Studies of 50 Hz circularly polarized magnetic fields of up to 350 microT on reproduction and embryo-fetal development in rats: exposure during organogenesis or during preimplantation

Groups of mated female Sprague-Dawley rats were simultaneously exposed to 0 (sham exposed), 7, 70, or 350 microT (rms) circularly polarized 50 Hz magnetic fields (MF) for 22 h/day on gestational day 8-15, the period of rat fetal organogenesis (organogenesis study) or from day 0 to day 7 of gestation, the rat preimplantation period (preimplantation study). Developmental toxicity was assessed on gestational day 20. Identical experiments were repeated to confirm reproducibility of both studies. In both studies, statistically significant differences between exposed and sham exposed animals were observed in several measured parameters; however, these differences only appeared in one, but not both replicate experiments and generally at only an isolated exposure level. Because these differences were not reproducible and did not show a dose response relationship, they were not considered related to MF exposure. In the organogenesis study, lower kidney weights of dams were seen at 70 and 350 microT in Experiment 1. Lower dam liver weights and lower mean body weights of viable female and male fetuses were seen at 70 microT in Experiment 2. Otherwise, there were no differences in these parameters or in group means for fetal loss after implantation, number of viable fetuses, fetal body weight and sex ratio, incidences of external, visceral, and skeletal abnormalities or variations, or tissue abnormalities after histopathological examination. In the preimplantation study, dam health and indices for reproduction and embryo-fetal development, including pre or postimplantation loss, number and body weight of live fetuses, and sex ratio, external, skeletal abnormalities and variations, and skeletal ossification did not differ. Dam inorganic phosphorous concentration at 350 microT was elevated in one experiment and depressed in another. In one experiment, visceral abnormalities, primarily thymic remnant in neck and accessory liver lobe, were increased in the 7 microT group. Based on these results from two studies, we conclude that circularly polarized 50 Hz MF exposure of up to 350 microT during the fetal organogenesis or during the preimplantation period does not affect reproduction and embryo-fetal development in Sprague-Dawley rats.     

Effects of low-frequency magnetic fields on fetal development in rats

We studied effects of alternating magnetic fields on the embryonic and fetal development of rats. Mated females of the Han:Wistar-strain were sham exposed or exposed continuously to a 50-Hz field or to a 20,000 pulse-per-second (pps) sawtooth magnetic field from day 0 to day 20 of pregnancy for 24 h/day until necropsied on day 20. The respective peak-to-peak intensities of the fields were 35.6 μT (sinewave) and 15.0 μT (sawtooth). Each treatment group contained 72 bred females. Control animals were kept under the same conditions without the magnetic field. No adverse effects were seen in the dams. The mean numbers of implantations and living fetuses per litter were statistically significantly increased in the 50-Hz group. There were, however, three total resorptions of litters in dams of the control group, which contributed to the difference in the number of living fetuses. The corrected body-mass gains (gains without uterine content) of dams were similar in all groups. Pregnancy rates, incidences of resorptions. late fetal deaths, and fetal body masses were similar in all groups. The incidence of fetuses with minor skeletal anomalies was statistically significantly increased in both exposed groups. Only one serious malformation (anophthalmia, sawtooth-exposed group) and a few minor visceral malformations were found. In conclusion, the magnetic fields used in this study did not increase the incidence of major malformations or resorptions in Wistar rats. The increased number of skeletal anomalies and implantations we observed indicates, however, that some developmental effects in rats may attend exposure to time-varying magnetic fields. © 1993 Wiley-Liss. Inc.     

