The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid,and not of isomerization to all-trans-retinoic acid

Previous studies suggested that the rabbit is much more susceptible to the teratogenic action of 13-cis-retinoic acid (13-cis-RA) than the mouse or the rat, while the teratogenicity of all-trans-RA was comparable in these species. In the present study we investigated if pharmacokinetics can explain these species- and structure-related differences. The embryotoxic and teratogenic potential of all-trans-retinoid acid (all-trans-RA) and 13-cis-RA were evaluated in the Swiss hare rabbit after oral administration of daily doses of the two drugs throughout organogenesis, from gestation day (GD) 6 to 18 (plug day=GD 0). All-trans-RA was given at dose levels of 0.7, 2 or 6 mg/kg body weight per day and 13-cis-RA at 3, 7.5 or 10 mg/kg per day. The doses needed to elicit a minimum teratogenic response were found to be 6 mg/kg per day for all-trans-RA and 10 mg/kg per day for 13-cis-RA. Using these doses, transplacental pharmacokinetics of all-trans- and 13-cis-RA were performed. Pregnant rabbits were treated once daily from GD 7 to 12 and plasma and embryo samples were collected for HPLC analysis at various time intervals after the final dose. The main plasma metabolites of all-trans-and 13-cis-RA were all-trans-β-glucuronide (all-trans-RAG) and 13-cis-4-oxo-RA, respectively. The elimination of 13-cis-RA and its metabolites from maternal plasma were much slower than of all-trans-RA resulting in accumulation of the 13-cis-isomers in plasma. Marked differences in the placental transfer of the two drugs and their metabolites were observed. All-trans-RA and all-trans-4-oxo-RA were efficiently transferred to the rabbit embryo, reaching concentrations similar to the plasma levels. On the contrary, the 13-cis-isomers reached the embryo to a lesser extent. Despite its limited placental transfer, a considerable embryonic exposure to 13-cis-RA and 13-cis-4-oxo-RA was noticed after treatment with isotretinoin, as indicated by their area-under-the-concentration-time-curve (AUC) values in the embryo, which were in the same range as the corresponding AUC value of all-trans-RA after treatment with the all-trans-isomer. Our results suggest that the high sensitivity of the rabbit to the teratogenic effects of 13-cis-RA can be attributed mainly to the 13-cis-isomers and not to isomerization to all-trans-RA. The significant exposure of the rabbit embryo to 13-cis-RA and its 4-oxo metabolite is a result of their very slow excretion rates from the maternal organism. Furthermore, this study supports the view that embryonic AUC values should be considered as the most suitable pharmacokinetic correlate to retinoid induced teratogenesis. Results of the pharmacokinetic study have been presented in part at the 20th Conference of the European Teratology Society, August 31–September 3, 1992, Würzburg, Germany, and have appeared in a short abstract (Tzimas et al. 1992) Supported by a grant from the Deutscher Akademischer Austauschdienst     

农药“渝—7802”对小鼠的致畸性实验研究

“渝-7802”是新研制的一种有机硫杀菌剂,作者对其进行了小鼠的致畸性及其胚胎毒性的研究。结果表明,即使高达37.00mg/kg的剂量(相当于1/3 LD_(50)),也未导致小鼠胎鼠的致畸效应及胚胎毒性。因此,认为该农药无致畸作用。     

Developmental and reproductive toxicity evaluation of toluene vapor in the rat II. Developmental toxicity

The developmental toxicity of toluene was evaluated via whole body inhalation exposure, in pregnant Sprague Dawley rats exposed to toluene (99.9% pure) from gestation day (GD) 6–15 inclusive, 6 h/day, at concentrations of 0, 250, 750, 1500 and 3000 ppm (0, 938, 2812, 5625 and 11250 mg/m³). Doses were selected from a preliminary study performed over a range of concentrations from 0 to 5000 ppm, in which maternal and fetal toxicity were observed at 2000 ppm and above. This study has been cited in various regulatory documents and is presented here to allow greater accessibility to results and conclusions.

