- Email: [email protected]
Low-dose radiation-induced adaptive response in bone marrow cells of mice
Mutation Research, 302 (1993) 83-89
83
~-5 1993 Elsevier Science Publishers B.V. All rights rescrved 0165-7992/93/$06.00
MUTLET 00792
Low-dose radiation-induced adaptive response in bone marrow cells of mice Zeba Farooqi and P.C. Kesavan School of Life Sciences, Jawaharlal Nehru Unicersity, New Delhi-110067, b~dia
(Received 21 July 1992) (Revision received 13 January 1993) (Accepted 20 January 1993)
Keywords: Radioadaptive response; Conditioning dose; Challenging dose: Gamma-rays
Summary Using bone marrow ceils of whole body irradiated mice, the cytogenetic adaptive response induced by low conditioning doses of gamma-rays was investigated. The conditioning doses (0.025 and 0.05 Gy) were given at a dose-rate of 1.67 G y / m i n . The challenging dose of 1 Gy was given at a dose-rate of 0.045 G y / s . The challenging dose was given at different time intervals after the conditioning dose. The time intervals between the conditioning dose and challenging dose were 2, 7.5, 13, 18.5 and 24 h. When the time interval between the conditioning dose and the challenging dose was 2 h, both conditioning doses (0.025 and 0.05 Gy) reduced the frequency of MNPCEs and chromosomal aberrations in the bone marrow cells. The data collected at different time intervals (7.5, 13, 18.5 h) reveal that the radioadaptivc response persisted for a longer time when the lower conditioning dose (0.025 Gy) was given. With the higher conditioning dose (0.05 Gy), the radioadaptive response disappeared after a time interval of 13 h. When the time interval between the conditioning dose and the challenging doses was 18.5 or 24 h, only the lower conditioning dose appeared effective in inducing the radioadaptive response.
The exposure to various DNA damaging stresses like mutagenic and clastogenic agents is known to induce an adaptive response in a variety of pro- and cukaryotic cells (Samson and Cairns, 1977; Samson and Schwartz, 1980; Reiger et al., 1982; Olivieri et al., 1984; Morimoto et ai., 1986; Shadley et al., 1987; Ikushima, 1987; Wolff et al., 1988). This response to environmental stress eventually enhances the resistance of the cells to
Correspondence: Prof. P.C. Kesavan, Schcx)lof Life Sciences, Jawaharlal Nehru University, New Delhi-f10067, India.
the damaging impact and provides additional capacities to repair and achieve better survival. The adaptive response was first reported by Samson and Cairns (1977) in E. coli. The bacterial cells that had been exposed to low levels of alkylating agents became less susceptible to the subsequent high doses of the same and other compounds. Adaptation after low level exposures has also been found to occur in Vicia faba exposed to chemical clastogens (Reiger ct al., 1982) and in E. coli exposed to hydrogen peroxide (Demple and Halbrook, 1983). Analogous responses are known for mammalian cells in vitro.
b;4
A pretreatment with low conditioning doses of either tritium /3-rays or X-rays significantly reduced the yicld of chromosomal aberrations induced by a subsequent higher challenging dose in human lymphocytcs (Olivieri et al., 1984; Shadley and Wolff, 1987; Sankaranarayanan ct al., 1989; Fan et al., 1990). A similar radioadaptivc response has also been observed with regard to induction of micronuclei and SCE in prolifcrativc Chinese hamster V79 ceils (Ikushima, 1987, 1989), survival and mutagenesis in human lymphocytes (Sandcrson and Morley, 1986). While all thcsc experiments had been performed using in vitro systems, the focus has now shifted to investigate the induction of the adaptivc rcsponsc in vivo. Wojcik and Tuschl (1990) have reported enhanced unscheduled DNA synthesis (UDS) and concomitant reduction in the frequencies of induced SCEs in mice pre-cxposed in vivo to low doses of ionizing radiations. The induction of the cytogenetic adaptive response in somatic and germ cells in mice exposed to whole body X-irradiation in vivo was reported by Cai and Liu (1990). The induction of thc cytogcnctic adaptive response was also reported in rabbits exposed to very low y-rays (Liu et al., 1992). The objective of the present study was to verify the induction of the radioadaptive response in terms of MNPCEs and chromosomal aberrations in bone marrow cells of micc givcn wholc body exposure to y-rays. Our ultimate aim is to understand the mechanism(s) underlying the adaptive response, if any, induced in in vivo systems. Materials and method
Test system Swiss albino male mice 9-12 weeks old and weighing 22-32 g wcrc used in the experiments. The animals were kept in polypropylene cages with ad libitum access to standard diet and drinking water. Experimental design The conditioning doses (0.025 and 0.05 Gy) were given as acute whole body exposure to y-rays with the help of a teletherapy unit located at the INMAS, New Delhi. The dose-rate as determined by Fricke's (Fe2+/Fe 3+) dosimetry was
1.67 G y / m i n . The experimental design took care to include appropriate controls in order to assess the cffccts, if any, of the low conditioning doses per se. The challenging dose of 1 Gy of y-rays was given 2, 7.5, 13, 18.5 or 24 h after the conditioning dose. The source of y-rays for the challenging dose was a gamma-chamber (204 TBq; BARC, India) located in our laboratory, and the dose-rate was 0.045 G y / s with the attenuator. For each treatment point, five animals were used. For MNPCEs different sampling times (24, 48, 72 h) and for chromosomal aberrations two sampling times (24, 48 h) were scored.
