Induction of specific-locus and dominant lethal mutations in male mice in the low dose range by Methyl Methanesulfonate (MMS)

Mutation Research, 230 (1990) 61-70

61

Elsevier MUT 04851

Induction of specific-locus and dominant lethal mutations in male mice in the low dose range by methyl methanesulfonate (MMS) U.H. Ehling and A. Neuh~iuser-Klaus GSF-Institut fiir Siiugetiergenetik, D-8042 Neuherberg (F.R.G.) (Received 7 August 1989) (Revision received 17 November 1989) (Accepted 23 November 1989)

Keywords: Specific-locus mutations; Dominant lethal mutations; Dose fractionation; Low dose range; Male mice; Methyl methanesulfonate

Summary Methyl methanesulfonate (MMS) induces specific-locus and dominant lethal mutations in spermatozoa and spermatids of mice. A dose of 15 m g / k g b.w. of MMS induces 9% dominant lethal mutations in the most sensitive germ-cell stages, corresponding to the mating intervals 5-8 and 9-12 days post treatment. A dose of 150 m g / k g b.w. of MMS in the same mating intervals induces 100% dominant lethal mutations. The sensitivity pattern for the induction of dominant lethal and specific-locus mutations is the same. In the mating interval 5-8 days a dose of 20 m g / k g b.w. of MMS induced 3.8 × 10 -5 mutations per locus per gamete. The yield of specific-locus and dominant lethal mutations in the low dose range increases proportionally with the dose. A dose given in 2, 4 or 5 fractions yields the same frequency of mutations as a single injection of the total dose. The additivity of small doses proves that the pre-mutational lesions are not or only partially repaired in these stages and that MMS is not or only partially detoxified. In addition, the frequency of dominant lethal and specific-locus mutations depends on the germ-cell stage.

The toxicological literature is replete with thresholded phenomena, and traditional thinking about dose-response relationships generally assumes a threshold until data indicate otherwise. In contrast, the experience with radiation mutagenesis has introduced theories and supporting data for the absence of a threshold. Chemical mutagens share the pharmacokinetics and metabolism of traditional toxins, as well as the potential for the

Correspondence: U.H. Ehling, GSF-Institut f'tir S~iugetiergenetik, D-8042 Neuherberg (F.R.G.).

stochastic inherently non-thresholded mechanism of radiation-induced mutations. Not surprisingly, there are strong differences of opinion as to whether, in the absence of data, one should assume a threshold or a non-thresholded response for the genotoxic effects of chemicals. For the clarification of these problems, there is no alternative at present but a case-by-case analysis based on the laborious acquisition of good dose-response data (Ehling et al., 1983). In this paper we investigate the induction of specific-locus and dominant lethal mutations by methyl methanesulfonate (MMS) in the low dose

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

62 range. MMS is an alkylating agent that induces specific-locus and dominant lethal mutations in male mice (Ehling, 1978; Kondo, 1981). At equimolar doses, MMS is about 4 times more effective than ethyl methanesulfonate in inducing dominant lethal mutations in spermatozoa and spermatids of mice. The lowest effective dose for the induction of dominant lethal mutations by MMS is 10 m g / k g b.w. (Ehling, 1977). This effect was detected with a sample size of 45 males and 45 females per 4-day mating interval. Increasing the sample size to 160 females per mating interval did not further increase the sensitivity of the dominant lethal assay. Therefore, it was not possible to decide whether a small increase in the frequency of dominant lethal mutations was due to the treatment of the male parent with 5 m g / k g b.w. of MMS or due to chance. Such results are sometimes used by toxicologists to claim that a threshold or a safe dose exists. If this supposition is correct, we have to assume that a dose of 5 m g / k g b.w. of MMS is completely detoxified or the induced genetic damage is completely repaired. These assumptions were tested in experiments where a total dose of 40, 20 or 15 m g / k g b.w. was given in 2, 4 or 5 fractions, 24 or 48 h apart. Similar experiments were carried out for the induction of specific-locus mutations in male mice. The detailed investigation of the induction of specific-locus mutations in germ cells of mice by MMS is essential for the evaluation for the risk estimation based on the parallelogram approach developed by Sobels (1984). This approach investigates whether it is possible to perform an estimation of the genetic risk on the basis of comparative mutagenesis in vitro and in vivo (Van Zeeland et al., 1985). Our investigation is part of the work on molecular dosimetry carried out by several laboratories within the third Environmental Programme of the Commission of the European Communities (Van Zeeland, 1988). Materials and methods

