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H Nau

Publications and source records attributed to H Nau.

At least 73 records · Page 4Linked to original sources
PubMed1996

All-trans-retinoyl-beta-glucuronide is a potent teratogen in the mouse because of extensive metabolism to all-trans-retinoic acid.

All-trans-retinoyl-beta-D-glucuronide (all-trans-RAG) is a water-soluble derivative of all-trans-retinoic acid (all-trans-RA) and has been characterized as an endogenous metabolite of vitamin A in rat bile and kidney. All-trans-RAG was previously demonstrated to be a major metabolite after application of all-trans-RA in several species (mouse, rat, rabbit, monkey); all-trans-RAG was described in these experiments to exhibit a very low placental transfer to the embryo. Because retinoid-like activity has been found after application of all-trans-RAG in vivo as well as in several in vitro systems, and because of its low placental transfer, this glycoconjugate appeared to be an interesting retinoid with possible therapeutic activity, but reduced teratogenicity. Here we investigated the teratogenic activity of all-trans-RAG in comparison to all-trans-RA in mice, and performed accompanying pharmacokinetic studies. Surprisingly, all-trans-RAG was more teratogenic than equimolar doses of all-trans-RA following subcutaneous application on day 11 of gestation in the mouse (20 mumol/kg body weight). Pharmacokinetic studies revealed that all-trans-RAG was extensively hydrolyzed to all-trans-RA and that the plasma area under the concentration-time curve (AUC) of all-trans-RA following all-trans-RAG application exceeded the plasma AUC value of all-trans-RA following application of all-trans-RA. Extensive hydrolysis of all-trans-RAG was also observed after intravenous application of this glycoconjugate. Transfer of all-trans-RAG to the embryo was low, but transfer was high to maternal organs such as the liver and kidney. These in vivo studies suggest that all-trans-RAG serves as a precursor of all-trans-RA by the intravenous and subcutaneous routes, and application of all-trans-RAG results in high and teratogenic in vivo exposure to all-trans-RA.

Animals↗
PubMed1996

Retinoid metabolism and transplacental pharmacokinetics in the cynomolgus monkey following a nonteratogenic dosing regimen with all-trans-retinoic acid.

Retinoids often exhibit a complex metabolic pattern and differential transplacental kinetics, which make it difficult to pinpoint the proximate compound responsible for the observed teratogenic effect. We have therefore studied the pharmacokinetics and metabolism of all-trans-retinoic acid (all-trans-RA) in cynomolgus monkeys following application of a nonteratogenic dosing regimen and compared the results with corresponding data from a previous study with a teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. All-trans-RA was administered to pregnant cynomolgus monkeys (Macaca fascicularis) by nasogastric intubation at a dose of 5 mg/kg body wt once daily from gestational day (GD) 16 to 26 and twice daily at 8-h intervals from GD 27 to 31. Examination of the fetuses of four dams on GD 100 +/- 2 showed no embryotoxic or teratogenic effects of the applied dosing regimen (Experiment 1). Maternal plasma retinoid pharmacokinetics on GD 16, 26, and 31 as well as embryonic retinoid profiles after the last drug administration on GD 31 were determined in thirteen further dams (Experiment 2). All-trans-RA reached much lower plasma concentrations after the last two treatments on GD 31 than after the first one on GD 16 and the eleventh one on GD 26 (0-24-h area-under-the-concentration-time-curve (AUC) values: 104 +/- 59 ng x h/ml (after the last treatment on GD 31), 189 +/- 110 (GD 16) and 393 +/- 305 ng x h/ml (GD 26). The predominant plasma metabolites of all-trans-RA were its beta-glucuronide and the beta-glucuronide of all-trans-4-oxo-RA. Both of these retinoids accumulated in the plasma during the period of treatment and displayed AUC values 5- to 30-fold higher than those of all-trans-RA. Embryonic concentrations of all-trans-RA were not increased over endogenous levels after the last administration on GD 31 when plasma concentrations were low. To evaluate the placental transport of all-trans-RA in the presence of high plasma concentrations, a further experiment was performed, in which a single dose of all-trans-RA (10 mg/kg body wt) was given to four pregnant monkeys on GD 31, and plasma pharmacokinetics as well as embryonic concentrations of retinoids at 4 h post-treatment were determined (Experiment 3). This dosing schedule yielded high plasma concentrations of all-trans-RA, while embryonic concentrations were about 40% of plasma levels. Based on the plasma AUC values on GDs 16 and 26 obtained in Experiment 2 and the degree of placental transfer, as determined on GD 31 in the presence of high plasma levels in Experiment 3, we estimated embryonic AUC values for the 24-h period following the nonteratogenic doses on GDs 16 and 26 in Experiment 2. These AUC values were similarly high to the embryonic AUC value of all-trans-RA obtained after application of the teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. In addition, plasma AUC values of all-trans-RA were 2- to 7-fold higher after all-trans-RA administration (present study) than after dosing with the teratogenic dose of 13-cis-RA. These results strengthen our recent suggestion that the teratogenic effects induced in cynomolgus monkeys by 13-cis-RA treatment cannot solely result from the action of all-trans-RA, but may involve 13-cis-RA and 13-cis-4-oxo-RA, which could act directly or function as transport vehicle.

