Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea

171

MutaUon Research, 177 (1987) 171-178 Elsevier MTR 04325

Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea T. N a g a o Hatano Research Instttute, Food and Drug Safety Center, Hadano, Kanagawa 257 (Japan) (Received 23 July 1986) (Revision received 5 November 1986) (Accepted 7 November 1986)

Key words: Frequency; Congenital defects; Dominant lethals; (Male mice, offspring); Methylnitrosourea.

Summary ICR strain male mice were injected intraperitoneally with daily doses of MNU (5-25 mg/kg) for 5 days and mated to untreated virgin females of the same strain on days 1-7, 8-14, 15-21 and 64-80 after the last dose. Copulations during these periods involve, respectively, spermatozoa, late spermatids, early spermatids, and spermatogonial stem cells at the time of the last treatment. The uterine contents were examined on day 18 of pregnancy for post-implantation losses (dominant lethality). Fetuses were examined for external and skeletal abnormalities. In contrast to the results reported for specific-locus mutations, MNU treatment of either postmeiotic cells or spermatogonial stem ceils caused dose-dependent significant increases in the incidence of congenital defects and of dominant lethals over the control levels. The relative sensitivity of germ cells sampled on days 1-7, 8-21 and 64-80 to MNU-induced congenital defects was 1:1.6:2. For the induction of dominant lethals, the sensitivity ratio was 1:1.8:0.5. It is proposed that congenital defects in the offspring of mice following paternal treatment with MNU may represent mostly chromosomal rather than genic changes. Cleft palate was the most frequent of the external abnormalities, which were significantly induced in every treatment series; fused ribs were the most frequent of the skeletal abnormalities, which were significantly induced in the treatment series for spermatogonial stem cells.

Congenital defects can be induced in the offspring of laboratory animals treated with a mutagen before copulation. Nomura (1975, 1979, 1982) showed that, in mice, (1) oocytes and postmeiotic male germ cells are highly susceptible to treatment with X-rays or urethane, (2) congenital defects are generally detected with a higher frequency in the prenatal than in the postnatal period, and (3) Correspondence: Dr. Tetsuji Nagao, Hatano Research Institute, Food and Drug Safety Center, Hadano, Kanagawa 257 (Japan).

some of the induced abnormalities are transmissible to the F2 generation. Subsequently, Kirk and Lyon (1984) demonstrated that, with respect to the induction of congenital defects, spermatogonial stem cells as well as postmeiotic cells are susceptible to X-ray treatment. Adams et al. (1980) and Trasler et al. (1985) described paternallymediated effects of cyclophosphamide-induced behavioral abnormalities and fetal abnormalities in rats. It thus seems that the incidence of congenital defects among the offspring of treated males may

0027-5107/87/$03.50 © 1987 Elsevier Scmnce Publishers B.V. (Biomedical Division)

172 provide a useful addition to the already available means of assessing the genetic hazards of physical and chemical agents, as proposed by Knudsen et al. (1977) and Kirk and Lyon (1982). More information is needed to properly evaluate the test. In the present study, the offspring of male mice treated with the alkylating agent methylnitrosourea (MNU) before copulation were examined for congenital defects. The defects were external or skeletal abnormalities detected among fetuses. MNU was used because, in mice and other organisms, it is capable of inducing both chromosome aberrations and point mutations. Parkin et al. (1973) reported that MNU caused a significant increase in dominant lethals over the control level at the postmeiotic but not at other stages of the spermatogenic cycle; and Generoso et al. (1984) showed that MNU was highly effective in causing heritable translocations in late spermatids. Russell et al. (1983) reported that for inducing specific locus mutations MNU was effective neither at postmeiotic stages nor at spermatogonial stem cell stages but was highly effective at a germ cell stage corresponding to differentiating spermatogonia or early spermatocytes. In the present study, treatment of either postmeiotic or spermatogonial stem cells with MNU caused dose-dependent significant increases in congenital defects in the offspring. Materials and methods

