Comparison of in vitro and in vivo Developmental Toxicity and Pharmacokinetics of Phenytoin in the Rat

Abstract

Abstract:The rat whole embryo culture was compared to an in vivo experiment with regard to embryotoxicity as well as exposure characteristics, using phenytoin as a model compound. Intra-embryonic concentrations and their embryotoxic effects were determined on gestation day 11 after in vitro administration of 50-150 mg/ml or in vivo gavage of 500-1500 mg/kg body-weight on gestation day 10. In addition, exposure kinetics were studied in vivo after a single oral dose on gestation day 10, and developmental defects on gestation day 21 were scored. The embryotoxic effects observed on gestation day 11 were more pronounced after in vitro exposure in comparison to in vivo exposure at similar intra-embryonic concentrations. Exposure of phenytoin on gestation day 10 in vitro via the culture medium resulted in general embryotoxicity on gestation day 11, whereas in vivo effects as determined on gestation day 11 were minimal. Plasma concentrations of phenytoin increased and plateaued around 35 mg/ml during the 48 hr monitoring period. Plasma concentration curves and pharmacokinetic parameters did not show remarkable differences between the dose groups, indicating that absorption is the limiting factor at the dose range used. Although the developmental effects were minimal as observed in vivo on gestation day 11, specific malformations (defects encompassing the urogenital, craniofacial and skeletal systems) were observed on gestation day 21. These findings show that with similar intra-embryonic concentrations of phenytoin the embryotoxicity in rat whole embryo culture was not comparable with the in vivo embryotoxicity as determined on gestation day 11. This discrepancy may at least partly be explained by differences in exposure characteristics.In embryotoxicity testing the in vitro test with closest resemblance to in vivo complexity available is the rodent whole embryo culture (New 1978). This system can be used to detect developmental effects of compounds on the isolated intact embryo during a developmentally critical 48 hr period (Schmid 1985; Bechter & Schö n 1988;Piersma et al. 1995Piersma et al. & 1996. For the interpretation of results of in vitro embryotoxicity testing, the nature of the effects observed and the lowest effective compound concentration are essential. As in vitro systems necessarily represent a small part of the living animal, extrapolation of these findings to the in vivo situation is not straightforward.The present study was carried out to compare an embryo culture experiment with an in vivo experiment in the rat with regard to exposure and effect characteristics, using phenytoin as a model compound. In man, phenytoin causes growth retardation, microcephaly, mental deficiency and craniofacial anomalies, known as the foetal hydantoin syndrome (Hanson & Smith 1975). Growth retardation and craniofacial defects have been produced in mice and rats in vivo (Lorente et al. 1981;Rowland et al. 1990; Finnell & Dansky 1991) and in embryo culture (Bruckner et al. 1983;Lindhout et al. 1987;Ozolins et al. 1995). T...

Dose–Response Effects of Diphenylhydantoin on Pregnant Dams and Embryo‐Fetal Development in Rats

“…The results of our study clearly show that oral administration of DPH is minimally embryotoxic at a minimal maternotoxic dose and significantly embryotoxic and teratogenic at an overt maternotoxic dose. Similar developmental toxicities have been demonstrated in a range of animal species including the rat (Rowland et al ; Finnell and Dansky ; Beekhuijzen et al, ). However, some human studies have cast doubt on the association between DPH exposure and teratologic risk (Livingston et al, ; Meyer, ; Goujard et al, ; Shepard and Lemire, ).…”

“…In contrast, the significant embryotoxicity and teratogenicity observed in the 300 mg/kg group was considered to be a direct effect of DPH. According to a report by Beekhuijzen et al (), developmental toxicity of DPH is specific and not mediated by maternal toxicity. In vitro experimental studies have also shown that treating rodent embryos with DPH directly causes malformations encompassing allantois circulation, crown‐rump length, somite number, and yolk sac diameter (Beekhuijzen et al, ; Abramov and Wells, ).…”

Developmental neurotoxicity role of cyclophosphamide on post‐neural tube closure of rodents in vitro and in vivo

“…Moreover, it is worthy to point out there are reports that CPA‐induced neurotoxicity was potented by activate excitotoxic mechanisms in vitro and in vivo (Rzeski et al, 2004; Jansen et al, 2006). Although evidence of CPA‐induced retardation in neurogrowth exists (Mammon et al, 2006; Beekhuijzen et al, 2000), further studies are needed to elucidate the mechanisms for CPA‐induced impairments process leading to malformation before and after neural tube closure.…”

Whole Embryo Culture: An Important Tool in Developmental Toxicology Today

“…Pharmacokinetic analysis revealed comparable intra-embryonic concentrations at this time point, but the comparison of the concentrationtime curves revealed that the AUC values for the in vivo embryos were much lower than the AUC values obtained in vitro. A subsequent in vitro experiment with lower AUC values comparable to the maximal AUC values obtained in vivo thus revealed no adverse effects in the cultured embryo [103]. A very similar study performed with hydroxyurea and the same focus had the same outcome [104].…”

“…It is reasonable to suggest that known pharmacokinetic drug parameters like peak concentration and half-life in vivo have to be mimicked in vitro in order to obtain a comparability of results [95]. For example, in a comparative study on the embryotoxicity of phenytoin [103], it was observed that the embryos exposed in vivo showed no significant effect on embryonic development on gestational day 11, whereas the embryos exposed in vitro did. Pharmacokinetic analysis revealed comparable intra-embryonic concentrations at this time point, but the comparison of the concentrationtime curves revealed that the AUC values for the in vivo embryos were much lower than the AUC values obtained in vitro.…”