Animals↗
PubMed1996

Teratogenic effects of sodium valproate in mice and rats at midgestation and at term.

This experiment was carried out with the aims of comparing the embryotoxic potential of valproic acid (VPA) in rats and mice, better defining the malformation pattern in these species, and comparing the embryotoxic effects detectable in mid-pregnancy to those observed in fetuses at term. Pregnant CD:Crl rats were treated subcutaneously (s.c.) at 08:00, 16:00, and 00:00 on day 9 of gestation with 0, 150, or 300 mg/ kg VPA; pregnant NMRI mice were treated s.c. at 00:00 on day 7 of gestation, and at 08:00 and 16:00 on day 8 of gestation with 0,75, 150, or 300 mg/kg VPA. Groups of females were killed on day 9 (mice) or day 11 (rats) of pregnancy and their embryos were carefully examined under a dissecting microscope. The remaining females were killed 1 day before parturition and their fetuses were examined for external, visceral, and skeletal malformations. A very high frequency (84%) of malformed embryos was recorded in the group of mice treated with 300 mg/kg, including open brain folds (73%), somite defects (36%), and heart malformations (20%). The rat embryos were less sensitive: only 43% of them were malformed after treatment with 300 mg/ kg, however, the pattern of malformations was quite similar to that observed in mice. The treatment with 150 mg/kg produced about 32% malformed embryos in mice and only 8.5% in rats. More than 84% of mouse fetuses from mothers treated with the highest dose showed a severe form of exencephaly. The axial skeleton was also severely affected. The postimplantation loss reached 52%. Exencephaly and skeletal malformations were also recorded in mouse fetuses from mothers exposed to 150 mg/kg. The dose of 75 mg/kg was without effects. Exencephaly was not observed in rat fetuses at term. In this species the axial skeleton was the most severely affected region at 300 mg/kg, while the lowest dose produced only sporadic malformations. These results confirm that the mouse is the more sensitive species for the teratogenic effects of VPA. Furthermore, it has been shown that, in both species, the axial skeleton is a system which is very sensitive to the teratogenic effects of VPA. The observed alterations show a possible link between axial specification and VPA and suggest further studies of embryos exposed to VPA for the expression of genes controlling the identity of vertebral segments.

Abnormalities, Drug-Induced↗
PubMed1996

Stereoselective distribution of the teratogenic thalidomide analogue EM12 in the early embryo of marmoset monkey, Wistar rat and NMRI mouse.

