Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.     

Inhibition of isotretinoin teratogenicity by acetylsalicylic acid pretreatment in mice.

Although isotretinoin (ITR) has been suggested to cause malformations via cytopathic effects on embryonic cells, the molecular mechanisms of ITR cytotoxicity in teratogenesis are not clear. Since ITR undergoes metabolism by prostaglandin synthase to a potentially cytotoxic peroxyl free radical, the possible role of prostaglandin synthase metabolism as a modulator of ITR teratogenicity was evaluated. Craniofacial and limb abnormalities were noted in fetuses on day 18.5 of gestation following administration of ITR to pregnant CD-1 mice in a three dose regimen of 100 mg/kg at 4 hr intervals on day 10.5 of gestation (plug day = day 0.5 of gestation). Mice were also treated with acetylsalicylic acid (ASA), an irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase, at doses of 20 and 60 mg/kg body weight 2 hr prior to each ITR dose. ASA pretreatment of mice receiving ITR treatment showed a dose-dependent decrease in the overall incidence of malformations, number of defects per fetus, and the incidence of specific craniofacial and limb defects. Equivalent doses of ASA given to control mice did not cause malformations or alter the incidence of resorptions. These results demonstrate that ASA is able to ameliorate the teratogenic effects of ITR observed in fetal mice near term and indicate that prostaglandin metabolism could play a mechanistic role in ITR teratogenicity.     

Strain differences in teratogenic susceptibility to trypan blue between WM and BDIX rat strains

Female rats of WM (Wistar-Mishima)/Nem strain were mated with WM/Nem (group W) or BDIX/Nem males (group WB), and BDIX/Nem females were mated with BDIX/Nem (group B) or WM/Nem males (group BW). On day 8 of gestation, pregnant females were treated intraperitoneally with 1% aqueous solution of trypan blue at a dose of between 20 and 120 mg/kg of body weight. On day 20 of gestation, fetuses were examined for external, visceral, and skeletal malformations. In group W, fetal mortality increased dose dependently at doses higher than 20 mg/kg, and incidences of external, visceral, and skeletal malformations were significantly higher than control at doses of 30 mg/kg and more. In group B, fetal mortality and the incidence of external malformations were significantly higher than control only in the group treated with 120 mg/kg, and no significant increase of visceral and skeletal malformations was shown. It was confirmed that BDIX strain is much more resistant to trypan blue teratogenicity than WM strain. In group BW, nearly the same teratogenic effects were shown as in group W in terms of fetal mortality and incidence of malformations. However, in group WB, teratogenic effects were not so remarkable as in group BW, suggesting patroclinous effects in teratogenic susceptibility to trypan blue. In group BW, sex differences in teratogenic susceptibility were found; male fetuses were more susceptible to trypan blue than females.     

Lack of teratogenicity of trans-2-ene-valproic acid compared to valproic acid in rats

The teratogenicity of trans-2-ene-valproic acid (300 and 400 mg/kg) was compared with that of valproic acid (VPA; 300 mg/kg) and controls (corn oil) administered by gavage to Sprague-Dawley CD rats on embryonic (E) days 7-18. At the 300 mg/kg dose, trans-2-ene-VPA produced no change in maternal weight, number of implantations, proportion of resorptions, proportion of malformations, or fetal weight. By contrast, the same dose of VPA (300 mg/kg) reduced maternal weight during gestation, increased malformations (12.0% vs. 0.7% in controls), and reduced fetal body weight by 25.1%. An even higher dose of trans-2-ene-VPA (400 mg/kg) produced a reduction in maternal body weight during treatment and reduced fetal body weight (by 7.9%), but did not increase resorptions or malformations in the fetuses. On day E18, maternal serum drug concentrations of VPA were higher in the VPA-treated group compared with those of trans-2-ene-VPA in the trans-2-ene-VPA-treated groups at 1 hr posttreatment. At 6 hr posttreatment the reverse was seen. trans-2-ene-VPA may be absorbed more rapidly and distributed differently than VPA. Overall, the data support the view that trans-2-ene-VPA at equal or higher doses than VPA is not teratogenic in rats.     

Action of the phenothiazine derivative methophenazine on prenatal development in rats.

Single doses of 100-400 mg/kg or multiple doses of 10 or 50 mg/kg of the phenothiazine derivative methophenzaine were given per osto Wistar rats at various times on the 7th-14th days fo gestation and the fetuses examined near term. Results indicated that methophenazine was mainly embryolethal when administered on the 8th-11th days, and was teratogenic at later times, producing types of malformations that depended on the day of treatment, the most susceptible period being the 13th and 14th days of gestation. Teratogenicity occurred only when the dosages were highly toxic to the pregnant rats. Ribovlavin given ip on the 14th day significantly reduced the embryolethal but not the teratogenic action of methophenazine.     

