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Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[a]pyrene in postmeiotic germ cells of male mice
Mutation Research, 94 (1982) 155-163 Elsevier Biomedical Press
155
Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[a]pyrene in postmeiotic germ cells of male mice * W . M . G e n e r o s o , K.T. Cain, C.$. H e l l w i g a n d N.L.A. Cacheiro Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) (Received 10 July 1981) (Accepted 21 October 1981)
Summary Benzo[a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatozoa. In contrast to the dominant-lethal effects observed the study showed no detectable increase in heritable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.
BaP has been clearly shown to be an effective mutagen in a number of mammalian and submammalian genetic test systems whose end points are presumed to be indicative of point-mutational events. With respect to chromosomal aberration effects, on the other hand, data on BaP are relatively scarce and ambiguous. Results of the only three published cytogenetic studies, which were performed in mammalian in vitro cell culture systems, are conflicting. Two of these studies (Abe and Sasaki, 1977; Popescu et al., 1977) showed no chromosomal aberration effects, but the third (Dean, 1981) showed positive effects. Chromosomal aberration effects of BaP have By acceptance of this article, the publishers or recipient acknowledges the right of the U.S. Government to retain a nonexclnsive, royalty-free license in and to any copyright covering the article. * Research jointly sponsored by the Environmental Protection Agency under Interagency Agreement DS-E681-AN and the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7~lO5-eng-26 with the Union Carbide Corporation. Abbreviation: BaP, benzo[a]pyrene. 0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press
156
also been studied in germ cells of mice by use of dominant-lethal tests, but data, which were presented in only two papers, have been inadequate. In one of these reports (Epstein et al., 1972) the qualitative question of mutagenic effectiveness was not answered. The other report (Generoso et al., 1979a) showed that BaP is highly effective in inducing dominant-lethal mutations in certain male postmeiotic germ cells, but, because of the nature of this report, details of the experiment and results were not included. The present paper provides unequivocal evidence for dominantlethal effects of BaP, and, important from a basic standpoint, it shows another case of a chemical mutagen that is effective in inducing dominant-lethal mutations but is relatively ineffective in inducing heritable translocations in postmeiotic male germ cells.
Materials and methods
A preliminary acute toxicity study indicated that a dose of 500 mg/kg BaP was the maximum tolerated dose for a single i.p. injection. Even though it affected the ability of treated male mice to mate, this dose was used in all dominant-lethal and heritable translocation experiments. BaP was dissolved in corn oil and given in a maximum volume of 0.5 ml. The volume of injected solution was adjusted according to the weight of each animal. Each control male was given 0.4 ml of corn oil. In all experiments males were of the (101 × C3H)F~ stock and about 12 weeks old at the time of treatment. 3 series of dominant-lethal experiments were performed. The first was aimed at determining the sensitivity pattern of various stages of spermatogenesis. 32 experimental and 20 control males were used. Each male was caged with two 10-12-weekold virgin (101 × C3H)F~ females late in the afternoon of the day of injection. Females were checked for presence of vaginal plugs every morning for 48 days, and each mated female was replaced by a fresh one. Females that did not show a plug after 5 days in the cage with the male were also replaced. Mated females were killed for uterine analysis between the 12th and 15th day after the plug appeared. The lburpose of the second series of dominant-lethal experiments was to verify the most sensitive stage and to better quantitate the dominant-lethal effect at this stage. Accordingly, 32 experimental and 24 control males were each caged with two 10-12-week-old virgin (C3H × 101)F~ females beginning the day of injection until immediately after plugs were checked on the 8th day after injection. The third series was designed to determine the yield of dominant-lethal mutations when treated males were mated to different stocks of untreated females. 2 experiments were conducted. In the first, all matings in the experimental and control groups took place from 3.5 to 7.5 days after treatment, and males were mated with untreated virgin (C3H × C57BL)Fm, (SEC × C57BL)F~, or (C3H × 101)F~ females. In the second experiment, the same 3 stocks plus T-stock females were used; the experimental males were mated 3.5-6.5 days after treatment. However, because this BaP dominant-lethal experiment was conducted simultaneously with an experiment testing another mutagen, and because males treated with these 2 mutagens were
157
mated at different intervals after .treatment, the single control group of males was mated during an interval that covered part of each interval in the experimental groups, i.e., 1.5-4.5 days after treatment. In the heritable translocation study, BaP-treated males were mated to 2 stocks of female mice, (SEC × C57BL)F1 and (C3H × 101)FI, 3.5-7.5 days after treatment. The sequential procedure described by Generoso et al. (1981) was used to screen male progeny for translocation heterozygosity. Suspect males were analyzed cytologically.