Thalidomide administration during early gestation results in specific and dramatic limb defects in primates, but not in laboratory rodents such as the rat and mouse. The thalidomide analogue EM12 [2-(2,6-dioxopiperidine-3-yl)-phthalimidine] was used in the present study because this compound is metabolically more stable and teratogenically more potent than thalidomide in the monkey. We have administered the pure enantiomers, since we have previously shown that S-EM12 proved to be much more teratogenic in the monkey than R-EM12. In maternal plasma, placenta and embryo of the pregnant marmoset monkey (Callithrix jacchus) and Wistar rat, the concentrations were investigated of the enantiomers and their metabolites after administration of R- and S-EM12. With whole body autoradiography the distribution in the embryo, including the target tissue, the embryonic limb bud was examined in the NMRI mouse and marmoset monkey. Our investigations showed that both the R- and the S-enantiomers were transferred to the embryo during organogenesis [monkey, gestation day (GD) 61; rat, GD 12; mouse, GD 10]. The gestation period chosen was toward the end of the thalidomide-sensitive stage, but yielded sufficient gestational material for analysis. Considerable amounts of the enantiomers were produced via racemization of the administered pure enantiomers and were present in maternal plasma as well as in placenta and embryo. In the monkey, the racemization were stereoselective: the S-enantiomer was eliminated more slowly in the monkey than the R-enantiomer, possibly because of stereospecific binding and metabolism. In the plasma and embryo of both rat and monkey, the metabolites were detected in considerably lower concentrations than EM12, emphasizing the importance of the parent drug in regard to the teratogenic effect. The whole-body autoradiography in marmoset and mouse showed high radioactivity in the embryonic CNS, the branchial apparatus and in the limb buds. The S-enantiomer of EM12 was more strongly concentrated than the R-enantiomer in these areas. In the limb buds, the highest concentrations of radioactivity were observed in the periphery, sometimes at the very tip of the buds. Accumulation of radioactivity in limb buds and neural epithelium relative to other areas of the embryo was much more pronounced in the monkey than in the mouse. Future studies must demonstrate if this accumulation has implications for the mechanism of thalidomide teratogenesis in primate species.

Animals↗
PubMed1996

Identification of 9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol in human plasma after liver consumption.

Vitamin A is a well-established teratogen in several animal species. Case reports as well as a recent epidemiological study suggest that vitamin A intake in excess of 25,000 or 10,000 IU respectively, can result in retinoid-specific defects in the offspring. A single meal of liver contains, on the average, a 10- to 20-fold higher amount of vitamin A than what is already suspected to be teratogenic. To evaluate the risk of liver consumption during pregnancy, we have studied levels of vitamin A and a number of potentially active retinoid metabolites in plasma of ten healthy male volunteers following consumption of fried turkey liver (2 g raw weight/kg body weight). HPLC, UV spectroscopy and mass spectrometry were used for identification and quantitation of retinoids in plasma. As shown previously, vitamin A intake via liver consumption resulted in greatly increased plasma levels of 13-cis-retinoic acid (13-cis-RA) and 13-cis-4-oxo-RA, and low levels of all-trans-RA and all-trans-4-oxo-RA. In our present investigation 9-cis-RA, 9,13-di-cis-RA, and 14-hydroxy-4,14-retro-retinol (14-HRR) were identified for the first time in humans as physiological metabolites of vitamin A. 9-cis-RA is a potent teratogen as well as a high affinity ligand of retinoid receptors, and 14-HRR was previously shown to promote lymphocyte activation in vitro. The present study bears on the issue of a possible teratogenic risk of liver consumption, as active retinoids were identified in human plasma, and their levels could be related to previous human studies as well as to experimental studies in sensitive animal species.

Animals↗
PubMed1996

Intracellular pH monkey embryos at various stages of organogenesis estimated by dimethadione distribution.

Previous experiments using the transplacental distribution of 14C-DMO (5,5-dimethyloxazolidine-2,4-dione or commonly known as dimethadione) have demonstrated that the pH of rat embryos and fluids progressively decreases during organogenesis. The aim of the present experiments was to similarly evaluate pH changes during organogenesis in the cynomolgus monkey, which is a model for human embryogenesis. Using DMO quantitated by gas chromatography-mass spectrometry as opposed to the counting of radiolabelled compound, cynomolgus monkey embryos were determined to undergo a similar decrease in embryonic pHi over an approximately comparable period of development (Days 24-36 of gestation). The ratio of DMO in chorionic fluid to DMO in maternal plasma in the cynomolgus monkey also displayed a decrease with advancing gestational age indicative of a pH decrease. The DMO transplacental distribution was found to be significantly slower in the cynomolgus monkey than that in rodents. The present investigation indicates that the magnitude of the reduction of pH in embryonic cells and in extra-embryonic fluids over a period of organogenesis in the cynomolgus monkey is similar to the reduction detected in rodent embryos and fluids over a comparable developmental period, but the relative gradient between maternal blood pH and embryonic intracellular pH is different. The difference in the pH gradient between the two species may lead to differential transplacental distribution of exogenous and endogenous substances.

Analysis of Variance↗
PubMed1996

Methionine reduces the valproic acid-induced spina bifida rate in mice without altering valproic acid kinetics.