Chromosomal aberration studies 24 and 48 h after the last irradiation, the animals were killed by cervical dislocation. Colchicine (4 m g / k g body weight) had been intraperitoneally injected 2 h prior to death to arrest the metaphases. After death, both femurs of the animals were removed. The bones were cleared of adhering muscle tissue. The bone marrow cells were flushed into a 10-ml centrifuge tube using a hypodermic syringe filled with 1 ml fetal calf serum. The cells were centrifuged and supcrnatant was aspirated. The cell pellet was resuspended in hypotonic solution (1% Na citrate) at 37°C. Twenty minutes after the hypotonic shock, the cells were fixed 3 times in a mixture of 3 parts methanol and 1 part acetic acid. The cell suspension was dropped on the slides and air-dried preparations were stained with Giemsa (Gurr, 2%). After keeping in Xylene for 20 rain, the slides were mounted in DPX. The coded slides were observed and scored for aberrations like breaks, rings, exchanges, fragments. 100 metaphases per animal were scored and their data pooled. MN in polychromatic erythrocytes (MNPCEs) The animals were killed at either 24, 48 or 72 h after the challenging dose irradiation. To study the persistence of the adaptive response with respect to induction of micronuclei only one sacrifice time of 24 h was used. The slides with the bone marrow cell smears were processed as described by Schmid (1975). All the slides were coded before scoring and for each experimental
85
animal 2500 polychromatic erythrocytes were scored from two slides to detect the presence of MNPCEs. The data were subjected to Student's t test to calculate the probability and significance level whcrcver required. Results
To test the possibility of a radioadaptivc response by external whole body acute exposure to low doses of y-rays, the animals exposed to a conditioning dose of either 0.025 Gy or 0.05 Gy of y-rays were subsequently challenged with 1 Gy of y-rays at intervals of 2 or 24 h. The yield of micronuclci in PCEs was measured in animals killed 24, 48, 72 h after the challenging dose. As shown in Table 1, a reduction of the incidence of MNPCEs following exposure to the challenging dose was observed in animals conditioned with either 0.025 or 0.05 Gy. This radioadaptive response was statistically significant ( p < 0.01 ) when the time interval between the conditioning dose and challenging dose was 2 h (Table 11. When the time interval between the conditioning dose and challenging dose was 24 h, the radioadaptive response was observed in only those animals which were conditioned with the lower dose (0.025 Gy). The higher inductive dose (0.05 Gy) failed to elicit the adaptive response when the time interval between the challenging and conditioning dose was 24 h (Table 1). When the lower conditioning dose of 0.(}25 Gy was used, the adaptive response (in terms of MN) persisted through all the time intervals. However, when the higher conditioning dose of 0.05 Gy was given to the animals, the radioadaptive response disappeared progressively as the time interval between the conditioning dose and challenging dose increased from 13 to 24 h (Fig. 1). The data (Table 2) illustrate the reduction of the frequency of chromosomal aberrations and the percentage aberrant cells when the mice had been pretreated with a conditioning dose of 0.025 Gy and 0.05 Gy. There was a decrease in the yield of chromosomal aberrations in mice when the time interval between conditioning dose and challenging dose was 2 h (Tables 2 and 3). However, when the time interval was 24 h, the lower dose of 0.025 Gy was
TABLE 1 EFFECT
OF
CONDITIONING
RAYS ON THE
FREOUENCY
DOSES
OF
GAMMA-
OF MNPCEs INDUCED
BY A C H A i . I . , E N G I N G D O S E IN M O U S E B O N E M A R ROW CELLS SAMPLED AT DIFFERENT TIME INTERVALS Condi-
Inter-
Challeng-
Fixation
Mean
tioning dose (Gy)
treatment time
ing dose ( 1 Gy)
time (h)
_+S E M "
-
24 48
[).72+0.19 1.[){5_+0.26
-
72
[).53.+_[).13
[).[)25
-
24 48 72
1.75 +_0.41 1.40 _+0.26 0.60+[).13
0.05
-
24 48 72
4.24 +_0.71 3.16 -,- 0.02 0.99 - [).29
0.025
2 2 2
+ + +
24 48 72
12.0 + 1.30 * 3.59 + 11.41 * 1.46,-[).133 *
[).[15
2 2 2
+ + +
24 48 72
11.3 _+0.48 * 4.93 + 0.23 * * 3.[16 _+[).48
0.[)25
24
+
24
18.12_+ 1.18 **
24 24
+ +
48 72
5.[)5 + {).25 * * 2.60 -+ 0.60
24 24 24
+ + +
24 48 72
25.{) -+ 1.8[) 8.95 -+ 1.911 4.20 _+[).26
+
24
25.32 -+ 1.90
+ +
48 72
9.75 _+ 1.30 3.70 _+[).25
(h) Control
0.05
-
-
-
" Mean_+ S E M for five mice, 2500 P C E s s c o r e d p e r a n i m a l a n d d a t a pooled. - . no t r e a t m e n t , + , t r e a t m e n t . * Significantly r e d u c e d f r e q u e n c y ( p < 0.011 in c o m p a r i s o n with that i n d u c e d by I G y alone. ** Significantly r e d u c e d f r e q u e n c y ( p < [).05) in c o m p a r i s o n with that i n d u c e d by 1 Gy.
more effective in reducing the yield of chromosomal aberrations than the higher inductive dose of 0.05 Gy (Tables 2 and 3). However, when computed in terms of expected frequency (sum of the yields induced by y-rays alone plus conditioning treatment alone minus control) and the observed frequency (Tables 4 and 5), the data show that the expected frequency is higher than the ob-
80
TABLE 2 O F C O N D I T I O N I N G DOSES OF G A M M A - R A Y S ON T I l E I N D U C T I O N OF C I I R O M O S O M A L A B E R R A T I O N S BY A C H A L L E N G I N G D O S E IN M O U S E BONE M A R R O W CELLS S A M P L E D AT 24 h EFFECT
('onditioning dose
Intertreatment
(Gy)
time (h)
Challenging dose ( I Gy)
% Aberrant cells
Aberrations/100 metaphases (mean) Ch'd Isoch'd R Frag Ch'l br br cxch
Ch'd
Mean aberrations per cell _+SE
cxch
Control
-
3
3
-
-
-
0.025
-
4.5
3
-
-
1
-
0.04 + 0.018
-
-
8.4
5
-
2
1
0 . 0 6 + (I.II24
0.025
2
+
16
7
1
I
8
1
1
(I.19-+0.028
0.05
2
+
23
9
3
1
18
1
I
0.32-+0.07
0.025
24
+
21
7
2
1
15
2
(I.26_+(I.(14 *
(I.(15
24
+
26
14
-
2
22
2
0.4(I _+ 0 . 0 3
-
+
31
18
3
4
24
3
(I.475:(I.05
11.115
0.03 + 0.017
*
I(X) mctaphases per animal scored; five animals per treatment used and data pooled. * Significantly lower than l Gy value ( p < O.O1), Student's t test.