Results
Induction of dominant-lethal mutations Results of the 3 series of dominant-lethal studies are shown in Tables 1-3. Data on the serial matings (Table 1) indicate dominant-lethal effects in the intervals 4.5-5.5 and 6.5-7.5 days, which correspond to early and middle spermatozoa, and possibly in the intervals 32.5-33.5 and 34.5-35.5 days, which correspond to preleptotene spermatocytes. Possible dominant-lethal effects are indicated by the lowerthan-control average number of living embryos (note that in the control the lowest average was 5.9), higher-than-control incidence of dead implantations, and the fact that the 2 earlier intervals and the 2 later intervals are consecutive. Because the dominant-lethal effects observed were low and because of the need for better quantitation of dominant-lethal effects for comparison with induction of heritable translocations, the second series of dominant-lethal experiments was conducted, in which only the spermatozoal stages were sampled (Table 2). Results show clear-cut dominant-lethal effects at matings in the interval 3.5-7.5 days after treatment. Additional evidence of dominant-lethal effects at these stages (series 3) is shown in Table 3. Furthermore, the data indicate that the yield of BaP-induced dominant-lethal mutations in these stages is affected by the stock of untreated females used. This phenomenon was explained in an earlier report (Generoso et al., 1979a) as possibly due to the differential ability of fertilized eggs to repair the BaP-induced lesions in the fertilizing sperm. Induction of heritable translocations Data for heritable translocations are shown in Table4. Of the total 548 male progeny analyzed from the 2 stocks of mothers, only 1 was found to be a carrier of a reciprocal translocation. This male came from an (SEC × C57BL)F~ mother. This incidence, even if based only on the number of progeny scored from this stock (i.e., 257), is not si~nificantly higher ( p = 0.11, Fischer's exact test) than the historical control frequency of 1 translocation in 4392 tested for this parental stock combination.
BaP, 500 m g / k g
Treatment
0.5- i.5 2.5- 3.5 4.5- 5.5 6.5- 7.5 8.5- 9.5 10.5-11.5 12.5-13.5 14.5-15.5 16.5-17.5 18.5-19.5 20.5-21.5 22.5-23.5 24.5-25.5 26.5-27.5 28.5-29.5 30.5-31.5 32.5-33.5 34.5-35.5 36.5-37.5 38.5-39.5 40.5-41.5
Treatment-tofertilization interval (days) 9 17 7 9 II 10 13 17 7 13 17 20 17 9 9 12 l0 7 6 19 10
Number of mated females
9 17 5 9 10 10 11 14 6 12 14 15 14 8 7 l0 l0 6 5" 17 8
N u m b e r of pregnant females
I N D U C T I O N OF D O M I N A N T - L E T H A L M U T A T I O N S IN M A L E G E R M CELLS a
TABLE I
7.3 7.5 5.8 6.7 7.9 7.0 8.8 7.4 7.0 7.3 6.7 7.3 6.6 6.6 7.1 6.5 6.9 6.0 6.2 6.2 6.3
N u m b e r of implantations per pregnant female 6.3 6.4 3.6 5.3 7.2 6.6 8.1 7.0 6.0 6.5 6.2 6.9 6.4 6.1 6.9 6.2 5.5 5.2 5.8 5.7 5.5
N u m b e r of living embryos per pregnant female
14 15 38 20 9 6 8 5 14 I1 8 6 3 8 4 5 20 14 7 9 12
Dead implants (~)
II 26 9 16 21 19 23 13 19 14 14 13 17 10 18 8 13 17 13 18 8 16 8 2
0.5- 1.5 2.5- 3.5 4.5- 5.5 6.5- 7.5 8 . 5 - 9.5 10.5-11.5 12.5-13.5 14.5-15.5 16.5-17.5 18.5-19.5 20.5-21.5 22.5-23.5 24.5-25.5 26.5-27.5 28.5-29.5 30.5-3 i.5 32.5-33.5" 34.5-35.5 36.5-37.5 38.5-39.5 4~.5-41.5 42.5-43.5 44.5-45.5 46.5-47.5
I1 26 9 16 19 19 20 13 18 14 14 13 17 10 16 8 12 15 13 14 8 16 8 2
11 9 4 7.7 8.2 7.2 7.8 7.7 7.3 7.8 8.0 7.7 7.6 7.6 7.4 7.0 7.5 6.8 6.6 6.4 6.8 6.7 7.0 6.8 7.4 7.0 6.5
7.3 6.3 7.8
a Males and females were of (101 XC3H)F~ stock. This experiment corresponds to series 1 in the text.