The antiepileptic drug valproic acid (VPA) is an established human teratogen causing spin bifida aperta. We recently developed a mouse model in which spina bifida aperta and occulta are induced with VPA. In a search for protection against neural tube defects, we investigated the effect of methionine on the incidence of VPA-induced spina bifida in the mouse. To induce spina bifida, we injected VPA (350 mg VPA-Na/kg body weight) subcutaneously three times on d 9 of gestation at 0, 6 and 12 h. In some mice, L-methionine (3 x 70 mg/kg body weight) was injected intraperitoneally 30 min before each VPA administration. When fetuses were examined on d 18, methionine treatment slightly reduced the VPA-induced spina bifida aperta rate from 5 to 1% (P > 0.05, no significant difference). The incidence of VPA-induced spina bifida occulta (90%) was significantly lower (28%) when methionine was also administered (P < 0.05). Examination on d 10 showed that the number of embryos in the mice administered VPA and methionine having an open neuroporus posterior was significantly lower than in mice administered VPA alone (P < 0.05). Pharmacokinetic studies indicated that VPA concentrations in maternal plasma and embryo did not differ between the two groups. Methionine reduces VPA-induced spina bifida in mice without altering VPA kinetics.

Animals↗
PubMed1996

Embryotoxic doses of vitamin A to rabbits result in low plasma but high embryonic concentrations of all-trans-retinoic acid: risk of vitamin A exposure in humans.

Retinoid pharmacokinetics were examined in plasma, placenta and embryos of gestational d 12 rabbits following application of an embryotoxic dosing regimen (10 mg retinyl palmitate/kg body wt per day from gestational d 7 to 12). Vehicle-treated or untreated rabbits served as controls. Physiological concentrations of all-trans-retinoic acid (all-trans-RA) and 13-cis-RA in rabbit plasma (5-8.33 nmol/L) were very close to the endogenous levels in human plasma. In addition, we identified endogenous all-trans-RA, 3,4-didehydroretinol and 3,4-didehydroretinoic acid in rabbit embryo. Following the last retinyl palmitate administration, apparent steady-state concentrations of all retinoids were reached in the examined compartments of rabbits. The major polar retinoid in plasma was 9, 13-di-cis-RA, but its embryonic concentrations were only about 6% of those in plasma. In the embryo, retinol and its esters were found at high concentrations; lower amounts of all-trans-4-oxo-RA and the newly identified 14-hydroxy-4, 14-retro-retinol could also be measured. Embryonic concentrations of all-trans-RA were about 100% higher than endogenous levels. The overall exposure of the embryo to this retinoid was, however, substantial. Embryonic area under the concentration time curve values strongly suggest that the embryotoxicity of the applied dosing regimen is mainly due to the action of all-trans-RA. A very remarkable finding of this study is the marginal increase of plasma concentrations of all-trans-RA over their endogenous levels, which is comparable to the human situation after vitamin A intake. This analogy indicates that high vitamin A intake may be associated with a higher risk for teratogenic effects in humans even in the absence of high elevation of plasma all-trans-RA levels.

Animals↗
PubMed1996

Topical retinaldehyde increases skin content of retinoic acid and exerts biologic activity in mouse skin.

Retinaldehyde, a natural metabolite of beta-carotene and retinol, has been proposed recently for topical use in humans. Because retinaldehyde does not bind to retinoid nuclear receptors, its biologic activity should result from enzymatic transformation by epidermal keratinocytes into ligands for these receptors, such as all-trans retinoic acid and 9-cis-retinoic acid. In this study, we analyzed by high performance liquid chromatography the type and amounts of tissue retinoids as well as several biologic activities resulting from topical application of either retinaldehyde or all-trans retinoic acid on mouse tail skin. Biologic activities of all-trans retinoic acid and retinaldehyde were qualitatively identical in metaplastic parameters (induction of orthokeratosis, reduction of keratin 65-kDa mRNA, increase in filaggrin and loricrin mRNAs) and hyperplastic parameters (increase in epidermal thickness, increase in bromodeoxyuridine (BrdU)-positive cells, increase in keratin 50-kDa mRNA, and reduction in keratin 70-kDa mRNA). Some quantitative differences, not all in favor of all-trans retinoic acid, were found in several indices. Cellular retinoic acid-binding protein II and cellular retinol-binding protein I mRNAs were increased by both topical retinaldehyde and all-trans retinoic acid. Whereas all-trans retinoic acid, 9-cis-retinoic acid, and 13-cis-retinoic acid were not detectable (limit 5 ng/g) in vehicle-treated skin, 0.05% retinaldehyde-treated skin contained 13 +/- 6.9 ng/g wet tissue of all-trans retinoic acid (mean +/- SD), 12.6 +/- 5.9 ng/g 13-cis-retinoic acid, and no 9-cis-retinoic acid. In contrast, 9-cis-retinoic acid was detectable in 0.05% of all-trans retinoic acid-treated skin, which also contained 25-fold more all-trans retinoic acid and 5-fold more 13-cis-retinoic acid than retinaldehyde-treated skin. Our results show that topical retinaldehyde is transformed in vivo into all-trans retinoic acid by mouse epidermis. The small amounts of ligand for retinoic acid nuclear receptors thus produced are sufficient to induce biologic effects similar to those resulting from the topical application of the ligand itself in much higher concentration.

