Mutation Research, 303 (1993) 157-161 157 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00

MUTLET 0838

Induction of an adaptive response to dominant lethality and to chromosome damage of mouse germ cells by low dose radiation

L u Cai *, J ie J iang , Bin W a n g , H a n Y a o and Xian l i W a n g Institute of Radiation Medicine, Norman Bethune University of Medical Sciences, Changchun 130021, People's Republic of China

(Received 31 March 1993) (Revision received 6 July 1993)

(Accepted 13 August 1993)

Keywords: Dominant lethality; Chromosome aberration; Adaptive response; Low dose X-rays; Germ cells; Reciprocal translocation

Summary

In the present paper, dominant lethal mutations, chromosome aberrations in spermatocytes and reciprocal translocations in stem spermatogonia were analyzed after whole body exposure of mice to X-radiation. Results both from chromosome aberrations in spermatocytes and for reciprocal transloca- tions in spermatogonia showed that pre-exposure to low doses up to 200 mGy could induce a significant dose-dependent reduction in adapted mice compared to the non-adapted mice; the lower the adaptive dose, the greater the reduction. For dominant lethal mutations, it was found that spermatogonia (both stem cells and differentiated cells) and spermatocytes adapted to 50 mGy X-rays could show an adaptive response, but spermatids and spermatozoa could not.

Human lymphocytes treated with a low dose of ionizing radiation prior to a high-dose exposure could have fewer than the expected number of chromatid aberrations (Olivieri et al., 1984; Wiencke et al., 1986; Shadley and Wolff, 1987). This phenomenon has been termed an adaptive response to ionizing radiation, and has been ob- served widely in human lymphocytes (Shadley and Wolff, 1987; Cai and Liu, 1990), rabbit lym- phocytes (Cai and Liu, 1990), cultured Chinese hamster cells (V79 cell line) (Ikushima, 1987, 1989), mouse splenocytes (Wojcik and Tuschl,

* Corresponding author.

Abbreviations: Dr, first low dose of radiation or adaptive radiation; D2, second large dose of radiation or challenge radiation.

1991), bone marrow cells (Cai and Liu, 1990), as well as in plant cells (Cortes et al., 1990). Those observations have demonstrated that the induc- tion of this response was dependent on the cell cycle stage during pre-irradiation (Shadley et al., 1987), and also was dependent on both the total dose of the pre-treatment and the rate at which the dose was given (Shadley and Wiencke, 1989). The induced adaptive response was observed to persist for at least three cell cycles (Shadley et al., 1987) and to be re-induced by an additional 10 mGy X-rays after the third cell cycle (Cai and Liu, 1990). It was found that human lymphocytes exposed to low doses of ionizing radiation be- came refractory to high doses of radiation as well as to chemical mutagens (bleomycin, mitomycin C) (Wolff et al., 1988). On the other hand a pre-treatment with low concentrations of bleomy-

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cin could also induce an adaptive response to subsequent treatment with bleomycin or X-rays (Vijayalaxmi and Burkart, 1989; Wolff et al., 1989).

To date, however, all the characteristics and conditions or requirements for the induction of this response were mostly obtained with somatic cells. Only limited studies, carried out by Cai and Liu (1990) and by Fritz-Niggli and Shaeppi-Buechi (1991), showed that an adaptive response could also be found in mouse spermatocytes and in Drosphila melanogaster oocytes after treatment with low-dose radiation. In the present paper, chromosome aberrations in spermatocytes a short time after exposure to different large doses, and reciprocal translocations in spermatogonia 60 days after exposure to different large doses of radia- tion, as well as the induction of dominant lethal- ity in different stages of mouse germ cells were analyzed to confirm and extend previous studies.

Materials and methods

Animals Kunming male mice (30 + 2 g) were used in

various experiments. In the experiments for cyto- genetic analysis, 4 h or 60 days after exposure to

the challenge or second radiation dose (D2), the mice were given an i.p. injection of 0.2 ml colchicine (4 /xg/g body weight) and were killed by decapitation 4 h later to process the testis specimens.

