Alternative toxicity tests.
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Animal Testing Alternatives↗
Biomedical subjects
H Spielmann
Publications and source records attributed to H Spielmann.
Genotoxic and embryotoxic effects of gonadotropin-hyperstimulated ovulation of murine oocytes, preimplantation embryos, and term fetuses.
Compared to spontaneous ovulation, gonadotropin-hyperstimulated ovulation (superovulation) in mice resulted in a fourfold increase in the number of preimplantation embryos 3 days post coitum, 50% of which died before term. Both in vitro development of embryos during the preimplantation period and transfer of morulae from superovulated females to pseudopregnant untreated foster mothers indicate that the prenatal loss occurring shortly before implantation up to term is due to maternal factors rather than to direct hormonal effects on oocytes or early embryos. Indeed, no genotoxic events could be observed in 4-cell to blastocyst stage embryos from superovulated female mice as revealed by the chromosomal aberration test and the sister chromatid exchange assay. Chromosome analysis of the pronuclei from mouse zygotes showed an increased rate of aberrations in oocyte-derived nuclei after superovulation in comparison to spontaneous ovulation. The present data suggest that aberrant murine oocytes may be fertilized, but they do not survive the first cleavage stages. The result is discussed with respect to the high incidence of chromosomal abnormalities found in human oocytes after gonadotropin-hyperstimulated ovulation.
Animals↗
Embryologic and cytogenetic effects of ethanol on preimplantation mouse embryos in vitro.
Ethanol and its primary metabolite acetaldehyde were studied in cultured preimplantation mouse embryos with respect to embryotoxicity, embryolethality, chromosome breaking activities, and ability to induce sister chromatid exchange (SCE). Analysis of differentiation and cell number of mouse morulae and blastocysts show that acetaldehyde is three orders of magnitude more toxic than ethanol, indicating that the metabolite is responsible for the embryotoxicity of ethanol in preimplantation embryos. Concentrations of ethanol that do not inhibit growth induce SCEs and chromosome aberrations. The SCE-inducing effect of ethanol disappears in the presence of 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH). These data suggest that preimplantation embryos are able to convert ethanol to acetaldehyde and that ADH is the enzyme involved. It is, furthermore, shown histochemically that mouse oocytes as well as morulae and blastocysts are able to oxidize ethanol in the presence of NAD+.
Alcohol Dehydrogenase↗
Cytotoxicity test using blastocyst-derived euploid embryonal stem cells: a new approach to in vitro teratogenesis screening.
To develop a mammalian in vitro system for teratogenicity testing, cytotoxicity of xenobiotics was evaluated in pluripotent euploid embryonal stem cells (ESC) derived from mouse blastocysts. The dimethyl-thiazol-diphenyl tetrazolium bromide (MTT) assay was the most appropriate test system for cytotoxicity determinations with ESC. Only compounds that do not require metabolic activation were selected for testing from the database for validation of in vitro teratogenesis assays by Smith et al. Results obtained with ESC were compared to corresponding data from fibroblasts from day-14 mouse embryos to detect differences in sensitivity between undifferentiated and differentiated cells. ESC showed a higher sensitivity to known teratogens than fibroblast cultures, which allows calculation of a sensitivity ratio of "adult" cells (differentiated fibroblasts) to embryonal cells (undifferentiated ESC) in a mammalian system similar to the hydra assay. Although some xenobiotics had to be classified as false negatives in our system, the ESC cytotoxicity assay holds promise as a new in vitro screening assay in teratology.
Animals↗
[Cocaine in pregnancy: a second Contergan?].
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Abnormalities, Drug-Induced↗
Teratogenicity of steady-state concentrations of etretinate and metabolite acitretin maintained in maternal plasma and embryo by intragastric infusion during organogenesis in the mouse: a possible model for the extended elimination phase in human therapy.
