An examination of respiratory distress and chromosomal abnormalities in the offspring of male mice treated with ethylnitrosourea

Mutation Research, 229 (1990) 115-122 Elsevier

115

MUT 02703

An examination of respiratory distress and chromosomal abnormalities in the offspring of male mice treated with ethylnitrosourea Taisei Nomura 1,3, Hiroko Gotoh 1,2 and Tatsuo Namba 1,. 1 Department of Radiation Biology, Faculty of Medicine, Osaka University, Nakanoshima, Kita-ku, Osaka 530 (Japan), 2 Department of Life Science, Saho Women's College, Rokuyaoncho, Nara 630 (Japan) and ~ Genetics, University of Wisconsin, Madison, 14"153706 (U.S.A.) (Received 2 October 1988) (Accepted 28 January 1989)

Keywords: Functional defects; Respiratory distress; Offspring; Paternal exposure; Ethylnitrosourea; Chromosomal abnormality; (Mouse)

Summary A functional defect (respiratory distress), in addition to morphological defects, was induced in the offspring of male ICR mice treated with ethylnitrosourea (ENU) before mating. ENU (100 and 50/~g/g) was injected intraperitoneally into adult male ICR mice that were then mated with untreated females. After the cesarian operation on the 18th day of gestation, fetuses were resuscitated. In the apneic fetuses showing respiratory distress, the lung was collapsed and the ductus arteriosus was not closed. The incidence of fetuses showing respiratory distress was significantly increased with the high dose (100/~g/g) of ENU, and it was higher after spermatogonial exposure than after postmeiotic exposure. There was no linearity in the dose-response relationship at the lower dose (50 /xg/g), as was the case with the specific-locus mutation. The frequency per/tg ENU of fetuses showing respiratory distress was 3.7 x 10 -4 for spermatogonial treatment (calculated at a dose of 100/tg/g), the value being about 10-20 times higher than that of ordinary mutations in mice. About half of the fetuses showing respiratory distress often had specific anomalies (dwarfism and gigantic thymus), but the remainder showed no morphological changes. Spermatogonial treatment produced a zero or very low incidence of translocations in the meiotic configurations of primary spermatocytes. G-band analysis of the affected F1 fetuses also revealed no visible chromosomal abnormalities (there could be small deletions or inversions) except that trisomy 19 was found in a dwarf fetus.

Correspondence: Dr. Taisei Nomura, Department of Radiation Biology, Faculty of Medicine, Osaka University, 3-57 Nakanoshima 4-chome, Kita-ku, Osaka 530 (Japan). * Present address: Saiseikai-kure General Hospital, Kure, Hiroshima-ken (Japan).

Abbreviations: ENU, N-ethyl-N-nitrosourea; RDS, respiratory distress syndrome.

Recently, radiation and chemicals have been proved to produce morphological abnormalities (e.g., tumors and malformations) in the F1 offspring of experimental animals after treatment of the male or female parent (Nomura et al., 1971; Nomura, 1975, 1978, 1982, 1986, 1988; Kirk and Lyon, 1982, 1984), in addition to the mutations having clear-cut endpoints such as recessive coat color mutations and dominant skeletal, cataract or

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

116 electrophoretic mutations (Russell et al., 1958, 1960, 1982; Bartsch-Sandhoff, 1974; Russell, 1984; Ehling, 1984; Charles and Pretsch, 1986, 1987). Information obtained from the work on morphological anomalies (malformations) will be especially valuable in assessing genetic risk from radiation and chemicals, because a majority of human genetic diseases shows this kind of irregular and uncertain inheritance and most of the induced malformations are similar to those found in humans (Nomura, 1978, 1982, 1988). In fact, similar results have been observed in the children of fathers exposed to herbicides in South Vietnam (Can, 1984). In comparison with these morphological defects, however, functional (e.g., physiological and biochemical) disorders are more commonly observed in human newborns. Among them, dyspnea and apnea are seen very often at birth (Avery, 1987). Consequently, we tested whether these respiratory distress disorders can be induced in the offspring of mice after parental exposure to toxic agents. Ethylnitrosourea (ENU) was selected in this study, because it is known to produce a high incidence of dominant mutations affecting enzyme activity (Charles and Pretsch, 1987) in addition to morphological changes (dominant lethals, coat color and cataract mutations, tumors, malformations, etc.) in the offspring of experimental animals after paternal treatment (Tomatis et al., 1982; Russell et al., 1982; Russell, 1984; Ehling, 1984; Nomura, 1988). Chromosomal abnormality was also examined in the fetuses having these disorders and their parents treated with ENU. Materials and methods

