Cell sensitivity to transplacental carcinogenesis by N-ethyl-N-nitrosourea is greatest in early post-implantation development.

In a clear demonstration of the changing sensitivity of the developing mammal to transplacental carcinogenesis, Ivankovic and Druckrey [S. Ivankovic, H. Druckrey, Transplacentare Erzeugung maligner Tumoren des Nervensystem: I. Athyl-nitroso-harnstoff (ANH) an BD IX-Ratten, Z. Krebsforsch. 71 (1968) 320-360] exposed pregnant BD IX rats to a pulse of N-ethyl-N-nitrosourea (ENU), a reactive carcinogen with a half-life of 20 min. No tumors were seen with ENU exposure before gestation day 12, but the multiplicity of neurogenic tumors increased steadily thereafter and was greatest with treatment on day 20, followed by a decline in sensitivity for the last three days of gestation. Similarly, a transplacental study of ENU in the Syrian hamster [B.A. Diwan, S. Rehm, J.M. Rice, Age- and dose-dependent transplacental carcinogenesis by N-nitrosoethylurea in Syrian golden hamsters, J. Cancer Res. Clin. Oncol. 122 (1996) 643-652] found that the numbers of tumors induced were greatest after exposure of late fetal stages. While these observations suggested that embryonic cells are refractory to carcinogenesis, an alternative explanation could be that a significant tumor yield was not observed because too few target cells were present in the embryo. I have resolved this issue by combining these published data with others on the numbers of neuroectodermal cells in the developing BD IX rat brain [R. Müller, M.F. Rajewsky, Elimination of O6-ethylguanine from the DNA of brain, liver, and other rat tissues exposed to ethylnitrosourea at different stages of prenatal development, Cancer Res. 43 (1983) 2897-2904] and total cell counts of successive developmental stages of the Syrian hamster fetus [P.J. Donovan, G.T. Smith, Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster, Mutation Res., 1999, this issue], allowing the risk per cell at different stages of gestation to be calculated. Sensitivity to carcinogenesis was found to be greatest early in gestation and to decrease as gestation proceeds. For the rat model, tumor frequency per cell changed from 1.3x10(-6) at day 12 exposure to 2.6x10(-8) at day 23 exposure, a 50-fold decrease. For the hamster model, the tumor-initiation rate decreased 1250-fold from 1.2x10(-5) at day 7 exposure to 9.6x10(-9) at day 13 exposure. Thus, two independent experiments with different rodent species demonstrate that sensitivity of individual cells to damage leading to transplacental carcinogenesis is greatest in the early fetus and lessens markedly as gestation proceeds, in parallel with changing sensitivity to mutation (Donovan et al., Mutat. Res., this issue).     

Hamster and rat fetal cells have low spontaneous mutation frequencies and rates

Somatic cells of whole Syrian hamster fetuses (gestation day 13) were isolated and tested by an in vivo/in vitro mutation assay for spontaneous mutation frequencies using independent 6-thioguanine (6-TG), diphtheria toxin (DT), and ouabain mutation selection systems. Optimum conditions were ascertained. For 6-TG mutants, a total of 21 mutants were found in cells from 24 litters on 1993 plates, for an overall mutant frequency of 1.8 x 10(-7) per viable cell with 12 positive litters. In all, 26 litters were tested using DT; 77 mutants were found in 840 plates, yielding an overall mutant frequency of 2.6 x 10(-7), with 20 positive litters. No correlations or familial effects were found among 23 litters tested for both DT and 6-TG. Of 14 litters which were tested for ouabain mutants, 4 were positive, with a total of 5 mutants found on 988 plates, for an overall mutant frequency of 7.6 x 10(-8). For 14 F344 rat fetuses, the overall 6-TG spontaneous mutation frequency was determined to be 1.6 x 10(-7). From the data, estimates of mutation rates were calculated. For mutation to 6-TG resistance the rate was 8.3 x 10(-8), for mutation to DT resistance the rate was 8.1 x 10(-8) and for ouabain, the spontaneous mutation rate was 5.7 x 10(-8). For F344 rat, the spontaneous mutation rate was 1.1 x 10(-7). Induced mutant frequencies after in utero exposure to 1 mmol/kg N-ethyl-N-nitrosourea (ENU) were 311, 135 and 200 times the spontaneous value for 6-TG, DT and ouabain, respectively, for Syrian hamster fetal cells and 125 times the spontaneous 6-TG value for fetal F344 rat cells. Both spontaneous mutation frequencies and underlying spontaneous mutation rates are low, consistent with the view that fetal cells exercise extremely tight control over DNA fidelity.     

