Archives of
Arch. Toxicol. 38, 1-11 (1977)
TOXICOLOGY 9 by Springer-Verlag 1977
Dominant Lethal Mutations in Male Mice* U. H. Ehling Abteilung ffir Genetik der Gesellschaft ffir Strahlen- und Umweltforschung, D-8042 Neuherberg, Federal Republic of Germany
Abstract. Dominant lethal mutations are due to chromosome aberrations as demonstrated by analysis of first cleavage. With a sample size of 4 0 - 4 5 mice per dose the induction of dominant lethal mutations by 10 mg/kg of methyl methanesulfonat (MMS) can be detected in spermatids in the mating interval 9 - 1 2 days posttreatment (6-11%). In the same mating interval a dose of 150 mg/kg of MMS induces 100% dominant lethal mutations. MMS and other chemical mutagens can be characterized by their different spermatogenic response. The germ cell stage specific induction of dominant lethal mutations by chemical agents is very likely due to their different pathways and therefore, to different effects on the structural and macromolecular changes during spermatogenesis. The feasibility of standardizing test protocol for the dominant lethal assay in mice, based on collaborative studies, is discussed. The reproducibility of results and the sensitivity of the induction of dominant lethal mutations in the collaborative studies demonstrate the usefullness of the method for mutagenicity screening. Key words: Dominant lethal mutations - Dose-response-relationship - Differential spermatogenic response - Standardization - Mouse.
Zusammenfassung. Dominante Letalmutationen sind eine Folge von Chromosomenaberrationen, die sich im ersten Teilungsstadium nachweisen lassen. Mit einer Kollektivgr6f3e von 40--45 M/iusen pro Dosis kann die Induktion von dominanten Letalmutationen mit 10 mg/kg Methylmethansulfonat (MMS) in Spermatiden im Paarungsintervall 9 - 1 2 Tage nach der Behandlung nachgewiesen werden (6-11%). Im gleichen Paarungsintervall induziert eine Dosis von 150 mg/kg MMS 100% dominante Letalmutationen. MMS und andere chemische Mutagene zeichnen sich durch eine unterschiedliche Wirkung auf verschiedene Keimzellstadien aus. Die keimzellspezifische Induktion von dominanten Letalmutationen ist bedingt durch die unterschiedlichen Wirkungsmecha* Presented at the 3rd Meeting of the Gesellschaft f/Jr Umwelt-Mutationsforschung, D-8042 Neuherberg, July 1-2, 1976
u. H. Ehling nismen der Mutagene, die verschiedenartige Biosyntheseprozesse blockieren oder verschiedenartige pr/imutative Sch~idigungen induzieren. Die M6glichkeiten der Standardisierung des dominanten Letaltestes aufgrund der Erfahrung von Ringversuchen werden diskutiert. Die Reproduzierbarkeit der Ergebnisse und die Empfindlichkeit der Methode in den Ringversuchen beweisen, daf3 der dominante Letaltest eine sehr brauchbare Methode fiir die Mutagenit/itspriifung ist.
The basic purpose of mutagenicity testing is to obtain data relevant to human beings. Mammalian test systems are recommended for mutagenicity screening because, in principle, it is most desirable to be able to extrapolate the results of those systems closest to man. The mammalian test systems, which are most relevant for the evaluation of chemicals, measure the induction of mutations in germ cells. One test system which fullfills these criteria is the dominant lethal assay. The wide use of the dominant lethal assay for mutagenicity screening is due to the relative ease of mutation detection and the economy of the method. The method consists essentially of sequentially mated treated and untreated male mice with untreated female mice. Females are inspected daily for vaginal plugs, dissected on the 14th-16th day of pregnancy, scored for corpora lutea and for total implants comprising early and late deaths and living foetuses. The induction of dominant lethal mutations is determined by the increase of pre- and postimplantation loss of zygotes over the control. This simple procedure is an essential advantage of the test method. Experience has shown that, due to the simplicity of the method, mutagenicity tests were performed without determining biological parameters, which are important for the reproducibility of the results (Green and Springer, 1973). For mutagenicity screening it is necessary to develop a test protocol that is not based on the virtue of one laboratory, but on a collaborative study of several laboratories. Such a collaborative study was supported by the Bundesministerium fiir Forschung und Technologie, Bonn. The aim of the collaborative study, in which methyl methanesulfonat (MMS) (Buselmaier et al., 1975), cyclophosphamid (Buselmaler et al., 1976) and benzol (Ehling et al., 1977) were tested, is the standardization of the dominant lethal assay in male mice. Before discussing the principles of the standardized test protocol it is necessary to describe the nature of dominant lethal mutations.
Dominant Lethal Mutations
The term "dominant lethal mutation" is used to describe embryonic death resulting from chromosome breakage in parental germ cells. Any induced changes which affect the germ cells themselves or render the gametes incapable of participating in fertilization are excluded. The pioneer studies of Hertwig (1935) and her coworker Schaefer (1939) already indicated that litters sired during the presterile period of irradiated male mice were found to be of reduced size. Since there was no effect on sperm mobility and since the number of fertilized eggs was normal it was concluded that the reduced littersize was due to death of embryos after fertilization. Finding various nuclear and chromosomal abnormalities in cleavage stages lead to the con-
Dominant Lethal Mutations in Male Mice
3
clusion that embryonic death was due to chromosomal abnormalities, induced by irradiation in spermatozoa. The mechanisms of dominant lethal mutations were discussed in detail by L. B. Russell (1962). She wrote: "Breakage of a single chromosome in the sperm is probably followed by formation of an acentric fragment and a dicentric. The acentric presumably fails to be incorporated into one of the nuclei resulting from the first cleavage and may lie naked in the cytoplasm, or form a micronucleus, or be lost from the cells altogether. The dicentric may also form a micronucleus; or it may form a bridge either at the first cleavage or - if it is included whole in one blastomere nucleus - at a later cleavage. Such bridges can theoretically produce mechanical interferences with cleavage divisions; or they may initiate bridge-breakage-fusion cycles which eventually lead to loss of the chromosome. Breakage of two chromosomes that leads to an asymmetrical exchange also results in fragments and usually dicentrics at cleavage, with the eventual loss of two chromosomes from the nuclear complement of the blastomeres." From this description it follows that the manifestation of dominant lethal mutations occurs shortly before or after implantation. This observation can be confirmed in every experiment where dominant lethal muta tions are induced. To study the cytogenetic basis of chemically induced dominant lethal mutations, Brewen et al. (1975) treated young adult male mice with MMS. These males were sequentially mated to superovulated females from the 1st to 23rd day after injection. The morning after mating the females were sacrified and ova flushed from the ampulla. The ova were cultured, in the presence of colchicine, for 26 h and metaphase preparations were made of the first cleavage division. The types of aberrations seen were predominantly double fragments, chromatid interchanges, some chromatid deletions as well as a shattering effect on the male complement at 100 mg/kg of MMS during the peak sensitivity to induction of dominant lethal mutations. The results of a dominant lethal experiment and the study of Brewen et al. (1975) are compared in Figure 1. The comparison indicates a good correlation between the 100. 90. o"
80. 70.
Fig. 1. The yield of dominant lethal mutations against time after injection of male mice with MMS. The broken line represents the observed frequency of mutations after 80 mg/kg (Ehling, 1975). The solid line is calculated from aberration data after 100 mg/kg (Brewen et al., 1975). ~ = Chromosome aberrations (100 mg/kg MMS), (3- O = Dominant lethals (80 mg/kg MMS)
0
o
60. 50.
