Increase In Embryolethality Research Articles

Evaluation of the maternal and developmental toxicity of 6-methylmercaptopurine riboside in rats

Thiopurine prodrugs (azathioprine, AZA, and 6-mercaptopurine, 6MP) are embryotoxic to rodents and rabbits. Little is known about the developmental toxicity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine drug metabolite that is thought to mediate its liver toxicity. A limb bud assay found that 6MMPr impairs the in vitro morphogenetic differentiation of mouse limb extremities, being more potent than 6MP in the assay. This study evaluated the embryotoxicity of 6MMPr (0, 7.5, 15, 30 mg/kg bw sc) in rats after single-dose exposure in mid organogenesis (GD10). One group of pregnant rats was similarly treated with 6MP (15 mg/kg bw sc). After C-section (GD21), fetuses were weighed, and examined for external abnormalities. One third of each litter was examined for soft-tissue abnormalities while the remaining fetuses were cleared and stained for skeleton evaluation. 6MMPr caused a dose-dependent maternal weight loss followed by recovery before term pregnancy. Except for a nonsignificant increase in embryolethality and slight reduction in fetal weight at 30 mg/kg bw, no indication of embryotoxicity was noted at this dose or at lower doses of 6MMPr. In contrast, 6MP led to nearly 98 % of post-implantation losses in the presence of slight-to-mild maternal toxicity. These results are consistent with the notion that maternal treatment with 6MMPr affects embryo development, causing a nonsignificant increase in embryolethality and a slight reduction in fetal weight at 30 mg/kg bw. However, there was no increase in abnormalities at this dose, which was severely toxic to the dams, as reflected in the maternal weight gain data.

LGL Can Partition the Cortex of One-Cell Caenorhabditis elegans Embryos into Two Domains

Many metazoan cell types are polarized by asymmetric partitioning of the conserved PAR (PAR-3/PAR-6/PKC-3) complex. Cortical domains containing this PAR complex are counterbalanced by opposing domains of varying composition. The tumor-suppressor protein LGL facilitates asymmetric localization of cell fate determinants, in part through modulating the activity of the PAR complex. However, the mechanisms by which LGL acts to maintain a cortical domain remain unclear. Here we identify Caenorhabditis elegans LGL in a biochemical complex with PAR proteins, which localize to the anterior cortex. But LGL itself localizes to the posterior cortex. We show that increasing the amounts of LGL can restrict localization of the PAR complex to an anterior cortical domain, even in the absence of PAR-2. Importantly, LGL must be phosphorylated on conserved residues to exert this function. LGL and the PAR complex can maintain two cortical domains that are sufficient to partition cell fate determinants. Our data suggest a mechanism of "mutual elimination" in which an LGL phosphorylation cycle regulates association of the PAR complex with the cortex: binding of LGL to the PAR complex at the interface of the two domains stimulates its phosphorylation by PKC-3, and the whole complex leaves the cortex.

Anticonvulsant activity, teratogenicity and pharmacokinetics of novel valproyltaurinamide derivatives in mice.