Etretinate (Tegison, Tigason), a retinoid used for the treatment of skin disorders such as psoriasis, was shown to teratogenic in the human. Because of the long terminal half-life of this drug (100 days), considerable plasma levels of etretinate and its main metabolite, etretin (acitretin), were observed for up to 2 years following discontinuation of therapy. We have therefore investigated, in a newly developed animal model, the potential teratogenic risk of such persisting levels of these aromatic retinoids. Etretinate was administered by intragastric infusion throughout organogenesis in the mouse (day 8-15) via subcutaneously implanted osmotic minipumps connected to external reservoirs containing oily solutions of the drug. Dose-dependent developmental effects were found, the fetal weight decreased and the resorption rate and incidence of major malformation increased. A dose of 0.84 mg/kg/day resulted in retinoid-specific defects, in particular shortening of the limbs and cleft palate. This low dose infused resulted in mean etretinate concentrations of 6.5 ng/ml maternal plasma and 12.5 ng/g embryo (measured on days 10 and 12 of gestation). The corresponding concentrations of the metabolite etretin were 38 ng/ml plasma and 95 ng/g embryo. Our results emphasize the high teratogenic risk of relatively low, persisting concentrations of etretinate and etretin such as those observed after discontinuation of human therapy, because the area of the concentration-time curve is likely to be the decisive parameter in regard to teratogenesis.
Abnormalities, Drug-Induced↗
Beneficial effects of ascorbic acid on preimplantation mouse embryos after exposure to cyclophosphamide in vivo.
To study mechanisms of embryotoxicity in early pregnancy, we have evaluated the genotoxic and embryolethal effects of ascorbic acid (AA) alone or in combination with cyclophosphamide (CPA). Female mice were exposed on day 3 of pregnancy. Embryotoxicity was investigated at term and genotoxicity shortly after treatment using the chromosomal aberration test and the sister chromatid exchange (SCE) assay as sensitive end points. Additionally, cytotoxic effects were determined by a proliferation test. AA was not found to be embryotoxic, cytotoxic, or genotoxic when given alone. In combination with 10 mg/kg CPA, however, which induced 50% aberrant metaphases, 100% increase SCE frequency, and a strong inhibition of cell proliferation, AA in a dose range of 25-1,600 mg/kg did not change SCE and proliferation, but reduced the rate of aberrant metaphases significantly. This anticlastogenic effect was clearly correlated to a beneficial effect on embryolethality at term when 200 mg/kg ascorbic acid was given in combination with 40 mg/kg CPA. The results suggest that during early pregnancy AA is not genotoxic even at so-called megadoses doses, but it seems to protect early embryos against damage induced by genotoxic agents like CPA.
Animals↗
Cytogenetic studies on preimplantation mouse embryos exposed to methylnitrosourea in vivo.
Since exposure of mice to methylnitrosourea (MNU) during the preimplantation period can induce malformations and an increased postnatal death rate, direct embryotoxic effects were studied in preimplantation embryos shortly after treatment of pregnant mice on days 2 and 3 of gestation with single i.p. injections of 2.5, 5.0, and 10.0 mg/kg MNU. Embryos exposed to MNU for 24 h after treatment on day 2 showed a significant reduction of cell number and induction of sister chromatid exchange (SCE) frequency, but no structural chromosomal aberrations or inhibition of development during culture. Embryos exposed to MNU in vivo for 3 h on day 3 showed significantly reduced cell numbers, a significant inhibition of development in culture, and an increase in structural chromosome aberrations. Due to the high cytotoxicity of MNU, determination of SCE was not possible. The results indicate that MNU reaches preimplantation mouse embryos shortly after maternal treatment and that malformations seen at term and postnatal effects are probably induced by the direct action of MNU on early embryos. Furthermore, the importance of the time interval chosen for evaluation of toxicologic endpoints in preimplantation embryos is demonstrated.
Animals↗
Abnormal development of mouse embryos exposed to methylnitrosourea before implantation.
