tretinoin and Abnormalities--Drug-Induced

Prenatal exposure to ethanol results in fetal alcohol spectrum disorder (FASD), a syndrome characterised by a broad range of clinical manifestations including craniofacial dysmorphologies and neurological defects. The characterisation of the mechanisms by which ethanol exerts its teratogenic effects is difficult due to the pleiotropic nature of its actions. Different experimental model systems have been employed to investigate the aetiology of FASD. Here, I will review studies using these different model organisms that have helped to elucidate how ethanol causes the craniofacial abnormalities characteristic of FASD. In these studies, ethanol was found to impair the prechordal plate-an important embryonic signalling centre-during gastrulation and to negatively affect the induction, migration and survival of the neural crest, a cell population that generates the cartilage and most of the bones of the skull. At the cellular level, ethanol appears to inhibit Sonic hedgehog signalling, alter levels of retionoic acid activity, trigger a Ca(2+)-CamKII-dependent pathway that antagonises WNT signalling, affect cytoskeletal dynamics and increase oxidative stress. Embryos of the domestic chick Gallus gallus domesticus have played a central role in developing a working model for the effects of ethanol on craniofacial development because they are easily accessible and because key steps in craniofacial development are particularly well established in the avian embryo. I will finish this review by highlighting some potential future avenues of fetal alcohol research.

Topics: Abnormalities, Drug-Induced; Animals; Calcium Signaling; Chick Embryo; Craniofacial Abnormalities; Disease Models, Animal; Embryo, Mammalian; Embryo, Nonmammalian; Endoderm; Ethanol; Face; Fetal Alcohol Spectrum Disorders; Gastrula; Genetic Predisposition to Disease; Hedgehog Proteins; Holoprosencephaly; Humans; Maxillofacial Development; Neural Crest; Signal Transduction; Skull; Species Specificity; Tretinoin; Wnt Signaling Pathway

Topics: Abnormalities, Drug-Induced; Adult; Alitretinoin; Anti-Inflammatory Agents; Capsules; Clinical Trials as Topic; Contraception; Contraindications; Drug Administration Routes; Drug Costs; Eczema; Female; France; Headache; Humans; Hyperlipidemias; Immunologic Factors; Male; Pregnancy; Retinoid X Receptors; Social Security; Tretinoin

Azole fungicides are widely used in agriculture and in human mycosis. Their antifungal activity is based on their ability to inhibit CYP51, a key enzyme in the formation of fungal wall. Several azole fungicides tested in laboratory animals have been found to possess a common teratogenic potential to induce facial, axial skeleton, and limb defects. The mechanism of the teratogenic effect has been hypothesized to be related to the capability of these substances to alter embryonic retinoic acid catabolism. Although a number of human epidemiological studies were unable to demonstrate a definite relationship between azole exposure during pregnancy and birth defects, some case reports indicate a possible teratogenic effect of high doses of azoles in humans. Because of their common mechanism of action, azole fungicides should be regarded with caution for use in pregnant women.

Topics: Abnormalities, Drug-Induced; Animals; Antifungal Agents; Embryonic Development; Female; Humans; Imidazoles; Pregnancy; Teratogens; Tretinoin; Triazoles

Because all-trans retinoic acid (atRA) is teratogenic in all species tested and many of the specific defects induced are common across the phylogenetic spectrum, it would be logical to predict that murine strain differences in teratology to this agent are minimal. However, for specific defects, strain susceptibilities are vastly different. Studies with atRA have shown stark differences between C57BL/6 and SWV mouse strains in susceptibility to postaxial forelimb ectrodactyly and ectopic hindlimb formation, with the C57 strain being more susceptible for both defects. Various approaches were used to determine why these strains differ in susceptibility, but the mechanisms remain unknown. Hindlimb duplications were hypothesized to be caused by the formation of ectopic posterior body axes. For forelimb ectrodactyly, a locus on chromosome 11, Rafar, has linkage to the strain difference, and mRNA localization has shown that specific genes (Fgf8, Dlx3, Bmp4, and Sp8) in the postaxial preAER (prior to formation of the apical ectodermal ridge) of the developing limb bud (the site of the defect) were downregulated hours after atRA administration more in the susceptible C57 than in the SWV strain. Because both atRA and divalent cadmium induce postaxial forelimb ectrodactyly (right-sided predominance) at a high rate in C57BL/6 and low in the SWV strain, there is debate as to whether they share a common mechanism. These teratogens cause a greater-than-additive level of forelimb ectrodactyly when coadministered at low doses, but cadmium does not induce ectopic hindlimb formation. The hypothesis is that these agents have separate molecular pathologic pathways that converge to perturb a common anatomic structure.

Topics: Abnormalities, Drug-Induced; Animals; Bone Morphogenetic Protein 4; Cadmium Chloride; DNA-Binding Proteins; Ectromelia; Female; Fibroblast Growth Factor 8; Fingers; Forelimb; Hand Deformities, Congenital; Hindlimb; Homeodomain Proteins; Limb Deformities, Congenital; Male; Mice; Mice, Inbred C57BL; Quantitative Trait Loci; RNA, Messenger; Teratogens; Transcription Factors; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Cricetinae; Dose-Response Relationship, Drug; Morphogenesis; Tretinoin

This review of the central nervous system (CNS) and behavioral teratology of the retinoids over the last 50 years is a commemorative retrospective organized by decade to show the prominent research focus within each period and the most salient findings. In the 1960s, research focused on the gross CNS malformations associated with exposure and the delineation of dose-response and stage-specific responses in rodent models. Relevant scientific events before and during the 1960s are also discussed to provide the zeitgeist in which the field of neurobehavioral teratology emerged in the 1970s. During this period, studies demonstrated that adverse effects on postnatal behavior could be produced in animals exposed to doses of vitamin A lower than those that were teratogenic or impacted growth. Work during the 1980s showed an overrepresentation of behavioral studies focused on the reliability of screening methods, while the marked effects of human exposure were illustrated in children born to women treated with isotretinoin during pregnancy. The human catastrophe invigorated research during the 1990s, a period when technological advances allowed more elegant examinations of the developing CNS, of biochemical, cellular, and molecular developmental events and regulatory actions, and of the effects of direct genetic manipulations. Likewise, research in the 1990s reflected a reinvigoration of research in neurobehavioral teratology evinced in studies that used animal models to try to better understand human vulnerability. These foci continued in the 2000-2010 period while examinations of the role of retinoids in brain development and lifelong functioning became increasingly sophisticated and broader in scope. This review of the work on retinoids also provides a lens on the more general ontogeny of the field of neurobehavioral teratology. Birth Defects Research (Part A), 2010. © 2010 Wiley-Liss, Inc.

Topics: Abnormalities, Drug-Induced; Animals; Brain; Central Nervous System; Female; History, 20th Century; History, 21st Century; Humans; Hypervitaminosis A; Isotretinoin; Male; Pregnancy; Retinoids; Teratogens; Teratology; Tretinoin

Management of the pregnant patient with acute promyelocytic leukemia (APL) is a challenge. Immediate treatment of APL is critical, as it is an oncologic emergency, with a high risk of morbidity and mortality associated with disseminated intravascular coagulation. However, administration of chemotherapy and differentiating agents in pregnancy is controversial because of potential teratogenic effects. In addition, complications associated with APL, including retinoic acid syndrome, add to the complexity of management. To better understand how to manage this complex patient care situation, we searched the PubMed database (January 1972-May 2008) for English-language articles about maternal and fetal outcomes resulting from APL treatment during pregnancy. A total of 42 cases from 35 articles were identified: 12 first-trimester, 21 second-trimester, and 9 third-trimester cases. The most commonly administered agents were all-trans-retinoic acid (ATRA), anthracyclines, and antimetabolites. Complete remission was reported in 35 (83%) of 42 patients. Administration of ATRA or chemotherapy in the first trimester was associated with an increased risk of fetal malformations and spontaneous abortion, whereas administration in the second and third trimesters was associated with relatively favorable fetal outcomes. The overall treatment of the pregnant patient with APL should include a discussion about pregnancy termination, especially if APL is diagnosed in the first trimester. If the pregnancy is to continue, then the appropriate chemotherapy regimen needs to be determined. Frequent fetal monitoring, along with aggressive management of potential APL-related complications, is necessary to allow for optimal maternal and fetal outcomes.

Topics: Abnormalities, Drug-Induced; Abortion, Therapeutic; Antineoplastic Agents; Female; Humans; Leukemia, Promyelocytic, Acute; Pregnancy; Pregnancy Complications, Hematologic; Pregnancy Complications, Neoplastic; Pregnancy Outcome; Tretinoin

All-trans retinoic acid (atRA) is the transcriptionally active product of vitamin A and induces gene expression via specific receptors at nM concentrations. Essential enzymes that regulate the local levels of atRA are the CYP26 members of the cytochrome P450 family, which catabolize atRA. Compounds that have been designed to inhibit these enzymes are known as Retinoic Acid Metabolic Blocking Agents (RAMBAs). Treatment with these compounds will raise endogenous atRA levels and may be therapeutic for the treatment of diseases that respond to high atRA concentrations, including several types of cancer as well as skin conditions such as psoriasis and acne. This review describes the mechanism of action of the RAMBAs and discusses the potential side effects of these compounds. atRA is highly teratogenic and the potential teratogenicity of the RAMBAs is described by comparison with the abnormalities resulting from null mutation of individual CYP26 genes. The possible effects of RAMBAs on the adult brain are also described that have the potential for harm but, in the right circumstances, may also be beneficial.

Topics: Abnormalities, Drug-Induced; Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Humans; Retinoic Acid 4-Hydroxylase; Teratology; Tetralones; Tretinoin

Histone deacetylases (HDACs) are nuclear and cytoplasmic enzymes that deacetylate a number of substrates, of which histones are the best known and described in the literature. HDACs are present in eukaryotic and bacteria cells, and are fundamental for a number of cellular functions, including correct gene expression. Surprisingly, only up to 20% of the whole genome is controlled by HDACs, but key processes for survival, proliferation, and differentiation have been strictly linked to HDAC enzyme functioning. The use of HDAC inhibitors (HDACi) has been proposed for the treatment of neoplastic diseases. Their effectiveness has been suggested for a number of liquid and solid tumors, particularly acute promyelocytic leukemia (APL). The role of HDACs in embryo development is currently under investigation. Published data indicate knockout phenotype analysis to be of particular interest, in which a number of HDACs play a key role during development. Little data have been published on the effects of HDACi on embryonic development, although for valproic acid (VPA), literature from the 1980s described its teratogenic effects in experimental animals and humans. To date, all tested HDACi have shown teratogenic effects similar to those described for VPA when tested in zebrafish, Xenopus laevis, and mice. HDACs were also able to alter embryo development in invertebrates and plants. A model, similar to that proposed in APL, involving retinoic acid receptors (RAR) and tissue specific Hox gene expression, is suggested to explain the HDAC effects on embryo development.

Topics: Abnormalities, Drug-Induced; Animals; Embryonic Development; Enzyme Inhibitors; Female; Genes, Homeobox; Histone Deacetylase Inhibitors; Humans; Models, Biological; Neoplasms; Pregnancy; Receptors, Retinoic Acid; Teratogens; Tretinoin

Acute promyelocytic leukemia (APL) is characterized by onset at a young age and a life-threatening hemorrhagic diathesis, which is attributed to a disseminated intravascular coagulation (DIC)-like coagulopathy. The discovery of all-trans-retinoic acid has changed the course of APL treatment by reducing the onset of DIC and inducing a complete and durable remission in more than 90% of patients. The occurrence of APL during pregnancy is not a frequent event, but the management of these patients raises many therapeutic and ethical dilemmas and requires a careful clinical case evaluation of fetal and maternal risk, coagulation status, the parents' wishes, and therapeutic options. Here we describe 3 patients with APL diagnosed during pregnancy. Clinical data and the therapeutic approaches are presented. In the discussion, we analyze clinical decisions and therapeutic options and compare our cases with those found in the literature.

Topics: Abnormalities, Drug-Induced; Abortion, Therapeutic; Adult; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Case Management; Cesarean Section; Fatal Outcome; Female; Gestational Age; Humans; Idarubicin; Infant, Newborn; Leukemia, Promyelocytic, Acute; Male; Pregnancy; Pregnancy Complications, Neoplastic; Remission Induction; Risk; Safety; Tretinoin

This paper reviews the teratogenicity of isotretinoin in regard to aspects of species variation, toxicokinetics, and metabolism. Particular emphasis is given to the hypothesis that most effects of isotretinoin (13-cis-retinoic acid) are mediated by isomerization to the all-trans-retinoic acid. This mechanism of action would provide a basis for the understanding of species differences and the extrapolation of experimental results to the human situation and thus improve drug development. The insensitive species (rat, mouse) eliminate the drug rapidly through detoxification to the beta-glucuronide; also, placental transfer is limited in these species. On the other hand, in sensitive species (primates), the drug is predominantly metabolized to the active 13-cis-4-oxo-retinoic acid; placental transfer is more extensive here. The beta-glucuronides showed limited placental transfer in all species examined; these metabolites exhibited very low, if any, measurable concentrations in the human. The 13-cis-retinoic acid is not appreciably bound to cellular retinoid-binding proteins or nuclear receptors and exhibits low tissue distribution and placental transfer. Its access to the nucleus may be extensive. Because of the long half life of 13-cis-retinoic acid, continuous isomerization results in significant area under the concentration-time curve levels of all-trans-retinoic acid in the mouse, monkey and the human; the all-trans-retinoic acid formed is extensively distributed across the placenta and may be an important factor that contributes to the teratogenic potency of 13-cis-retinoic acid. Isomerization cannot explain the teratogenic effects of 13-cis-retinoic acid in the rat and rabbit. It is concluded that the high teratogenic activity of isotretinoin in sensitive species (human, monkey) is related to slow elimination of the 13-cis-isomer, to metabolism to the 4-oxo-derivative, to increased placental transfer, to continuous isomerization and significant exposure of the target tissue to all-trans-retinoic acid; and to lack of binding to cytoplasmic retinoid binding proteins that could possibly result in ready access to the nucleus.