MMS was purchased from Schuchart (Munich, F.R.G.). It was dissolved in distilled water immediately before use. The solution was injected intraperitoneally (i.p.) within 30 min of prepara-

tion. The administered volume was 1 ml per animal, except that 0.5 ml per animal was administered in the 6 0 - m g / k g b.w. dose group of the specific-locus experiments. Control males were injected with an equal volume of the solvent. The historical control group for specific-locus mutations has been built up in the laboratory since 1974. The weights of the male mice ranged from 25 to 29 g. The amount of mutagen injected did not vary from the nominal value for the particular animal by more than 5%. Dominant lethal mutations were tested in (102/El × C 3 H / E I ) F 1 male mice 13-14 weeks old. After the last injection each male was caged with 1 hybrid virgin female mouse 13-14 weeks old. A 4-day sequential mating procedure was used over a total of 5 mating intervals for all dose groups. Timing of the mating intervals relates to the last treatment. Successful matings were detected by daily examination of females for vaginal plugs. The frequency of induced dominant lethal mutations was calculated as follows: Frequency of dominant lethal mutations = 1 _ [ Live embryos of experimental g r o u p / $ Live embryos of control g r o u p / $

1

For the statistical analysis of pre-implantation, post-implantation and total losses, the Wilcoxon signed rank test as modified by Krauth (1971) was used. F o r the specific-locus test ( 1 0 2 / E l x C 3 H / E 1 ) F 1 male mice, 10-13 weeks old, were treated and immediately after the last treatment, each male was caged with an untreated test-stock female, 10-15 weeks old. A 4-day sequential mating procedure was used over a total of 5 mating intervals. The test-stock females were homozygous for the following 7 recessive markers: a (nonagouti), b (brown), c ch (chinchilla), d (dilute), p (pink-eyed dilution), s (piebald spotting) and se (short-ear). These 7 loci are distributed among 5 autosomes. The c and p loci are linked on chrom o s o m e 7, with an average recombination frequency of 12-157o, and the d and s e loci are closely linked, being only 0.16 cM apart on chromosome 9 (Davisson and Roderick, 1981). The offspring were counted, sexed and carefully examined externally at birth. The litters were

63 examined again when cages were changed, the final examination being at 20-21 days of age. The mutations were tested and confirmed by an allelism test. Viability tests of 3 p mutations are still in progress. Four mutants (1 b, 2 p, 1 s) could not be tested because of dominant deleterious effects expressed by the mutants. The specific-locus mosaic mutant was confirmed according to the procedure described recently (Ehling and Neuh~iuser-Klaus, 1988b). The calculation of the probabilities was based on Fisher's exact treatment of a 4-fold contingency table employing computer routines available from BMDP (Dixon, 1983). For the calculation of the confidence limits the tables of Crow and Gardner (1959) were used. Results

Different stages in gametogenesis may be scored for induced mutations depending upon the interval between treatment and fertilization. If spermatogenesis is not affected by the treatment, the various gametogenic stages are sampled in the mouse during the following post-treatment mating intervals: spermatozoa (1-7 days), spermatids (821 days), spermatocytes (22-35 days), followed by differentiating spermatogonia and spermatogonial stem cells (Oakberg, 1975). The germ-cell stages correspond only in the single treatment experiments with these time intervals. Dominant lethal mutations