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< _z
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l F I
i t t
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o
.o
---.o J
10.
i
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;
i
10
12
14
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CONCEPTION IN DAYS AFTER TREATMENT
_
~
18
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u.H. Ehling
frequency of cells containing a cytologically visible aberration and the frequency of dominant lethal mutations. These data strongly suggest that chromosome aberrations observed at the first cleavage division are the basis of MMS-induced dominant lethal mutations.
MMS-lndueed Dominant Lethal Mutations
In the collaborative study the dose-effect-relation for MMS-induced dominant lethal mutations was studied with 0, 20, 40 and 80 mg/kg. The results of these experiments were discussed in detail by Machemer (1975). Additional experiments were performed in our laboratory to test the dose range of 5 - 2 0 mg/kg of MMS. In both sets of experiments the same procedure was used except for the mating scheme. In the collaborative study one male was mated to two untreated females, in the experiment summarized in Table 1, one male was mated with one untreated female. The experiments summarized in Table 1 were carried out as follows: (101 • C3H)Fl-male mice were injected intraperitoneally with a single dose of MMS in 1 ml distilled water per animal. The control group was given distilled water only. Immediately after the injection each male was mated with one untreated (101 • C3H)F 1 virgin female for 4 days. Additional three matings were made in 4day intervals. The age of the mice when first mated was 12-14 weeks. The statistical analysis of the data was performed by Vollmar according to a procedure described recently (Vollmar and Stucky, 1975). The mutation frequency was calculated as follows: frequency of dominant lethal mutations = 1 - live embryos per female in the experimental group divided by live embryos per female in the control group. If the treatment is ineffective this calculation should give a zero value but the sample size will, of course, result in statistically insignificant deviation from zero in positive and negative directions. The maximal deviation in the controls between different mating intervals is < 2.7%. A dose of 10 mg/kg of MMS in our laboratory is on the borderline of detectability. It does induce dominant lethal mutations in the mating interval 9 - 1 2 days posttreatment in a sample size of 4 0 - 4 5 animals. In the first experiment the increase is mainly due to postimplantation loss (P < 0.04), in the second experiment mainly to preimplantation loss (P < 0.03). In the sample size of 160 animals per mating interval the control data ( 1 - 4 days) are compared with the experimental data 5 - 8 days posttreatment. For comparison of the experimental data 9 - 1 2 days posttreatment the control data from the 13--16 day interval were used. This procedure did not increase the sensitivity of the assay. The induction of dominant lethal mutations with 20 mg/kg of MMS was demonstrated earlier (Ehling, 1975). The difference between the mating interval 9 - 1 2 days in the earlier study and Table 1 is due to the different mating schedule used in both experiments. Mating schedules influence the distribution of pregnant females. Drastic changes in the frequency of MMS-induced dominant lethal mutations during spermatogenesis are illustrated by Figure 1. The manifestation of dominant lethal mutations occurs shortly before or after implantation. The relation between pre- and postimplantation loss is dose dependent. In the low dose range the majority of dominant lethal mutations is due to postimplantation loss. With increasing doses the postimplantation loss decreases, whereas
1- 4 5- 8 9--12 13--16
1- 4 5- 8 9--12 13--16
I- 4 5-- 8 9--12 13--16
1-- 4 5-- 8 9--12 13--16
1-- 4 5-- 8
9--12 13--16
1-- 4 5-- 8
9--12 13--16
10
20
0
10
0 5
5 0
0 10
10 0
160 158
160 160
160 160
160 160
45 45 45 45
45 45 45 45
40 40 40 40
40 40 40 40
40 40 40 40
Number of females
Calculation is based on absolute figures
1-- 4 5- 8 9--12 13--16
Mating intervals (days)
0
Dose (mg/kg)
98.1 94.9
95.6 97.5
96.3 97.5
97.5 95.0
100.0 97.8 93.3 97.8
95.6 97.8 93.3 93.3
95.0 100.0 97.5 97.5
95.0 97.5 97.5 92.5
95.0 97.5 95,0 97.5
Fertile matings (%)
12.3 12.0
11.8 12.1
12.4 12.3
12.2 12.2
12.2 12.2 11.6 12.2
12.1 11.9 12.2 11.8
11.7 11.7 11.1 11.8
12.0 12.4 11.5 12.0
12.0 12.2 12.0 11.8
Corpora lutea per female
Table 1. MMS induction of dominant lethal mutations in (101 x C 3 H ) F 1 male mice
10.7 10.7
10.7 10.9
11.1 10.9
10.8 10.9
11.1 11.1 10.5 11.0
10.7 10.9 11.1 11.0
10.6 10.6 10.3 11.1
10.8 11.5 10.3 11.1
10.9 11.0 11.0 11.1
Implants per female
9.8 9.6
9.7 9.8
10.2 I0.0
9.9 9.9
9.9 10.0 9.3 9.8
9.8 9.8 10.0 9.9
9.3 9.0 8.4 9.9
9.9 10.1 9.1 9.7
9.9 10.2 10.2 10.0
Live embryos per female
9.1 9.9
9.6 10.1
8.4 8.4
8.7 9.4
10.8 9.4 11.1 11.0
8.9 9.6 10.1 10.0
12.2 15.4 18.5 11.1
8.0 12.3 12.2 12.2
-- 1.4 0
0 -- 1.7
-- 1.6 0
0 -- 0.04
-- 1.2 -- 1.8 6.7 1.1
0 0 0 0
6.6 12.1 17.9 1.3
0 1.0 10.9 3.2
0 0 0 0
(%)
(%) 8.9 7.5 7.4 9.5
D o m i n a n t lethal mutations
Dead implants
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~7
6
u.H. Ehling
the preimplantation loss increases drastically (Fig. 2). A dose of 150 mg/kg of MMS induced only preimplantation loss. The preimplantation loss was determined by Kratochvil (1975). She developed a method to distinguish between fertilized and unfertilized ova in the dominant lethal assay. The frequency of preimplantation loss agrees well with the frequency of highly damaged cells in the experiment of Brewen et al. (1975). The total induction of dominant lethal mutations is clearly dose-dependent as shown in Figure 2; also, the dose-effect-relationship is based on three different experiments (Table 1:10 and 20 mg/kg; Ehling, 1975:40 and 80 mg/kg; Ehling et al., 1968:150 mg/kg). The slight increase in total frequency of dominant lethal mutations between doses of 20 and 40 mg/kg may be due to differences in the proportion of pregnant females on a day to day basis. The induction of dominant lethal mutations by 10 mg/kg of MMS (Table 1) and by 20 mg/kg of MMS in the collaborative study (Machemer, 1975) demonstrates the sensitivity of this assay. In contrast to the dominant lethal results the determination of the lowest effective concentration with other methods is based on the results of a single laboratory. The frequency of micronuclei in mouse bone-marrow cells was significantly increased by 25 mg/kg of MMS (Matter and Grauwiler, 1974). Lang and Adler (1977) reported that heritable translocations in male mice are induced by 40 mg/kg of MMS. However, no other dose was tested in this study. Presumed somatic mutations in mice are induced by a dose of 50 mg/kg of MMS (Fahrig, 1975), but this could not be confirmed by L. B. Russell (1977).