Toluene induced clinical signs in pregnant dams (ataxia, hyper-responsivity, increased water intake, decreased food consumption) at 3000 ppm, ataxia and hyper-responsivity at 1500 ppm, and reduced maternal body weight gain at 1500 during the exposure period only and at 3000 ppm from initiation of exposure to GD20. At Caesarean section on GD20, no adverse effects on implantation, number and viability of fetuses, or fetal sex distribution were observed. Litter weight and mean fetal weight was reduced at 3000 ppm and mean fetal weight was reduced at 1500 ppm. Instances of reduced or unossified skeletal elements occurred at the same dose levels. Mean fetal weight was also reduced at 250 ppm but not at 750 ppm. Extensive statistical analysis of fetal body weight data support the conclusion that there is no toxicologically significant dose-related effect on fetal body weight at or below 750 ppm. Low incidences (≤2.5%) of various malformations occurred in the 250, 1500, and 3000 ppm groups; there was no increase in the incidence of specific or total malformations with increased exposure and thus these were not attributed to toluene.

In this Toluene study, the maternal toxicity NOAEL was 750 ppm with a defined maternal and developmental toxicity LOAEL of 1500 ppm.     

轧制油及其添加剂对大白鼠致畸胎实验研究

本文报告了轧制油及其添加剂十二醇、2,6-二叔丁基对甲酚对大白鼠致畸胎实验观察。本实验采用大白鼠经口灌胃染毒。结果表明,轧制油1213mg/kg剂量时导致胎鼠骨骼发育异常;十二醇307.5mg/kg剂量时出现多例死胎、胎鼠骨骼的严重畸形;2,6-二叔丁基对甲酚无致畸效应。     

硫酸铟生殖毒性的研究

采用常规致畸实验方法,给予妊娠大鼠432.80 mg/kg、135.25 mg/kg、43.28 mg/kg（1/5-1/50 LD50）硫酸铟经口灌胃染毒,以探讨硫酸铟的致畸效应以及其对雌性大鼠的生殖毒性作用。结果染毒组孕鼠孕末增重明显降低,吸收胎数均显著高于对照组,存活胎仔仅见个别有卷尾、波状肋、少肋等,未见到腭裂、脑外露、器官缺失等典型畸变;各染毒组成形胎仔的生长发育与对照组比较差异无统计学意义。提示硫酸铟具有明显早期胚胎毒性作用,可导致早期胚胎大量死亡并形成吸收胎。其致畸作用及对成形胎仔的生长发育影响并不明显。     

Use of low-molecular-weight heparin from the first trimester of pregnancy: a retrospective study of 111 consecutive pregnancies

BACKGROUND: During the first trimester of pregnancy, unfractionated heparin is the standard anticoagulant treatment for pregnant women at high risk of thrombosis. OBJECTIVE: To observe maternal and fetal tolerance for low-molecular-weight heparin begun in the first trimester of pregnancy. METHODS: Observational study conducted from 1 January 1997 to 31 May 2001. All patients began treatment before the 15th week of pregnancy. The outcome measures were the incidence and causality of adverse events in mother and fetus. RESULTS: The study included 97 patients (and 111 pregnancies) at very high risk for thrombosis. Seven fetal losses (6.3%) were observed: three early spontaneous abortions, three late spontaneous abortions and one medically indicated abortion. Twenty-five (22.5%) bleeding events occurred during pregnancy, seven (6.3%) of which required medical intervention: five curettages for first trimester spontaneous abortions, one late abortion at 21 weeks and one placental abruption at 25 weeks. Of nine (8.1%) primary postpartum hemorrhages involving a blood loss > or = 500 mL, three involved losses of 1000 mL or more and one required embolization of the uterine arteries. Five patients had thrombocytopenia, but none was treatment-related. Local cutaneous reactions occurred in 33 (29.7%) patients. Six (5.4%) maternal thromboembolic complications occurred during pregnancy or postpartum. At birth, two children had non-chromosomal congenital malformations (pyelectasia, cleft lip and palate). No fetal or neonatal complication was attributed to the treatment. CONCLUSION: The use of low-molecular-weight heparin (LMWH) for patients requiring anticoagulant treatment from the first trimester appears safe for mother and fetus.     

Evaluation of stereoselective anticonvulsant,teratogenic, and pharmacokinetic profile of valnoctylurea (capuride): A chiral stereoisomer of valproic acid urea derivative