served frequency. The difference between the two for the Iowcr conditioning dose (0.025 Gy) is quite significant. For the higher conditioning dose of 0.05 Gy this difference is small which indicates that there could be a slight adaptive response which could be interpreted as the fraction of the progressively diminishing adaptive response. Furthcr experiments arc in progress to find out the optimal conditioning dose for the induction of the adaptive response in mice when the time
interval between conditioning dose and challenging dose varies from 2 to 24 h. Discussion One of the impressive manifestations of low dose irradiation is the adaptive response. Since the demonstration by Samson and Cairns (1972) that pretreatment with low doses confers enhanced resistance against the damaging effects of
TABLE 3 EFFECT OF C O N D I T I O N I N G DOSES OF G A M M A - R A Y S ON T H E I N D U C T I O N OF C H R O M O S O M A l , A B E R R A T I O N S BY A C H A L I . E N G I N G D O S E IN M O U S E B O N E M A R R O W CEI.LS S A M P L E D AT 48 h Conditioning dose (Gy)
Intertreatment time (h)
Challenging dose ( 1 Gy)
% Aberrant cells
Aberrations/100 metaphases (mean) Isoch'd R Frag Ch'l br cxch
Ch'd br
Mean aberrations Ch'd
per cell _+SE
exch
Control
-
3
3
-
-
(I.(13 _+ 0 . 0 1 7
0.025
-
"~
1
-
1
-
0 . 0 2 _+ 0 . 0 0 9
0.05
-
8
6
-
2
-
0 . 0 8 5:I).(123
I).1125
2
+
12
9
(I.(15
2
+
12
8
-
4
-
1
-
5
-
1
(I.(125
24
+
14
7
(I.(15
24
+
18
+
22
1
7
1
0.17_+0.(11
12
1
2
9
-
2
I).25_+(I.04
10
1
-
15
2
0 . 2 5 + 11.(15
100 mctaphases per animal scored; five animals per treatment uscd and data l:V,x)lcd. * Significantly lower than 1 Gy value (p < 0.05), Student's t test.
0.12+0.03 0.16-t-0.03
*
87 TABLE 5
• MNI'K)O0
30 i 26
26
oiili
,, 12
0
2
n
I Gy
!
, 11
I
i',
lib
0 . 0 2 6 O y • 1 Ely
~
1i11.
] I[
7.5 13 18.5 Intertreltnlent time Interval (hr)
T H E E X P E C T E D F R E Q U E N C Y A N D O B S E R V E D FREQUENCY OF ABERRATIONS
24
I
0.06 Gy * I Gy
Fig. 1. Influence of conditioning dose on the persistence of the adaptive response.
a subsequent (challenging) higher dose, there have been several interesting studies (Olivieri et al., 1984; lkushima, 1987; Shadley et al., 1987; Wolff et al., 1988). The radioadaptive response now stands clearly demonstrated in a wide range of in vitro (Shadlcy et al., 1987; Wolff et al., 1989) and in vivo mammalian systems (Cai and Liu, 1990; Wojcik and Tuschl, 1990; Liu et al., 1992). Data presented in this study (Tables 1-3) also confirm the induction of the radioadaptivc response in mice by conditioning doses of 0.025 and 0.05 Gy. When the time interval between the conditioning dose and the challenging dose was 2 h, both inductive doses (0.025 and 0.05 Gy) induced the adaptive response; however, when the intcrtreatTABLE 4 TIIE E X P E C T E D F R E Q U E N C Y A N D O B S E R V E D FREQ U E N C Y O F MNPCEs Expected frequency ~
Observed frequcncy
I.
0.025 + 1 Gy (24 h intertreatment time)
26.32
18.12
2.
0.05 + I Gy (24 h intertreatment time)
28.84
25.0
Expected frequency ~
Observed frequency (mean)
1.
0.48
0.26
0.50
0.40
24
oondlllonl~ CIOILel:O.C)26 • 000 Oy. ghallenoInO dole* 1 QY0 " Meen from 0 plae, 28O0 PCIEI eoo~d ~ r animal
Treatment
Treatmcnt '
a Sum of the yields of Mn induced by y-rays alone + conditioning treatment alone minus control.
2.
0.025 + 1 Gy (24 h intertreatment time) 0.05 + 1 Gy (24 h intertreatment time)
" Sum of the yields of aberrations induced by y-rays alone plus conditioning treatment alone minus control.
mcnt time was 24 h, only the lower dose (0.025 Gy) was effective in inducing the adaptive response. However, when the data were computed as the difference between the expected frequency (sum of the yields induced by T-rays alone plus conditioning treatment alone minus control) and thc observed frequency (Tables 4 and 5), the difference between the two frequencies was significant for the lower conditioning dosc (0.025 Gy). For the higher conditioning dosc this difference was small, which could be an indication of disappearing adaptive response. The different time intervals between the conditioning dose and challenging dose (2, 7.5, 13, 18.5 and 24 h) reveal the persistence of the adaptive response. With the lower conditioning dose of 0.025 Gy the adaptive response persisted for a longer time. With the higher conditioning dose the radioadaptivc response disappeared as the time interval between the conditioning dose and challenging dose increased. In the literature, there are conflicting reports regarding the optimal range of conditioning doses to induce maximal response in a variety of test systems (Samson and Schwartz, 1980; Shadley and Wolff, 1987; Shadley and Wiencke, 1989; Vijayalaxmi and Burkart, 1989; Ikushima, 1989). Cai and Liu (1990) have reported a negative correlation between the magnitude of the adaptive response and the size of induction dose in the mouse in vivo. Furthermore, Liu et al. (1992)
88 have reported that the i n d u c t i o n of the cytogcnetic adaptive response in terms of a reduction in a b e r r a t i o n s occurred only for fragments, indicating that the a d a p t a t i o n was chiefly effective for one-hit events. O n the o t h e r hand, Olivieri et al. (1984) using [3H]TdR in c o n d i t i o n i n g of h u m a n lymphocytes observed that a larger dose (3.7 k B q / m l ) s e e m e d to induce a b e t t e r adaptive response to a challenging dose of X - i r r a d i a t i o n than a smaller dose of (I.37 k B q / m l . W i e n c k e ct al. (1986) also found an increase in the inductive effect with an increasing p r i m i n g dose of tritiated thymidine within a certain range. Y o n e z a w a et al. (1992) have shown an e n h a n c e d survival rate in mice pre-exposed to low doses of 2.5-15 cGy, 2 m o n t h s prior to the second exposure to a sublethal dose. T h e optimal a n d significant increase was observed with 5 - 1 0 cGy. In the context of i n d u c i n g an adaptive response, the possibility that the i m m u n e system is being activatcd significantly at a particular low dose range is currently receiving c o n s i d e r a b l e attention. The observation made by G r e e n s t o c k et al. (1992) is of interest. They observed that low doses ( < 0.005 Gy) stimulate an increase in I F N - y or IL-2 receptors ( h u m a n cytokine receptors) in isolated h u m a n lymphocytes. This stimulation sccn at low doses was not a p p a r e n t at high doses ((I.2 Gy). T h e data p r e s e n t e d here (Tables 1-3; Fig. l) reveal that the lower c o n d i t i o n i n g dose of 0.025 Gy induces r a d i o a d a p t a t i o n for a longer period of time in mice than the higher dose of 0.(15 Gy. Studies with p r o t e i n synthesis inhibitors ( Y o u n g b l o m ct al., 1989) and 2-D gel clcctrophoresis (Wolff et al., 1989) havc indicated the r e q u i r e m e n t of synthesis of new proteins possibly through the involvement of gene expression at low doses of radiation. T h e r e is, therefore, scope to assume that in our system the small c o n d i t i o n ing dose induces a largcr yicld a n d / o r longerlived adaptive proteins. T h e signals for gene expression could consist of d a m a g e to D N A and n u c l c o p r o t e i n s ( W e i c h s e l b a u m et al., 1991) or the formation of r e d u c t i o n products of oxygen as d e m o n s t r a t e d by Singh a n d Kesavan (1991). Further work is in progress to elucidate the low dose irradiation i n d u c e d principle(s) responsible for the m a n i f e s t a t i o n of the adaptive responsc in the mouse system.
