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Tests for dominant-lethal effects of 1,2-dibromo-3-chloropropane (DBCP) in male and female mice
Mutation Research, 156 (1985) 103-108
103
Elsevier
MTR 00960
Tests for dominant-lethal effects of 1,2-dibromo-3-chloropropane (DBCP) in male and female mice * W.M. Generoso, K.T. Cain and L.A. Hughes Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (U.S.A.) (Received 26 September 1984) (Accepted 15 November 1984)
Summary DBCP was studied for dominant-lethal effects in male and female mice and for total reproductive effects in females. In males it was administered either intraperitoneally or subcutaneously while in females it was given only by the former route. No DBCP-related response was observed in either males or females indicating its ineffectiveness in inducing chromosomal aberrations or cytotoxicity in mouse germ cells. These findings differ markedly from the observations made in rats by other investigators. Thus, the probable existence of a species difference in germ cell response to DBCP has been strengthened by the availability of the present results. It should be noted, however, that only two stocks of male mice have been studied so far for dominant-lethal and germ cell cytotoxicity effects.
Three separate studies in male rats indicate that 1,2-dibromo-3-chloropropane (DBCP) is clearly an inducer of dominant-lethal mutations in this species (Teramoto et al., 1980; Saito-Suzuki et al., 1982; Rao et al., 1983). Thus, when the only published study in male mice reported no evidence of a dominant-lethal response (Teramoto et al., 1980), one wonders if there is truly a large species difference or if a clear positive response in mice might be observed under different experimental conditions. With respect to the latter possibility, * Research sponsored jointly by the National Toxicology Program under NIEHS Interagency Agreement Y01-ES-20085 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-AC05-84OR21400 with the Martin Marietta Energy Systems, Inc. By acceptance of this article, the publisher or recipient acknowledges the U.S. Government's tight to retain a nonexclusive, royalty-free license in and to any copyright coveting the article.
two factors to be considered are the strains of male and female mice (Generoso et al., 1983) and routes of administration. With respect to the first possibility it should be emphasized that, although the number of mutagenic chemicals that have been studied to date for dominant-lethal effects in both male mice and rats has been small, there had been no case in which a clear-cut dominant-lethal inducer in one species was totally ineffective in the other. Teramoto et al. (1980) suggested that the mouse-rat difference they observed in dominantlethal response to DBCP may be attributable to a species-specific metabolic pathway. This could very well be true if, indeed, there is species difference in dominant-lethal response but this possibility needs to be established more firmly before any attempt to study the basis for the difference is made. Accordingly, we investigated the response of the mouse, both male and female, to dominant-lethal and reproductive effects of DBCP.
0165-1218/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
104
Materials and methods
DBCP was provided by the National Toxicology Program chemical repository, the Radian Corporation, Austin, TX. In the male dominantlethal study DBCP was prepared in corn oil and administered either intraperitoneally or subcutaneously in a maximum volume of 0.5 ml or 0.2 ml, respectively. In the female dominant-lethal study DBCP was prepared in tricaprylin (Eastman) and injected intraperitoneally in a maximum volume of 0.2 ml per mouse. For the total reproductive capacity experiment, DBCP was prepared in corn oil and injected intraperitoneally to females in a maximum volume of 0.5 ml. Prior to conducting these experiments, a 30-day acute toxicity study was performed in order to determine the maximum tolerated dose. In the male dominant-lethal study, two experiments were conducted. In one experiment, 48 (101 x C3H)F 1 male mice, about 12 weeks old, were given daily injections (intraperitoneally) of 80 m g / k g DBCP for 5 consecutive days. Of these, 1 mouse died within 1 day after receiving the last dose. Immediately after the last injection, 42 of the survivors and the same number of control males (injected with 0.5 ml of corn oil) were caged individually with 2 virgin females (also about 12 weeks of age) from one of the following stocks: (SEC x C57BL)F1, (SEE x 101)F~, (101 x C3H)F1, or T stock. Females were checked for vaginal plugs each morning for 42 days following the last day of treatment. Uterine analysis was performed 12-15 days after observation of the vaginal plug. In the other experiment, 30 (C3H × 101)F1 males were given single subcutaneous injection of 200 m g / k g DBCP in corn oil. The treated males and the same number of control males were each caged with 1 T-stock and I (SEC x C57BL)F1 female during the period 14-30 days after treatment. Females mated during this period were replaced with fresh ones. Germ cell effects in females were studied using the total reproductive capacity and the dominantlethal tests. In the former test, 72 (C3H x C57BL)F1 females were divided randomly into two equal groups and given a single intraperitoneal injection of either 110 m g / k g DBCP or 0.5 ml corn oil. Each female was caged on the day of injection with a male from (SEC x C57BL)F~ stock.
