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.

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.

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.

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.

Validation project of alternatives for the Draize eye test.

The Federal Health Office (BGA) has started in June 1988 a national interlaboratory study to validate alternatives to the Draize test. It is supported by grants of the Department of Research and Technology (BMFT) of West Germany. The aim of this collaborative study is to validate the classification of chemicals with regard to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg test-chorioallantoic membrane (HET-CAM) assay, according to Lüpke.

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.

Embryotoxicity of stable isotopes and use of stable isotopes in studies of teratogenetic mechanisms.

Experiments on teratogenic effects of stable isotopes from our own and other laboratories are evaluated. In the first series of investigations, the enrichment of the stable isotope 13C derived from U-13C-glucose was studied in mouse embryos at various stages of development, including limb buds in organ culture. Preimplantation mouse embryos incubated in vitro in 13C-enriched medium for 48 hours showed normal development during subsequent differentiation in vitro and also in vivo after embryo transfer to faster mothers.

[Evaluation of the embryotoxic risk of industrial chemicals in pregnancy].

For the first time exposure levels during pregnancy have been evaluated for industrial chemicals in the German list of "Maximal occupational exposure limits and biological tolerance levels of occupational chemicals 1985" (MAK-Werte-Liste). According to this evaluation only a single substance (methylmercury) is embryotoxic in man, a prenatal risk cannot be excluded for eight chemicals, and 18 chemicals are safe at occupational exposure limits (MAK-Werte). Furthermore, pregnant women should avoid exposure to any of the 112 carcinogenic chemicals of the list and to 26 substances which are under evaluation for embryotoxic properties.

Studies on the embryotoxic risk of drug treatment during the preimplantation period in the mouse.

For risk evaluation of exposure to drugs in early pregnancy the dose-response relationship for embryotoxicity was determined in mice during the preimplantation period using cytotoxic drugs (cyclophosphamide, mitomycin, vinblastine) and therapeutic drugs which are embryotoxic in laboratory animals or in humans during organogenesis (diazepam, doxycycline, phenobarbital, rifamycin, tolbutamide). No malformations but only signs of retardation and a dose related increase in the resorption rate could be detected after treatment with some of the drugs at term even at dose levels close to the maternal LD50 (MLD50). A comparison of the embryolethal dose during the preimplantation period (ELD50) with the MLD50 revealed no risk for the therapeutic drugs, a slight risk for mytomycin and vinblastine and an unusually high risk for cyclophosphamide.

Preimplantation mouse embryos cultured in mouse, rat and human sera: differentiation and sister chromatid exchange.

Development of 4-cell and of 8-cell mouse embryos and of morulae and blastocysts is inhibited in vitro by mouse serum but not by rat or human sera which also do not influence sister chromatid exchange in cultured morulae and blastocysts.

Development and sister chromatid exchange of mouse morulae and blastocysts cultured in rat serum containing active metabolites of cyclophosphamide.

To establish an in vitro test system in which sera from animals treated with various chemicals can be tested for embryotoxic effects during the preimplantation period, mouse morulae and blastocysts were cultured in the presence of rat serum (RS) from animals which had been treated with cyclophosphamide (CPA). Development during in vitro culture for 96 h, cell number, chromosomal aberrations and sister chromatid exchange (SCE) were the end-points tested in exposed embryos. SCE frequency was the most sensitive parameter, indicating embryotoxic effects in preimplantation mouse embryos after only 1 h of exposure to RS-CPA.

Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization.

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program.

[In-vitro-fertilization: chromosome analysis of unfertilized human oocytes].

Chromosomal abnormalities and abnormal embryonic development have been observed after human in vitro fertilisation (IVF). Such investigations are not permitted according to the ethical guidelines approved by the IVF team in Berlin ("Berliner Modell"). However, the chromosomal status was studied in oocytes which remained unfertilised in a human IVF programme.

Changes of the adenine ribonucleotide content during preimplantation development of mouse embryos in vivo and in vitro.

The amount of ATP, ADP and AMP and also the adenylate energy charge and the ATP/ADP ratio were determined in preimplantation mouse embryos (strain NMRI) in vivo. The ATP content decreased from 0.64 pmol at fertilization to 0.

Normal adenylate ribonucleotide content in mouse embryos homozygous for the t12 mutation.

The recently improved firefly luciferase assay was used to determine ATP, ADP or AMP in single preimplantation mouse embryos from crosses yielding lethal t12/t12 embryos. Normal values of the three adenylate ribonucleotides were found in freshly collected 2-cell and 4-cell embryos and during in vitro culture to the blastocyst stage. A decrease in adenylate ribonucleotide content was seen in putative t12/t12 embryos only when they were degenerating.

Mutagenic, teratogenic and pharmacokinetic properties of cyclophosphamide and some of its deuterated derivatives.

To elucidate which metabolic pathway leads to the ultimate mutagenic and teratogenic metabolite of cyclophosphamide (CPA), the mutagenicity in vitro as well as the teratogenicity in vivo and the pharmacokinetics of CPA and several deuterated analogs (5,5-d2-CPA, 4,4-d2-CPA and 4,4-6,6-d4-CPA) were compared. 5,5-d2-CPA was less mutagenic (isotope effects between 1.7 and 12.

Teratogenic effects of cyproterone acetate and medroxyprogesterone treatment during the pre- and postimplantation period of mouse embryos. II. Cyproterone acetate and medroxyprogesterone acetate treatment before implantation in vivo and in vitro.

The study was performed to investigate direct embryotoxic effects of maternal progestin treatment during the preimplantation period. In the first experiment pregnant mice received a single subcutaneous injection of either cyproterone acetate (CA) or medroxyprogesterone acetate (MPA) on day 2 of pregnancy (5-600 mg/kg). In a second experiment four-cell embryos were exposed to CA or MPA in vitro (3 or 30 micrograms/ml medium).

Teratogenic effects of cyproterone acetate and medroxyprogesterone treatment during the pre- and postimplantation period of mouse embryos. I.

Pregnant mice were treated with a single subcutaneous injection of either cyproterone acetate (CA) or medroxyprogesterone acetate (MPA). In the first experiment the animals received 5-900 mg/kg of the hormone before implantation (day 2 of pregnancy). CA treatment on day 2 caused a dose-dependent decrease in fetal weight and a significant dose-dependent increase in the rates of cleft palate and urinary tract abnormalities.