Congenital defects in the offspring of male mice treated with ethylnitrosourea

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 mg/kg 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.     

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.     

X-ray- and chemically induced germ-line mutation causing phenotypical anomalies in mice

A large and significant increase of phenotypical anomalies was observed in the progeny of ICR parent mice treated before mating with X-rays, urethane, 7,12-dimethylbenz[a]anthracene, ethylnitrosourea (ENU), and 4-nitroquinoline 1-oxide, but the increase was not significant with furylfuramide. Major types of induced anomalies were cleft palate, dwarf, open eyelid, tail anomalies, and exencephalus. Dwarf, open eyelid and tail anomalies were predominant types of viable anomalies and were inherited as if they were dominant mutations with varying expressivity or penetrance. Incidence of prenatal anomalies increased with treated doses of X-rays, urethan, or ENU for both spermatozoa and spermatogonia. Spermatogonia were less sensitive to X-rays and urethane than spermatozoa, while ENU induced a very high incidence of prenatal anomalies by the spermatogonial treatment. In contrast to the previous works with X-rays, there was a clear, almost linear increase of anomalies in the dose range from 0 to 216 rad after spermatogonial exposure. For treatment of oocytes, there was also a clear increase with doses of X-rays and urethane. Doubling doses of X-rays for prenatal anomalies were 12 rad for spermatozoa, 27 rad for spermatogonia, and 19 rad for mature oocytes. These values are similar to those for ordinary mouse mutations. However, the mean rate of prenatal anomalies per rad (1.2 X 10(-4), 6.6 X 10(-5) and 9.1 X 10(-5) for spermatozoa, spermatogonia and mature oocytes, respectively) and that for 1 micrograms/g of ENU (3.4 X 10(-4) for spermatogonia) were 4-40 times higher than that of ordinary mutation in mice, because overall phenotypical abnormalities were scored in this study. Information obtained from the work on phenotypical anomalies is valuable to assess genetic risk of radiation and chemicals, because a majority of human genetic diseases show this kind of irregular and uncertain inheritance and most of the induced anomalies are similar to those found in humans.     

Comparison of the incidence of 5-azacytidine-induced exencephaly between MT/HokIdr and Slc:ICR mice.

The incidence of 5-azacytidine-induced exencephaly was compared between MT/HokIdr strain (MT) and Slc:ICR strain (ICR) mice. MT mice have a genetic predisposition for exencephaly, but ICR mice do not. Pregnant mice were given 5-azacytidine (1 mg/kg to 100 micrograms/kg) injected intraperitoneally on Day 7.5 of gestation (vaginal plug day = Day 0.5), and fetuses were observed for external malformations on Day 18.5 of gestation. One hundred micrograms/kg 5-azacytidine induced exencephaly in MT mice but not in ICR mice, and 1 mg/kg 5-azacytidine resulted in resorptions in MT mice but caused exencephaly in ICR mice. These results indicated that MT mice had 10-fold more sensitivity to 5-azacytidine than ICR mice. It seems likely that less than effective doses of teratogens for animals without genetic predispositions are still effective in inducing malformations in animals with a genetic predisposition for malformations. When 4-somite-stage embryos of both MT and ICR mice were cultured in rat serum supplemented with 5-azacytidine, 0.02 micrograms/ml 5-azacytidine induced the failure of closure of cephalic neural tube in MT embryos but not in ICR embryos, and 0.2 micrograms/ml 5-azacytidine induced severe growth retardation in MT embryos but in ICR embryos it only induced embryos with smaller heads and fewer somites than in control. These results indicated that MT mouse embryos in culture also had a 10-fold-increased sensitivity to 5-azacytidine compared with ICR mouse embryos, suggesting maternal effects play no significant role in their increased sensitivity to 5-azacytidine.     

