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Induction of specific-locus and dominant-lethal mutations in male mice by diethyl sulfate (DES)
Mutation Research, 199 (1988) 191-198
191
Elsevier MTR04596
Induction of specific-locus and dominant-lethal mutations in male mice by diethyl sulfate (DES) U.H. Ehling and A. Neuh~iuser-Klaus lnstitut fiir Siiugetiergenetik, Gesellschaft]'fir Strahlen- und Umweltforschung D-8042 Neuherberg (F.R.G.)
(Received 23 September1987) (Revision received30 November 1987) (Accepted 7 December 1987)
Keywords: Specific-locusmutations; Dominant-lethal mutations; Male mice; Diethyl sulfate (DES).
Summary Diethyl sulfate (DES), a monofunctional alkylating agent, induces mutations and chromosomal aberrations in many different organisms and cell systems, including dominant-lethal mutations in male mice. However, until now it could not be demonstrated that DES induces specific-locus mutations in mice. This observation would contradict the close correlation observed between the induction of dominant-lethal mutations and specific-locus mutations in mice with other chemicals. DES induces dominant-lethal and specific-locus mutations in spermatozoa and late spermatids of mice. The mutation frequency for dominant-lethal mutations is dose-dependent, while for specific-locus mutations it is independent of the dose. In the mating interval 5-8 days post-treatment the mutation frequency for 200 m g / k g DES is 17.0 x 10 -5 and for 300 m g / k g 7.5 × 10 -5 mutations per locus. The dose-dependent increase of dominant-lethal mutations probably reduced the chance of recovering specific-locus mutations. The importance of these findings for mutagenicity testing is discussed.
Diethyl sulfate (DES) is an active alkylating agent. It has been produced commercially for at least 60 years and is mainly used as an alkylating agent to convert active-hydrogen compounds such as phenols, amines and thiols to the corresponding ethyl derivatives (IARC, 1974). This monofunctional alkylating agent induces mutations and
Correspondence: Dr. U.H. Ehling, Institut for S~tugetiergenetik, Gesellschaft fiir Strahlen- und Umweltforschung, D-8042 Neuherberg (Federal Republic of Germany). Abbreviations: bw, body weight; DES, diethyl sulfate; DMSO, dimethyl sulfoxide;i.p., intraperitoneal; MMS, methyl methanesulfonate.
chromosomal aberrations in many different organisms and cell systems (Hoffmann, 1980). The induction of dominant-lethal mutations in male mice was reported by Malashenko (1971), whereas Russell (1984) claims that DES does not induce specific-locus mutations in mice. These findings contradict the observation that mutagens are capable of inducing dominant-lethal and specificlocus mutations in the same germ cell stages (Ehling, 1974). It is the purpose of this paper to investigate this contradiction. In addition, the induction of specific-locus mutations in germ cells of mice is a cornerstone for the evaluation of the risk estimation based on the parallelogram approach developed by Sobels
0027-5107/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (Biomedical Division)
192 (1984). This approach investigates the possibility if the estimation of the genetic risk can ultimately be performed on the basis of comparative mutagenesis in vitro and in vivo (van Zeeland et al., 1985). DES is one of the compounds which was chosen for such a comparison.
Matings were detected by examination of females for vaginal plugs on working days. The uterine contents of fertilized females were determined 14-16 days after conception. The frequency of induced dominant-lethal mutations was calculated as follows: Frequency of dominant-lethal mutations
Materials and methods
Diethyl sulfate (DES) is commercially available from Fluka (Buchs, Switzerland) and was kindly provided by the Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, The Netherlands, in the framework of the EC Molecular Dosimetry Program. 100, 200 or 300 mM stock solutions of DES were prepared with 40% dimethyl sulfoxide (DMSO) in phosphate-buffered saline, cooled on ice. The emulsion was stirred until a clear solution was obtained. The compound was injected intraperitoneally (i.p.) within 30 min of preparation. The maximum administered volume of the test solution was 0.25 ml per animal. The higher doses were toxic. For the highest dose the lethality ranged from 8 to 33% in the different experiments. The weight of the treated (101/El x C3H/E1)F 1 males ranged from 25 to 31 g. The amount of mutagen injected did not vary from the nominal value for the particular animal by more than 5%. The concurrent controls in the dominant-lethal experiment received an equal volume of DMSO in phosphate-buffered saline. The control mice for the specific-locus experiment received an equal volume of phosphate solvent, physiological saline or were untreated. The historical control was built up in the laboratory from 1974 to the present. Dominant lethality was tested in (101/El × C3H/E1)F 1 hybrid male mice. At the time of treatment the males were 80-98 days old. After injection each male was caged with one (101/El × C3H/E1)F 1 hybrid virgin female mouse 89-98 days old. A 4-day sequential mating procedure was used over a total of 12 mating intervals for the dose groups 0, 100, 200 and 300 m g / k g body weight of DES. Two additional experiments were performed with a larger sample size of 50 males for 0 and 100 m g / k g DES or 0 and 200 m g / k g DES for 5 mating intervals only.
= 1 _ [ Live embryos of experimentalgroup/~ ] Live embryos of control group/ For the statistical analysis of pre-implantation, post-implantation, and total loss, the Wilcoxon signed rank test as modified by Krauth (1971) was used. For the specific-locus test (101/El × C 3 H / E1)F1 male mice, 10-14 weeks old, were treated and immediately after treatment each male was caged with an untreated virgin test-stock female, 10-12 weeks old. A 4-day sequential mating procedure was used for 5 intervals. Twenty-one days after treatment a permanent monogamic mating schema was used. The test-stock females were homozygous for the following 7 recessive markers: a (non-agouti), b (brown), c ch (chinchilla), d (dilute), p (pink-eyed dilution), s (piebald spotting) and s e (short-ear). These 7 loci are distributed among 5 autosomes. The c ch and p loci are linked on chromosome 7, with an average recombination frequency of about 14%, and the d a n d s e loci are closely linked, being only 0.16 cM apart on chromosome 9 (Ehling, 1978). The offspring were counted, sexed and carefully examined externally at birth. The litters were examined again when cages were changed, the final examination being at 20-21 days of age. The reliability of classification by phenotype was generally confirmed by an allelism test. Six out of 8 recovered mutations were tested for homozygous viability. From the 2 untested mutants 1 with a dilute phenotype was semisterile and 1 with a brown phenotype died prior to the allelism test. 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.
