P-2b-69

Cytogenetic adaptive response induced by pre-exposure

in human lymphocytes and marrow cells of mice
Ziqiang Meng1, Lianzhen Zhang2
1 Institute of Environmental Medicine and Toxicology,
Department of Life Science, Shanxi University,Taiyuan 030006,
People's Republic of China
2 China Institute for Radiation Protection, Taiyuan 030006,
People's Republic of China

ABSTRACT
The frequencies of chromosomal aberrations both in human lymphocytes and in mouse marrow cells
exposed to low-level radiation were higher than in their unexposed controls. However, the frequencies of
adaptive response in two kinds of cells pre-exposed to low-level radiation induced by a subsequent high dose of
X-rays or -rays were lower than those of the groups only exposed to high-level radiation. This implies that
adaptive response for cytogenetic indicators might be induced by pre-exposure to low-level radiation. The results
also show the existence of possible variations between individual lymphocytes.
Keywords: low-level radiation; adaptive response; adaptive response; human lymphocytes

INTRODUCTION
In 1977, Samson and Cairns found an inducible DNA repair process, the "adaptive repair response" in
Escherichia coli. Human lymphocytes exposed to low doses of X-rays, -ray irradiation, or endogenous
irradiation in the form of incorporated thymidine(3H-TdR), become less susceptible to chromatid damage
induced by a subsequent high dose of X-rays or -rays. This adaptive response to ionizing radiation is believed to
be caused by the induction of a chromosomal break repair system (Olivieri et al.,1984; Vijayalaxmi and Burkart,
1989; Sankaranarayanan et al., 1989). Olivieri et al. (1984) first reported that human blood lymphocytes pre-
exposed to low levels of 3H-TdR became less susceptible to chromatid break induced by a subsquent high dose
of X-rays. In the present studies,we examined the question of whether an adaptive response can be elicited by
low-level irradiation from 3H-TdR and by low doses of X-rays and -rays.

MATERIALS AND METHODS

Animals and irradiation
Swiss mice (6-8 weeks) were obtained from the Animal House of the China Institute for Radiation
Protection. Their weight ranged from 25 to 30 g at the start of experiments. The numbers of males and females
were the same. The mice were maintained at a constant temperature and humidity (233, 502%), with a
12-h light-dark cycle. They were fed commercial pellet and given water and libitum.
The animals were exposed to X-rays generated at 200kV, 10mA and with 0.5 mm Cu and 1.0mm Al
filtration. The inductive dose (D1) was 0.005, 0.01, or 0.1 Gy at a dose rate of 0.14 Gy/min for the in vivo
experiment. Three hours later the mice were irradiated with 1.5 Gy X-rays (D2) at the above dose rate.
Lymphocytes and irradiation
Venous blood was collected from nine healthy, non-smoking males. 0.3 ml whole blood was added to 5ml
RPMI 1640 medium containing 20% newborn calf serum (Hangzhou), 50ul/mlPHA(Shanghai Bio. Co.),
penicillin 100IU/ml and streptomycin 100g/ml. The cells were cultured in a CO2 incubator at 37 in 5% CO2.
Conditions of irradiation:(1) 6h after culture initiation, 3H-TdR(specific activity: 5 Ci/mol, purchased from
the Shanghai Nuclear Research Institute ) was added at 307102 Bq/ml in cultures(D1); 30h after culture
initiation, the cells were irradiated with 0.5 Gy X-rays, the dose rate used as mentioned above. The cells were
exposed to 3H-TdR for 48h from the first 6h of culture initiation to cell harvest. (2) X-rays irradiation: first
irradiation with 0.005Gy (D1) and second exposure to 1.50 Gy (D2). X-rays were generated and the dose rate
used as mentioned above. The interval between the D1 and D2 irradiation was 48h. (3) Gamma-ray irradiation:
first irradiation with 0.05 Gy or 0.1 Gy (D1),second exposure to 1.50 Gy (D2). The source of -rays was 60Co
with a dose rate of 0.80 Gy/min. The cells received the second irradiation of -rays at the higher dosage 48h after
the first irradiation.
Slide preparation
For human blood lymphocytes, the cells were cultured for 54h. 0.1ml of colcemid (final concentration:2.7
10-5M) was added to each culture to arrest cells in mitosis 4-6 h before harvest. Hypotonic treatment was
performed with prewarmed 0.075 M KCl for 15 min at 37. The air-dried slides were stained with Giemsa.
For animals, colcemid (4mg/g b.w.) was injected intraperitoneally immediately following the second
irradiation, the mice were killed 3h later, both femurs and tibias were isolated, and the adherent muscle was