References Cai, L., and S.Z. Liu (199{)) Induction of cytogenetic adaptive response of somatic and germ cells in vivo and in vitro by low dose X-irradiation, Int. J. Radiat. Biol., 58, 187-194. Demple, B., and J. Halbrook (1983) Inducible repair of oxidative DNA damage in Escherichia coli, Nature, 304, 466468. Fan, S., Vijayalaxmi,G. Mindek and W. Burkart (1991))Adaptive response to low doses of X-rays in human blood lymphocytes, Mutation Res., 243, 53-56. Greenstock, C.I,., Y. Xu, R.E.J. Mitchell and A. Trivedi (1992) Stimulation of human cytokine receptor expression by low dose irradiation, in: 40th Annual Meeting of the Radiation Research Society, 14-18 March, Salt Lake City, UT. lkushima, T. (1987) Chromosomal responses to ionising radiation reminiscent of an adaptive response in cultured Chinese hamster cells, Mutation Res., 180, 215-221. lkushima. T. (1989) Radioadaptive response: Characterisation of a cytogenetic repair induced by low-level ionising radiation in cultured Chinese hamster cells, Mutation Res., 227, 241 - 246. Liu, S.Z., L. Cai and S.Q. Sun (1992) Induction of a cytogenetic adaptive response by exposure of rabbits to very low dose-rate gamma radiation, Int. J. Radiat. Biol., 62, 187190. Morimoto, K.M., Sato-Mizuno and A. Koizumi (1986) Adaptation-like response to the chemical induction of sister chromatid exchanges in human lymphocytes,llum. Genet., 73, 81-85. Olivieri. G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine, Science. 223. 594-597. Reiger, R., A. Michaeli find H. Nicoloff (1982) Inducible repair processes in plant root tip meristem? "Below additivity effects' of unequally fractionated clastogen concentrations, Biol. Zcntralbl., 101. 125-138. Samson, L.. and J. Cairns (1977) A new pathway fi)r DNA repair in E. coli. Nature, 267. 281-283. Samson, I_,., and J.l,. Schwartz (1981)1 Evidence for an adaptive DNA repair pathway in CH() and human skin fibroblast cell lines, Nature, 287, 861-863. Sanderson, B.J.S.. and A.A. Morley (1986) Exposure of human lymphocytes to ionising radiation reduces mutagenesis by subsequent ionising radiation, Mutation Res., 164, 347-351. Sankaranarayanan. K.. A. van Duyn, M.J. Lcx~s and A.T. Natarajan (1989) Adaptive response of human lymphocytes to low level radiation from radioisotopes or X-rays, Mutation Res., 211, 7-12. Schmid, W. (1975)The micronucleus test, Mutation Res., 31, 0-15. Shadley, J.D., and S. Wolff (1987) Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation, Mutagcnesis, 2, 95-06. Shadlcy, J.D., and J.K. Wiencke (1989) Induction of the adaptive response by X-rays is dependent on radiation intensity. Int. J. Radiat. Biol., 56, 1117 118.
89 Shadley, J.D., V. Afzal and S. Wolff (1987) Characterisation of the adaptive response to ionizing radiation induced by low doses of X-rays to human lymphocytes, Radiat. Res., 111,511-517. Singh, S.P., and P.C. Kesavan (1991) Biochemical effects of heat shock and caffeine on post-irradiation oxic and anoxic damage in barley seeds of low and high water content, Int. J. Radiat. Biol., 59, 1227-1236. Vijayalaxmi, and W. Burkart (1989) Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin, Mutation Res., 21 I, 1-5. Weichselbaum, R.R., D.E. Hallahan, V. Sukhatme, A. Dritschilo, M.L. Sherman and D.W. Kufe (1991) Biological consequences of gene regulation after ionizing radiation exposure, J. Natl. Cancer Inst., 83, 480-484. Wiencke, J.K., V. Afzal, G. Olivieri and S. Wolff (1986) Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism, Mutagenesis, I, 375-3811. Wojcik, A., and H. Tuschl (1990) Indication of an adaptive response in C57BL mice pre-exposed in vivo to low doses of ionising radiations, Mutation Res., 243, 67-73.