Effects of low-frequency magnetic fields on fetal development in CBA/Ca mice

Effects of alternating magnetic fields (MFs) on the embryonic and fetal development in CBA/Ca mice were studied. Mated females were exposed continuously to a sinusoidal 50 Hz (13 μT or 0.13 mT root mean square) or a sawtooth 20 kHz (15 μT peak-to-peak) MF from day 0 to day 18 of pregnancy for 24 h/day until necropsied on day 18. Control animals were kept under the same conditions without the MF. MFs did not cause maternal toxicity. No adverse effects were seen in maternal hematology and the frequency of micronuclei in maternal bone marrow erythrocytes did not change. The MFs did not increase the number of resorptions or fetuses with major or minor malformations in any exposure group. The mean number of implantations and living fetuses per litter were similar in all groups. The corrected weight gain (weight gain without uterine content) of dams, pregnancy rates, incidences of resorptions and late fetal deaths, and fetal body weights were similar in all groups. There was, however, a statistically significant increase in the incidence of fetuses with at least three skeletal variations in all groups exposed to MFs. In conclusion, the 50 Hz or 20 kHz MFs did not increase incidences of malformations or resorptions in CBA/Ca mice, but increased skeletal variations consistently in all exposure groups. Bioelectromagnetics 19:477–485, 1998. © 1998 Wiley-Liss, Inc.     

Malformations in rat fetuses induced by trypan blue

Malformations of fetuses obtained from Wistar rat dams treated with trypan blue during gestation were studied. Fetuses were examined on day 20 of gestation. One hundred and twenty-seven fetuses showed abnormalities of the external features, skeleton and internal organs, separately or in combination. External malformations were found in 108 fetuses. The most frequent external malformation was anomaly of tail. Spina bifida, club foot, exencephaly and anal atresia were also observed frequently. Skeletal malformations were detected in 48 fetuses. Deformity of vertebrae in the lumbar, sacral and/or caudal regions was found in 46 fetuses. Internal malformations were observed in 27 fetuses. Anomaly of heart and/or great vessels, hydrocephaly and micro- or anophthalmia were observed frequently. About 90% of the fetuses with skeletal malformations also showed some external malformations. In contrast, about 48% of the fetuses with internal malformations also had some external malformations. These results suggest that, for teratological study, internal examination is more important in detecting malformations of fetuses than skeletal examination.     

Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy.

Bisphenol A (BPA) is an essential component of epoxy resins used in the lacquer lining of metal food cans, as a component of polycarbonates, and in dental sealants. The present study was conducted in an attempt to evaluate the adverse effects of the environmental estrogen BPA on initiation and maintenance of pregnancy and embryofetal development after maternal exposure during the entire period of pregnancy in Sprague-Dawley rats. The test chemical was administered by gavage to mated females from days 1 to 20 of gestation (sperm in varginal lavage = day 0) at dose levels of 0, 100, 300, and 1000 mg/kg. All females were subjected to caesarean section on day 21 of gestation and their fetuses were examined for external, visceral and skeletal abnormalities. In the 1000 mg/kg group, significant toxic effects including abnormal clinical signs, decreased maternal body weight and body weight gain, and reduced food consumption were observed in pregnant rats. An increase in pregnancy failure was also found in the successfully mated females. In addition, increased number of embryonal deaths, increased postimplantation loss, reduced litter size and fetal body weight, and decreased number of fetal ossification centers of several skeletal districts were seen. On the contrary, no significant changes induced by BPA were detected in the number of corpora lutea and implantation sites and by fetal morphological examinations. In the 300 mg/kg group, suppressed maternal body weight and body weight gain, decreased food intake and reduced body weight of male fetuses were seen. There were no adverse signs of either maternal toxicity or developmental toxicity in the 100 mg/kg group. It was concluded that BPA administration during the entire period of pregnancy in rats produced pregnancy failure, pre- and postimplantation loss, fetal developmental delay and severe maternal toxicity, but no embryo-fetal dysmorphogenesis at an oral exposure level of 1000 mg/kg.     