Topics: Abnormalities, Drug-Induced; Animals; Dermatologic Agents; Female; Half-Life; Humans; Isomerism; Isotretinoin; Keratolytic Agents; Maternal-Fetal Exchange; Mice; Pregnancy; Primates; Rabbits; Rats; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Female; Fetus; Humans; Infant, Newborn; Isotretinoin; Neural Crest; Pregnancy; Prenatal Exposure Delayed Effects; Tretinoin

A still unresolved public health concern is that excessive vitamin A intake, like vitamin A deficiency, possibly causes birth defects not only in animals but also in man. Due to the low incidence of possibly vitamin A-related malformations in man, available data cannot convincingly define the upper safe limit of periconceptional vitamin A intake. Direct human intervention studies are not feasible for ethical reasons. Therefore, a novel approach in addressing this issue was chosen by combining teratogenicity data from a validated animal model with data on systemic exposure to vitamin A and its major metabolites in female volunteers. In a study in pregnant women endogenous plasma concentrations of vitamin A metabolites during early pregnancy ranged from 0.26 to 7.72 ng/ml. Since they did not cause any foetal malformations, retinoid plasma levels in this range can be considered non-teratogenic. Results of a trial in non-pregnant women document that daily oral vitamin A supplements of 4000, 10,000 and 30,000 IU given for 3 weeks were in the range or slightly above the range of endogenous plasma levels seen in early pregnancy. Even after a 3-week treatment with 30,000 IU/day, peak plasma levels of retinoic acid and isotretinoin were within or just slightly above the range of their physiological levels. In cynomolgus monkeys (average weight: 3-4 kg), a NOAEL (no observed adverse effect level) of 7500 IU per kg body weight and a LOAEL (lowest observed adverse effect level) for developmental toxicity of 20,000 IU/kg was found. Considering these results in the cynomolgus monkey, a dose of 30,000 IU/day should also be considered as non-teratogenic in man.

Topics: Abnormalities, Drug-Induced; Animals; Clinical Trials as Topic; Dietary Supplements; Female; Humans; Isotretinoin; Liver; Macaca fascicularis; Pregnancy; Tretinoin; Vitamin A

Apart from oral drug treatment, drug therapy in acne vulgaris comprises topical treatment with agents with a primarily keratolytic action (e.g. tretinoin and benzoylperoxide), and with antibiotics (clindamycin, erythromycin, and erythromycin-zinc complex). The acne grade in the particular patient usually determines the selection of the preferred route of administration, viz. topical or oral, or a combination of both, and topical treatment is usually preferred in mild to moderate acne. The fact that a topically applied compound may also become systemically available to a quantifiable extent, is not generally considered.. The present paper reviews the clinical data on transdermal uptake of anti-acne agents in man, also with respect to their relevance for daily clinical practice.. The majority of published data on transdermal penetration of topical anti-acne agents focuses on the retinoid tretinoin, and on the antimicrobial agent clindamycin. This interest emerges from the fact that these agents have been associated with embryotoxicity/teratogenicity, and pseudomembranous colitis, respectively. For both compounds the extent of systemic availability after topical application is low, viz. 5-7% and 8%, respectively, at its highest. The height and variability in endogenous retinoid levels is very likely to outweigh any contribution of exogenously applied tretinoin, but a full consensus on the safe use of topical tretinoin in pregnancy is still lacking. With respect to clindamycin, the suggested association between its topical use and the occurrence of pseudomembranous colitis appears not to be of clinical relevance. In order to reduce systemic exposure to clindamycin as much as possible, topical application of clindamycin phosphate is to be preferred over clindamycin hydrochloride salt. Regarding other topical anti-acne agents, it has been suggested that topical zinc-erythromycin is to be preferred over erythromycin, both from clinical efficacy and safety viewpoints. With respect to the currently used compounds like benzoylperoxide, azelaic acid, and adapalene, available clinical pharmacokinetic data are scarce, and significant safety concerns did not emerge as yet.. The limited transdermal uptake of topical anti-acne agents underpins their safe use in daily clinical practice. With respect to topical retinoids, formal consensus is lacking regarding their use in pregnancy.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Administration, Cutaneous; Administration, Oral; Anti-Bacterial Agents; Clindamycin; Dermatologic Agents; Drug Therapy, Combination; Enterocolitis, Pseudomembranous; Female; Humans; Keratolytic Agents; Pregnancy; Skin Absorption; Tretinoin

Tretinoin has been thoroughly evaluated for its potential as an embryofetal developmental toxicant. Oral tretinoin produces developmental anomalies in animal models; the minimal teratogenic dose is consistently 2.5 to 10 mg/kg. In contrast, topical application does not induce developmental malformations in laboratory animals. A structurally related compound, isotretinoin, is a potent toxicant in humans and animals; the lowest systemic dose that induces fetal anomalies varies more than 100-fold depending on the model. Oral isotretinoin is a more potent developmental toxicant than oral tretinoin in monkeys. Between-drug differences in the metabolism and transplacental transfer of the two retinoids account for the differences in toxicant potency. Pharmacokinetic studies reveal that absorption of tretinoin from the skin is poor and yields maternal plasma concentrations below the developmentally toxic threshold established after oral administration. Analysis of outcomes of developmental toxicology and pharmacokinetic studies suggests that the human risk of fetal anomalies is negligible after therapeutic application of topical tretinoin.

Topics: Abnormalities, Drug-Induced; Animals; Humans; Isotretinoin; Keratolytic Agents; Tretinoin

Vitamin A (retinol) is a fat-soluble vitamin that is necessary for cell growth and differentiation. Excess vitamin A has been associated with teratogenic effects in animals and humans. Because vitamin A deficiency is very uncommon in the industrialized world, the current recommendation is that routine vitamin A supplementation is not necessary. If vitamin A supplements are used, they should be limited to less than 5,000 IU per day. Systemic administration of the naturally occurring retinoid tretinoin has been associated with birth defects, fetal resorption, and stillbirths in animals; however, topical use is not associated with increased birth defects and is classified as a category B drug during pregnancy. The synthetic retinoids isotretinoin, etretinate, and etretin are strictly contraindicated during pregnancy (category X) as they have been associated with teratogenic syndromes in humans. In addition, owing to the prolonged elimination half-life of aromatic retinoids, effective contraception should be used for at least 2 years following discontinuation of treatment with these drugs.

Topics: Abnormalities, Drug-Induced; Acitretin; Etretinate; Female; Humans; Isotretinoin; Pregnancy; Retinoids; Tretinoin; Vitamin A

Retinoyl beta-glucuronide is a naturally occurring, biologically active metabolite of vitamin A. Although retinoyl beta-glucuronide is regarded as a detoxification product of retinoic acid, it plays several roles in the functions of vitamin A. It can serve as a source of retinoic acid, and it may be a vehicle for transport of retinoic acid to target tissues. Topically applied retinoyl beta-glucuronide is comparable in efficacy to retinoic acid in the treatment of acne in humans, without the same side effects. Retinoyl beta-glucuronide may or may not be teratogenic, depending on the mode of administration and the species in which it is used. It may be a valuable therapeutic compound for the treatment of skin disorders and certain types of cancers.

Topics: Abnormalities, Drug-Induced; Animals; Female; Intestinal Absorption; Pregnancy; Tretinoin; Vitamin A

In this paper I have tried to bring together work that highlights the role of homeobox genes in generating craniofacial form. I review both normal and disrupted embryogenesis and ask whether mis-expression of the homeobox genes outside their normal domains could be contributing to congenital facial abnormalities arising from either genetic or teratogenic actions. Experimentally generated transgenic mice carrying loss- or gain-of-function mutations in homeobox genes, in combination with their normal expression patterns, have allowed us to compile and test models of embryonic specification based around a Hox/homeobox code. These models form the basis on which the functional questions are considered. There are four major sections covering different experimental approaches designed to ectopically induce homeobox genes in the head. Transgenic mice, where heterologous promoters drive a given Hox gene in the head, have shown that the more posteriorly expressed Hox genes tend to have a significant effect only on the skull bones of mesodermal origin whereas those normally expressed more anteriorly, in the hindbrain and branchial arches, can affect more anterior branchial arch and neural crest-derived structures. Manipulation experiments which can induce homeobox genes in small, localised regions of the facial precursors show clear and dramatic effects of this expression on facial development. Null mutations in predicted repressors of Hox gene expression, however, do not appear to give rise to substantial craniofacial abnormalities. Retinoic acid, on the other hand, is well known for its teratogenic actions and its ability to induce Hox gene expression. Evidence is now accumulating that at least some of its teratogenic actions may be mediated by its regulation of the Hox and other homeobox genes in the head.

Topics: Abnormalities, Drug-Induced; Animals; Brain; Chick Embryo; Craniofacial Abnormalities; Face; Facial Bones; Gene Expression Regulation, Developmental; Genes, Homeobox; Head; Homeodomain Proteins; Mice; Mice, Knockout; Mice, Transgenic; Morphogenesis; Neural Crest; Organ Specificity; Promoter Regions, Genetic; Recombinant Fusion Proteins; Tretinoin

Both a conceptual and a practical borderland between teratology and mutagenesis is early embryogenesis, the period between fertilization and gastrulation. Radiation and a variety of chemicals adversely affect the early conceptus leading to in utero mortality and malformations. The post-fertilization period of susceptibility differs from exposures of gametes, the later producing excessive pre- and peri-implantational death and low rates of fetal anomalies predominated by growth retardation. In contrast mutagen exposure of the zygote induces peri-implantational death, pan-gestational death and fetal anomalies predominated by hydrops, abdominal wall defects, and eye aberrations. The mechanism for this pathology remains unclear. These same agents produce a broader range of phenotypic anomalies during the remainder of pre-gastrulation development with anomalies overlapping those induced during organogenesis. Retinoic acid and 5-azacytidine administered prior to gastrulation produce novel malformation syndromes indicative of gene expression modification. The rates and types of defects from mutagen treatment of both gametes and the early conceptus contrast with those resulting from embryonic treatment during organogenesis, and the mechanisms are likely to differ. The pre-gastrulation period has not been explored to the extent reported during gametogenesis or organogenesis. Pre-gastrulation teratology is a new area of investigation with relevance both to reproductive toxicology and to mammalian developmental biology.

Topics: Abnormalities, Drug-Induced; Animals; Azacitidine; Blastocyst; Embryonic and Fetal Development; Gene Expression Regulation, Developmental; Mammals; Mice; Mutagens; Teratogens; Tretinoin; Xenobiotics; Zygote

Retinoic acid-dependent homeobox Hoxb-1 gene expression offers an unanticipated example of both a positive and a negative transcriptional activity of RA, as exerted at different times during embryogenesis. A paradigm for the transduction of positive and negative signaling is the discovery that retinoic acid response elements (RAREs), positioned in the 3' enhancer and 5' promoter of the Hoxb-1 gene, may function respectively as positive and negative regulators, thereby permitting a diffused (early positive) as well as segmentally specified and limited (late negative) expression of the gene. This molecular action of retinoic acid may provide a mechanism for our understanding of normal embryogenesis and of the interference with this process by ectopic retinoic acid, thereby leading to teratogenesis.