ICR strain mice purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals were used. MNU (from Nakarai Chemicals Ltd.) was dissolved in phosphate buffer (pH 5.8) immediately before use. The chemical solution was injected intraperitoneally into 10week old male mice (weighing 42 _+ 3 g) at daily doses of 5, 15 or 25 m g / k g for 5 days. Males used as controls received a 5-day i.p. dose of phosphate buffer (5 ml/kg). Each male was caged with two untreated virgin females of the same strain during the periods 1-7, 8-14, 15-21 and 64-80 days after the last dose. Copulations during these periods involve, respectively, spermatozoa, late spermatids, early spermatids, and spermatogonial stem cells at the time of the last treatment. The presence of a vaginal plug was taken as day 0 of

pregnancy. The females were humanely killed on day 18 of pregnancy. Corpora lutea and implantation sites in the uterus were counted, and the number of live and dead fetuses, and the number of moles were recorded. All live fetuses were coded, weighed, and examined for external abnormalitle,s including cleft palate and dwarfism under a dkssecting microscope at 5.6 × magnification. A fetus weighing less than 70% of the average of the rest of the litter was classified as a dwarf. This criterion is more severe than that (i.e., 75% of the average weight of the litter mates) employed in the studies by Kirk and Lyon (1982, 1984) and Nomura (1982). Finally, live fetuses were processed for skeletal examination using the original Dawson's technique (Dawson, 1926). Cervical and lumbar ribs were not scored because they occur spontaneously with an appreciable frequency in fetuses of the ICR strain (author's unpublished observation), The frequency of abnormal fetuses was calculated as the number of fetuses with external or skeletal abnormalities divided by the number of live fetuses. The frequency of pre-implantation losses was calculated as the number of corpora lutea minus the number of implantations divided by the number of corpora lutea. The frequency of post-implantation losses was calculated as the number of moles plus the number of dead fetuses divided by the number of implantations. An increase of post-implantation losses over the control level was taken as an indication of dominant lethals caused by MNU in the germ cells. Since the number of abnormal fetuses and of post-implantation losses detected were too small for the purpose of determining whether germ cells sampled on days 8-14 and 15-21 after the last dose differ in their sensitivities to MNU effect, the data from these two mating periods were combined. For comparison, control data from all mating periods were pooled since they were found to be statistically homogeneous. Results

The frequencies of pre- and post-implantation losses observed after treating male mice with various doses of MNU are shown in Table 1. When treated postmeiotic cells were sampled on days

173 TABLE 1 F R E Q U E N C I E S OF PRE- A N D P O S T - I M P L A N T A T I O N LOSSES A M O N G T H E C O N C E P T U S E S OF M A L E M I C E T R E A T E D W I T H V A R I O U S DOSES OF M N U

Treatment a

MatinS interval (days)

Females

Corpora lutea [A ]

Total implants [B ]

Freq. of pre-impl. losses

5 × 5 mg/kg

1- 7

42 b (40) c

655

564

5 x15 mg/kg

21

(20)

323

266

5×25 mg/kg

34

(32)

511

435

13.7 (11.1-16.8) e 17.6 * (13.5-22.5) 14.9 (11.5-18.4)

56

(54)

894

771

5×15 mg/kg

32

(30)

423

379

5×25 mg/kg

45

(42)

638

501

13.8 (11.4-16.2) 10.4 (7.5-13.9) 21.5 ** (18.0-25.2)

38

(35)

560

500

5x15 mg/kg

31

(30)

393

369

5 x25 mg/kg

45

(43)

674

573

55

(51)

833

727

5x5 mg/kg

5x5 mg/kg

Concurrent control

8-21

64-80

1-80

10.7 (8.1-13.5) 6.1 (3.8-8.8) 15.0 (12.1-18.0)

12.7 (10.3-15.2)

Moles [C]

Dead fetuses [D ]

Freq. of post-impl. losses

71 (0) d

19

26 (0)

11

72 (0)

24

16.0 ** (12.6-19.4) c 13.9 * (9.9-19.0) 22.1 ** (17.7-26.6)

57 (0)

33

63 (0)

18

153 (0)

11

36 (0)

22

21 (0)

20

56 (16)

31

39 (0)

26

11.7 (9.2-14.2) 21.4 ** (16.8-26.2) 32.7 ** (27.6-37.9) 11.6 (8.9-14.8) 11.1 (7.9-14.9) 15.2 ** (12.2-18.5)

8.9 (6.9-11.2)

" Daily i.p. injection of MNU into male mice at 5, 15 or 25 m g / k g for 5 days. b N u m b e r of females with plug. ¢ N u m b e r of pregnant females. d N u m b e r of small moles. e 95% confidence interval calculated using Crow and Gardner's table (1959). * Significantly different from the control by X 2 test at P < 0.05. ** Significantly different from the control by X 2 test at P < 0.01.