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removed. Each bone was cleaned with 70% ethyl alcohol, and epiphyses of the bone were cut off with scissors.
The marrow was flushed out with physiological saline into a centrifuge tube with a 25-G needle, followed by
centrifugation at 1000 g for 5 min, treated with 0.075 M KCl, and air-dried slide were stained with Giemsa.
Chromosomal aberration analysis
At least 300 cells from each treatment group were scored. Only cells with 46 chromosomes for human and
40 chromosomes for mice were included in the analysis. The frequencies of chromatid breaks, chromatid gaps
and of the occasional dicentries and rings were recorded (Buckton and Evans, 1973). Since the frequencies of
gaps were nearly the same in the relevant comparison groups, they are not given in the tables. In view of the
relatively low yields of chromatid exchanges, dicentrics and rings, these data are not shown. The expected
frequencies of chromatid breaks in the combined treatment groups were computed as the sum of the effects of
the individual treatment minus the frequency in control. The statistical significance of reductions in the
frequencies of chromatid breaks (relative to the expected ones) was evaluated using the x2-test.

Table 1
Effects of 0.005, 0.01, 0.05, or 0.10 Gy of X-rays given to mice on the frequencies of chromosomal
aberrations induced by a subsequent X-ray dose of 0.75 Gy given after 3 h
D1 D2 Number of Chromatid breaks
(Gy) (Gy) cells scored Observed Expected
N % %
0 0 435 6 1.37
0 0.75 442 176 39.82
0.005 0 400 7 1.75
0.005 0.75 354 87 24.58** 40.20
0.01 0 476 14 2.94
0.01 0.75 462 116 25.11** 41.39
0.05 0 400 14 3.50
0.05 0.75 496 122 24.60** 41.95
0.10 0 478 28 5.86
0.10 0.75 626 146 23.32** 44.31
** The observed frequency is very significantly lower than expected (P<0.01).

RESULTS AND DISCUSSION

Table 1 summarizes the results of the analysis of CA in bone marrow cells of irradiated mice. The observed
frequency of CA in the marrow cells pre-exposed to low-level X-rays is very significantly lower than expected.
However, Jacobson-Kram and Williams (1988) reported that in the bone marrow cells of mice given an
"adaptive" dose of 0.125 Gy and, 24h later, a challenge dose of 1.50 Gy of -irradiation., the frequencies of
chromatid breaks per cells were similiar to those in the groups which received the challenge dose only. The
differences between our and their results may be due to differences in irradiation and experimental conditions.

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Table 2
Effects of continuous treatment with 3H-TdR (3.7102 Bq/ml) on the frequencies of chromosomal
aberrations obtained in human lymphocytes treated with with 0.5 Gy of X-rays
Donor Age(years) Treatment group Number of Chromatid breaks
cells scored Observed Expected
N % %
A 25 control 400 1 0.25
3
H-TdR 300 17 5.67
0.50Gy of X-rays 400 123 30.75
3
H-TdR+0.50Gy of X- 344 101 29.36* 36.17
rays
B 23 control 400 2 0.5
3
H-TdR 400 28 7.00
0.50Gy of X-rays 400 138 34.50
3
H-TdR+0.50Gy of X- 400 136 34.00* 41.00
rays
C 30 control 450 2 0.44
3
H-TdR 400 19 4.75
0.50Gy of X-rays 400 106 26.50
3
H-TdR+0.50Gy of X- 400 141 35.25NS 30.81
rays
D 25 control 600 3 0.50
3
H-TdR 400 44 11.00
0.50Gy of X-rays 600 258 43.00
3
H-TdR+0.50Gy of X- 600 246 41.00** 53.50
rays
E 20 control 600 2 0.33
3
H-TdR 400 38 9.50
0.50Gy of X-rays 400 128 32.00
3
H-TdR+0.50Gy of X- 400 90 22.50** 41.17
rays
* The observed frequency is significantly lower than expected (P<0.05).
** The observed frequency is very significantly lower than expected (P<0.01).
NS
The observed frequency is not significantly lower than expected (P>0.05)

The results on human blood lymphocytes are indicated in Table 2-4. These data confirm that human
peripheral blood lymphocytes can become adapted to low-level irradiation from 3H-TdR or exposure to a low
dose of X- or -irradiation. Frequencies of CA in the lymphocytes pre-exposed to low-dose irradiation were
significantly low than those in the lymphocytes receiving the challenge dose only.
It is also shown in Table 2 and 3 that the lymphocytes from one donor (donor C) did not show an adaptive
response after prior treatment with 3H-TdR or X-ray irradiation. These findings suggest the existence of
variations between individuals with respect to the induction of adaptive respose in their lymphocytes. A similiar
conclusion was also arrived at by Bosi and Olivieri (1988). It is possible that some of this variability is related to
differences between individuals in the degree of catabolism of the nucleosides (Bodycote and Wolff, 1986).
In conclusion, the results of our study support the view that human blood lymphocytes in vitro and mouse
marrow cells in vivo, pre-exposed to low-level irradiation, can become adapted so that they become less
sensitive to the chromosomal damage effects of subsequent radiation exposures. However, there are inter-
individual differences with respect to the magnitude of the adaptive effect. The mechanism of the adaptive
response to low-level irradiation remains unclear, and further study is needed.

