Masato Naya

Development of Rabbit Whole Embryo Culture during Organogenesis

Abstract The rodent embryo culture has widely been spread as a technique in teratogenicity studies. However, the whole embryo culture of rabbits has not been developed. Pregnant rabbits are usually used in a teratogenicity test as a non‐rodent animal to examine the species specificity. Rabbit embryos of the Japanese White strain on day 10 of gestation (the time of copulation = day 0) were cultured at 38°C for 48 hours in 100% rabbit serum. The 35 ml bottles with 5 ml serum were gassed with 95% 02 and 5%70 CO2 continuously. The yolk sac and amniotic sac were opened 24 hours after the beginning of the culture. At the end of the culture, embryos were examined for somite number, as well as protein content and crown‐rump length. Embryos cultured for 48 hours from day 10 seemed to be well developed as well as in vivo embryos. These results showed that rabbit embryo culture became a useful system for the study of reproductive and developmental toxicology, teratology and developmental biology.

Protective Effects of Glutathione on the Teratogenicity of 5‐Fluorouracil in Mice and Rats

Abstract The influence of endogenous or exogenous glutathione (GSH) on the teratogenicity of 5‐fluorouracil (5‐FU) was studied after a single intraperitoneal injection of 5‐FU at a teratogenic dose (20 or 30 mg/kg) to ICR mice on day 11 of gestation. Pretreatment with intravenous GSH at 300 mg/kg, 5 minutes before 5‐FU administration, decreased the incidence of oligodactyly induced with 5‐FU and lowered the fetal mortality, but it did not prevent the decrease in body weight of fetuses. Pretreatment with intraperitoneal diethylmaleate, which decreases the level of endogenous GSH, at 350 mg/kg 6 hours before 5‐FU administration increased the incidence of oligodactyly. Pretreatment orally with GSH at a dose of 300 mg/kg 30 minutes before intraperitoneal injection of 5‐FU at 30 mg/kg to Wistar rats on day 13 of gestation decreased the frequency of limb malformations, but it did not prevent the decrease in body weight of fetuses.

Effects of N‐acetyl‐L‐cysteine on Teratogenicity of 5‐Fluorouracil in Mice

ABSTRACT Embryotoxicity and teratogenicity of 5‐fluorouracil (5‐FU) and modulation of its effect by N‐acetyl‐L‐cysteine (NAC) andor phorone were evaluated in mice. Pregnant ICR mice were intraperitoneally injected with 25 mglkg of 5‐FU on day 11 of gestation (vaginal plug = day 0). Pregnant mice were pretreated with NAC at dose levels of 80, 160, and 320 mg/kg injected intravenously 2 hours before dosing with 5‐FU. Pregnant mice were killed on day 17 of gestation. Fetuses were examined for external malformations, especially limb malformations. Pretreatment with 160 mg/kg and 320 mg/ kg of NAC decreased the incidence and severity of oligodactyly induced by 5‐FU. There was little difference in maternal body weight gain, fetal mortality, and fetal weight between the 5‐FU group and the 5‐FU plus NAC groups. Pretreatment with phorone, a glutathione depleting agent, at dose levels of 160 and 320 mg/kg injected intraperitoneally, 4 hours before dosing with 5‐FU, increased the incidence and severity of oligodactyly induced by 5‐FU. Cotreatment with NAC at dose levels of 160 and 320 mg/kg decreased the incidence and severity of oligodactyly induced by 5‐FU and 80 mg/kg of phorone. Cotreatment with NAC 160 mg/kg could not suppress the augmentative effect of phorone on 5‐FU teratogenicity under the severe condition, that is, the excess amount of phorone such as 320 mg/kg. These results indicate that the teratogenicity of 5‐FU is mitigated with NAC pretreatment, and also the level of endogenous glutathione is one of the factors which significantly affects teratogenicity of 5‐FU.

Effects of Glutathione and Related Compounds on Teratogenicity of 5-Fluorouracil or Cadmium Hydrochloride in Mice*

Glutathione (GSH) is a tripeptide consisting of cysteine, glutamic acid and glycine, and plays an important role in detoxification reactions. In this report, we describe (1) the effects of the depleting agents of GSH such as diethylmaleate (DEM), phorone, and buthionine sulfoximine (BSO) on teratogenicity of 5‐fluorouracil (5‐FU) in mice, (2) the effects of GSH or N‐acetyl‐L‐cysteine (NAC), a precursor of GSH on teratogenicity of 5‐FU or cadmium hydrochloride (Cd) in mice. Pregnant ICR mice were injected intra‐peritoneally (i.p.) with 5‐FU at dose levels of 20, 25, and 30 mg/kg on day 11 of gestation (vaginal plug = day 0). Mice were injected i.p. with DEM, phorone, or BSO 4 to 6 hours before dosing with 5‐FU. Mice were also pretreated intravenously (i.v.) with GSH at dose levels of 150, 300 and 600 mg/kg, or NAC at dose levels of 80, 160, and 320 mg/kg 0.5 to 2 hours before dosing with 5‐FU. In the Cd‐teratogenicity study, mice were injected i.v. with GSH or NAC before dosing with Cd at 3.5 mg/kg i.p. on day 11 of gestation. Pretreatment with DEM, phorone or BSO increased the incidence of oligodactyly induced by 5‐FU, while pretreatment with GSH or NAC decreased the incidences. Pretreatment with GSH or NAC decreased the incidence of cleft palate and abnormal palatal rugae induced by Cd. The results suggest that cysteine plays a key role in the teratogenicity of 5‐FU or Cd in mice.

Effects of N‐acetyl‐L‐cysteine on Teratogenicity of Cadmium in Mice

ABSTRACT Embryotoxicity and teratogenicity of cadmium (Cd) and modulation of its effects by N‐acetyl‐L‐cysteine (NAC) were evaluated in mice. Pregnant ICR mice were intraperitoneally injected with 3.5 mg/kg of CdCl2 on day 10 or 11 of gestation (vaginal plug = day 0). Pregnant mice were pretreated with 160 mg/kg of NAC intravenously 2 hours before dosing with CdCl2. Pregnant mice were killed on day 17 of gestation. Fetuses were examined for external malformations, especially limb malformations, cleft palate and abnormal palatal rugae. There was little difference in body weight gain of dams during the gestation period in the groups treated with NAC plus Cd as compared with the groups treated with Cd alone. Pretreatment with 160 mg/kg of NAC decreased the fetal mortality, incidence of cleft palate and abnormal palatal rugae induced by Cd on day 11. On day 10, pretreatment with NAC decreased the incidence of Cd induced abnormal palatal rugae. These results clearly indicate that NAC exerts protective effects against embryotoxicity and teratogenicity of Cd.