Topics: Abnormalities, Drug-Induced; Animals; Gene Expression Regulation; Genes, Homeobox; Humans; Regulatory Sequences, Nucleic Acid; Repetitive Sequences, Nucleic Acid; Tretinoin

DiGeorge syndrome (DGS) is a developmental defect which associates hypo- or aplasia of the thymus and parathyroids, facial dysmorphism and conotruncal cardiac malformations. The etiological factor in a great majority of DGS patients is monosomy for the 22q11.2 chromosomal region either through a large interstitial deletion of that region (inherited or de novo) or through an unbalanced translocation involving chromosome 22. In one instance, a balanced translocation of chromosome 22 was associated with a DGS phenotype. Extensive analyses of this region of chromosome 22 has led to the obtention of precise physical maps of the corresponding genomic region, to the cloning of the balanced translocation breakpoint and to the isolation of different genes from the minimal critical deleted region.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Chromosome Aberrations; Chromosome Deletion; Chromosome Disorders; Chromosome Mapping; Chromosomes, Human, Pair 22; DiGeorge Syndrome; Female; Genes; Humans; Infant, Newborn; Male; Pregnancy; Pregnancy Complications; Rats; Syndrome; Translocation, Genetic; Tretinoin

Limb outgrowth is arguably the most fundamental aspect of limb development. It begins with the emergence of buds from the embryo's lateral body wall. More rapid growth along each bud's proximodistal axis than along its anteroposterior or dorsoventral axes yields the limb's basic elongated shape. Many processes that generate refinements of this basic limb form are now being explored at the molecular level. Yet, there remain gaps in our understanding of basic limb outgrowth itself. This review examines the pivotal role of the apical ectodermal ridge in promoting and maintaining limb-bud outgrowth. It discusses the interplay between the apical ectodermal ridge and the subridge limb mesenchyme. It examines evidence that the pattern of limb anomalies in the offspring of mothers exposed to exogenous retinoids such as retinoic acid strongly suggests interference with apical ectodermal ridge function. It covers evidence that cellular retinoic acid-binding protein in the cytoplasm of the cells under the apical ectodermal ridge limits the effects of retinoic acid, a potent retinoid and teratogen, on retinoic acid-driven gene transcription. It explains that retinoic acid generally enhances differentiation in a variety of cell types. On the basis of the information presented, it is suggested that the limb ectoderm promotes cellular retinoic acid-binding protein expression in the subridge mesenchyme and thereby limits the access of retinoic acid to its nuclear receptors in these cells. Cellular-retinoic-acid-binding-protein-mediated, local sequestration or inactivation of free retinoic acid is suggested as a prerequisite not only for the continued responsiveness of the distal mesenchyme to growth promotion by the apical ectodermal ridge, but to the maintenance of the apical ectodermal ridge itself by the subridge mesenchyme.

Topics: Abnormalities, Drug-Induced; Animals; Chick Embryo; Ectoderm; Extremities; Gene Expression Regulation, Developmental; Limb Deformities, Congenital; Mesoderm; Rats; Receptors, Retinoic Acid; Tretinoin

Retinoic acid (RA) plays an important role in normal embryogenesis; however, excessive doses are teratogenic. At present, the molecular mechanisms responsible for these effects of RA are not well understood. The action of retinoids are believed to be mediated by two classes of proteins, nuclear receptors (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) and small cellular retinol-binding and retinoic acid-binding proteins (CRBP-I, CRBP-II, CRABP-I and CRABP-II). Teratogenic doses of RA increase the level of RAR-beta 2 mRNA, RAR-alpha 2 mRNA, CRBP-I mRNA and CRABP-II mRNA in mouse conceptuses and embryos. The elevation in the level of only RAR-beta 2 mRNA correlates with the target tissues, as well as developmental stages that are sensitive to the teratogenic effects of RA. In addition, we have screened a few other natural and synthetic retinoids with similar results. These results are consistent with the possibility that RAR-beta 2 may mediate at least some of the effects of retinoids during abnormal development.

Topics: Abnormalities, Drug-Induced; Animals; Carrier Proteins; Mice; Receptors, Cell Surface; Receptors, Retinoic Acid; Retinoid X Receptors; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Messenger; Transcription Factors; Tretinoin

We consider the evidence that RA, the vitamin A metabolite, is involved in three fundamental aspects of the development of the CNS: 1) the stimulation of axon outgrowth in particular neuronal sub-types; 2) the migration of the neural crest; and 3) the specification of rostrocaudal position in the developing CNS (forebrain, midbrain, hindbrain, spinal cord). The evidence we discuss involves RA-induction of neurites in cell cultures and explants of neural tissue; the teratological effects of RA on the embryo's nervous system; the observation that RA can be detected endogenously in the spinal cord; and the fact that the receptors and binding proteins for RA are expressed in precise domains and neuronal cell types within the nervous system.

Topics: Abnormalities, Drug-Induced; Amphibians; Animals; Carrier Proteins; Cell Differentiation; Central Nervous System; Chick Embryo; Gene Expression Regulation; Genes, Homeobox; Humans; Mice; Morphogenesis; Neural Crest; Neurites; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Acitretin was introduced as a replacement for etretinate, the ethyl ester of acitretin. Acitretin is eliminated at a much faster rate than etretinate. Although both drugs are teratogens, the replacement was important especially as it allowed for a much shorter post-medication period in which pregnancy should be precluded. Recent findings showed the presence of etretinate in the plasma of acitretin-treated patients. This article gives a review of known metabolic pathways of the retinoids and tries to elucidate the possible conversion of acitretin into etretinate after acitretin ingestion.

Topics: Abnormalities, Drug-Induced; Acitretin; Animals; Biotransformation; Etretinate; Humans; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Anticonvulsants; Dose-Response Relationship, Drug; Ethanol; Female; Humans; Lithium; Maternal-Fetal Exchange; Pregnancy; Pregnancy in Diabetics; Radiotherapy; Tretinoin; Warfarin

This is a review of the efficacy of etretinate/acitretin in the treatment of psoriasis and of the currently reported side-effects. The data indicate that retinoids bring significant improvement (if not total clearing) with frequent low-morbidity but rarely serious side-effects. The most serious side-effect of etretinate/acitretin is teratogenicity.

Topics: Abnormalities, Drug-Induced; Acitretin; Adult; Aged; Bone Diseases; Chemical and Drug Induced Liver Injury; Etretinate; Eye Diseases; Female; Humans; Lipids; Male; Middle Aged; Psoriasis; Risk Factors; Skin Diseases; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Ethanol; Heart Defects, Congenital; Humans; Teratogens; Tretinoin; Vitamin A

The retinoids, a group of compounds consisting of vitamin A and its derivatives, have been the subject of intense investigation over the past 30 years. These molecules have shown beneficial effects in the areas of acne, psoriasis, neoplastic processes and, most recently, reversal of extrinsically aged skin. Additional retinoids are currently under development. Adverse reactions to these drugs include mucocutaneous irritation, hyperlipidemia, and profound teratogenicity. Appropriate patient selection is imperative before beginning therapy with these medications. An overview of retinoid metabolism and the currently available compounds is presented. The newest class of retinoids, the arotinoids, is also discussed.

Topics: Abnormalities, Drug-Induced; Etretinate; Female; Humans; Isotretinoin; Pregnancy; Retinoids; Skin Diseases; Tretinoin

Normal anterior segment embryogenesis is summarized followed by a review of syndromes of spontaneous and inherited conditions of abnormal development in humans and animals. The study of teratogen-induced malformations in animal models has provided valuable information about critical periods during gestation for the initiation of anterior segment dysgenesis. Although the major developmental events leading to iridocorneal angle formation occur during the third trimester, it appears that embryonic insult much earlier in human gestation (during the first three to five weeks post fertilization) can induce an abnormal sequence of events leading to anterior segment dysgenesis.

Topics: Abnormalities, Drug-Induced; Animals; Anterior Eye Segment; Cataract; Disease Models, Animal; Ethanol; Female; Glaucoma; Humans; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Ochratoxins; Pregnancy; Syndrome; Teratogens; Trabecular Meshwork; Tretinoin

Dermatologists frequently are consulted by a pregnant patient or a woman of childbearing age who desires acne therapy. Because there are no published studies in which women took acne medications throughout pregnancy, information about safety must be obtained indirectly from studies in which the agents were taken for another indication during some portion of pregnancy. Oral tetracycline is associated with maternal liver toxicity and deciduous tooth staining in the infant, and tetracycline occasionally has been associated with other congenital anomalies. Maternal isotretinoin ingestion is associated with major craniofacial and cardiac deformities, as well as other congenital anomalies. Erythromycin, however, appears to be safe. Topical acne medications never have been implicated as a cause of fetal deformities in human beings. Dermatologists should be aware of potential toxic and teratogenic effects of acne medicines before prescribing them to women of childbearing age. Prompt reporting of adverse effects is encouraged.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Administration, Cutaneous; Administration, Oral; Benzoyl Peroxide; Clindamycin; Erythromycin; Female; Humans; Pregnancy; Pregnancy Complications; Salicylates; Tetracyclines; Tretinoin

Between October 1982 and June 1985 the Adverse Drug Reaction Reporting System received reports of 104 suspected adverse reactions occurring in 93 patients who took isotretinoin. Adverse reactions involving the skin and mucous membranes (29 reports), central nervous system (23), musculoskeletal system (12), pregnancy (11), and eyes (8) were most commonly reported. Severe headache was the most frequently reported adverse reaction (15 reports). In four cases headaches were attributed to pseudotumor cerebri. Some of the reported reactions, for example, a disulfiram (Antabuse)-like reaction and oculogyric crisis, have not been described previously in the literature. Other reports, such as congenital malformations, serve to emphasize some of the serious reactions that are known to occur. These spontaneous reports of adverse reactions associated with isotretinoin use, together with the literature we review, may help alert physicians to the diverse spectrum of adverse reactions that may develop in patients taking isotretinoin.

Topics: Abnormalities, Drug-Induced; Adolescent; Adult; Central Nervous System; Eye; Female; Humans; Isotretinoin; Male; Musculoskeletal System; Pregnancy; Skin; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Animals; Female; Humans; Infant, Newborn; Isotretinoin; Kinetics; Precancerous Conditions; Pregnancy; Skin Neoplasms; Tretinoin

Topics: Abnormalities, Drug-Induced; Adolescent; Adult; Female; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Teratogens; Tretinoin

As a teratogenic agent retinoic acid (RA) produces severe limb reduction defects if administered at a certain stage of embryonic development. In vitro, RA is able to prevent chondrogenesis and this inhibitory effect is accompanied by the absence of cartilage specific proteoglycans in treated cultures. Such an effect is ruled out as a direct causative factor in teratogenesis for two reasons. First, the limbs of treated embryos show extensive chondrogenesis and this cartilage is normal as far as the expression of biochemical markers of differentiation are concerned. Second, the morphogenetic effects of a mutant gene, cmd, where there is a functional deficit of the proteoglycan core protein are very different from those associated with RA-induced teratogenesis. The differences between the two are not wholly reconciled by the fact that the effects of the mutant gene are cumulative and progressive while those of the RA insult are transitory. There are a number of developmental events which are, however, altered by RA in the mesenchymal cells of the early limb bud such as cell proliferation, cell death, and hyaluronic acid metabolism. Not only any one or more of these factors may secondarily inhibit chondrogenesis but, more importantly, may also have a number of other consequences in the developing embryo. Since a number of cell types besides mesenchymal cells respond to RA by altering their pattern of differentiation, it is conceivable that some fundamental molecular step in the process of differentiation provides a target for its action. In a recent review, Sporn and Roberts (1983) have suggested that to be compatible with the wide ranging effects of retinoids documented so far, any hypothesis put forward for its molecular mechanism of action must include a role in gene expression. No experimental work has yet directly addressed how retinoids might modify gene expression. We believe that along with teratocarcinoma stem cell lines, the use of retinoids as selective teratogens may open up another avenue in search of molecular mechanisms of cell differentiation.

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cell Differentiation; Ectromelia; Extremities; Female; Glycosaminoglycans; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Mice, Mutant Strains; Pregnancy; Rodent Diseases; Tretinoin

Approximately 120,000 women of childbearing age used isotretinoin in the first 16 months after its release for the treatment of cystic acne. In September, 1983, the American Academy of Dermatology requested its members to relate the outcome of pregnancies of women inadvertently exposed to isotretinoin ( Accutane ) during pregnancy to its Adverse Drug Reaction Reporting System ( ADRRS ). Of nine pregnancies reported, seven ended in spontaneous abortion or the birth of an infant with birth defects. Of thirty-five pregnancies with isotretinoin exposure reported to the ADRRS or the U.S. Food and Drug Administration, twenty-nine (83%) resulted in spontaneous abortion or infants with birth defects. The most frequently reported severe birth defects involved the central nervous system (microcephaly or hydrocephalus) and the cardiovascular system (anomalies of the great vessels). Microtia or absence of external ears were also noted in a majority of cases. These findings illustrate the usefulness of specialty-based reporting of adverse drug effects and emphasize the teratogenic risk of isotretinoin in humans. Physicians need to fully and carefully inform women of childbearing age of these risks.