Calculation of Dominant Lethal Mutations
Two different approaches are used to determine the frequency of dominant lethal mutations in male mice. One is based on postimplantation death, the other on both pre- and postimplantation loss. The index of dominant lethality based on postimplantation death alone was advocated by Bateman (1958), Epstein and Shafner (1968), and recently again by Searle and Beechey (1974). Support for this index comes from irradiation experiments in which Searle and Beechey (1974) found the decrease in implantations per female to be mainly due to failure of fertilization. This finding cannot, however, be generalized to apply to chemically induced dominant lethal mutations. The results of the MMS experiment (Fig. 2) clearly demonstrate that 100% preimplantation death of fertilized ova is observed in the mating interval 9-12 day posttreatment. The postimplantation index underestimates even the frequency of radiation induced dominant lethal mutations, as was already pointed out by L. B. Russell (1962). Certainly, for the detection of a chemical mutagen, possible underestimation of the mutation frequency should be avoided. Calculations based on comparisons of treated and control groups with respect to the ratio of living plus recently dead embryos to corpora lutea, used by W. L. Russell et al. (1954), or with respect to the number of live embryos per females (Ehling et al., 1968), include the pre- and postimplantation loss. The disadvantage of such calculations of dominant lethal frequency is that the formula cannot discriminate between preimplantation loss and unfertilized ova. However, no formula, by itself, can achieve such a differentiation: for the exact determination of dominant
Dominant Lethal Mutations in Male Mice
7
80
o 60
~
40
g 20
;o
~o
~o
'
~o
iso
' (rag/kg)
Fig. 2. Dose-effect-relationshipof MMS-induced dominant lethal mutations in spermatids of mice (9-12 days posttreatment).The dark area represents the postimplantationloss. The differencebetween total frequency(upper line) and postimplantation loss is due to the manifestationof lethal mutations before implantation
lethal frequency it is necessary to determine the rate of fertilized ova (Kratochvil, 1975). Increased frequency of non-fertilization may also be a useful indicator of possible hazard.
Differential Spermatogenie R e s p o n s e
One fascinating aspect of the study of induction of dominant lethal mutations in male mice is the similarity of drug action and spermatogenic response. The relationship between the yield of induced dominant lethal mutations in different spermatogenic stages after intraperitoneal injection of MMS was described in detail by Ehling et al. (1968). The two humped distribution of frequency of dominant lethal mutations in different spermatogenic stages (Fig. 1) was also observed in earlier experiments. The first hump ( 1 - 8 days) is very similar to the induction of dominant lethal mutations by Mitomen | (Ehling, 1974). The possible cause for the second hump ( 8 - 1 3 days) has not yet been identified. Chemical mutagens can be characterized by their different spermatogenic response. The germ cell stage specific induction of dominant lethal mutations with compounds examined in our laboratory is summarized in Table 2. The germ cell stage specific induction of dominant lethal mutations by chemical agents is very likely due to their different pathways and therefore, to their different effects on the structural and macromolecular changes during spermatogenesis. Distinct spermatogenic response can be used to predict possible interference of a c o m -
8
U. H. Ehling
Table 2. Spermatogenic response of mice to chemical mutagens Spermatogenic cell stage a) Compound
Structure
Spermatozoa Spermatids 1 7 days 8 2 days
Spermatocytes 22 35 days
Spermatogonia > 35 days
/CH2 CHz-CI CH 3 N~O "xCHz CH2 CI
Mitomen|
MMS
CH3-SOz-O-CH3
EMS
CH3-SO:-O CH2 CH3
I n PMS
Cyclophosphamid
CH3-SO2-O CH2-CH2-CH3
C1 CH2 CH,N ~_NoH C/HcH2 -~ ~CI CH2 CH~ "XO CH2 OH
5-FI . . . . . . . . i]
QHF
H o.~, IT F
o~;~c.~oH deoxyuridine CO NH CH CH3)2 Natulan| CH NH-NH CH Mitomy~dnC
i PMS
Busulfan
IH2N-,~((~H~(~HO/ NH2 J I [
./CH3
CH3 SO; 0 CB ~ . ~.CH 3 CH~ CH2-O SO2 CH3 / CH2 C H 2 0 SO2 (THa
a)
IIIIIIllll]ll]D~
..... ioo~
IIIIIILI!i i i i iJ III I! IIIIIIIIIl iiiiiiiii iiiiii IlllllIIIIIIII IIII iiii i i i i i i IIIIIIIIIIII lilllllllliii!ii ii _ _
Failure of fertilization
pound with specific inhibition of a biochemical synthesis during spermatogenesis (Ehling et al., 1972). A detailed description of the biochemical mechanisms involved in the germ cell specific induction of mutations was given elsewhere (Ehling, 1974). Similar observations on the germ cell specific induction of dominant lethal mutations with other compounds were reported by Frohberg and Schulze-Schencking (1974), Generoso (1974), R6hrborn (1970) and Malashenko (1971). From the differential spermatogenic response to induction of dominant lethal mutations it follows that, for the practical testing procedure for chemical mutagens, all germ cell stages must be tested. Furthermore, taking into account the drastic changes in the frequency of dominant lethal mutations even within a 24 h interval it is mandatory to use a sequential mating schedule of only a few days, preferably a 4 day mating interval. The 4 day mating interval corresponds with the average length of the estrous cycle of female mice (Bronson et al., 1966).
Dominant Lethal Mutations in Male Mice
9
Standardization of the Method
Evaluation of the collaborative studies with MMS (Machemer, 1975), cyclophosphamid (Buselmaier et al., 1976) and benzol (Ehling, 1977) lead to differentiation of criteria, which must be standardized, and criteria which can be only determined by the individual investigator. Standardization of the dominant lethal assay should include the following recommendations: In sequential matings of one control male with one or more females the highest frequency of fertile matings was observed in all laboratories when one male was caged with one female (Buselmaier et al., 1975; Ehling, 1977). The pair mating system is also the optimal choice for statistical reasons. Therefore, a I : 1 mating ratio is recommended. Comparing a 4 day sequential mating interval with a 7 day schedule, the collaborative study demonstrates that very few matings occur on the 5th, 6th and 7th day (Machemer, 1975) because the average length of the estrous cycle of the females is 4 days (Bronson et al., 1966). To achieve an approximately similar number of fertilized females each day after treatment, the 4 day sequential mating schedule is recommended. Other recommendations include the length of an experiment (48 days), the homogenity of the weights of the males, the handling of escaped or dead animals, the presentation of the results, the calculation of the frequency of dominant lethal mutations and the sensitivity of the experimental animals. For another feature of the dominant lethal assay, i.e. the optimal age of untreated females, it is impossible to give a general recommendation because the development of the embryos depends not only on the mother's genotype (Ehling, 1964; Frohberg, 1971) but also on the conditions in the animal quarters (Ehling, 1976). The endogenous and exogenous factors that are optimal for the dominant lethal assay must be determined for each independent screening unit. The diverse breeding conditions and the different genotypes used in various laboratories make it inadvisable to suggest a fixed number of mice for a dominant lethal test. It is mandatory to take the quality of the controls into account. To assure a reasonable sample size for mutagenicity testing it is necessary to develop statistical criteria (Vollmar, 1977) or to demand a sensitive positive control. It is insufficient to use a sample size that can detect the induction of dominant lethal mutations by 100 mg/kg of MMS. The positive control should be able to detect induction of dominant lethals by 20 mg/kg of MMS or less. Formulation of guidelines for the dominant lethal assay based on the results of the collaborative studies (Machemer, 1975; Buselmaier et al., 1976; Ehling, 1977), is still in progress. Some of the principles of the guidelines are mentioned above. The detailed version of the guidelines will be published in the near future in the Archives of Toxicology.