193 TABLE 1 INDUCTION Dose (mg/kg)
OF
DOMINANT-LETHAL
MUTATIONS
IN
MALE
MICE
BY
DIETHYL
SULFATE
Mating interval (days)
Number of females mated/fertilized (n)
Corpora lutea per female
Implants per female
Live embryos per female
Dead implants (%)
0
1-4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
25/24 25/24 25/25 25/24 25/24 25/25 25/24 25/25 25/24 25/24 25/25 25/25
11.8 12.3 11.9 12.2 12.2 12.1 12.0 12.0 12.2 12.1 12.3 11.9
10.9 11.7 10.8 10.8 11.5 11.4 11.2 11.3 11,5 11.7 11.5 11.0
10.2 10.7 10.0 9.9 10.4 10.2 10.4 10.3 10.7 10.8 10.4 10.4
6,9 8.2 8.1 8.1 9.5 10.5 7.4 8.9 6.5 7.9 9.7 5.1
0 0 0 0 0 0 0 0 0 0 0 0
100
1- 4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
25/21 23/20 23/21 23/22 23/22 23/22 23/22 23/22 23/23 23/23 23/21 23/23
t2.0 12.1 12.5 11.9 11.8 12.2 12.4 12.3 11.8 12.0 12.2 12.0
11.2 11.2 11.1 11.0 11.2 11.5 11.7 11.6 11.0 11.0 11.1 11.1
9.9 10.1 10,4 9,7 10.3 10.7 10.9 10.4 10.1 9.9 10.0 10.6
11.5 9.8 6.8 11.6 8.1 6.3 7.0 10.2 8.3 9.5 10.7 4.3
2.6 5.7 -4.2 2.4 0.5 -5.2 -4.7 - 1.3 5.4 7.8 4.3 - 1.6
200
1- 4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
25/22 25/23 25/24 25/23 25/25 25/25 25/25 25/25 25/23 25/24 25/25 25/24
11.7 12.0 12.0 12.0 11.8 11.9 12.3 12.0 12.1 12.2 12.2 11.9
11.1 11.0 10.9 11.0 11.2 11.4 11.3 11.2 10.9 11.6 11.2 11.2
10,1 9.6 9.7 10.0 10.2 10,6 10.4 10.3 9.8 10.8 10.2 10.2
9.4 12.3 11.1 8.7 8.2 7.0 8.2 8.5 10.0 7.2 8.9 8.9
0.7 10.3 2.9 - 0.8 1.3 - 3.5 0.1 0.0 8.6 0.0 1.5 2.2
300
1- 4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
24/18 23/21 22/22 22/22 22/21 22/22 22 /22 22 /21 22 /22 22 /22 22 /21 22 /21
11.9 11.9 12.5 12.1 12.2 12.1 11.9 12.0 12.1 12.1 11.9
11.6 10.8 11.7 10.9 10.8 11.5 11.4 11.4 11.3 11.5 11.4
12.5
11.3
10.4 9.4 10.5 9.5 9.8 10.4 10.2 10.6 10.5 10.0 11.0 10.5
10.0 12.8 10.1 13.3 9.7 9.5 10,4 7.5 6.9 13.8 4.2 7.6
- 2.7 12.0 - 5.4 4.7 5.9 - 1.6 1.4 - 2.8 1.9 7.4 - 5.3 - 0.3
a Calculation is based on absolute figures.
Induced dominant lethals a (%)
(DES)
194
Results D i f f e r e n t stages in gametogenesis m a y b e s c o r e d for i n d u c e d m u t a t i o n s d e p e n d i n g u p o n the interval b e t w e e n t r e a t m e n t a n d f e r t i l i z a t i o n . If s p e r m a t o g e n e s i s is n o t affected b y the t r e a t m e n t , the various g a m e t o g e n i c stages are s a m p l e d in the m o u s e d u r i n g the following p o s t - t r e a t m e n t m a t i n g intervals: s p e r m a t o z o a ( 1 - 7 days), s p e r m a t i d s ( 8 - 2 1 days), s p e r m a t o c y t e s ( 2 2 - 3 5 days), followed b y d i f f e r e n t i a t i n g s p e r m a t o g o n i a a n d g o n i a l stem cells (Oakberg, 1975).
Dominant-lethal mutations D o m i n a n t - l e t h a l m u t a t i o n s i n c l u d e the loss o f fertilized eggs b e f o r e a n d after i m p l a n t a t i o n . T h e frequency of i n d u c e d d o m i n a n t - l e t h a l m u t a t i o n s as c a l c u l a t e d w o u l d i n c l u d e a n y increase over the c o n t r o l value in unfertilized eggs. A d e c r e a s e in i m p l a n t a t i o n s p e r female f r o m the c o n t r o l value indicates i n d u c e d p r e - i m p l a n t a t i o n d e a t h o r an
excess of u n f e r t i l i z e d eggs. T h e difference b e t w e e n i m p l a n t a t i o n s p e r f e m a l e a n d live e m b r y o s p e r female r e p r e s e n t s p o s t - i m p l a n t a t i o n death. All females that h a d i m p l a n t s were classified as fertile. W i t h a s a m p l e size of 19 ( 1 0 1 / E l x C 3 H / E 1 ) F 1 m a l e mice a n d a 1 : 1 m a t i n g r a t i o we can detect a 15% increase in the m u t a t i o n frequency, with a s a m p l e size of 45 a n i m a l s we can detect an increase of 10% (Vollmar, 1977). U s i n g a s a m p l e size of 25 mice we tested 0, 100, 200 a n d 300 m g / k g b w of D E S ( T a b l e 1). I n the m a t i n g interval 5 - 8 d a y s the pre- a n d p o s t - i m p l a n t a t i o n losses were significantly d i f f e r e n t f r o m the c o n t r o l value for the 200- a n d the 3 0 0 - m g / k g group. T h e prei m p l a n t a t i o n loss after 100 m g / k g b w was at the b o r d e r l i n e of significance ( P = 0.079). F r o m these d a t a it can be c o n c l u d e d that D E S induces d o m i n a n t - l e t h a l m u t a t i o n s m a i n l y in s p e r m a t o z o a . T o v a l i d a t e the conclusion, we tested in indep e n d e n t e x p e r i m e n t s the i n d u c t i o n of d o m i n a n t lethal m u t a t i o n s for 100 a n d 200 m g / k g b w of
TABLE 2 INDUCTION OF DOMINANT-LETHAL MUTATIONS IN MALE MICE BY DIETHYL SULFATE (DES) Dose (mg/kg)
Mating interval (days)
Number of females mated/fertilized (n)
Corpora lutea per female
Implants per female
Live embryos per female
Dead implants (,%)
0
1- 4 5- 8 9-12 13-16 17-20
50/49 50/48 50/49 50/50 50/50
12.0 12.2 12.0 12.5 12.1
11.4 11.2 11.0 11.5 11.3
10.2 10.3 10.0 10.6 10.3
10.6 7.6 9.5 7.8 9.0
0 0 0 0 0
100
1- 4 5- 8 9-12 13-16 17-20
50/45 50/47 50/45 50/47 50/47
11.8 11.8 12.3 12.3 12.0
11.3 10.7 11.1 11.3 11.3
10.1 9.2 10.1 10.4 10.3
10.4 13.7 9.2 8.3 8.6
0.7 10.7 - 1.5 2.2 - 0.8
0
1- 4 5- 8 9-12 13-16 17-20
50/48 50/50 50/48 50/49 50/50
12.3 12.3 12.3 11.8 11.4
11.5 11.8 11.0 11.3 11.1
10.5 10.7 10.1 10.5 10.2
8.7 9.5 8.3 6.5 8.2
0 0 0 0 0
200
1- 4 5- 8 9-12 13-16 17-20
50/43 49/48 49/46 49/47 49/47
12.0 11.9 11.7 11.6 11.7
11.4 11.3 10.7 11.2 11.2
10.2 9.3 9.2 10.3 10.5
11.2 17.9 14.2 7.8 6.6
2.8 13.7 9.0 2.2 - 3.2
a Calculation is based on absolute figures.
