Fumio Ariyuki

In vitro developmental toxicity of concanavalin A in rat embryos : Analysis of neural crest cell migration using monoclonal antibody HNK-1

The developmental toxicity of concanavalin A (Con A) was evaluated in vitro using a rat whole embryo culture system, and the distributions of the neural crest cells were immunohistochemically investigated in embryos with monoclonal antibody HNK-1. In addition, binding sites of Con A in the embryos were observed according to the avidin-biotin peroxidase complex method with biotin-labeled anti-Con A. The rat embryos on embryonic day 8 were exposed to a final concentration of 12.5, 25, 50, or 100 micrograms/ml of Con A for a 72 hr culture period. Exposure to Con A concentration-dependently resulted in lower viability, decreases in yolk sac diameter, crown-rump length, and number of somites, and increases in the incidence of morphological abnormalities characterized by neural tube defects in the embryos. In the Con A-treated embryos, the distributions of the neural crest cells were restricted in the dorsal and cranial regions, and the migration into the interventricular chamber was delayed in the cardiac region. The Con A-treated embryos were confirmed to have Con A binding on the wall of the outflow tract in the cardiac region and in the mesenchyme of the cranial region, which are thought to be migration pathways of neural crest cells. These findings suggested that Con A inhibited the early migration of neural crest cells by binding directly to some substrates distributed along the pathways in the embryos, so that the neural crest cells could not punctually reach the locations where they would proliferate and differentiate into the destined cell types.

A New in Vitro Screening Method for Teratogens Using Human Embryonic Palatal Mesenchymal Cells

Abstract In order to establish an in vitro screening assay system for cleft palate‐inducing teratogens, we tested 31 teratogenic and 10 tionteratogenic compounds using human embryonic cultured cells. We examined whether cleft palate‐inducing ability can be detected by differential growth inhibition between human embryonic palatal mesenchymal (HEPM) cells and human embryonic fibroblasts (MRC‐5). Thirty one compounds with proven cleft palate‐inductive effects in vivo preferentially inhibited the proliferation of HEPM cells. The average of the relative resistant rates (rate of IC50 value for HEPM cells to MRC‐5 cells) of teratogens was 0.53. In contrast, almost all nonterato‐gens identically inhibited the proliferation of both cell lines and the average of the relative resistant rates was 1.01. These results show that teratogens which induce cleft palate in vivo preferentially inhibit the proliferation of embryonic palatal mesenchymal cells.

Behavioral Effects of Hydroxyurea Exposure during Organogenetic Period of the Sprague‐Dawley Rats

Abstract As an attempt to establish the methodology of behavioral teratology, this study was carried out to investigate whether the effects of HU would be different between Wistar and Sprague‐Dawley (SD) rats. Hydroxyurea (HU) was intraperitoneally injected to pregnant SD rats at does of 50 or 100 mg/kg/day during the organogenetic period (days 9 to 12 of gestation). Male offspring from these dams were observed for their morphological and behavioral developments. Microphthalmia was found in only 2 pups at the 21st day of the 100 mg/kgyday group. No adverse effect of postnatal growth was observed in all treated groups. The number of ambulation in the open field test at 3 and 4 weeks of age in the 50 mg/kg/day group and at 5 weeks of age in the 100 mg/kg/day group was decreased. No inhibition of reflex development, traction response and rotorod performance was observed in all treated groups. These findings suggested that the morphological and behavioral effects of HU in SD male rats are less severe than those in Wistar male rats as was in our previous study. The strain differences in the exploratory behavior seemed to be more severe, because the specific behavioral type in both strains might be potentiated by HU treatment.

Relationship between Teratogenic Effects and Tissue Binding Pattern of Concanavalin A in Rat Embryos

ABSTRACT This study was conducted to examine the effects of concanavalin A (Con A) on rat neurulation. Con A was injected intravenously to pregnant rats at a single dose of 5, 10, or 20 mg/kg between gestational days (GD) 7.5 and 10.0, and the embryos were macroscopically examined on GD 11.5. Con A induced a high mortality rate, the treatment with 10 and 20 mg/kg on GD 9.5 was completely lethal. Con A caused a high incidence of neural tube defects (NTD), the incidence in the 10 mg/kg on GD 8.0 and 8.5 groups and in the 20 mg/kg on GD 8.0, 8.5 and 9.0 groups being significantly higher than that in the control group. Histological evaluation revealed that Con A inhibited neural tube expansion and altered neural crest cell shape. In the Con A‐treated embryos, the stratified structure of the neuroepithelium was irregular and its extracellular space was expanded. In rat embryos from intact dams, the distribution of receptors for Con A was demonstrated histochemically on the cell surfaces around neurulation sites between GD 8.5 and 10.5, but not on GD 7.5. These findings indicate that Con A bound around the neurulation sites in rat embryos may disturb the cell‐cell communication required to form the neural tube, resulting in NTD.