Congenital defects in the offspring of male mice treated with ethylnitrosourea

Mutation Research, 202 (1988) 25-33 Elsevier

25

MTR 04629

Congenital defects in the offspring of male mice treated with ethylnitrosourea Tetsuji Nagao Hatano Research Institute, Food and Drug Safety Center, Hadano, Kanagawa 257 (Japan) (Received 20 March 1987) (Revision received 16 February 1988) (Accepted 3 March 1988)

Keywords: Congenital defects, male mice offspring; Ethyl nitrosourea; Cleft palate; Vertebrae, malformed

Summa W

Daily doses of ENU (25-100 mg/kg) were injected intraperitoneally into ICR strain male mice for 5 days. The males were mated to untreated virgin females of the same strain on days 1-16 and 64-80 after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The uterine contents were examined on day 18 of pregnancy for evidence of dominant lethal effects. The fetuses were examined for external and skeletal abnormalities. ENU treatment of either postmeiotic cells or spermatogonial stem cells caused dose-dependent significant increases in the incidence of abnormal fetuses over the control level. The induction rate per live fetus per unit dose in m g / k g by treating spermatogonial stem cells was estimated to be 1.0 x 10 - 4 , which is 3-fold lower than the rate previously estimated for the same endpoint at the same germ cell stage with MNU. Cleft palate was the most frequent external abnormality in the ENU-treated and the control series. Malformed vertebrae was the most frequent skeletal abnormality in the treated series. Rib fusion was the only skeletal malformation seen in the control series. Dominant lethals were clearly induced when germ cells were treated as postmeiotic cells.

The mutagenicity of the simple alkylating agent, ethylnitrosourea (ENU), has been well documented in mice. Of special importance in this context are the findings by Russell and coworkers that ENU among a number of chemical agents tested is the most highly efficient mutagen for inducing gene mutations in spermatogonial stem cells when assayed using the specific locus method

Correspondence: Dr. Tetsuji Nagao, Hatano Research Institute, Food and Drug Safety Center, Hadano, Kanagawa 257 (Japan). Telex 0463(82)9627; tel. 0463(82)4751.

(Russell et al., 1979, 1982a,b; Hitotsumachi et al., 1985). The high genotoxic activity at the stem cell stage has been further demonstrated using dominant skeletal mutations (Selby and Lee, 1981; Selby and Niemann, 1984), dominant cataract mutations (Ehling et al., 1982; Favor, 1983), proteincharge mutations (Johnson and Lewis, 1981; Ehling et al., 1985), and enzyme-activity mutations (Ehling et al., 1985) as endpoints. The dominant lethal effect of ENU has been shown in postmeiotic cells (Generoso et al., 1984). In the present study, male mice were treated with ENU before they copulated and their off-

0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

26 spring were inspected for congenital defects. The congenital defects scored were skeletal and external abnormalities in fetuses. Johnson and Lewis (1981), Lovell et al. (1985), and Lyon and Renshaw (1986) reported that E N U treatment of spermatogonial stem cells was ineffective in causing congenital defects in offspring. In this report, I describe experimental evidence that E N U treatment of either postmeiotic cells or spermatogonial stem cells can produce congenital defects in the subsequent generation. Materials and methods

I C R strain mice purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan) were used. E N U (from Nakarai Chem., Tokyo, Japan) was dissolved in phosphate buffer at p H 5.8 immediately before use. The solution was injected intraperitoneally into 10-week-old male mice (weighing 42 + 3 g) at a daily dose of 25, 50, 75, or 100 m g / k g for 5 days. Males used as controls received a 5-day i.p. dose of phosphate buffer (5 ml/kg). Treated and control males were individually mated with two 10-12-week-old untreated virgin females of the same strain during the periods 1-16 and 64-80 days after the last dose. Copulations during these periods involve, respectively, treated postmeiotic cells and spermatogonial stem cells. The females were examined for the presence of a vaginal plug each morning. Mated females were replaced with new females. Mated females were humanely killed on day 18 after a vaginal plug was observed and their uterine contents were examined. Females with no implants were classified as non-pregnant and discarded from further analyses. In the females with implants, corpora lutea and implantation sites 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 abnormalities including cleft palate and dwarfism under a dissecting microscope at 5.6 x 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 N o m u r a (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 these abnormalities occur spontaneously with an appreciable frequency in fetuses of the I C R strain (author's unpublished observation). 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. Both these frequencies were expressed as per cent mean per litter. An increase of post-implantation losses over the control level was taken as an indication of dominant lethals caused by E N U in the germ cells. The frequency of congenital defects was calculated as the number of live fetuses with external or skeletal abnormality divided by the number of live fetuses. This frequency was expressed as per cent mean per fetus. The control data from the two mating periods were pooled since they were found to be statistically homogeneous when compared at P = 0.05 using Fisher's exact test for abnormal fetuses and M a n n - W h i t n e y U test for pre- and post-implantation losses. Results