Induced dominant lethals a (,%)
195
DES with a sample size of 50 males and females per mating interval. The sensitivity pattern for the induction of dominant-lethal mutations in both experiments is similar (Tables 1 and 2). After i.p. injection of 200 mg/kg bw DES (Table 2) the frequency of dominant-lethal mutations is highly significantly different from the control value in the mating interval 5-8 days post treatment ( P = 0.0003). In the mating interval 9-12 days only the post-implantation loss is significantly different from the control value (P = 0.0049). After i.p. injection of 100 mg/kg bw DES dominant-lethal mutations were induced only in the mating interval 5-8 days ( P = 0.009). It is very likely that lower doses of DES induce dominant-lethal mutations only in spermatozoa of mice and that higher doses of DES induce dominantlethal mutations in spermatozoa and late spermatids. A relatively high frequency of dominant lethals is observed in the mating intervals 33-40 days post injection (Table 1). However, it is unclear whether the effect is due to the induction of dominant-lethal mutations or a pseudo-dominant-
lethal effect. Three reasons can be given for the latter interpretation: (1) the sensitivity pattern is not constant; (2) the effect is not dose-dependent; (3) the effect is mainly due to loss before implantation. It is possible that the observed effect is due to a small reduction in the fertilization rate of ova. To clarify this point additional experiments to examine the ova have to be performed (Kratochvilova, 1978).
Specific-locus mutations The differential spermatogenic response for dominant-lethal mutations can also be observed for the induction of specific-locus mutations (Table 3). The reduction of the average litter size indicates the induction of dominant lethals. The sensitivity pattern for the induction of dominantlethal mutations in Tables 1 and 2 corresponds well with the reduction of the average litter size in Table 3. The highest frequency of specific-locus mutations in the 200-mg/kg bw group of DES corresponds with the highest frequency of dominant lethals in the mating interval 5-8 days post treatment.
TABLE 3 I N D U C T I O N OF S P E C I F I C - L O C U S M U T A T I O N S IN M A L E M I C E BY D I E T H Y L S U L F A T E (DES) Dose (mg/kg) 0
Mating interval (days)
N u m b e r of offspring
Average fitter size
N u m b e r of mutations
Confidence limits 95%
-
248413
1.3
0.8- 1.9
200
1- 4 5- 8 9 -1 2 13-16 17-20 21-4 2 >43
3441 3371 3788 3714 3809 2 609 13551
7.5 7.0 7.4 7.9 8.3 8.0 8.0
0 4b 2c 0 0 0 0
0 17.0 7.5 0 0 0 0
0 -13.6 5.8-40.7 1.3-25.2 0 -12.6 0 -12.3 0 -18.0 0 - 3.5
300
1- 4 5- 8 9-1 2 13-16 17-20
2176 1900 2260 2661 2885
6.9 5.8 6.6 7.7 8.0
0 1d 0 e 0 1f
0 7.5 0 0 5.0
0 -21.6 0.4--40.0 0 -20.8 0 -17.6 0.3-26.4
a b c d
22 a
Mutations per locus per 105 gametes
Includes one d u s t e r of 2 s e and one cluster of 6 s mutations. p < 0.001. Additionally a gonosomal pd mosaic was recovered. p = 0.05. p = 0.16.
e One X-autosomal translocation was recovered. f P = 0.23.
196 TABLE 4 S P E C T R U M OF SPECIFIC-LOCUS M U T A T I O N S I N D U C E D BY D I E T H Y L S U L F A T E (DES) IN S P E R M A T O Z O A A N D SPERMATIDS Dose
Locus
(mg/kg)
a
b
c
d
dse
se
p
s
Total
200
0
1
1
1
0
0
2
1
6
300
0
0
0
0
0
0
2
0
2
value, the observed mutation frequency is lower in the 300-mg/kg than in the 200-mg/kg group (Table 3). Considering the wide confidence limits it seems problematic to conclude that the increased frequency of dominant-lethal mutations lowers the chance of recovering specific-locus mutations. However, comparable results were obtained for methyl and ethyl methanesulfonate (Ehling, 1978; Ehling and Neuhauser-Klaus, 1984). It will be interesting to test the induction of specific-locus mutations with 100 m g / k g bw DES. The distribution of mutations induced by DES among specific loci is summarized in Table 4. Considering the sample size of the experiments the distribution agrees with observations from earlier experiments with chemical mutagens (Ehling and Neuh~iuser-Klaus, 1984). All mutations that as homozygotes cause death before maturity have been classified as lethals (Table 5). The observed frequency of 83% lethals agrees well with the results of other experiments (Ehling and Neuh~iuser-Klaus, 1984). The sensitivity pattern of methyl methanesulfonate (MMS) for the induction of specific-locus mutations is comparable with the differential spermatogenic response of DES. In the MMS experiments we
A highly significant difference between the control mutation rate and the treated group was observed for specific-locus mutations (Table 3) and for dominant-lethal mutations (Table 2) only 5-8 days post treatment. For both assays the induction of mutations in the mating interval 9-12 days is less pronounced. The difficulty in establishing a dose-effect relationship for specific-locus mutations is clearly demonstrated by a comparison of the mutation rates for 200 and 300 mg/kg bw DES (Table 3). The sensitivity pattern for the induction of mutations is the same. The mutation rate for the 200mg/kg group for the 5-8-day mating interval is highly significantly different from the control value (P < 0.001). In contrast, if one had only the information of the experiments with 300 mg/kg, one would have concluded that the induction of specific-locus mutations is due to chance (P = 0.16). Only the reduction of the average litter size, an indication for the induction of dominant-lethal mutations, increases with the dose, not the frequency of specific-locus mutations. It is likely that the induction of dominant-lethal mutations eliminates partly the induced specific-locus mutations. Therefore, taking the mutation rates at face
TABLE 5 V I A B I L I T Y OF H O M O Z Y G O U S S P E C I F I C - L O C U S M U T A T I O N S I N D U C E D BY D I E T H Y L S U L F A T E IN S P E R M A T O Z O A AND SPERMATIDS Dose
N u m b e r l e t h a l / n u m b e r tested per locus
(mg/kg)
a
200 300
.