Magnetic field exposure and prognostic factors in childhood leukemia

We examined the association between magnetic field (MF) exposure and the presence of prognostic risk factors among 482 children diagnosed with acute lymphoblastic leukemia (ALL) between 1996 and 2001. Personal 24-h MF measurements were obtained for 412 children; 386 children were included in analyses. There were no trends seen between increasing exposure to MF and the presence of adverse clinical and tumor-specific prognostic factors. Our results suggest that exposure to MF is not associated with the presence of unfavorable cytogenetic abnormalities in leukemic blast cells or with clinical factors at the time of diagnosis that predict poor survival.     

Developmental toxicity of pentostatin (2'-deoxycoformycin) in rats and rabbits

The developmental toxicity of the potent adenosine deaminase (ADA) inhibitor, pentostatin (2'-deoxycoformycin), was investigated in pregnant rats and rabbits administered daily iv doses during organogenesis. Rats received 0, 0.01, 0.10, or 0.75 mg/kg on gestation days 6-15 and rabbits received 0, 0.005, 0.01, or 0.02 mg/kg on gestation days 6-18 and maternal and fetal parameters were evaluated on gestation day 21 (rats) or 30 (rabbits). Live fetuses were examined for external, visceral, and skeletal malformations and variations. In rats, maternal body weight gain and food consumption were significantly suppressed at doses of 0.10 and 0.75 mg/kg during the treatment period but returned to control levels during posttreatment. Increased postimplantation loss and decreased numbers of live fetuses, litter size, and fetal body weight were observed at 0.75 mg/kg. A statistically significant increase in the incidence of vertebral malformations occurred at 0.75 mg/kg. The incidence of certain skeletal variations (extra presacral vertebrae, extra ribs, hypoplastic vertebrae) was also increased at 0.75 mg/kg. Ossification of cervical centra was reduced at 0.75 mg/kg compared with controls. In rabbits, marked maternal toxicity (death, body weight loss, and decreased food consumption) and reproductive toxicity (abortion and premature delivery) occurred in all pentostatin-treated groups. However, there were no significant effects on number of live fetuses, pre- or postimplantation loss, litter size, or fetal body weights in the animals with live litters. There was also no apparent increase in the incidence of malformations or variations in the live fetuses of pentostatin-treated rabbits. Thus, these studies demonstrate developmental toxicity of pentostatin in rats and rabbits, and teratogenicity in rats, at maternally toxic doses.     

Teratological studies of prenatal exposure of mice to a 20 kHz sawtooth magnetic field

In order to evaluate the importance of gestational age in possible effects due to exposure to a 20 kHz sawtooth magnetic field, pregnant ICR mice at gestational 2.5-15.5 days post-coitus, which is the most sensitive stage for the induction of major congenital malformations, were exposed in a carrousel irradiator. The mice were exposed to a 20 kHz intermediate frequency (IF) sawtooth magnetic field had a 6.5 microT peak intensity for 8 h/day. The animals were sacrificed on the 18th day of gestation; and the fetuses were examined for mortality, growth retardation, changes in head size, and other morphological abnormalities. From the above conditions, it is concluded that the exposure to a 20 kHz sawtooth magnetic field with 6.5 microT peak intensity does not inflict any adverse effect on fetuses of pregnant mice.     

Developmental toxicity evaluation of ELF magnetic fields in Sprague-Dawley rats

To identify possible effects of horizontally polarized magnetic field (MF) exposure on maintenance of pregnancy and embryo-fetal development, an MF exposure system was designed and constructed and 96 time-mated female Sprague-Dawley (SD) rats (24/group) received continuous exposure to 60 Hz MF at field strengths of 0 (sham control) and 5, 83.3, or 500 microT (50, 833, or 5000 mG). Dams received MF or sham exposures for 22 h/day on gestational day 6-20. MF was monitored continuously throughout the study. There were no evidences of maternal toxicity or developmental toxicity in any MF exposed groups. Mean maternal body weight, organ weights, and hematological and serum biochemical parameters in groups exposed to MF did not differ from those in sham control. No exposure related differences in fetal deaths, fetal body weight, and placental weight were observed between MF exposed groups and sham control. External, visceral, and skeletal examination of fetuses demonstrated no significant differences in the incidence of fetal malformations between MF exposed and sham control groups. In conclusion, exposure of pregnant rats to 60 Hz at MF strengths up to 500 microT during gestation day 6-20 did not produce any biologically significant effect in either dams or fetuses.     