The results obtained for pre- and post-implantation losses are summarized in Table 1. When treated postmeiotic cells were sampled on days 1-16 after the completion of the treatment, postimplantation losses showed a dose-dependent increase over the control level. The frequency at any dose higher than 5 x 25 m g / k g differed significantly from the control frequency. These data indicate that E N U treatment caused dominant lethals in postmeiotic cells. All the moles recorded in the control series and the treatment series for postmeiotic cells were large ones, most probably representing trophoblasts without fetus. The great majority of dead fetuses seen in the control and the treatment series were very similar in size and shape to fetuses around days 12-13 of gestation. The average frequencies per total implants of moles

27 TABLE 1 FREQUENCIES OF PRE- A N D POST-IMPLANTATION LOSSES AMONG THE CONCEPTUSES OF MALE MICE TREATED WITH VARIOUS DOSES OF ENU Treatment (mg/kg) a

Pregnancy rate

Total number of Corpora Implants lutea

Freq. of pre-impl. losses e

Total number of Moles Dead fetuses

Freq. of post-impl. losses e

1 598 1379 1451 690

1 401 1213 1268 540

12.1_+ 12.2_+ 12.2_+ 16.6-+

1.6 1.7 1.7 3.2 ##

136 126 162 83

59 46 46 27

13.6_+1.1 14.6_+1.1 # 17.4-+1.4 ## 21.7_+2.2 ##

Spermatogonial stem cells c 5 x 25 0.96 (73/76) 5 x 50 0.89 (78/88) 5× 75 0.67 * (45/67) 5×100 0.24 ** (7/29)

1 169 1258 655 122

1065 1 102 515 53

9.3_+ 1.5 11.7-+ 2.2 22.1 _+ 4.1 #~ 60.8 _+13.2 *~

80 96 51 6

30 42 22 1

10.4-+1.1 13.6-+1.5 14.5 _+1.7 7.6 _+4.0

Concurrent control

1807

1630

121

59

11.2_+0.9

Postmeiotic cells b 5 × 25 0.97 5X 50 0.91 5 × 75 0.90 5 × 100 0.98

b c d e * ** # ##

0.96

(99/102) 0 (89/98) (94/104) (45/46)

(112/117)

8.7_+ 1.3

Daily i.p. injection of ENU into male mice at 0, 25, 50, 75, or 100 mg/kg for 5 days. Number of males treated with these doses was, respectively, 30, 38, 39, 24, and 15. Germ cells sampled in matings on days 1-16 after the last dose. Germ cells sampled in matings on days 64-80 after the last dose. Number of females with implants/number of females with plug. Mean per litter+S.E. Significantly different from the control by X2 test at P < 0.05. Significantly different from the control by X2 test at P < 0.01. Significantly different from the control by Mann-Whitney U test at P < 0.05. Significantly different from the control by Mann-Whitney U test at P < 0.01.