200,300
-
b
.
c
d
1/1
-
. -
se
. 1/1
-
-
Lethals %
p
s
total
2/2 1/2 a
1/1 -
4/4 1/2
100 50
3/4
1/ 1
5/6
83
a The homozygous viable p mutant was recovered from the mating interval 17-20 days post treatment.
197 observed 10 lethals out of 14 tested mutations. The high frequency of homozygous lethal mutations induced in post-spermatogonial germ cell stages suggests that small deficiencies are the main cause for mutations induced in these germ cell stages. This observation explains likewise the correlation between the induction of dominant-lethal and specific-locus mutations.
Discussion Malashenko and Surkova (1973) reported that (C3H x 101)F 1 male mice were resistant to the induction of dominant-lethal mutations by DES with a dose of 150 m g / k g bw. The authors mated the treated males only in the second and third week after treatment. According to our results the induction of dominant-lethal mutations occurred only in the mating interval 5-8 days post treatment (Table 2). It is unlikely that we would have detected the induction of dominant-lethal mutations with the mating schedule used by the authors. However, Malashenko and Surkova (1973) described the induction of dominant-lethal mutations using the same dose and mating schedule for C 5 7 B L / 6 and CBA males indicating strain differences for the response to DES. The induction of specific-locus mutations was tested by Malashenko (1976). He injected i.p. 5 m g / k g bw DES twice weekly for 10 weeks. The total administered dose was 100 mg/kg. In 2842 offspring of the control group he found no mutation and in 5042 offspring derived from spermatogonia treated with DES he observed 1 mutation at the dilute locus ( P = 0.64). The results summarized in Table 3 are a good example of the difficulty in evaluating specificlocus data if one used only a high dose point (de Serres, 1986). With the conventional permanent mating scheme after treatment (Searle, 1975) the great majority of all females would have conceived 1 - 4 days post treatment and a second litter would have been conceived 20-24 days after treatment. With such a mating scheme one would be unable to detect the mutagenicity of DES. Therefore it is not surprising that Russell (1984) came to the wrong conclusion that 'diethyl sulfate ... produced no mutations in this test'. For the evaluation of specific-locus data it is
important to have information of the mutation rate in all spermatogenic cell stages. In addition, it is necessary to have a sufficient number of offspring derived from the different germ cell stages. For the statistical evaluation it is mandatory to consider the results of each mating interval separately. Grouping all the data of the post-spermatogonial germ cell stages together for statistical analysis is not always meaningful and may lead to a wrong conclusion. Specific-locus mutations were 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, 1986). Knowing the resources necessary to determine the doubling dose in a specific-locus experiment it is worth while to compare the specific-locus results with other genetic endpoints. Based on the results of the 200-mg/kg group and the 5-8-day mating interval the doubling dose for specific-locus mutations for DES is 17 mg/kg. Using the regression coefficient for the same germ cell stage for the dominant-lethal assay (Table 1) gives a doubling dose of 22 mg/kg. The doubling dose for dominant lethals agrees well with the doubling dose for specific-locus mutations. Similar comparisons were made with other mutagens (Ehling, 1982). In general, these comparisons are encouraging. For a final recommendation, however, it is necessary to increase the data base for the determination of the doubling dose with different genetic endpoints.
Acknowledgements Experiments were supported by Contract ENV637-D(B) of the Commission of the European Communities. The able assistance of S. Frischholz, A. Kt~hler, A. Lauterbach, B. May, G. Schenke and K. Weiser is greatly appreciated.
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198 Ehling, U.H. (1974) Differential spermatogenic response of mice to the induction of mutations by antineoplastic drugs, Mutation Res., 26, 285-295. Ehling, U.H. (1978) Specific-locus mutations in mice, in: A. Hollaender and F.J. de Serres (Eds.), Chemical Mutagens, Vol. 5, Plenum, New York, pp. 233-256. Ehling, U.H. (1982) Risk estimations based on germ-cell mutations in mice, in: T. Sugimura, S. Kondo and H. Takebe (Eds.), Environmental Mutagens and Carcinogens, University of Tokyo Press, Tokyo/Alan R. Liss, Inc., New York, pp. 709-719. Ehling, U.H., and A. Neuh~iuser-Klaus (1984) Dose-effect relationships of germ-cell mutations in mice, in: Y. Tazima, S. Kondo and Y. Kuroda (Eds.), Problems of Threshold in Chemical Mutagenesis, The Environmental Mutagen Society of Japan, Kokusai-bunken, Ltd., Tokyo, pp. 15-25. Hoffmann, G.R. (1980) Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds, Mutation Res., 75, 63-129. IARC (1974) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man: Some aromatic amines, hydrazine and related substances, N-nitroso compounds and miscellaneous alkylating agents, Vol. 4, International Agency for Research on Cancer, Lyon, pp. 277-281. Kratochvilova, J. (1978) Evaluation of pre-implantation loss in dominant-lethal assay in the mouse, Mutation Res., 54, 47-54. Krauth, J. (1971) A locally most powerful tied rank test in a Wilcoxon situation, Ann. Math. Statist., 42, 1949-1956. Malashenko, A.M. (1971) Sensitivity of mouse testis ceils to the induction of dominant lethals by diethylsulphate, Genetika, 7/1, 84-91.