Developmental toxicity of 1,6-hexamethylene diisocyanate (HDI) in the Sprague-Dawley rat

BACKGROUND: 1,6-Hexamethylene diisocyanate (HDI), a widely used chemical in commercial polyurethane manufacture, has been shown to affect the respiratory tract of experimental animals. However, its potential to affect neonatal development, particularly after inhalation exposure, is less well described. The present study was conducted to assess the developmental toxicity of HDI. METHODS: Gravid Sprague-Dawley rats were exposed to concentrations of 0, 0. 005, 0.050, or 0.300 ppm HDI via inhalation (whole-body exposure) on days 0-19 of gestation. Maternal toxicity, as demonstrated by clinical signs and changes in body weight gain during gestation, was characterized. Dams were sacrificed on gestation day 20, at which time fetuses were removed by cesarean section, the uterus was examined, and a gross maternal necropsy was performed. Maternal evaluation also included lung weight and a detailed histopathologic assessment of the nasal turbinates, larynx, trachea, and lungs. All fetuses were evaluated for external anomalies. Approximately one-half of each litter was examined for visceral effects, the other half underwent a skeletal (bone and cartilage) examination. RESULTS: Maternal toxicity was demonstrated in the 0.300- and, to a lesser extent, in the 0.050-ppm exposure groups. No maternal effects were noted in the 0.005-ppm group. Test compound-related maternal effects were restricted to histopathological findings and included acanthosis, hyperkeratosis, inflammation of the nasal turbinates, and, more seriously, degeneration of the olfactory epithelium. No pathological alterations were noted in the larynx, trachea, or lungs in any dose group. No test compound-related effects were observed on any reproductive parameters, or any embryonic endpoints, including pre/postimplantation loss and resorption. There were no effects on litter size or the number of fetuses per implantation site and no effects on fetal or placental weights were observed. No test compound-related fetal external, visceral, or skeletal findings were observed. No effect on the fetal or litter incidence of total malformations or variations was observed, and there was no difference in the incidence of malformations between males and females. CONCLUSIONS: Administered as described in this study, 1, 6-HDI produced maternal effects (nasal turbinate histopathology) at concentrations of 0.050 and 0.300 ppm with no developmental toxicity observed at any concentration.     

Structural teratogenicity evaluation of methyl chloride in rats and mice after inhalation exposure

One hundred bred Fischer-344 female rats were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 25 females per exposure concentration, from gestation day (gd) 7 through gd 19. On gd 20, the females were sacrificed for evaluation of maternal reproductive and fetal parameters. Maternal and fetal toxicity was apparent at the highest exposure concentration. There were no methyl chloride-induced external, skeletal, or visceral abnormalities seen in the fetuses. One hundred thirty-two C57BL/6 female mice bred to C3H males to produce B6C3F1 offspring were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 33 females per exposure concentration, from gd 6 through gd 17. Exposure to the entire 1,500-ppm group was terminated on gd 10-14, with the animals killed in extremis. Selective necrosis of neurons in the internal granular layer of the cerebellum, ranging from individual cell involvement to focal areas comprising large numbers of neurons, was found in all females. On gd 18, the females from the other treatment groups, all of which survived, were killed for evaluation of maternal reproductive and fetal parameters. No evidence was seen of maternal or fetal toxicity in these exposure groups. There were no significant alterations in external appearance in fetuses from any of the exposure groups. Visceral examination of mouse fetuses revealed a small, but statistically significant, incidence of heart defects in litters of the 500-ppm group. The anomaly, a reduction or absence of the atrioventricular valve, chordae tendineae, and papillary muscle, was observed on the left side (bicuspid valve) in three fetuses and the right side (tricuspid valve) in six fetuses: three males and six females. It is concluded that methyl chloride inhalation exposure in pregnant rats, during critical periods of embryo and fetal development, is not teratogenic at concentrations which elicit maternal and fetal toxicity. In pregnant mice, methyl chloride was severely toxic to dams following 4 days or more of exposure to 1,500 ppm in air. Methyl chloride, at 500, but not 100 ppm, was teratogenic in mice, leading to a malformation in the heart. No embryo-fetal toxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 100 ppm of ethyl chloride.     