Topics: Abnormalities, Drug-Induced; Abortion, Spontaneous; Acne Vulgaris; Female; Heart Defects, Congenital; Humans; Hydrocephalus; Infant, Newborn; Isotretinoin; Microcephaly; Pregnancy; Pregnancy Complications; Pregnancy Trimester, First; Tretinoin

In the one year since isotretinoin has been available in the United States for the treatment of severe, recalcitrant, nodulocystic acne, there has been extensive clinical verification of the reports of its dramatic efficacy in the treatment of this troublesome disease. Proper selection of patients, as well as treatment with adequate doses of drug for 3 to 5 months, will most often result in significant clinical improvement or total clearing. Although dosages of less than 1 mg/kg/day may produce a nearly equivalent degree of improvement with somewhat fewer or less severe side effects, the recommended daily dose remains 1 mg/kg/day because lower dosages are associated with more frequent relapses. In severe cases, the daily dosage may be increased to 2 mg/kg/day. Teratogenicity, elevation of serum triglycerides, liver function abnormalities, pancreatitis, and pseudotumor cerebri may all be associated with isotretinoin therapy and require close monitoring of the patient.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Animals; Central Nervous System; Chemical and Drug Induced Liver Injury; Folliculitis; Humans; Isotretinoin; Middle Aged; Mucous Membrane; Musculoskeletal System; Rats; Skin Diseases; Sweat Gland Diseases; Tretinoin

Alitretinoin, like all retinoids, is teratogenic, and can only be given to women of childbearing potential if pregnancy is excluded and a strict contraceptive programme is followed.. This study was designed to determine whether alitretinoin in the semen of men treated with alitretinoin poses a teratogenic risk to their female partners.. In total, 24 healthy men aged 18-45 years received alitretinoin 20 mg (n = 12) or 40 mg (n = 12), once daily for 14 days. Subjects in the 40 mg dose group provided ejaculate at baseline, on day 1, before and approximately 4 h after dosing on day 2, and at follow-up on study day 21 (± 2).. Alitretinoin and 4-oxo-alitretinoin were detected in 11 of the 12 semen samples. The highest level of alitretinoin in semen was 7.92 ng/mL. Assuming an ejaculate volume of 10 mL, the amount of drug transferred in semen would be about 80 ng, 1/375,000 of a single 30 mg capsule. Complete absorption of 80 ng of alitretinoin from semen, presuming a volume of distribution confined to 5 L of circulating blood in the partner, would lead to an increase in plasma alitretinoin concentration of 0.016 ng/mL, which appears to be negligible compared with measured endogenous plasma levels. Increases in plasma levels of related retinoids are also negligible.. Alitretinoin in the semen of men receiving up to 40 mg of oral alitretinoin per day is unlikely to be associated with teratogenic risk in their female partners. Barrier contraception is therefore not required for men taking alitretinoin.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Adolescent; Adult; Alitretinoin; Dermatologic Agents; Dose-Response Relationship, Drug; Drug Administration Schedule; Humans; Male; Middle Aged; Risk Assessment; Semen; Tretinoin; Young Adult

Since 1982, systemic Roaccutan has been used to treat acne that is resistant to traditional therapy. The treatment produces certain side effects and a potential for teratogenesis. The result has been worldwide reports of a number of malformed and abnormal infants. In the present questionnaire presented to 94 Norwegian women of child-bearing potential, no pregnancy occurred during the treatment period. Before start of treatment, all patients agreed to have an abortion if conception occurred during treatment. The article discusses the type of contraception, the quality of information and the effect of treatment upon the acne condition. The study confirms the importance of Roaccutan in the treatment of cystic acne, and shows that the Norwegian prescription routine for Roaccutan is adequate.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Clinical Trials as Topic; Female; Humans; Middle Aged; Pregnancy; Risk Factors; Tretinoin

Synthetic retinoids were first evaluated 15 years ago for systemic treatment of psoriasis in the Federal Republic of Germany. Etretinate was introduced 2 years ago into the market for systemic treatment of all severe types of the disease. Today etretinate is administered as monotherapy and/or combined with other modalities (anthralin, tar, topical corticosteroids, selective UV therapy, RePUVA), which leads to successful clearing in most cases. Nevertheless, thorough consideration of the risk-benefit ratio is required in each individual patient. The advantages and disadvantages are presented that should be taken into consideration. As a rule, severe cases of psoriasis are admitted to the hospital; initial treatment is given and then continued on an outpatient basis. In some patients, particularly those with pustular eruptions and/or erythroderma, low-dosage oral etretinate may be continued for prophylactic reasons over several months or years. Since the amount of hospitalization is reduced, the overall treatment costs are reduced in spite of the high cost of the drug. The main disadvantage of oral retinoids is their teratogenicity, although no severe cases of retinoid toxicity have been reported in the last 2 years in the Federal Republic of Germany since their introduction. As a successor drug to etretinate, its free aromatic acid, Ro 10-1670 is now under clinical investigation. It seems to be clinically effective, is rapidly eliminated, and requires only 4 weeks contraception after discontinuation of oral administration. Arotinoids then follow.

Topics: Abnormalities, Drug-Induced; Acitretin; Benzoates; Chemical and Drug Induced Liver Injury; Clinical Trials as Topic; Etretinate; Humans; Lipids; Long-Term Care; Prognosis; Psoriasis; Retinoids; Risk; Skin; Tretinoin

Topics: Abnormalities, Drug-Induced; Australia; Clinical Trials as Topic; Etretinate; Female; Humans; Isotretinoin; Neoplasms; Pregnancy; Skin Diseases; Tretinoin; Vitamin A

Retinoic acid signaling plays a critical role during embryogenesis and requires tight regulation. Exposure to exogenous retinoic acid during fetal development is known to have teratogenic effects, producing a recognizable embryopathy.. We describe a case of retinoic acid embryopathy secondary to maternal isotretinoin use until the ninth week of gestation and expand the phenotype to include the rare features of parietal bone agenesis and athelia. Histology of the parietal region showed fibrous tissue with no intramembranous ossification. The fetus also had multiple craniofacial dysmorphisms, thymic agenesis, and transposition of the great arteries with double outlet right ventricle and subaortic perimembranous ventricular septal defect. Neuropathology revealed enlarged ventricles with agenesis of the cerebellar vermis, focal duplication of the central canal and scattered parenchymal ependymal rests, and possible cerebral heterotopias with associated abnormal neuronal lamination. A chromosomal microarray was normal.. Parietal bone agenesis and athelia are both rare congenital anomalies not previously reported in retinoic acid embryopathy. However, retinoic acid or its degrading enzyme has been demonstrated to exert effects in both of these developmental pathways, offering biological plausibility. We propose that this case may represent an expansion of the phenotype of retinoic embryopathy.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Breast Diseases; Congenital Microtia; Female; Fetal Diseases; Humans; Parietal Bone; Phenotype; Tamoxifen; Transposition of Great Vessels; Tretinoin

The purpose of the study is to develop an algorithm to identify pregnancies in administrative databases and apply it to assess pregnancy rates and outcomes in women prescribed isotretinoin or tretinoin.. Using the 2011 to 2015 Truven Health MarketScan Database, we identified pregnancies, including losses and terminations. In a cohort design, nonpregnant women filling a prescription for isotretinoin or tretinoin were matched to five women without either prescription. Women were followed for 365 days or until conception, medication discontinuation, or enrollment discontinuation ("prescription episode"). Rates of pregnancy, risks of pregnancy losses, and prevalence of infant malformations at birth were assessed by exposure.. We identified 2 179 192 livebirths, 8434 stillbirths, 2521 mixed births, 415 110 spontaneous abortions, 124 556 elective terminations, and 8974 unspecified abortions. There were 86 834 isotretinoin and 973 587 tretinoin episodes, matched to 5 302 105 unexposed women. Pregnancy rates were 3 (isotretinoin), 19 (tretinoin), and 34 (unexposed) per 1000 person-years. Risk of spontaneous pregnancy losses were similar; however, terminations were more common in the isotretinoin-exposed (28% [95% CI: 21%-36%]) than the tretinoin-exposed (10% [95% CI: 9%-11%]) or unexposed pregnancies (6%). Malformations occurred in 4.5% (95% CI: 3.5%-5.6%) of the tretinoin-exposed pregnancies and 4.2% of the unexposed pregnancies (adjusted odds ratio: 1.16 [95% CI: 0.85-1.58]); isotretinoin-exposed births were too few to assess malformations.. Administrative databases can complement risk evaluation and mitigation strategies (REMS) for known teratogens and contribute to safety surveillance for other medications. Here, isotretinoin-exposed pregnancy rates were low, but existent, and many pregnancies were terminated. Tretinoin exposure was not associated with a meaningfully elevated risk of losses or malformations as compared with unexposed pregnancies.

Topics: Abnormalities, Drug-Induced; Administrative Claims, Healthcare; Adolescent; Adult; Databases, Factual; Drug Prescriptions; Female; Humans; Infant, Newborn; Isotretinoin; Maternal Exposure; Middle Aged; Pregnancy; Pregnancy Outcome; Pregnancy Rate; Prevalence; Product Surveillance, Postmarketing; Tretinoin; United States; Young Adult

We previously reported that Wnt5a/CaMKIIδ (calcium/calmodulin-dependent protein kinase II delta) pathway was involved in the embryonic tongue deformity induced by excess retinoic acid (RA). Our latest study found that the expression of miR-31-5p, which was predicted to target the 3'UTR of CamkIIδ, was raised in the RA-treated embryonic tongue. Thus, we hypothesized that the excess RA regulated Wnt5a/CaMKIIδ pathway through miR-31-5p in embryonic tongue.. C2C12 myoblast line was employed as an in vitro model to examine the suppression of miR-31-5p on CamkIIδ expression, through which RA impaired the myoblast proliferation and differentiation in embryonic tongue.. RA stimulated the expression of miR-31-5p in both embryonic tongue and C2C12 myoblasts. Luciferase reporter assay confirmed that the 3'UTR of CamkIIδ was a target of miR-31-5p. MiR-31-5p mimics disrupted CamkIIδ expression, C2C12 proliferation and differentiation as excess RA did, while miR-31-5p inhibitor partially rescued these defects in the presence of RA.. Excess RA can stimulate miR-31-5p expression to suppress CamkIIδ, which represses the proliferation and differentiation of tongue myoblasts.

Topics: 3' Untranslated Regions; Abnormalities, Drug-Induced; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Differentiation; Cell Proliferation; HEK293 Cells; Humans; Mice; MicroRNAs; Muscle Development; Myoblasts; Tongue; Tretinoin

Although numerous studies have confirmed that consumption of folic acid (FA) during early pregnancy reduces the risk of oral facial clefts in newborn infants, the optimal dose of FA for reducing this risk remains unknown. We evaluated various doses of FA for their ability to reduce the incidence of all-trans retinoic acid (ATRA)-induced cleft palate in mice.. Pregnant C57BL/6J mice were randomly assigned to eight groups dosed with corn oil (control group), ATRA (80 mg/kg), FA (40 mg/kg), or ATRA (80 mg/kg) + FA (2.5 mg, 5 mg, 10 mg, 20 mg, or 40 mg/kg body weight) on gestation day 11 (GD11), after which samples of maternal blood obtained on GD 11 were analyzed for serum folate levels. After receiving the doses, randomly selected mice in each dose group were sacrificed on GDs 13.5, 14.5, and 15.5, and the fetuses were removed for examination by light microscopy and scanning electron microscopy to detect the incidence of cleft palate.. Among the pregnant mice dosed with ATRA+FA, those dosed with 5 mg/kg FA had fetuses with the lowest incidence of cleft palate. In addition, the eight groups of pregnant mice had significantly different serum folate concentrations (P < .001).. When administered to pregnant mice at a specific dose and on the proper gestation day, FA showed an antiteratogenic effect by reducing the incidence of ATRA-induced cleft palate in fetal mice.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Female; Folic Acid; Maternal Exposure; Mice; Mice, Inbred C57BL; Pregnancy; Prenatal Care; Teratogens; Tretinoin

To investigate the protective effect of folinic acid on the hatching ability and developmental defects in a retinoic acid-induced teratogenic model of chick embryo.. The experimental study was conducted at the Department of Anatomy, Regional Centre of the College of Physicians and Surgeons Pakistan, Islamabad, from February 2009 to February 2010. Chicken eggs were divided into two experimental groups and a control group. The first experimental group was injected with retinoic acid to induce a defective model, while the second experimental group was concomitantly injected folinic acid to observe its protective effects on retinoic acid-induced defects in the development and hatching process. Both groups were compared with the age-matched control group.. A total of 90 fertilised eggs were divided into three groups. The experimental groups had significantly more delayed and assisted hatchings compared to the control group (p<0.05) but the difference between the experimental groups regarding the mode and day of hatching was insignificant (p>0.05).. Irrespective of the presence of folinic acid, prenatal retinoic acid exposure significantly altered the hatchability characteristics in the experimental groups compared to the control.

