocytes received the adaptive dose at 32 h, were challenged at 48 h and fixed at 54 h. Tritiated thymidine was added to all cultures after the challenge treatment. The first blood

sample of donor 5 showed a lower frequency of chromosome aberrations and second division metaphases in cultures without fetal calf serum. This result suggested an influence of fetal calf serum on cell proliferation kinetics through the G 2 phase and the aberration yield (Table 3, blood sample 1). One month later another blood sample was taken from donor 5 to repeat the experiment. Additionally four aliquots of the second blood sample were challenged with 0.5 Gy at 48 h after PHA stimulation and fixed at 50 h (Table 3). No tritiated thymidine was added to these cultures because the cells were fixed 2 h after the challenge dose at which time point none of the irradiated S phase cells could have reached metaphase. In the experiments with the second blood sample the influence of the fetal calf serum suggested by the first experiment could not be corroborated. The numbers of chromosome aberrations in cultures with and without fetal calf serum were

TABLE 3 TEST F O R A D A P T I V E RESPONSE W I T H TWO BLOOD SAMPLES F R O M D O N O R 5 (MALE, A G E 28), C O N D I T I O N S VARIED Treatment

Gaps

0.01 Gy (10% FCS) 0.01 Gy (no FCS)

Exchanges

Deletions

Number of cells

Damaged cells (%)

Aberrations in 100 cells

[3H]dThd labeled metaphases * (%)

1 -

N

2 2

100 100

2 2

2 2

30 32

46 56 27 23

1 1 5

113 88 61 48

214 201 187 191

44 37 28 25

54 44 34 30

21 26 14 13

28 20 23 29 76 78 48 26

1 ~2 9 12 1 -

43 55 51 57 274 256 150 146

100 100 100 100 200 200 100 100

39 47 47 47 80 78 81 81

45 55 55 57 146 140 152 146

23 17 28 25 -

Blood sample 1 1.5 0.01 + 1.5 1.5 0.01 + 1.5

Gy Gy Gy Gy

(10% FCS) (10% FCS) (no FCS) (no FCS)

Blood sample 2 1.5 0.01 + 1.5 1.5 0.01 + 1.5 0.5 0.01 + 0.5 0.5 0.01 + 0.5

Gy Gy Gy Gy Gy Gy Gy Gy

(10% FCS) (10% FCS) (no FCS) (no FCS) (10% FCS) (10% FCS) (no FCS) (no FCS)

Cultures were incubated either with or without FCS. The adaptive dose was given at 32 h after PHA stimulation and the challenge dose was given at 48 h after PHA stimulation. Cultures challenged with 0.5 Gy were fixed at 50 h and cultures challenged with 1.5 Gy were fixed at 54 h. Unirradiated controls showed no aberrations in 100 cells. * After challenge treatment of 1.5 Gy 0.02 /zCi/ml [3H]dThd was added to the cultures to label those cells which had been irradiated during S phase.

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similar (Table 3, blood sample 2). In none of the experiments did blood samples of donor 5 display an adaptive response. Discussion

Our primary intention to investigate the cell cycle dependence of the adaptive response could not be carried out, because none of the blood cultures from five different donors expressed a protective adaptive response. This finding agrees with results of three other investigations with entirely negative results (Schmid et al., 1989; Bauchinger et al., 1989; Aghamohammadi and Savage, 1991). In contrast to these findings most of the published data point out that an adaptive response to low doses of ionizing radiation exists in human lymphocytes (Olivieri, 1984, 1990; Sanderson, 1986; Wiencke, 1986, 1987; Shadley, 1987; Wolff, 1988, 1989, 1990; Bosi and Olivieri, 1989; Sankaranarayanan et al., 1989; Shadley and Wiencke, 1989; Vijayalaxmi and Burkart, 1989; Youngblom et al., 1989; Burkart and Vijayalaxmi, 1990; Fan et al., 1990; Wolff et al., 1990, 1991; Khandogina et al., 1991; Kelsey et al., 1991; Wang et al., 1991). However, even in these investigations occasionally a lack of adaptive response or enhanced aberration yield has been recorded. In our experiments blood cultures of donor 1 expressed a synergistic response when challenged in the G 2 phase (Table 1). No adaptive response was found when aliquots of the same blood sample were challenged in the late G 1 phase (22 or 26 h after PHA). These findings could be explained by the results of Wang et al. (1991), who found that adaptation in the GI phase occurs only when the challenge dose is given in the interval from 5 h to 9 h following the adaptive dose which has been given in the GI phase (5 h after PHA). Notably the response of donor 1 blood cultures to challenge in the G 2 phase was synergistic, thus there is not even an indication of adaptive response. In the experiments with three different donors (Table 2) donor 2 showed a synergistic response to challenge in the G 2 phase, but only in one out of two experiments with adaptive doses of 0.01 Gy or 0.02 Gy (both of these doses lie within the dose range described to induce adaptive response). Investigations at-

tempting to determine the reasons for the variability in the adaptive response by supplying the culture medium with hormones whose concentration can vary during different physiological conditions in humans, did not yield clear results (Olivieri, 1990). Even if the hormonal status of the donor had an influence, the observation with donor 2 could not be explained by an alteration in hormonal status, because two aliquots of the same blood sample were used. To date there is neither an explanation of why certain donors show a protective adaptive response and others a synergistic one or none, nor an explanation of why certain donors sometimes express a protective adaptive response and sometimes a synergistic one (Bosi and Olivieri, 1989; Olivieri, 1990). Because of the time consuming cytogenetic methods involved, only few investigations with small numbers of donors have been worked out to evaluate the consistency of an adaptation phenomenon to ionizing radiation throughout the human population. Bosi and Olivieri (1989) tried to evoke the adaptive response in blood cultures from 18 different donors with X-rays or with tritiated thymidine. Four donors did not show an adaptive response or showed a synergistic one. One donor displayed adaptation only when adapted with X-rays and two others only when adapted with tritiated thymidine. Three of the donors who showed no adaptive response and three of the donors who did were investigated again (Olivieri, 1990). Opposite results were found in four out of the six experiments which had been carried out under essentially the same conditions. Previous investigations from our own laboratory finding adaptive responses (Vijayalaxmi and Burkart, 1989; Burkart and Vijayalaxmi, 1990; Fan et al., 1990) were performed with different donors who are not available any more. Our results shown in this article indicate a lower frequency of occurrence of the adaptive response throughout the human population than might be expected from the published data. For a more precise assessment of this frequency it will be necessary to investigate a larger number of blood samples by automated analysis. Differential staining of metaphases which allows comparison of adaptive response experiments independent of mitotic perturbation might be useful for this pur-

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pose, too (Aghamohammadi and Savage, 1991). Only if more uniform cell populations showing a more consistent adaptive response than the heterogeneous human iymphocytes can be found will the phenomenon become accessible to biochemical investigations.

Acknowledgements We thank Prof. F.E. Wfirgler, ETH Ziirich for constructive discussions during our work and for critical reading of the manuscript. This work was supported by Swiss National Science Foundation Grant No. 31-26617.89.

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