Animals Virgin ICR mice were used (Nomura, 1988). Mice were maintained with mouse diet CA-1 (CLEA Japan, Tokyo) and chlorinated tap water in the barrier-conditioned mouse room at 2325 o C. The spontaneous rate of anomalies has not changed noticeably (Nomura, 1975, 1978, 1982, 1988). Treatment with E N U Adult male mice (63-65 days old) received a single intraperitoneal injection of N-ethyl-N-

nitrosourea (ENU, Sigma, St. Louis, MO). ENU (stabilized with 20 w/w% of 5% acetic acid solution and kept at - 2 0 ° C ) was dissolved in 0.1 M sodium phosphate buffer solution (pH 5.8)just before injection. Doses used were 50 and 100 /~g/g body weight. The maximum tolerated dose of ENU in adult male ICR was 120 #g/g. Control groups received the same volume of the solvent.

Examination of functional and morphological defects An estrous female ICR was selected by the appearance of the vaginal orifice, and mated with ENU-treated or untreated males in the evening at various intervals 1-21 days or 90-180 days after treatment, so that the postmeiotic or spermatogonial stage, respectively, was treated. The next morning, we examined whether a vaginal plug was present to determine the day of conception (day 0) (Nomura and Okamoto, 1972; Nomura, 1974). The plug day (day 0) corresponds to day 1 in previous papers (Nomura and Okamoto, 1972; Nomura, 1974, 1978, 1982). The mouse room was lit from 4.00 to 18.00 h so that fertilization occurred at about 2.00 h on day 0 (Nomura et al., 1987). Pregnant mice were killed on day 18 of gestation by cervical dislocation, and then fetuses were taken out by cesarian operation. Fetuses were resuscitated just after the cesarian operation by patting very gently with tissue paper and wiping amniotic fluid from nose, mouth and body surface. Fetuses were then classified as apneic or pneic. Numbers of corpora lutea, implants, early deaths, late (fetal) deaths, living fetuses, and fetuses with morphological anomalies were recorded as described previously (Nomura, 1975, 1978). Fetuses with viable anomalies were fostered by lactating mothers to examine the heritability of the anomaly, and those with functionally and morphologically lethal defects were submitted to chromosomal examination. Usually, pregnant mice are killed around the 12th day of gestation to detect dominant lethals. In the present study, however, mice were killed on the 18th day in order to detect functional and morphological abnormalities at the same time (Nomura, 1975, 1978). Skeletal anomalies were examined by soft X-ray apparatus (SOFRON SRO-MSO, Soken, Tokyo), and then

117

abnormalities of the internal organs were examined under a dissecting microscope. The total incidence of morphological anomalies has been published previously (Nomura, 1988). For statistical analysis, the t-test was applied for dominant lethals after testing variance ratio (Table 1), and the x2-test with Yates' correction was applied for malformations (Table 2).

G-band preparation of chromosomes from fetuses The whole fetal spleen and one third of the fetal liver were minced with slide glasses, and suspended in about 10 ml of 0.9% NaC1 solution containing 3 /~g/ml of colchicine (Sigma). Cells were dispersed by repeated pipetting with a Pasteur pipette and passed through a tissue screen. After centrifuging the cell suspension at 900 rpm (radius of centrifuge head 15 cm) for 10 rain, cells were treated with 1 ml of hypotonic (75 mM) KC1 solution containing 3/~g/ml of colchicine for 25 rain at 37 ° C, and fixed in methanol-acetic acid (1:1) solution. Cells were washed twice with methanol-acetic acid (1 : 1) solution and then airdried on the slide. The slides were heated at 60 ° C overnight, and treated with trypsin (0.0125% in Hanks' buffer solution, Osaka Univ. Microbial Dis. Co., Osaka) for 30-35 s at 37°C for G-banding (Cowell, 1984). They were washed with ice-cold phosphate-buffered saline solution (pH 7.4) and tap water, and then stained for 8 rain in a 4% Giemsa solution (Merck, Darmstadt) diluted with S~Srensen's phosphate buffer solution (1/15 M, pH 6.8).