Administration, Topical↗
PubMed1996

Metabolism of topical retinaldehyde and retinol by mouse skin in vivo: predominant formation of retinyl esters and identification of 14-hydroxy-4, 14-retro-retinol.

We have previously shown that retinaldehyde (RAL), a natural metabolite of beta-carotene and retinol (ROL), can be used topically in human skin and exerts biological activity; it may be a convenient way to deliver multipotential vitamin A activity in epidermis. RAL can be converted enzymatically into 2 pathways: one leads to ROL (and then retinyl esters), the other to retinoic acid (RA). The aim of the present study was 2-fold: (i) to see if RAL is metabolised in vivo when topically applied on mouse skin, and (ii) if so, to analyse the occurrence and relative importance of the 2 metabolic pathways as compared to ROL. We studied by HPLC the metabolites detectable in mouse tail skin upon topical application of RAL and ROL. As compared to vehicle-treated controls, RAL-treated mouse skin contained low amounts of all-trans RA and 13-cis-RA, whereas ROL content increased 10-fold and retinyl esters 30-fold after RAL application. As compared to RAL, ROL-treated mouse skin showed no detectable RA, slightly less retinyl esters but a significant amount of 14-hydroxy-4, 14-retro-ROL (14-HRR), a metabolite not previously reported in the skin. 14-HRR was the predominant polar metabolite of ROL. These data indicate that keratinocytes metabolise topical RAL, thus confirming the concept of using RAL as a precursor. Both pathways are used but in significantly different proportions. Thus, only a low proportion of RAL is metabolised into all-trans-RA, which may explain the low irritancy profile of topical RAL and supports the concept of a controlled delivery of ligands. That keratinocytes predominantly channel RAL into storage forms indicates that RAL should also be considered as a convenient way to load the epidermis with vitamin A. The detection of 14-HRR, a metabolite not previously reported in skin, that promotes growth of B Iymphocytes and activation of T Iymphocytes, suggests distinct potentials of topical ROL and RAL.

Administration, Topical↗
PubMed1996

Antiepileptic drug treatment in pregnancy: drug side effects in the neonate and neurological outcome.

Antiepileptic drugs taken by pregnant epileptic women are known human teratogens. They may also cause pharmacological side effects in the newborn, i.e. sedation and or withdrawal symptoms. We examined the relationship between the maternal antiepileptic therapy, neonatal behaviour and later neurological functions in infancy. The study comprised 40 children exposed in utero to a single antiepileptic drug (phenobarbitone, phenytoin, valproic acid). Valproic-acid-exposed children were the highest compromised, except for apathy, which was most profound in phenobarbitone-exposed neonates. Valproic acid serum concentrations at birth correlated with the degree of neonatal hyper-excitability and neurological dysfunction when children were re-examined 6 years later. We suggest that valproic acid may not only cause malformations but also cerebral dysfunction immediate and long term.

Anticonvulsants↗
PubMed1996

Plasma retinoids after topical use of retinaldehyde on human skin.