Irradiation Mice were exposed to X-rays generated at 200

kpV, 10 mA and 0.5 mm Cu and 1.0 mm A1 filtration. The adaptive or first low dose of radia- tion (D 1) was 50 mGy for the dominant lethal experiment and 10, 50, 100, 150, 200, 300 mGy for cytogenetic experiments. D 1 was delivered at a dose rate of 50 mGy/min. The challenge dose (D 2) was 1.5 Gy for the cytogenetic experiments and 2.0 Gy for the dominant lethal experiment at a dose rate of 0.44 Gy/min. The interval between D 1 and D 2 irradiations was 3 h. The detailed radiation schedules for various experiments are described in the Results section.

Preparations for chromosome analysis Germ cells were treated with 1% sodium ci-

trate for 30 min and fixed in acetic acid : methanol (1 : 3); the fixed cells were dropped onto wet glass slides and stained with Giemsa for 20 min.

TABLE 1

RESULTS OF DOMINANT LETHAL TEST FOR NINE CONSECUTIVE WEEKS OF MATING

Group Stage (week) Number of Number of total Number of living pregnant fetuses fetuses females n x_+S

Frequency of dominant lethal mutations (%)

n x + S observed expected

D 2 Spermatozoa 33 346 10.48 +- 2.04 D 1 + D e (lst week) 35 357 10.11 +- 2.88 D 2 Spermatids 88 843 9.56 +- 2.12 D 1 + D 2 (2-3 weeks) 116 1090 9.35+-2.13 D 2 Spermatocytes 77 715 9.29 +- 2.46 D 1 + D 2 (4-5 weeks) 106 1021 9.63 +- 2.69 D 2 Differentiated

spermatogonia 50 408 7.85 + 3.25 D 1 + D 2 (6th week) 23 207 8.91 J: 3.27 D 2 Stem cells 160 1463 8.96 +- 2.48 D x + D 2 (7-9 weeks) 170 1677 9.64+-2.31

Control All stages 58 531 D 1 (1-9 weeks) 44 412 D 2 (1-7 weeks) 408 3 775 D 1 + D 2 450 4352

293 8.88 +- 1.85 308 8.75 +- 2.75 644 7.33 + 2.42 828 7.14+-2.31 545 7.35 + 2.55 815 7.62+-2.75

356 196

1 323 1578

500 384

3161 3 725

6.81 + 3.41 8.52 + 3.23 8.05 _+ 2.49 9.17+_2.33

15.32 13.75 16.28 23.64 24.47 24.60 23.78 20.17 * 24.74

12.75 5.31 ** 13.71 9.57 5.90 * * 10.53

5.84 6.80

16.26 14.44 * 17.22

* P < 0.05, ** P < 0.01, vs. expected.

Dominant lethality Male mice were irradiated with 50 mGy X-rays

and 3 h later with 2.0 Gy; then 2 days later they were caged with female mature virgin mice in a proportion of one male to three females. The irradiated male mice were in the cages with fe- male mice for 5 days a week and out of the cages for 2 days and then were put into cages with other female mice once more; this procedure was repeated until the ninth week after Da irradia- tion. The female mice caged with irradiated mice were killed on day 19 after the beginning of caging with the males. The numbers of implanta- tions, living fetuses, early deaths, and late deaths were observed.

Results

Dominant lethality The Kunming male mice were divided into

four groups. One group was irradiated with 50 mGy X-rays and 3 h later with 2.0 Gy X-rays, i.e., D 1 + D 2 group. One control group was not irra- diated. The other two groups were irradiated only with D~ or only with D 2 and were called the D 1

group or D 2 group respectively. The D 1 treat- ment did not markedly increase the rate of domi- nant lethal mutations (Table 1). The D 1 + D 2 groups, in general, showed fewer than the ex- pected number (the effects of D 1 group plus D 2 group minus control group). To study possible differences during spermatogenesis, two groups were compared separately in different stages. The results indicated that the frequencies of dominant lethal mutations were significantly reduced (P < 0.05 or P < 0.01) in the D 1 -I- D 2 groups of stem spermatogonia, differentiated spermatogonia and spermatocytes, but not in D~ + D 2 groups of spermatids and spermatozoa.