Control (corn oil)
12 9 4
42.5-43.5 44.5-45.5 46.5-47.5 7.2 7.7 7.1 7.3 7.1 6.6 7.2 7.5 7.1 7.3 7.2 6.7 6.1 6.9 6.3 6.0 5.9 6.3 6.0 6.2 6.0 6.7 6.6 6.5
7.0 5.8 7.0 7 7 2 7 9 9 8 7 8 4 6 9 14 8 7 9 8 8 10 11 11 l0 5 0
4 9 l0
0.5-2.5 3.5-7.5
0.5-2.5 3.5-7.5
BaP, 500 m g / k g
Control (corn oil)
33 46
32 39
Number of mated females
32 46
31 38
Number of pregnant females
8.0 7.8
7.9 5.9
Number of implantations per pregnant female
7.7 7.4
7.2 4.3
Number of living embryos per pregnant female
$g dominant-lethal mutations = [ I - living embryos per fertile female in experimental group ] [ living embryos per fertile female in control group J × 100.
b
a Males and females were of (101 ×C3H)Ft and ( C 3 H × 101)Ft stocks. This experiment corresponds to series 2 in the text. Percentage of induced dominant-lethal mutations was calculated as:
Treatment-tofertilization interval (days)
Treatment
INDUCTION OF DOMINANT-LETHAL MUTATIONS IN SPERMATOZOA a
TABLE 2
3 6
9 28
(g)
Dead implants
6 42
(~)
Dominantlethal mutations b
3.5-7.5 3.5-7.5 3.5-7.5 3.5-7.5 3.5-7.5 3.5-7.5 3.5-6.5 3.5-6.5 3.5-6.5 3.5-6.5 1.5-4.5 1.5-4.5 1.5-4.5 !.5-4.5
(C3H × C57BL)F~ (SEC × C57BL)FI (C3H× 101)F~
(C3HXC57BL)F I (SEC × C57BL)F~ (C3H × 101)F~
(C3H × C57BL)F1 (SEC × C57BL)F~ (C3HX 101)F~ T stock
(C3H × C57BL)F~ (SEC × C57BL)F~ (C3H× 101)F~ T stock
BaP, 500 mg/kg
Control (corn oil)
Bap, 500 mg/kg
Control (corn oil)
33 34 22 35
27 27 14 26
35 33 31
25 25 15
Number of mated females
33 23 22 34
26 24 13 19
34 27 28
23 20 14
Number of pregnant females
11.2 10.5 7.8 9.6
10.0 8.4 6.1 5.8
10.1 10.1 8.4
9.7 8.4 6.1
Number of implantations per pregnant female
• . [ riving embryos per fertile female in experimental group ] % dominant-lethal mutataons= [I living embryos per fertile female in control group J × 100.
a Males were of (101 ×C3H)F 1 stock. This experiment corresponds to series 3 in the text. b Percentage of induced dominant-lethal mutations was calculated as:
Experiment H
Experiment I
Treatment-tofertilization interval (days)
Stock of untreated females
Treatment
10.4 10.2 7.0 7.7
8.0 6.6 4.1 3.8
9.5 9.6 7.8
7.5 7.0 3.9
Number of living embryos per pregnant female
7 3 Il 20
20 2I 33 34
6 5 6
23 17 36
Dead implants (%)
23 35 41 51
21 27 50
Dominantlethal mutations b (%)
YIELD, OF DOMINANT-LETHAL MUTATIONS INDUCED IN MALE GERM CELLS IN DIFFERENT STOCKS OF UNTREATED FEMALE MICE a
TABLE 3
162 TABLE 4 I N D U C T I O N O F H E R I T A B L E T R A N S L O C A T I O N S IN M A L E POSTMEIOTIC G E R M CELLS a Stock of females
N u m b e r of progeny tested
N u m b e r of partially sterile progeny
N u m b e r of sterile progeny
(SEC × C57BL)F~ ( C 3 H × 101)F 1
257 291
0 0
2b 4c
a Male (101 × C3H)F~ mice were given a single i.p. injection of 500 mg B a P / k g and mated with untreated females 3.5-7.5 days after treatment. b 1 male was found cytologically to be a carrier of a reciprocal translocation; the other was cytologically normal. c These males were cytologically normal.