1 The purpose of this study was to synthesize novel valproyltaurine (VTA) derivatives including valproyltaurinamide (VTD), N-methyl-valproyltaurinamide (M-VTD), N,N-dimethyl-valproyltaurinamide (DM-VTD) and N-isopropyl-valproyltaurinamide (I-VTD) and evaluate their structure-pharmacokinetic-pharmacodynamic relationships with respect to anticonvulsant activity and teratogenic potential. However, their hepatotoxic potential could not be evaluated. The metabolism and pharmacokinetics of these derivatives in mice were also studied. 2 VTA lacked anticonvulsant activity, but VTD, DM-VTD and I-VTD possessed anticonvulsant activity in the Frings audiogenic seizure susceptible mice (ED(50) values of 52, 134 and 126 mg kg(-1), respectively). 3 VTA did not have any adverse effect on the reproductive outcome in the Swiss Vancouver/Fnn mice following a single i.p. injection of 600 mg kg(-1) on gestational day (GD) 8.5. VTD (600 mg kg(-1) at GD 8.5) produced an increase in embryolethality, but unlike valproic acid, it did not induce congenital malformations. DM-VTD and I-VTD (600 mg kg(-1) at GD 8.5) produced a significant increase in the incidence of gross malformations. The incidence of birth defects increased when the length of the alkyl substituent or the degree of N-alkylation increased. 4 In mice, N-alkylated VTDs underwent metabolic N-dealkylation to VTD. DM-VTD was first biotransformed to M-VTD and subsequently to VTD. I-VTD's fraction metabolized to VTD was 29%. The observed metabolic pathways suggest that active metabolites may contribute to the anticonvulsant activity of the N-alkylated VTDs and reactive intermediates may be formed during their metabolism. In mice, VTD had five to 10 times lower clearance (CL), and three times longer half-life than I-VTD and DM-VTD, making it a more attractive compound than DM-VTD and I-VTD for further development. VTD's extent of brain penetration was only half that observed for the N-alkylated taurinamides suggesting that it has a higher intrinsic activity that DM-VTD and I-VTD. 5 In conclusion, from this series of compounds, although VTD caused embryolethality, this compound emerged as the most promising new antiepileptic drug, having a preclinical spectrum characterized by the highest anticonvulsant potential, lowest potential for teratogenicity and favorable pharmacokinetics.

Effect of oral dosing vehicles on the developmental toxicity of flubendazole in rats

Flubendazole was suspended in deionized water or olive oil and administered by gavage once daily to pregnant rats on Days 8–15 of pregnancy to examine if the embryolethal and teratogenic doses were affected by the vehicles used. Flubendazole in olive oil caused a statistically significant increase in embryolethality at doses of 7.83 mg/kg per day and higher, with complete resorption in all dams at 31.33 mg/kg per day. When flubendazole was suspended in deionized water, a significant increase in embryolethality occurred only at a maternal dose of 125.32 mg/kg per day. The proportion of litters with anomalous fetuses was significantly increased at doses of 31.33 mg/kg per day and above when flubendazole was administered in deionized water, but increased at doses at four times lower when flubendazole was administered as in olive oil. Administered as a single dose in olive oil on any one of Days 6–12 of pregnancy, a flubendazole dose of 31.33 mg/kg caused significant increases in embryolethality and decreased fetal body weights on Days 7–9, with an 82.7% incidence of embryolethality on Day 8, with complete resorption in 5 of the 8 dams. The critical periods for teratogenic effects were between Days 8 and 11 of pregnancy, with Day 9 being the most critical. Fetuses with gross, skeletal, or internal anomalies were seen in dams given a single dose of as low as 7.83 mg/kg.

Evaluation of the developmental toxicity of annatto in the rat

Annatto, a dye extracted from Bixa orellana seeds, is used as a color additive in butter, cheese and in a variety of other foods as well as in drugs and cosmetics. Toxicological data on annatto and on its main carotenoid pigment bixin are still scarce. In this study we evaluated the developmental toxicity of annatto (28% of bixin). Annatto (0, 31.2, 62.5, 125, 250 and 500 mg/kg body weight/day) was given by gavage to Wistar rats on days 6–15 of pregnancy. Ceasarean sections were performed on day 21. Implantations, living and dead fetuses and resorptions were recorded. Fetuses were weighed and examined for externally-visible anomalies. One-third of fetuses from each litter was examined for visceral anomalies by a microsectioning technique. The remaining fetuses were cleared and stained with Alizarin Red S for skeleton evaluation. No adverse effect of annatto on the mothers was noted. No increase in embryolethality and no reduction of fetal body weight were observed among annatto-exposed rats. Annatto did not induce any increase in the incidence of externally-visible, visceral or skeletal anomalies in the exposed offspring. These findings suggest that annatto was neither maternally toxic nor embryotoxic in the rat. Therefore, the no-observed-adverse-effect level (NOAEL) for annatto-induced maternal and developmental toxicity was 500 mg/kg body weight/day or greater (or ⩾140 mg bixin/kg body weight/day) by the oral route.