BACKGROUND: Retinaldehyde (RAL), a natural metabolite of beta-carotene and retinol (ROL), is tolerated by human skin after topical application. PURPOSE: To see if topical application of a large quantity of RAL on human skin is associated with a detectable alteration of constitutive levels of plasma retinoids resulting from metabolism of RAL in the skin. METHODS: Plasma retinoids [ROL, all-trans-retinoic acid (all-trans-RA), RAL, retinyl palmitate/oleate, 13-cis-RA and 4-oxo-13-cis-RA] were analyzed by high-pressure liquid chromatography. Determinations were done in 10 healthy male volunteers kept on a vitamin-A-poor diet before, during and after daily topical application of 7 mg of RAL to 40% of the body surface for 14 days. RESULTS: The introduction of a restricted vitamin A diet before RAL application resulted in a decrease in the plasma levels of ROL, all-trans-RA and retinyl palmitate/oleate. Topical application of RAL did not induce an alteration of the plasma levels of retinoid metabolites. No RAL was detectable in any of the plasma samples. CONCLUSION: The skin metabolism of topically applied RAL does not result in detectable alterations of constitutive levels of plasma retinoids in humans.

Administration, Topical↗
PubMed1995

Physiologically based pharmacokinetics of methoxyacetic acid: dose-effect considerations in C57BL/6 mice.

Methoxyacetic acid (MAA), a weak acid with a pKa of 3.57, was used to test the broad hypothesis that distribution of weak acids in maternal and fetal tissues is determined principally by the pKa of the acid and the pH values of tissue and fluid compartments and to examine tissue dose-teratogenesis relationships, as well as administered dose-teratogenesis relationships. Five related experimental studies were conducted in pregnant C57BL/6CrIBR mice: a conventional dose-response study of developmental toxicity and transplacental pharmacokinetics in mice, a second dose-response study in which reproductive outcomes in litters from individual dams were related to individual pharmacokinetic behavior, a protein-binding experiment, an embryo tissue localization study, and determination of pH in maternal and embryonic compartments after exposure to MAA. MAA was administered intraperitoneally at 9:00 a.m. on day 10 of gestation, at doses ranging from 88 to 164 mg/kg. Localization within the forelimb bud of the embryo, an MAA target site, was determined by computerized image analysis of the distribution of radiolabeled MAA. The kinetic predictions of a physiologically based model incorporating tissue pH values and MAA pKa agreed well with observed concentrations at the lowest dose. However, at intermediate and higher doses, concentrations in both maternal and embryonic tissues were consistently underestimated. MAA was bound neither to maternal plasma proteins nor to embryonic proteins. Intermediate and higher doses of MAA caused dose-dependent transient depressions in tissue pH, but these were not of sufficient duration to bring predicted tissue concentrations into congruence with the concentrations observed. Distribution of MAA within the forelimb bud was broadly consistent with the pH hypothesis, but MAA concentration was not increased in the distal postaxial sector that is the site of the precursor cells of the missing digits. Internal exposure to MAA, defined as the area under the maternal plasma or embryo concentration curve (AUC), was not proportional to administered dose, but AUC-response relationships generated by the group and individual dose-response studies were comparable. While AUC may be a useful measure of effective MAA dose, it cannot be accurately predicted at teratogenic doses of this agent by the model as it is presently structured.

Abnormalities, Drug-Induced↗
PubMed1995

Single versus multiple dose administration of all-trans-retinoic acid during organogenesis: differential metabolism and transplacental kinetics in rat and rabbit.

Standard teratogenicity testing is usually performed by administration of a test compound daily throughout an extended period of organogenesis (e.g., between Days 6 and 15 in rat and 6 and 18 in rabbit). On the other hand, single dose experiments during a specific period were often demonstrated to be more effective in unveiling a particular teratogenic effect. We have assessed here if toxicokinetics is an important factor for the interpretation of the differences between two administration regimens of all-trans-retinoic acid (all-trans-RA) in two species. The transplacental pharmacokinetics of a low teratogenic dose of all-trans-RA administered orally were compared in a single versus multiple dose regimen in both the Wistar rat and the Swiss hare rabbit. In both species, the single dose animals were treated on Gestational Day 12, while the multiple dose animals received daily doses from Gestational Days 7 through 12. Pharmacokinetic profiles were determined for maternal plasma and embryo after dosing on Gestational Day 12 (for both the single and multiple dose regimens) and analyzed by reverse-phase HPLC. The dose used for both species was 6 mg/kg body wt/day which has recently been reported to be a marginal to low teratogenic dose when administered daily throughout organogenesis. In both rat and rabbit, the AUC of all-trans-retinoic acid in maternal plasma was much reduced (factor of 9 in the rat, factor of 2 in the rabbit) after multiple application as compared to the single administration, presumably due to enzyme induction. A similar, but not as pronounced effect was also observed in the embryo of both species. This diminished effect in the embryo indicates a relative increase of placental transfer at the lower maternal plasma concentration observed after multiple dosing, which may possibly be due to an increased availability of binding sites such as cytosolic retinoic acid binding protein and nuclear receptors in the embryo. In the rat, also the metabolite levels were reduced, while in the rabbit, the metabolites of the 13-cis-configuration were concomitantly increased. Our results suggest that multiple administration of a drug such as retinoic acid, which induces its own elimination pathways, results in substantially lowered drug levels in maternal plasma and embryo.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗
PubMed1995