Dominant lethal mutations include the death of fertilized eggs before and after implantation. The calculated frequency of induced dominant lethal mutations would include any increase over the control value in unfertilized eggs. A decrease in implantations per female from the control value indicates induced pre-implantation death or an excess of unfertilized eggs. The difference between implantations per female and live embryos per female represents post-implantation death. The results of the MMS experiments are summarized in Tables 1 and 2. They indicate that the induction of dominant lethal mutations is due to loss before and after implantation. If the treatment is ineffective the calculation of the frequency of dominant lethal mutations should give a zero

value but the sample size will result in statistically insignificant deviations from zero in positive and negative directions. The minus values are a function of the sample size and the quality of the experimental animals. The low minus values ( < 2.5) in Table 1 indicate a low variability between animals. The high minus value of 6.3% in Table 2 is due to chance. Neither the frequency of dead implantations ( P = 0.97) nor the loss before implantation ( P = 0.13) is significantly different from the corresponding control value for this particular group. A variation in the frequency of dominant lethals in the mating intervals 5-8 and 9-12 days for the single injection of 20 m g / k g b.w. of MMS was seen in different experiments (Table 1). It was also observed in the coordinated study of several laboratories for the induction of dominant lethal mutations by MMS (Ehling, 1975). This variation is due to the changing numbers of successful matings per day during these specific intervals. The important observation of these experiments is the effectiveness in inducing dominant lethal mutations by fractionation of the dose into 4 injections of 5 m g / k g b.w. of MMS, even if the fractionation interval between injections is 48 h (Table 1). This observation is especially remarkable because of the drastic changes in mutation frequency in different spermatogenic stages (Ehling et al., 1968). It indicates that MMS is not or only partially detoxified and the pre-mutational lesions are not or only partially repaired during the chosen fractionation interval. In all MMS experiments there is no indication that the fractionation of the dose reduced the yield of induced mutations. The results of an experiment with 15 m g / k g b.w. of MMS given as a single i.p. injection or in 5 injections of 3 m g / k g b.w. of MMS separated by 24 h intervals is summarized in Table 2. A dose of 3 m g / k g b.w. is 0.015 of the LDs0, which is 200 m g / k g b.w. Only in the mating intervals 5 - 8 and 9-12 days post treatment is the total loss highly significantly different from the control values ( P = 0.0002-0.03). Kratochvilova (1978) demonstrated that the pre-implantation losses after treatment of male mice with alkylating agents in the mating intervals 5-12 days post treatment are due to dominant lethal mutations. In all MMS experiments there was no indication that the fractiona-

64 TABL E 1 I N D U C T I O N OF D O M I N A N T L E T H A L M U T A T I O N S IN ( 1 0 2 / E I x C 3 H / E 1 ) F 1 M A L E M I C E BY M E T H Y L M E T H A N E S U L F O N A T E (MMS) Dose (mg/kg)

Interval between injections (h)

0

0

40

0

2X20

24

4 X 10

24

0

0

20

0

2 X 10

24

4x 5

24

0

0

20

0

Mating interval (days)

Fertile matings a (%)

Corpora lutea per female

Implants per female

Live embryos per female

Dead implants b (%)

Induced domi na nt lethals b (%)

1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20

100 96 92 96 100 100 100 92 96 92 100 100 96 96 100 92 88 96 96 92

12.3 12.4 12.6 12.5 12.9 12.2 11.4 11.4 12.5 12.2 12.3 11.5 10.9 12.4 12.8 12.0 11.7 11.1 12.3 12.2

10.9 11.0 11.2 11.0 11.7 10.5 9.9 9.6 11.2 11.3 10.6 9.7 9.2 11.2 11.6 10.6 10.2 9.6 11.0 11.6

10.1 10.0 10.4 10.1 10.6 8.6 6.0 5.7 9.1 10.1 8.8 6.0 4.8 9.5 10.3 8.2 5.7 6.5 9.8 10.8

7.4 9.1 7.0 8.0 9.2 17.9 39.1 40.0 18.3 10.8 17.0 38.0 48.2 14.6 10.7 23.0 44.2 32.6 10.6 6.4

0 0 0 0 0 14.3 39.3 45.0 9.9 4.8 13.1 39.7 54.5 5.8 2.6 18.9 42.9 38.1 2.9 - 2.1

1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20

96 96 100 96 100 92 96 80 96 80 100 92 100 100 96 100 100 100 100 100

11.7 12.5 12.4 12.5 11.2 12.5 12.1 11.8 12.1 11.9 12.4 11.9 12.2 12.4 11.5 11.7 12.3 11.8 12.1 11.7

10.8 11.8 11.0 10.9 10.3 11.5 11.6 10.7 10.6 10.7 11.0 11.2 10.8 11.0 10.4 10.8 11.2 10.5 11.1 10.4