Malashenko, A.M. (1976) Investigation of the effect of diethylsulphate applied at low doses in laboratory mice using the method of specific loci, Genetika, 12/3, 163-165. Malashenko, A.M., and N.I. Surkova (1973) Induction of dominant lethals with diethylsulphate in male mice of different genotypes, Genetika, 9/4, 147-149. Oakberg, E.F. (1975) Effects of radiation on the testis, in: Handbook of Physiology, Section 7, Endocrinology 5, pp. 233-243. Russell, W.L. (1984) Dose response, repair, and no-effect dose levels in mouse germ-cell mutagenesis, in: Y. Tazima, S. Kondo and Y. Kuroda (Eds.), Problems of Threshold in Chemical Mutagenesis, The Environmental Mutagen Society of Japan, Kokusai-bunken, Ltd., Tokyo, pp. 153-160 (Quotation p. 157). Searle, A.G. (1975) The specific locus test in the mouse, Mutation Res., 31,277-290. Sobels, F.H. (1984) Problems and perspectives in genetic toxicology, in: G. Obe (Ed.), Mutations in Man, Springer, Berlin-Heidelberg, pp. 1-19. UNSCEAR (1986) (United Nations Scientific Committee on the Effects of Atomic Radiation) Genetic and Somatic Effects of Ionizing Radiation, United Nations, New York. Vollmar, J. (1977) Statistical problems in mutagenicity tests, Arch. Toxicol., 38, 13-25. Zeeland, A.A. van, G.R. Mohn, A. Neuh~iuser-Klaus and U.H. Ehling (1985) Quantitative comparison of genetic effects of ethylating agents on the basis of DNA adduct formation. Use of O6-ethylguanine as molecular dosimeter for extrapolation from cells in culture to the mouse. Environ. Health Perspect., 62, 163-169.
191
Elsevier MTR04596
Induction of specific-locus and dominant-lethal mutations in male mice by diethyl sulfate (DES) U.H. Ehling and A. Neuh~iuser-Klaus lnstitut fiir Siiugetiergenetik, Gesellschaft]'fir Strahlen- und Umweltforschung D-8042 Neuherberg (F.R.G.)
(Received 23 September1987) (Revision received30 November 1987) (Accepted 7 December 1987)
Keywords: Specific-locusmutations; Dominant-lethal mutations; Male mice; Diethyl sulfate (DES).
Summary Diethyl sulfate (DES), a monofunctional alkylating agent, induces mutations and chromosomal aberrations in many different organisms and cell systems, including dominant-lethal mutations in male mice. However, until now it could not be demonstrated that DES induces specific-locus mutations in mice. This observation would contradict the close correlation observed between the induction of dominant-lethal mutations and specific-locus mutations in mice with other chemicals. DES induces dominant-lethal and specific-locus mutations in spermatozoa and late spermatids of mice. The mutation frequency for dominant-lethal mutations is dose-dependent, while for specific-locus mutations it is independent of the dose. In the mating interval 5-8 days post-treatment the mutation frequency for 200 m g / k g DES is 17.0 x 10 -5 and for 300 m g / k g 7.5 × 10 -5 mutations per locus. The dose-dependent increase of dominant-lethal mutations probably reduced the chance of recovering specific-locus mutations. The importance of these findings for mutagenicity testing is discussed.
Diethyl sulfate (DES) is an active alkylating agent. It has been produced commercially for at least 60 years and is mainly used as an alkylating agent to convert active-hydrogen compounds such as phenols, amines and thiols to the corresponding ethyl derivatives (IARC, 1974). This monofunctional alkylating agent induces mutations and
Correspondence: Dr. U.H. Ehling, Institut for S~tugetiergenetik, Gesellschaft fiir Strahlen- und Umweltforschung, D-8042 Neuherberg (Federal Republic of Germany). Abbreviations: bw, body weight; DES, diethyl sulfate; DMSO, dimethyl sulfoxide;i.p., intraperitoneal; MMS, methyl methanesulfonate.
chromosomal aberrations in many different organisms and cell systems (Hoffmann, 1980). The induction of dominant-lethal mutations in male mice was reported by Malashenko (1971), whereas Russell (1984) claims that DES does not induce specific-locus mutations in mice. These findings contradict the observation that mutagens are capable of inducing dominant-lethal and specificlocus mutations in the same germ cell stages (Ehling, 1974). It is the purpose of this paper to investigate this contradiction. In addition, the induction of specific-locus mutations in germ cells of mice is a cornerstone for the evaluation of the risk estimation based on the parallelogram approach developed by Sobels
0027-5107/88/$03.50 © 1988 ElsevierSciencePublishers B.V. (Biomedical Division)
192 (1984). This approach investigates the possibility if the estimation of the genetic risk can ultimately be performed on the basis of comparative mutagenesis in vitro and in vivo (van Zeeland et al., 1985). DES is one of the compounds which was chosen for such a comparison.
Matings were detected by examination of females for vaginal plugs on working days. The uterine contents of fertilized females were determined 14-16 days after conception. The frequency of induced dominant-lethal mutations was calculated as follows: Frequency of dominant-lethal mutations
Materials and methods
Diethyl sulfate (DES) is commercially available from Fluka (Buchs, Switzerland) and was kindly provided by the Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, The Netherlands, in the framework of the EC Molecular Dosimetry Program. 100, 200 or 300 mM stock solutions of DES were prepared with 40% dimethyl sulfoxide (DMSO) in phosphate-buffered saline, cooled on ice. The emulsion was stirred until a clear solution was obtained. The compound was injected intraperitoneally (i.p.) within 30 min of preparation. The maximum administered volume of the test solution was 0.25 ml per animal. The higher doses were toxic. For the highest dose the lethality ranged from 8 to 33% in the different experiments. The weight of the treated (101/El x C3H/E1)F 1 males ranged from 25 to 31 g. The amount of mutagen injected did not vary from the nominal value for the particular animal by more than 5%. The concurrent controls in the dominant-lethal experiment received an equal volume of DMSO in phosphate-buffered saline. The control mice for the specific-locus experiment received an equal volume of phosphate solvent, physiological saline or were untreated. The historical control was built up in the laboratory from 1974 to the present. Dominant lethality was tested in (101/El × C3H/E1)F 1 hybrid male mice. At the time of treatment the males were 80-98 days old. After injection each male was caged with one (101/El × C3H/E1)F 1 hybrid virgin female mouse 89-98 days old. A 4-day sequential mating procedure was used over a total of 12 mating intervals for the dose groups 0, 100, 200 and 300 m g / k g body weight of DES. Two additional experiments were performed with a larger sample size of 50 males for 0 and 100 m g / k g DES or 0 and 200 m g / k g DES for 5 mating intervals only.