Validation of an in vitro teratology system using chiral substances: stereoselective teratogenicity of 4-yn-valproic acid in cultured mouse embryos.

In vitro systems are important for toxicity testing as well as for investigating the mechanism of action of xenobiotics. The validation of such in vitro systems is often incomplete and extrapolation to the in vivo situation is equivocal. In the present study, we studied the effects of enantiomers of an analogue of the antiepileptic drug valproic acid (VPA): R(+)- and S(-)-4-yn-VPA (R- and S-2-n-propyl-4-pentynoic acid), which have previously been shown to induce selective teratogenicity in mice after in vivo administration, in mouse whole-embryo culture (WEC). Aqueous solutions of the sodium salts of the pure R- and S-enantiomers as well as R,S-4-yn-VPA (racemic mixture) or VPA itself were added to the culture medium at 0, 0.075, 0.15, 0.3, 0.6, or 1.2 mmol/liter and embryos were evaluated 24 hr later. The S-4-yn-VPA enantiomer induced clear concentration-dependent dysmorphogenesis that was evident even at the lowest concentration. The primary anomalies were neural tube defects, erratic neural seams, blisters, and rotational defects. Embryolethality was observed at 1.2 mmol/liter. The R-4-yn-VPA enantiomer was neither embryotoxic nor dysmorphogenic at any tested concentration. The lack of biological activity over 24 hr in WEC with the R-enantiomer suggests also that, as previously shown in vivo, there was no racemization of this isomer to the more active S-enantiomer. The racemic mixture of R and S isomers appeared to be slightly more embryolethal and dysmorphogenic than VPA. Overall, the potency of the S-enantiomer was approximately four times that of VPA. Therefore, the rank order of the four chemicals tested was S(-) >> S(-), R(+) > VPA >>> R(+), which is in agreement with the effects observed in in vivo exposed mice. These data demonstrate a direct stereoselective effect of these compounds on the embryo. This is the first illustration of the stereoselectivity of a xenobiotic in the WEC in vitro test system. Pure and stable enantiomers, which induce stereoselective toxicity in vivo, are demonstrated to be valuable for validation of this in vitro system.

Animals↗
PubMed1995

Developmental stage-associated differences in the transplacental distribution of 13-cis- and all-trans-retinoic acid as well as their glucuronides in rats and mice.

In mice and rats, 13-cis-retinoic acid (13-cis-RA) has a much lower teratogenic potency than all-trans-retinoic acid (all-trans-RA). Previous studies on Gestational Day (GD) 11 or 12 (in mice or rats, respectively) showed a limited transfer of 13-cis-RA to the embryo, in contrast to the efficient transplacental passage of all-trans-RA. In the present study we examined if the distribution between maternal circulation and embryo of 13-cis- and all-trans-RA as well as their metabolites is different at gestational times when the placenta is developing from a choriovitelline to a chorioallantoic type. In the first experiment, 13-cis-RA was administered orally to pregnant rats (75 mg/kg/day) daily, from either GD 7 to 12 or 11 to 16. In the second experiment, 13-cis-RA or all-trans-RA was given orally to pregnant mice once on either GD 11 or 14, at two dose levels (10 and 100 mg/kg). HPLC analysis of plasma and embryo samples collected at various time points post-treatment showed that 13-cis-RA was predominantly metabolized to its beta-glucuronide (13-cis-RAG) while all-trans-RA was primarily biotransformed to all-trans-4-oxo-RA and to a lesser extent to all-trans-RAG. 13-cis-RA showed a more efficient transplacental passage to the rat embryo on GD 16 than on GD 12, as indicated by higher ratios of embryonic to maternal plasma concentrations (E/M concentration ratio) on GD 16 vs. GD 12 and its E/M ratio of area-under-the-concentration-time-curve values (E/M AUC ratio; twofold higher on GD 16 vs. GD 12). In the mouse, the E/M concentration ratio of 13-cis-RA was significantly higher on GD 14 than on GD 11 only at the low dose. On the other hand, all-trans-RA and all-trans-4-oxo-RA showed an efficient transfer to the mouse embryo at both gestational times. In both species, 13-cis- and all-trans-RAG embryonic concentrations on the later gestational days exceeded significantly the corresponding ones on the earlier gestational days which resulted in far higher E/M concentration and AUC ratios for each of these metabolites on the later vs earlier gestational days. This may result from a more efficient placental transfer of the RAGs during later gestational stages and/or higher capacity of the late embryo or other conceptal tissues to biotransform RAs in situ to their glucuronides.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Antiepileptic drugs alter endogenous retinoid concentrations: a possible mechanism of teratogenesis of anticonvulsant therapy.