1 - 7 or 8-21 after the completion of the treatment, pre-implantation losses were significantly induced at 5 x 15 m g / k g and 5 × 25 mg/kg, respectively. However, the link between M N U dose and the incidence of pre-implantation losses was not clear in either sample. In contrast, post-implantation losses showed a dramatic dose-dependent significant increase over the control level when treated postmeiotic cells were sampled on days 8-21. The increase was less striking when the treated postmeiotic cells were sampled on days 1-7, and much less so when treated spermatogonial stem cells

were sampled on days 64-80. At the highest dose level tested, the frequencies of post-implantation losses in the three mating periods significantly differed from each other by the X z test at P < 0.05. After Abbott's correction of the observed frequencies of post-implantation losses, the frequencies of dominant lethals induced with 5 x 25 m g / k g M N U were calculated to be 14.1%, 26.1% and 6.9% for germ cells sampled on days 1-7, 8-21 and 64-80, respectively. The ratio of these frequencies, 1:1.8:0.5, implies that spermatids are most sensitive to the induction of dominant lethals

174 followed by spermatozoa, and spermatogonial stem cells. This pattern of stage sensitivity agrees well with the experimental data reported by Parkin et al. (1972) for MNU-induced dominant lethals. However, it should be noted that the ratio value does not hold at the lower dose levels because germ cells sampled on days 1-7 and days 64-80 were practically insensitive to a change in M N U dose from 5 × 5 m g / k g to 5 x 15 mg/kg, whereas germ cells sampled on days 8-21 exhibited a clear dose-dependent response. The failure to detect a dose-response relation in the data from the mating period 1-7 days may be related to the fact that, with the treatment schedule and mating procedure employed, a large fraction of germ cells sampled in this period comprised cells treated at both the spermatozoa and late spermatid stages. Further experiments with a single i.p. injection method, various doses of MNU, and more refined mating intervals are needed to determine a precise picture of the dose-response curve for MNU-induced dominant lethals in germ cells at various stages. The average frequency of dead fetuses over the three different dose levels was 4.3% (54/1265) when treated postmeiotic cells were sampled on days 1-7, 3.8% (62/1651) when treated postmeiotic cells were sampled on days 8-21, and 5.1% (73/1442) when treated spermatogonial stem cells were sampled on days 64-80. None of these frequencies differ significantly by the X2 test ( P > 0.05) from the control frequency of 3.6% (26/727). The average frequency of moles was, respectively, 14.1% (169/1265), 16.5% (273/1651), and 7.8% (113/1442). All these frequencies significantly differed by the X2 test at P < 0.05 from the control frequency of 5.4% (39/727). All moles detected in the treatment series for postmeiotic cells and in the control series were large ones, which represent death at the late embryonic stages. Small moles representing death soon after implantation were detected with an appreciable frequency (i.e., 16/573) only when spermatogonial stem cells were treated with 5 × 25 mg/kg. The results of the analysis for fetal abnormalities are shown in Table 2. Irrespective of the mating interval, the frequencies of abnormal fetuses increased over the control level approximately linearly as the dose of MNU increased.