Topics: Abdominal Wall; Abnormalities, Drug-Induced; Animals; Chick Embryo; Leucovorin; Limb Deformities, Congenital; Teratogenesis; Teratogens; Tretinoin; Vitamin B Complex

To characterize the abnormal metabolic profile of all-trans-retinoic acid (ATRA)-induced craniofacial development in mouse embryos using proton magnetic resonance spectroscopy (1H-MRS).. Timed-pregnant mice were treated by oral gavage on the morning of embryonic gestation day 11 (E11) with all-trans-retinoic acid (ATRA). Dosing solutions were adjusted by maternal body weight to provide 30, 70, or 100 mg/kg RA. The control group was given an equivalent volume of the carrier alone. Using an Agilent 7.0 T MR system and a combination of surface coil coils, a 3 mm×3 mm×3 mm 1H-MRS voxel was selected along the embryonic craniofacial tissue. 1H-MRS was performed with a single-voxel method using PRESS sequence and analyzed using LCModel software. Hematoxylin and eosin was used to detect and confirm cleft palate.. 1H-MRS revealed elevated choline levels in embryonic craniofacial tissue in the RA70 and RA100 groups compared to controls (P<0.05). Increased choline levels were also found in the RA70 and RA100 groups compared with the RA30 group (P<0.01). High intra-myocellular lipids at 1.30 ppm (IMCL13) in the RA100 group compared to the RA30 group were found (P<0.01). There were no significant changes in taurine, intra-myocellular lipids at 2.10 ppm (IMCL21), and extra-myocellular lipids at 2.30 ppm (EMCL23). Cleft palate formation was observed in all fetuses carried by mice administered 70 and 100 mg/kg RA.. This novel study suggests that the elevated choline and lipid levels found by 1H-MRS may represent early biomarkers of craniofacial defects. Further studies will determine performance of this test and pathogenetic mechanisms of craniofacial malformation.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Choline; Cleft Palate; Craniofacial Abnormalities; Creatine; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Lipids; Male; Mice; Pregnancy; Proton Magnetic Resonance Spectroscopy; Taurine; Tretinoin

Xenopus tropicalis embryos were exposed for 48 hr to the mixtures of 5 μg Sn/L triphenyltin (TPT), which is a well-known endocrine disruptor, and 0.25-5 μg/L 9-cis retinoic acid (9c-RA), which is the natural ligand of retinoid X receptor. The phenotypes induced by combined exposure were more variable than those resulting from single exposure to either TPT or 9c-RA. The prominent phenotypes included underdeveloped head structures, abnormal eyes, narrow fins, enlarged proctodaeum, etc. Especially, combined exposure induced unexpected notochord malformations, which ranged from small swellings of the surface of the tails to the extension and extrusion of notochord out of the posterior tails. Compared with the 5 μg Sn/L TPT-treated group, the index of fin deficiency was not affected, and the index of axis deficiency was significantly increased with increasing RA concentrations in the mixtures. Our results suggest that combined exposure to TPT and 9c-RA induced not only more variable phenotypes of malformations than exposure to single compound but also some new and unexpected phenotypes.

Topics: Abnormalities, Drug-Induced; Alitretinoin; Animals; Drug Interactions; Embryo, Nonmammalian; Embryonic Development; Female; Male; Organotin Compounds; Phenotype; Retinoid X Receptors; Teratogenesis; Tretinoin; Xenopus

Various biochemical and physiological stimuli may interfere with endoplasmic reticulum (ER) homeostasis, causing aggregation and accumulation of unfolded or malfolded proteins in the ER and resulting in ER stress (ERS), and the ER attenuates ERS through unfolded protein response (UPR). All-trans-retinoic acid (ATRA) can enhance or attenuate some ERS-induced physiological or pathological changes. Our previous studies showed that there was UPR in short forelimbs induced by ATRA. Many kinds of malformations induced by ATRA may correlate with cell death, and knowing that UPR is closely associated with cell death, so we speculate that the short forelimbs may caused by UPR-induced cell death. To test this hypothesis, the present study investigated the expression of UPR-related genes and proteins in the same short forelimb malformation model to determine whether ATRA-induced short forelimb malformation occurred through UPR-induced cell death. Subsequently, we further observed the differentiation and proliferation of chondrocytes and the expression of related genes and/or proteins. It was found that ATRA evoked UPR in this short forelimb model, thus activating the anti-cell death pathway and inhibiting the cell death-promoting pathway. Cell death was not evident in short forelimb, and ATRA inhibited the expression of Ccnb1 and Ccna1, thus retarding chondrocyte maturation. As a result, the number of immature chondrocytes in short forelimb was greater than the normal level. We therefore believe that ATRA induces short forelimb malformation most likely by inhibiting chondrocyte maturation rather than by evoking excess cell death.

Topics: Abnormalities, Drug-Induced; Animals; Blotting, Western; Cell Survival; Chondrocytes; Cyclin A1; Cyclin B1; Embryonic Development; Endoplasmic Reticulum; Female; Forelimb; Histocytochemistry; Mice; Mice, Inbred ICR; Pregnancy; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Unfolded Protein Response

Retinoic acid, an active metabolite of vitamin A, plays essential signaling roles in mammalian embryogenesis. Nevertheless, it has long been recognized that overexposure to vitamin A or retinoic acid causes widespread teratogenesis in rodents as well as humans. Although it has a short half-life, exposure to high levels of retinoic acid can disrupt development of yet-to-be formed organs, including the metanephros, the embryonic organ which normally differentiates into the mature kidney. Paradoxically, it is known that either an excess or a deficiency of retinoic acid results in similar malformations in some organs, including the mammalian kidney. Accordingly, we hypothesized that excess retinoic acid is teratogenic by inducing a longer lasting, local retinoic acid deficiency. This idea was tested in an established in vivo mouse model in which exposure to excess retinoic acid well before metanephric rudiments exist leads to failure of kidney formation several days later. Results showed that teratogen exposure was followed by decreased levels of Raldh transcripts encoding retinoic acid-synthesizing enzymes and increased levels of Cyp26a1 and Cyp26b1 mRNAs encoding enzymes that catabolize retinoic acid. Concomitantly, there was significant reduction in retinoic acid levels in whole embryos and kidney rudiments. Restoration of retinoic acid levels by maternal supplementation with low doses of retinoic acid following the teratogenic insult rescued metanephric kidney development and abrogated several extrarenal developmental defects. This previously undescribed and unsuspected mechanism provides insight into the molecular pathway of retinoic acid-induced teratogenesis.

Topics: Abnormalities, Drug-Induced; Animals; Cytochrome P-450 Enzyme System; Female; Gene Expression Regulation, Developmental; Kidney; Maternal Exposure; Mice; Pregnancy; Pregnancy, Animal; Retinoic Acid 4-Hydroxylase; RNA, Messenger; Signal Transduction; Teratogens; Time Factors; Tretinoin

Most known human teratogens are associated with a unique or characteristic pattern of major and minor malformations and this pattern helps to establish the causal link between the teratogenic exposure and the outcome. Although traditional case-control and cohort study designs can help identify potential teratogens, there is an important role for small cohort studies that include a dysmorphological examination of exposed and unexposed infants for minor structural defects. In combination with other study design approaches, the small cohort study with a specialized physical examination fulfills a necessary function in screening for new potential teratogens and can help to better delineate the spectrum and magnitude of risk for known teratogens.

Topics: Abnormalities, Drug-Induced; Anticonvulsants; Antidepressive Agents; Arthritis, Rheumatoid; Carbamazepine; Case-Control Studies; Cohort Studies; Female; Fever; Fluoxetine; Humans; Isoxazoles; Leflunomide; Maternal Exposure; Neural Tube Defects; Physical Examination; Pregnancy; Prospective Studies; Research Design; Teratogens; Tretinoin

Microphthalmia (reduced eye size), generally accompanied by vision defects, is a hallmark of fetal alcohol spectrum disorder (FASD) in humans. In zebrafish, embryonic ethanol exposure over the time of retinal neurogenesis also results in microphthalmia. This microphthalmia is in part the consequence of reduced retinal cell differentiation, including photoreceptors. Here we pursue 2 signaling pathways implicated in other aspects of FASD pathogenesis: retinoic acid (RA) and Sonic hedgehog (Shh).. We evaluated markers for RA and Shh signaling within the eyes of embryos treated with ethanol during the period of retinal neurogenesis. We also performed rescue experiments using administration of exogenous RA and microinjection of cholesterol, which augments Shh signaling.. Using sequential or co-treatments, RA did not rescue ethanol-induced microphthalmia at any concentration tested. In addition, RA itself caused microphthalmia, although the underlying mechanisms were distinct from those of ethanol. Interestingly, RA treatment appeared to recover photoreceptor differentiation in a concentration-dependent manner. This may be an independent effect of exogenous RA, as ethanol treatment alone did not alter RA signaling in the eye. Cholesterol injection also did not rescue ethanol-induced microphthalmia at any concentration tested, and ethanol treatments did not alter expression of shh, or of ptc-2, which is normally regulated by Shh signaling.. Together these findings indicate that, during the time of retinal neurogenesis, effects of ethanol on eye development are likely independent of the RA and Shh signaling pathways. These studies suggest that FASD intervention strategies based upon augmentation of RA or Shh signaling may not prevent ethanol-induced microphthalmia.

Topics: Abnormalities, Drug-Induced; Acridine Orange; Animals; Central Nervous System Depressants; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Ethanol; Female; Hedgehog Proteins; Microphthalmos; Neurogenesis; Retina; Signal Transduction; Transgenes; Tretinoin; Vitamins; Zebrafish

Retinoid-mediated signal transduction plays a crucial role in the embryogenesis of various organs. We previously reported the successful induction of anorectal malformations in mice using retinoic acid (RA). Retinoic acid controls the expression of essential target genes for cell differentiation, morphogenesis, and apoptosis through a complicated interaction in which RA receptors form heterodimers with retinoid X receptors. In the present study, we investigated whether the retinoid antagonist, LE135, could prevent the induction of anorectal malformations (ARMs) in mice.. Retinoic acid was intraperitoneally administered as 100 mg/kg of all-trans RA on E9; and then the retinoid antagonist, LE135, was intraperitoneally administered to pregnant ICR strain mice on the eighth gestational day (E8), 1 day before administration of RA (group B) or on E9, simultaneously (group C) with RA administration. All of the embryos were obtained from the uteri on E18. Frozen sections were evaluated for concentric layers around the endodermal epithelium by hematoxylin and eosin staining.. In group A, all of the embryos demonstrated ARM with rectoprostatic urethral fistula, or rectocloacal fistula, and all of the embryos showed the absence of a tail. In group B, 36% of the embryos could be rescued from ARM. However, all of the rescued embryos had a short tail that was shorter than their hind limb. The ARM rescue rates in group B were significantly improved compared to those in group A (P < .01). In group C, 45% of the embryos were rescued from ARM, but all of the rescued embryos had short tail. The ARM rescue rate in group C was significantly improved compared to that in group A (P < .01). However, there was no significant difference in the ARM rescue rate between group B and Group C.. The present study provides evidence that in the hindgut region, RAR selective retinoid antagonist, LE135, could rescue embryos from ARM. However, the disturbance of all-trans RA acid was limited to the caudal region. Further study to establish an appropriate rescue program for ARM in a mouse model might suggest a step toward protection against human ARM in the future.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Anal Canal; Animals; Cloaca; Dibenzazepines; Drug Administration Schedule; Drug Evaluation, Preclinical; Female; Fistula; Gene Expression Regulation, Developmental; Humans; Injections, Intraperitoneal; Male; Mice; Mice, Inbred ICR; Models, Animal; Pregnancy; Prostatic Diseases; Random Allocation; Receptors, Retinoic Acid; Rectal Fistula; Rectum; Species Specificity; Tail; Tretinoin

Topics: Abnormalities, Drug-Induced; Adrenal Cortex Hormones; Cosmetics; Female; Humans; Hydroquinones; Mercury; Pregnancy; Risk; Skin Pigmentation; Tretinoin; Wound Healing

Retinoic acid (RA) has been shown to induce congenital clubfoot in animal models, but it is unknown whether the effect of RA on the formation of clubfoot in vivo results from generalized growth retardation or from the specific effects of hindlimb skeletal development. Our experimental research was based on a clubfoot model treated by maternal administration of RA (120, 130 or 140 mg/kg body weight) as an intragastric dose on embryonic day 10 (E10), and a control group was administered with an equivalent dose of solvent. Prenatal RA exposure reduced fetal body weight, length and skeletal ossification of the hindlimb compared with the control fetuses in a dose-dependent manner. The normal development curves indicated that the RA-exposed fetuses showed delayed increase in body weight and skeletal ossification development. However, there was no uniform effect on the skeletons of the hindlimb, not least retardation in ossification and induction malformation on the talus and calcaneus. Our results demonstrated that prenatal RA exposure had retardation effects on the developing hindlimb skeleton that was independent of those on the overall fetal growth. The normal skeletal ossification showed that the talus and calcaneus were poorly ossified and they were delayed by almost one day in the RA 120 mg/kg group. Therefore, during the susceptible stages, different regions of the limb bud responded differently to the teratogenic effects of RA.

Topics: Abnormalities, Drug-Induced; Animals; Body Patterning; Bone Development; Clubfoot; Disease Models, Animal; Female; Fetal Development; Hindlimb; Osteogenesis; Pregnancy; Rats; Rats, Sprague-Dawley; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Cricetinae; History, 20th Century; Morphogenesis; Teratology; Tretinoin

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1-15 min) pulses of low doses of retinoic acid (RA: 0.25-2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6-hr window (developmental stages NF15-NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA-dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes.

Topics: Abnormalities, Drug-Induced; Animals; Benzoates; Drug Antagonism; Embryo, Nonmammalian; Female; Gene Expression Regulation, Developmental; Homeodomain Proteins; In Situ Hybridization; Jaw; Jaw Abnormalities; Keratolytic Agents; Leucovorin; Morphogenesis; Neural Crest; Pulse Therapy, Drug; Retinoids; Time Factors; Transcription Factors; Tretinoin; Vitamin B Complex; Xenopus laevis; Xenopus Proteins

Dominant hemimelia (Dh) is an autosomal dominant mutation that arose spontaneously in mice. Dh animals are asplenic and they exhibit asymmetric hindlimb defects in association with reduced numbers of lumbar vertebrae. These defects suggest that Dh acts early in embryonic development to affect patterning of the anterior-posterior (A-P) and left-right axes. This study was undertaken to determine whether retinoic acid (RA), which is involved in A-P patterning and coordination of bilaterally synchronized somitogenesis, affects phenotypic expression of the Dh gene.. Thirty-four pregnant females were given, by oral intubation, a single dose of 50 or 75 mg all-trans RA per kilogram body weight at GD 9, 10, or 11. The pregnant females were then euthanized at GD 18 and fetuses removed by cesarean section. A total of 326 fetuses were identified by phenotype and linked DNA and their skeletons were analyzed.. There was a differential effect of RA on the axial skeleton and hindlimb of Dh/+ mice as compared to their wild-type littermates. Dose- and stage-specific effects on sternebrae and vertebrae were observed.. The effects of RA dosing on numbers of sternebrae and vertebrae suggest that Dh embryos have a primary defect in retinoid-mediated A-P patterning. Dosing with RA may produce the observed effects on phenotypic expression of Dh/+ by indirectly or directly modifying an already existing altered Hox expression pattern. As the relationship between axial patterning and the asymmetric limb is unknown, Dh is an important model for studying this relationship.