9.8 11.1 10.3 10.0 9.7 10.0 10.1 9.1 9.8 9.7 9.7 9.3 9.2 9.7 9.3 9.4 9.2 9.2 10.0 9.7

9.2 5.7 6.9 8.8 6.2 12.8 12.9 14.6 7.1 8.9 12.0 17.1 15.1 12.3 10.0 13.3 18.5 11.8 9.7 6.9

0 0 0 0 0 -2.1 9.4 11.5 1.3 -0.2 1.6 16.4 10.5 2.8 3.6 4.8 17.7 10.1 -0.4 - 0.4

1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20

96 96 100 100 96 100 92 96 92 100

12.3 12.8 12.3 12.1 12.0 12.4 11.8 12.0 11.9 12.1

11.2 11.1 10.6 10.8 10.8 11.0 10.8 10.8 10.7 10.8

9.9 10.4 9.7 10.0 9.9 10.1 8.9 9.5 9.5 10.0

11.2 6.4 8.0 7.8 8.5 8.7 17.7 11.2 11.4 7.4

0 0 0 0 0 - 1.6 14.1 1.8 4.8 - 1.2

65 TABLE 1 (continued) Dose (mg/kg)

Interval between injections

Mating interval (days)

Fertile matings " (%)

Corpora lutea per female

Implants per female

Live embryos per female

Dead implants b (%)

Induced dominant lethals b (%)

1- 4 5- 8 9-12 13-16 17-20 1- 4 5- 8 9-12 13-16 17-20

92 96 100 100 96 96 100 92 100 96

12.6 12.1 12.0 11.8 12.1 12.4 12.1 12.3 12.1 12.0

11.5

10.0

12.5

-

10.6 10.3 10.4 10.9 11.4 10.9 10.6 10.5 10.9

9.1 9.0 9.8 9.9 9.8 9.3 9.4 9.7 10.2

13.8 12.1 5.7 9.5 13.9 14.3 10.7 7.6 6.9

(h) 2 × 10

4x5

48

48

1.3 12.0 7.0 1.2 0.4 1.3 10.2 2.9 2.8 -2.5

25 females per mating interval. b Calculation is based on absolute figures.

tion of the dose reduced the yield of induced mutations. The effect of the different doses is additive. The frequency of dominant lethal mutations is dose-dependent.

Specific-locus mutations The results of specific-locus experiments after single and fractionated treatment with MMS are summarized in Table 3. The decrease in the aver-

TABLE 2 I N D U C T I O N OF D O M I N A N T LETHAL MUTATIONS IN (102/El x C3H/E1)F 1 MALE MICE BY METHYL METHANES U L F O N A T E (MMS) Dose (mg/kg)

Interval between injections (h)

Mating interval (days)

Fertile matings a (%)

Corpora lutea per female

Implants per female

Live embryos per female

Dead implants b (%)

Induced dominant lethals b (%)

0

0

1- 4 5- 8 9-12 13-16 17-20

96.7 96.7 96.7 96.7 96.7

12.9 12.7 12.9 12.8 12.9

11.7 11.6 11.7 11.6 11.9

10.4 10.7 10.7 10.7 10.9

10.7 8.3 9.1 7.7 8.7

0 0 0 0 0

15

0

1- 4 5- 8 9-12 13-16 17-20

96.7 100.0 96.7 100.0 93.3

13.5 12.4 12.3 12.7 12.4

11.8 10.9 10.9 11.3 11.3

11.1 9.7 9.7 10.4 10.1

6.1 10.7 10.8 7.7 10.4

-6.3 9.0 9.1 2.7 7.2

24

1- 4 5- 8 9-12 13-16 17-20

100.0 96.7 93.3 96.7 100.0

12.4 12.7 12.7 12.8 12.5

10.8 11.1 10.5 11.5 11.2

10.0 9.7 9.4 10.3 10.2

7.1 12.4 11.2 9.9 9.0

3.7 8.7 12.2 3.2 6.7

5 X3

" 30 females per mating interval. b Calculation is based on absolute figures.

