Hiromitsu Ohmori

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum.

Abstract: Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2‐4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5‐bromo‐2′‐deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5‐trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.

Pathogenesis of cleft palate in mouse embryos exposed to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate in mouse embryos. It has been believed that TCDD inhibits palatal fusion by suppression of disappearance of medial edge epithelial (MEE) cells on palatal shelves. However, we found that exencephalic mouse embryos were resistant to the cleft palate-inducing action of TCDD. In the present study, we examined cell kinetics in MEE and palatal mesenchyme in embryos exposed to TCDD with or without exencephaly for elucidation of pathogenesis of cleft palate by TCDD. Pregnant Jcl:ICR mice were given TCDD orally at 40 microg/kg at gestation day (GD) 12.5. Embryos were harvested between GD 13.5 and GD 14.5 and examined for cell kinetics by bromodeoxyuridine (BrdU) and TUNEL methods. Exencephaly was induced by intraperitoneal injection of CdCl(2) at 6 mg/kg at GD 7.5. BrdU-positive cells were decreased in TCDD-treated embryos in MEE and mesenchymal cells. TUNEL-positive cells were detected in MEE both in TCDD-treated and untreated control embryos, as well as in embryos with or without exencephaly. We also measured the gap between shelves between GD 14. 0 and GD 14.5. There were no differences at GD 14.0 between control and TCDD-exposed embryos, but at GD 14.25 and GD 14.5, TCDD-exposed embryos had wider gaps than controls. These findings indicate that cleft palate by TCDD results from poor development of palatal shelves. Teratogenesis Carcinog. Mutagen. 20:73-86, 2000.

Neurotoxic effects of phenytoin on postnatal mouse brain development following neonatal administration.

Phenytoin (PHT) is a commonly used anticonvulsant drug. It has been reported that children exposed prenatally to PHT have brain malformations and psychomotor dysfunction. The neonatal development of the central nervous system (CNS) in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT on postnatal brain development, we administered PHT at doses of 10, 17.5, 25, or 35 mg/kg to newborn mice once a day during postnatal days (PD) 2-4. These dose levels result in plasma levels corresponding to the therapeutic ranges in humans. We measured the weight of total brain, cerebrum, cerebellum, and brain stem on PD 5 through 21, and examined early motor functions including head elevation, elevation of pelvis, pivoting, crawling, and righting reflex . Total brain weight, cerebral weight, and cerebellar weight in the group treated with 25 or 35 mg/kg were significantly reduced compared to controls from PD 5 to 21. Mice treated with PHT at 25 or 35 mg/kg showed decreased locomotor abilities and righting reflex on PD 5. In all phenytoin treatment groups, phenytoin levels in the brain were higher than those in the plasma on the third day of PHT treatment. We thus observed neurotoxic effects of PHT on postnatal brain development in mice. Our present data may provide useful implications for the management of PHT-induced developmental neurotoxicity and evaluation of psychomotor development in children exposed to PHT during the late fetal period.

Effects of low-dose phenytoin administered to newborn mice on developing cerebellum

To examine correlations between dose levels of phenytoin (PHT) and neurotoxic effects on cerebellar development, we administered 10, 17.5, 25, and 35 mg/kg PHT suspended in sesame oil orally to newborn Jcl:ICR mice once a day during postnatal days 2-4 and determined plasma PHT concentrations during the administration period. Mortality rates were 12.5% and 35.2% in males and 15.3% and 33.3% in females for the 25 and 35 mg/kg PHT-treated groups during the PHT treatment, respectively. In the 25 and 35 mg/kg PHT-treated groups, total brain weight, the size of the cerebellum, and cerebellar weight were significantly reduced on postnatal day 21. However, in the 10 and 17.5 mg/kg PHT-treated groups, total brain weight and the size and weight of the cerebellum did not differ from those of the control group. Histologically, the number of pyknotic cells in the external granular layer (EGL) in the 25 and 35 mg/kg PHT-treated groups was increased on postnatal day 5, and the EGL was thicker than in the control group on postnatal day 14. Some of the Purkinje cells in the 35 mg/kg PHT-treated group showed degeneration. Plasma PHT levels were 10.7 +/- 2.2 and 24.6 +/- 2.6 micrograms/ml in the 25 and 35 mg/kg PHT groups on the third day of PHT treatment, respectively. In the 25 mg/kg PHT group, plasma PHT level was found to be in the therapeutic range for humans, 10-20 micrograms/ml. Accordingly, during pregnancy, epileptic women should be carefully given PHT at the lowest effective dose while plasma PHT levels are monitored properly. These findings emphasize the importance of pharmacokinetics in evaluating of phenytoin-induced developmental neurotoxicity.

Neurotoxicity of phenytoin administered to newborn mice on developing cerebellum

To examine the neurotoxic effects of phenytoin (PHT) on cerebellar development, we administered 50 mg/kg PHT suspended in sesame oil orally to newborn Jcl:ICR mice once a day during postnatal days 2-14 and determined plasma PHT concentrations at designated intervals during the administration period. In the treated group, walking reflex and negative geotaxis were poorly developed on postnatal day 14. Pyknotic cells in the external granular layer (EGL) significantly increased and were prominent in the vermis area compared with controls on postnatal day 14. Plasma PHT levels were 34-36 micrograms/ml on the 3rd day of PHT treatment and approached a steady-state situation. Total brain weight, size of the cerebellum, and cerebellar weight were significantly reduced in the treated group on postnatal day 56. Accordingly, oral administration of PHT in the neonatal period induced neurotoxic damage on the developing cerebellum.