Wolff, S., V. Afzal, J.K. Wiencke, G. Olivieri and A. Michaeli (1988) Human lymphc~ytes exposed to low doses of ionising radiation become refractory to high doses of radiation as well as to chemical mutagens that induce double strand breaks in DNA, Int. J. Radiat. Biol., 53, 39-48. Wolff, S., J.K. Wiencke, V. Afzal, J. Youngblom and F. Cortes (1989) The adaptive response of human lymphocytcs to very low doses of ionising radiation: a case of induced chromosome repair with the induction of specific proteins, in: Lx~wDose Radiation: Biological Basis of Risk Assessment, Taylor & Francis, London, pp. 446-454. Yonezawa, M., J. Misonoh and A. Takeda (1990) Modification of radiosensitivity by lov,' dose irradiation, Abstract, Annual Meeting of the Japan Radiation Research Society, Sendai, 24-26 October. Youngblom, J.lt., J.K. Wicnckc and S. Wolff (1989) Inhibition of the adaptive response of human lymphocytes to very low doses of ionising radiation by the protein synthesis inhibitor cyclohcximide, Mutation Res., 227, 257-261 Communicated by F.H. Sobels
83
~-5 1993 Elsevier Science Publishers B.V. All rights rescrved 0165-7992/93/$06.00
MUTLET 00792
Low-dose radiation-induced adaptive response in bone marrow cells of mice Zeba Farooqi and P.C. Kesavan School of Life Sciences, Jawaharlal Nehru Unicersity, New Delhi-110067, b~dia
(Received 21 July 1992) (Revision received 13 January 1993) (Accepted 20 January 1993)
Keywords: Radioadaptive response; Conditioning dose; Challenging dose: Gamma-rays
Summary Using bone marrow ceils of whole body irradiated mice, the cytogenetic adaptive response induced by low conditioning doses of gamma-rays was investigated. The conditioning doses (0.025 and 0.05 Gy) were given at a dose-rate of 1.67 G y / m i n . The challenging dose of 1 Gy was given at a dose-rate of 0.045 G y / s . The challenging dose was given at different time intervals after the conditioning dose. The time intervals between the conditioning dose and challenging dose were 2, 7.5, 13, 18.5 and 24 h. When the time interval between the conditioning dose and the challenging dose was 2 h, both conditioning doses (0.025 and 0.05 Gy) reduced the frequency of MNPCEs and chromosomal aberrations in the bone marrow cells. The data collected at different time intervals (7.5, 13, 18.5 h) reveal that the radioadaptivc response persisted for a longer time when the lower conditioning dose (0.025 Gy) was given. With the higher conditioning dose (0.05 Gy), the radioadaptive response disappeared after a time interval of 13 h. When the time interval between the conditioning dose and the challenging doses was 18.5 or 24 h, only the lower conditioning dose appeared effective in inducing the radioadaptive response.
The exposure to various DNA damaging stresses like mutagenic and clastogenic agents is known to induce an adaptive response in a variety of pro- and cukaryotic cells (Samson and Cairns, 1977; Samson and Schwartz, 1980; Reiger et al., 1982; Olivieri et al., 1984; Morimoto et ai., 1986; Shadley et al., 1987; Ikushima, 1987; Wolff et al., 1988). This response to environmental stress eventually enhances the resistance of the cells to
Correspondence: Prof. P.C. Kesavan, Schcx)lof Life Sciences, Jawaharlal Nehru University, New Delhi-f10067, India.
the damaging impact and provides additional capacities to repair and achieve better survival. The adaptive response was first reported by Samson and Cairns (1977) in E. coli. The bacterial cells that had been exposed to low levels of alkylating agents became less susceptible to the subsequent high doses of the same and other compounds. Adaptation after low level exposures has also been found to occur in Vicia faba exposed to chemical clastogens (Reiger ct al., 1982) and in E. coli exposed to hydrogen peroxide (Demple and Halbrook, 1983). Analogous responses are known for mammalian cells in vitro.
b;4
A pretreatment with low conditioning doses of either tritium /3-rays or X-rays significantly reduced the yicld of chromosomal aberrations induced by a subsequent higher challenging dose in human lymphocytcs (Olivieri et al., 1984; Shadley and Wolff, 1987; Sankaranarayanan ct al., 1989; Fan et al., 1990). A similar radioadaptivc response has also been observed with regard to induction of micronuclei and SCE in prolifcrativc Chinese hamster V79 ceils (Ikushima, 1987, 1989), survival and mutagenesis in human lymphocytes (Sandcrson and Morley, 1986). While all thcsc experiments had been performed using in vitro systems, the focus has now shifted to investigate the induction of the adaptivc rcsponsc in vivo. Wojcik and Tuschl (1990) have reported enhanced unscheduled DNA synthesis (UDS) and concomitant reduction in the frequencies of induced SCEs in mice pre-cxposed in vivo to low doses of ionizing radiations. The induction of the cytogenetic adaptive response in somatic and germ cells in mice exposed to whole body X-irradiation in vivo was reported by Cai and Liu (1990). The induction of thc cytogcnctic adaptive response was also reported in rabbits exposed to very low y-rays (Liu et al., 1992). The objective of the present study was to verify the induction of the radioadaptive response in terms of MNPCEs and chromosomal aberrations in bone marrow cells of micc givcn wholc body exposure to y-rays. Our ultimate aim is to understand the mechanism(s) underlying the adaptive response, if any, induced in in vivo systems. Materials and method
Test system Swiss albino male mice 9-12 weeks old and weighing 22-32 g wcrc used in the experiments. The animals were kept in polypropylene cages with ad libitum access to standard diet and drinking water. Experimental design The conditioning doses (0.025 and 0.05 Gy) were given as acute whole body exposure to y-rays with the help of a teletherapy unit located at the INMAS, New Delhi. The dose-rate as determined by Fricke's (Fe2+/Fe 3+) dosimetry was
1.67 G y / m i n . The experimental design took care to include appropriate controls in order to assess the cffccts, if any, of the low conditioning doses per se. The challenging dose of 1 Gy of y-rays was given 2, 7.5, 13, 18.5 or 24 h after the conditioning dose. The source of y-rays for the challenging dose was a gamma-chamber (204 TBq; BARC, India) located in our laboratory, and the dose-rate was 0.045 G y / s with the attenuator. For each treatment point, five animals were used. For MNPCEs different sampling times (24, 48, 72 h) and for chromosomal aberrations two sampling times (24, 48 h) were scored.