Litters produced during 1 year after treatment were recorded soon after birth and discarded immediately. In the dominant-lethal study T-stock and (C3H x C57BL)F1 females were given single intraperitoneal injection of 110 m g / k g DBCP in tricaprylin. Control females received 0.2 ml of tricaprylin. 48 females were used in each group. They were mated to (C3H x C57BL)F 1 males during 6 days post treatment. It should be noted that the sensitivity of oocytes to dominant-lethal effects of mutagenic chemicals increases with advancing follicular development (Generoso, 1969, 1973). Results
The various doses used in the experiments were based on the results of 30-day acute toxicity studies shown in Table 1. In the treatment group in which 80 m g / k g dose was given intraperitoneally daily for 5 consecutive days, 1 mouse out of 12 died immediately after the second dose. In the male dominant-lethal experiment in which this treatment regimen was used 1 died out of 48 males treated shortly after receiving the last dose. In the male dominant-lethal experiment in which DBCP was injected subcutaneously, the dose of 200 m g / k g administered once, killed 6 out of 36 mice treated although in the acute toxicity study none TABLE 1 SURVIVAL OF DBCP-TREATED MALE MICE Dose ~ (mg/kg)
Route of administration
Number of treated males
Number of survivors b
300 250 200 150 100
Subcutaneous Subcutaneous Subcutaneous Subcutaneous Subcutaneous
12 12 18 12 12
0 5 18 12 12
150 130 110 90 80 x 5 a
Intraperitoneally Intraperitoneally Intraperitoneally Intraperitoneally Intraperitoneally
12 12 12 12 12
1 6 12 12 11 c
a Male (C3H x 101)F 1 mice were given single injection except those given 80 m g / k g per day for 5 consecutive days (80 x 5). b Survival was scored during a 30-day post-treatment observation period. ¢ The only death was recorded immediately after the injection of the second dose.
105 TABLE 2 D O M I N A N T - L E T H A L STUDY IN MALES WITH DBCP A D M I N I S T E R E D I N T R A P E R I T O N E A L L Y Treatment a
Strain of females
Treatment to fertilization interval (days)
Number of mated females
Number of pregnant females
Number of implantations per pregant female
DBCP
(SEC x C57BL)F1
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5 20.5-23.5 24.5-27.5 28.5-31.5 32.5-35.5 36.5-39.5 40.5-41.5
48 25 31 31 41 34 38 53 33 43 12
38 22 23 26 32 27 30 42 20 28 8
9.9 9.5 10.2 9.3 9.8 10.2 9.6 9.0 8.5 8.9 9.3
9.7 9.1 9.7 9.0 9.4 9.6 9.3 8.7 8.2 8.5 8.6
2.1 3.8 4.7 3.3 3.8 6.2 3.1 3.4 3.0 5.2 6.8
Corn oil
(SEC x C57BL)F 1
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5 20.5-23.5 24.5-27.5 28.5-31.5 32.5-35.5 36.5-39.5 40.5-41.5
42 29 34 36 29 31 41 45 39 51 18
36 25 27 29 23 23 28 34 28 34 17
10.0 9.8 10.4 9.7 10.3 9.5 8.8 9.1 9.9 9.2 8.2
9.7 9.5 10.0 9.4 10.1 9.2 8.5 8.7 9.5 8.5 7.3
2.5 2.9 4.3 2.8 2.1 3.2 3.2 3.9 4.0 7.4 10.8
DBCP
T stock
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5
40 28 51 42 45
34 28 48 39 43
9.1 9.2 9.2 9.3 9.1
7.6 7.4 7.6 8.0 7.5
17.1 19.5 17.9 14.0 17.4
(SEC × 101)F1
20.5-23.5 24.5-27.5 28.5-30.5
48 31 22
33 23 16
8.4 7.5 6.9
8.1 7.1 6.9
4.0 5.8 0.9
(101 × C3H)F l
31.5-33.5 34.5-37.5 38.5-41.5
24 31 25
19 28 23
7.4 7.8 7.2
6.7 6.9 6.4
8.6 11.5 10.9
T stock
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5
48 33 46 38 43
42 31 45 37 39
9.4 9.1 9.3 8.8 9.4
7.4 7.3 7.2 7.3 7.8
21.3 20.1 22.5 17.5 16.4
(SEC x 101)F1
20.5-23.5 24.5-27.5 28.5-30.5
44 34 18
30 25 14
8.5 7.4 6.9
8.3 7.2 6.6
3.1 3.2 4.1
(101 × C3H)F 1
31.5-33.5 34.5-37.5 38.5-41.5
28 39 27
22 34 23
7.5 7.8 7.5
6.7 7.1 6.5
11.4 8.7 13.3
Corn oil
Number of living embryos per pregnant female
Dead implants (%)
a 42 (101 X C3H)F 1 males were given daily injections of 80 m g / k g DBCP for 5 consecutive days. The same number of control males were given 0.5 ml of corn oil daily.