Urethane and N-nitrosodiethylamine are mutagenic for the Syrian hamster fetus

Urethane and N-nitrosodiethylamine are soluble environmental carcinogens that initiate tumors transplacentally, but have a mixed history of effectiveness in mutagenesis assays in vitro or in vivo with adult rodents. To test for their transplacental mutagenicity, Syrian hamster fetuses at 12 days in gestation were exposed transplacentally to urethane or N-nitrosodiethylamine at 0.5 or 1.0 mM/kg. The fetal cells were isolated on day 13 of gestation and tested for diphtheria toxin resistance as a mutation marker. Both compounds were significantly mutagenic, at both doses, causing 6- to 20-fold increases in mutations compared with controls. Compared with N-nitrosodiethylamine, urethane was somewhat more effective as a mutagen with a more marked dose-response. These results are consistent with mutagenesis as part of the mechanism of transplacental carcinogenicity of urethane and N-nitrosodiethylamine.     

Hypoploidy and hyperplasia in the developing brain exposed to alcohol in utero

Prenatal effects of acute maternal alcohol ingestion on chromosome segregation and mitotic frequency in the brain cells of the fetus were evaluated in mice by direct chromosome and mitotic counts and by flow cytometry. Fetuses were exposed to acute transplacental doses of alcohol for 4 days and killed on the fifth day. Those litters in which the fetuses were developed to the equivalent of normal 16th-17th-day gestation age were analyzed. A marked increase in the number of hypoploid metaphases was observed in direct proportion to the dose ingested by the mother. An over 30% increase in hypoploidy over controls was measured in the fetuses exposed to the highest dose. Counts of mitotic cells showed an over tenfold increase in the mitotic index of the fetal brain exposed to alcohol. Flow cytometric measurements of DNA content in isolated fetal brain cell nuclei showed a shift from a single G0/G1 peak in controls to a bimodal G0/G1-G2 + M distribution in alcohol-exposed fetuses of the same developmental age.     

Urethane and N-nitrosodiethylamine are mutagenic for the Syrian hamster fetus

Urethane and N-nitrosodiethylamine are soluble environmental carcinogens that initiate tumors transplacentally, but have a mixed history of effectiveness in mutagenesis assays in vitro or in vivo with adult rodents. To test for their transplacental mutagenicity, Syrian hamster fetuses at 12 days in gestation were exposed transplacentally to urethane or N-nitrosodiethylamine at 0.5 or 1.0 mM/kg. The fetal cells were isolated on day 13 of gestation and tested for diphtheria toxin resistance as a mutation marker. Both compounds were significantly mutagenic, at both doses, causing 6- to 20-fold increases in mutations compared with controls. Compared with N-nitrosodiethylamine, urethane was somewhat more effective as a mutagen with a more marked dose–response. These results are consistent with mutagenesis as part of the mechanism of transplacental carcinogenicity of urethane and N-nitrosodiethylamine.     

X-ray induced mutation in Syrian hamster fetal cells

Transabdominal X-rays are a risk factor for childhood leukemia, and X-ray exposure of mouse fetuses has led to increases in both mutations and initiated tumors in offspring. However, fetal sensitivity and dose-response characteristics with regard to transplacental mutagenesis by X-rays have never been quantified. In the current experiment, pregnant Syrian hamsters at day 12 of gestation were irradiated with 300-kV X-rays. Twenty-four hours later, the fetuses were removed and their cells were allowed a 5 day expression time in culture. They were then seeded for colony formation and also for mutation selection by 6-thioguanine (6-TG). Mutation frequency was linear over the entire dose range, 10-600 R. The average induced 6-TG mutant frequency was 4.7 x 10(-7) per R. These results suggest that fetal cells are highly sensitive to induction of mutations by X-rays, and that a no-effect threshold is not likely. The 10 R dose caused a 25-fold increase in mutation frequency over the historical control, 45 x 10(-7) versus 1.8 x 10(-7), an increase per R of 2.5-fold. Increased risk of childhood cancer related to obstetrical transabdominal X-ray has also been estimated at 2.5-fold per R. Thus, our results are consistent with mutation contributing to this effect.     