Conclusion
The recommended systems for mutagenicity testing are generally those used to demonstrate the effectiveness of a certain mutagen in a single laboratory (Committee 17,
10
U.H. Ehling
1975; Ramel, 1973; WHO, 1971). For mutagenicity screening it is necessary to develop a test protocol that is not based on the virtue of one laboratory, but on the collaborative study of several laboratories. Standardization, as far as possible, of the dominant lethal assay is an important aspect of the progress of mutagenicity testing. Standardization of the method should include determination of the sensitivity and the costs of testing a compound. Standardization of testing procedures based on collaborative studies are required for all mutagen screening systems. The standardized test protocols should form the foundation for an intra- and interspecies comparison of different methods with coded compounds. Such a comparison is mandatory for a rational selection of mutagen screening systems. The reproducibility of the results and the sensitivity of the induction of dominant lethal mutations in the collaborative studies demonstrate that the dominant lethal assay is a very useful method for mutagenicity screening.
Acknowledgements. The author wishes to acknowledge the able technical assistance of Miss S. Sitta and Miss M. Kr6hnert. The author is also indebted to Dipl. Math. J. Vollmar for statistical advice. The experimental work was supported by Forschungsvorhaben MT 407 b of the Bundesministerium fiir Forschung und Technologie and contract No. 066-74-1 ENVD of the E.C. Environmental Research Programme.
References Bateman, A. J.: Mutagenic sensitivity of maturing germ cells in the male mouse. Heredity 12, 213-232 (1958) Brewen, J. G., Payne, H. S., Jones, K. P., Preston, R. J.: Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic male germ cells. Mutation Res. 33, 239-250 (1975) Bronson, F. H., Dagg, C. P., Snell, G. D.: Reproduction. In: Biology of the laboratory mouse (E. L. Green, Ed.), pp. 187--204. New York: McGraw-Hill 1966 Buselmaier, W., Ehling, U. H., Frohberg, H., Lang, R., Lorke, D., Machemer, L., Matter, B. E., M/iller, D., RShrborn, G., Roll, R., Schulze-Schencking, M.: Methodik der Mutagenit~itspr/ifung I. GSFBericht B 564 (1975) Buselmaier, W., Dycka, J., Ehling, U. H., Fahrig, R., Frohberg, H., Lang, R., Lorke, D., Machemer, L., Matter, B. E., M/iller, D., R6hrborn, G., Roll, R., Schulze-Schencking, M.: Methodik der Mutagenit~itspriifung III. GSF-Bericht B 630 (1976) Committee 17: Environmental mutagenic hazards. Science 187, 503-514 (1975) Ehling, U. H.: Strain variation in reproductive capacity and radiation response of female mice. Radiat. Res. 23, 603-610 (1964) Ehling, U. H.: Differential spermatogenic response of mice to the induction of mutations by antineoplastic drugs. Mutation Res. 26, 285--295 (1974) Ehling, U. H.: Induktion dominanter Letalmutationen bei m~innlichen M~iusen durch Methylmethansulfonat (MMS). GSF-Bericht B 564, 29--31 (1975) Ehling, U. H.: Mutagenicity testing and risk estimation with mammals. Mutation Res. 41, 113-122 (1976) Ehling, U. H.: Mutagenitfitspr/ifung von Benzol im dominanten Letaltest der Maus. GSF-Bericht B 667 (1977) Ehling, U. H., Cumming, R. B., Mailing, H. V.: Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res. 5, 417--428 (1968) Ehling, U. H., Doherty, D. G., Malling, H. V.: Differential spermatogenic response of mice to the induction of dominant-lethal mutations by n-propyl methanesulfonate and isopropyl methanesulfonate. Mutation Res. 15, 175-184 (1972)
Dominant Lethal Mutations in Male Mice
11
Epstein, S. S., Shafner, H.: Chemical mutagens in the human environment. Nature (Lond.) 219, 385-387 (1968) Fahrig, R.: A mammalian spot test: Induction of genetic alterations in pigment cells of mouse embryos with X-rays and chemical mutagens. Molec. gen. Genet. 138, 309-314 (1975) Frohberg, H.: The evaluation of mutagenicity tests within the scope of toxicological trials. Arch. Toxikol. 28, 135-148 (1971) Frohberg, H., Schulze-Schencking, M.: Recent findings concerning dose response relationship in mutagenicity testing of chemicals. Arch. Toxicol. 32, 1-17 (1974) Generoso, W. M.: Chromosomal aberration effects of chemicals in mouse germ cells. In: Molecular and environmental aspects of mutagenesis (L. Prakash et al., Eds.), pp. 253-267. Springfield: Thomas 1974 Green, S., Springer, J. A.: The dominant-lethal test: Potential limitations and statistical considerations for safety evaluation. Environ. Hlth Perspect. 6, 37--50 (1973) Hertwig, P.: Sterilitfitserscheinungen bei rtntgenbestrahlten M~iusen. Z. indukt. Abstamm.- u. Vererb.L. 70, 517-523 (1935) Kratochvil, J.: Pr/iimplantativer Verlust dominanter Letalmutationen nach Behandlung der m~innlichen Maus mit Methylmethansulfonat. GSF-Bericht B 565, 5-17 (1975) Lang, R., Adler, I.-D.: Heritable translocation test and dominant-lethal assay in male mice with methyl methanesulfonate. Mutation Res. 48, 75--88 (1977) Machemer, L.: Collaborative study of methyl methanesulfonate mutagenicity with the dominant-lethal assay in mice. GSF-Bericht B 564, 7-28 (1975) Malashenko, A. M.: Sensitivity of mouse testis cells to the induction of dominant lethals by diethylsulphate. Genetika (USSR) 7, No. 3, 84-91 (1971) Matter, B. E., Grauwiler, J.: Micronuclei in mouse bone-narrow cells. A simple in vivo model for the evaluation of drug-induced chromosomal aberrations. Mutation Res. 23, 239-249 (1974) Ramel, C. (Ed.): Evaluation of genetic risks of environmental chemicals. Ambio Special Report 3 (1973) Rthrborn, G.: The activity of alkylating agents. I. Sensitive mutable stages in spermatogenesis and oogenesis. In: Chemical mutagenesis in mammals and man (F. Vogel, G. Rthrborn, Eds.), pp. 294-316. Berlin-Heidelberg-New York: Springer 1970 Russell, L. B.: Chromosome aberrations in experimental mammals. In: Progress in medical genetics, Vol. 2 (A. G. Steinberg, A. G. Bearn, Eds.), pp. 230--294. New York: Grune and Stratton 1962 Russell, L. B.: Validation of the in vivo somatic mutation method in the mouse as a prescreen for germinal point mutations, Arch. Toxicol. 38, 75-85 (1977) Russell, W. L., Russell, L. B., Kimball, A. W.: The relative effectiveness of neutrons from a nuclear detonation and from a cyclotron in inducing dominant lethals in the mouse. The Amer. Naturalist 88, 269-286 (1954) Schaefer, H.: Die Fertilit/it von M~iusem~innchen nach Bestrahlung mit 200 r. Z. mikr.-anat. Forsch. 46, 121-152 (1939) Searle, A. G., Beechey, C. V.: Sperm-count, egg-fertilization and dominant lethality after X-irradiation of mice. Mutation Res. 22, 63-72 (1974) Vollmar, J.: Statistical problems in mutagenicity tests. Arch. Toxicol. 38, 13-25 (1977) Vollmar, J., Stucky, W.: Zur statistischen Auswertung des dominanten Letaltests bei der m~innlichen Maus. GSF-Bericht B 565, 24-30 (1975) WHO (World Health Organization): Evaluation and testing of drugs for mutagenicity: Principles and problems. Techn. Rep. Ser. (Geneva) 482 (1971) Received December 21, 1976
Arch. Toxicol. 38, 1-11 (1977)
TOXICOLOGY 9 by Springer-Verlag 1977
Dominant Lethal Mutations in Male Mice* U. H. Ehling Abteilung ffir Genetik der Gesellschaft ffir Strahlen- und Umweltforschung, D-8042 Neuherberg, Federal Republic of Germany
Abstract. Dominant lethal mutations are due to chromosome aberrations as demonstrated by analysis of first cleavage. With a sample size of 4 0 - 4 5 mice per dose the induction of dominant lethal mutations by 10 mg/kg of methyl methanesulfonat (MMS) can be detected in spermatids in the mating interval 9 - 1 2 days posttreatment (6-11%). In the same mating interval a dose of 150 mg/kg of MMS induces 100% dominant lethal mutations. MMS and other chemical mutagens can be characterized by their different spermatogenic response. The germ cell stage specific induction of dominant lethal mutations by chemical agents is very likely due to their different pathways and therefore, to different effects on the structural and macromolecular changes during spermatogenesis. The feasibility of standardizing test protocol for the dominant lethal assay in mice, based on collaborative studies, is discussed. The reproducibility of results and the sensitivity of the induction of dominant lethal mutations in the collaborative studies demonstrate the usefullness of the method for mutagenicity screening. Key words: Dominant lethal mutations - Dose-response-relationship - Differential spermatogenic response - Standardization - Mouse.
