Saju Kawauchi

Susceptibility of Day-12.5 and Day-13.5 Fetal Mouse Palates Cultured in Vitro to 5-Fluorouracil and Hydroxyurea

Abstract The maxillary regions of day‐12.5 and day‐13.5 ICR mouse fetuses were cultivated in a chemically‐defined serumless medium by a suspension culture technique to examine the toxic effects of 5‐fluorouracil (5‐FU) and hydroxyurea (HU) on cultured palates and to compare the sensitivity of fetal mouse palates at different stages of development. The palates of day‐12.5 and day‐13.5 fetal mice were explanted and exposed in vitro for 72 hr to 0.1–50 μg 5‐FU/ml or to 5–76 μg HU/ml. 5‐FU inhibited the growth and fusion of day‐12.5 palatal shelves in vitro dependently on its concentrations. Day‐13.5 palates were significantly less sensitive to 5‐FU than day‐12.5 palates, and the minimal toxic concentrations (MTCs) of 5‐FU were 0.1 and 10 μg/ml for day‐12.5 and day‐13.5 fetal palates, respectively. HU inhibited the in vitro growth and fusion of day‐12.5 fetal palatal shelves in a concentration dependent manner, but only slightly suppressed the growth of day‐13.5 fetal palates. The MTCs of HU were 19 and 76 μg/ml for day‐12.5 and day‐13.5 fetal palates, respectively. Therefore, day‐12.5 fetal mouse palates (at stage‐1 or earlier stages of palatogenesis) seemed significantly more susceptible to these teratogenic chemicals than day‐13.5 fetal palates (at stages 2–3 of palatogenesis). The palates of day‐12.5 ICR fetal mice may be more suitable than day‐13.5 palates for in vitro teratogen screening and for the study of mechanisms of normal and abnormal palatogenesis.

In Vitro Developmental Toxicity of Aspirin and Its Major Metabolites on Cultured Fetal Mouse Palates

Palatal primordia of day‐12.5 ICR mouse fetuses were cultured in a chemically‐defined serumless medium by a suspension culture technique, and the developmental toxicity of aspirin and its metabolites on in vitro palatogenesis was studied. Explanted fetal palates were exposed in vitro for 72 hr to 0.5‐2 mM aspirin (ASP), 0.25‐2 mM salicylic acid (SA), 0.5‐2 mM salicyluric acid (SUA), 1–2 mM 2,3‐dihydroxybenzoic acid (3DHB), or 1–2 mM 2,5‐dihydroxybenzoic acid (5DHB). After 72 hr culture, ASP at 2 mM and SA at 0.25 mM inhibited the growth and fusion of palatal shelves, and SUA at 1 mM prevented palatal fusion. On the other hand, 3DHB and 5DHB did not exert any significant toxic effects on cultured palates at concentrations up to 2 mM. Judging from the 50% inhibitory concentration (IC50), SA (IC50= 0.9 mM) was the most toxic of the 5 compounds tested, with a decreasing order of ASP (IC50= 1.5 mM), SUA (IC50= 1.6 mM), and DHBs (IC50= over 2 mM for both 3DHB and 5DHB). With respect to developmental toxicity, cultured fetal mouse palates showed the susceptibility to aspirin and its metabolites which is intermediate between the susceptibility of rat embryos in vivo and that of postimplantation rat embryos cultured in vitro. The significance of fetal organ culture for evaluating developmental toxicity of chemicals is also discussed.

Residues of Clopidol (3, 5-Dichloro-2, 6-dimethyl-4-pyridinol) in Tissues and Eggs of Chickens

Residues of clopidol (3, 5-dichloro-2, 6-dimethyl-4-pyridinol) in chicken tissues and eggs were studied. Clopidol isolated from various tissues was determined by gas chromatography. Chickens of 10 weeks of age were given feed containing 250, 375 and 750ppm of clopidol for 10 days before laying eggs. The average contents of clopidol in the eggs were 6ppm, 10ppm and 19ppm, respectively. The concentration ratio (clopidol in whole egg/clopidol in feed ×100) of clopidol was found to be 2.64% on average, and clopidol in the egg disappeared within approximately one week.In other experiment, chickens of 4 weeks of age were given clopidol-containing feed (250, 375 and 750ppm) for 8 weeks. High concentrations of clopidol were found in the liver, blood and kidneys as compared with the breast, thigh muscle and fat. After cessation of medication, the clopidol concentration in each tissue fell below 0.1ppm within 3 days.