66 TABLE 3 INDUCTION OF SPECIFIC-LOCUS MUTATIONS BY METHYL METHANESULFONATE (MMS) IN GERM CELLS OF MICE Dose (mg/kg)

Mating intervals (days)

Number of offspring

0

-

20

1- 4 5- 8 9-12 13-16 17-20

5638 5593 5518 5785 5759

40

1- 4 5- 8 9--12 13--16 17-20

4×10 0

60

a b c d e f

Average litter size

248413

Number of mutations at 7 loci

Mutations per locus per 10 5 gametes

95% Confidence limits

22 a

1.3

0.8-

1.9

6.6 6.6 6.7 7.0 6.9

1 2 2 1 0

2.5 5.1 5.2 2.5

0.10.90.90.10

13.5 17.1 17.3 13.1 8.1

2902 1716 1750 2799 3072

6.0 4.0 4.1 6.0 6.6

2b 3b 3c 1 0

9.8 25.0 24.5 5.1 --

1.76.86.70.30 -

32.9 67.4 66.1 27.2 15.3

1- 4 5-- 8 9--12 13--16 17-20

1426 898 1 186 1638 1692

6.6 4.4 5.6 7.7 7.6

0 le 1 0 0

15.9 12.0 -

0 0.80.60 0 -

32.9 84.7 64.1 28.6 27.7

1- 4 5- 8 9-12 13-16 17-20

2323 515 516 2524 2956

6.5 2.4 2.4 6.9 7.9

1 1 2 2 0f

6.1 27.7 55.4 11.3 -

0.3- 32.7 1.4--147.7 9.8--185.1 2.0- 37.8 0 -- 15.9

Includes one cluster of 2 s e and one of 6 s mutants. One pink-eyed dilution mutant included which died prior to weaning. One b or p mutant died perinatally and is not included. Interval between injections 24 h. Additionally 1 X-autosomal translocation (Adler and Neuhiiuser-Klaus, 1987). One d mosaic not included.

a g e l i t t e r size is a n i n d i c a t i o n o f t h e i n d u c t i o n o f dominant lethal mutations. The highest rate of specific-locus mutations was observed in the mating intervals 5-12 days. In these mating intervals the mutation frequency induced by the lowest t e s t e d d o s e o f M M S (20 m g / k g b . w . ) is s i g n i f i cantly different from the control frequency (P = 0.023). T h e s e m a t i n g i n t e r v a l s a r e a l s o t h e m o s t sensitive ones for the induction of dominant lethal m u t a t i o n s ( T a b l e s 1 a n d 2). I n t h e l o w d o s e r a n g e u p t o 60 m g / k g b . w . o f M M S t h e i n d u c t i o n o f s p e c i f i c - l o c u s m u t a t i o n s is d o s e - d e p e n d e n t (Fig. 1). A m o r e d e t a i l e d a n a l y s i s o f t h e d o s e - e f f e c t c u r v e is n o t w a r r a n t e d , b e c a u s e o f t h e w i d e c o n f i dence limits of the point estimates for the muta-

t i o n f r e q u e n c i e s ( T a b l e 3). T h e m u t a t i o n r a t e s o f s p e c i f i c loci i n t h e m a t i n g i n t e r v a l s 5 - 1 2 d a y s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t ( P = 0.7) b e t w e e n a s i n g l e i n j e c t i o n o f 4 0 m g / k g b.w. o f M M S a n d 4 d a i l y i n j e c t i o n s o f 10 m g / k g b . w . o f M M S . T h e statistical analysis indicates that we observe no differences in the frequency of induced specificlocus mutations after single treatment and fractionation of the total dose. In the higher dose r a n g e o f m o r e t h a n 6 0 m g / k g b.w. o f M M S a larger fraction of mutagenized spermatozoa and spermatids could lead to a selective elimination of zygotes, which could lead to an underestimation of the specific-locus mutation yield at high doses ( E h l i n g , 1978).

67

100-

9O 80 ¸ ~D

706050-

~0_ 400 :,x 0

50

"~-

20-

2'0

~0 (mg/kg)

4'0

Fig. 1. Dose-effect relationship for the induction of specific-locus mutations in male mice after i.p. injection of MMS, with 90% confidence intervals. The data are collected from the most sensitive mating intervals for the induction of mutations: 5 - 1 2 days post treatment.

spectra of chlormethine and earlier experiments with other mutagens acting in post-spermatogonial germ cells (Ehling and Neuh~iuser-Klaus, 1989), except that the frequency of p mutations is higher, 48% vs. 22% in all other experiments whereas the frequency of c and s mutations is lower (c: 0 vs. 12%; s: 22 vs. 36%).