Zusammenfassung. Dominante Letalmutationen sind eine Folge von Chromosomenaberrationen, die sich im ersten Teilungsstadium nachweisen lassen. Mit einer Kollektivgr6f3e von 40--45 M/iusen pro Dosis kann die Induktion von dominanten Letalmutationen mit 10 mg/kg Methylmethansulfonat (MMS) in Spermatiden im Paarungsintervall 9 - 1 2 Tage nach der Behandlung nachgewiesen werden (6-11%). Im gleichen Paarungsintervall induziert eine Dosis von 150 mg/kg MMS 100% dominante Letalmutationen. MMS und andere chemische Mutagene zeichnen sich durch eine unterschiedliche Wirkung auf verschiedene Keimzellstadien aus. Die keimzellspezifische Induktion von dominanten Letalmutationen ist bedingt durch die unterschiedlichen Wirkungsmecha* Presented at the 3rd Meeting of the Gesellschaft f/Jr Umwelt-Mutationsforschung, D-8042 Neuherberg, July 1-2, 1976
u. H. Ehling nismen der Mutagene, die verschiedenartige Biosyntheseprozesse blockieren oder verschiedenartige pr/imutative Sch~idigungen induzieren. Die M6glichkeiten der Standardisierung des dominanten Letaltestes aufgrund der Erfahrung von Ringversuchen werden diskutiert. Die Reproduzierbarkeit der Ergebnisse und die Empfindlichkeit der Methode in den Ringversuchen beweisen, daf3 der dominante Letaltest eine sehr brauchbare Methode fiir die Mutagenit/itspriifung ist.
The basic purpose of mutagenicity testing is to obtain data relevant to human beings. Mammalian test systems are recommended for mutagenicity screening because, in principle, it is most desirable to be able to extrapolate the results of those systems closest to man. The mammalian test systems, which are most relevant for the evaluation of chemicals, measure the induction of mutations in germ cells. One test system which fullfills these criteria is the dominant lethal assay. The wide use of the dominant lethal assay for mutagenicity screening is due to the relative ease of mutation detection and the economy of the method. The method consists essentially of sequentially mated treated and untreated male mice with untreated female mice. Females are inspected daily for vaginal plugs, dissected on the 14th-16th day of pregnancy, scored for corpora lutea and for total implants comprising early and late deaths and living foetuses. The induction of dominant lethal mutations is determined by the increase of pre- and postimplantation loss of zygotes over the control. This simple procedure is an essential advantage of the test method. Experience has shown that, due to the simplicity of the method, mutagenicity tests were performed without determining biological parameters, which are important for the reproducibility of the results (Green and Springer, 1973). For mutagenicity screening it is necessary to develop a test protocol that is not based on the virtue of one laboratory, but on a collaborative study of several laboratories. Such a collaborative study was supported by the Bundesministerium fiir Forschung und Technologie, Bonn. The aim of the collaborative study, in which methyl methanesulfonat (MMS) (Buselmaier et al., 1975), cyclophosphamid (Buselmaler et al., 1976) and benzol (Ehling et al., 1977) were tested, is the standardization of the dominant lethal assay in male mice. Before discussing the principles of the standardized test protocol it is necessary to describe the nature of dominant lethal mutations.
Dominant Lethal Mutations
The term "dominant lethal mutation" is used to describe embryonic death resulting from chromosome breakage in parental germ cells. Any induced changes which affect the germ cells themselves or render the gametes incapable of participating in fertilization are excluded. The pioneer studies of Hertwig (1935) and her coworker Schaefer (1939) already indicated that litters sired during the presterile period of irradiated male mice were found to be of reduced size. Since there was no effect on sperm mobility and since the number of fertilized eggs was normal it was concluded that the reduced littersize was due to death of embryos after fertilization. Finding various nuclear and chromosomal abnormalities in cleavage stages lead to the con-
Dominant Lethal Mutations in Male Mice
3
clusion that embryonic death was due to chromosomal abnormalities, induced by irradiation in spermatozoa. The mechanisms of dominant lethal mutations were discussed in detail by L. B. Russell (1962). She wrote: "Breakage of a single chromosome in the sperm is probably followed by formation of an acentric fragment and a dicentric. The acentric presumably fails to be incorporated into one of the nuclei resulting from the first cleavage and may lie naked in the cytoplasm, or form a micronucleus, or be lost from the cells altogether. The dicentric may also form a micronucleus; or it may form a bridge either at the first cleavage or - if it is included whole in one blastomere nucleus - at a later cleavage. Such bridges can theoretically produce mechanical interferences with cleavage divisions; or they may initiate bridge-breakage-fusion cycles which eventually lead to loss of the chromosome. Breakage of two chromosomes that leads to an asymmetrical exchange also results in fragments and usually dicentrics at cleavage, with the eventual loss of two chromosomes from the nuclear complement of the blastomeres." From this description it follows that the manifestation of dominant lethal mutations occurs shortly before or after implantation. This observation can be confirmed in every experiment where dominant lethal muta tions are induced. To study the cytogenetic basis of chemically induced dominant lethal mutations, Brewen et al. (1975) treated young adult male mice with MMS. These males were sequentially mated to superovulated females from the 1st to 23rd day after injection. The morning after mating the females were sacrified and ova flushed from the ampulla. The ova were cultured, in the presence of colchicine, for 26 h and metaphase preparations were made of the first cleavage division. The types of aberrations seen were predominantly double fragments, chromatid interchanges, some chromatid deletions as well as a shattering effect on the male complement at 100 mg/kg of MMS during the peak sensitivity to induction of dominant lethal mutations. The results of a dominant lethal experiment and the study of Brewen et al. (1975) are compared in Figure 1. The comparison indicates a good correlation between the 100. 90. o"
80. 70.
Fig. 1. The yield of dominant lethal mutations against time after injection of male mice with MMS. The broken line represents the observed frequency of mutations after 80 mg/kg (Ehling, 1975). The solid line is calculated from aberration data after 100 mg/kg (Brewen et al., 1975). ~ = Chromosome aberrations (100 mg/kg MMS), (3- O = Dominant lethals (80 mg/kg MMS)
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10
12
14
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CONCEPTION IN DAYS AFTER TREATMENT
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frequency of cells containing a cytologically visible aberration and the frequency of dominant lethal mutations. These data strongly suggest that chromosome aberrations observed at the first cleavage division are the basis of MMS-induced dominant lethal mutations.