Teratological evaluation of mouse fetuses exposed to a 20 kHz EMF

As a continuation of our previous study, we performed a teratological evaluation of the importance of gestational age with regards to the exposure of 20 kHz intermediate frequency magnetic field (IF) on pregnant ICR mice. The pregnant mice were exposed to a 20 kHz IF magnetic field for 8 h/day in a carousel irradiator at 30 µT which is the limit standard for occupational population in Korea. The animals were sacrificed on the 18th day of gestation and the fetuses were examined for mortality, growth retardation, changes in head size and other morphological abnormalities. We concluded that exposure to 30 µT with 20 kHz IF did not cause any observable adverse effects on mouse fetuses. Bioelectromagnetics 30:330–333. © 2009 Wiley‐Liss, Inc.     

Susceptible period of nitrous oxide teratogenicity in Sprague-Dawley rats

The susceptible period of nitrous oxide (N2O) teratogenicity was studied in 170 Sprague-Dawley rats. Seven groups of 20 timed-pregnant rats were exposed to 60% N2O for 24 hours on each of days 6-12 of gestation; a control group of 30 timed-pregnant rats was exposed to air on day 9. On day 20 of gestation, dams were killed and reproductive indices were determined; their fetuses were subsequently examined for external, skeletal, and visceral abnormalities. There were no differences among the groups in the number of implantations and live fetuses, mean fetal weight, and sex ratio. The incidence of fetal wastage was higher than control in N2O-treated groups exposed on days 8 and 11 of gestation. Skeletal malformations of the ribs and vertebrae were increased following exposure on day 9 of gestation. However, the specific minor anomaly, cervical rib, was increased only following exposure on day 8 of gestation. The incidences of right-sided aortic arch and left-sided umbilical artery, abnormalities indicative of altered laterality, were increased following exposure on day 8 of gestation. Nitrous oxide administration during organogenesis causes several reproductive defects by mechanisms which remain to be determined.     

Effects of pulsed magnetic fields on the developing mouse embryo

The influence of a pulsed magnetic field (PMF; sawtooth with 45-μs linear rise time and 5-μs decay time, peak strength of 15 μT, and frequency 20 pps) on the embryogenesis of CBA/S mice was investigated in five experiments based on a total of 707 exposed and 543 unexposed primigravidas. Sham and PMF exposures began on day 1 of gestation (experiments 1 and 2), on day 2 (experiment 3), on day 5 (experiment 4). and on day 7 (experiment 5): all exposures continued until day 19 post conception (p.c.) when they were terminated, at which time the following variables were measured: number of implants; number of placental resorptions; number of living fetuses; number of dead fetuses; number of malformations in living and dead fetuses; and length and body mass of living fetuses. Control dams were sham-exposed concurrently with corresponding. PMF-exposed dams. With the exception of experiment 5, in which exposure to PMF started on day 7 p.c., all groups of exposed mice had significantly more placental resorptions when compared with concurrent controls. The increased resorption rate was not reflected in a reduction in litter size or in the number of litters. A significant increase in malformed fetuses was not seen in any of the exposed groups, or when groups were pooled. Only in experiment 1 was the number of dead fetuses affected by exposure to PMF. The effect of PMF on the implantation rate was not significant. Body mass and length of exposed fetuses were significantly reduced only when the PMF treatment began on day 7 p.c. That PMF-treated mice had significantly more placental resorptions when exposure began on day 5 p.c. or earlier (before implantation), but not when exposure began on day 7 (after implantation), may indicate a causative pre-implantation effect. Because a PMF-induced increase in the number of resorptions has not been observed in other strains of mice, the effect might be strain-related. © 1993 Wiley-Liss, Inc.     