Prior exposure to indole-3-carbinol decreases the incidence of specific cyclophosphamide-induced developmental defects in mice

BACKGROUND: Indole-3-carbinol (I3C) is a product of the hydrolysis of glucobrassicin that is found in cruciferous vegetables. I3C can intervene in toxic processes that are mediated by oxidative mechanisms because it possesses the chemical and pharmacokinetic properties necessary to provide a free radical trap. Cyclophosphamide (CP) is a bifunctional alkylating agent known to produce DNA damage and to cause developmental toxicity, including malformations, in laboratory animals. METHODS: Pregnant CD-1 mice were given a 100 mg/kg dose of I3C 24 or 48 hr before administration of 20 mg/kg CP on gestation day 10 (GD 10). Controls were given the vehicle (DMSO), I3C, or CP. This regimen was carried out to determine if I3C could protect against the developmental toxicity of alkylating agents, such as CP. Dams were sacrificed on GD 17 and their litters were examined for adverse effects. RESULTS: Treatment with I3C 48 hr before CP administration was associated with decreased fetal limb and tail malformations. Limb malformation incidences were reduced from 42% litters affected in the CP control to 16% in the I3C/CP 48-hr treatment group, and tail malformations were reduced from 45% in the CP control to 16% in the I3C/CP 48-hr treatment group, indicating a protective effect of prior exposure to I3C. I3C given 24 hr before CP had no significant protective effect, while having an apparently adverse consequence with regard to the incidence of talipes. CONCLUSIONS: Exposure of a developing mammal to indole-3-carbinol before exposure to cyclophosphamide during organogenesis can influence the teratogenicity of cyclophosphamide.     

Pharmacokinetic assessment of teratologically effective concentrations of an endogenous retinoic acid metabolite

Retinoic acid is a natural vitamin A derivative that undergoes oxidative metabolism in the body to yield several metabolites, which apparently represent the products of a detoxification pathway. To assess if such metabolic conversions diminished teratogenic potency, one of the major metabolites (4-oxo-all-trans-retinoic acid) was tested for its teratogenic activity in pregnant ICR mice and further investigated for its pharmacokinetic features to determine if it accumulated in the embryo in concentrations sufficient to elicit a teratogenic response. Administration of single oral doses (10, 25, 50, or 100 mg/kg) of the compound to ICR mice on day 11 of gestation (plug day = day 0) produced dose-dependent frequencies of serious fetal anomalies of the type usually associated with the use of retinoic acid and other retinoids. The metabolite was equivalent in teratogenic potency to retinoic acid, and, in the instance of cleft palate frequency, it was even more active. Concentrations of 4-oxo-all-trans-retinoic acid and its 13-cis isomer were measured in the maternal plasma and whole embryos at 30 min to 10 hr after administration of the lowest (10 mg/kg) and the highest (100 mg/kg) teratogenic dose of 4-oxo-all-trans-retinoic acid by means of high-performance liquid chromatography methodology. Distribution of the compound in the maternal system and transfer to the embryo occurred rapidly with either dose. Peak concentration in the maternal plasma and the embryo persisted for 3-4 hr after the higher dose but not with the lower dose; however, elimination kinetics for the two dose levels were similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different embryo-fetal toxicity effects for three VLA-4 antagonists

BACKGROUND: VLA‐4 (Very late antigen 4, integrin α₄β₁) plays an important role in cell‐cell interactions that are critical for development. Homozygous null knockouts of the α₄subunit of VLA‐4 or VCAM‐1 (cell surface ligand to VLA‐4) in mice result in abnormal placental and cardiac development and embryo lethality. Objectives of the current study were to assess and compare the teratogenic potential of three VLA‐4 antagonists. METHODS: IVL745, HMR1031, and IVL984 were each evaluated by the subcutaneous route in standard embryo‐fetal developmental toxicity studies in rats and rabbits. IVL984 was also evaluated in mice. Fetuses were examined externally, viscerally, and skeletally. RESULTS: IVL745 did not cause significant maternal or fetal effects at doses up to 100 or 250 mg/kg/day in rats or rabbits, respectively. HMR1031 treatment resulted in marked maternal toxicity and slight fetal toxicity at the highest tested doses of 200 and 75 mg/kg/day in rats and rabbits, respectively. HMR1031 embryo‐fetal effects consisted of slightly lower body weight and crown‐rump length in rats and minor sternebral defects in rabbits. IVL984 treatment resulted in minimal maternal effects at doses up to 40, 15, and 100 mg/kg/day in rats, rabbits, and mice, respectively (excluding abortions in rabbits). However, marked developmental effects were observed at the lowest tested IVL984 doses, 1, 0.2, and 3 mg/kg/day in rats, rabbits, and mice, respectively. IVL984 embryo‐fetal effects consisted of increased total post‐implantation loss due to early resorptions and high incidences of cardiac malformations and skeletal malformations and/or variations. Notably, spiral septal defects were observed in up to 76% of rat fetuses and up to 58% of rabbit fetuses. CONCLUSIONS: Dramatic differences in teratogenic potential were observed: IVL745 was not teratogenic, HMR1031 caused slight embryo‐fetal effects at maternally‐toxic doses, and IVL984 was a potent teratogen at doses where direct maternal toxicity was limited to abortions in rabbits. Prominent effects of IVL984 included embryo lethality and cardiac malformations including spiral septal defects in three species. Birth Defects Res B 71:55–68, 2004. © 2004 Wiley‐Liss, Inc.     

