Use of two validated in vitro tests to assess the embryotoxic potential of mycophenolic acid.

Mycophenolate mofetil is a widely used immunosuppressive drug that recently has been categorized as a human teratogen. Animal experiments indicate a teratogenic potential of the drug, but so far, it has not been studied in embryotoxicity in vitro assays. The aim of this study was to evaluate the in vitro embryotoxic potential of mycophenolic acid and investigate the ability of such tests to detect its embryotoxic potential. We used two validated assays: the rat whole embryo culture and the murine embryonic stem cell test. Rat embryos cultured from gestational day 9.5 for 48 h with the drug showed dysmorphogenic development already at a concentration of 250 microg mycophenolic acid/l medium. At concentrations of 750 microg/l and more, all rat embryos exhibited malformations. The main effects were defective yolk sac blood circulation, neural tube defects (open cranial neural pore), malformations of the head with missing eye anlagen and heart anomalies. Moreover, the exposed embryos showed a concentration-dependent decrease in protein content, crown-rump length, number of somites and morphological score. The murine embryonic stem cell test was slightly more sensitive. Proliferation and differentiation of the ES-D3-cells were significantly impaired at concentrations of 31 and 125 microg mycophenolic acid/l medium, respectively. In the differentiation assay, at a concentration of 125 microg mycophenolic acid/l medium and more, the number of wells with differentiated cardiomyocytes significantly decreased. Additionally, a cytotoxicity assay with balb/c 3T3 mouse fibroblasts was used to compare the proliferation and vitality of embryonic cells with adult cells. In the balb/c 3T3 cytotoxicity assay, the number of vital mouse fibroblasts significantly decreased at a mycophenolic acid concentration of 62 microg/l and more. In conclusion, by using the two validated in vitro tests, we showed that mycophenolic acid exhibits a pronounced embryotoxic potential at cytotoxic concentrations. This result from validated in vitro tests is of special interest, because it supports the use of the tests to detect human teratogens.

Is metformin therapy for polycystic ovary syndrome safe during pregnancy?

Polycystic ovary syndrome is characterized among other things by oligo-amenorrhea and may account for more than 75% of cases with anoluvatory infertility. Due to its positive effects on polycystic ovary syndrome-induced infertility metformin has become one of the most common drugs used in this group of patients. The efficacy of the drug as well as the first reports on metformin used in pregnancy has encouraged the continued use of the drug after conception. This MiniReview reviews the current pros and cons of metformin use in pregnancy while awaiting the results of ongoing randomised, controlled clinical trials addressing the subject.

Maternal and fetal toxicity of N-methylmorpholine by oral administration in rats.

N-methylmorpholine, which is used as a catalyst in polyurethane foams producing, in solvents, stabilizing agents, and corrosion inhibitors, was administered to female rats by gavage at 100, 200, 600, and 900 mg/kg during organogenesis. It did not exhibit selective toxicity toward the developing conceptus. This compound administered to pregnant females was fetotoxic and teratogenic in the presence of maternal toxicity. N-methylmorpholine induced anophthalmia, internal hydrocephalus, and hydronephrosis but only at one dose which was also maternotoxic. Teratogenesis Carcinog. Mutagen. 19:369-376, 1999.

Anophthalmia in litters of female rats treated with the food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Anophthalmia in litters of pregnant rats treated with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a food-derived carcinogen, was incidentally encountered in a risk-assessment study with 2-generation exposure to PhIP. Female Fischer 344 animals were given 200 ppm PhIP in the diet for 4 wk before mating with nontreated males and also during gestation and lactation periods. Mean numbers of newborn rats per litter in control and PhIP-treated groups were 7.9 +/- 2.9 and 7.1 +/- 1.6 in trial 1 and 8.3 +/- 1.9 and 6.1 +/- 2.4 in trial 2. Among 49 (trial 1) and 63 (trial 2) offspring from PhIP-treated dams, 9 (18.4%) and 32 (50.8%) demonstrated anophthalmia, and 1 (2.0%) and 8 (12.7%) demonstrated hydrocephaly. Five of 7 (71.4%) and 13 of 14 (92.9%) dams delivered pups with malformations in trials 1 and 2, respectively. Also, in a previous study that was carried out with the same protocol and that used the Sprague-Dawley strain of rats, anophthalmia and hydrocephaly were observed in 2 and 1 out of 175 pups, respectively, from 100 ppm PhIP-treated dams. No congenital malformations were found in control groups of the same size in either experiment. In addition to having been previously identified as a cause of carcinogenic activity, our findings suggest that PhIP is capable of causing anophthalmia in rats when administered during the gestational period.

A comparison of the in vivo and in vitro response of rat embryos to 5-fluorouracil.

This study serves to further define the capabilities of the whole embryo culture system using the well-known teratogen, 5-fluorouracil (5-FU), an antineoplastic agent. An initial in vivo study was performed whereby pregnant rats were injected intraperitoneally with 10-30 mg/kg 5-FU on day 9 of gestation. On day 20 of gestation, the effects of this drug on the growth and development of embryos were evaluated. The number of externally malformed fetuses increased in a dose-related manner, and the most common defect was micro-/anophthalmos in fetuses of dams treated with 5-FU. Growth retardation was also noted in the 5-FU treated groups. An in vitro study was performed in which drug concentrations were varied (0.15-0.30 microg/ml). Externally abnormal embryos were observed in whole embryo culture system from embryonic day 9 to 11. The most common defect was hypoplastic optic vesicles. In the whole embryo culture system, crown-rump length, somite number, protein contents, and morphological score were decreased in a dose-dependent fashion. Finally, histological evaluation and observation of the pattern of cell death of the optic vesicle of 11-day-old embryos in in vivo and in vitro were performed. These parameters revealed no differences in response between in vivo and in vitro embryos treated with 5-FU, suggesting that the whole embryo culture system was an appropriate model for developmental toxicity studies of 5-FU.

Normal and abnormal midfacial development in the cadmium-treated hamster.

Clefts of the midface ranging in severity from a notched lip to complete facial disorganization were observed in the offspring of golden hamsters injected with cadmium chloride on the morning of the eighth day of gestation. Other malformations frequently observed were microphthalmia, anophthalmia, and diencephalic encephaloceles. Histologic examination of embryos on the 10th, 11th, 12th, and 13th days of gestation revealed a marked deficiency of mesenchyme in the frontonasal process, which led to a foreshortened and deformed nasal septum. These data confirm that the teratogenicity of cadmium is highly specific for the region of the anterior neural segment on the eighth day of gestation. The deficiency of mesenchyme in the frontonasal process may be the result of disruption in neural crest cell development in this region.

Eye malformations in rats: induction by prenatal exposure to nickel carbonyl.

Exposure of pregnant rats to inhalation of nickel carbonyl on days 7 or 8 of gestation frequently causes the progeny to develop ocular anomalies, including anophthalmia and microphthalmia. The incidence of extraocular anomalies is very low. The specificity of nickel carbonyl for induction of ocular anomalies in rats appears to be unique among known teratogenic agents.