a n d o f d e a d fetuses o v e r the 4 d i f f e r e n t d o s e s in this t r e a t m e n t series were, r e s p e c t i v e l y , 11.5% ( 5 0 7 / 4 4 2 2 ) a n d 4.0% ( 1 7 8 / 4 4 2 2 ) . T h e f o r m e r f r e q u e n c y w a s s i g n i f i c a n t l y h i g h e r b y X 2 test at P < 0.05 t h a n the c o r r e s p o n d i n g c o n t r o l f r e q u e n c y o f 7.4% ( 1 2 1 / 1 6 3 0 ) , b u t t h e l a t t e r was n o t significantly different from the corresponding control f r e q u e n c y o f 3.6% ( 5 9 / 1 6 3 0 ) . T h u s , d o m i n a n t l e t h a l s i n d u c e d b y E N U in p a t e r n a l p o s t m e i o t i c cells a f f e c t e d p o s t - i m p l a n t a t i o n e m b r y o n i c d e v e l o p m e n t . P r e - i m p l a n t a t i o n loss was s i g n i f i c a n t l y i n c r e a s e d o v e r t h e c o n t r o l level o n l y at 5 x 100 m g / k g . T h e r a t e o f p r e g n a n c y was n o t a f f e c t e d s i g n i f i c a n t l y at a n y dose. T h e s e results i m p l y t h a t p r e - i m p l a n t a t i o n loss i n d u c e d a f t e r t r e a t i n g p o s t m e i o t i c cells w i t h 5 x 100 m g / k g E N U m a y b e r e l a t e d to t h e d o m i n a n t l e t h a l p h e n o m e n a . W h e n t r e a t e d s p e r m a t o g o n i a l s t e m cells w e r e s a m p l e d o n d a y s 6 4 - 8 0 a f t e r t h e last dose, p o s t i m p l a n t a t i o n loss was slightly i n c r e a s e d o v e r the

c o n t r o l level at d o s e s h i g h e r t h a n 5 × 25 m g / k g ( T a b l e 1). H o w e v e r , n o n e o f t h e i n c r e a s e s w e r e s t a t i s t i c a l l y real. I n c o n t r a s t , p r e - i m p l a n t a t i o n loss was s i g n i f i c a n t l y i n d u c e d in a d o s e - d e p e n d e n t m a n n e r , a n d the r a t e o f p r e g n a n c y , r e p r e s e n t e d b y a proportion of females with implants among those w i t h plug, was d e c r e a s e d d r a s t i c a l l y as t h e d o s e i n c r e a s e d . P r o b a b l y , p r e - i m p l a n t a t i o n losses ind u c e d a f t e r t r e a t i n g s p e r m a t o g o n i a l s t e m cells m a y b e m a i n l y d u e to o l i g o s p e r m y o r n o n f u n c t i o n i n g sperm, reflecting a strong cytotoxic effect of ENU at the s t e m cell s t a g e ( F i c s o r et al., 1984; O a k b e r g a n d C r o s t h w a i t , 1983). I n this c o n t e x t , it s h o u l d b e n o t e d t h a t the f r e q u e n c i e s o f p r e - i m p l a n t a t i o n losses g i v e n in T a b l e 1 for s p e r m a t o g o n i a l s t e m cells are fairly u n d e r e s t i m a t e d b e c a u s e the n u m b e r o f c o r p o r a l u t e a w a s n o t c o u n t e d in f e m a l e s w i t h no implants. A s s h o w n in T a b l e 2, t h e f r e q u e n c i e s o f a b n o r m a l fetuses o b s e r v e d a f t e r t r e a t i n g p o s t -

28 TABLE 2 F R E Q U E N C I E S OF ABNORMAL FETUSES A M O N G O F F S P R I N G OF MALE MICE TREATED WITH VARIOUS DOSES OF ENU Treatment

Live

(mg/kg) a

fetuses

External

Skeletal

Frequency

(A)

(B)

(C)

[B+CxIo0 ~

1206

13

4

5 × 50

1041

7

1

5x

75

1060

17

6

5 x 100

430

12

1

955

10

0

5 x 50

964

24 *

5×

442

15

4

Concurrent control

1450

7

0

Historical control

5 086

23

5

Postmeiotic cells b 5 x 25

Spermatogonial stem cells c 5 × 25

75

Abnormals

13 ~

)

1.41" (0.80--2.18) a 0.77 (0.32-1.43) 2.17 ** (1.41-3.21) 3.02 * * (1.55-4.97)