MMS-lndueed Dominant Lethal Mutations
In the collaborative study the dose-effect-relation for MMS-induced dominant lethal mutations was studied with 0, 20, 40 and 80 mg/kg. The results of these experiments were discussed in detail by Machemer (1975). Additional experiments were performed in our laboratory to test the dose range of 5 - 2 0 mg/kg of MMS. In both sets of experiments the same procedure was used except for the mating scheme. In the collaborative study one male was mated to two untreated females, in the experiment summarized in Table 1, one male was mated with one untreated female. The experiments summarized in Table 1 were carried out as follows: (101 • C3H)Fl-male mice were injected intraperitoneally with a single dose of MMS in 1 ml distilled water per animal. The control group was given distilled water only. Immediately after the injection each male was mated with one untreated (101 • C3H)F 1 virgin female for 4 days. Additional three matings were made in 4day intervals. The age of the mice when first mated was 12-14 weeks. The statistical analysis of the data was performed by Vollmar according to a procedure described recently (Vollmar and Stucky, 1975). The mutation frequency was calculated as follows: frequency of dominant lethal mutations = 1 - live embryos per female in the experimental group divided by live embryos per female in the control group. If the treatment is ineffective this calculation should give a zero value but the sample size will, of course, result in statistically insignificant deviation from zero in positive and negative directions. The maximal deviation in the controls between different mating intervals is < 2.7%. A dose of 10 mg/kg of MMS in our laboratory is on the borderline of detectability. It does induce dominant lethal mutations in the mating interval 9 - 1 2 days posttreatment in a sample size of 4 0 - 4 5 animals. In the first experiment the increase is mainly due to postimplantation loss (P < 0.04), in the second experiment mainly to preimplantation loss (P < 0.03). In the sample size of 160 animals per mating interval the control data ( 1 - 4 days) are compared with the experimental data 5 - 8 days posttreatment. For comparison of the experimental data 9 - 1 2 days posttreatment the control data from the 13--16 day interval were used. This procedure did not increase the sensitivity of the assay. The induction of dominant lethal mutations with 20 mg/kg of MMS was demonstrated earlier (Ehling, 1975). The difference between the mating interval 9 - 1 2 days in the earlier study and Table 1 is due to the different mating schedule used in both experiments. Mating schedules influence the distribution of pregnant females. Drastic changes in the frequency of MMS-induced dominant lethal mutations during spermatogenesis are illustrated by Figure 1. The manifestation of dominant lethal mutations occurs shortly before or after implantation. The relation between pre- and postimplantation loss is dose dependent. In the low dose range the majority of dominant lethal mutations is due to postimplantation loss. With increasing doses the postimplantation loss decreases, whereas
1- 4 5- 8 9--12 13--16
1- 4 5- 8 9--12 13--16
I- 4 5-- 8 9--12 13--16
1-- 4 5-- 8 9--12 13--16
1-- 4 5-- 8
9--12 13--16
1-- 4 5-- 8
9--12 13--16
10
20
0
10
0 5
5 0
0 10
10 0
160 158
160 160
160 160
160 160
45 45 45 45
45 45 45 45
40 40 40 40
40 40 40 40
40 40 40 40
Number of females
Calculation is based on absolute figures
1-- 4 5- 8 9--12 13--16
Mating intervals (days)
0
Dose (mg/kg)
98.1 94.9
95.6 97.5
96.3 97.5
97.5 95.0
100.0 97.8 93.3 97.8
95.6 97.8 93.3 93.3
95.0 100.0 97.5 97.5
95.0 97.5 97.5 92.5
95.0 97.5 95,0 97.5
Fertile matings (%)
12.3 12.0
11.8 12.1
12.4 12.3
12.2 12.2
12.2 12.2 11.6 12.2
12.1 11.9 12.2 11.8
11.7 11.7 11.1 11.8
12.0 12.4 11.5 12.0
12.0 12.2 12.0 11.8
Corpora lutea per female
Table 1. MMS induction of dominant lethal mutations in (101 x C 3 H ) F 1 male mice
10.7 10.7
10.7 10.9
11.1 10.9
10.8 10.9
11.1 11.1 10.5 11.0
10.7 10.9 11.1 11.0
10.6 10.6 10.3 11.1
10.8 11.5 10.3 11.1
10.9 11.0 11.0 11.1
Implants per female
9.8 9.6
9.7 9.8
10.2 I0.0
9.9 9.9
9.9 10.0 9.3 9.8
9.8 9.8 10.0 9.9
9.3 9.0 8.4 9.9
9.9 10.1 9.1 9.7
9.9 10.2 10.2 10.0
Live embryos per female
9.1 9.9
9.6 10.1
8.4 8.4
8.7 9.4
10.8 9.4 11.1 11.0
8.9 9.6 10.1 10.0
12.2 15.4 18.5 11.1
8.0 12.3 12.2 12.2
-- 1.4 0
0 -- 1.7
-- 1.6 0
0 -- 0.04
-- 1.2 -- 1.8 6.7 1.1
0 0 0 0
6.6 12.1 17.9 1.3
0 1.0 10.9 3.2
0 0 0 0
(%)
(%) 8.9 7.5 7.4 9.5
D o m i n a n t lethal mutations
Dead implants
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6
u.H. Ehling
the preimplantation loss increases drastically (Fig. 2). A dose of 150 mg/kg of MMS induced only preimplantation loss. The preimplantation loss was determined by Kratochvil (1975). She developed a method to distinguish between fertilized and unfertilized ova in the dominant lethal assay. The frequency of preimplantation loss agrees well with the frequency of highly damaged cells in the experiment of Brewen et al. (1975). The total induction of dominant lethal mutations is clearly dose-dependent as shown in Figure 2; also, the dose-effect-relationship is based on three different experiments (Table 1:10 and 20 mg/kg; Ehling, 1975:40 and 80 mg/kg; Ehling et al., 1968:150 mg/kg). The slight increase in total frequency of dominant lethal mutations between doses of 20 and 40 mg/kg may be due to differences in the proportion of pregnant females on a day to day basis. The induction of dominant lethal mutations by 10 mg/kg of MMS (Table 1) and by 20 mg/kg of MMS in the collaborative study (Machemer, 1975) demonstrates the sensitivity of this assay. In contrast to the dominant lethal results the determination of the lowest effective concentration with other methods is based on the results of a single laboratory. The frequency of micronuclei in mouse bone-marrow cells was significantly increased by 25 mg/kg of MMS (Matter and Grauwiler, 1974). Lang and Adler (1977) reported that heritable translocations in male mice are induced by 40 mg/kg of MMS. However, no other dose was tested in this study. Presumed somatic mutations in mice are induced by a dose of 50 mg/kg of MMS (Fahrig, 1975), but this could not be confirmed by L. B. Russell (1977).