The numbers of mutations observed at each of the 7 loci are listed in Table 4. Of the 23 mutations included, 21 mutations were genetically tested for allelism, 2 p mutants died prior to weaning. One additional presumed mutant with reduced eye pigment died perinatally. The mutation spectrum of MMS is similar to the mutation TABLE 4

S P E C T R U M O F M M S - I N D U C E D SPECIFIC-LOCUS M U T A T I O N S Total

Locus

Dose (mg/kg)

a

20 40 4×10 60

0 0 0 0

Total (n) (%)

0

b

-

c

d

d , se

se

1 2 0 1

0 0 0 0

0 0 0 1a

1 0 0 0

1 0 0 0

2 4 1 4

1 3 1 0

6 9 2 6

4

0

1~

1

1

4

4

4

11 48

5 22

23

-

17

p

s

Additionally one confirmed specific-locus mosaic mutation at the d locus.

TABLE 5 H O M O Z Y G O U S VIABILITY O F SPECIFIC-LOCUS M U T A T I O N S D E R I V E D F R O M M M S - T R E A T E D POST-SPERMATOG O N I A L G E R M - C E L L STAGES N u m b e r l e t h a l / N u m b e r tested per locus a

b

c

d

d, se

se

p

s

-

3/3

-

1/1

1/1

1/1

5/6

1/4

Total

Percent lethal

12/16

75

68

45to

m

~

40-

35

g~3o

~-4

5-8

9-12

13-16

17~20

(days post freatment) [2~ domTnant rethols specific locus mutations

Fig. 2. Frequency of dominant lethal and specific-locus mutations in different mating intervals after i.p. injection of male mice with 40 mg/kg b.w. of methyl methanesulfonate (MMS).

Viability tests are still in progress. Preliminary results are summarized in Table 5. All mutations that as homozygotes cause death before maturity have been classified as lethals. The observed frequency of 75% lethals is slightly higher than the overall frequency of 69% observed in earlier experiments with other chemical mutagens in postspermatogonial stages (Ehling and Neuh~iuserKlaus, 1984, 1988a,b). The high frequency of homozygous lethal mutations induced in spermatozoa and spermatids suggests that small deficiencies are the main cause for specific-locus mutations in these germ-cell stages. This observation explains the correlation in stage sensitivity for the induction of dominant lethal and specific-locus mutations (Fig. 2). The specific-locus mutations induced in post-spermatogonial germ-cell stages are qualitatively different from specific-locus mutations induced in spermatogonia (Ehling, 1989a). Discussion

A dose of 15 m g / k g b.w. of MMS induces 9% dominant lethal mutations in the most sensitive mating intervals, 5-12 days post treatment (Table 2). A dose of 150 m g / k g b.w. of MMS in the same mating intervals induces 100% dominant lethal mutations (Ehling et al., 1968). The sensitivity pattern for the induction of dominant lethal and

specific-locus mutations is the same (Fig. 2), therefore, a dose-dependent induction of specific-locus mutations may lead to an underestimation of the yield at high doses (Ehling, 1978). Such a dose-effect relationship was observed for diethyl sulfate (DES). In the mating interval 5-8 days post treatment the mutation frequency for 200 m g / k g b.w. of DES was 17.0 x 10 -5 and for 300 m g / k g b.w. 7.5 x 10 -5 mutations per locus. The dose-dependent increase in dominant lethal mutations probably reduced the chance of recovering specificlocus mutations (Ehling and Neuh~iuser-Klaus, 1988a). Up to 60 m g / k g b.w. of MMS dominant lethal and specific-locus mutations increase proportionally with the dose. In a small sample of 1155 offspring conceived in all mating intervals post treatment with 80 m g / k g b.w. of MMS zero mutations were observed (Ehling, 1978). The mutation yield depends on the dose (Fig. 1, Tables 1-3). However, the impact of the germ-cell stages on the yield of dominant lethal and specific-locus mutations is more pronounced than the dose-effect relationship (Fig. 2). The frequency of dominant lethal and specific-locus mutations is medium in the mating interval 1 - 4 days post treatment, high in the mating intervals 5 - 8 and 9-12 days post treatment and low in the mating interval 13-16 days post treatment, only 1 mosaic mutation was observed in the mating interval 17-