1.05 (0.56-1.85) 3.73 ** (2.63-5.06) 4.30 ** (2.53-6.55) 0.48 (0.23-0.95) 0.55 (0.37-0.78)

Daily i.p. injection of ENU into male mice at 0, 25, 50, 75, or 100 m g / k g for 5 days. Number of males treated with these doses was, respectively, 30, 38, 39, 24, and 15. Germ cells sampled in matings on days 1-16 after the last dose. Germ cells sampled in matings on days 64-80 after the last dose. The frequency of abnormal fetuses at 5 × 100 m g / k g was not determined for these germ cells because the number of live fetuses was too small (see Table 1). a 95% confidence interval calculated using Crow and Gardner's table (1959). Including one fetus with both external and skeletal abnormalities (see Table 3). * 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. a

meiotic cells with 5 x 2 5 , 5 x 7 5 , and 5 x 1 0 0 m g / k g E N U and those after treating spermatogonial stem cells with 5 x 50 and 5 x 75 m g / k g were significantly higher than the concurrent control frequency of 0.48%. In both the treatment series, the incidence of fetal abnormality was roughly related with E N U dose. The control frequency determined concurrently was in good agreement with the historical control frequency of 0.55%. Thus, treatment of either postmeiotic cells or spermatogonial stem cells with E N U caused fetal abnormality in the offspring. The highest frequency was 3.0% at 5 × 100 m g / k g in the treat-

ment series for postmeiotic cells and 4.3% at 5 × 75 m g / k g in the treatment series for spermatogonial stem cells, suggesting that, under the conditions used, spermatogonial stem cells were relatively more susceptible to ENU, with respect to the induction of fetal abnormalities. To compare these data quantitatively with the dose-response data previously reported for congenital defects induced by treating spermatogonial stem cells with M N U (Nagao, 1987), the comparable data set given in Table 2 was approximated to a linear regression equation Y = a + bD ( Y = frequency per live fetus of abnormals, a =

29 TABLE 3 TYPES OF FETAL ABNORMALITIES

AMONG OFFSPRING

Type

OF MALE MICE TREATED WITH ENU N u m b e r (relative frequency) of a b n o r m a l i t i e s ENU-treated a

Control b

Cleft p a l a t e +dwarfism + open eyelids/hypognathia/anal atresia/dwarfism + brain hernia + exencephalus

52 (53.1) 9 (9.2) 1 (1.0) - ¢ -

19 (63) _ c 1 (3) 1 (3)

Dwarfism + o p e n eyelids + e x e n c e p h a l u s / o p e n eyelids

18 (18.4) 4 (4.1) 1 d (1,0)

3 (10) -

External abnormalities

Exencephahis + o p e n eyelids + spina bifida + open eyelids/spina bifida/polydactyly

3 2 1 1

(3,1) (2,0) (1.0) (1.0)

2 2 -

Polydactyly + o p e n eyelids

3 1

(3,1) (1.0)

-

Abdominal hernia

2

(2.0)

-

Microphthalmus

-

Total

2

98 (100)

(7) (7)

(7)

30 (100)

Skeletal abnormalities F u s i o n or absence of vertebrae + fusion of ribs

12 (41.4) 1 (3.4)

-

B e n d i n g of a p p e n d i c u l a r skeleton + w a v y ribs + fusion of r i b s / f u s i o n of m a x i l l a e

5 (17.2) 3 (10.3) 1 d (3.4)

-

T h i c k e n i n g of ribs

3 (10.3)

-

F u s i o n of ribs

2

(6.9)

5 (100)

W a v y ribs

2

(6.9)

-

Total a b c o

29 (100)

5 (100)

D a t a p o o l e d from all experiments. D a t a p o o l e d from the c o n c u r r e n t a n d the historical controls. N o t detected. A fetus with b o t h external a n d skeletal abnormalities.