Calculation of Dominant Lethal Mutations
Two different approaches are used to determine the frequency of dominant lethal mutations in male mice. One is based on postimplantation death, the other on both pre- and postimplantation loss. The index of dominant lethality based on postimplantation death alone was advocated by Bateman (1958), Epstein and Shafner (1968), and recently again by Searle and Beechey (1974). Support for this index comes from irradiation experiments in which Searle and Beechey (1974) found the decrease in implantations per female to be mainly due to failure of fertilization. This finding cannot, however, be generalized to apply to chemically induced dominant lethal mutations. The results of the MMS experiment (Fig. 2) clearly demonstrate that 100% preimplantation death of fertilized ova is observed in the mating interval 9-12 day posttreatment. The postimplantation index underestimates even the frequency of radiation induced dominant lethal mutations, as was already pointed out by L. B. Russell (1962). Certainly, for the detection of a chemical mutagen, possible underestimation of the mutation frequency should be avoided. Calculations based on comparisons of treated and control groups with respect to the ratio of living plus recently dead embryos to corpora lutea, used by W. L. Russell et al. (1954), or with respect to the number of live embryos per females (Ehling et al., 1968), include the pre- and postimplantation loss. The disadvantage of such calculations of dominant lethal frequency is that the formula cannot discriminate between preimplantation loss and unfertilized ova. However, no formula, by itself, can achieve such a differentiation: for the exact determination of dominant
Dominant Lethal Mutations in Male Mice
7
80
o 60
~
40
g 20
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~o
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Fig. 2. Dose-effect-relationshipof MMS-induced dominant lethal mutations in spermatids of mice (9-12 days posttreatment).The dark area represents the postimplantationloss. The differencebetween total frequency(upper line) and postimplantation loss is due to the manifestationof lethal mutations before implantation
lethal frequency it is necessary to determine the rate of fertilized ova (Kratochvil, 1975). Increased frequency of non-fertilization may also be a useful indicator of possible hazard.
Differential Spermatogenie R e s p o n s e
One fascinating aspect of the study of induction of dominant lethal mutations in male mice is the similarity of drug action and spermatogenic response. The relationship between the yield of induced dominant lethal mutations in different spermatogenic stages after intraperitoneal injection of MMS was described in detail by Ehling et al. (1968). The two humped distribution of frequency of dominant lethal mutations in different spermatogenic stages (Fig. 1) was also observed in earlier experiments. The first hump ( 1 - 8 days) is very similar to the induction of dominant lethal mutations by Mitomen | (Ehling, 1974). The possible cause for the second hump ( 8 - 1 3 days) has not yet been identified. Chemical mutagens can be characterized by their different spermatogenic response. The germ cell stage specific induction of dominant lethal mutations with compounds examined in our laboratory is summarized in Table 2. The germ cell stage specific induction of dominant lethal mutations by chemical agents is very likely due to their different pathways and therefore, to their different effects on the structural and macromolecular changes during spermatogenesis. Distinct spermatogenic response can be used to predict possible interference of a c o m -
8
U. H. Ehling
Table 2. Spermatogenic response of mice to chemical mutagens Spermatogenic cell stage a) Compound
Structure
Spermatozoa Spermatids 1 7 days 8 2 days
Spermatocytes 22 35 days
Spermatogonia > 35 days
/CH2 CHz-CI CH 3 N~O "xCHz CH2 CI
Mitomen|
MMS
CH3-SOz-O-CH3
EMS
CH3-SO:-O CH2 CH3
I n PMS
Cyclophosphamid
CH3-SO2-O CH2-CH2-CH3
C1 CH2 CH,N ~_NoH C/HcH2 -~ ~CI CH2 CH~ "XO CH2 OH
5-FI . . . . . . . . i]
QHF
H o.~, IT F
o~;~c.~oH deoxyuridine CO NH CH CH3)2 Natulan| CH NH-NH CH Mitomy~dnC
i PMS
Busulfan
IH2N-,~((~H~(~HO/ NH2 J I [
./CH3
CH3 SO; 0 CB ~ . ~.CH 3 CH~ CH2-O SO2 CH3 / CH2 C H 2 0 SO2 (THa
a)
IIIIIIllll]ll]D~
..... ioo~
IIIIIILI!i i i i iJ III I! IIIIIIIIIl iiiiiiiii iiiiii IlllllIIIIIIII IIII iiii i i i i i i IIIIIIIIIIII lilllllllliii!ii ii _ _
Failure of fertilization
pound with specific inhibition of a biochemical synthesis during spermatogenesis (Ehling et al., 1972). A detailed description of the biochemical mechanisms involved in the germ cell specific induction of mutations was given elsewhere (Ehling, 1974). Similar observations on the germ cell specific induction of dominant lethal mutations with other compounds were reported by Frohberg and Schulze-Schencking (1974), Generoso (1974), R6hrborn (1970) and Malashenko (1971). From the differential spermatogenic response to induction of dominant lethal mutations it follows that, for the practical testing procedure for chemical mutagens, all germ cell stages must be tested. Furthermore, taking into account the drastic changes in the frequency of dominant lethal mutations even within a 24 h interval it is mandatory to use a sequential mating schedule of only a few days, preferably a 4 day mating interval. The 4 day mating interval corresponds with the average length of the estrous cycle of female mice (Bronson et al., 1966).
Dominant Lethal Mutations in Male Mice
9
Standardization of the Method
Evaluation of the collaborative studies with MMS (Machemer, 1975), cyclophosphamid (Buselmaier et al., 1976) and benzol (Ehling, 1977) lead to differentiation of criteria, which must be standardized, and criteria which can be only determined by the individual investigator. Standardization of the dominant lethal assay should include the following recommendations: In sequential matings of one control male with one or more females the highest frequency of fertile matings was observed in all laboratories when one male was caged with one female (Buselmaier et al., 1975; Ehling, 1977). The pair mating system is also the optimal choice for statistical reasons. Therefore, a I : 1 mating ratio is recommended. Comparing a 4 day sequential mating interval with a 7 day schedule, the collaborative study demonstrates that very few matings occur on the 5th, 6th and 7th day (Machemer, 1975) because the average length of the estrous cycle of the females is 4 days (Bronson et al., 1966). To achieve an approximately similar number of fertilized females each day after treatment, the 4 day sequential mating schedule is recommended. Other recommendations include the length of an experiment (48 days), the homogenity of the weights of the males, the handling of escaped or dead animals, the presentation of the results, the calculation of the frequency of dominant lethal mutations and the sensitivity of the experimental animals. For another feature of the dominant lethal assay, i.e. the optimal age of untreated females, it is impossible to give a general recommendation because the development of the embryos depends not only on the mother's genotype (Ehling, 1964; Frohberg, 1971) but also on the conditions in the animal quarters (Ehling, 1976). The endogenous and exogenous factors that are optimal for the dominant lethal assay must be determined for each independent screening unit. The diverse breeding conditions and the different genotypes used in various laboratories make it inadvisable to suggest a fixed number of mice for a dominant lethal test. It is mandatory to take the quality of the controls into account. To assure a reasonable sample size for mutagenicity testing it is necessary to develop statistical criteria (Vollmar, 1977) or to demand a sensitive positive control. It is insufficient to use a sample size that can detect the induction of dominant lethal mutations by 100 mg/kg of MMS. The positive control should be able to detect induction of dominant lethals by 20 mg/kg of MMS or less. Formulation of guidelines for the dominant lethal assay based on the results of the collaborative studies (Machemer, 1975; Buselmaier et al., 1976; Ehling, 1977), is still in progress. Some of the principles of the guidelines are mentioned above. The detailed version of the guidelines will be published in the near future in the Archives of Toxicology.