= 1 _ [ Live embryos of experimentalgroup/~ ] Live embryos of control group/ For the statistical analysis of pre-implantation, post-implantation, and total loss, the Wilcoxon signed rank test as modified by Krauth (1971) was used. For the specific-locus test (101/El × C 3 H / E1)F1 male mice, 10-14 weeks old, were treated and immediately after treatment each male was caged with an untreated virgin test-stock female, 10-12 weeks old. A 4-day sequential mating procedure was used for 5 intervals. Twenty-one days after treatment a permanent monogamic mating schema was used. The test-stock females were homozygous for the following 7 recessive markers: a (non-agouti), b (brown), c ch (chinchilla), d (dilute), p (pink-eyed dilution), s (piebald spotting) and s e (short-ear). These 7 loci are distributed among 5 autosomes. The c ch and p loci are linked on chromosome 7, with an average recombination frequency of about 14%, and the d a n d s e loci are closely linked, being only 0.16 cM apart on chromosome 9 (Ehling, 1978). The offspring were counted, sexed and carefully examined externally at birth. The litters were examined again when cages were changed, the final examination being at 20-21 days of age. The reliability of classification by phenotype was generally confirmed by an allelism test. Six out of 8 recovered mutations were tested for homozygous viability. From the 2 untested mutants 1 with a dilute phenotype was semisterile and 1 with a brown phenotype died prior to the allelism test. 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.
193 TABLE 1 INDUCTION Dose (mg/kg)
OF
DOMINANT-LETHAL
MUTATIONS
IN
MALE
MICE
BY
DIETHYL
SULFATE
Mating interval (days)
Number of females mated/fertilized (n)
Corpora lutea per female
Implants per female
Live embryos per female
Dead implants (%)
0
1-4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
25/24 25/24 25/25 25/24 25/24 25/25 25/24 25/25 25/24 25/24 25/25 25/25
11.8 12.3 11.9 12.2 12.2 12.1 12.0 12.0 12.2 12.1 12.3 11.9
10.9 11.7 10.8 10.8 11.5 11.4 11.2 11.3 11,5 11.7 11.5 11.0
10.2 10.7 10.0 9.9 10.4 10.2 10.4 10.3 10.7 10.8 10.4 10.4
6,9 8.2 8.1 8.1 9.5 10.5 7.4 8.9 6.5 7.9 9.7 5.1
0 0 0 0 0 0 0 0 0 0 0 0
100
1- 4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
25/21 23/20 23/21 23/22 23/22 23/22 23/22 23/22 23/23 23/23 23/21 23/23
t2.0 12.1 12.5 11.9 11.8 12.2 12.4 12.3 11.8 12.0 12.2 12.0
11.2 11.2 11.1 11.0 11.2 11.5 11.7 11.6 11.0 11.0 11.1 11.1
9.9 10.1 10,4 9,7 10.3 10.7 10.9 10.4 10.1 9.9 10.0 10.6
11.5 9.8 6.8 11.6 8.1 6.3 7.0 10.2 8.3 9.5 10.7 4.3
2.6 5.7 -4.2 2.4 0.5 -5.2 -4.7 - 1.3 5.4 7.8 4.3 - 1.6
200
1- 4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
25/22 25/23 25/24 25/23 25/25 25/25 25/25 25/25 25/23 25/24 25/25 25/24
11.7 12.0 12.0 12.0 11.8 11.9 12.3 12.0 12.1 12.2 12.2 11.9
11.1 11.0 10.9 11.0 11.2 11.4 11.3 11.2 10.9 11.6 11.2 11.2
10,1 9.6 9.7 10.0 10.2 10,6 10.4 10.3 9.8 10.8 10.2 10.2
9.4 12.3 11.1 8.7 8.2 7.0 8.2 8.5 10.0 7.2 8.9 8.9
0.7 10.3 2.9 - 0.8 1.3 - 3.5 0.1 0.0 8.6 0.0 1.5 2.2
300
1- 4 5-8 9-12 13-16 17-20 21-24 25-28 29-32 33-36 37-40 41-44 45-48
24/18 23/21 22/22 22/22 22/21 22/22 22 /22 22 /21 22 /22 22 /22 22 /21 22 /21
11.9 11.9 12.5 12.1 12.2 12.1 11.9 12.0 12.1 12.1 11.9
11.6 10.8 11.7 10.9 10.8 11.5 11.4 11.4 11.3 11.5 11.4
12.5
11.3
10.4 9.4 10.5 9.5 9.8 10.4 10.2 10.6 10.5 10.0 11.0 10.5
10.0 12.8 10.1 13.3 9.7 9.5 10,4 7.5 6.9 13.8 4.2 7.6
- 2.7 12.0 - 5.4 4.7 5.9 - 1.6 1.4 - 2.8 1.9 7.4 - 5.3 - 0.3
a Calculation is based on absolute figures.
Induced dominant lethals a (%)
(DES)
194
Results D i f f e r e n t stages in gametogenesis m a y b e s c o r e d for i n d u c e d m u t a t i o n s d e p e n d i n g u p o n the interval b e t w e e n t r e a t m e n t a n d f e r t i l i z a t i o n . If s p e r m a t o g e n e s i s is n o t affected b y the t r e a t m e n t , the various g a m e t o g e n i c stages are s a m p l e d in the m o u s e d u r i n g the following p o s t - t r e a t m e n t m a t i n g intervals: s p e r m a t o z o a ( 1 - 7 days), s p e r m a t i d s ( 8 - 2 1 days), s p e r m a t o c y t e s ( 2 2 - 3 5 days), followed b y d i f f e r e n t i a t i n g s p e r m a t o g o n i a a n d g o n i a l stem cells (Oakberg, 1975).