constant, b = regression coefficient, and D = total E N U dose in m g / k g ) by the least square method with the reciprocal of variance of the frequency observed used as a weighting factor. The weighted regression analysis at P = 0.05 indicates that the dose-response data do not deviate significantly from linearity. The linear equation computed was Y = 0.004 + 1.0(+0.3)10-4D. The X 2 value from

the test of goodness for fit was 5.43 (df= 2, P > 0.05). The regression coefficient in the equation means that treating spermatogonial stem cells with 1 m g / k g E N U induces one abnormal among 1 0 4 live fetuses in the subsequent generation. The average frequencies of externally and of skeletally abnormal fetuses were, respectively, 1.3% (49/3737) and 0.3% (12/3737) among fetuses aris-

30

ing from treated postmeiotic cells and 2.1% (49/2361) and 0.7% (17/2361) among fetuses arising from treated spermatogonial stem cells. All these frequencies were significantly higher by Fisher's exact test at P < 0.05 than the corresponding control frequencies, i.e., 0.5% (7/1450) for externally abnormal fetuses and 0% (0/1450) for skeletally abnormal fetuses. When the data from the treated germ cells were summed, there were 98 fetuses with external abnormalities and 29 with skeletal abnormalities among 6098 live fetuses. When the concurrent control and the historical control data were summed, there were 30 fetuses with external abnormalities and 5 with skeletal abnormalities among 6536 live fetuses. The spectra of these two classes of fetal abnormalities are shown in Table 3. Cleft palate, dwarfism, open eyelids, hypognathia, anal atresia, exencephalus, spina bifida, polydactyly, and abdominal hernia were recorded as external abnormalities in the treated series. Among these, cleft palate was the most common, followed by dwarfism, open eyelids, and exencephalus. The frequencies of these 4 types of abnormalities relative to total fetuses with external abnormality were, respectively, 63% (-- 53.1 + 9.2 + 1.0, Table 3), 34% (-- 9.2 + 1.0 + 18.4 + 4.1 + 1.0), 10% ( = 1.0 + 4.1 + 1.0 + 2.0 + 1.0 + 1.0), and 8% ( = 1.0 + 3.1 + 2.0 + 1.0 + 1.0). These 4 types were also observed in the control series. The most common type was also cleft palate, present in 69% of fetuses with external abnormality. Skeletal abnormalities recorded in the treated series, except fusion of maxillae, can be categorized into 3 subclasses: malformed vertebrae, bending of the appendicular skeleton, and malformed ribs. The most common subclass was malformed vertebrae, present in 45% ( = 41.4 + 3.4) of the fetuses with skeletal abnormality. In the control series, rib fusion was the only skeletal abnormality found. Discussion

The results of the present study show that ENU induces congenital defects in the offspring of male mice treated before they copulate. The effect was clearly seen for both external and skeletal abnormalities in fetuses, when the germ cells were treated either as postmeiotic cells or as spermato-

gonial stem cells (Table 2). Dominant lethals were clearly induced only when postmeiotic cells were treated (Table 1). Presumably, any dominant lethals induced at the stem cell stage may be eliminated before or during spermatogenesis. Ehling et al. (1982) and Favor (1983) reported that for inducing specific locus mutations ENU was clearly effective at the spermatogonial stem cell stage but was practically ineffective at postspermatogonial stages. This finding contrasts with the present results obtained for congenital defects, suggesting that the nature of genetic lesions responsible for the production of congenital defects may be quite different from that associated with specific locus mutations. As pointed out by Favor (1983), however, the negative data for specific locus mutations were inconclusive because mosaic-type specific locus mutations were recovered with an appreciable frequency among offspring derived from ENU-treated postspermatogonia (Neuh~user-Klaus and Ehling, cited by Favor, 1983). More information on the response of postmeiotic cells to ENU induction of specific locus mutations is needed in order to determine whether the stage-sensitivity pattern observed in the present study for congenital defects is specific to the endpoint scored. Despite this uncertainty, the data from the present study lend further support to the notion that the testing of congenital defects among the offspring of male animals treated with a chemical agent may provide a useful adjunct to the already available means of genotoxicity testing (Nomura, 1975, 1982; Knudsen et al., 1977; Adams et al., 1981; Kirk and Lyon, 1982, 1984; Nagao, 1987). The observation that treating spermatogonial stem cells with ENU induces congenital defects in the subsequent generation (Table 2) agrees with previous findings that ENU is a potent inducer of dominant skeletal mutations in spermatogonial stem cells of treated mice (Selby and Lee, 1981; Selby and Niemann, 1984). However, contrary findings have been reported. Johnson and Lewis (1981) failed to detect an increase in the incidence of external malformations among the offspring of reciprocal crosses of D B A / 2 J and C 5 7 B L / 6 J mice in which the spermatogonial stem cells of the sires were treated with 250 m g / k g ENU. With the same strains of mice and the same treatment con-