The dose-response data were fitted to a linear equation Y = a + b D (Y = frequency per live fetu,~ of abnormal fetuses, a = constant, b = regression coefficient, and D = total dose in mg/kg) by the method of least squares with the reciprocal of the variance of the observed frequency as weighting factor. The control data were included in the fitting at zero dose. The weighted regression analysis at P = 0.05 indicates that the data from each mating period do not deviate significantly from the expected linear dose response relation, the regression coefficient being significantly different from zero. The linear regression equations obtained for the data sets from matings on days 1- 7, 8-21 and 64-80 were, respectively, Y = 0.005 + 1.6(+0.3)10--4D, Y = 0.005 + 2.6(_+0.2)10 4/) and Y=0.003 + 3.1(+0.6)10- 4D. X 2 value from the test of goodness for fit was, respectively, 0.05 ( d . f . = l , P > 0 . 7 ) , 3.22 ( d . f . = 2 , P > 0 . 0 5 ) , and 1.29 (d.f. = 2, P > 0.2). The parallelism test at P = 0.05 indicates that the regression coefficient computed for the data from matings on days 1 -7 significantly differs from each of those for the data from matings on days 8--21 and 64 80, and that the coefficients for the data from the later two mating periods do not differ significantly from each other. The relative sensitivity of germ cells sampled on days 1-7, 8-21 and 64-80 is 1"1.6:2. As reflected in the frequency of congenital defects, these results imply that spermatids and spermatozoa differ in sensitivity, while spermatogonial stem cells and spermatids have similar sensitivities; however, considering the treatment schedule and the mating procedure employed in the present study, other interpretations are possible. The average frequency of external abnormahties over the three different dose levels was 1.7% (40/2358) among fetuses arising from treated postmeiotic cells and 1.8% (22/1256) among fetuses arising from treated spermatogonial stem cells. These frequencies are significantly higher by Fisher's exact test at P < 0.05 than the control frequency of 0.45% (3/662). The average frequency of skeletal abnormalities was, respectively, 0.2% (5/2358) and 1.0% (12/1256). The later frequency is significantly higher ( P = 0.006) than the control frequency of 0% (0/662). Thus, under the present experimental conditions, external abnormalities

175 TABLE 2 FREQUENCIES OF ABNORMAL FETUSES AMONG OFFSPRING OF MALE MICE TREATED WITH VARIOUS DOSES OF MNU Treatment a

5 x 5 mg/kg

Interval (days)

Abnormals External [B]

Skeletal [C]

Frequency

1.05 (0.42-2.36) b 1.31 (0.36-3.54) 2.65 ** (1.32-4.95)

474

3

2

5 g 15 mg/kg

229

3

0

5 g 25 mg/kg

339

8

1

681

7

1

5 × 15 mg/kg

298

8

0

5 x 25 mg/kg

337

11

1

442

1

2

5 × 15 mg/kg

328

3

5

5 X 25 mg/kg

486

18

5

662

3

0

0.45 (0.12-1.22)

4 424

20

5

0,57 (0.38-0.81)

5 X 5 mg/kg

5 x 5 mg/kg

Concurrent control Historical control

1-7

Live fetuses [A]

8-21

64-80

1-80

1.17 (0.48-2.19) 2.68 ** (1.10-5.01) 3.56 ** (1.98-6.03) 0.68 (0.19-1.83) 2.44 ** (1.00-4.55) 4.73 ** (3.07-7.01)

a Daily i.p. injection of MNU into male mice at 5, 15 or 25 mg/kg for 5 days. b 95% confidence interval calculated using Crow and Gardner's table (1959). * Significantly different from the concurrent control by Fisher's exact test at P < 0.05. ** Significantly different from the concurrent control by Fisher's exact test at P < 0.01.

were i n d u c e d in every treatment series, whereas skeletal a b n o r m a l i t i e s were i n d u c e d only in the t r e a t m e n t series for spermatogonial stem cells. W h e n the data from the treated germ cells were s u m m e d , there were 62 fetuses with external a b n o r m a l i t i e s a n d 17 with skeletal abnormalities a m o n g 3614 live fetuses. W h e n the c o n c u r r e n t control a n d the historical control data (Table 2) were s u m m e d , there were 23 fetuses with external a b n o r m a l i t i e s a n d 5 with skeletal a b n o r m a l i t i e s a m o n g 5086 live fetuses. T h e spectra of these two classes of fetal a b n o r m a l i t i e s are shown in T a b l e 3. Cleft palate, dwarfism, a n d syndactyly were