Histological Alterations in Gestational Day 13 Rat Embryos from Albendazole‐Treated Dams

AbstractGestational day (GD) 13 rat embryos from dams treated p. o. with albendazole (ABZ) on GD 10–12 were examined as an experimental model for the pathogenesis of embryotoxicity induced by benzimidazole compounds. Dosage levels were 0, 10, 20 and 30 mg/kg b. w. of ABZ. At 10 mg/kg b. w., most embryos appeared macroscopically normal. However, the histological examination of a representative sample showed subtle alterations including discrete necrotic foci in the lateral and rostral telencephalon, delayed differentiation of lens, increased wavyness of the notochord caudad to the forelimb bud and absent or poorly defined apical ectodermal ridge in the forelimb bud. At 20 and 30 mg/kg b. w. a sharp, dose‐related increase in embryolethality, developmental delays and macroscopic abnormalities were accompanied histologically by marked necrotic alterations of a number of embryonic tissues. The telencephalon and neural tube were severely affected, showing also a regeneration attempt through the disordered formation of tubules. Besides neuroectoderm, necrosis and tissue depletion were mostly evident in neural crest and mesoderm‐derived structures, such as branchial bars, limb buds and paraxial mesoderm. Lesions in the somites showed a head‐to‐tail increasing gradient; the sclerotomes were consistently more affected than the dermatomes. Wavyness of the notochord cephalad to the forelimb bud and fragmented/abnormal nuclei in the erythroblasts were also noted. Examination of embryos during in vivo organogenesis may be a useful tool for investigating the pathogenesis of embryotoxicity.

Lowering pH increases embryonic sensitivity to formate in whole embryo culture

The effects of formate exposure on mammalian embryo development were investigated using the rat whole embryo culture system as a model. Day 9.5 (presomite) rat embryos were explanted and cultured for 48 hr in rotating bottles containing rat serum with 0, 0.2, 0.4, 0.8, 1.2 or 1.6 mg sodium formate/ml culture medium at pH 8.13, 7.75, 7.00, 6.50 or 6.00 to determine whether the pH of the culture medium affects the in vitro developmental toxicity of formate. Several parameters of embryonic development decreased in the presence of decreasing pH, suggesting that altered pH alone could have a negative impact on embryo development. Exposure to 1.6 mg formate/ml affected protein concentration, somite number (SN), head length (HL), developmental score (DS), crown-rump length (CRL) and yolk-sac diameter of embryos at all pH levels. Formate became more toxic with decreasing pH of the culture media. There was an apparent pH-dependent increase in embryolethality at 1.6 mg formate/ml and 100% lethality at pH 6.00. The 1.2-mg/ml formate concentration affected DS, CR, HL and protein content at the pH 7.75 level whereas 0.8 mg formate/ml resulted in reduced DS, HL, CR, SN and protein content at pH levels of 7.00 and lower. At pH 6.5, embryos that were not exposed to formate were not significantly different from the other control groups except in reduced CR but at this pH, all exposure levels of formate resulted in microcephaly and reduction in embryonic protein as well as reduced CR. These data demonstrate that sensitivity to formate-induced embryo toxicity and dysmorphogenesis in whole embryo culture is increased in the presence of lower pH.