Chromosome aberrations Kunming male mice were irradiated with 50

mGy X-rays and then 3 h later with 1.5 Gy X-rays. Four hours after exposure to D2, chromo- some aberrations in spermatocytes were analyzed and compared in the D~ + D 2 group to the ex- pected (Table 2). In the data of Table 2, a signifi- cant cytogenetic adaptive response in spermato- cytes was found in the D t + D 2 groups with 50-

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T A B L E 2

I N D U C T I O N OF THE A D A P T I V E RESPONSE BY DIF- F E R E N T L O W DOSES OF X-RAYS IN M O U S E SPER- M A T O C Y T E S

Trea tment Number Chromatid and isochromatid of cells (%)

scored Observed Expected

None 2500 0.90 _+ 0.33 50 mGy 700 2.00 + 0.82 50 mGy + 1.5 Gy 500 3.80 + 2.39 * * 10.24 100 mGy 800 1.13 + 0.84 100 mGy + 1.5 Gy 600 3.50 +_ 2.07 * * 9.37 150 mGy 800 2.00 +_ 0.82 150 m G y + 1.5 Gy 800 4.38+_2.07 ** 10.24 200 mGy 700 2.29 + 1.60 200 mGy + 1.5 Gy 800 7.25 +_ 3.90 * 10.53 300 mGy 700 2.29 + 1.25 300 mGy + 1.5 Gy 800 9.75 +_ 5.01 10.53 1.5 Gy 700 9.14_+3.85

* P < 0.05, ** P < 0.01 vs. expected.

200 mGy treatment as D 1. In the D 1 (300 mGy) + D z group, the chromatid and isochromatid breaks were decreased, but this was not statistically sig- nificant. Based on the results in all D 1+ D e groups, the reduction (or adaptive response) showed a dose-dependent response: the lower the D~ dose level, the greater the reduction in chro- matid aberrations following the challenge treat- ment.

TABLE 3

I N D U C T I O N OF A D A P T I V E RESPONSE FOR SPER- M A T O G O N I A L T R A N S L O C A T I O N BY D I F F E R E N T D 1

Trea tment (Gy) Number Spermatogonial translocation of cells (%)

Observed Expected

0.00 2 000 0.05 + 0.22 0.10 1700 0.24+_0.43 0.15 1586 0.35_+0.51 0.20 1800 0.56 +_ 0.71 0.30 1 271 0.57:1:0.69 1.50 700 2.00 + 0.82 0.01+1.5 900 0.33_+0.50 ** 0.05+1.5 900 0.44+-0.72 ** 0.10+ 1.5 800 0.63+-0.74 ** 0.15 + 1.5 900 0.67 + 0.71 * * 0.20 + 1.5 800 1.00 +- 1.07 0.30 + 1.5 700 1.70 + 1.11

2.00 2.00 2.19 2.17 2.15 2.15

** P < 0.01 vs. expected.

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Reciprocal translocations in spermatogonia Analysis of multivalents in primary spermato-

cytes (MI) after treatment of spermatogonia is called the 'translocation test' (Adler, 1982). Therefore, the multivalents in MI at 60 days after treatment of spermatogonia with D1, D 2 and D 1 + D 2 were analyzed; they are summarized in Table 3. From those data, it can be seen that pre-exposure to 10-150 mGy X-rays could cause a significant reduction of reciprocal transloca- tions. In all "D~ + D 2 groups, the magnitude of the adaptive response was found to be dose-de- pendent.