31 ditions, Lovell et al. (1985) reported that there were no significant differences between the offspring of the treated and the control males in the frequencies of skeletal abnormalities that could be attributed to induced mutations. Similarly, Lyon and Renshaw (1986) did not observe any significant induction of external abnormalities in the offspring of an intra-strain cross of ( C 3 H / H e H x 1 0 1 / H ) F 1 mice in which the stem cell stage of the sires was treated with 250 m g / k g ENU. These negative results contrast sharply with the positive data obtained in the present study for ex'ternal and skeletal abnormalities in ICR strain mice treated with comparable doses (Table 2). More than 80% of the fetuses recorded in the present study as external abnormals were associated with dwarfism or cleft palate (see Table 3). If these two types were not scored, no evidence for the ENU effect would be obtained in the present study as it was in the previous study by Johnson and Lewis (1981). In the study by Lovell et al. (1985), the frequency of skeletally abnormal fetuses was 1.8% (4/225) in the ENU-treated series and 0.8% (2/253) in the control series. These data can be classified as inconclusive when assayed statistically by the method proposed by Selby and Olson (1981). That is to say that the number of fetuses they scored is too small to draw a conclusion. In the study by Lyon and Renshaw (1986), the control frequencies of externally abnormal fetuses from different experiments varied in a range of 1.1-2.0%. The frequency in the ENU-treated series was 2.5% (22/866) which did not differ much not only from the corresponding control frequency of 2.0% (8/396) but also from a positive frequency of 3.1% (35/1096) in the EMS-treated series. Hence, I suppose that, in the study by Lyon and Renshaw (1986), a high variability of the spontaneous frequency might have masked a significant effect of the ENU treatment. In addition, differences in the strains of mice used may merit serious consideration in seeking a possible reason for the discrepancy between the present results and the previously reported data; the sensitivity of spermatogonial stem cells to ENU induction of dominant visible mutations may exhibit strain variation. From these arguments, it seems that genotoxicity testing of a chemical agent with the use of congenital defects as endpoints requires the choice

of a suitable strain of mice and types of abnormality, and a proper experimental design with respect to the number of fetuses to be inspected. The rate of congenital defects (i.e., number of abnormals/live fetus/unit dose in m g / k g ) induced by treating spermatogonial stem cells with ENU was 1.0 x 10 -4. When the methyl analog of ENU, MNU, was applied at the same germ cell stage using the same treatment schedule and mating scheme employed in the present study, the induction rate was 3.0 x 10 -4 (Nagao, 1987). Thus, when spermatogonial stem cells are treated, ENU is 0.3-fold as effective as M N U in inducing congenital defects in the offspring. Assuming a linear dose-response relation, practically the same value of the relative effectiveness of ENU (on the basis of m g / k g unit) in inducing somatic recessive mutations was estimated from the data published by Russell and Montgomery (1982). These workers injected ENU or MNU i.p. into females on day 10.5 of pregnancy and scored fur color spots as somatic recessive mutations and polydactyly, kinky tail, and other external abnormalities as congenital defects in the newborn offspring. Using the same assumption, I estimate from their data on congenital defects that the teratogenic effectiveness of ENU relative to M N U is 0.07. This value is considerably lower than the relative genotoxic effectiveness estimated for paternally-transmitted congenital defects given above. These comparative analyses lead to the conclusion that congenital defects induced indirectly in the offspring by treating the paternal genome with ENU before copulation and those induced directly by treating embryos with ENU at the organogenic stage represent quite different entities. Although the genotoxic potency of ENU and M N U differ by a factor of 3, the spectra of the male-transmitted fetal abnormalities induced by these agents do not differ much, suggesting a qualitative similarity in their genotoxic effects. The relative number ratio of externally abnormal fetuses and skeletally abnormal fetuses was 1 : 0.3 in the offspring of ENU-treated males (see Table 3). The ratio in the offspring of MNU-treated males was also 1:0.3 (data from Nagao, 1987). The ratio of cleft palate, dwarfism, exencephalus and open eyelids in the ENU-treated series was 1 . 9 : 1 : 0 . 2 : 0 . 3 , which is very close to that of