Chromosomal aberration studies 24 and 48 h after the last irradiation, the animals were killed by cervical dislocation. Colchicine (4 m g / k g body weight) had been intraperitoneally injected 2 h prior to death to arrest the metaphases. After death, both femurs of the animals were removed. The bones were cleared of adhering muscle tissue. The bone marrow cells were flushed into a 10-ml centrifuge tube using a hypodermic syringe filled with 1 ml fetal calf serum. The cells were centrifuged and supcrnatant was aspirated. The cell pellet was resuspended in hypotonic solution (1% Na citrate) at 37°C. Twenty minutes after the hypotonic shock, the cells were fixed 3 times in a mixture of 3 parts methanol and 1 part acetic acid. The cell suspension was dropped on the slides and air-dried preparations were stained with Giemsa (Gurr, 2%). After keeping in Xylene for 20 rain, the slides were mounted in DPX. The coded slides were observed and scored for aberrations like breaks, rings, exchanges, fragments. 100 metaphases per animal were scored and their data pooled. MN in polychromatic erythrocytes (MNPCEs) The animals were killed at either 24, 48 or 72 h after the challenging dose irradiation. To study the persistence of the adaptive response with respect to induction of micronuclei only one sacrifice time of 24 h was used. The slides with the bone marrow cell smears were processed as described by Schmid (1975). All the slides were coded before scoring and for each experimental
85
animal 2500 polychromatic erythrocytes were scored from two slides to detect the presence of MNPCEs. The data were subjected to Student's t test to calculate the probability and significance level whcrcver required. Results
To test the possibility of a radioadaptivc response by external whole body acute exposure to low doses of y-rays, the animals exposed to a conditioning dose of either 0.025 Gy or 0.05 Gy of y-rays were subsequently challenged with 1 Gy of y-rays at intervals of 2 or 24 h. The yield of micronuclci in PCEs was measured in animals killed 24, 48, 72 h after the challenging dose. As shown in Table 1, a reduction of the incidence of MNPCEs following exposure to the challenging dose was observed in animals conditioned with either 0.025 or 0.05 Gy. This radioadaptive response was statistically significant ( p < 0.01 ) when the time interval between the conditioning dose and challenging dose was 2 h (Table 11. When the time interval between the conditioning dose and challenging dose was 24 h, the radioadaptive response was observed in only those animals which were conditioned with the lower dose (0.025 Gy). The higher inductive dose (0.05 Gy) failed to elicit the adaptive response when the time interval between the challenging and conditioning dose was 24 h (Table 1). When the lower conditioning dose of 0.(}25 Gy was used, the adaptive response (in terms of MN) persisted through all the time intervals. However, when the higher conditioning dose of 0.05 Gy was given to the animals, the radioadaptive response disappeared progressively as the time interval between the conditioning dose and challenging dose increased from 13 to 24 h (Fig. 1). The data (Table 2) illustrate the reduction of the frequency of chromosomal aberrations and the percentage aberrant cells when the mice had been pretreated with a conditioning dose of 0.025 Gy and 0.05 Gy. There was a decrease in the yield of chromosomal aberrations in mice when the time interval between conditioning dose and challenging dose was 2 h (Tables 2 and 3). However, when the time interval was 24 h, the lower dose of 0.025 Gy was
TABLE 1 EFFECT
OF
CONDITIONING
RAYS ON THE
FREOUENCY
DOSES
OF
GAMMA-
OF MNPCEs INDUCED
BY A C H A i . I . , E N G I N G D O S E IN M O U S E B O N E M A R ROW CELLS SAMPLED AT DIFFERENT TIME INTERVALS Condi-
Inter-
Challeng-
Fixation
Mean
tioning dose (Gy)
treatment time
ing dose ( 1 Gy)
time (h)
_+S E M "
-
24 48
[).72+0.19 1.[){5_+0.26
-
72
[).53.+_[).13
[).[)25
-
24 48 72
1.75 +_0.41 1.40 _+0.26 0.60+[).13
0.05
-
24 48 72
4.24 +_0.71 3.16 -,- 0.02 0.99 - [).29
0.025
2 2 2
+ + +
24 48 72
12.0 + 1.30 * 3.59 + 11.41 * 1.46,-[).133 *
[).[15
2 2 2
+ + +
24 48 72
11.3 _+0.48 * 4.93 + 0.23 * * 3.[16 _+[).48
0.[)25
24
+
24
18.12_+ 1.18 **
24 24
+ +
48 72
5.[)5 + {).25 * * 2.60 -+ 0.60
24 24 24
+ + +
24 48 72
25.{) -+ 1.8[) 8.95 -+ 1.911 4.20 _+[).26
+
24
25.32 -+ 1.90
+ +
48 72
9.75 _+ 1.30 3.70 _+[).25
(h) Control
0.05
-
-
-
" Mean_+ S E M for five mice, 2500 P C E s s c o r e d p e r a n i m a l a n d d a t a pooled. - . no t r e a t m e n t , + , t r e a t m e n t . * Significantly r e d u c e d f r e q u e n c y ( p < 0.011 in c o m p a r i s o n with that i n d u c e d by I G y alone. ** Significantly r e d u c e d f r e q u e n c y ( p < [).05) in c o m p a r i s o n with that i n d u c e d by 1 Gy.
more effective in reducing the yield of chromosomal aberrations than the higher inductive dose of 0.05 Gy (Tables 2 and 3). However, when computed in terms of expected frequency (sum of the yields induced by y-rays alone plus conditioning treatment alone minus control) and the observed frequency (Tables 4 and 5), the data show that the expected frequency is higher than the ob-
80
TABLE 2 O F C O N D I T I O N I N G DOSES OF G A M M A - R A Y S ON T I l E I N D U C T I O N OF C I I R O M O S O M A L A B E R R A T I O N S BY A C H A L L E N G I N G D O S E IN M O U S E BONE M A R R O W CELLS S A M P L E D AT 24 h EFFECT
('onditioning dose
Intertreatment
(Gy)
time (h)
Challenging dose ( I Gy)
% Aberrant cells
Aberrations/100 metaphases (mean) Ch'd Isoch'd R Frag Ch'l br br cxch
Ch'd
Mean aberrations per cell _+SE
cxch
Control
-
3
3
-
-
-
0.025
-
4.5
3
-
-
1
-
0.04 + 0.018
-
-
8.4
5
-
2
1
0 . 0 6 + (I.II24
0.025
2
+
16
7
1
I
8
1
1
(I.19-+0.028
0.05
2
+
23
9
3
1
18
1
I
0.32-+0.07
0.025
24
+
21
7
2
1
15
2
(I.26_+(I.(14 *
(I.(15
24
+
26
14
-
2
22
2
0.4(I _+ 0 . 0 3
-
+
31
18
3
4
24
3
(I.475:(I.05
11.115
0.03 + 0.017
*
I(X) mctaphases per animal scored; five animals per treatment used and data pooled. * Significantly lower than l Gy value ( p < O.O1), Student's t test.