c o m m o n types a m o n g the external a b n o r m a l i t i e s seen in the treated series. Cleft palate was the most c o m m o n ; it was present in 55% ( = 43.5 + 8.1 + 1.6 + 1.6, see T a b l e 3) of all live fetuses with external abnormalities, a n d was occasionally a c c o m p a n i e d b y dwarfism a n d other a b n o r m a l i ties. Cleft palate was the most c o m m o n a m o n g fetuses i n the control series as well; it represented 69% ( = 61 + 4 + 4) of all external abnormalities. R i b fusion was the most c o m m o n a m o n g six different types of skeletal abnormalities f o u n d in the treated series. I n the control series, it was the only skeletal a b n o r m a l i t y found.

176 TABLE 3 TYPES OF FETAL ABNORMALITIES AMONG OFFSPRING OF MALE MICE TREATED WITH VARIOUS DOSES OF MNU Type

Number (relative frequency) of abnormalities MNU-treated a Control b

External abnormahtles Cleft palate 27 (43 5) + dwarfism 5 (8 1) + dwarfism/open eyelids l (1.6) +abdominal herma/syndactyly 1 (1.6) + brain berma + exencephalus Dwarfism Syndactyly Exencephalus +open eyelids + kinky tail Open eyehds Umbdical hernia Microphthalmus Total Skeletal abnormalities Fusion of ribs Thickemng of ribs Fusion of vertebrae Wavy ribs Fusion of the exoccipital and atlas Absence of cervical vertebra Total

11 (17 7) 8 (12 9) 3 (4 8) 1 (1 6) 1 (1.6) 3 1

14 (61)

1 1

(4) (4)

1

(4)

2 2

(9) (9)

2

(9)

(4 8) (1 6)

62 (100)

23 (100)

9 (53) 3 (18) 2 (12)

5 (100)

1

(6)

1 1

(6) (6)

17 (100)

5 (100)

a Data pooled from all experiments b Data pooled from concurrent and historical controls

Discussion T h e results of this s t u d y show that treating m a l e mice with M N U before they c o p u l a t e inc r e a s e d the incidence of congenital defects a m o n g their offspring. T h e effect was n o t likely to have b e e n m e d i a t e d t h r o u g h the seminal fluid as M N U has a short half life in aqueous solution (i.e., a b o u t 1 h at p H 7.0 a c c o r d i n g to D r u c k e r e y et al., 1964). F u r t h e r m o r e , if M N U - c o n t a m i n a t e d seminal fluid was r e s p o n s i b l e for i n d u c i n g congenital defects, the effect o f M N U t r e a t m e n t w o u l d have been m o r e p r o n o u n c e d a m o n g fetuses in the first m a t -