A teratogenicity study on hydroxyurea and diphenylhydantoin in cats

Hydroxyurea, an antitumor drug and known teratogen in rat, miniature swine and dog, and diphenylhydantoin, a teratogen in mouse and rat, were assessed for teratogenic effects in cat. Pregnancies were induced, by synchronizing gonadotropin-stimulated estrus and ovulation with natural copulations. Hydroxyurea at 50 or 100 mg/kg, and sodium diphenylhydantoin at 1 or 2 mg/kg dosages, were administered orally in single daily doses from gestation days 10-22. Appropriate controls given empty capsules, were included for each drug. Cats were necropsied on gestation day 43. Fetuses were examined for external, visceral and skeletal malformations. Hydroxyurea at 50 mg/kg dose produced a low teratogenic activity and at 100 mg/kg a high incidence of non-pregnancy and resorptions with, consequently, fewer live fetuses. Diphenylhydantoin gave no clear evidence of teratogenicity at any test dose but was embryolethal at the maternally toxic dose of 2 mg/kg. So far, studies conducted suggest that the cat is a useful species for screening drugs and chemicals for their teratogenic potential.     

Cocaine as a cause of congenital malformations of vascular origin: experimental evidence in the rat

Cocaine hydrochloride was administered to pregnant Sprague-Dawley rats as a single intraperitoneal dose or as two doses 1-4 hours apart. A single dose administered on day 16 of gestation was teratogenic in a dose-dependent manner, with 40 mg/kg being a no-effect dose and 50 mg/kg the lowest teratogenic dose; 80 mg/kg was lethal to the dam. Forty-eight hours after exposure to a teratogenic dose on day 16 of pregnancy, the fetuses showed severe hemorrhage and edema in the their extremities, particularly the footplates, tail, genital tubercle, and upper lip/nose. When the fetuses were examined on day 21 of gestation, the main externally visible malformations were reduction deformities of the limbs and tail. When two doses of cocaine were administered 1-4 hours apart, the incidence of affected fetuses increased as the time interval between the two doses decreased. Two doses of cocaine administered 2 hours apart were not teratogenic on day 9, 10, 11, 12, 13, or 14 of gestation but did induce reduction deformities on days 15, 16, 17, 18, or 19. The same dose administered 1 hour apart was teratogenic on days 14-19. In general, cocaine administration on gestational days 14, 15, or 16 induced more severe and more widespread hemorrhage and edema than administration on days 17, 18, or 19. In the latter cases, damage was restricted to the distal parts of the hindlimb digits and the tail. The results show that in the rat cocaine is only teratogenic during the late organogenic or postorganogenic period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryo‐Fetal Developmental Toxicity Studies with Pregabalin in Mice and Rabbits

Pregabalin was evaluated for potential developmental toxicity in mice and rabbits. Pregabalin was administered once daily by oral gavage to female albino mice (500, 1250, or 2500 mg/kg) and New Zealand White rabbits (250, 500, or 1250 mg/kg) during organogenesis (gestation day 6 through 15 [mice] or 6 through 20 [rabbits]). Fetuses were evaluated for viability, growth, and morphological development. Pregabalin administration to mice did not induce maternal or developmental toxicity at doses up to 2500 mg/kg, which was associated with a maternal plasma exposure (AUC_0–24) of 3790 μg?hr/ml, ≥30 times the expected human exposure at the maximum recommended daily dose (MRD; 600 mg/day). In rabbits, treatment‐related clinical signs occurred at all doses (AUC_0–24 of 1397, 2023, and 4803 μg?hr/ml at 250, 500, and 1250 mg/kg, respectively). Maternal toxicity was evident at all doses and included ataxia, hypoactivity, and cool to touch. In addition, abortion and females euthanized moribund with total resorption occurred at 1250 mg/kg. There were no treatment‐related malformations at any dose. At 1250 mg/kg, compared with study and historical controls, the percentage of fetuses with retarded ossification was significantly increased and the mean number of ossification sites was decreased, which correlated with decreased fetal and placental weights, consistent with in utero growth retardation. Therefore, the no‐effect dose for developmental toxicity in rabbits was 500 mg/kg, which produced systemic exposure approximately 16‐times human exposure at the MRD. These findings indicate that pregabalin, at the highest dose tested, was not teratogenic in mice or rabbits     