The induction of recessive mutations in mouse primordial germ cells with N-ethyl-N-nitrosourea

A specific-locus test was carried out to examine the mutagenic activity of N-ethyl-N-nitrosourea (ENU) on mouse primordial germ cells (PGC). Embryos of C3H/He mice were treated transplacentally with 30 or 50 mg ENU per kg of maternal body weight on day 8.5, 10.5, or 13.5 of gestation (G8.5 day, G10.5 day, or G13.5 day). Male and female mice that had been treated with ENU in embryonic stages were mated with female or male tester PW mice to detect recessive mutations induced in PGC.

ENU induced recessive mutations at a relatively high rate in PGC at these developmental stages. The most sensitive stage was G10.5 day. On G8.5 day, the induced mutation rate in males and females was not significantly different. Cluster mutations, which originate from the limited number of PGC and cell killing, were more frequently induced at an earlier developmental stage. The induced mutation rate per unit dose of ENU (1 mg/kg) was higher in G8.5 and G10.5 day PGC than in stem-cell spermatogonia. It can be concluded that mouse PGC are more sensitive than stem-cell spermatogonia to the induction of recessive mutations by ENU.     

Aberrant fetal growth and development after in vitro culture of sheep zygotes.

The effects of in vitro culture systems for sheep zygotes on subsequent fetal growth and development to day 61 and day 125 of gestation were studied. Zygotes recovered from superovulated Scottish Blackface ewes approximately 36 h after intrauterine insemination using semen from a single Suffolk sire were cultured for 5 days in (a) a granulosa cell co-culture system (co-culture); (b) synthetic oviductal fluid medium without serum (SOF-); and (c) synthetic oviductal fluid medium supplemented with human serum (SOF+). Control embryos were recovered from superovulated donor ewes at day 6 after oestrus. Embryos were transferred at day 6 to synchronous Scottish Blackface recipient ewes. In total, 146 gravid uteri were recovered, comprising 97 at day 61 (20 co-culture, 27 SOF-, 25 SOF+ and 25 control) and 49 at day 125 (13 co-culture, 8 SOF-, 6 SOF+ and 22 control) of gestation. Fetuses derived from co-cultured embryos were 14% heavier (P < 0.01) by day 61 of gestation than those derived from control embryos. Growth coefficients derived from the linear allometric equation logey = logea + b logex (where y = organ mass; x = fetal mass) were significantly greater (P < 0.05) for liver, heart, kidneys and plantaris muscle in fetuses derived from co-cultured embryos, and for liver in fetuses derived from SOF+ embryos than those for control fetuses. Fetuses derived from co-cultured embryos were 34% heavier (P < 0.001) and fetuses derived from SOF+ embryos were 18% heavier (P < 0.01) by day 125 of gestation than those derived from control embryos. Growth coefficients for liver and heart for fetuses derived from co-culture and SOF+ embryos were also significantly greater (P < 0.05) at this stage of gestation than those for control group fetuses. In contrast, allometric coefficients for these organs in fetuses derived from embryos cultured in SOF without serum supplementation were not different from those for controls. Excessive volumes of amniotic fluid (polyhydramnios) were observed in 23% of conceptuses derived from co-cultured embryos. In vitro embryo culture can significantly influence fetal growth and this study provides quantitative evidence of major shifts in the patterns of organ and tissue development.     

Growth retardation and microcephaly induced in mice by placental infection with murine cytomegalovirus

BACKGROUND: The placenta is regarded as a site of congenital cytomegalovirus (CMV) infection. The placental infection of fetuses with murine CMV (MCMV) was investigated in a mouse model. METHODS: The placentas and fetuses were examined using the polymerase chain reaction (PCR) and Southern blotting for viral DNA and immunostaining for viral antigen. Since the transplacental infection rarely occurs, the placentas were directly injected with MCMV at day 12.5 of gestation; the embryos were then allowed to develop until day 18.5 of gestation. RESULTS: Formation of infected foci at day 18. 5 of gestation was found in more than 60% of the injected placentas. Infection of about 50% of the embryos occurred from the infected placentas. The frequency of infection in the brain was 27%, which was the same as that in the liver and higher than that in the lungs. In the brains, infected cells were often observed in the ventricular zone of the cerebrum and sometimes in the cortical plate and the hippocampus. Developmental retardation with microcephaly was observed in about 25% of offspring exposed to infection in utero. CONCLUSIONS: These results suggest that formation of infected foci in the placenta is important for embryonic congenital infection, and that the cerebral ventricular zone is one of the most susceptible sites for CMV infection in the embryonic stage.     