Preparation of meiotic configuration from mouse testis The male parents were killed 6 months after intraperitoneal treatment with ENU, and the testes removed and placed in isotonic (2.2%) sodium citrate solution. The tubules were pulled out gently and their contents were teased out by fine straight forceps. The cell suspension was passed through a tissue screen and centrifuged at 550 rpm for 5 min and the supernatant (mostly containing spermatozoa and spermatids) was discarded. Sedimented cells were suspended in 1 ml of hypotonic (1%) sodium citrate solution and incubated at 37 ° C for 12 rain. Cells were fixed by gently adding 9 ml of methanol-acetic acid (1:1) solution, and centri-

fuged at 1200 rpm for 10 rain. Cells were washed with fixative solution, and then air-dried on the slide (Evans et al., 1964). The slides were stained in a 4% Giemsa solution for 8 rain. Results and discussion

Respiratory distress and combined anomalies Paternal treatment with ENU at the postmeiotic stage decreased t h e numbers of living fetuses due to the increased dominant lethals. However, the incidence of late (fetal) deaths was not significantly elevated by parental treatment (Table 1). This was the case with radiation and other chemicals, although late deaths were easily induced by the treatment during pregnancy (Nomura, 1987). Nonetheless, various types of malformations were observed (legend to Table 1) in the fetuses which died at late stages (days 16-18). The incidence of fetuses showing respiratory distress syndrome (RDS) increased significantly with the high dose (100/~g/g) of ENU (Table 2). The incidence was higher after spermatogonial exposure than after postmeiotic exposure. The resuits were compatible with those of specific-locus mutations (Russell, 1984) and morphological abnormalities (Nomura, 1988). Germ-line alterations may lead to respiratory distress in F1 offspring. Although there was no linearity in the dose-response relationship at the lower dose (50 #g/g), this was the case in specific-locus mutations (Russell et al., 1982). The frequency per/~g ENU of RDS was 3.7 X 10 -4 for spermatogonia (calculated at a dose of 100 #g/g). The value was similar to that of morphological abnormalities (Nomura, 1988) but about 10-20 times higher than those of ordinary mutations in mice (Russell et al., 1982; Ehling, 1984; Charles and Pretsch, 1987). However, the spontaneous frequency of functional defects was also high (Table 2). The ratio of the induced and spontaneous frequencies was not very different from that for specific-locus mutations. In all fetuses showing respiratory distress, patent ductus arteriosus and collapsed lung were observed under the dissecting microscope (Fig. 1), as was the case in humans (Sadler, 1985; Avery, 1987). About half of them also had specific mor-

118

Fig. 1. Cardiovascular abnormalities in the fetuses showing respiratory distress. After the cesarian operation, fetuses were resuscitated as described in Materials and methods. In the normal pneic fetus (A), the lung is filled with air and the ductus arteriosus (arrow) is closed, while in the apneic fetus (B), the lung is collapsed (without air) and the ductus arteriosus is still open (arrow). Ao, aorta; P, pulmonary artery; RA, right atrium; LA, left atrium; RV, right ventricle; L, lung.

119 TABLE 1 IMPLANTS, LETHALS AND LIVING FETUSES AFTER PATERNAL EXPOSURE TO ENU Treated stage

Postmeiotic Spermatogonia Control

Dose

Number of mice with

Number

Early deaths

Late deaths

Living fetuses

(#g/g)

plug

implants

of implants

N (%) a

N (%) a

N (%) a

50 100 50 100 0

36 33 17 49 45

32 27 16 37 40

414 331 202 379 516

25 (5.9 + 2.3) 67 (20.5 + 6.0) * 7 (3.1 + 2.1) 17 (5.6+3.5) 25 (4.8 5:2.1)

8 (1.9) b 8 (2.4) 5 (2.5) 14 (3.7) c 16 (3.1)

381 (92.0) 256 (77.3) * 190 (94.1) 348 (92.1) 475 (92.1)

a Percent of implants. For early deaths, % early deaths averaged over litter values and 90% confidence limits of the mean computed from t-distribution were given as an indicator of dominant lethals. b Accompanied by 1 cleft palate, 1 tail anomaly, 1 gigantic thymus, and 1 diaphragmatic hernia. c Accompanied by 1 exencephalus + open eyelid. * p < 0.001. The value of dominant lethals at 100 # g / g of ENU was published in a previous paper (Nomura, 1988). TABLE2 INCIDENCE OF FETUSES SHOWING RESPIRATORY DISTRESS AND MORPHOLOGICAL ANOMALIES AFTER PATERNAL EXPOSURE TO ENU Treated stage Postmeiotic Spermatogonia