32

2 : 1 : 0 . 3 : 0 . 3 in the MNU-treated series. A substantial difference in the spectrum was noticed for skeletal abnormalities. The number ratio of malformed ribs, malformed vertebrae, and bending of the appendicular skeleton was 1 2 : 1 3 : 9 in the ENU-treated series and 13 : 3 : 0 in the M N U treated series (X 2 = 10.3, df= 2, P < 0.01). When the small number of skeletally abnormal fetuses detected (e.g., 17 with M N U ) is considered, however, the difference may be due to an unknown fluctuation in the experimental conditions. Further scoring of skeletally abnormal fetuses in further experiments with ENU, M N U and other mutagens is needed to determine whether the difference in reactivity with DNA or the ability to induce chromosome aberrations, as exists between ENU and MNU, results in different spectra of skeletal abnormalities. More important at this moment is the fact that congenital defects can be induced not only by paternal treatment with a mutagen of high clastogenic activity (e.g., M N U , Nagao, 1987; X-rays, Nomura, 1982) but also by paternal treatment with a mutagen of relatively low clastogenic activity (e.g., ENU, the present study; urethane, Nomura, 1982). Further evidence supporting this fact is accumulating in my laboratory using the ICR strain mice and other genotoxins such as triethylenemelamine, mitomycin C, procarbazine, and propyl methanesulfonate. Acknowledgement I thank K. Fujikawa who gave continuous advice throughout the present study and read very early drafts of the manuscript, J.R. Miller and S. Hitotsumachi who also read the manuscript and provided many helpful comments, and Y. Ishizuka and A. Wada who provided skilful technical assistance. Special thanks go to M. Yasuda, K. Tatsumi and M. Mizutani for encouragement. References Adams, P.M., J.D. Fabricant and M.S. Legator (1981) Cyclophosphamide-induced spermatogenic effects detected in the F t generation by behavioral testing, Science, 211, 80-82. Crow, E.L., and R.S. Gardner (1959) Confidence intervals for the expectation of a poisson variable, Biometrika, 46, 441-453.