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

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 micrograms/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 micrograms/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 micrograms/g), as was the case with the specific-locus mutation. The frequency per microgram ENU of fetuses showing respiratory distress was 3.7 X 10(-4) for spermatogonial treatment (calculated at a dose of 100 micrograms/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.     

Acetazolamide: maternal toxicity, pattern of malformations, and litter effect

Thirty litters of C57BL 6J mice were administered intraperitoneally one of four doses (0, 500, 750, and 1,000 mg/kg maternal weight) of acetazolamide on day 9 of gestation. The fetuses were removed on day 18 and fixed, stained, cleared, and examined for the pattern of malformations. The forelimb postaxial limb deficiency was the most common abnormality, but forelimb postaxial polydactyly and a postaxial deficiency in the hindlimb were also observed. Males were significantly more likely to be malformed than females at all doses, in contrast to the predominance of females observed in rat fetuses exposed to acetazolamide (Scott et al.: Teratology 6:239-240, '73). The occurrence of limb malformations did not correlate with maternal weight loss, the birth weight of the fetus, or the position of the fetus in the uterus. A "litter effect" was demonstrated in that there was a nonuniform distribution of litters with different proportions of malformed fetuses.     

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.     

Ethylnitrosourea (ENU)-induced apoptosis in the rat fetal tissues

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs. In this study, pregnant rats were treated with 60 mg/kg ENU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue for clarifying the mechanisms of the ENU fetotoxicity and teratogenicity. From 3 to 12 HAT, the moderate to marked increase in the number of pyknotic cells was detected in the fetal CNS, craniofacial mesenchymal tissues, gonads and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results strongly suggest that excess cell death by apoptosis in the fetal CNS, craniofacial tissues and gonads may have a close relation to the later occurrence of anomalies reported in these tissues following ENU-administration.     

Sex chromosome complement and developmental diversity in pre-and post-hatching porcine embryos

To examine sex and development relationships in porcine embryos in early gestation, 10 gilts were killed on Day 4, 5, or 6 post mating (first day of standing estrus = Day 0). Embryos recovered immediately after slaughter were cultured in Medium 199 with colcemid (0.05mug/ml), fixed on slides, and stained with 4% Giemsa. The number of cells in each specimen was counted from the slides, and, whenever cell dispersion allowed, sex was determined by presence or absence of the Y-chromosome in at least 2 spreads from each embryo. Three gilts slaughtered on Day 4 yielded 2- and 4-cell stage embryos (n = 38), but no data on sex could be obtained due to lack of mitosis or readable metaphase spreads. Three Day 5 litters had individual specimens ranging from 8 to 14 cells (n = 8), 32 to 64 cells (n = 10), and 13 to 31 cells (n = 11), with the sex determined in 15 of these. Cell numbers ranged from 18 to 165 (n = 14), 16 to 32 (n = 9), 36 to 82 (n = 12), and 16 to 30 (n = 9) in the 4 gilts slaughtered on Day 6, with the sex determined in 26 of these. Embryos within each litter were divided into low, medium and high cell numbers by 3 equal divisions of the range of cell numbers. Three Day-5 embryos and 1 Day-6 embryo were lost during preparation; neither the cell numbers nor the sex could be determined in 4 Day-5 and in 3 Day-6 embryos. The overall sex ratio approximated 1:1, but on Day 5, the ratios for males to females were 0:5, 1:3 and 6:0 for the low, medium and high cell number groups, respectively. Embryos of undetermined sex in these same groups numbered 3, 1 and 3, respectively. On Day 6 the distribution was 1:11, 4:2 and 8:0 in favor of the males, while embryos of undetermined sex in the low, medium and high cell number groups numbered 5, 7 and 2, respectively. Chi-square analysis of the combined Day-5 and Day-6 results indicated the presence of significantly more females among embryos with low cell numbers and more males in the high cell number group (P < 0.01).     

Paternal exposure to cyclophosphamide alters cell-cell contacts and activation of embryonic transcription in the preimplantation rat embryo