Conclusion
The recommended systems for mutagenicity testing are generally those used to demonstrate the effectiveness of a certain mutagen in a single laboratory (Committee 17,
10
U.H. Ehling
1975; Ramel, 1973; WHO, 1971). For mutagenicity screening it is necessary to develop a test protocol that is not based on the virtue of one laboratory, but on the collaborative study of several laboratories. Standardization, as far as possible, of the dominant lethal assay is an important aspect of the progress of mutagenicity testing. Standardization of the method should include determination of the sensitivity and the costs of testing a compound. Standardization of testing procedures based on collaborative studies are required for all mutagen screening systems. The standardized test protocols should form the foundation for an intra- and interspecies comparison of different methods with coded compounds. Such a comparison is mandatory for a rational selection of mutagen screening systems. The reproducibility of the results and the sensitivity of the induction of dominant lethal mutations in the collaborative studies demonstrate that the dominant lethal assay is a very useful method for mutagenicity screening.
Acknowledgements. The author wishes to acknowledge the able technical assistance of Miss S. Sitta and Miss M. Kr6hnert. The author is also indebted to Dipl. Math. J. Vollmar for statistical advice. The experimental work was supported by Forschungsvorhaben MT 407 b of the Bundesministerium fiir Forschung und Technologie and contract No. 066-74-1 ENVD of the E.C. Environmental Research Programme.
References Bateman, A. J.: Mutagenic sensitivity of maturing germ cells in the male mouse. Heredity 12, 213-232 (1958) Brewen, J. G., Payne, H. S., Jones, K. P., Preston, R. J.: Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic male germ cells. Mutation Res. 33, 239-250 (1975) Bronson, F. H., Dagg, C. P., Snell, G. D.: Reproduction. In: Biology of the laboratory mouse (E. L. Green, Ed.), pp. 187--204. New York: McGraw-Hill 1966 Buselmaier, W., Ehling, U. H., Frohberg, H., Lang, R., Lorke, D., Machemer, L., Matter, B. E., M/iller, D., RShrborn, G., Roll, R., Schulze-Schencking, M.: Methodik der Mutagenit~itspr/ifung I. GSFBericht B 564 (1975) Buselmaier, W., Dycka, J., Ehling, U. H., Fahrig, R., Frohberg, H., Lang, R., Lorke, D., Machemer, L., Matter, B. E., M/iller, D., R6hrborn, G., Roll, R., Schulze-Schencking, M.: Methodik der Mutagenit~itspriifung III. GSF-Bericht B 630 (1976) Committee 17: Environmental mutagenic hazards. Science 187, 503-514 (1975) Ehling, U. H.: Strain variation in reproductive capacity and radiation response of female mice. Radiat. Res. 23, 603-610 (1964) Ehling, U. H.: Differential spermatogenic response of mice to the induction of mutations by antineoplastic drugs. Mutation Res. 26, 285--295 (1974) Ehling, U. H.: Induktion dominanter Letalmutationen bei m~innlichen M~iusen durch Methylmethansulfonat (MMS). GSF-Bericht B 564, 29--31 (1975) Ehling, U. H.: Mutagenicity testing and risk estimation with mammals. Mutation Res. 41, 113-122 (1976) Ehling, U. H.: Mutagenitfitspr/ifung von Benzol im dominanten Letaltest der Maus. GSF-Bericht B 667 (1977) Ehling, U. H., Cumming, R. B., Mailing, H. V.: Induction of dominant lethal mutations by alkylating agents in male mice. Mutation Res. 5, 417--428 (1968) Ehling, U. H., Doherty, D. G., Malling, H. V.: Differential spermatogenic response of mice to the induction of dominant-lethal mutations by n-propyl methanesulfonate and isopropyl methanesulfonate. Mutation Res. 15, 175-184 (1972)
Dominant Lethal Mutations in Male Mice
11
Epstein, S. S., Shafner, H.: Chemical mutagens in the human environment. Nature (Lond.) 219, 385-387 (1968) Fahrig, R.: A mammalian spot test: Induction of genetic alterations in pigment cells of mouse embryos with X-rays and chemical mutagens. Molec. gen. Genet. 138, 309-314 (1975) Frohberg, H.: The evaluation of mutagenicity tests within the scope of toxicological trials. Arch. Toxikol. 28, 135-148 (1971) Frohberg, H., Schulze-Schencking, M.: Recent findings concerning dose response relationship in mutagenicity testing of chemicals. Arch. Toxicol. 32, 1-17 (1974) Generoso, W. M.: Chromosomal aberration effects of chemicals in mouse germ cells. In: Molecular and environmental aspects of mutagenesis (L. Prakash et al., Eds.), pp. 253-267. Springfield: Thomas 1974 Green, S., Springer, J. A.: The dominant-lethal test: Potential limitations and statistical considerations for safety evaluation. Environ. Hlth Perspect. 6, 37--50 (1973) Hertwig, P.: Sterilitfitserscheinungen bei rtntgenbestrahlten M~iusen. Z. indukt. Abstamm.- u. Vererb.L. 70, 517-523 (1935) Kratochvil, J.: Pr/iimplantativer Verlust dominanter Letalmutationen nach Behandlung der m~innlichen Maus mit Methylmethansulfonat. GSF-Bericht B 565, 5-17 (1975) Lang, R., Adler, I.-D.: Heritable translocation test and dominant-lethal assay in male mice with methyl methanesulfonate. Mutation Res. 48, 75--88 (1977) Machemer, L.: Collaborative study of methyl methanesulfonate mutagenicity with the dominant-lethal assay in mice. GSF-Bericht B 564, 7-28 (1975) Malashenko, A. M.: Sensitivity of mouse testis cells to the induction of dominant lethals by diethylsulphate. Genetika (USSR) 7, No. 3, 84-91 (1971) Matter, B. E., Grauwiler, J.: Micronuclei in mouse bone-narrow cells. A simple in vivo model for the evaluation of drug-induced chromosomal aberrations. Mutation Res. 23, 239-249 (1974) Ramel, C. (Ed.): Evaluation of genetic risks of environmental chemicals. Ambio Special Report 3 (1973) Rthrborn, G.: The activity of alkylating agents. I. Sensitive mutable stages in spermatogenesis and oogenesis. In: Chemical mutagenesis in mammals and man (F. Vogel, G. Rthrborn, Eds.), pp. 294-316. Berlin-Heidelberg-New York: Springer 1970 Russell, L. B.: Chromosome aberrations in experimental mammals. In: Progress in medical genetics, Vol. 2 (A. G. Steinberg, A. G. Bearn, Eds.), pp. 230--294. New York: Grune and Stratton 1962 Russell, L. B.: Validation of the in vivo somatic mutation method in the mouse as a prescreen for germinal point mutations, Arch. Toxicol. 38, 75-85 (1977) Russell, W. L., Russell, L. B., Kimball, A. W.: The relative effectiveness of neutrons from a nuclear detonation and from a cyclotron in inducing dominant lethals in the mouse. The Amer. Naturalist 88, 269-286 (1954) Schaefer, H.: Die Fertilit/it von M~iusem~innchen nach Bestrahlung mit 200 r. Z. mikr.-anat. Forsch. 46, 121-152 (1939) Searle, A. G., Beechey, C. V.: Sperm-count, egg-fertilization and dominant lethality after X-irradiation of mice. Mutation Res. 22, 63-72 (1974) Vollmar, J.: Statistical problems in mutagenicity tests. Arch. Toxicol. 38, 13-25 (1977) Vollmar, J., Stucky, W.: Zur statistischen Auswertung des dominanten Letaltests bei der m~innlichen Maus. GSF-Bericht B 565, 24-30 (1975) WHO (World Health Organization): Evaluation and testing of drugs for mutagenicity: Principles and problems. Techn. Rep. Ser. (Geneva) 482 (1971) Received December 21, 1976