Dominant-lethal mutations D o m i n a n t - l e t h a l m u t a t i o n s i n c l u d e the loss o f fertilized eggs b e f o r e a n d after i m p l a n t a t i o n . T h e frequency of i n d u c e d d o m i n a n t - l e t h a l m u t a t i o n s as c a l c u l a t e d w o u l d i n c l u d e a n y increase over the c o n t r o l value in unfertilized eggs. A d e c r e a s e in i m p l a n t a t i o n s p e r female f r o m the c o n t r o l value indicates i n d u c e d p r e - i m p l a n t a t i o n d e a t h o r an
excess of u n f e r t i l i z e d eggs. T h e difference b e t w e e n i m p l a n t a t i o n s p e r f e m a l e a n d live e m b r y o s p e r female r e p r e s e n t s p o s t - i m p l a n t a t i o n death. All females that h a d i m p l a n t s were classified as fertile. W i t h a s a m p l e size of 19 ( 1 0 1 / E l x C 3 H / E 1 ) F 1 m a l e mice a n d a 1 : 1 m a t i n g r a t i o we can detect a 15% increase in the m u t a t i o n frequency, with a s a m p l e size of 45 a n i m a l s we can detect an increase of 10% (Vollmar, 1977). U s i n g a s a m p l e size of 25 mice we tested 0, 100, 200 a n d 300 m g / k g b w of D E S ( T a b l e 1). I n the m a t i n g interval 5 - 8 d a y s the pre- a n d p o s t - i m p l a n t a t i o n losses were significantly d i f f e r e n t f r o m the c o n t r o l value for the 200- a n d the 3 0 0 - m g / k g group. T h e prei m p l a n t a t i o n loss after 100 m g / k g b w was at the b o r d e r l i n e of significance ( P = 0.079). F r o m these d a t a it can be c o n c l u d e d that D E S induces d o m i n a n t - l e t h a l m u t a t i o n s m a i n l y in s p e r m a t o z o a . T o v a l i d a t e the conclusion, we tested in indep e n d e n t e x p e r i m e n t s the i n d u c t i o n of d o m i n a n t lethal m u t a t i o n s for 100 a n d 200 m g / k g b w of
TABLE 2 INDUCTION OF DOMINANT-LETHAL MUTATIONS IN MALE MICE BY DIETHYL SULFATE (DES) Dose (mg/kg)
Mating interval (days)
Number of females mated/fertilized (n)
Corpora lutea per female
Implants per female
Live embryos per female
Dead implants (,%)
0
1- 4 5- 8 9-12 13-16 17-20
50/49 50/48 50/49 50/50 50/50
12.0 12.2 12.0 12.5 12.1
11.4 11.2 11.0 11.5 11.3
10.2 10.3 10.0 10.6 10.3
10.6 7.6 9.5 7.8 9.0
0 0 0 0 0
100
1- 4 5- 8 9-12 13-16 17-20
50/45 50/47 50/45 50/47 50/47
11.8 11.8 12.3 12.3 12.0
11.3 10.7 11.1 11.3 11.3
10.1 9.2 10.1 10.4 10.3
10.4 13.7 9.2 8.3 8.6
0.7 10.7 - 1.5 2.2 - 0.8
0
1- 4 5- 8 9-12 13-16 17-20
50/48 50/50 50/48 50/49 50/50
12.3 12.3 12.3 11.8 11.4
11.5 11.8 11.0 11.3 11.1
10.5 10.7 10.1 10.5 10.2
8.7 9.5 8.3 6.5 8.2
0 0 0 0 0
200
1- 4 5- 8 9-12 13-16 17-20
50/43 49/48 49/46 49/47 49/47
12.0 11.9 11.7 11.6 11.7
11.4 11.3 10.7 11.2 11.2
10.2 9.3 9.2 10.3 10.5
11.2 17.9 14.2 7.8 6.6
2.8 13.7 9.0 2.2 - 3.2
a Calculation is based on absolute figures.
Induced dominant lethals a (,%)
195
DES with a sample size of 50 males and females per mating interval. The sensitivity pattern for the induction of dominant-lethal mutations in both experiments is similar (Tables 1 and 2). After i.p. injection of 200 mg/kg bw DES (Table 2) the frequency of dominant-lethal mutations is highly significantly different from the control value in the mating interval 5-8 days post treatment ( P = 0.0003). In the mating interval 9-12 days only the post-implantation loss is significantly different from the control value (P = 0.0049). After i.p. injection of 100 mg/kg bw DES dominant-lethal mutations were induced only in the mating interval 5-8 days ( P = 0.009). It is very likely that lower doses of DES induce dominant-lethal mutations only in spermatozoa of mice and that higher doses of DES induce dominantlethal mutations in spermatozoa and late spermatids. A relatively high frequency of dominant lethals is observed in the mating intervals 33-40 days post injection (Table 1). However, it is unclear whether the effect is due to the induction of dominant-lethal mutations or a pseudo-dominant-
lethal effect. Three reasons can be given for the latter interpretation: (1) the sensitivity pattern is not constant; (2) the effect is not dose-dependent; (3) the effect is mainly due to loss before implantation. It is possible that the observed effect is due to a small reduction in the fertilization rate of ova. To clarify this point additional experiments to examine the ova have to be performed (Kratochvilova, 1978).
Specific-locus mutations The differential spermatogenic response for dominant-lethal mutations can also be observed for the induction of specific-locus mutations (Table 3). The reduction of the average litter size indicates the induction of dominant lethals. The sensitivity pattern for the induction of dominantlethal mutations in Tables 1 and 2 corresponds well with the reduction of the average litter size in Table 3. The highest frequency of specific-locus mutations in the 200-mg/kg bw group of DES corresponds with the highest frequency of dominant lethals in the mating interval 5-8 days post treatment.
TABLE 3 I N D U C T I O N OF S P E C I F I C - L O C U S M U T A T I O N S IN M A L E M I C E BY D I E T H Y L S U L F A T E (DES) Dose (mg/kg) 0
Mating interval (days)
N u m b e r of offspring
Average fitter size
N u m b e r of mutations
Confidence limits 95%
-
248413
1.3
0.8- 1.9
200
1- 4 5- 8 9 -1 2 13-16 17-20 21-4 2 >43
3441 3371 3788 3714 3809 2 609 13551
7.5 7.0 7.4 7.9 8.3 8.0 8.0
0 4b 2c 0 0 0 0
0 17.0 7.5 0 0 0 0
0 -13.6 5.8-40.7 1.3-25.2 0 -12.6 0 -12.3 0 -18.0 0 - 3.5
300
1- 4 5- 8 9-1 2 13-16 17-20
2176 1900 2260 2661 2885
6.9 5.8 6.6 7.7 8.0
0 1d 0 e 0 1f
0 7.5 0 0 5.0
0 -21.6 0.4--40.0 0 -20.8 0 -17.6 0.3-26.4
a b c d
22 a
Mutations per locus per 105 gametes
Includes one d u s t e r of 2 s e and one cluster of 6 s mutations. p < 0.001. Additionally a gonosomal pd mosaic was recovered. p = 0.05. p = 0.16.
e One X-autosomal translocation was recovered. f P = 0.23.