Purpose: The purpose of this study was to evaluate the stereoselective anticonvulsant activity, neurotoxicity, pharmacokinetics, and teratogenic potential of two stereoisomers of valnoctylurea (VCU), a central nervous system (CNS)–active urea derivative of valnoctic acid, which is a constitutional isomer of valproic acid (VPA). Methods: VCU stereoisomers (2S,3S)‐VCU and (2R,3S)‐VCU were synthesized. Their anticonvulsant activity was determined and compared to VPA and racemic‐VCU in rats utilizing the maximal electroshock seizure (MES) and the subcutaneous pentylenetetrazole (scMet) tests. Neurotoxicity was determined in rats using the positional sense test, muscle tone test, and gait and stance test. The induction of neural tube defects (NTDs) by VCU stereoisomers was evaluated in a mouse strain highly susceptible to VPA‐induced teratogenicity. The pharmacokinetics of VCU was studied in a stereoselective manner following intraperitoneal (i.p.) administration to rats. Results: (2S,3S)‐VCU and (2R,3S)‐VCU median effective dose ED₅₀ values were 29 mg/kg [95% confidence interval (CI) = 8–60 mg/kg] and 42 mg/kg (95% CI = 36–51 mg/kg) (MES) and 22 mg/kg (95% CI = 13–51 mg/kg) and 12 mg/kg (95%CI = 7–21 mg/kg) (scMet), respectively. (2S,3S)‐VCU was more potent and had a wider safety margin (p < 0.05), defined as the protective index (PI = TD₅₀/ED₅₀), at both the MES (PI > 17) and scMet (PI > 23) tests than racemic‐VCU or (R,S)‐VCU (PI = 2.8 and 9.9, respectively). VCU stereoisomers caused NTDs at doses >4 times that of their anticonvulsant ED₅₀ values. At a dose of 112 mg/kg, (2R,3S)‐VCU was nonteratogenic and less embryotoxic than its stereoisomer (2S,3S)‐VCU. No stereoselective pharmacokinetics was observed following intraperitoneal dosing of racemic‐VCU to rats. VCU was mainly eliminated by metabolism with a half‐life of 2 h. Conclusions: VCU anticonvulsant activity and wide PI values make it a potential candidate for development as a new, potent antiepileptic drug (AED).     

Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies

Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2-/-) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2-/- and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.     

Valproic acid and its derivatives enhanced estrogenic activity but not androgenic activity in a structure dependent manner

Steroid hormones affect metabolic pathways and cellular functions. Valproic acid (VPA), used as antiepileptic drug, inhibits histone deacetylases and interacts with intracellular receptors. We analyzed the impact of VPA and VPA derivatives on activation of estrogen and androgen receptors (ER and AR) using reporter gene assays. VPA and its long-chain derivatives 2-(2-propynyl)-hexanoic acid [butyl-4-yn-VPA], 2-(2-propynyl)-heptanoic acid [S-pentyl-4-yn-VPA] and 2-(2-propynyl)-nonanoic acid [heptyl-4-yn-VPA] enhanced 17β-estradiol-induced ERα and ERβ activation partly synergistically with a structure–activity correlation. The extent of this effect regarding to ERα activation increased with prolongation of the aliphatic side chain. Regarding AR activation, VPA, S-pentyl-4-yn- and heptyl-4-yn-VPA slightly induced AR activity when tested alone. In combination with the AR agonist 5α-dihydrotestosterone, VPA, S-pentyl-4-yn- and heptyl-4-yn-VPA showed anti-androgenic effects without an apparent structural relation. Our results indicate that VPA and its derivatives affect estrogen signaling with a structural specificity, while the (anti-)androgenic effects of these compounds are not structurally correlated.     

Limb bud cell cultures for estimating the teratogenic potential of compounds

Mesenchyme cells, derived from embryonic limb buds, cultured at high cell density, multiply and differentiate into chondrocytes. Using alcian blue, a stain specific for cartilage proteoglycans, the degree of chondrogenesis can be visualized in the micromass cultures as well as quantified by extraction of the stain and spectrophotometric determination of its absorbance. In the presence of active retinoids chondrogenesis is concentration-dependently inhibited. For comparison of the activity of the various retinoids the concentration needed to reduce alcian blue staining by 50% was estimated. In order to validate whether the activity in limb bud cells can predict the teratogenic potential in vivo, the in vitro activity of 25 retinoids was compared with their in vivo teratogenicity observed mainly in rodents. For retinoids which were already in the biologically active form like those with a free carboxylic acid endgroup, there was a good quantitative correlation between the in vitro and in vivo activity. In contrast, the ethylester analog etretinate was slightly active and the ethylamine analog motretinide inactive in vitro but both were teratogenic in vivo. This finding may indicate that these retionoids were not metabolized to the active form in vitro. In conclusion, these results suggest that the limb bud cell culture system may be useful for a preliminary testing to select non-teratogenic retinoids. For the risk assessment in humans, however, the in vitro result should be verified in animal studies.