69 20 days post treatment (Tables 1 and 3). The relatively high dominant lethal value in the mating interval 17-20 days in Table 2 is due to the high frequency of live embryos in this control group (10.9). It is unlikely that mutations are induced in spermatocytes and spermatogonia by MMS. Because of the high germ-cell stage sensitivity for the induction of mutations it is surprising that the dose given in 2, 4 or 5 fractions yields the same frequency of mutations as the single injection of the total dose. The additivity of small doses proves that the pre-mutational lesions are not or only partially repaired and that MMS is not or only partially detoxified. Similar results were obtained by us with ethylnitrosourea (ENU) in spermatogonia of mice. A single i.p. injection of 160 m g / k g b.w. of ENU yielded 26 specific-locus mutations in 7373 offspring. Fractionation of the total dose in 4 x 40 m g / k g b.w. of ENU injected at intervals of 24 h yielded 25 specific-locus mutations in 7730 offspring (Ehling and Neuh~iuser-Klaus, 1984). However, in these experiments the time interval between injections is critical. Russell et al. (1982) reported that 10 × 10 m g / k g b.w. of ENU injected in weekly intervals yielded only 1 / 6 - 1 / 8 of the frequency of specific-locus mutations observed after a single i.p. injection of 100 m g / k g b.w. of ENU. Taking the time factor into account the fractionation experiments in the low dose range are useful to study the effectiveness of small doses. The cumulation of truly zero responses should remain zero, while cumulation of small unresolvable effects should add up to an observable increase. The usefulness of this strategy was demonstrated for the induction of dominant lethal and specific-locus mutations. From the fractionation experiments (Tables 1-3) at low doses one can conclude that the induced frequency of mutations by MMS is additive, indicating a non-thresholded response for MMS. For the determination of the specific-locus mutation rate in mice by MMS it is essential to use a sequential mating procedure, because of the high specificity of the compound to induce mutations mainly in the mating intervals 5-12 days post treatment. Specific-locus mutations in male mice have been

used in radiation genetics to determine the doubling dose. The doubling dose is a useful indicator for the evaluation of the potential human hazard with a given exposure (UNSCEAR, 1988). The same principles are recommended by the Environmental Protection Agency (EPA) for the quantification of genetic risk of chemical mutagens. In the 'Guidelines for Mutagenicity Risk Assessment' published by EPA (1986) it is stated: 'Any risk assessment should clearly delineate the strengths and weaknesses of the data, the assumptions made, the uncertainties in the methodology, and the rationale used in reaching the conclusions, e.g., similar or different routes of exposure and metabolic differences between humans and test animals. When possible, quantitative risk assessments should be expressed in terms of the estimated increase of genetic disease per generation, or the fractional increase in the assumed background spontaneous mutation rate of humans (BEIR, 1972). Examples of quantitative risk estimates have been published (BEIR, 1972; Ehling and Neuh~iuser, 1979; UNSCEAR, 1982); these examples may be of use in performing quantitative risk assessments for mutagens'. According to the EPA concept it is useful to determine the doubling dose for specific-locus mutations. The doubling dose for MMS is 2-3 m g / k g b.w. using the regression coefficient of the MMS-induced mutation response for the mating intervals in which the most sensitive germ-cell stages are sampled (Table 3). Using the same procedure for the determination of the doubling dose of dominant lethal mutations gives a value of 19-20 m g / k g b.w. The doubling dose for specific-locus mutations is 1 order of magnitude lower than the doubling dose for dominant lethal mutations. Similar comparisons were made with other mutagens (Ehling, 1989b). In general, the comparisons are encouraging. For a final recommendation, however, it is necessary to increase the data base for the determination of the doubling dose for specific-locus mutations.

Acknowledgements Experiments were supported by Contract EV4V-0064-D(B) of the Commission of the European Communities. The able assistance of S.

70 F r i s c h h o l z , B. M a y , G . S c h e n k e a n d K . W e i s e r is greatly appreciated.

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