Discussion Data presented here strongly indicate that BaP has chromosomal aberration effects in certain male postmeiotic germ cells. The dominant-lethal effects found in treated spermatozoa seem unequivocal. It also seems that dominant-lethal mutations were detected in matings that occurred in the interval 32.5-35.5 days after treatment, although no further study was performed to verify these effects. If the BaP treatment had no effect on germ-cell development, it would appear from the data of Oakberg (1960) that this interval corresponds to spermatocytes which are in the preleptotene stage of meiosis. From the standpoint of mechanism, the significant result of the present study is the lack of association between the production of dominant-lethal mutations and the production of heritable translocations in BaP-treated middle and early spermatozoa. The yield of induced dominant-lethal mutations in the 2 stocks of females used in the corresponding heritable translocation experiments ranged from 27 to 35% for (SEC × C57BL)FI females and from 41 to 50% for (C3H × 101)FI females. On the other hand, the same BaP treatment did not produce a significant increase in the frequency of heritable translocations over background level. In the cases of ethyl methanesulfonate or triethylenemelamine, at least 10% of offspring could be expected to have reciprocal translocations at levels of dominant-lethal effects similar to those produced by BaP (Generoso et al., 1978). Thus, the present BaP results provide a second example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells. The first example was the alkylating chemical isopropyl methanesulfonate (Generoso et al., 1979b). We postulated earlier (Generoso et al., 1979b) that the relative rates at which dominant-lethal mutations and heritable translocations are produced from chemical treatment of postmeiotic male germ cells depend upon the longevity of induced
163
premutational lesions. Heritable translocations are produced at a high rate relative to dominant-lethal mutations when premutational lesions are converted into deletions and interchanges prior to the first postfertilization chromosome replication. Thus, the converse might explain the relative ineffectiveness of BaP in producing heritable translocations, i.e., the induced premutational lesions persisted to the time of pronuclear chromosome replication and possibly even to subsequent early cleavage divisions and led to dominant lethality.
Acknowledgements The authors acknowledge Drs. E.F. Oakberg, R.J. Preston and P.B. Selby for reviewing the manuscript.
References Abe, S., and M. Sasaki (1977) Studies in chromosomal aberrations and sister chromatid exchanges induced by chemicals, Proc. Jpn. Acad., 53, 46-49. Dean, B.J. (1981) The activity of 27 coded compounds in the RL t chromosome assay, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Ch. 52, Elsevier/North-Holland, New York, pp. 570-579. Epstein, S.S., E. Arnold, J. Andrea, W. Bass and Y. Bishop (1972) Detection of chemical mutagens by the dominant lethal assay in the mouse, .Toxicol. Appl. Pharmacol., 23, 288-325. Generoso, W.M., K.T. Cain, S.W. Huff and D.G. Gosslee (1978) Inducibility by chemical mutagens of heritable transloctions in male and female germ cells of mice, in: W.G. Flamm and M.A. Mehlman (Eds.), Advances in Modern Toxicology, Vol. 5, Hemisphere, Washington, DC, pp. 109-129. Generoso, W.M., K.T. Cain, M. Krishna and S.W. Huff (1979a) Genetic lesions induced by chemicals in spermatozoa and spermatids of mice are repaired in the egg, Proc. Natl. Acad. Sci. (U.S.A.), 76, 435-437. Generoso, W.M., S.W. Huff and K.T. Cain (1979b) Relative rates at which dominant-lethal mutations and heritable translocations are induced by alkylating chemicals in postmeiotic male germ cells of mice, Genetics, 93, 163-171. Generoso, W.M., M. Krishna, K.T. Cain and C.W. Sheu (1981) Comparison of two methods for detecting translocation heterozygotes in mice, Mutation Res., 81, ! 77-186. Oakberg, E.F. (1960) Irradiation damage to animals and its effect on their reproductive capacity, J. Dairy Sci., 43, Suppl., 54-67. Popescu, N.C., D. Turnbull and J.A. DiPaolo (1977) Sister chromatid exchanges/chromosome aberration analysis with the use of several carcinogens and noncarcinogens, J. Natl. Cancer Inst., 59, 289-293.