Topics: Abnormalities, Drug-Induced; Animals; Body Patterning; Dose-Response Relationship, Drug; Ectromelia; Female; Gene Expression Regulation, Developmental; Genes, Dominant; Genes, Homeobox; Homeodomain Proteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Pregnancy; Spine; Tretinoin

Retinoic acid is a widely used drug in the treatment of cystic acne. It has teratogenic effects that depend on the gestational period in which it is used. We report a seven months old female whose mother was exposed to retinoic acid in both pre-gestational and gestational periods. She had a retardation of psychomotor development and a brain MRI showed frontal atrophy and a malformation of the posterior fossa. We discuss the mechanisms of the teratogenic effects of retinoic acid.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Acne Vulgaris; Atrophy; Cranial Fossa, Posterior; Craniofacial Abnormalities; Female; Frontal Lobe; Humans; Infant; Isotretinoin; Keratolytic Agents; Maternal Exposure; Pregnancy; Prenatal Exposure Delayed Effects; Psychomotor Disorders; Teratogens; Tretinoin

Retinoic acid (RA) is essential for inner ear development. However, exposure to excess RA at a critical period leads to inner ear defects. These defects are associated with disruption in epithelial-mesenchymal interactions.. This study investigates the role of Dlx5 in the epithelial-mesenchymal interactions that guide otic capsule chondrogenesis, as well as the effect of excess in utero RA exposure on Dlx5 expression in the developing mouse inner ear. Control of Dlx5 by Fgf3 and Fgf10 under excess RA conditions is investigated by examining the developmental window during which Fgf3 and Fgf10 are altered by in utero RA exposure and by testing the ability of Fgf3 and Fgf10 to mitigate the reduction in chondrogenesis and Dlx5 expression mediated by RA in high-density cultures of periotic mesenchyme containing otic epithelium, a model of epithelial-mesenchymal interactions in which chondrogenic differentiation of periotic mesenchyme ensues in response to induction by otic epithelium.. Dlx5 deletion alters expression of TGFbeta(1), important for otic capsule chondrogenesis, in the developing inner ear and compromises the ability of cultured periotic mesenchyme containing otic epithelium, harvested from Dlx5 null embryos, to differentiate into cartilage when compared with control cultures. Downregulation in Dlx5 ensues as a consequence of in utero RA exposure in association with inner ear dysmorphogenesis. This change in Dlx5 is noted at embryonic day 10.5 (E10.5), but not at E9.5, suggesting that Dlx5 is not a direct RA target. Before Dlx5 downregulation, Fgf3 and Fgf10 expression is modified in the inner ear by excess RA, with the ability of exogenous Fgf3 and Fgf10 to rescue chondrogenesis and Dlx5 expression in RA-treated cultures of periotic mesenchyme containing otic epithelium supporting these fibroblast growth factors (FGFs) as intermediary genes by which RA mediates its effects.. Disruption in an Fgf3, -10/Dlx5 signaling cascade is operant in molecular mechanisms of inner ear teratogenesis by excess RA.

Topics: Abnormalities, Drug-Induced; Animals; Cell Communication; Cells, Cultured; Chondrogenesis; Ear, Inner; Epithelium; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 3; Homeodomain Proteins; Maternal Exposure; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Pregnancy; Signal Transduction; Tretinoin

To observe the morphological features of the lumbosacral neural tube defects (NTDs) induced by all-trans retinoic acid (atRA) and to explore the pathogenesis of these defects.. Rat embryos with lumbosacral NTDs were obtained by treating pregnant rats with administration of atRA. Rat embryos were obtained by cesarean. Fetuses were sectioned and stained with hematoxylin-eosin (H&E). Relevant structures including caudal neural tube were examined. In the atRA-treated rats, about 48% embryos showed lumbosacral NTDs. There appeared a dorsally and rostrally situated, neural-plate-like structure (myeloschisis) and a ventrally and caudally located cell mass containing multiple canals (hamartoma) in the lumbosacral NTDs induced by atRA.. Retinoic acid could disturb the notochord and tail bud development in the process of primary and secondary neurulation in rat embryos, which cause lumbosacral NTDs including myeloschisis and hamartoma. The morphology is very similar to that happens in humans.

Topics: Abnormalities, Drug-Induced; Animals; Embryonic Development; Female; Gestational Age; Hamartoma; Keratolytic Agents; Neural Tube Defects; Notochord; Pregnancy; Rats; Rats, Wistar; Spinal Cord; Spinal Diseases; Spine; Teratogens; Tretinoin

Defects in digit number or fusion as a teratogenic response are well documented in humans and intensively studied in various mouse models. Maternal exposure to excess levels of all-trans-retinoic acid (RA) at gestational day 9.5 induces postaxial ectrodactyly (digit loss) in the murine C57BL/6N strain but not in the SWV/Fnn strain.. Whole-mount in situ hybridization was used to examine the differential expression of limb patterning genes at the transcriptional level between the two mouse strains following the maternal exposure to a teratogenic level of RA. The detection of a gene with altered expression was followed by either the evaluation of other genes that were synexpressed or with an assessment of downstream genes.. In the C57BL/6N limb bud following maternal RA administration, gene-specific perturbations were observed within hours of the RA injection in the posterior pre-AER (apical ectodermal ridge) (Fgf8, Dlx3, Bmp4, Sp8, but not Dlx2 or p63), whereas these genes were normally expressed in the SWV/Fnn limb bud. Furthermore, although RA caused comparable reductions of Shh expression between the strains in the 12 h after administration, some Shh downstream genes were differentially expressed (e.g., Gli1, Ptc, and Hoxd13), whereas others were not (e.g., Fgf4, Bmp4, and Gremlin).. It is proposed that altered gene expression in both pre-AER and mesoderm is involved in the pathogenesis of postaxial digit loss, and that because the alterations in the pre-AER occur relatively early in the temporal sequence of events, those changes are candidates for an initiating factor in the malformation.

Topics: Abnormalities, Drug-Induced; Animals; Ectoderm; Female; Forelimb; Gene Expression Regulation, Developmental; In Situ Hybridization; Limb Buds; Limb Deformities, Congenital; Male; Mesoderm; Mice; Mice, Inbred C57BL; Species Specificity; Teratogens; Tretinoin

Apoptosis is an essential physiological process in embryonic development. In the developing eye of vertebrates, three periods of developmental apoptosis can be distinguished: early, intermediate and later. Within the apoptosis pathway, caspases play a crucial role. It has also been shown that HSP110 may have a potential role in apoptosis. The aim of this research was to study the expression of HSP110, caspase-3 and -9 in physiological, retinoic- or irradiation-induced apoptosis during early eye development. Seven pregnant C57Bl/6J mice received 80 mg kg(-1) of all-trans retinoic acid mixed with sesame oil. Seven pregnant NMRI mice received 2 Gy irradiation at the same gestational day. Control mice of both strains (seven mice of each) were not submitted to any treatment. Embryos were harvested at 3, 6, 12 and 24 h after exposition, fixed, dehydrated and embedded. Coronal sections (5 microm) were made. Slide staining occurred alternatively using anti-caspase-3, anti-caspase-9 and anti-HSP110 immunohistochemistry. HSP110 and caspase-3 expression presented similar topographic and chronological patterns, whereas expression of HSP110 was more precocious in retinoic acid-treated embryos. After retinoic exposure, caspase-3- and HSP110-positive cells were increased in the region of the optic vesicle. By contrast, after irradiation, caspase-3- and HSP110-positive cells were noticeably increased in the optic vesicle, peri-optical mesoderm but less in lens placode. HSP110 was expressed before caspase-3. By contrast, caspase-9 was expressed by a very small number of cells in the optic vesicle either under physiological or under teratogenic conditions. Thus, it seems that activation of caspase-9 is dispensable in early eye developmental apoptosis.

Topics: Abnormalities, Drug-Induced; Abnormalities, Radiation-Induced; Animals; Apoptosis; Caspase 3; Caspase 9; Embryonic Development; Eye; Female; Gestational Age; HSP110 Heat-Shock Proteins; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Models, Animal; Pregnancy; Teratogens; Tretinoin

A proposed mechanism for ethanol teratogenicity entails ethanol-mediated reductions in retinoic acid (RA). This premise was investigated utilizing a mouse model, with limb reduction defects as the teratogenic end point.. Ethanol, Disulfiram, or BMS-189453 was administered to C57BL/6J mice on the 9(th) day of pregnancy. Forelimb morphology was assessed on gestation day 18 using Alcian blue and Alizarin red staining. Nile blue sulfate or LysoTracker Red (LTR) vital staining identified cell death in the limb bud. The ability of RA to prevent ethanol-induced cell death was assessed by coadministration followed by laser scanning confocal microscopic examination of LTR-staining. In situ hybridization and qPCR were used to examine gene expression in treated limb buds.. Ethanol, Disulfiram, and BMS-189453 resulted in postaxial ectrodactyly, intermediate ectrodactyly, and other digital defects. Excessive Nile blue sulfate staining was evident in the presumptive AER following each of the three exposures. Ethanol-induced LTR staining was prevented by RA supplementation. Both in situ hybridization and qPCR illustrated decreases in Shh and Tbx5 in ethanol-exposed embryos as compared to control.. Contrary to studies of prolonged RA deficiency, acute exposure to functional antagonists of RA results in limb defects that are morphologically similar to those caused by ethanol. The rescue of ethanol-induced cell death by RA and similar changes in Shh transcription further suggest that RA contributes to ethanol-induced limb dysmorphology. Moreover, the repression of key mediators of limb development soon after ethanol exposure adds to the existing knowledge of the pathogenic effects of ethanol.

Topics: Abnormalities, Drug-Induced; Animals; Cell Death; Ethanol; Female; Hedgehog Proteins; In Situ Hybridization; Limb Deformities, Congenital; Mice; Polymerase Chain Reaction; Pregnancy; RNA, Messenger; Signal Transduction; T-Box Domain Proteins; Teratogens; Tretinoin

The response to exposure to all-trans-retinoic acid (RA) during embryogenesis varies from physiologic to severe teratogenic effects and is dependent upon the dose and the stage of development in all species. Vangl2 and Dishevelled genes play key roles in establishing planar cell polarity and regulating convergent extension movements during the neurula period. The effects of RA-mediated teratogenesis might be due to its misregulation of Vangl2 and Dishevelled genes. The aim of this study is to monitor the modulation of Vangl2 and Dishevelled in Kunming mouse embryos following maternal treatment with a single oral dose of 30 mg/(kg body weight) of RA during the neurula period. Exposure of 7.75 d embryos to RA induced characteristic morphological changes. The most obvious external effect was the failure of neural tube closure in the midbrain and forebrain regions in 10 d embryos, resulting in exencephaly in later embryos. RA treatment also led to a pronounced decrease of Vangl2 mRNA at 4 and 18 h and a pronounced increase at 66 h after maternal treatment, as detected by reverse transcription-polymerase chain reaction. Western blot analysis showed a marked decrease of Vangl2 protein at 18 and 42 h and a marked increase at 66 and 90 h after maternal treatment. Dishevelled1/2/3 mRNA was significantly down-regulated at 4 and 18 h and up-regulated at 42 h in the fetus after RA treatment, except for an up-regulation of Dishevelled3 at 66 h. The Dishevelled2 mRNA and its protein matched each other. These results hinted that Vangl2 and Dishevelled genes might take part in RA teratogenesis of mouse embryos.

Topics: Abnormalities, Drug-Induced; Adaptor Proteins, Signal Transducing; Animals; Central Nervous System; Dishevelled Proteins; Female; Gene Expression Regulation, Developmental; Mice; Mice, Inbred Strains; Nerve Tissue Proteins; Phosphoproteins; Tretinoin

Nitrofen is a diphenyl ether that induces a spectrum of birth defects subsequent to administration to pregnant rodents, in which the molecular etiology of these defects are poorly characterized. Because previous reports showed that nitrofen induced apoptosis in undifferentiated P19 teratocarcinoma cells, we hypothesized that undifferentiated fetal cells have greater susceptibility to nitrofen-induced apoptosis than their differentiated derivatives.. To investigate this hypothesis, cell lines including P19 and F9 were differentiated with retinoic acid into neuronal and endodermal derivatives respectively. Apoptosis was characterized by caspase-3 cleavage and Terminal transferase dUTP nick end labeling (TUNEL) assays.. Both differentiated cell-types had reduced nitrofen-induced caspase-3 cleavage and DNA fragmentation compared with the naive controls, strongly suggesting that differentiation of these cells protects against nitrofen-induced apoptosis. In addition, resistance to apoptotic induction was proportional to the expression levels of the differentiation marker, p27 (kip1) while direct proportionality was not observed for the antiapoptotic protein Bcl-2.. These studies show that nitrofen may induce its associated birth defects via a mechanism involving apoptosis of undifferentiated fetal cells.