ing p e r i o d than a m o n g those in the subsequent m a t i n g periods. Clearly, this was not the case as the yields of a b n o r m a l fetuses and of d o m i n a n t lethals were a b o u t 2-fold higher when treated germ cells were s a m p l e d in the m a t i n g p e r i o d 8- 2t days than when treated germ cells were s a m p l e d in the m a t i n g p e r i o d 1 - 7 d a y s (Tables 1 and 2). Theme results are c o m p a t i b l e with the finding of Sega et al. (1981) that M N U injected i.p. into mice prod u c e d more a l k y l a t i o n s m s p e r m a t i d s than 111 spermatozoa. A clear d o s e - d e p e n d e n t increase in fetal abn o r m a l i t y was also observed for s p e r m a t o g o m a l stem cells (Table 2). This implies that the M N U effect is transmissible from cell to cell and remains u n c h a n g e d d u r i n g the extended, c o m p l e x process of spermatogenesis. Thus, it seems r e a s o n a b l e to suggest that the m a i n target for the induction of congenital defect b y p a t e r n a l M N U t r e a t m e n t ma> be the genetic m a t e r i a l of the germ cells, as prop o s e d by Sega et al. (1981) for d o m i n a n t lethals i n d u c e d at p o s t m e i o t i c stages by M N U . D o m i n a n t lethals were i n d u c e d in s p e r m a t o g o n i a l stem cells as well as in p o s t m e i o t i c cells (Table 1). But the i n d u c t i o n of d o m i n a n t lethals in s p e r m a t o g o n i a l stem cells is not as clear as it is in p o s t m e i o t i c cells. This finding c o n t r a s t s to that suggesting that s p e r m a t o g o n i a l stem cells and s p e r m a t i d s have similar sensitivity when congenital defects a m o n g fetuses were used to m e a s u r e genetic d a m a g e (Table 2). Probably, the m a j o r i t y of d o m i n a n t lethals i n d u c e d at the stem cell stage are e l i m i n a t e d before or d u r i n g s p e r m a t o g e n e s i s proceeds. Russell et al. (1983) r e p o r t e d that 75 m g / k g M N U inj e c t e d i.p. into mice was not effective either at the stem cell stage or at p o s t m e i o t i c stages in inducing specific-locus mutations. Their finding contrasts with the p r e s e n t results. S u m m i n g up all these results and c o n s i d e r a t i o n s , I am inclined to believe that the m a j o r i t y of congenital defects i n d u c e d by p a t e r n a l t r e a t m e n t with M N U m a y represent genetic d a m a g e s at the c h r o m o s o m e level. This p o s s i b i l i t y will be tested in subsequent studies using p r o p e r cytogenetic techmques. T h e results of studies by N o m u r a (1982) with I C R mice a n d by K i r k a n d L y o n (1984) with ( C 3 H / H e H × 1 0 1 / H ) F 1 h y b r i d s showed that external a b n o r m a l i t i e s including d w a r f i s m a n d cleft p a l a t e were i n d u c e d by p a t e r n a l t r e a t m e n t with

177

X-rays. Paternal treatment with urethane was also effective in inducing external abnormalities (Nomura, 1982). The results of the present study with ICR strain show that external abnormalities were induced by paternal treatment with MNU (Table 3). From these results it appears that external abnormalities may serve as fairly stable endpoints in the genotoxicity testing of physical and chemical agents. Differences in strains of mice, mutagen used, and varying experimental conditions may result in different spectra of external abnormalities. Cleft palate, the most common type of abnormality (55% of total external abnormalities) seen in the present study, was rare in Kirk and Lyon's study (3%, 1984), in which dwarfism was the predominant abnormality (74%); the frequency of dwarfism in the present study was 27% (= 17.7 + 8.1 + 1.6, see Table 3). The use of skeletal abnormalities as endpoints may also merit serious consideration in developing a test protocol for measuring male-transmitted genetic damage because this class of defect was clearly induced when spermatogonial stem cells were exposed to MNU (Table 3) despite the fact that these germ cells were seemingly refractory to the induction of specific locus mutations by MNU (Russell et al., 1983). It should be pointed out in this context that the test for dominant mutations affecting the skeleton has been successfully employed to monitor the genotoxic effects of X-rays and ethylnitrosourea on spermatogonial stem cells (Ehling, 1966; Selby and Lee, 1981). Evidence suggesting that paternal exposure to drugs or environmental pollutants may lead to birth defects in the offspring of man has been accumulating (see Schardein, 1985). If a spermatogonial stem cell carries a stable genetic lesion, then gametes bearing the change will be produced continually throughout the stem cell's life span. This means that the stem cell is the most important germ cell stage for risk consideration in men. The present observation that treatment of male mice with MNU at the stem cell stage caused a clear increase in fetal abnormality demonstrates the vulnerability of the stem cell.

Acknowledgements The author thanks Dr. K. Fujikawa for stimulating interest and Drs. J.R. Miller, K. Fujikawa

and W.M. Generoso for critical reading of the manuscript and suggestions. Sincere gratitude is also expressed to Dr. M. Mizutani for encouragement throughout the present study.

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methyl methanesulfonate, m the germ cells of male m,~c, Chem-Biol. Interact, 33, 253-269 Selby, P B., and S.S. Lee (1981) Sensitive-indicator result,, show the ethylnitrosourea ~s also a supermutagen for dominant skeletal mutations, Environ Mutagen, 3, 373 Trasler, J.M., B.F Hales and B Robalre (19851 Paternal cyclophospham~de treatment of rats causes fetal loss and malformations without affecting male fertdity. Nature (London), 316, 144-146