Effects of feed restriction during organogenesis on embryo-fetal development in rabbit

BACKGROUND: Appropriate maternal nutrition and body weight gain during pregnancy is well established as a major factor in healthy prenatal development in humans. Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate developmental toxicity data that may be challenging to interpret. To aid with this, the effects of feed restriction, and subsequent reduction in maternal body weight gain, on embryo-fetal development was investigated in the rabbit. METHODS: Groups of 15 pregnant New Zealand White rabbits were offered 150 (control), 110, 75, 55, 35, and 15 g feed/day from gestation day (GD) 7-19. Cesarean sections were carried out on GD 29 and fetuses were examined for external, visceral, and skeletal development. RESULTS: Maternal body weights at the end of the feed restriction period (GD 20) were 0.97, 0.98, 0.93, 0.94, and 0.86 x control for the 110, 75, 55, 35, and 15 g feed/day groups, respectively. Only at 15 g feed/day was there a net maternal body weight loss (the GD 20 body weight was 0.93 x the GD 6 body weight) at the end of the feed restriction period. Six does aborted in the 15 g feed/day group; there were no other abortions associated with feed restriction. Fetal body weight was significantly reduced at 75, 55, 35, and 15 g feed/day (0.95, 0.90, 0.86, and 0.84 x control, respectively). There were no external or visceral malformations or variations, and no skeletal malformations associated with feed restriction. The incidence of fetuses with sternebrae 5 or 6 unossified was increased at feed levels < or = 75 g/day. At a feed level of 35 g/day there was an increase in unossified metatarsals and metacarpals, and an increase in the number of fetuses with a reduced number of caudal vertebrae ossified. Although these findings were not increased at a feed level of 15 g/day, the lack of dose response was likely due to increased abortion and subsequent decrease in fetuses available for evaluation at 15 g feed/day. CONCLUSION: These data demonstrate that feed restriction to feed levels that produce substantial reductions in maternal body weight gain can result in developmental toxicity expressed by abortion, reduced fetal weight, and alterations in ossification. Abortion only occurred when feed was restricted to an amount that produced maternal body weight loss (15 g feed/day) whereas reduced fetal weight and increased incidence of fetuses with unossified sternebrae, metatarsals, metacarpals, or caudal vertebrae were noted at feed levels of < or = 75 g/day. There were no fetal malformations associated with feed restriction.     

Chick embryo development can be irreversibly altered by early exposure to weak extremely-low-frequency magnetic fields

Several reports have shown that weak, extremely-low-frequency (ELF), pulsed magnetic fields (PMFs) can adversely affect the early embryonic development of the chick. In this study, freshly fertilized chicken eggs were exposed during the first 48 h of postlaying incubation to PMFs with 100 Hz repetition rate, 1.0 μT peak-to-peak amplitude, and 500 μs pulse duration. Two different pulse waveforms were used, having rise and fall times of 85 μs (PMF-A) or 2.1 μs (PMF-B). It has been reported that, with 2 day exposure, these fields significantly increase the proportion of developmental abnormalities. In the present study, following exposure, the eggs were allowed to incubate for an additional 9 days in the absence of the PMFs. The embryos were taken out of the eggs and studied blind. Each of the two PMF-exposed groups showed an excess in the percentage of developmental anomalies compared with the respective sham-exposed samples. This excess of anomalies was not significant for the PMF-A-treated embryos (P = 0.173), whereas it was significant for the PMF-B-exposed group (P = 0.007), which showed a particularly high rate of early embryonic death. These results reveal that PMFs can induce irreversible developmental alterations and confirm that the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields. The data also validate previous work based on the study of PMFs' effects at day 2 of embryonic development under field exposure. © 1994 Wiley-Liss, Inc.     