Control

Dose

Living

Fetuses showing respiratory distress

Other morphological

(#g/g)

fetuses

N (%)

Combined morphological anomalies

anomalies

50 100 50 100

381 256 190 348

3 (0.8) 6 (2.3) * 1 (0.5) 13(3.7)**

2 D, 1 M-T, 1 CP 1 CP 60E 2 CP, 2 0 E , 1Ex

0

475

2D 2 D, 1 C P + D none 3 D, 1CP, 1 C P + G T , 1 E x + D 1 CP+ D + G T 1D

1 (0.2)

10E

D, dwarf; OE, open eyelid; M, meningocele; T, tail anomalies (kinky and/or short); CP, cleft palate; GT, gigantic thymus; Ex, exencephalus. * p < 0.02, ** p < 0.001.

phological anomalies, dwarfism and gigantic t h y m u s , in c o m b i n a t i o n w i t h c l e f t p a l a t e a n d exe n c e p h a l u s ( T a b l e 2), b u t t h e r e m a i n d e r s h o w e d

TABLE 3 MEIOTIC CONFIGURATIONS OF PRIMARY SPERMATOCYTES OF MALE PARENTS TREATED WITH ENU Dose (#g/g)

Number of metaphases examined a

Translocations No. (%) Details b

100 50 0

500 500 500

1 (0.2) 0 (0.0) 0 (0.0)

1811 + IV(C)

a First metaphases from 5 males in each experiment. b II, bivalent; IV (C), quadrivalent (chain).

no morphological changes. Furthermore, some f e t u s e s w i t h l e t h a l a n o m a l i e s (cleft p a l a t e , e x e n c e p h a l u s , etc.) d i d n o t s h o w r e s p i r a t o r y distress, although they died shortly after birth because of misswallowing, bleeding, and cerebral damage. O t h e r b i o c h e m i c a l o r p h y s i o l o g i c a l d e f e c t s in the respiratory system might be involved.

Chromosomal abnormality in the fetuses and their parents T o f i n d a p o s s i b l e c a u s e o f g e r m - l i n e altera t i o n s l e a d i n g to f u n c t i o n a l a n d m o r p h o l o g i c a l anomalies, chromosomes were examined for c h a n g e s in t h e f e t u s e s s h o w i n g t h e s e d e f e c t s a n d t h e i r p a r e n t s t r e a t e d w i t h E N U . A l t h o u g h treatm e n t o f t h e p o s t m e i o t i c s t a g e s r e s u l t e d in a sig-

120 TABLE 4 ANALYSIS OF G - B A N D E D STRUCTURE OF CHROMOSOMES IN THE F 1 FETUSES OF MALE ICR MICE TREATED W I T H E N U AT THE S P E R M A T O G O N I A L STAGE Dose of ENU (/~g/g)

Defects a

Number of fetuses

Chromosome abnormality

100

RDS RDS + CP CP D + CP D D + Ex OE None

5 1 3 1 1 1 1 15

None None None None Trisomy 19 b None None None

50

RDS OE None

1 3 9

None None None

0

None

34

None

75.0%), 24/36 (15/23, 9/13) (65.2-69.2%), 26/36 (8/12, 18/24) (66.7-75.0%), 7/21 (6/18, 1/3) (33.3%), 8/11 (3/6, 5/5) (50-100%), 22/45 (10/ 21, 12/24) (47.6-50%), 5/8 (62.5%) from F2 to FIe, respectively. However, chromosomal changes were not detected at present by G-band analysis. Small deletions and inversions or gene mutations could be involved in germ-line transmission. Alternatively, numerical changes might be involved in some of the congenital malformations, because trisomy 19 was found in a dwarf fetus (Fig. 2).

Perspectives Epidemiologically, the incidence of untoward outcomes (abortion, stillbirth, major congenital defects, etc.) was not significantly higher in the children of atomic bomb survivors than those of unirradiated parents (Schull et al., 1981), while such defects can be induced in the offspring of

For abbreviations see the legend to Table 2. b Fig. 2.