106 TABLE 3 DOMINANT-LETHAL STUDY IN MALE MICE WITH DBCP ADMINISTERED SUBCUTANEOUSLY Treatment a
Strain of females
Treatment to fertilization interval (days)
Number of mated females
Number of pregnant females
Number of implantations per pregnant female
Number of living embryos per pregnant female
Dead implants (%)
DBCP
T stock
14.5-17.5 18.5-21.5 22.5-25.5 26.5-29.5
30 21 28 18
30 21 26 18
8.9 9.1 8.1 7.9
7.8 7.0 6.0 6.9
12.0 22.5 26.2 13.3
(SEC × C57BL)F 1
14.5-17.5 18.5-21.5 22.5-25.5 26.5-29.5
32 30 29 19
23 24 22 14
9.3 8.6 9.0 9.4
9.0 8.3 8.8 9.2
4.2 4.3 2.5 2.3
T stock
14.5-17.5 18.5-21.5 22.5-25.5 26.5-29.5
31 25 27 14
27 24 25 13
8.7 8.2 8.4 8.0
7.1 7.2 7.0 6.5
17.9 11.7 16.6 19.2
(SEC × C57BL)F 1
14.5-17.5 18.5-21.5 22.5-25.5 26.5 -29.5
36 21 18 18
30 14 12 11
9.8 9.1 9.3 7.2
9.3 8.9 9.1 6.9
4.8 2.3 1.8 3.8
Corn oil
a 30 (C3H x 101)F1 males were given single subcutaneous injection of 200 mg/kg DBCP. The same number of control males were given 0.2 ml of corn oil each.
d i e d a m o n g t h e 12 t r e a t e d . I n t h e f e m a l e d o m i -
d a t a t h a t D B C P d i d n o t i n d u c e a d e t e c t a b l e in-
nant-lethal and total reproductive capacity experi-
crease in d o m i n a n t - l e t h a l m u t a t i o n s irrespective of
m e n t s n o n e died as a result of D B C P toxicity out o f 144 a n d 36 t r e a t e d , r e s p e c t i v e l y .
w h e t h e r it w a s a d m i n i s t e r e d i n t r a p e r i t o n e a l l y o r
Results of the d o m i n a n t - l e t h a l studies in m a l e s
a n d total r e p r o d u c t i v e c a p a c i t y results in female m i c e ( T a b l e s 4 a n d 5) i n d i c a t e a l a c k o f e f f e c t i n
a r e s h o w n i n T a b l e s 2 a n d 3. It is c l e a r f r o m t h e
subcutaneously.
Similarly,
both
dominant-lethal
TABLE 4 DOMINANT-LETHAL STUDIES IN FEMALE MICE Expt.
Treatment a
Stock of females
Number of mated females b
Number of pregnant females
Number of implantations per pregnant female
Number of living embryos per pregnant female
Dead implants (%)
I
DBCP Control
T stock T stock
42 44
36 41
8.5 8.4
6.9 7.3
19.2 13.4
II
DBCP Control DBCP Control
(C3H x C57BL)F~ (C3H x C57BL)F 1 T stock T stock
46 37 45 47
33 29 41 42
9.4 9.4 8.5 8.2
8.6 8.9 6.9 6.9
8.4 5.5 19.4 15.9
a DBCP at 110 mg/kg was given as a single i.p. injection. Controls received 0.2 ml tricaprylin. b All females were mated to (C3H × C57BL)F~ males. All matings occurred during the post-treatment interval 0.5-5.5 days.
107 TABLE 5 REPRODUCTIVE PERFORMANCE OF DBCP-TREATED FEMALES a Post-
DBCP b
Control ¢
treatment interval (days)
Number of fecund females
Number of young born per females
Number of fecund females
Number of young born per female
18-24 25-43 44-62 63-81 82-100 101-119 120-138 139-157 158-176 177-195 196-214 215-233 234-252 253-271 272-290 291-309 310-328 329-347 348-366
35 26 28 31 23 34 32 32 33 28 26 26 29 11 20 12 9 8 3
9.2 9.3 9.8 10.0 9.6 10.7 10.9 10.2 9.8 9.4 7.8 7.0 5.3 4.2 4.3 3.7 2.4 2.9 3.0
34 27 30 32 28 29 33 30 34 31 28 31 26 25 19 17 15 9 4
8.8 9.5 9.9 9.7 10.6 10.1 10.4 9.5 10.4 9.8 8.3 6.7 5.3 4.6 3.6 3.0 2.8 2.9 2.5
a 36 (C3H :,
female germ cells. The total reproductive capacity test in female mice detects d o m i n a n t - l e t h a l a n d oocyte killing effects a n d reproductive effects associated with i n d u c e d changes in the m a t e r n a l physiology.