Topics: Abnormalities, Drug-Induced; Animals; Apoptosis; Caspase 3; Cell Differentiation; Cell Line; Herbicides; Mice; Phenyl Ethers; Proto-Oncogene Proteins c-bcl-2; Tretinoin

Mouse strain differences in teratologic response are well documented. However, because retinoids cause similar malformation syndromes across many species, the strain differences may be predicted to be minimal. The goals of this study were to characterize and explain the differences between the C57BL/6N and SWV mouse strains in terms of all-trans-retinoic acid (RA)-induced teratologic effects at the time of gestation that cause postaxial forelimb ectrodactyly.. Visceral and skeletal malformations were determined by Wilson's sectioning and double-staining techniques, respectively; developmental staging was performed according to the somite count; and retinoid concentrations were assessed by HPLC.. C57BL/6N mice were more susceptible than SWV mice to induction of embryolethality, cardiovascular defects, and forelimb ectrodactyly, whereas the opposite was true for the induction of ear, thymus, and tail agenesis, and cleft palate, gastroschisis, and anal atresia. As determined by somite counts, 1 strain intercross was developmentally advanced compared to the parental strains and the reciprocal cross. Retinoid susceptibility was equivalent between the reciprocal crosses for some malformations and determined by the maternal genotype for others. Toxicokinetic experiments showed that whole-embryo peak retinoid concentrations did not differ between the strains, but the area under the curve (AUC) for all-trans-RA was 1.3 times higher in C57BL/6N than in SWV embryos.. The malformation spectrum induced by RA was strain-specific, and the strain sensitivity for forelimb ectrodactyly was consistent with all previously tested teratogenic agents (i.e., C57BL/6N was more sensitive than SWV). The strain differences in teratologic effects were not explained by developmental timing differences or toxicokinetic differences at the whole-embryo level.

Topics: Abnormalities, Drug-Induced; Animals; Drug Administration Schedule; Female; Limb Deformities, Congenital; Male; Mice; Mice, Inbred C57BL; Time Factors; Tretinoin

To analyze the expression and role of three proteins (HSP110, caspase-3 and caspase-9) during craniofacial development.. Seven pregnant C57Bl/6J mice received, by force-feeding at gestation day 9 (E9), 80 mg/kg of all-trans retinoic acid mixed to sesame oil. Seven pregnant NMRI mice received two grays irradiation at the same gestation day. Control mice of both strains (seven mice for each strain) were not submitted to any treatment. Embryos were obtained at various stages after exposition (3, 6, 12 and 24 h), fixed, dehydrated and embedded. Coronal sections (5 microm) were made. Slide staining occurred alternatively using anti-Hsp110, anti-caspase-3 and anti-caspase-9 immunohistochemistry.. Expression of HSP110, caspase-3 and caspase-9 was found in cells of well-known locations of programmed cell death. After retinoic acid exposure, expressions were increased especially in neural crest cells of mandibular and hyoid arches. Quantification of positive cells shows that caspase-9 and Hsp110 were expressed before caspase-3. After irradiation, the expression of the three proteins quickly increased with a maximum 3 h after irradiation. For all three models of apoptosis (physiological, retinoic-induced and irradiation-induced) HSP110 positive cells were more numerous than caspase-3 positive cells. Caspase-3 positive cells were more numerous than caspase-9 positive cells especially in mesectodermal irradiation-induced apoptotic cells.. The findings show a potential function of HSP110 in apoptosis during embryo development. Caspase-3-expressing cells are more numerous than cells expressing caspase-9, especially irradiation-induced apoptotic neural crest cells. This suggests that other caspases, still to be identified, may activate caspase-3 in this model.

Topics: Abnormalities, Drug-Induced; Abnormalities, Radiation-Induced; Animals; Apoptosis; Caspase 3; Caspase 9; Caspases; Craniofacial Abnormalities; Embryonic Development; Female; HSP110 Heat-Shock Proteins; Immunohistochemistry; Maxillofacial Development; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Neural Crest; Pregnancy; Tretinoin

Prenatal rat embryo exposure to retinoids induces severe malformations in various organs; the most active and teratogenic metabolite is all-trans-retinoic acid (atRA). The mechanisms of this embryopathy are only partly known. In the present study, the influence of glycine on the teratogenicity of atRA was investigated.. Embryos from 5 groups of white rats were studied: Group 1 remained untreated; Group 2 received glycine 2% in drinking water ad libitum from the first gestational day (GD 1); Group 3 was administered vehicle (corn oil); Group 4 was treated with atRA (50 mg/kg of body weight) injected (IP); and Group 5 was treated with atRA (50 mg/kg of body weight IP) plus glycine 2% in drinking water ad libitum from GD 1. atRA was administrated daily from GD 8-10. Dams were killed on the 21st day of pregnancy, and their fetuses were examined to detect external, visceral, and skeletal malformations.. The results show that the atRA-administered dose is not toxic for the dams, and that although fetal death was not observed, it produced abnormalities in the fetuses. Glycine reduced atRA-induced teratogenic effects (external and skeletal defects).. The results indicate that glycine effectively reduces the teratogenic effects of atRA. Thus, glycine might be useful for the prevention of vitamin A teratogenicity.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Drug Antagonism; Female; Fetal Death; Glycine; Glycine Agents; Maternal-Fetal Exchange; Pregnancy; Rats; Rats, Wistar; Teratogens; Tretinoin

Retinoic acid (RA) is a vitamin A derivative that participates in patterning and regulation of inner ear development. Either excess RA or RA deficiency during a critical stage of inner ear development can produce teratogenic effects. Previous studies have shown that in utero exposure of the developing mouse inner ear to a high dose of all-trans RA (atRA) results in severe malformations of the inner ear that are associated with diminished levels of endogenous transforming growth factor-beta1 (TGF-beta(1)) protein.. In this study, the effects of a teratogenic level of atRA on levels and patterns of expression of TGFbeta receptor II (TGFbetaRII) and Smad2, a downstream component of the TGFbeta signal transduction pathway, are investigated in the developing mouse inner ear. The expression pattern of endogenous RA receptor alpha (RARalpha) and the ability of an RARalpha(1)-specific antisense oligonucleotide (AS) to modulate otic capsule chondrogenesis are demonstrated in the inner ear and in culture.. Endogenous TGFbetaRII and Smad2 are downregulated in the inner ear following in utero atRA treatment. In addition, a reduction in endogenous TGFbeta(1) and a marked suppression of chondrogenesis occur in RARalpha(1) AS-treated cultures in comparison to untreated or oligonucleotide-treated control cultures. This chondrogenic suppression can be partially overcome by supplementation of RARalpha(1) AS-treated cultures with exogenous TGFbeta(1) protein.. Our findings support a role for TGFbeta in the physiological and pathological effects of RA on inner ear development.

Topics: Abnormalities, Drug-Induced; Animals; Chondrogenesis; DNA-Binding Proteins; Down-Regulation; Ear, Inner; Epithelium; Female; Gene Expression; Male; Mesoderm; Mice; Oligonucleotides, Antisense; Pregnancy; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Retinoic Acid; Receptors, Transforming Growth Factor beta; Retinoic Acid Receptor alpha; Signal Transduction; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans-retinoic acid (RA) was associated with altered expression of TGFalpha, EGF receptor, and binding of EGF. This study uses knockout (KO) mice to examine the roles of EGF and TGFalpha in teratogenic responses of embryos exposed to RA.. Pregnant wild-type (WT) mice of mixed genetic background, EGF KO, C57BL/6J, and TGFalpha KO mice were given a single oral dose of RA (100 mg/kg, 10 ml/kg) or corn oil on GD 10 at 12 PM, GD 11 at 12 PM or 4 PM, or GD 12 at 8 AM or 12 PM (plug day = GD 0). GD 18 fetuses were examined for external, visceral, and skeletal effects.. After exposure to RA on GD 12, the incidence of CP in EGF KO was significantly reduced relative to WT. In TGFalpha KO fetuses, RA exposure on GD 10 increased the incidence of CP versus C57BL/6J. The incidence of skeletal defects in the limbs, vertebrae, sternebrae, and ribs were also affected by lack of expression of EGF or TGFalpha with region-specific amelioration or exacerbation of the effects of RA. In TGFalpha KO fetuses, incidences of forelimb long bone and digit defects increased relative to C57BL/6J. In EGF KO fetuses, relative to WT, the incidence of hindlimb oligodactyly was increased. In EGF KO, but not WT, RA produced short, bent radius, humerus, and ulna. Both TGFalpha and EGF KO mice had increased incidences of dilation of the renal pelvis and this was reduced by RA.. RA exposure produced skeletal and visceral defects in all genotypes; however, EGF or TGFalpha KO influenced the incidence and severity of defects. This study supports a role for EGF and TGFalpha in the response to RA.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Cleft Palate; Epidermal Growth Factor; Female; Fetus; Kidney Pelvis; Mice; Mice, Knockout; Phenotype; Pregnancy; Prenatal Injuries; Transforming Growth Factor alpha; Tretinoin

The prevention of human neural tube defects by folic acid administration and the potential for fetal surgical intervention for myelomeningocele (MMC) have renewed interest in the molecular pathways and pathophysiology of spina bifida. Animal models for assessment of the early developmental biology and pathophysiology of this lesion are needed. The goal of this study was to develop and characterize a non-surgical rat model of MMC. Time-dated Sprague-Dawley rats were gavage fed different doses of retinoic acid (RA) dissolved in olive oil at E10 (maternal n = 55, fetal n = 505). Control animals received olive oil alone (maternal n = 20, fetal n = 265) or were untreated (maternal n = 5, fetal n = 63). Fetuses were analyzed by detailed histopathology and MRI. Overall, isolated MMC occurred in 60.7% (307/505) of RA-exposed fetuses and no controls. Histopathology confirmed the entire spectrum of severity observed in human MMC, ranging from exposure of the cord with intact neural elements to complete cord destruction. MRI of the brain of MMC fetuses confirmed structural changes similar to humans with Arnold-Chiari malformation, including downward displacement of the cerebellum to just above the foramen magnum and compression of the developing medulla into a small posterior fossa. In conclusion, the RA-induced rat model of MMC is developmentally and anatomically analogous to human MMC. This relatively efficient and cost-effective model of MMC should facilitate investigation of the developmental biology and pathophysiology of MMC, and may be useful for the evaluation of further strategies for prenatal treatment.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Arnold-Chiari Malformation; Brain; Cranial Fossa, Posterior; Disease Models, Animal; Female; Fetus; Magnetic Resonance Imaging; Meningomyelocele; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Dysraphism; Tretinoin

To explore the possible correlations between clinical and experimental pathological changes of congenital clubfoot and the pathodynamic developmental procedure.. Eighty-three female Wistar rats were administered with retinoic acid on the 10th day after pregnancy. And from February 2001 to February 2004, 48 patients were analyzed with electropysiological examination.. There was clubfoot-like deformity in 53.7% of the experimental fetuses. Persistence of the embryonic position of the talus and tibia in fetuses was observed. Poor overlapping between talus and calcaneus was seen. Cell apoptosis at the anterior corner of spinal cord were seen. Of all the patients, 68.3% were abnormal with electropysiological examination. The pathological sites were frequently localized in lumbarsacral region.. Congenital clubfoot is correlated closely with defects of neural tube and spinal cord.

Topics: Abnormalities, Drug-Induced; Animals; Anterior Horn Cells; Apoptosis; Child; Child, Preschool; Clubfoot; Female; Humans; Infant; Pregnancy; Rats; Rats, Wistar; Tretinoin

Topical tretinoin (Retin-A) is used to treat acne and photodamaged skin. Its teratogenic potential is of concern due to its similarity to isotretinoin (Accutane), a recognized human teratogen. Through the California Teratogen Information Service and Clinical Research Program, between 1983 and 2003, 106 pregnant women with first-trimester exposure to topical tretinoin were prospectively ascertained and followed. Birth outcomes, including pregnancy loss, major structural defects, and pre- and postnatal growth were compared to 389 similarly and prospectively ascertained women with no topical tretinoin exposure during pregnancy. Because a distinct pattern of malformation had already been described for isotretinoin, we also compared exposed (n = 62) and unexposed (n = 191) infants on the prevalence of a specific subset of minor malformations selected to represent the spectrum of defects comprising the retinoic acid embyopathy. There were no significant differences between groups in the proportion of pregnancies ending in spontaneous abortion (6.6% in exposed vs. 8.5% in unexposed; P = 0.53), or infants with major structural defects (2.2% in exposed vs. 1.2% in unexposed; P = 0.62). In addition, the groups were similar in birth weight, length and head circumference, and there were no significant differences between groups in length of gestation. Furthermore, the prevalence of one or more retinoic acid-specific minor malformations did not differ significantly between groups (12.9% in exposed vs. 9.9% in unexposed; P = 0.51). First-trimester topical tretinoin exposure in this study was not associated with an increased risk of any adverse pregnancy outcome evaluated. Specifically, there was no indication that topical tretinoin is associated with an increased risk for minor malformations that are consistent with the retinoic acid embryopathy. Although it is impossible to exclude the possibility that some women/infants may be uniquely susceptible to topical tretinoin exposure, this study provides further reassurance for women who are inadvertently exposed early in pregnancy.