155
Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[a]pyrene in postmeiotic germ cells of male mice * W . M . G e n e r o s o , K.T. Cain, C.$. H e l l w i g a n d N.L.A. Cacheiro Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) (Received 10 July 1981) (Accepted 21 October 1981)
Summary Benzo[a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatozoa. In contrast to the dominant-lethal effects observed the study showed no detectable increase in heritable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.
BaP has been clearly shown to be an effective mutagen in a number of mammalian and submammalian genetic test systems whose end points are presumed to be indicative of point-mutational events. With respect to chromosomal aberration effects, on the other hand, data on BaP are relatively scarce and ambiguous. Results of the only three published cytogenetic studies, which were performed in mammalian in vitro cell culture systems, are conflicting. Two of these studies (Abe and Sasaki, 1977; Popescu et al., 1977) showed no chromosomal aberration effects, but the third (Dean, 1981) showed positive effects. Chromosomal aberration effects of BaP have By acceptance of this article, the publishers or recipient acknowledges the right of the U.S. Government to retain a nonexclnsive, royalty-free license in and to any copyright covering the article. * Research jointly sponsored by the Environmental Protection Agency under Interagency Agreement DS-E681-AN and the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7~lO5-eng-26 with the Union Carbide Corporation. Abbreviation: BaP, benzo[a]pyrene. 0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press
156
also been studied in germ cells of mice by use of dominant-lethal tests, but data, which were presented in only two papers, have been inadequate. In one of these reports (Epstein et al., 1972) the qualitative question of mutagenic effectiveness was not answered. The other report (Generoso et al., 1979a) showed that BaP is highly effective in inducing dominant-lethal mutations in certain male postmeiotic germ cells, but, because of the nature of this report, details of the experiment and results were not included. The present paper provides unequivocal evidence for dominantlethal effects of BaP, and, important from a basic standpoint, it shows another case of a chemical mutagen that is effective in inducing dominant-lethal mutations but is relatively ineffective in inducing heritable translocations in postmeiotic male germ cells.
Materials and methods
A preliminary acute toxicity study indicated that a dose of 500 mg/kg BaP was the maximum tolerated dose for a single i.p. injection. Even though it affected the ability of treated male mice to mate, this dose was used in all dominant-lethal and heritable translocation experiments. BaP was dissolved in corn oil and given in a maximum volume of 0.5 ml. The volume of injected solution was adjusted according to the weight of each animal. Each control male was given 0.4 ml of corn oil. In all experiments males were of the (101 × C3H)F~ stock and about 12 weeks old at the time of treatment. 3 series of dominant-lethal experiments were performed. The first was aimed at determining the sensitivity pattern of various stages of spermatogenesis. 32 experimental and 20 control males were used. Each male was caged with two 10-12-weekold virgin (101 × C3H)F~ females late in the afternoon of the day of injection. Females were checked for presence of vaginal plugs every morning for 48 days, and each mated female was replaced by a fresh one. Females that did not show a plug after 5 days in the cage with the male were also replaced. Mated females were killed for uterine analysis between the 12th and 15th day after the plug appeared. The lburpose of the second series of dominant-lethal experiments was to verify the most sensitive stage and to better quantitate the dominant-lethal effect at this stage. Accordingly, 32 experimental and 24 control males were each caged with two 10-12-week-old virgin (C3H × 101)F~ females beginning the day of injection until immediately after plugs were checked on the 8th day after injection. The third series was designed to determine the yield of dominant-lethal mutations when treated males were mated to different stocks of untreated females. 2 experiments were conducted. In the first, all matings in the experimental and control groups took place from 3.5 to 7.5 days after treatment, and males were mated with untreated virgin (C3H × C57BL)Fm, (SEC × C57BL)F~, or (C3H × 101)F~ females. In the second experiment, the same 3 stocks plus T-stock females were used; the experimental males were mated 3.5-6.5 days after treatment. However, because this BaP dominant-lethal experiment was conducted simultaneously with an experiment testing another mutagen, and because males treated with these 2 mutagens were
157
mated at different intervals after .treatment, the single control group of males was mated during an interval that covered part of each interval in the experimental groups, i.e., 1.5-4.5 days after treatment. In the heritable translocation study, BaP-treated males were mated to 2 stocks of female mice, (SEC × C57BL)F1 and (C3H × 101)FI, 3.5-7.5 days after treatment. The sequential procedure described by Generoso et al. (1981) was used to screen male progeny for translocation heterozygosity. Suspect males were analyzed cytologically.