Teratogenicity of all‐trans retinoic acid during early embryonic development in the cynomolgus monkey (Macaca fascicularis)

The embryotoxic and teratogenic potential of all-trans retinoic acid was assessed following exposure prior to and during early organogenesis in the cynomolgus monkey (Macaca fascicularis). Sixteen pregnant females were orally administered all-trans retinoic acid (Tretinoin, Hoffmann-La Roche) once daily from GD 10-20 and twice daily from GD 21-24 at three different dosages, 5 (n = 9), 10 (n = 6) and 20 mg/kg (n = 1). Adverse clinical signs resembling hypervitaminosis A were observed in one animal at 5 mg/kg, in three animals at 10 mg/kg, and in the animal treated with 20 mg/kg all-trans retinoic acid. Maternal weight loss was observed in the 10- and 20-mg/kg groups. A dose-dependent increase in embryolethality was observed, with 22% (2/9), 50% (3/6), and 100% (1/1) occurring at 5, 10, and 20 mg/kg, respectively. The majority of embryonic deaths occurred between GD 16 and 20; the incidence of these early losses was higher than in historical and concurrent controls. No malformations, but a single growth-retarded fetus, was observed in the 5-mg/kg group. Craniofacial malformations, consisting of external ear defects, mandibular hypoplasia, cleft palate, and temporal bone abnormalities, were seen in three viable fetuses in the 10-mg/kg group. Skeletal variations were common to the majority (70%, 7/10) of viable fetuses in both dose groups and were increased relative to historical controls (32%, 25/77). Unlike previous studies with 13-cis-retinoic acid during the pre- and early organogenic stages of development (Hummler et al., Teratology 42:263-272, 1990), no thymic hypo- or aplasia or heart anomalies were observed, which may be attributable to the slightly longer 13-cis retinoic acid treatment period, i.e., GD 10-27. However, external ear and temporal bone defects were common to both all-trans and 13-cis retinoic acid. The similarity observed in the malformation syndrome induced by both all-trans and 13-cis retinoic acid in the cynomolgus monkey and 13-cis retinoic acid embryopathy in humans supports this macaque species as a model for further developmental toxicity studies of vitamin A-related compounds.

Vitamin A effects on fetal mouse cephalic acetylcholinesterase and choline acetyltransferase.

Pregnant C3H mice were exposed to vitamin A on the 7th to 10th day after copulation. Fetuses were examined at 18 or 19 days gestation for gross abnormalities, and assayed for total brain acetylcholinesterase (EC 3.1.1.7., AChE) activity and choline acetyltransferase (EC 2.3.1.6., ChAT) activity. A dose-related increase in embryolethality was found after treatment with vitamin A on day 9. Earlier administration of vitamin A on day 7 or 8 was associated with a higher fetal mortality. A marked increase in the incidence of gross abnormalities was found with an increase in dose of the teratogen. Administration of 5,000 IU vitamin A on day 9 was not associated with an alteration in fetal weight, brain weight or brain AChE activity. Similarly, there was no change in brain weight or AChE activity after 10,000 IU on day 9, although fetal weight was reduced. The administration of 10,000 IU on day 10, however, was associated with an elevated brain AChE activity, but no alteration in fetal weight, brain total protein content, or brain ChAT activity. The increased brain AChE activity was due predominantly to an increase in one of the isoenzymes. The results indicate that an alteration in brain AChE activity depends on the dose and timing of administration of vitamin A, and does not necessarily correlate with morphological abnormalities, ChAT activity, or brain protein content.

Amniotic fluid cholinesterase of valproate-induced exencephaly in the mouse: an animal model for prenatal diagnosis of neural tube defects

After a single administration of the antiepileptic drug valproic acid (VPA; i.p.:600 mg/kg) on day 8 of gestation in the mouse embryotoxicity and amniotic fluid (AF) cholinesterase (ChE) were evaluated on day 16 of gestation. VPA treatment induced an increase in embryolethality, neural tube defects (exencephaly), cleft palate, deformed vertebrae, open eyes, and a reduction in fetal weight. In VPA-exposed fetuses AF total ChE (TChE) activity of exencephalic fetuses was higher than that of normal fetuses. However, in 3 out of 110 normal fetuses of the control group TChE activity was found in the AF. There was no correlation between blood contamination of AF and its TChE activity, either in non-exencephalic control or treated embryos. Using ethopropazine as a "pseudo"-ChE inhibitor in vitro, the percentage of acetyl-ChE in blood-contaminated AF was similar to that of fetal rather than maternal serum, indicating that AF was contaminated with fetal and not with maternal blood. VPA-induced exencephaly in mice may provide an animal model to further investigate biochemical markers for prenatal diagnosis of neural tube defects.