Discussion T h e question of whether or n o t D B C P is capable of p r o d u c i n g genetic a n d cytotoxic damage i n m o u s e germ cells has b e e n clarified further with the availability of the present data. T h e difference b e t w e e n mice a n d rats in this respect was reviewed recently by O a k b e r g a n d C u m m i n g s (1984). While it appears that D B C P clearly induces d o m i n a n t lethals i n male rats ( T e r a m o t o et al., 1980; SaitoSuzuki, 1982; R a o et al., 1983), the present results add strength to the likelihood that it has n o effect in male mice. Also the lack of a n y D B C P - i n d u c e d reproductive response observed d u r i n g the 42 days
p o s t - t r e a t m e n t m a t i n g period is consistent with the cytological data of Oakberg a n d C u m m i n g s (1984). I n the rat studies, D B C P was a d m i n i s t e r e d either orally or b y inhalation. I n the mouse, the T e r a m o t o et al. (1980) study had D B C P administered orally a n d in the present study it was a d m i n i s t e r e d intraperitoneally or subcutaneously. The most sensitive rat germ cells to D B C P are the early spermatids. I n all mouse studies the sensitivity of these germ cells was also studied. C o n t r a r y to the results in males, data in females do n o t suggest large species difference b e t w e e n mice a n d rats. I n the present m o u s e study, DBCP, given i n t r a p e r i t o n e a l l y at the m a x i m u m tolerated c o n c e n t r a t i o n , does n o t appear to have either d o m i n a n t - l e t h a l or oocyte cytotoxic effects. Similarly, in female rats, in which D B C P was given by i n h a l a t i o n at a c o n c e n t r a t i o n a n d t r e a t m e n t regim e n s that p r o d u c e d d o m i n a n t - l e t h a l response in males, n o r e d u c t i o n in litter sizes was observed
108
suggesting the lack of substantial dominant-lethal or cytotoxic effects (Rao et al., 1983). Thus, it appears that there is a marked sex difference in the response of rats to dominant-lethal and germ cell cytotoxicity effects. Similar sex differences in response to a mutagenic chemical, EMS, have been observed before in mice (Generoso and Russell, 1969). The intriguing question is why do male mice and rats differ greatly in their response to DBCP. Perhaps it is significant that in the rat both dominant-lethal and cytotoxic effects were observed. The germ cell stages involved for these two types of DBCP-induced damage are presumably different. One explanation was offered by Teramoto et al. (1980) - - i.e., there exist species-dependent pathways in DBCP metabolism. Indeed, evidence seems to suggest that DBCP is an indirect mutagen that is converted in vivo into a reactive alkylating intermediate (see review by Whorton and Foliart, 1983). Another possible explanation could be that young spermatids and earlier spermatogenic stages of mice can repair DBCP-induced D N A lesions more effectively than corresponding stages in the rat. However, while there is an indication that premeiotic germ cells of male mice exposed to DBCP are capable of carrying out unscheduled D N A synthesis (Lee and Suzuki, 1979), no comparable information is available in the rat. Thus, neither explanation is supported by evidence. It should be emphasized that only two stocks of male mice have been studied so far for dominant-lethal and cytotoxicity effects.
References Generoso, W.M. (1969) Chemical induction of dominant lethals
in female mice, Genetics, 61,461-470. Generoso, W.M. (1973) Total reproductive capacity procedure in female mice, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 3, Plenum, New York, pp. 241-258. Generoso, W.M., and W.L. Russell (1969) Strain and sex variations in the sensitivity of mice to dominant-lethal induction with ethyl methanesulfonate, Mutation Res., 8,
589-598. Generoso, W.M., K.T. Cain and A.J. Bandy (1983) Some factors affecting the mutagenic response of mouse germ cells to chemicals, in: F.J. de Serres and W. Sheridan (Eds), Environmental Science Research, Utilization of Mammalian Specific Locus Studies in Hazard Evaluation and Estimation of Genetic Risk, Vol. 28, Plenum, New York, pp. 227-239. Lee, I.P., and K. Suzuki (1979) Induction of unscheduled DNA synthesis in mouse germ cells following 1,2-dibromo-3-chloropropane (DBCP) exposure, Mutation Res., 68, 169-173. Oakberg, E.F., and C.C. Cummings (1984) Lack of effect of dibromochloropropane on the mouse testis, Environ. Mutagen., 6, 621-625. Rao, K.S., J.D. Burek, J.A. John, B.A. Schwetz, T.J. Bell, W.J. Potts and C.M. Parker (1983) Toxicologic and reproductive effects of inhaled 1,2-dibromo-3-chloropropane in rats, Fund. Appl. Toxicol., 3, 104-110. Saito-Suzuki, R., S. Teramoto and Y. Shirasu (1982) Dominant-lethal studies in rats with 1,2-dibromo-3-chloropropane and its structurally related compounds, Mutation Res., 101, 321-327. Teramoto, S., R. Saito, H. Aoyama and Y. Shirasu (1980) Dominant-lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane (DBCP), Mutation Res., 77, 71-78. Whorton, M.D., and D.E. Foliart (1983) Mutagenicity, carcinogenicity and reproductive effect of dibromochloropropane (DBCP), Mutation Res., 123, 13-30.