Lack of adaptive response of gamma radiation for protection against neutron-induced teratogenesis

BACKGROUND: Although there are some reports on neutron teratology, there is little information on the adaptive response of gamma radiation for protection against neutron‐induced teratogenesis. This study examined whether or not a low dose of gamma radiation can induce an adaptive response in mouse fetuses exposed to a subsequent dose of neutrons in vivo. METHODS: Pregnant ICR mice were exposed to a priming dose of 0.3 Gy (0.9 Gy/min) of gamma rays on day 10.5 of gestation and challenged with 0.8 Gy (0.94 Gy/minute) of neutrons 24 hlater. The mice were sacrificed on day 18.5 of gestation. The fetuses were examined for mortality, growth retardation, and other morphologic abnormalities. RESULTS: The tail length in the 0.3 Gy of gamma rays + 0.8 Gy of neutrons group was significantly shorter than in the 0.8 Gy of neutrons group. Although there was no significant difference compared with the 0.8 Gy of neutrons group, the number of live fetuses in the 0.3 Gy of gamma rays +0.8 Gy of neutrons group was lower. There was no evidence of primed exposure‐related reductions in the malformed fetuses. Although there was no significant difference compared with the unprimed group, the number of malformed offspring in the primed group was higher. Furthermore, the incidence of kinked tail and adactyly was significantly higher in the primed mice than in the unprimed mice. CONCLUSIONS: Overall, this study shows that exposure to 0.3 Gy of gamma rays failed to induce an adaptive response of fetogenesis to a neutron challenge dose. Birth Defects Res (Part B) 83:502‐506, 2008. © 2008 Wiley‐Liss, Inc.     

Embryotoxicity of oral administered chlorothalonil in mice

BACKGROUND: Chlorothalonil (2,4,5,6-tetrachloroisophthalonitril), the nephrotoxic fungicide, was examined for its potential to produce developmental toxicity in mice after oral administration. METHODS: Pregnant ICR (CD-1) mice were given sublethal doses of 0 (corn oil), 100, 400, and 600 mg/kg/day chlorothalonil by gavage on gestation days (GD) 6-15. RESULTS: Maternal effects in 400 and 600 mg/kg/day dose groups included signs of toxicity such as weakness and depression in the maternal activity, and reduction in body weight and weight gain. No maternal toxicity was apparent in the 100 mg/kg/day dose group. Maternal exposure to chlorothalonil during organogenesis significantly affected the number of live fetuses, early resorption, and mean fetal weight in the 400 and 600 mg/kg/day dose groups. No external, visceral, and skeletal abnormalities were observed among any of the treated groups compared to the control. CONCLUSIONS: On the basis of the present results chlorothalonil can produce clinical signs of toxicity and fetotoxicity without teratogenic effects at 400 and 600 mg/kg/day dose groups.     

Induction of external abnormalities in offspring of male mice irradiated with 252Cf neutron.

To assess the genetic effects of fission neutron, the induction of external malformations was studied in F1 fetuses after F0 male mice were irradiated. Male mice of the ICR:MCH strain were irradiated with 252Cf neutron at doses of 0.238, 0.475, 0.95 and 1.9 Gy. They were mated with non-irradiated female mice at 71-120 days after the irradiation. Pregnant females were autopsied on day 18 of gestation and their fetuses were examined for deaths and external abnormalities. No increases of pre- and post-implantation losses were noted at any dose. External abnormalities were observed at rates of 1.40% in the 0.238 Gy, 2.23% in the 0.475 Gy, 3.36% in the 0.95 Gy and 3.26% in the 1.9 Gy groups; the rate in the control group was 1.65%. The dose-response curve was linear up to 0.95 Gy, and then flattened out; the induction rate of external abnormalities was 2.7 x 10(-4)/gamete/cGy based on the linear regression. These results indicated that fission neutron effectively induces external abnormalities in F1 fetuses after spermatogonial irradiation.     

ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea

The mouse electrophoretic specific-locus test for induced germ-cell mutations, was used to determine the response of spermatogonial stem cells to a series of doses of the germ cell mutagen N-ethyl-N-nitrosourea (ENU). Male DBA/2J and C57B1/6J mice were treated with doses of 50, 100, 200 or 250 mg/kg ENU and their progeny screened for electrophoretically-detectable mutations at 32 separate loci. As expected, increasing doses of ENU led to increasing mutant frequencies. The differences in mutant frequencies between treated DBA/2J and C57B1/6J males were not statistically significant.     

Teratogenic interaction of ethanol and hyperthermia in mice

Alcohol and maternal hyperthermia have been implicated in human birth defects. Both ethanol and heat can induce neural tube defects (NTDs) and other developmental abnormalities in mice when large doses are given during pregnancy. To explore the teratogenic interaction of both agents, pregnant ICR mice were injected with a single dose of 25% ethanol and/or were heat-stressed in a water bath at 42 degrees C on the morning of Day 8 of gestation. Combined treatment with ethanol (0.01-0.02 ml/g) and heat (10 min), when they were given concurrently or 1 hr apart, resulted in a significant increase of resorptions and externally malformed fetuses. Skeletal malformations and visceral variations also increased significantly following a concurrent exposure to both agents. These results indicate that ethanol and heat can be synergistically teratogenic in mice when the doses of each agent are below the teratogenic threshold. It was also suggested that pretreatment with a small dose of ethanol may not enhance the teratogenicity of heat when the hyperthermic stress is strong enough and teratogenic by itself.     

乙酰基亚硝基脲诱导小鼠突变的初步研究

目的　探索乙酰基亚硝基脲 (ENU)诱导小鼠突变的效率 ,筛查并获得能显性遗传的突变型小鼠。方法　采用 8～ 10周龄的雄性C57BL 6小鼠 33只、DBA 2小鼠 18只 ,腹腔注射ENU10 0mg kg ,每周一次共三次 ,与同品系母鼠配种 ,在后代小鼠中针对可见表型筛查突变个体。结果　处理雄鼠有 9至 13周的不育期 ;在已经筛查的12 4 1只小鼠中得到眼睛异常、多趾、少趾及腹部白斑、矮小等突变个体 6 1只 ,突变率约 5 % ;获得单基因显性遗传的突变品系 2种。结论　ENU为小鼠的强诱突变剂 ;通过诱变可以得到遗传突变小鼠 ,为建立人类疾病动物模型提供条件 ;大规模诱变实验对小鼠功能基因组的研究有重要意义     

Threshold-like dose of local beta irradiation repeated throughout the life span of mice for induction of skin and bone tumors

The backs of female ICR mice were irradiated with beta rays from 90Sr-90Y three times a week throughout life. Previously we observed 100% tumor incidence at five different dose levels ranging from 1.5 to 11.8 Gy per exposure, but no tumor on repeated irradiation with 1.35 Gy for 300 days (Radiat. Res. 115, 488, 1988). In the present study, delay of tumor development was again seen at a dose of 1.5 Gy per exposure, with further delay at 1.0 Gy. The final tumor incidence was 100% with these two doses. At 0.75 Gy per exposure, no tumor appeared within 790 days after the start of irradiation, but one osteosarcoma and one squamous cell carcinoma did finally appear. These findings indicate a threshold-like response of tumor induction in this repeated irradiation system and further suggest that the apparent threshold may be somewhat less than 0.75 Gy per exposure.     

Developmental effects of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in teratogenicity

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)     

In utero morphological effects of hydroxyurea on the fetal development in Sprague-Dawley rats