served frequency. The difference between the two for the Iowcr conditioning dose (0.025 Gy) is quite significant. For the higher conditioning dose of 0.05 Gy this difference is small which indicates that there could be a slight adaptive response which could be interpreted as the fraction of the progressively diminishing adaptive response. Furthcr experiments arc in progress to find out the optimal conditioning dose for the induction of the adaptive response in mice when the time
interval between conditioning dose and challenging dose varies from 2 to 24 h. Discussion One of the impressive manifestations of low dose irradiation is the adaptive response. Since the demonstration by Samson and Cairns (1972) that pretreatment with low doses confers enhanced resistance against the damaging effects of
TABLE 3 EFFECT OF C O N D I T I O N I N G DOSES OF G A M M A - R A Y S ON T H E I N D U C T I O N OF C H R O M O S O M A l , A B E R R A T I O N S BY A C H A L I . E N G I N G D O S E IN M O U S E B O N E M A R R O W CEI.LS S A M P L E D AT 48 h Conditioning dose (Gy)
Intertreatment time (h)
Challenging dose ( 1 Gy)
% Aberrant cells
Aberrations/100 metaphases (mean) Isoch'd R Frag Ch'l br cxch
Ch'd br
Mean aberrations Ch'd
per cell _+SE
exch
Control
-
3
3
-
-
(I.(13 _+ 0 . 0 1 7
0.025
-
"~
1
-
1
-
0 . 0 2 _+ 0 . 0 0 9
0.05
-
8
6
-
2
-
0 . 0 8 5:I).(123
I).1125
2
+
12
9
(I.(15
2
+
12
8
-
4
-
1
-
5
-
1
(I.(125
24
+
14
7
(I.(15
24
+
18
+
22
1
7
1
0.17_+0.(11
12
1
2
9
-
2
I).25_+(I.04
10
1
-
15
2
0 . 2 5 + 11.(15
100 mctaphases per animal scored; five animals per treatment uscd and data l:V,x)lcd. * Significantly lower than 1 Gy value (p < 0.05), Student's t test.
0.12+0.03 0.16-t-0.03
*
87 TABLE 5
• MNI'K)O0
30 i 26
26
oiili
,, 12
0
2
n
I Gy
!
, 11
I
i',
lib
0 . 0 2 6 O y • 1 Ely
~
1i11.
] I[
7.5 13 18.5 Intertreltnlent time Interval (hr)
T H E E X P E C T E D F R E Q U E N C Y A N D O B S E R V E D FREQUENCY OF ABERRATIONS
24
I
0.06 Gy * I Gy
Fig. 1. Influence of conditioning dose on the persistence of the adaptive response.
a subsequent (challenging) higher dose, there have been several interesting studies (Olivieri et al., 1984; lkushima, 1987; Shadley et al., 1987; Wolff et al., 1988). The radioadaptive response now stands clearly demonstrated in a wide range of in vitro (Shadlcy et al., 1987; Wolff et al., 1989) and in vivo mammalian systems (Cai and Liu, 1990; Wojcik and Tuschl, 1990; Liu et al., 1992). Data presented in this study (Tables 1-3) also confirm the induction of the radioadaptivc response in mice by conditioning doses of 0.025 and 0.05 Gy. When the time interval between the conditioning dose and the challenging dose was 2 h, both inductive doses (0.025 and 0.05 Gy) induced the adaptive response; however, when the intcrtreatTABLE 4 TIIE E X P E C T E D F R E Q U E N C Y A N D O B S E R V E D FREQ U E N C Y O F MNPCEs Expected frequency ~
Observed frequcncy
I.
0.025 + 1 Gy (24 h intertreatment time)
26.32
18.12
2.
0.05 + I Gy (24 h intertreatment time)
28.84
25.0
Expected frequency ~
Observed frequency (mean)
1.
0.48
0.26
0.50
0.40
24
oondlllonl~ CIOILel:O.C)26 • 000 Oy. ghallenoInO dole* 1 QY0 " Meen from 0 plae, 28O0 PCIEI eoo~d ~ r animal
Treatment
Treatmcnt '
a Sum of the yields of Mn induced by y-rays alone + conditioning treatment alone minus control.
2.
0.025 + 1 Gy (24 h intertreatment time) 0.05 + 1 Gy (24 h intertreatment time)
" Sum of the yields of aberrations induced by y-rays alone plus conditioning treatment alone minus control.
mcnt time was 24 h, only the lower dose (0.025 Gy) was effective in inducing the adaptive response. However, when the data were computed as the difference between the expected frequency (sum of the yields induced by T-rays alone plus conditioning treatment alone minus control) and thc observed frequency (Tables 4 and 5), the difference between the two frequencies was significant for the lower conditioning dosc (0.025 Gy). For the higher conditioning dosc this difference was small, which could be an indication of disappearing adaptive response. The different time intervals between the conditioning dose and challenging dose (2, 7.5, 13, 18.5 and 24 h) reveal the persistence of the adaptive response. With the lower conditioning dose of 0.025 Gy the adaptive response persisted for a longer time. With the higher conditioning dose the radioadaptivc response disappeared as the time interval between the conditioning dose and challenging dose increased. In the literature, there are conflicting reports regarding the optimal range of conditioning doses to induce maximal response in a variety of test systems (Samson and Schwartz, 1980; Shadley and Wolff, 1987; Shadley and Wiencke, 1989; Vijayalaxmi and Burkart, 1989; Ikushima, 1989). Cai and Liu (1990) have reported a negative correlation between the magnitude of the adaptive response and the size of induction dose in the mouse in vivo. Furthermore, Liu et al. (1992)
88 have reported that the i n d u c t i o n of the cytogcnetic adaptive response in terms of a reduction in a b e r r a t i o n s occurred only for fragments, indicating that the a d a p t a t i o n was chiefly effective for one-hit events. O n the o t h e r hand, Olivieri et al. (1984) using [3H]TdR in c o n d i t i o n i n g of h u m a n lymphocytes observed that a larger dose (3.7 k B q / m l ) s e e m e d to induce a b e t t e r adaptive response to a challenging dose of X - i r r a d i a t i o n than a smaller dose of (I.37 k B q / m l . W i e n c k e ct al. (1986) also found an increase in the inductive effect with an increasing p r i m i n g dose of tritiated thymidine within a certain range. Y o n e z a w a et al. (1992) have shown an e n h a n c e d survival rate in mice pre-exposed to low doses of 2.5-15 cGy, 2 m o n t h s prior to the second exposure to a sublethal dose. T h e optimal a n d significant increase was observed with 5 - 1 0 cGy. In the context of i n d u c i n g an adaptive response, the possibility that the i m m u n e system is being activatcd significantly at a particular low dose range is currently receiving c o n s i d e r a b l e attention. The observation made by G r e e n s t o c k et al. (1992) is of interest. They observed that low doses ( < 0.005 Gy) stimulate an increase in I F N - y or IL-2 receptors ( h u m a n cytokine receptors) in isolated h u m a n lymphocytes. This stimulation sccn at low doses was not a p p a r e n t at high doses ((I.2 Gy). T h e data p r e s e n t e d here (Tables 1-3; Fig. l) reveal that the lower c o n d i t i o n i n g dose of 0.025 Gy induces r a d i o a d a p t a t i o n for a longer period of time in mice than the higher dose of 0.(15 Gy. Studies with p r o t e i n synthesis inhibitors ( Y o u n g b l o m ct al., 1989) and 2-D gel clcctrophoresis (Wolff et al., 1989) havc indicated the r e q u i r e m e n t of synthesis of new proteins possibly through the involvement of gene expression at low doses of radiation. T h e r e is, therefore, scope to assume that in our system the small c o n d i t i o n ing dose induces a largcr yicld a n d / o r longerlived adaptive proteins. T h e signals for gene expression could consist of d a m a g e to D N A and n u c l c o p r o t e i n s ( W e i c h s e l b a u m et al., 1991) or the formation of r e d u c t i o n products of oxygen as d e m o n s t r a t e d by Singh a n d Kesavan (1991). Further work is in progress to elucidate the low dose irradiation i n d u c e d principle(s) responsible for the m a n i f e s t a t i o n of the adaptive responsc in the mouse system.