103
Elsevier
MTR 00960
Tests for dominant-lethal effects of 1,2-dibromo-3-chloropropane (DBCP) in male and female mice * W.M. Generoso, K.T. Cain and L.A. Hughes Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (U.S.A.) (Received 26 September 1984) (Accepted 15 November 1984)
Summary DBCP was studied for dominant-lethal effects in male and female mice and for total reproductive effects in females. In males it was administered either intraperitoneally or subcutaneously while in females it was given only by the former route. No DBCP-related response was observed in either males or females indicating its ineffectiveness in inducing chromosomal aberrations or cytotoxicity in mouse germ cells. These findings differ markedly from the observations made in rats by other investigators. Thus, the probable existence of a species difference in germ cell response to DBCP has been strengthened by the availability of the present results. It should be noted, however, that only two stocks of male mice have been studied so far for dominant-lethal and germ cell cytotoxicity effects.
Three separate studies in male rats indicate that 1,2-dibromo-3-chloropropane (DBCP) is clearly an inducer of dominant-lethal mutations in this species (Teramoto et al., 1980; Saito-Suzuki et al., 1982; Rao et al., 1983). Thus, when the only published study in male mice reported no evidence of a dominant-lethal response (Teramoto et al., 1980), one wonders if there is truly a large species difference or if a clear positive response in mice might be observed under different experimental conditions. With respect to the latter possibility, * Research sponsored jointly by the National Toxicology Program under NIEHS Interagency Agreement Y01-ES-20085 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-AC05-84OR21400 with the Martin Marietta Energy Systems, Inc. By acceptance of this article, the publisher or recipient acknowledges the U.S. Government's tight to retain a nonexclusive, royalty-free license in and to any copyright coveting the article.
two factors to be considered are the strains of male and female mice (Generoso et al., 1983) and routes of administration. With respect to the first possibility it should be emphasized that, although the number of mutagenic chemicals that have been studied to date for dominant-lethal effects in both male mice and rats has been small, there had been no case in which a clear-cut dominant-lethal inducer in one species was totally ineffective in the other. Teramoto et al. (1980) suggested that the mouse-rat difference they observed in dominantlethal response to DBCP may be attributable to a species-specific metabolic pathway. This could very well be true if, indeed, there is species difference in dominant-lethal response but this possibility needs to be established more firmly before any attempt to study the basis for the difference is made. Accordingly, we investigated the response of the mouse, both male and female, to dominant-lethal and reproductive effects of DBCP.
0165-1218/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
104
Materials and methods
DBCP was provided by the National Toxicology Program chemical repository, the Radian Corporation, Austin, TX. In the male dominantlethal study DBCP was prepared in corn oil and administered either intraperitoneally or subcutaneously in a maximum volume of 0.5 ml or 0.2 ml, respectively. In the female dominant-lethal study DBCP was prepared in tricaprylin (Eastman) and injected intraperitoneally in a maximum volume of 0.2 ml per mouse. For the total reproductive capacity experiment, DBCP was prepared in corn oil and injected intraperitoneally to females in a maximum volume of 0.5 ml. Prior to conducting these experiments, a 30-day acute toxicity study was performed in order to determine the maximum tolerated dose. In the male dominant-lethal study, two experiments were conducted. In one experiment, 48 (101 x C3H)F 1 male mice, about 12 weeks old, were given daily injections (intraperitoneally) of 80 m g / k g DBCP for 5 consecutive days. Of these, 1 mouse died within 1 day after receiving the last dose. Immediately after the last injection, 42 of the survivors and the same number of control males (injected with 0.5 ml of corn oil) were caged individually with 2 virgin females (also about 12 weeks of age) from one of the following stocks: (SEC x C57BL)F1, (SEE x 101)F~, (101 x C3H)F1, or T stock. Females were checked for vaginal plugs each morning for 42 days following the last day of treatment. Uterine analysis was performed 12-15 days after observation of the vaginal plug. In the other experiment, 30 (C3H × 101)F1 males were given single subcutaneous injection of 200 m g / k g DBCP in corn oil. The treated males and the same number of control males were each caged with 1 T-stock and I (SEC x C57BL)F1 female during the period 14-30 days after treatment. Females mated during this period were replaced with fresh ones. Germ cell effects in females were studied using the total reproductive capacity and the dominantlethal tests. In the former test, 72 (C3H x C57BL)F1 females were divided randomly into two equal groups and given a single intraperitoneal injection of either 110 m g / k g DBCP or 0.5 ml corn oil. Each female was caged on the day of injection with a male from (SEC x C57BL)F~ stock.