Paternal exposure to chronic low doses of cyclophosphamide, an anticancer agent, results in aberrant embryonic development of the progeny. We hypothesized that paternal exposure to cyclophosphamide disturbs zygotic gene activity regulating proper progression through preimplantation development and that this disturbance results in improper cell-cell interactions. To test this hypothesis, we analyzed cell-cell interactions and the expression of cytoskeletal elements in preimplantation embryos sired by male rats gavaged with saline or 6 mg kg(-1) day(-1) cyclophosphamide for 5 wk. Embryos from control litters had 4-12 cells on Day 2 of gestation; cell-cell contacts were observed consistently. Embryos from litters sired by cyclophosphamide-treated males were frequently abnormal and had lower cell numbers and decreased cell-cell contacts. Steady state concentrations of the mRNAs for cell adhesion molecules (cadherins and connexin 43) and structural proteins (beta-actin, collagen, and vimentin) were low in two- and four-cell control embryos; expression increased dramatically by the eight-cell stage. In contrast, embryos sired by cyclophosphamide-treated males displayed the highest expression of most trancripts at the two-cell stage. In parallel with the mRNA profiles, E-cadherin immmunoreactivity was nearly absent in two-cell control embryos and was strong by the eight-cell stage; immunoreactivity in embryos sired by drug-treated fathers was strong at the two-cell stage but absent at later stages. Thus, drug exposure of the paternal genome led to dysregulated expression of structural elements and decreased cell interactions during preimplantation embryonic development.     

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.     

Lead exposure on critical days of fetal life affects fertility in the female mouse

Female mice were exposed to lead in utero by intravenous injection of lead chloride into the mothers at different stages of pregnancy. At a mature age the mice exposed as fetuses (F1 generation) conceived at a normal rate, but the litter size and fetal survival varied significantly. Small litters and increased numbers of fetal deaths were observed in mice exposed to lead on day 8 of intrauterine life. The live fetuses in this group were normal with respect to weight and morphological appearance. Serum levels of estradiol and progesterone, measured on day 17 of pregnancy, did not differ significantly between F1 mice of a control, unexposed group and of the group exposed to lead on day 8 of intrauterine life. Ovarian follicle counts revealed a significantly smaller number of primordial follicles in the latter group. It is suggested that the exposure to lead at a time of early organogenesis caused the observed fertility decrease by interfering with the development of the female germ cells.     

Teratologic studies on rat perinates and offspring from dams treated with ethylnitrosourea (ENU).

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues, limbs and male reproductive organs. Recently we clarified that excess cell death caused by apoptosis occurred in these organs and tissues of rat fetuses from dams treated with ENU at day 13 of gestation (GD13). In this study, we examined fetuses at GD21 and offspring at 10 weeks of age after ENU administration to pregnant rats at GD13 in order to clarify the relationship between ENU-induced apoptosis in the fetal tissues and teratogenicity of ENU. Severe intrauterine growth retardation was observed in the ENU group, and the body weight of the offspring in the ENU group was significantly lower than that of the control group throughout the experiment. In addition, a high incidence of microencephaly, ectrodactyly and curved caudal vertebrae was observed in the offspring from dams treated with ENU at GD13. Judging from the results of our previous and present studies, it was strongly suggested that ENU-induced apoptosis in rat fetal tissues may play an important role in the induction of anomalies in the corresponding tissues.     

Possible mechanism of congenital malformations induced by exposure of mouse preimplantation embryos to mitomycin C

ICR mice were treated intraperitoneally with mitomycin C at 5 mg/kg on day 3 of gestation. On day 18 of gestation, fetuses of treated dams were inspected for external, skeletal and visceral malformations. At 6 or 12 hr after mitomycin C treatment, the blastocysts were obtained from the uteri of treated dams and the degenerated cells within inner cell mass (ICM) and trophectoderm (TE) tissues were examined microscopically. On day 5, 8, 11, or 18 of gestation, the uteri of treated dams were obtained and those including embryos/fetuses and placentae were examined histologically. Finally, on each of gestational days 5-14, the blood of the treated dams was collected and the hematological parameters determined. Pre- and postimplantation losses in the dams treated with mitomycin C were significantly increased; increased frequency of abdominal wall defects and lumbar ribs in term fetuses, decreased fetal weight, and increased placental weight were noted as well. No significant increase in visceral malformations was found in term fetuses treated with mitomycin C. Frequency of degenerated cells within ICM and TE of blastocysts from dams treated with mitomycin C was significantly increased as compared with the controls. In dams treated with mitomycin C, decidua developed insufficiently and the trophoblast giant cell layer was not separated from the uterine lumen by maternal components; hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted. The number of erythrocytes, as well as hemoglobin concentration, hematocrit, and the percentage of reticulocytes in blood of dams treated with mitomycin C were significantly lower from days 6-12 of gestation, as compared with controls. The results of the present study showed that an increase in number of degenerated cells within blastocysts results in preimplantation loss and both maternal and embryonic hypoxia during major organogenesis results in postimplantation loss and congenital fetal malformations.