a

nificant increase in dominant lethals caused by large chromosomal changes, spermatogonial treatment induced a very low, if not zero, incidence of dominant lethals in the F a (Table 1) and translocations in the meiotic configurations of primary spermatocytes of parents (Table 3). G-band analysis did not show chromosomal abnormalities in 17 abnormal fetuses of ENUtreated male parents (Table 4). X-rays, however, induce a high incidence of translocations in the parental germ cells (Leonard and Deknudt, 1969) and in the resultant offspring (Nomura, 1978, 1982, 1986). Consequently, large visible chromosomal changes (translocations, large deletions and inversions, etc.) may not necessarily cause functional defects in the offspring. Although heritability of RDS could not be examined because of its lethality, some morphological abnormalities (e.g., dwarfism, open eyelid, etc.) are known to be heritable (Nomura 19Z8, 1982, 1 = 88). Wulctkve mating of the offspring bearing open eyelid produced a higher incidence of open eyelid in the next generations: 2/36 (5.6%), 8/37 (21.6%), 2 3 / 63 (16/44, 7/19) (36.4-36.8%), 16/25 (10/16, 6/9) (62.5-66.7%), 14/27 (8/19, 6/8) (42.1-

Fig. 2. G-band pattern of the chromosome from a dwarf fetus of a male ENU-treated parent. Trisomy 19 was found in all 10 metaphases examined (for details, see text and Table 4).

121 mice exposed to radiation and chemicals (Nomura, 1975, 1978, 1982, 1988; Kirk and Lyon, 1982, 1984; this paper). However, the result does not indicate that human male germ cells are more resistant to radiation and chemicals than those of experimental animals, although human oocytes are much more resistant at least to radiation killing than mouse oocytes. Abortion (embryonic death) as a consequence of germ-ceU exposure can be induced only after postmeiotic exposure to radiations and chemicals but not after spermatogonial exposure in the mouse experiments (e.g., doubling dose is about 1000 rad for spermatogonial treatment) (Ltining and Searle, 1971; Nomura, 1982, 1988). There is an extremely low chance of achieving pregnancy just after atomic bomb exposure. Furthermore, most of the major congenital malformations are lethal shortly after birth and there is no significant increase in congenital malformations in the five offspring of mice after spermatogonial exposure to moderate doses (36216 rad) of X-rays (Nomura, 1982, 1988). It is also well known that parental exposure to radiations was not effective in increasing the incidence of stillbirths (late death) in contrast to gestational exposure (Nomura, 1978, 1982, 1987, 1988). As for mutations, there was no increase in base-substitution mutation affecting electrophoretic mobility of serum proteins in the children of atomic bomb survivors (Satoh and N e d , 1988), This was the case in mice (Pretsch, 1986). Consequently, adequate genetic markers have not been examined in the past epidemiological studies in Hiroshima and Nagasaki. In contrast, specific-locus recessive mutations affecting coat color and dominant mutations affecting enzyme activity (mostly caused by deletion mutation) are significantly induced by radiations in mice (Russell et al., 1960; Charles and Pretsch, 1986). It is noted that one enzyme activity mutation was detected in about 5000 children of atomic bomb survivors (Satoh and Neel, 1988) and the mutation frequency per rad per locus does not differ from that in mice (1.2 × 10 -7, 2.2 × 10 -7 and 1.7 × 10 -7, for enzyme activity and specific-locus mutations in mice and enzyme activity mutation in man, respectively) (Nomura, 1989). As for chemicals, presumed exposure to herbicides resulted in a significant increase in malfor-

mations (deft palate, anencephaly, etc.) in children of war veterans who had fought in the sprayed area of South Vietnam and married unexposed North Vietnam women (28/1801, 1.6%) compared to the children of war veterans who had never been in the South and married North Vietnam women (118/16570, 0.7%) (Cfm, 1984). Consequently, germ-fine transmission of birth defects may occur in man as a consequence of parental exposure. Several human populations, e.g., offspring of the atomic bomb survivors, heavy cigarette smokers, specific industrial workers (Nomura, 1979), heavily medicated or irradiated patients, etc., are of particular interest. Since functional defects like RDS are more commonly observed in human newborns than major morphological defects (Avery, 1987), the study should be extended to radiation and various chemicals and also to the variety of physiological and biochemical defects.

Acknowledgements The work was supported by Grants-in-Aid for Environmental Science 59030071 (1984), 60030052 (1985) and 61030048 (1986), and for Scientific Research on Priority 62602015 ( 1 9 8 7 ) f r o m the Japanese Ministry of Education, Science, and Culture. We thank Drs. H. Tanaka, S. Hata, T. Enomoto and T. Ohhashi for their help, Misses K. Shibata, S. Kida, Y. Murakami, M. Okamoto and M. Maeda for their assistance, and Professors K. Moriwaki and M.S. Sasaki for their advice in G-band analysis.

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