Litters produced during 1 year after treatment were recorded soon after birth and discarded immediately. In the dominant-lethal study T-stock and (C3H x C57BL)F1 females were given single intraperitoneal injection of 110 m g / k g DBCP in tricaprylin. Control females received 0.2 ml of tricaprylin. 48 females were used in each group. They were mated to (C3H x C57BL)F 1 males during 6 days post treatment. It should be noted that the sensitivity of oocytes to dominant-lethal effects of mutagenic chemicals increases with advancing follicular development (Generoso, 1969, 1973). Results
The various doses used in the experiments were based on the results of 30-day acute toxicity studies shown in Table 1. In the treatment group in which 80 m g / k g dose was given intraperitoneally daily for 5 consecutive days, 1 mouse out of 12 died immediately after the second dose. In the male dominant-lethal experiment in which this treatment regimen was used 1 died out of 48 males treated shortly after receiving the last dose. In the male dominant-lethal experiment in which DBCP was injected subcutaneously, the dose of 200 m g / k g administered once, killed 6 out of 36 mice treated although in the acute toxicity study none TABLE 1 SURVIVAL OF DBCP-TREATED MALE MICE Dose ~ (mg/kg)
Route of administration
Number of treated males
Number of survivors b
300 250 200 150 100
Subcutaneous Subcutaneous Subcutaneous Subcutaneous Subcutaneous
12 12 18 12 12
0 5 18 12 12
150 130 110 90 80 x 5 a
Intraperitoneally Intraperitoneally Intraperitoneally Intraperitoneally Intraperitoneally
12 12 12 12 12
1 6 12 12 11 c
a Male (C3H x 101)F 1 mice were given single injection except those given 80 m g / k g per day for 5 consecutive days (80 x 5). b Survival was scored during a 30-day post-treatment observation period. ¢ The only death was recorded immediately after the injection of the second dose.
105 TABLE 2 D O M I N A N T - L E T H A L STUDY IN MALES WITH DBCP A D M I N I S T E R E D I N T R A P E R I T O N E A L L Y Treatment a
Strain of females
Treatment to fertilization interval (days)
Number of mated females
Number of pregnant females
Number of implantations per pregant female
DBCP
(SEC x C57BL)F1
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5 20.5-23.5 24.5-27.5 28.5-31.5 32.5-35.5 36.5-39.5 40.5-41.5
48 25 31 31 41 34 38 53 33 43 12
38 22 23 26 32 27 30 42 20 28 8
9.9 9.5 10.2 9.3 9.8 10.2 9.6 9.0 8.5 8.9 9.3
9.7 9.1 9.7 9.0 9.4 9.6 9.3 8.7 8.2 8.5 8.6
2.1 3.8 4.7 3.3 3.8 6.2 3.1 3.4 3.0 5.2 6.8
Corn oil
(SEC x C57BL)F 1
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5 20.5-23.5 24.5-27.5 28.5-31.5 32.5-35.5 36.5-39.5 40.5-41.5
42 29 34 36 29 31 41 45 39 51 18
36 25 27 29 23 23 28 34 28 34 17
10.0 9.8 10.4 9.7 10.3 9.5 8.8 9.1 9.9 9.2 8.2
9.7 9.5 10.0 9.4 10.1 9.2 8.5 8.7 9.5 8.5 7.3
2.5 2.9 4.3 2.8 2.1 3.2 3.2 3.9 4.0 7.4 10.8
DBCP
T stock
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5
40 28 51 42 45
34 28 48 39 43
9.1 9.2 9.2 9.3 9.1
7.6 7.4 7.6 8.0 7.5
17.1 19.5 17.9 14.0 17.4
(SEC × 101)F1
20.5-23.5 24.5-27.5 28.5-30.5
48 31 22
33 23 16
8.4 7.5 6.9
8.1 7.1 6.9
4.0 5.8 0.9
(101 × C3H)F l
31.5-33.5 34.5-37.5 38.5-41.5
24 31 25
19 28 23
7.4 7.8 7.2
6.7 6.9 6.4
8.6 11.5 10.9
T stock
0.5-3.5 4.5-7.5 8.5-11.5 12.5-15.5 16.5-19.5
48 33 46 38 43
42 31 45 37 39
9.4 9.1 9.3 8.8 9.4
7.4 7.3 7.2 7.3 7.8
21.3 20.1 22.5 17.5 16.4
(SEC x 101)F1
20.5-23.5 24.5-27.5 28.5-30.5
44 34 18
30 25 14
8.5 7.4 6.9
8.3 7.2 6.6
3.1 3.2 4.1
(101 × C3H)F 1
31.5-33.5 34.5-37.5 38.5-41.5
28 39 27
22 34 23
7.5 7.8 7.5
6.7 7.1 6.5
11.4 8.7 13.3
Corn oil
Number of living embryos per pregnant female
Dead implants (%)
a 42 (101 X C3H)F 1 males were given daily injections of 80 m g / k g DBCP for 5 consecutive days. The same number of control males were given 0.5 ml of corn oil daily.