Teratogenicity study of ammonium glycyrrhizinate in the Sprague-Dawley rat

Ammonium glycyrrhizinate (AG), a commercially used salt of glycyrrhizic acid, was administered in the drinking-water to Sprague-Dawley rats on days 7–17 of pregnancy. The actual intakes were 0, 21.33 ± 1.22, 238.75 ± 17.50 and 679.94 ± 69.87 mg AG/kg body weight/day for groups 0, 1, 2 and 3, respectively. AG caused polydipsia in the dams. Foetuses from the treated litters did not present an increase in external malformations, a decrease in weight or a decrease in the degree of ossification. However, there was a slight but significant increase in embryolethality and in the prevalence of external haemorrhages. Skeletal examination revealed a dose-related increase in minor anomalies, especially in the sternebral variants. Renal ectopy also increased significantly at the highest dose. These results indicate that the possible embryotoxicity of aromatizing compounds should be considered.

Embryotoxicity induced by diethylstilbestrol in vitro.

The embryotoxic potential of diethylstilbestrol (DES) was examined in a whole embryo culture system containing a P-450-dependent bioactivating system. Sprague-Dawley rat embryos were explanted on day 10 and cultured for 24 hours. Concentration-dependent effects of DES on embryonic growth parameters, viability, and embryotoxicity were observed. Concentrations of DES greater than 0.26 mM (final concentration) produced 100% embryolethality, while those below 0.15 mM were without significant effects. At a final concentration of 0.19 mM, DES produced only a slight increase in embryolethality. The same concentration elicited a marked increase in observed embryotoxicity, including prosencephalic hypoplasia, incomplete axial rotation, and open neural tubes. In addition, reductions in embryonic length, somite number, and protein and DNA content were observed. An exogenous P-450-dependent hepatic biotransforming (catechol-generating) system failed to alter either the incidence of observed toxic effects or measured growth parameters. Likewise, exposure of cultured embryos to 20% carbon monoxide (CO) failed to reduce DES-induced embryotoxicity, indicating a lack of participation of an endogenous P-450-dependent embryonic bioactivating system. Arachidonic acid (0.20 mM) and/or indomethacin (0.50 mM) also had no observable effect on DES-induced embryotoxicity, suggesting that prostaglandin synthase was not involved in the embryotoxic activity of DES, as has been proposed to explain its carcinogenic effect. The antioxidants N-acetylcysteine (1.14 mM) and alpha-tocopherol (0.08 mM) failed to protect against DES-induced embryotoxicity, while the anti-estrogen tamoxifen (up to 0.85 mM) actually enhanced this effect of DES in culture. The DES analogs Z,Z-dienestrol (DIES, 0.10 mM) and hexestrol (HES, 0.48 mM) were both embryotoxic in vitro. The presence of an exogenous P-450-dependent hepatic biotransforming system appeared to protect against HES-induced embryolethality but had little effect upon DIES-induced embryotoxicity. The results were consistent with a direct effect of DES independent of either estrogenicity or exogenously generated metabolites.

Embryotoxicity of cisplatin in rats and mice

Cisplatin [ cis-dichlorodiammineplatinum (II)], a broad spectrum antitumor agent, was tested for possible teratogenic and embryolethal effects on Wistar rats and Swiss Webster mice. Rats were given a single ip injection of 0.3, 1.0, 2.5, or 3.0 mg/kg cisplatin on Day 6, 8, 11, or 14 of gestation, whereas mice were given a single ip injection of 0.3, 3.0, 6.0, 8,0, or 13.0 mg/kg on Day 8 only. The embryonic LD50's in the rat were 2.88, 1.28, and 1.0 mg/kg for day 6, 8, and 11, respectively. There was no significant increase in embryolethality at any of the doses given on Day 14. The embryonic LD50 for mice was 5.24 mg/kg. An increase in the incidence of growth retardation or gross malformations was not discernable in the surviving fetuses with the number of dams used in this study. Cisplatin is highly embryolethal in rats and mice at dosages well below the adult therapeutic dosage in humans. This embryolethality is gestational stage-specific with the highest mortality corresponding to the period of rapid DNA replication in early organogenesis.