Results
Induction of dominant-lethal mutations Results of the 3 series of dominant-lethal studies are shown in Tables 1-3. Data on the serial matings (Table 1) indicate dominant-lethal effects in the intervals 4.5-5.5 and 6.5-7.5 days, which correspond to early and middle spermatozoa, and possibly in the intervals 32.5-33.5 and 34.5-35.5 days, which correspond to preleptotene spermatocytes. Possible dominant-lethal effects are indicated by the lowerthan-control average number of living embryos (note that in the control the lowest average was 5.9), higher-than-control incidence of dead implantations, and the fact that the 2 earlier intervals and the 2 later intervals are consecutive. Because the dominant-lethal effects observed were low and because of the need for better quantitation of dominant-lethal effects for comparison with induction of heritable translocations, the second series of dominant-lethal experiments was conducted, in which only the spermatozoal stages were sampled (Table 2). Results show clear-cut dominant-lethal effects at matings in the interval 3.5-7.5 days after treatment. Additional evidence of dominant-lethal effects at these stages (series 3) is shown in Table 3. Furthermore, the data indicate that the yield of BaP-induced dominant-lethal mutations in these stages is affected by the stock of untreated females used. This phenomenon was explained in an earlier report (Generoso et al., 1979a) as possibly due to the differential ability of fertilized eggs to repair the BaP-induced lesions in the fertilizing sperm. Induction of heritable translocations Data for heritable translocations are shown in Table4. Of the total 548 male progeny analyzed from the 2 stocks of mothers, only 1 was found to be a carrier of a reciprocal translocation. This male came from an (SEC × C57BL)F~ mother. This incidence, even if based only on the number of progeny scored from this stock (i.e., 257), is not si~nificantly higher ( p = 0.11, Fischer's exact test) than the historical control frequency of 1 translocation in 4392 tested for this parental stock combination.
BaP, 500 m g / k g
Treatment
0.5- i.5 2.5- 3.5 4.5- 5.5 6.5- 7.5 8.5- 9.5 10.5-11.5 12.5-13.5 14.5-15.5 16.5-17.5 18.5-19.5 20.5-21.5 22.5-23.5 24.5-25.5 26.5-27.5 28.5-29.5 30.5-31.5 32.5-33.5 34.5-35.5 36.5-37.5 38.5-39.5 40.5-41.5
Treatment-tofertilization interval (days) 9 17 7 9 II 10 13 17 7 13 17 20 17 9 9 12 l0 7 6 19 10
Number of mated females
9 17 5 9 10 10 11 14 6 12 14 15 14 8 7 l0 l0 6 5" 17 8
N u m b e r of pregnant females
I N D U C T I O N OF D O M I N A N T - L E T H A L M U T A T I O N S IN M A L E G E R M CELLS a
TABLE I
7.3 7.5 5.8 6.7 7.9 7.0 8.8 7.4 7.0 7.3 6.7 7.3 6.6 6.6 7.1 6.5 6.9 6.0 6.2 6.2 6.3
N u m b e r of implantations per pregnant female 6.3 6.4 3.6 5.3 7.2 6.6 8.1 7.0 6.0 6.5 6.2 6.9 6.4 6.1 6.9 6.2 5.5 5.2 5.8 5.7 5.5
N u m b e r of living embryos per pregnant female
14 15 38 20 9 6 8 5 14 I1 8 6 3 8 4 5 20 14 7 9 12
Dead implants (~)
II 26 9 16 21 19 23 13 19 14 14 13 17 10 18 8 13 17 13 18 8 16 8 2
0.5- 1.5 2.5- 3.5 4.5- 5.5 6.5- 7.5 8 . 5 - 9.5 10.5-11.5 12.5-13.5 14.5-15.5 16.5-17.5 18.5-19.5 20.5-21.5 22.5-23.5 24.5-25.5 26.5-27.5 28.5-29.5 30.5-3 i.5 32.5-33.5" 34.5-35.5 36.5-37.5 38.5-39.5 4~.5-41.5 42.5-43.5 44.5-45.5 46.5-47.5
I1 26 9 16 19 19 20 13 18 14 14 13 17 10 16 8 12 15 13 14 8 16 8 2
11 9 4 7.7 8.2 7.2 7.8 7.7 7.3 7.8 8.0 7.7 7.6 7.6 7.4 7.0 7.5 6.8 6.6 6.4 6.8 6.7 7.0 6.8 7.4 7.0 6.5
7.3 6.3 7.8
a Males and females were of (101 XC3H)F~ stock. This experiment corresponds to series 1 in the text.