106 TABLE 3 DOMINANT-LETHAL STUDY IN MALE MICE WITH DBCP ADMINISTERED SUBCUTANEOUSLY Treatment a
Strain of females
Treatment to fertilization interval (days)
Number of mated females
Number of pregnant females
Number of implantations per pregnant female
Number of living embryos per pregnant female
Dead implants (%)
DBCP
T stock
14.5-17.5 18.5-21.5 22.5-25.5 26.5-29.5
30 21 28 18
30 21 26 18
8.9 9.1 8.1 7.9
7.8 7.0 6.0 6.9
12.0 22.5 26.2 13.3
(SEC × C57BL)F 1
14.5-17.5 18.5-21.5 22.5-25.5 26.5-29.5
32 30 29 19
23 24 22 14
9.3 8.6 9.0 9.4
9.0 8.3 8.8 9.2
4.2 4.3 2.5 2.3
T stock
14.5-17.5 18.5-21.5 22.5-25.5 26.5-29.5
31 25 27 14
27 24 25 13
8.7 8.2 8.4 8.0
7.1 7.2 7.0 6.5
17.9 11.7 16.6 19.2
(SEC × C57BL)F 1
14.5-17.5 18.5-21.5 22.5-25.5 26.5 -29.5
36 21 18 18
30 14 12 11
9.8 9.1 9.3 7.2
9.3 8.9 9.1 6.9
4.8 2.3 1.8 3.8
Corn oil
a 30 (C3H x 101)F1 males were given single subcutaneous injection of 200 mg/kg DBCP. The same number of control males were given 0.2 ml of corn oil each.
d i e d a m o n g t h e 12 t r e a t e d . I n t h e f e m a l e d o m i -
d a t a t h a t D B C P d i d n o t i n d u c e a d e t e c t a b l e in-
nant-lethal and total reproductive capacity experi-
crease in d o m i n a n t - l e t h a l m u t a t i o n s irrespective of
m e n t s n o n e died as a result of D B C P toxicity out o f 144 a n d 36 t r e a t e d , r e s p e c t i v e l y .
w h e t h e r it w a s a d m i n i s t e r e d i n t r a p e r i t o n e a l l y o r
Results of the d o m i n a n t - l e t h a l studies in m a l e s
a n d total r e p r o d u c t i v e c a p a c i t y results in female m i c e ( T a b l e s 4 a n d 5) i n d i c a t e a l a c k o f e f f e c t i n
a r e s h o w n i n T a b l e s 2 a n d 3. It is c l e a r f r o m t h e
subcutaneously.
Similarly,
both
dominant-lethal
TABLE 4 DOMINANT-LETHAL STUDIES IN FEMALE MICE Expt.
Treatment a
Stock of females
Number of mated females b
Number of pregnant females
Number of implantations per pregnant female
Number of living embryos per pregnant female
Dead implants (%)
I
DBCP Control
T stock T stock
42 44
36 41
8.5 8.4
6.9 7.3
19.2 13.4
II
DBCP Control DBCP Control
(C3H x C57BL)F~ (C3H x C57BL)F 1 T stock T stock
46 37 45 47
33 29 41 42
9.4 9.4 8.5 8.2
8.6 8.9 6.9 6.9
8.4 5.5 19.4 15.9
a DBCP at 110 mg/kg was given as a single i.p. injection. Controls received 0.2 ml tricaprylin. b All females were mated to (C3H × C57BL)F~ males. All matings occurred during the post-treatment interval 0.5-5.5 days.
107 TABLE 5 REPRODUCTIVE PERFORMANCE OF DBCP-TREATED FEMALES a Post-
DBCP b
Control ¢
treatment interval (days)
Number of fecund females
Number of young born per females
Number of fecund females
Number of young born per female
18-24 25-43 44-62 63-81 82-100 101-119 120-138 139-157 158-176 177-195 196-214 215-233 234-252 253-271 272-290 291-309 310-328 329-347 348-366
35 26 28 31 23 34 32 32 33 28 26 26 29 11 20 12 9 8 3
9.2 9.3 9.8 10.0 9.6 10.7 10.9 10.2 9.8 9.4 7.8 7.0 5.3 4.2 4.3 3.7 2.4 2.9 3.0
34 27 30 32 28 29 33 30 34 31 28 31 26 25 19 17 15 9 4
8.8 9.5 9.9 9.7 10.6 10.1 10.4 9.5 10.4 9.8 8.3 6.7 5.3 4.6 3.6 3.0 2.8 2.9 2.5
a 36 (C3H :,
female germ cells. The total reproductive capacity test in female mice detects d o m i n a n t - l e t h a l a n d oocyte killing effects a n d reproductive effects associated with i n d u c e d changes in the m a t e r n a l physiology.