References Cai, L., and S.Z. Liu (199{)) Induction of cytogenetic adaptive response of somatic and germ cells in vivo and in vitro by low dose X-irradiation, Int. J. Radiat. Biol., 58, 187-194. Demple, B., and J. Halbrook (1983) Inducible repair of oxidative DNA damage in Escherichia coli, Nature, 304, 466468. Fan, S., Vijayalaxmi,G. Mindek and W. Burkart (1991))Adaptive response to low doses of X-rays in human blood lymphocytes, Mutation Res., 243, 53-56. Greenstock, C.I,., Y. Xu, R.E.J. Mitchell and A. Trivedi (1992) Stimulation of human cytokine receptor expression by low dose irradiation, in: 40th Annual Meeting of the Radiation Research Society, 14-18 March, Salt Lake City, UT. lkushima, T. (1987) Chromosomal responses to ionising radiation reminiscent of an adaptive response in cultured Chinese hamster cells, Mutation Res., 180, 215-221. lkushima. T. (1989) Radioadaptive response: Characterisation of a cytogenetic repair induced by low-level ionising radiation in cultured Chinese hamster cells, Mutation Res., 227, 241 - 246. Liu, S.Z., L. Cai and S.Q. Sun (1992) Induction of a cytogenetic adaptive response by exposure of rabbits to very low dose-rate gamma radiation, Int. J. Radiat. Biol., 62, 187190. Morimoto, K.M., Sato-Mizuno and A. Koizumi (1986) Adaptation-like response to the chemical induction of sister chromatid exchanges in human lymphocytes,llum. Genet., 73, 81-85. Olivieri. G., J. Bodycote and S. Wolff (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine, Science. 223. 594-597. Reiger, R., A. Michaeli find H. Nicoloff (1982) Inducible repair processes in plant root tip meristem? "Below additivity effects' of unequally fractionated clastogen concentrations, Biol. Zcntralbl., 101. 125-138. Samson, L.. and J. Cairns (1977) A new pathway fi)r DNA repair in E. coli. Nature, 267. 281-283. Samson, I_,., and J.l,. Schwartz (1981)1 Evidence for an adaptive DNA repair pathway in CH() and human skin fibroblast cell lines, Nature, 287, 861-863. Sanderson, B.J.S.. and A.A. Morley (1986) Exposure of human lymphocytes to ionising radiation reduces mutagenesis by subsequent ionising radiation, Mutation Res., 164, 347-351. Sankaranarayanan. K.. A. van Duyn, M.J. Lcx~s and A.T. Natarajan (1989) Adaptive response of human lymphocytes to low level radiation from radioisotopes or X-rays, Mutation Res., 211, 7-12. Schmid, W. (1975)The micronucleus test, Mutation Res., 31, 0-15. Shadley, J.D., and S. Wolff (1987) Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation, Mutagcnesis, 2, 95-06. Shadlcy, J.D., and J.K. Wiencke (1989) Induction of the adaptive response by X-rays is dependent on radiation intensity. Int. J. Radiat. Biol., 56, 1117 118.
89 Shadley, J.D., V. Afzal and S. Wolff (1987) Characterisation of the adaptive response to ionizing radiation induced by low doses of X-rays to human lymphocytes, Radiat. Res., 111,511-517. Singh, S.P., and P.C. Kesavan (1991) Biochemical effects of heat shock and caffeine on post-irradiation oxic and anoxic damage in barley seeds of low and high water content, Int. J. Radiat. Biol., 59, 1227-1236. Vijayalaxmi, and W. Burkart (1989) Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin, Mutation Res., 21 I, 1-5. Weichselbaum, R.R., D.E. Hallahan, V. Sukhatme, A. Dritschilo, M.L. Sherman and D.W. Kufe (1991) Biological consequences of gene regulation after ionizing radiation exposure, J. Natl. Cancer Inst., 83, 480-484. Wiencke, J.K., V. Afzal, G. Olivieri and S. Wolff (1986) Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism, Mutagenesis, I, 375-3811. Wojcik, A., and H. Tuschl (1990) Indication of an adaptive response in C57BL mice pre-exposed in vivo to low doses of ionising radiations, Mutation Res., 243, 67-73.
Wolff, S., V. Afzal, J.K. Wiencke, G. Olivieri and A. Michaeli (1988) Human lymphc~ytes exposed to low doses of ionising radiation become refractory to high doses of radiation as well as to chemical mutagens that induce double strand breaks in DNA, Int. J. Radiat. Biol., 53, 39-48. Wolff, S., J.K. Wiencke, V. Afzal, J. Youngblom and F. Cortes (1989) The adaptive response of human lymphocytcs to very low doses of ionising radiation: a case of induced chromosome repair with the induction of specific proteins, in: Lx~wDose Radiation: Biological Basis of Risk Assessment, Taylor & Francis, London, pp. 446-454. Yonezawa, M., J. Misonoh and A. Takeda (1990) Modification of radiosensitivity by lov,' dose irradiation, Abstract, Annual Meeting of the Japan Radiation Research Society, Sendai, 24-26 October. Youngblom, J.lt., J.K. Wicnckc and S. Wolff (1989) Inhibition of the adaptive response of human lymphocytes to very low doses of ionising radiation by the protein synthesis inhibitor cyclohcximide, Mutation Res., 227, 257-261 Communicated by F.H. Sobels