Evaluation of developmental toxicity induced by anticholinesterase insecticide,diazinon in female rats

Developmental toxicities, including birth defects, are significant public health problems. This study was planned to assess the cholinergic and developmental potentials of diazinon that is widely used as an organophosphate insecticide. Pregnant female Sprague‐Dawley rats were given diazinon orally at doses of 0, 1.9, 3.8, and 7.6 mg/kg body weight (b.w.)/day on gestation days 6 to 15. Maternal brain acetylcholinesterase activities, measured on gestation day20, were significantly decreased at 3.8 and 7.6 mg/kg b.w./day, but fetal acetylcholinesterase activity was not altered. Maternal toxicities, as evidenced by cholinergic symptoms including diarrhea, tremors, weakness, salivation, and decreased activities, were observed at the 3.8 and 7.6 mg/kg b.w./day dose groups. Net gravid uterine weight was decreased at a dose of 7.6 mg/kg b.w./day. No maternal effects were apparent in the 1.9 mg/kg b.w./day dose group. Maternal toxicity at a dose of 3.8 mg/kg b.w./day did not induce fetotoxicity or teratogeneicity. However, 7.6 mg/kg b.w./day doses significantly resulted in fetal toxicity and malformations in addition to maternal toxicity in animals. In conclusion, teratogenic disorders only outlined by doses that produced marked maternal toxicity. Since the malformations were not morphologically related, they were considered to be secondary to maternal toxicity; hence, the malformations were not related to cholinesterase inhibition. Birth Defects Res (Part B) 92:534–542, 2011. © 2011 Wiley Periodicals, Inc.     

Teratogenic effects of a single oral administration of methylmercuric chloride in mice.

The teratogenic effects of methylmercuric chloride (MMC) given orally as a single dose to pregnant ICR mice on day 10 of gestation were examined. The doses tested were 25, 20, 15 and 10 mg/kg. Controls received distilled water orally. Each group consisted of 20 females. Fetuses were taken on day 18 of gestation for teratological study. The number of resorbed or dead embryos was moderately increased in the 25 mg/kg group. Fetuses from dams given 25, 20 and 15 mg/kg MMC weighed significantly less than those in the control group. Many fetuses with malformations were observed in the treated groups; cleft palate occurred in 100, 58.6 and 28.0% of fetuses from dams given 25, 20 and 15 mg/kg MMC, respectively (statistically significant). Hydronephrosis appeared in 23.8 and 18.5% of fetuses from dams given 25 and 20 mg/kg MMC, respectively (statistically significant). Skeletal variations, incomplete ossification of sternebrae, for example, were also observed in the treated groups. These results indicate that MMC is teratogenic so far as cleft palate is concerned and embryotoxic in ICR mice.     

Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme a reductase

Mevinolin is a fungal metabolite, and in the hydroxyacid form, mevinolinic acid, it is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase, an enzyme essential in cholesterol biosynthesis. Oral administration of 800 mg/kg/day of mevinolin to rats from days 6 through 17 of gestation produced fetal malformations of the vertebrae and ribs in 29% of the litters, and there was a treatment-related increase in the incidence of gastroschisis. Mevinolinic acid at 60 and 90 mg/kg/day also produced fetal malformations of the vertebrae and ribs, and these teratogenic manifestations were markedly suppressed by coadministration of the product of HMG-Co A reductase, mevalonic acid, at a dosage level of 500 mg/kg b.i.d. A diet supplemented with 0.5% or 1.0% cholesterol had no effect on the teratogenicity of mevinolinic acid. Teratology studies in rats with a dihydroxyheptanoic acid derivative of mevinolin, a compound 1/700 as potent as mevinolinic acid as an inhibitor of HMG-Co A reductase, and dihydromevinolinic acid, an inhibitor of this enzyme comparable in activity to mevinolinic acid, indicated that the teratogenicity of these compounds was related to their relative enzyme inhibitory activity. The dihydroxyheptanoic acid derivative was not teratogenic at doses as high as 150 mg/kg b.i.d.; in contrast, when dihydromevinolinic acid was administered at 50 and 100 mg/kg/day, its potency as a teratogenic agent was comparable to that of mevinolinic acid. These studies demonstrated that inhibitors of HMG-CoA reductase produced terata in rats and that the teratogenic effects could be antagonized by coadministration of the enzyme product, mevalonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of formaldehyde and Sonacide (potentiated acid glutaraldehyde) on embryo and fetal development in mice