Discussion T h e question of whether or n o t D B C P is capable of p r o d u c i n g genetic a n d cytotoxic damage i n m o u s e germ cells has b e e n clarified further with the availability of the present data. T h e difference b e t w e e n mice a n d rats in this respect was reviewed recently by O a k b e r g a n d C u m m i n g s (1984). While it appears that D B C P clearly induces d o m i n a n t lethals i n male rats ( T e r a m o t o et al., 1980; SaitoSuzuki, 1982; R a o et al., 1983), the present results add strength to the likelihood that it has n o effect in male mice. Also the lack of a n y D B C P - i n d u c e d reproductive response observed d u r i n g the 42 days
p o s t - t r e a t m e n t m a t i n g period is consistent with the cytological data of Oakberg a n d C u m m i n g s (1984). I n the rat studies, D B C P was a d m i n i s t e r e d either orally or b y inhalation. I n the mouse, the T e r a m o t o et al. (1980) study had D B C P administered orally a n d in the present study it was a d m i n i s t e r e d intraperitoneally or subcutaneously. The most sensitive rat germ cells to D B C P are the early spermatids. I n all mouse studies the sensitivity of these germ cells was also studied. C o n t r a r y to the results in males, data in females do n o t suggest large species difference b e t w e e n mice a n d rats. I n the present m o u s e study, DBCP, given i n t r a p e r i t o n e a l l y at the m a x i m u m tolerated c o n c e n t r a t i o n , does n o t appear to have either d o m i n a n t - l e t h a l or oocyte cytotoxic effects. Similarly, in female rats, in which D B C P was given by i n h a l a t i o n at a c o n c e n t r a t i o n a n d t r e a t m e n t regim e n s that p r o d u c e d d o m i n a n t - l e t h a l response in males, n o r e d u c t i o n in litter sizes was observed
108
suggesting the lack of substantial dominant-lethal or cytotoxic effects (Rao et al., 1983). Thus, it appears that there is a marked sex difference in the response of rats to dominant-lethal and germ cell cytotoxicity effects. Similar sex differences in response to a mutagenic chemical, EMS, have been observed before in mice (Generoso and Russell, 1969). The intriguing question is why do male mice and rats differ greatly in their response to DBCP. Perhaps it is significant that in the rat both dominant-lethal and cytotoxic effects were observed. The germ cell stages involved for these two types of DBCP-induced damage are presumably different. One explanation was offered by Teramoto et al. (1980) - - i.e., there exist species-dependent pathways in DBCP metabolism. Indeed, evidence seems to suggest that DBCP is an indirect mutagen that is converted in vivo into a reactive alkylating intermediate (see review by Whorton and Foliart, 1983). Another possible explanation could be that young spermatids and earlier spermatogenic stages of mice can repair DBCP-induced D N A lesions more effectively than corresponding stages in the rat. However, while there is an indication that premeiotic germ cells of male mice exposed to DBCP are capable of carrying out unscheduled D N A synthesis (Lee and Suzuki, 1979), no comparable information is available in the rat. Thus, neither explanation is supported by evidence. It should be emphasized that only two stocks of male mice have been studied so far for dominant-lethal and cytotoxicity effects.
References Generoso, W.M. (1969) Chemical induction of dominant lethals
in female mice, Genetics, 61,461-470. Generoso, W.M. (1973) Total reproductive capacity procedure in female mice, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 3, Plenum, New York, pp. 241-258. Generoso, W.M., and W.L. Russell (1969) Strain and sex variations in the sensitivity of mice to dominant-lethal induction with ethyl methanesulfonate, Mutation Res., 8,
589-598. Generoso, W.M., K.T. Cain and A.J. Bandy (1983) Some factors affecting the mutagenic response of mouse germ cells to chemicals, in: F.J. de Serres and W. Sheridan (Eds), Environmental Science Research, Utilization of Mammalian Specific Locus Studies in Hazard Evaluation and Estimation of Genetic Risk, Vol. 28, Plenum, New York, pp. 227-239. Lee, I.P., and K. Suzuki (1979) Induction of unscheduled DNA synthesis in mouse germ cells following 1,2-dibromo-3-chloropropane (DBCP) exposure, Mutation Res., 68, 169-173. Oakberg, E.F., and C.C. Cummings (1984) Lack of effect of dibromochloropropane on the mouse testis, Environ. Mutagen., 6, 621-625. Rao, K.S., J.D. Burek, J.A. John, B.A. Schwetz, T.J. Bell, W.J. Potts and C.M. Parker (1983) Toxicologic and reproductive effects of inhaled 1,2-dibromo-3-chloropropane in rats, Fund. Appl. Toxicol., 3, 104-110. Saito-Suzuki, R., S. Teramoto and Y. Shirasu (1982) Dominant-lethal studies in rats with 1,2-dibromo-3-chloropropane and its structurally related compounds, Mutation Res., 101, 321-327. Teramoto, S., R. Saito, H. Aoyama and Y. Shirasu (1980) Dominant-lethal mutation induced in male rats by 1,2-dibromo-3-chloropropane (DBCP), Mutation Res., 77, 71-78. Whorton, M.D., and D.E. Foliart (1983) Mutagenicity, carcinogenicity and reproductive effect of dibromochloropropane (DBCP), Mutation Res., 123, 13-30.