196 TABLE 4 S P E C T R U M OF SPECIFIC-LOCUS M U T A T I O N S I N D U C E D BY D I E T H Y L S U L F A T E (DES) IN S P E R M A T O Z O A A N D SPERMATIDS Dose
Locus
(mg/kg)
a
b
c
d
dse
se
p
s
Total
200
0
1
1
1
0
0
2
1
6
300
0
0
0
0
0
0
2
0
2
value, the observed mutation frequency is lower in the 300-mg/kg than in the 200-mg/kg group (Table 3). Considering the wide confidence limits it seems problematic to conclude that the increased frequency of dominant-lethal mutations lowers the chance of recovering specific-locus mutations. However, comparable results were obtained for methyl and ethyl methanesulfonate (Ehling, 1978; Ehling and Neuhauser-Klaus, 1984). It will be interesting to test the induction of specific-locus mutations with 100 m g / k g bw DES. The distribution of mutations induced by DES among specific loci is summarized in Table 4. Considering the sample size of the experiments the distribution agrees with observations from earlier experiments with chemical mutagens (Ehling and Neuh~iuser-Klaus, 1984). All mutations that as homozygotes cause death before maturity have been classified as lethals (Table 5). The observed frequency of 83% lethals agrees well with the results of other experiments (Ehling and Neuh~iuser-Klaus, 1984). The sensitivity pattern of methyl methanesulfonate (MMS) for the induction of specific-locus mutations is comparable with the differential spermatogenic response of DES. In the MMS experiments we
A highly significant difference between the control mutation rate and the treated group was observed for specific-locus mutations (Table 3) and for dominant-lethal mutations (Table 2) only 5-8 days post treatment. For both assays the induction of mutations in the mating interval 9-12 days is less pronounced. The difficulty in establishing a dose-effect relationship for specific-locus mutations is clearly demonstrated by a comparison of the mutation rates for 200 and 300 mg/kg bw DES (Table 3). The sensitivity pattern for the induction of mutations is the same. The mutation rate for the 200mg/kg group for the 5-8-day mating interval is highly significantly different from the control value (P < 0.001). In contrast, if one had only the information of the experiments with 300 mg/kg, one would have concluded that the induction of specific-locus mutations is due to chance (P = 0.16). Only the reduction of the average litter size, an indication for the induction of dominant-lethal mutations, increases with the dose, not the frequency of specific-locus mutations. It is likely that the induction of dominant-lethal mutations eliminates partly the induced specific-locus mutations. Therefore, taking the mutation rates at face
TABLE 5 V I A B I L I T Y OF H O M O Z Y G O U S S P E C I F I C - L O C U S M U T A T I O N S I N D U C E D BY D I E T H Y L S U L F A T E IN S P E R M A T O Z O A AND SPERMATIDS Dose
N u m b e r l e t h a l / n u m b e r tested per locus
(mg/kg)
a
200 300
.
200,300
-
b
.
c
d
1/1
-
. -
se
. 1/1
-
-
Lethals %
p
s
total
2/2 1/2 a
1/1 -
4/4 1/2
100 50
3/4
1/ 1
5/6
83
a The homozygous viable p mutant was recovered from the mating interval 17-20 days post treatment.
197 observed 10 lethals out of 14 tested mutations. The high frequency of homozygous lethal mutations induced in post-spermatogonial germ cell stages suggests that small deficiencies are the main cause for mutations induced in these germ cell stages. This observation explains likewise the correlation between the induction of dominant-lethal and specific-locus mutations.
Discussion Malashenko and Surkova (1973) reported that (C3H x 101)F 1 male mice were resistant to the induction of dominant-lethal mutations by DES with a dose of 150 m g / k g bw. The authors mated the treated males only in the second and third week after treatment. According to our results the induction of dominant-lethal mutations occurred only in the mating interval 5-8 days post treatment (Table 2). It is unlikely that we would have detected the induction of dominant-lethal mutations with the mating schedule used by the authors. However, Malashenko and Surkova (1973) described the induction of dominant-lethal mutations using the same dose and mating schedule for C 5 7 B L / 6 and CBA males indicating strain differences for the response to DES. The induction of specific-locus mutations was tested by Malashenko (1976). He injected i.p. 5 m g / k g bw DES twice weekly for 10 weeks. The total administered dose was 100 mg/kg. In 2842 offspring of the control group he found no mutation and in 5042 offspring derived from spermatogonia treated with DES he observed 1 mutation at the dilute locus ( P = 0.64). The results summarized in Table 3 are a good example of the difficulty in evaluating specificlocus data if one used only a high dose point (de Serres, 1986). With the conventional permanent mating scheme after treatment (Searle, 1975) the great majority of all females would have conceived 1 - 4 days post treatment and a second litter would have been conceived 20-24 days after treatment. With such a mating scheme one would be unable to detect the mutagenicity of DES. Therefore it is not surprising that Russell (1984) came to the wrong conclusion that 'diethyl sulfate ... produced no mutations in this test'. For the evaluation of specific-locus data it is
important to have information of the mutation rate in all spermatogenic cell stages. In addition, it is necessary to have a sufficient number of offspring derived from the different germ cell stages. For the statistical evaluation it is mandatory to consider the results of each mating interval separately. Grouping all the data of the post-spermatogonial germ cell stages together for statistical analysis is not always meaningful and may lead to a wrong conclusion. Specific-locus mutations were 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, 1986). Knowing the resources necessary to determine the doubling dose in a specific-locus experiment it is worth while to compare the specific-locus results with other genetic endpoints. Based on the results of the 200-mg/kg group and the 5-8-day mating interval the doubling dose for specific-locus mutations for DES is 17 mg/kg. Using the regression coefficient for the same germ cell stage for the dominant-lethal assay (Table 1) gives a doubling dose of 22 mg/kg. The doubling dose for dominant lethals agrees well with the doubling dose for specific-locus mutations. Similar comparisons were made with other mutagens (Ehling, 1982). In general, these comparisons are encouraging. For a final recommendation, however, it is necessary to increase the data base for the determination of the doubling dose with different genetic endpoints.
Acknowledgements Experiments were supported by Contract ENV637-D(B) of the Commission of the European Communities. The able assistance of S. Frischholz, A. Kt~hler, A. Lauterbach, B. May, G. Schenke and K. Weiser is greatly appreciated.
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