Pregnant outbred albino mice were given formaldehyde or Sonacide (potentiated acid glutaraldehyde) by gavage on days 6--15 of gestation. The mice were killed on day 18, the general health and reproductive status of the dam evaluated, and the fetuses examined and processed in order to characterize external, visceral, and skeletal malformations. Although formaldehyde (stock solution containing 12--15% methanol as a preservative) was lethal to 22 of 34 dams treated with 185 mg/kg/day, and one of 35 dams treated with 148 mg/kg/day, these doses did not produce statistically significant (two-sided p < 0.05 versus controls) teratogenic effects in the fetuses of the surviving dams. Sonacide was also judged not to be teratogenic to the mice employed in this study, in spite of the fact that relatively high doses were employed. The highest doses of Sonacide studied (5.0 ml/kg/day, which is equivalent to 100 mg/kg/day of glutaraldehyde) killed 19 of 35 dams and caused a significant reduction in the mean weight gain of the surviving mothers. In addition, this dose produced a significant increase in the number of stunted fetuses.     

Efficiency of Coleus aromaticus extract in modifying cyclophosphamide and mitomycin-C induced clastogenicity in mouse bone marrow cells

The anticlastogenic potency of the ethanolic extract of a medicinal plant, C. aromaticus was investigated by taking bone marrow chromosomal aberration assay and micronucleus (MN) test as the test parameters. Swiss albino mice were fed orally with different doses (10,15, 25, 50 and 100 mg/kg body weight) of ethanolic extract for 7 days and on the 7th day, two doses each of anticancer drugs cyclophosphamide (CP; 25 and 50 mg/kg body weight) and mitomycin-C (MMC; 4 and 8 mg/kg body weight) were injected, ip, to different groups of animals. Bone marrow MN preparations were made at 24 and 48 hr time intervals. Coleus extract reduced CP and MMC induced MN and lower doses of the extract were found to be more effective than higher doses. The effective doses of extract in MN test were selected to study the anticlastogenic effects against CP (25 and 50 mg/kg body weight) and MMC (2 and 4 mg/kg body weight) induced chromosomal aberrations. The results indicate the protective effect of C. aromaticus against CP and MMC induced cytogenetic damage.     

Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

Induction of malformations in the cynomolgus monkey with 13-cis retinoic acid

The embryotoxic and teratogenic potential of 13-cis retinoic acid was assessed in the cynomolgus macaque (Macaca fascicularis). A total of 41 animals was orally administered 13-cis retinoic acid in four sequential experiments. In Exp. 1 three dose levels, 2, 10, and 25 mg/kg, were administered on gestational day (GD) 18-28; 5 mg/kg was administered as an equally divided dose twice daily in Exp. 2 and 3 on GD 21-24 and on GD 25-27, respectively; in Exp. 4 the drug was administered at 2.5 mg/kg once daily from GD 10 to 25 and twice daily (2 x 2.5 mg/kg) on GD 26 and 27. Maternal death and toxicity, manifested as reduction in maternal weight and food consumption, and diarrhea, was observed in Exp. 1 in all three dose groups. No significant maternal toxicity was observed in the treatment groups in Exp. 2, 3, and 4 or in the control group. The primary manifestation of developmental toxicity was embryolethality in Exp. 1 and 2. The incidence of embryonic deaths in Exp. 3 was comparable to the historical controls. No malformations in GD 100 fetuses were observed in Exp. 1, 2, and 3. In Exp. 4, five of seven fetuses (71%) had malformations of both external ears, four of seven fetuses (57%) exhibited hypo- or aplasia of the thymus, and two of seven (29%) had malformations (transposition of the great vessels, ventricular septal defect) of the heart. The teratogenic dose for the cynomolgus monkey in the present study was lower than that reported for all other experimental species. Although central nervous system and craniofacial defects were not observed, the incidence of ear and thymus defects was similar to that reported for the human. The cardiovascular defects resembled those reported clinically, but the incidence was lower in the cynomolgus monkey. The similarity in teratogenic sensitivity to humans supports the use of the monkey as a model for developmental toxicity studies of vitamin A-related compounds.     

Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice

BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.