Control (corn oil)
12 9 4
42.5-43.5 44.5-45.5 46.5-47.5 7.2 7.7 7.1 7.3 7.1 6.6 7.2 7.5 7.1 7.3 7.2 6.7 6.1 6.9 6.3 6.0 5.9 6.3 6.0 6.2 6.0 6.7 6.6 6.5
7.0 5.8 7.0 7 7 2 7 9 9 8 7 8 4 6 9 14 8 7 9 8 8 10 11 11 l0 5 0
4 9 l0
0.5-2.5 3.5-7.5
0.5-2.5 3.5-7.5
BaP, 500 m g / k g
Control (corn oil)
33 46
32 39
Number of mated females
32 46
31 38
Number of pregnant females
8.0 7.8
7.9 5.9
Number of implantations per pregnant female
7.7 7.4
7.2 4.3
Number of living embryos per pregnant female
$g dominant-lethal mutations = [ I - living embryos per fertile female in experimental group ] [ living embryos per fertile female in control group J × 100.
b
a Males and females were of (101 ×C3H)Ft and ( C 3 H × 101)Ft stocks. This experiment corresponds to series 2 in the text. Percentage of induced dominant-lethal mutations was calculated as:
Treatment-tofertilization interval (days)
Treatment
INDUCTION OF DOMINANT-LETHAL MUTATIONS IN SPERMATOZOA a
TABLE 2
3 6
9 28
(g)
Dead implants
6 42
(~)
Dominantlethal mutations b
3.5-7.5 3.5-7.5 3.5-7.5 3.5-7.5 3.5-7.5 3.5-7.5 3.5-6.5 3.5-6.5 3.5-6.5 3.5-6.5 1.5-4.5 1.5-4.5 1.5-4.5 !.5-4.5
(C3H × C57BL)F~ (SEC × C57BL)FI (C3H× 101)F~
(C3HXC57BL)F I (SEC × C57BL)F~ (C3H × 101)F~
(C3H × C57BL)F1 (SEC × C57BL)F~ (C3HX 101)F~ T stock
(C3H × C57BL)F~ (SEC × C57BL)F~ (C3H× 101)F~ T stock
BaP, 500 mg/kg
Control (corn oil)
Bap, 500 mg/kg
Control (corn oil)
33 34 22 35
27 27 14 26
35 33 31
25 25 15
Number of mated females
33 23 22 34
26 24 13 19
34 27 28
23 20 14
Number of pregnant females
11.2 10.5 7.8 9.6
10.0 8.4 6.1 5.8
10.1 10.1 8.4
9.7 8.4 6.1
Number of implantations per pregnant female
• . [ riving embryos per fertile female in experimental group ] % dominant-lethal mutataons= [I living embryos per fertile female in control group J × 100.
a Males were of (101 ×C3H)F 1 stock. This experiment corresponds to series 3 in the text. b Percentage of induced dominant-lethal mutations was calculated as:
Experiment H
Experiment I
Treatment-tofertilization interval (days)
Stock of untreated females
Treatment
10.4 10.2 7.0 7.7
8.0 6.6 4.1 3.8
9.5 9.6 7.8
7.5 7.0 3.9
Number of living embryos per pregnant female
7 3 Il 20
20 2I 33 34
6 5 6
23 17 36
Dead implants (%)
23 35 41 51
21 27 50
Dominantlethal mutations b (%)
YIELD, OF DOMINANT-LETHAL MUTATIONS INDUCED IN MALE GERM CELLS IN DIFFERENT STOCKS OF UNTREATED FEMALE MICE a
TABLE 3
162 TABLE 4 I N D U C T I O N O F H E R I T A B L E T R A N S L O C A T I O N S IN M A L E POSTMEIOTIC G E R M CELLS a Stock of females
N u m b e r of progeny tested
N u m b e r of partially sterile progeny
N u m b e r of sterile progeny
(SEC × C57BL)F~ ( C 3 H × 101)F 1
257 291
0 0
2b 4c
a Male (101 × C3H)F~ mice were given a single i.p. injection of 500 mg B a P / k g and mated with untreated females 3.5-7.5 days after treatment. b 1 male was found cytologically to be a carrier of a reciprocal translocation; the other was cytologically normal. c These males were cytologically normal.