In order to investigate in utero morphological effect of hydroxyurea (HU) in Sprague-Dawley rats, HU was intraperitoneally injected to pregnant Sprague-Dawley rats at a dose of 100 or 200 mg/kg/day during the organogenetic period (days 9-12 of gestation). A dose of 200 mg/kg/day induced growth retardation, high mortality and high incidence of malformations, although a dose of 100 mg/kg/day produced no adverse effects in the next generation. In the HU 200 mg/kg/day group the incidence of malformations in pups at 4 days of age was low as compared with that in fetuses and pups at 21 days of age. Increasing perinatal mortality in fetuses and pups due to severe central nervous system (CNS) malformations and disappearance of some cases of ventricular septal defect after delivery were considered as the possible causes to induce difference in malformation rate in various stage of development. Latent effect on the development of CNS malformations was observed between 4 and 21 days of age. There was no sex difference in teratogenic effect. These findings were compared with those in Wistar rats exposed to HU 200 mg/kg/day. The incidence of perinatal malformations and the stillbirths were significantly higher in the Wistar rats as compared with those in the Sprague-Dawley rats. In addition, such morphological effects of HU as the exencephaly, dilatation of lateral ventricle, anophthalmia, cleft palate and micrognathia are less severe in Sprague-Dawley rat fetuses than in Wistar rat fetuses.     

Lack of a co-promotion effect of 60 Hz rotating magnetic fields on N-ethyl-N-nitrosourea induced neurogenic tumors in F344 rats

The present study was conducted to investigate the possible effect of 60 Hz magnetic fields as promoters of brain tumors initiated transplacentally by ethylnitrosourea (ENU) in F344 rats. One hundred twenty mated animals were divided into six different groups and exposed in utero on day 18 of gestation to a single intravenous dose of either Saline (vehicle control, Group I), or ENU 10 mg/kg (Groups II-VI). In the present study, a total of 480 offspring was used. The offspring in group II were given no further treatment while the offspring in Groups III-VI were exposed to four different intensities of magnetic fields. Animals received exposure to 60 Hz magnetic field at field strengths of 0 Tesla (sham control, T1, Group III), 5 muT (T2, Group IV), 83.3 muT (T3, Group V), or 500 muT (T4, Group VI), for 21 h/day from the age of 4 weeks to the age of 32 or 42 weeks. At histopathological examination, tumors of the nervous system were seen in all the ENU-treated groups. The tumor incidence of the ENU group at 32nd and 42nd week necropsy was higher than that of the vehicle control group. The incidence of glial tumors at 42nd week necropsy was higher than the 32nd week necropsy. However, there were no differences in the tumor incidence between the sham control (T1) and ENU + magnetic field exposure groups (T2-T4). In conclusion, there was no evidence that exposure of offspring to 60 Hz at magnetic field strengths up to 500 muT to the age of 32 or 42 weeks promoted ENU-initiated brain tumors in rats.     

The effects of Scutellaria baicalensis extract on embryonic development in mice

BACKGROUND : Scutellaria baicalensis is a perennial herbaceous plant widely distributed in Oriental areas. Its roots, a commonly used medicinal source, reputedly calm fetuses in pregnant women; however, there is no sufficient evidence to date to assess its safety during pregnancy. This study aims to evaluate the effects of S. baicalensis aqueous extract on embryonic development in ICR mice. METHODS : Aqueous extract of S. baicalensis roots was prepared in accordance with clinical application. Pregnant mice were randomly divided into four groups, i.e., mice treated by gavage with water as negative control, with aqueous extract of 2 (1.8 times of human daily dose), 8 or 32 g/kg/day from gestation day (Gd) 6 to 15 as low-, middle-, and high-dose groups, respectively. The parameters of live and dead fetuses, resorptions, external and skeletal malformed fetuses, maternal body weight, maternal liver, kidneys, and heart weights were evaluated on Gd 18. RESULTS : There was no significant difference in fetal parameters among four groups. Maternal absolute liver and kidneys weights in the high-dose group were significantly higher than those in negative control (p<0.05). Relative liver and kidneys weights in this group were significantly higher than those in any other group (p<0.05). CONCLUSIONS : Oral administration of aqueous extract of S. baicalensis roots at or below 32 g/kg/day to ICR mice during organogenesis did not cause significant fetal external or skeletal malformations. However, 32 g/kg/day presented potential maternal toxicity. Birth Defects Res (Part B)86: 79-84, 2009. © 2009 Wiley-Liss, Inc.     

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.     