Dawson, A.B. (1926) Note on staining of the skeleton of cleared specimens with alizarin red S, Stain Technol., 1, 123-124. Ehling, U.H., J. Favor, J. Kratochvilova and A. Neuh~iuserKlaus (1982) Dominant cataract mutations and specificlocus mutations in mice induced by radiation or ethylnitrosourea, Mutation Res., 92, 181-192. Ehling, U.H., D.J. Charles, J. Favor, J. Graw, J. Kratochvilova, A. Neuh~iuser-Klaus and W. Pretsch (1985) Induction of gene mutations in mice: The multiple endpoint approach, Mutation Res., 150, 393-401. Favor, J. (1983) A comparison of the dominant cataract and recessive specific-locus mutation rates induced by treatment of male mice with ethylnitrosourea, Mutation Res., 110, 367-382. Ficsor, G., G.M. Oldford, K.R. Loughlin, B.B. Panda, J.L. Dubien and L.C. Ginsberg (1984) Comparison of methods for detecting mitomycin C- and ethyl nitrosourea-induced germ cell damage in mice: Sperm enzyme activity, sperm motility, and testis weight, Environ. Mutagen., 6, 287-298. Generoso, W.M., K.T. Chain, C.C. Cornett and N.L.A. Carcheiro (1984) DNA target sites associated with chemical induction of dominant-lethal mutations and heritable translocations in mice, in: V.L. Chopra, B.C. Joshi, R.P. Sharma and H.C. Barsal (Eds.), Genetics: New Frontiers, Vol. 1, Oxford and IBH, New Delhi, pp. 347-355. Hitotsumachi, S., D.A. Carpenter and W.L. Russell (1985) Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia, Proc. Natl. Acad. Sci. (U.S.A.), 82, 6619-6621. Johnson, F.M., and S.E. Lewis (1981) Electrophoretically detected germinal mutations induced in the mouse by ethylnitrosourea, Proc. Natl. Acad. Sci. (U.S.A.), 78, 3138-3141. Kirk, K.M., and M.F. Lyon (1982) Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays, Mutation Res., 106, 73-83. Kirk, K.M., and M.F. Lyon (1984) Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages, Mutation Res., 125, 75-85. Knudsen, 1., E.V. Hansen, O.A. Meyer and E. Poulsen (1977) A proposed method for the simultaneous detection of germ-cell mutations leading to fetal death (dominant lethality) and of malformations (male teratogenicity) in mammals, Mutation Res., 48, 267-270. Lovell, D.P., D.B. Willis and F.M. Johnson (1985) Lack of evidence for skeletal abnormalities in offspring of mice exposed to ethylnitrosourea, Proc. Natl. Acad. Sci. (U.S.A.), 82, 2852-2856. Lyon, M.F., and R. Renshaw (1986) Induction of congenital malformations in the offspring of mutagen treated mice, in: B. Bonne-Tamir, T. Cohen and R.M. Goodman (Eds.), Genetic Toxicology of Environmental Chemicals, Vol. 209, Part B, Genetic Effects and Applied Mutagenesis, Liss, New York, pp. 449-458. Nagao, T. (1987) Frequency of congenital defects and dominant lethals in the offspring of male mice treated with methylnitrosourea, Mutation Res., 177, 171-178.

33 Nomura, T. (1975) Transmission of tumors and malformations to the next generation of mice subsequent to urethan treatment, Cancer Res., 35, 264-266. Nomura, T. (1982) Paternal exposure to X-rays and chemicals induces heritable tumors and anomalies in mice, Nature (London), 296, 575-577. Oakberg, E.F., and C.D. Crosthwait (1983) The effect of ethyl-, methyl- and hydroxyethyl-nitrosourea on the mouse testis, Mutation Res., 108, 337-344. Russell, L.B., and C.S. Montgomery (1982) Supermutagenicity of ethylnitrosourea in the mouse spot test, Comparisons with methylnitrosourea and ethylnitrosourethane, Mutation Res., 92, 193-204. Russell, W.L., E.M. Kelly, P.R. Hunsicker, J.W. Bangham, S.C. Maddux and E.L. Phipps (1979) Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse, Proc. Natl. Acad. Sci. (U.S.A.), 76, 5818-5819. Russell, W.L., P.R. Hunsicker, D.A. Carpenter, C.V. Cornett and G.M. Guinn (1982a) Effect of dose fractionation on

the ethylnitrosourea induction of specific-locus mutations in mouse spermatogonia, Proc. Natl. Acad. Sci. (U.S.A.), 79, 3592-3593. Russell, W.L., P.R. Hunsicker, G.D. Raymer, M.H. Steele, K.F. Stelzner and H.M, Thompson (1982b) Dose-response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia, Proc. Natl. Acad. Sci. (U.S.A.), 79, 3589-3591. Selby, P.B., and S.S. Lee (1981) Sensitive-indicator results show that ethylnitrosourea is also a supermutagen for dominant skeletal mutations, Environ. Mutagen., 3, 373. Selby, P.B., and S.L. Niemann (1984) Non-breeding-test methods for dominant skeletal mutations shown by ethylnitrosourea to be easily applicable to offspring examined in specific-locus experiments, Mutation Res., 127, 93-105. Selby, P.B., and W.H. Olson (1981) Methods and criteria for deciding whether specific-locus mutation-rate data in mice indicate a positive, negative, or inconclusive result, Mutation Res., 83, 403-418.