The major antiepileptic drugs used for the control of seizures can induce developmental toxicity when administered during pregnancy. Vitamin A and retinoids are thought to control many processes of embryonic development including growth, differentiation and morphogenesis. We have therefore studied if the teratogenic action of antiepileptic agents could be mediated via alteration of the endogenous vitamin A--retinoid metabolism. Retinol and its oxidative metabolites all-trans-, 13-cis- and 13-cis-4-oxo-retinoic acid were measured in the plasma of 75 infants and children treated with various antiepileptic drugs for the control of seizures, and in 29 untreated controls of comparable age. Retinol levels increased with age, while the concentrations of retinoic acid compounds did not exhibit age-dependency. Valproic acid monotherapy increased retinol levels in the young age group and a trend toward increased retinol concentrations was also observed in all other patient groups. The plasma levels of the oxidative metabolites 13-cis- and 13-cis-4-oxo-retinoic acids were strongly decreased in all patient groups treated with phenytoin, phenobarbital, carbamazepine and ethosuximide, in combination with valproic acid, to levels which were below 1/3rd and 1/10th of corresponding control values, respectively. Little changes were observed with all-trans-retinoic acid except in one patient group treated with valproic acid/ethosuximide cotherapy where increased levels of this retinoid were found. Our study indicates that therapy with antiepileptic agents can have a profound effect on the endogenous retinoid metabolism. Because of the importance of retinoids for the signaling of crucial biological events during embryonic development, such altered retinoid metabolism may be highly significant in regard to antiepileptic drug teratogenesis.

Abnormalities, Drug-Induced↗
PubMed1995

Transplacental pharmacokinetics of a synthetic retinoid which is not bound by mouse embryonic cellular retinoic acid-binding protein.

Teratogenicity is a major side effect of retinoids, a class of compounds used in dermatology and oncology. The binding of retinoids to cellular retinoic acid-binding protein (CRABP) has been suggested to be important for the mechanism of retinoid embryopathy. Here data are presented on the transplacental pharmacokinetics of CD394 (4-[3-(1-adamantyl)-4-methoxybenzamido] benzoic acid) which does not bind to murine embryonic CRABP, although it is active in rat whole embryo culture and teratogenic in the rabbit in vivo. A single intragastric dose of CD394 (10 mg/kg) was administered to mice on day 11 of gestation. The extent of placental transfer of CD394, determined by HPLC, resembled more that of 13-cis-retinoic acid which also does not bind to CRABP, than that of the CRABP-binding all-trans-retinoic acid. CMax values of CD394 obtained after 1-2 h were: 1368 +/- 652 ng/ml for plasma, 203 +/- 132 ng/g for embryo and 856 +/- 563 ng/g for placenta. AUC (area-under-the-concentration-time-curve) values (0-12 h) were: 4319 ng x h/ml for plasma, 751 ng x h/g for embryo and 3163 ng x h/g for placenta. Thus, CD394 reached the embryo, although embryonic AUC values were less than one fifth of the maternal plasma AUC values. CD394 did not alter endogenous retinol concentrations in plasma, embryo, yolk sac or placenta. Our results indicate that CD394 reaches the embryo in vivo without binding to CRABP, although embryonic concentrations stayed well below plasma levels. This supports the opinion that binding to embryonic CRABP is not a prerequisite for reaching effective embryo concentrations and for the teratogenicity of retinoids.

Animals↗