Dose-response for retinoic acid-induced forelimb malformations and cleft palate: a comparison of computerized image analysis and visual inspection

BACKGROUND: The objectives of this study were to (1) compare two techniques (computerized image analysis and visual morphological evaluation) for the assessment of fetal forelimb malformations and (2) increase the robustness of the dose-response curve for forelimb and cleft palate malformations resulting from all-trans retinoic acid (RA) exposure in GD 11 mice. METHODS: Pregnant CD-1 mice were administered a single oral dose of all-trans RA (0, 2.5, 10, 30, 60, or 100 mg/kg) on GD 11. GD 18 fetuses were examined for malformations using visual morphological scoring and computerized image analysis. RESULTS: Dose-dependent changes occurred in the size and shape of the humerus, radius, and ulna based on both assessment methodologies. The most sensitive indicators for the lowest effect level (10 mg/kg) on forelimbs were roundness, a shape measurement determined by image analysis, and visual morphological scoring. For all other bone measurements (proximal and distal width, area, length, and perimeter), the lowest effect level was 30 mg/kg. The maximum effect for limb defects and total malformed fetuses was seen at 60 mg/kg and higher. Incidence of cleft palate increased over the entire range of administered doses reaching a maximum of 74% (100 mg/kg). CONCLUSIONS: Overall, results indicate that computerized image analysis was no more sensitive in detecting changes in the humerus, radius, and ulna than gross visual examination. Dose-response modeling of developmental endpoints yielded comparable benchmark dose levels for long bones and cleft palate that ranged from 0.24 to 7.6 mg/kg all-trans RA. Birth Defects Res B 71:289-295, 2004. Copyright 2004 Wiley-Liss, Inc.     

A study of the frequency of sister-chromatid exchange and of thioguanine-resistant cells in mouse spleen lymphocytes after in vivo exposure to ethylnitrosourea

As part of the development of a multi-endpoint, in vivo, mouse model for mutagenesis we have measured the frequency of sister-chromatid exchange (SCE) and the frequency of thioguanine-resistant (TGr) cells among the lymphocytes of the mouse spleen following acute, intraperitoneal exposure to ethylnitrosourea (ENU). The responses of these two endpoints have been monitored both as a function of the dose of ENU injected, ranging from 0 to 70 mg/kg, and as a function of time after injection, from 1 day to 72 days. The SCE frequency response was highest 1 day after the ENU was injected, increasing 2.5-fold over control values for mice that received 70 mg/kg, and declined to control values in all animals by 72 days. SCE showed a linear dose response both at 1 day and 8 days after injection. The frequency of TGr cells was at control levels at 1 day, but at 15, 36 and 72 days after ENU injection the frequency of TGr cells showed a linear dose response. In addition, the frequency of TGr cells increased linearly with time for both the 35 and 70 mg/kg doses. The frequency of TGr cells for mice that had received 70 mg ENU/kg 72 days previously, was 100-fold higher than in control animals, giving a frequency of 1.4 X 10(-4).     

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.     

Comparative study of blocking effects of various retinoids on the occurrence of permanent proliferation of vaginal epithelium in mice treated neonatally with estrogen

Neonatal injections of 20 micrograms 17 beta-estradiol (E2) induced persistent proliferation and cornification of the vaginal epithelium in adult ovariectomized C57 Black/Tw mice. However, permanent vaginal changes were prevented by various retinoids when, simultaneously with E2 treatment, the animals were given injections of 100 micrograms daily dose of retinol, retinol acetate, retinal or of 200 micrograms daily dose of retinol palmitate (RoP). Neonatal injections of a 100 micrograms daily dose of RoP had no preventive effect on the occurrence of E2-induced permanent vaginal changes. This finding suggests that the preventive effect of RoP is weaker than that of other retinoids showing approximately the same degree of prevention. Combined treatment with E2 plus retinoic acid (even a small dose of 20 micrograms) had such a toxic effect on newborn mice that they died within 7 days after birth, while the animals given neonatal injections of 20 micrograms retinoic acid alone survived until the termination of the experiment.