Discussion

To date, only limited evidence for the induc- tion of a cytogenetic adaptive response in mam- malian germ cells has been formed in a prelimi- nary study by Cai and Liu (1990). In that experi- ment, chromatid aberrations in the spermatogo- nia and spermatocytes of mice, pre-exposed to 50 mGy X-rays and then 3 h later challenged with 0.75 Gy X-rays, were lower (13.04%, 8.40%) in the D I + D 2 groups than in the D 2 groups (16.85%, 14.90%). In the present study, we found that treatment of mouse germ cells with 10-200 mGy X-rays could induce a significant reduction in the chromatid and isochromatid breaks found in spermatocytes 4 h after D2, and for the recip- rocal translocations 60 days after D 2 treatment. The induced adaptive response was also found to be dose-dependent, which verifies earlier works in human lymphocytes (Shadley and Wolff, 1987) and in mouse bone marrow cells (Cai and Liu, 1990).

The results in Table 1 illustrated that pre- treatment of germ cells with 50 mGy could also induce a significant adaptive response to radia- tion-induced dominant lethality in general. How- ever, the induced adaptive response was influ- enced by the cell stage, that is, pre-meiotic cells (stem spermatogonia, differentiated spermatogo- nia and spermatocytes) adapted to D 1 could show the adaptive response, but post-meiotic cells (spermatid and spermatozoa) could not. This may be due to the metabolism difference between the pre-meiotic and post-meiotic cells. Fritz-Niggli and Schaeppi-Buechi (1991) used mature and im- mature oocytes of Drosophila melanogaster to

demonstrate the existence of an adaptive re- sponse of a low dose of X-rays (20 mGy) for the induction of dominant mutation lethality by a subsequent large dose of X-rays (2 Gy) at various time intervals.

The mechanism of the cytogenetic adaptive response is not clear. Some workers considered that repair enzymes, such as poly(ADP-ribose)- polymerase, stimulated by low-dose radiation may play an important part in this response because they found that the adaptive response could be negated when 3-aminobenzamide, an inhibitor of poly(ADP-ribose)polymerase, was added to the lymphocyte cultures (Wiencke et al., 1986; Shadley et al., 1987; Ikushima, 1987). Another hypothesis was also put forward that the adaptive response induced by low-dose radiation may be related to the production of new proteins such as heat shock proteins or stress proteins, which to a certain extent protect cells from damage caused by a subsequent large dose of radiation; it was previously shown by Wolff et al. (1989) that lym- phocytes after exposure to 10 mGy X-rays synthe- sized some novel proteins. Later, Youngbloom et al. (1989) and Cai and Liu (1992) also provided some confirming studies that the adaptive re- sponse induced by low-dose radiation could be abolished by the inhibitor of protein synthesis cycloheximide.

Using the first idea mentioned above, some experimental results were difficult to explain. If the repair enzymes were stimulated by D 1, they would facilitate the repair of chromosome breaks leading to an increase in correct repair as well as misrepair which would increase the number of exchange aberrations in D1 + D 2 groups. Experi- ments carried out by Wang et al. (1991) and Shadley and Dai (1992), however, illustrated that exchange aberrations induced by 1.5 Gy X-rays in G 1 phase of human lymphocytes were lower in DI + D 2 groups than in D 2 groups, suggesting that the ability for misrepair for chromosome breaks was not increased. The present paper also demonstrated that spermatogonial reciprocal translocations, a kind of exchange aberrations, which result from misrepair, were decreased in groups exposed to low-dose radiation (Table 3). Those results seem to support the idea that low- dose radiation may induce some protective mate-

rials which protect the chromosomes from dam- age caused by D 2, that is, the original chromo- some breaks induced by D 2 in the group with D 1 treatment are lower than those in the group without D 1 treatment, resulting in a low inci- dence of the induction of exchange aberrations in the D1 + D 2 group. In other words, the adaptive response may not only be related to the role of enzymes, such as poly(ADP-ribose)polymerase stimulated by D 1, but other mechanisms, includ- ing the induction of some proteins by D 1, must also be considered. Therefore, further studies should be carried out in order to answer the question concerning the role of enzymes or pro- tective proteins caused by D1.

Finally, we can conclude that mammalian germ cells could also show the adaptive response for chromosome breaks, reciprocal translocations and dominant lethals, and conclude that the adaptive response can be influenced by germ cell stages.

Acknowledgement

This work was supported by the National Nat- ural Foundation of China, CA: 39000031.

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Communicated by K. Sankaranarayanan