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Table 3
Effect of 0.005 Gy of X-rays given at 32 h after PHA stimulation of human lymphocytes on the frequencies
of chromosomal aberrations by a subsequent X-ray dose of 1.50 Gy at 48 h
Donor Age Treatment group Number of Chromatid breaks
(years) cells scored Observed Expected
N % %
A 25 control 400 1 0.25
0.005 Gy 400 35 8.75
1.5 Gy 400 350 87.50
0.005+1.50 Gy 400 321 80.25** 96.00
B 23 control 400 2 0.50
0.005 Gy 400 14 3.50
1.5 Gy 400 386 96.50
0.005+1.50 Gy 400 376 94.00** 99.50
C 30 control 450 2 0.44
0.005 Gy 400 28 7.00
1.5 Gy 400 338 84.5
0.005+1.50 Gy 400 372 93.00NS 91.06
D 25 control 600 3 0.50
0.005 Gy 400 18 4.50
1.5 Gy 600 472 78.67
0.005+1.50 Gy 400 218 54.50** 82.67
E 20 control 600 2 0.33
0.005 Gy 500 62 12.14
1.5 Gy 400 194 48.5
0.005+1.50 Gy 400 140 35.00** 59.57
* The observed frequency is significantly lower than expected (P<0.05).
** The observed frequency is very significantly lower than expected (P<0.01).
NS
The observed frequency is not significantly lower than expected (P>0.05)
Table 4
Effect of 0.05 Gy or 0.10 Gy -rays given st 32 h after PHA stimulation of human lymphocytes on the
frequencies of chromosomal aberrations induced by a subsequent -rays dose of 1.50 Gy given at 48 h.
Donor Age Treatment group Number of cells Chromatid breaks
(years) scored Observed Expected
D1(Gy) D2(Gy) N % %
G 22 0 0 400 3 0.75
0 1.50 400 142 35.5
0.05 0 400 15 3.75
0.05 1.50 400 113 28.25** 38.50
0.10 0 400 18 4.5
0.10 1.50 400 119 29.75** 39.25
H 20 0 0 450 3 0.67
0 1.50 400 139 34.75
0.05 0 400 13 3.25
0.05 1.50 400 108 27.00** 37.33
0.10 0 400 16 4.00
0.10 1.50 400 110 27.50** 38.08
I 25 0 0 489 4 0.82
0 1.50 400 148 37.00
0.05 0 400 17 4.25
0.05 1.50 400 98 24.5** 40.43
0.10 0 450 28 6.22
0.10 1.50 460 129 28.04** 42.40
** The observed frequency is very significantly lower than expected (P<0.01).

References
1. Bodycote, J. and S. Wolff (1986) Metabolic breakdown of [3H]thymidine and the inability to measure human
lymphocyte proliferation by incorporated radioactivity, Proc. Natl. Acad. Sci. USA.
2. Bosi, R. and G. Olivieri (1988) Variability of the adaptive response to ionizing radiation in humans, Mutation

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Res., 211, 13-17.

3. Buckton, K. E. and H. J. Evans (Eds) (1973) Methods for the Analysis of Human Chromosome Aberrations,
WHO, Geneva, pp.1-51.
4. Jacobson-Kram, D. and J.R> Williams (1988) Failure to observe adaptive response to ionzing radiation in
mouse bone marrow cells in vivo, Environ. Mol. Mutagen. (Suppl. 11), Abstracts of the 19th Ann. Meeting of
EMS, pp. 49-50.
5. Olivieri, G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations
of radioactive thymidine, Science,223, 594-597.
6. Samson, L. and J. Cairns (1977) A new pathway for DNA repair in Escherichia coli, Nature, 267, 287-283.
7. Sankaranarayanan, K., A. van Duyn, M.J. Loos and a A.T. Natarajan (1988) Adaptive response of human
lymphocytes to low-level radiation from radioisotopes or X-rays, Mutation Res., 211, 7-12.
8. Vijayalaxmi and W. Burkart (1989) Resistance and cross-resistance to chromosome damage in human blood
lymphocytes adapted to bloomycin, Mutation Res., 211, 1-5.