Induction of prenatal toxicity in the rat by diethylstilbestrol, zeranol, 3,4,3',4',-tetrachlorobiphenyl, cadmium, and lead.

A teratological study was conducted in pregnant Sprague-Dawley rats dosed orally with diethylstilbestrol (DES), zeranol (ZN), 3,3',4,4'-tetrachlorobiphenyl (4CB), cadmium, or lead on days 6-18 of gestation. Fetuses were examined on day 19 for growth retardation, resorption, gross malformations, and organ-level anomalies. Synthesis of protein, DNA, and proteoglycan in fetal limb cartilage was also studied by measuring the incorporation of labeled precursors in vitro. DES, ZN, and 4CB produced a dose-dependent increase in embryolethality. Treatment with DES caused an increase in cryptorchidism and edema, and reduced average fetal weight. Zeranol also decreased fetal weight, but was not teratogenic nor did it alter rates of synthesis of macromolecules in cartilage. 4CB caused severe intestinal lesions that were associated with accumulation of blood in the digestive tract and amniotic fluid. 4CB also decreased overall fetal size and total and ossified tibia lengths. Cadmium produced no malformations although incorporation of [3H]amino acids by limb cartilage was slightly increased. Lead was not teratogenic.

Embryotoxic effects in mice treated wtih 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

1. The purest sample of 2,4,5-T available is capable of producing embryotoxic effects in NMRI mice when given orally on day 6–15 of gestation. A cleft palate frequency exceeding that of controls is observed with doses higher than 20 mg/kg-about 1/6 of the LD 50. Reductions in fetal weight may be observed with doses as low as 10–15 mg/kg. No clear-cut increase in embryolethality over that of controls is seen with these doses. 2. Preliminary studies with 2,4,5-T-butyl ester showed this drug to have embryotoxic effects on mice comparable to those seen after administration of 2,4,5-T when given during day 6–15 of gestation. 3. Using high doses (150–300 mg/kg) cleft palates can be produced with a single oral dose of 2,4,5-T (A). A maximal teratogenic effect is seen when the drug is administered on day 12 or 13 of gestation. 4. Oral administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDBD) at doses exceeding 1 μg/kg (day 6–15 of gestation) also produces cleft palate in the fetuses of the same strain of mice. Embryomortality is drastically increased as compared with controls when doses of 4.5 μg/kg or more are given. 5. Cleft palate without a pronounced embryolethal effect can be produced in NMRI mice with high (20–50 μg/kg) single doses of TCDBD given between day 7 and 13 of pregnancy. A maximal teratogenic effect is obtained on day 11 of gestation but another peak in cleft palate frequency seems to occur when the drug is given on day 8 of pregnancy. 6. A potentiation of the teratogenic effects (cleft palate) of 2,4,5-T and TCDBD can be obtained when teratogenic doses of one of the substances are combined with threshold doses of the other. But for a clear-cut potentiation of the effect of 30–60 mg/kg 2,4,5-T more than 1.5 ppm TCDBD are required. A TCDBD content of <1 ppm, therefore, is unlikely to contribute to the embryotoxic effects of 2,4,5-T (cleft palate) seen in NMRI mice. 7. Some of the 2,4,5-T preparations available must contain other contaminants which exaggerate the teratogenic effect to some extent. Such contaminants have not necessarily to be present in trace amounts. The more pronounced teratogenic effect is not due to 2,4,5-T-phenol.