Developmental toxicity of soman in rats and rabbits

Soman (GD; phosphonofluoridic acid, methyl-,1,2,2-trimethylpropyl ester) is an organophosphate compound with potent anticholinesterase activity. To determine developmental toxicity, soman was administered orally to CD rats on days 6 through 15 of gestation at dose levels of 0, 37.5, 75, 150, or 165 micrograms/kg/day and to New Zealand White (NZW) rabbits on days 6 through 19 of gestation at dose levels of 0, 2.5, 5, 10, or 15 micrograms/kg/day. At sacrifice, gravid uteri were weighed and examined for number and status of implants. Individual fetal body weights and external, visceral, and skeletal malformations were recorded. Mean maternal weight changes, fetal implantation status/litter, fetal weight, and fetal malformations/litter were compared between dose groups. Monitors for maternal toxicity were net body weight change, treatment weight change, mortality, and clinical signs of toxicity such as lethargy, ataxia, and tremors. Maternal rats and rabbits in the high-dose groups exhibited statistically significant increases in toxicity and mortality when compared to controls. There were no significant dose-related effects among dose groups in the prevalence of postimplantation loss, malformations, or in average body weight of live fetuses per litter. There was no evidence of increased prenatal mortality or fetal toxicity in the CD rat or NZW rabbit following exposure to soman, even at a dose that produced significant maternal toxicity.     

Axial skeletal malformations induced by acetazolamide in rabbits.

In order to evaluate the teratogenic potential of acetazolamide in rabbits, three groups of 18 artificially inseminated females were treated orally with 50, 100, or 150 mg/kg/day of acetazolamide on days 6-18 of gestation. These doses induced maternal acidosis and electrolyte changes, consistent with those reported in rats and considered to be a result of carbonic anhydrase inhibition, as well as reductions in maternal body weight gain. At cesarean sections, average fetal body weights in the acetazolamide groups were dose-dependently decreased compared with controls. There were no effects of acetazolamide on embryonic survival or external morphology of live fetuses. In the fetal skeletal examination, thoracic and lumbar vertebral malformations occurred in 0.7%, 3.9%, and 6.1% of fetuses in the 50, 100, and 150 mg/kg/day groups, respectively, compared with none in the control group. In addition, missing vertebra was seen in a small number of fetuses in the 100 and 150 mg/kg/day groups. These axial skeletal malformations were, in some cases, associated with costal malformations. These results indicate that acetazolamide at maternotoxic doses can produce axial skeletal malformations in the rabbit.     

Genotoxic potency in mouse spermatogonial stem cells of triethylenemelamine, mitomycin C, ethylnitrosourea, procarbazine, and propyl methanesulfonate as measured by F1 congenital defects

Male ICR mice were intraperitoneally injected with TEM, MMC, ENU, PCZ, or PMS and mated to untreated females of the same strain on days 64-80 after the treatment. Copulations during this period involve sperm that were spermatogonial stem cells at the time of the treatment. The fetuses were examined on day 18 of pregnancy for external and skeletal abnormalities. The 5 mutagens tested all caused significant increases in the incidence of abnormal fetuses over the control level. The genotoxically effective dose, in mmole/kg, for producing fetal abnormalities with a frequency of 2% was estimated to be 0.007 for TEM and MMC, 0.6 for ENU, 1.8 for PCZ, and 3.0 for PMS. These values correlate well with the mutagenic potency estimated from the data reported for inducing specific-locus mutations in spermatogonial stem cells. Irrespective of the kind of mutagen used, external abnormalities represented by cleft palate and dwarfism occurred more frequently than skeletal abnormalities represented by rib malformations. It is concluded from these data that F1 fetal abnormalities can serve as sensitive indicators for quantitatively assessing the genotoxicity of a chemical agent in spermatogonial stem cells.     

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.