On day 2 of gestation mice were exposed to single i.p. injections of 5, 10, 20, and 40 mg/kg methylnitrosourea (MNU). Evaluation at term revealed 100% embryolethality in the 40 mg/kg group but no signs of maternal toxicity (LD50 = 400 mg/kg). In mice treated with 5 and 10 mg/kg, no malformations could be detected at term. In contrast, 40% of the live fetuses exposed to 20 mg/kg MNU showed developmental abnormalities of vertebrae, ribs, long bones, and kidneys. Analysis of postnatal development 3 weeks after birth indicated a significant increase in mortality in the offspring of all animals exposed to MNU on day 2 of pregnancy. Further developmental or morphologic anomalies could not be detected in the offspring up to the age of 6 months, when autopsy was performed. The data show that exposure to MNU before implantation has embryolethal and teratogenic effects in a dose range one order of magnitude lower than the toxic dose range for adult animals.
Abnormalities, Drug-Induced↗
Maternal factors influencing development of embryos from mice superovulated with gonadotropins.
In NMRI mice superovulation with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) increased mating rate, number of implantation sites, rate of advanced and delayed resorptions, as well as retarded sternebral ossification and cleft palate. On day 3 of gestation in preimplantation embryos, cell number and mitotic index were lower after superovulation than after spontaneous ovulation. However, when preimplantation embryos from superovulated and control females were transferred on day 3 of pregnancy to pseudopregnant recipients (10 embryos per female) no differences could be detected between the two groups of fetuses at term. The results of the embryo transfer experiments indicate that abnormal embryonic development after superovulation with gonadotropins is predominantly induced by effects of the hormone treatment on the maternal uterine environment.
Abnormalities, Drug-Induced↗
Co-administration of ascorbic acid with cyclophosphamide (CPA) to pregnant mice inhibits the clastogenic activity of CPA in preimplantation murine blastocysts.
Cyclophosphamide (CPA) administered at a dose of 15 mg/kg body weight to pregnant inbred CBA/CaH mice 60 h after copulation significantly elevated the incidence of structural chromosomal aberrations and sister-chromatid exchanges in 96 h blastocysts. When ascorbic acid (800 mg/kg body weight) was co-administered with CPA (15 mg/kg) using different injection sites, the incidence of structural chromosomal aberrations and the number of aberrant metaphases were significantly lower as compared to the CPA-exposed embryos, but still significantly higher than untreated controls. Sister-chromatid exchanges were not significantly different in embryos exposed to CPA only when compared to those exposed to CPA and ascorbic acid. In terms of the developmental potential of embryos, pregnant mice injected with both CPA and ascorbic acid had significantly fewer resorptions per mouse, and a significantly higher number of viable fetuses retrieved on the 18th day of gestation when compared to mice injected with CPA alone. The data in this study demonstrate that the co-administration of ascorbic acid with CPA to pregnant mice ameliorates the CPA-induced clastogenicity and improves the developmental potential of these embryos. This study suggests that some of the CPA-induced genotoxicity is due to the generation of reactive oxygen molecules, but further research is necessary before this can be firmly concluded.
Animals↗
Unique role of studies on preimplantation embryos to understand mechanisms of embryotoxicity in early pregnancy.
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Animals↗
Amniotic fluid cholinesterase of valproate-induced exencephaly in the mouse: an animal model for prenatal diagnosis of neural tube defects.
After a single administration of the antiepileptic drug valproic acid (VPA; i.p.:600 mg/kg) on day 8 of gestation in the mouse embryotoxicity and amniotic fluid (AF) cholinesterase (ChE) were evaluated on day 16 of gestation. VPA treatment induced an increase in embryolethality, neural tube defects (exencephaly), cleft palate, deformed vertebrae, open eyes, and a reduction in fetal weight. In VPA-exposed fetuses AF total ChE (TChE) activity of exencephalic fetuses was higher than that of normal fetuses. However, in 3 out of 110 normal fetuses of the control group TChE activity was found in the AF. There was no correlation between blood contamination of AF and its TChE activity, either in non-exencephalic control or treated embryos. Using ethopropazine as a "pseudo"-ChE inhibitor in vitro, the percentage of acetyl-ChE in blood-contaminated AF was similar to that of fetal rather than maternal serum, indicating that AF was contaminated with fetal and not with maternal blood. VPA-induced exencephaly in mice may provide an animal model to further investigate biochemical markers for prenatal diagnosis of neural tube defects.