Discussion Data presented here strongly indicate that BaP has chromosomal aberration effects in certain male postmeiotic germ cells. The dominant-lethal effects found in treated spermatozoa seem unequivocal. It also seems that dominant-lethal mutations were detected in matings that occurred in the interval 32.5-35.5 days after treatment, although no further study was performed to verify these effects. If the BaP treatment had no effect on germ-cell development, it would appear from the data of Oakberg (1960) that this interval corresponds to spermatocytes which are in the preleptotene stage of meiosis. From the standpoint of mechanism, the significant result of the present study is the lack of association between the production of dominant-lethal mutations and the production of heritable translocations in BaP-treated middle and early spermatozoa. The yield of induced dominant-lethal mutations in the 2 stocks of females used in the corresponding heritable translocation experiments ranged from 27 to 35% for (SEC × C57BL)FI females and from 41 to 50% for (C3H × 101)FI females. On the other hand, the same BaP treatment did not produce a significant increase in the frequency of heritable translocations over background level. In the cases of ethyl methanesulfonate or triethylenemelamine, at least 10% of offspring could be expected to have reciprocal translocations at levels of dominant-lethal effects similar to those produced by BaP (Generoso et al., 1978). Thus, the present BaP results provide a second example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells. The first example was the alkylating chemical isopropyl methanesulfonate (Generoso et al., 1979b). We postulated earlier (Generoso et al., 1979b) that the relative rates at which dominant-lethal mutations and heritable translocations are produced from chemical treatment of postmeiotic male germ cells depend upon the longevity of induced
163
premutational lesions. Heritable translocations are produced at a high rate relative to dominant-lethal mutations when premutational lesions are converted into deletions and interchanges prior to the first postfertilization chromosome replication. Thus, the converse might explain the relative ineffectiveness of BaP in producing heritable translocations, i.e., the induced premutational lesions persisted to the time of pronuclear chromosome replication and possibly even to subsequent early cleavage divisions and led to dominant lethality.
Acknowledgements The authors acknowledge Drs. E.F. Oakberg, R.J. Preston and P.B. Selby for reviewing the manuscript.
References Abe, S., and M. Sasaki (1977) Studies in chromosomal aberrations and sister chromatid exchanges induced by chemicals, Proc. Jpn. Acad., 53, 46-49. Dean, B.J. (1981) The activity of 27 coded compounds in the RL t chromosome assay, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Ch. 52, Elsevier/North-Holland, New York, pp. 570-579. Epstein, S.S., E. Arnold, J. Andrea, W. Bass and Y. Bishop (1972) Detection of chemical mutagens by the dominant lethal assay in the mouse, .Toxicol. Appl. Pharmacol., 23, 288-325. Generoso, W.M., K.T. Cain, S.W. Huff and D.G. Gosslee (1978) Inducibility by chemical mutagens of heritable transloctions in male and female germ cells of mice, in: W.G. Flamm and M.A. Mehlman (Eds.), Advances in Modern Toxicology, Vol. 5, Hemisphere, Washington, DC, pp. 109-129. Generoso, W.M., K.T. Cain, M. Krishna and S.W. Huff (1979a) Genetic lesions induced by chemicals in spermatozoa and spermatids of mice are repaired in the egg, Proc. Natl. Acad. Sci. (U.S.A.), 76, 435-437. Generoso, W.M., S.W. Huff and K.T. Cain (1979b) Relative rates at which dominant-lethal mutations and heritable translocations are induced by alkylating chemicals in postmeiotic male germ cells of mice, Genetics, 93, 163-171. Generoso, W.M., M. Krishna, K.T. Cain and C.W. Sheu (1981) Comparison of two methods for detecting translocation heterozygotes in mice, Mutation Res., 81, ! 77-186. Oakberg, E.F. (1960) Irradiation damage to animals and its effect on their reproductive capacity, J. Dairy Sci., 43, Suppl., 54-67. Popescu, N.C., D. Turnbull and J.A. DiPaolo (1977) Sister chromatid exchanges/chromosome aberration analysis with the use of several carcinogens and noncarcinogens, J. Natl. Cancer Inst., 59, 289-293.