Amniotic Fluid↗
[Recommendations for the administration of vitamin A in pregnancy].
During pregnancy vitamin A supplementation cannot generally be recommended, since an average diet contains a sufficient amount (7500 IE/day) of vitamin A, since a typical malformation pattern has been observed after excessive doses of vitamin A during human pregnancy, and since some vitamin A derivatives are embryotoxic and teratogenic both in animals and humans. Particular reference is given to the recommendations of the American Teratology Society for the use of vitamin A during pregnancy.
Abnormalities, Drug-Induced↗
Potentiating effect of caffeine on embryotoxicity of cyclophosphamide treatment in vivo during the preimplantation period.
Since it is unknown if chemicals which are generally safe in pregnancy can potentiate the embryotoxicity of cytotoxic drugs before implantation, the combined effects of cyclophosphamide (CPA) and caffeine (CF) were studied in the mouse (day 2) with CPA at 20 mg/kg, which induces a 18% resorption rate, and CF at 100 mg/kg, which does not increase embryolethality. The embryotoxicity of CPA did not increase when CF was either given 6 h before or simultaneously with CPA on day 2. However, when CF was given 6 h after CPA, a potentiated increase of embryolethality was observed at term and of structural chromosomal aberrations in embryos even before implantation, while sister chromatid exchange (SCE) frequency was less increased than after single CPA treatment. The results demonstrate the importance of pharmacokinetics in pregnancy even before implantation. Furthermore, since SCE may not only indicate DNA damage but also repair, the reduced SCE rate and the increase in structural chromosome aberrations suggest that CF can inhibit the repair of CPA-induced DNA damage in preimplantation embryos. Finally, for the first time the data are providing evidence that CF at subthreshold doses can potentiate the embryotoxicity of an alkylating agent in vivo even before implantation.
Animals↗
Spontaneous and cyclophosphamide-induced sister-chromatid exchanges in diploid and endoreduplicated tetraploid metaphases of preimplantation mouse embryos.
Endoreduplicated tetraploid metaphases could for the first time be induced in preimplantation mouse embryos by culture in the suboptimum medium MEM. In such endomitoses sister-chromatid exchange (SCE) frequency was approximately the same during the first and the second cell cycle. However, when morulae and blastocysts were cultured in the presence of cyclophosphamide metabolites SCE frequency was increased predominantly during the second cell cycle. Compared to diploid metaphases a decreased SCE frequency was found under both conditions of endomitoses induction, which may be related to DNA-repair processes.
Animals↗
Test guideline. Behavioral toxicity testing in animal experiments according to section 9, para. 1, No. 2 of the Chemicals Act (Chemikaliengesetz) of the Federal Republic of Germany.
The German Chemicals Act requires that chemicals are tested for behavioral toxicity at stage 2 of the testing procedure, i.e. if more than 1000 annual tons are produced. For this purpose a guideline was developed according to which data on behavioral toxicity are to be collected, which are based on cageside observations during longterm exposure. The protocol covers outer appearance, as well as motor, sensory, autonomic and central nervous system functions. Data are to be reported in tabular form and should be evaluated by taking all aspects of the toxicological profile into account.
Animals↗
Studies on the embryotoxic risk of exposure to caffeine and ethanol during the preimplantation period in the mouse.
Pregnant mice were exposed before implantation to caffeine and ethanol to determine the dose-response relation for embryolethality during the preimplantation period. For risk estimation the embryotoxicity was evaluated at term and also 24 h after implantation. For ethanol no embryotoxic risk could be detected. Caffeine unexpectedly exhibited a high risk for embryoethality when compared to the maternal LD50. However, when taking into account realistic exposure levels an embryotoxic risk in early pregnancy can be excluded in humans for both caffeine and ethanol.
Animals↗