Topics: Abnormalities, Drug-Induced; Adult; California; Cohort Studies; Female; Gestational Age; Humans; Infant; Infant, Newborn; Keratolytic Agents; Pregnancy; Pregnancy Trimester, First; Prenatal Exposure Delayed Effects; Prevalence; Prospective Studies; Tretinoin

(1) In 1998 several cases of malformations similar to those induced by oral retinoids were reported in children exposed in utero to topical retinoids (adapalene and tretinoin). The results of two somewhat flawed epidemiological studies were reassuring. (2) New cases of birth defects were subsequently reported in children exposed in utero to topical tretinoin. (3) Epidemiological data are still scant and unconvincing: they neither confirm this risk nor rule it out completely. (4) It is best to avoid using topical retinoids altogether in early pregnancy. Women of child-bearing age must be fully informed of the risks and the importance of effective contraception. This also applies to patients with moderate forms of psoriasis, for which topical tazaroten is indicated.

Topics: Abnormalities, Drug-Induced; Acitretin; Administration, Topical; Craniofacial Abnormalities; Female; France; Humans; Infant, Newborn; Maternal Exposure; Naphthalenes; Pregnancy; Prenatal Exposure Delayed Effects; Retinoids; Tretinoin

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.

Topics: Abnormalities, Drug-Induced; Aging; Aldehyde Oxidoreductases; Animals; Animals, Newborn; Body Patterning; Bromodeoxyuridine; Carcinoma; Cell Death; Cell Differentiation; Cell Line, Tumor; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Embryo, Mammalian; Embryonic Induction; Extremities; Female; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Galactosides; Gene Expression Regulation, Developmental; High Mobility Group Proteins; Homeodomain Proteins; In Situ Hybridization; In Situ Nick-End Labeling; Indoles; Limb Buds; Male; Mesoderm; Mice; Mice, Knockout; Models, Biological; Pregnancy; Retinoic Acid 4-Hydroxylase; SOX9 Transcription Factor; Time Factors; Trans-Activators; Transcription Factors; Transfection; Tretinoin

In humans, the presence of cervical and lumbar ribs is of particular clinical significance. However, the relevance of their occurrence in the offspring of experimental animals in reproductive toxicologic studies is poorly understood. Maternal toxicity has been implicated in the etiology but conclusive evidence is lacking. The present study was undertaken to determine the incidence of supernumerary ribs (SNR) in mouse fetuses prenatally exposed to valproic acid (VPA) and retinoic acid (RA), and to compare their differential developmental susceptibility and morphological association with other axial skeletal anomalies. Single doses of valproic acid (VPA) or retinoic acid (RA) were administered to groups of mice on one of gestation days (GD) 7-12. Fetuses were collected on GD 18 and their skeletons examined for SNR. VPA treatment on GD 7 and GD 8 resulted in a high incidence of cervical and lumbar ribs, respectively. Cervical neural arch anomalies in the GD 7 group, and eight pairs of sternal ribs and seven sternebrae in the GD 8 group were observed in excess of the background SNR suggesting a direct effect of VPA on the developing mouse skeletal system. In the RA groups, GD 8-12 were susceptible for lumbar rib induction but increased incidence of cervical ribs was observed only from GD 9-12. Peak incidence of cervical ribs was found in the GD 10 and 11 groups and that of the lumbar ribs in the GD 8 and 11 groups. Although SNR incidence generally increased with increasing dose of RA, a strict dose-response relationship was lacking. Cervical arch anomalies were observed in as many embryos as those with cervical ribs, but eight pairs of sternal ribs and seven sternebrae did not correlate well with the lumbar ribs in the peak day groups. Interrupted cervical neural arches correlated well with lumbar ribs. The reduction in the frequency of presacral vertebrae from 26 to 25 in the VPA groups was limited to GD 7 (30%) and 8 (18%) groups. RA-induced reduction in presacral vertebral number extended to GD 9 and was greater in the GD 8 than in the GD 9 groups. Sternal anomalies occurred both in VPA and RA experiments and did not strictly correlate with the frequency of SNR. VPA had a narrow window of susceptibility, whereas RA effects on sternum extended from GD 9-12. The incidence of sternal anomalies generally increased with increasing dose and advancing developmental stage at which RA exposure occurred. These developmental susceptibility windows and associated malf

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Mammalian; Fetus; Mice; Radiography; Ribs; Teratogens; Tretinoin; Valproic Acid

The objectives of this study were to (1) compare two techniques (computerized image analysis and visual morphological evaluation) for the assessment of fetal forelimb malformations and (2) increase the robustness of the dose-response curve for forelimb and cleft palate malformations resulting from all-trans retinoic acid (RA) exposure in GD 11 mice.. Pregnant CD-1 mice were administered a single oral dose of all-trans RA (0, 2.5, 10, 30, 60, or 100 mg/kg) on GD 11. GD 18 fetuses were examined for malformations using visual morphological scoring and computerized image analysis.. Dose-dependent changes occurred in the size and shape of the humerus, radius, and ulna based on both assessment methodologies. The most sensitive indicators for the lowest effect level (10 mg/kg) on forelimbs were roundness, a shape measurement determined by image analysis, and visual morphological scoring. For all other bone measurements (proximal and distal width, area, length, and perimeter), the lowest effect level was 30 mg/kg. The maximum effect for limb defects and total malformed fetuses was seen at 60 mg/kg and higher. Incidence of cleft palate increased over the entire range of administered doses reaching a maximum of 74% (100 mg/kg).. Overall, results indicate that computerized image analysis was no more sensitive in detecting changes in the humerus, radius, and ulna than gross visual examination. Dose-response modeling of developmental endpoints yielded comparable benchmark dose levels for long bones and cleft palate that ranged from 0.24 to 7.6 mg/kg all-trans RA. Birth Defects Res B 71:289-295, 2004.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Dose-Response Relationship, Drug; Female; Fetus; Forelimb; Image Processing, Computer-Assisted; Mice; Pregnancy; Tretinoin

The question of how alterations in cell behavior produced by retinoic acid (RA) influenced the development of skeletogenic mesenchyme of the limb bud was examined in this study. Our established model was employed, which involves treatment of pregnant mice with a teratogenic dose of RA (100 mg/kg) on 11 days postcoitum (dpc) resulting in a severe truncation of all long bones of the forelimbs in virtually every exposed fetus. It is shown that RA, administered at a stage to induce phocomelia in virtually all exposed embryos, resulted in immediate appearance of enhanced cell death within the mesenchyme in the central core of the limb bud, an area destined for chondrogenesis. The central core mesenchyme, which in the untreated limb buds experiences a sharp decline in cell proliferation heralding the onset of chondrogenesis, demonstrated a reversal of the process; this mesenchyme maintained a higher rate of cell proliferation upon RA exposure. These events resulted in a truncation and disorganization of the chondrogenic anlage, more pronounced in zeugopodal mesenchyme than in the autopod. We conclude that an inhibition of chondrogenesis was secondary to a disruption in cellular behavior caused by RA, a likely consequence of misregulation in the growth factor signaling cascade.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Apoptosis; Cell Proliferation; Chondrogenesis; Disease Models, Animal; Ectromelia; Female; Forelimb; Limb Buds; Male; Mesoderm; Mice; Mice, Inbred Strains; Pregnancy; Teratogens; Tretinoin

The fetal rat models with congenital clubfoot were constructed by treating 24 Wistar rats with all trans retinoic acid (ATRA). The MC-3T3-E1 cells were cultured with ATRA, 17 beta-estrogen (E2) or combinations of the two chemicals. The flow cytometer was used to determine the cell proliferation. The insulin-like growth factor-II (IGF-II ) and IGF conjugated protein-6 (IGFBP-6) mRNA level in rat calvaria bone tissue and MC-3T3-E1 cells were detected by northern blotting analysis and reverse transcription polymerase chain reaction. The congenital clubfoot of fetal rat was induced by ATRA in concentration of 100 approximately 140 mg/kg with dosage-dependence effect. The expression of IGF-II mRNA and cell proliferation were enhanced by E2(1 x 10(-6) mol/L) in rat calvaria bone tissue and MC-3T3-E1 cells, whereas the IGFBP-6 mRNA was increased. ATRA(1 x 10(-6) mol/L), however, inhibited the effect of E2 on regulation of IGF- II gene and IGFBP-6 gene as well as MC-3T3-E1 cell proliferation. These findings provide the evidence that ATRA can induce congenital skeleton malformation and congenital clubfoot in pregnant Wistar rats. IGF-II and IGFBP-6 are important regulative factors for skeleton development and osteoblast proliferation in rat.

Topics: Abnormalities, Drug-Induced; Animals; Cell Division; Clubfoot; Female; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor II; Male; Rats; Rats, Wistar; RNA, Messenger; Tretinoin

Previous studies in our laboratory identified retinoid-induced defects that are mediated by RAR-RXR heterodimerization using interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in mice (Elmazar et al. 1997, Toxicol Appl Pharmacol 146:21-28; Elmazar et al. 2001, Toxicol Appl Pharmacol 170:2-9; Nau and Elmazar 1999, Handbook of experimental pharmacology, vol 139, Retinoids, Springer-Verlag, pp 465-487). The present study was designed to investigate whether these RAR-RXR heterodimer-mediated defects can be also induced by interactions of natural and synthetic ligands for retinoid receptors. A non-teratogenic dose of the natural RXR agonist phytanic acid (100 mg/kg orally) or its precursor phytol (500 mg/kg orally) was coadministered with a synthetic RARalpha-agonist (Am580; 5 mg/kg orally) to NMRI mice on day 8.25 of gestation (GD8.25). Furthermore, a non-teratogenic dose of the synthetic RXR agonist LGD1069 (20 mg/kg orally) was also coadministered with the natural RAR agonist, all- trans-retinoic acid (atRA, 20 mg/kg orally) or its precursor retinol (ROH, 50 mg/kg orally) to NMRI mice on GD8.25. The teratogenic outcome was scored in day-18 fetuses. The incidence of Am580-induced resorptions, spina bifida aperta, micrognathia, anotia, kidney hypoplasia, dilated bladder, undescended testis, atresia ani, short and absent tail, fused ribs and fetal weight retardation were potentiated by coadministration of phytanic acid or its precursor phytol. Am580-induced exencephaly and cleft palate, which were not potentiated by coadministration with the synthetic RXR agonists, were also not potentiated by coadministration with either phytanic acid or its precursor phytol. LGD1069 potentiated atRA- and ROH-induced resorption, exencephaly, spina bifida, aperta, ear anotia and microtia, macroglossia, kidney hypoplasia, undescended testis, atresia ani, tail defects and fetal weight retardation, but not cleft palate. These results suggest that synergistic teratogenesis can be induced by coadministration of a natural RXR ligand (phytanic acid) with a synthetic RAR agonist (Am580). Thus, certain potentially useful therapeutic agents or nutritional factors such as phytanic acid should be tested for teratogenic risk by coadministration with other retinoid receptor agonists.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Benzoates; Bexarotene; Dose-Response Relationship, Drug; Drug Synergism; Female; Gestational Age; Ligands; Mice; Mice, Inbred Strains; Phytanic Acid; Phytol; Pregnancy; Prenatal Exposure Delayed Effects; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Teratogens; Tetrahydronaphthalenes; Tretinoin; Vitamin A

This study investigated the effects of dietary retinoic acid (RA) on frog hindlimb development. Xenopus laevis (African clawed frog) tadpoles were fed a diet supplemented with 0, 1, 10, or 100 microg of RA/g of food for 2 or 5 d at different stages of metamorphosis. Hindlimb deformities were induced in the group fed 100 microg of RA/g of diet for 5 d. Exposures beginning at mid-hindlimb bud development induced bilaterally bent tibiafibula (bony triangles), while exposures later in hindlimb development induced deformities of the feet, including fusion of the 1st and 2nd clawed digits and reduced length of the 4th and 5th digits (due to reduced, missing, or misplaced phalanges). There were also cases of extra phalanges in the 5th digit. The eye was another target of RA exposure. In one experiment, 58% of the tadpoles fed 10 microg of RA/g had a smaller or absent right eye. Additionally, 11% of the tadpoles fed 100 microg of RA/g of diet developed a smaller or absent left eye. Waterborne heavy metals (Zn or Cu) modified RA effects on the hindlimb and eye. Co-exposure to metals and RA resulted in cases of unilateral bony triangles and reduced rates of smaller eyes. There were also cases of extra hindlimb digits in Zn-exposed animals. Dietary RA exposure in tadpoles can cause some deformities that differ from waterborne RA exposures in previous studies. RA also induced deformities that resemble those in affected wild frog populations (bony triangles), although the patterns of other deformities and missing segments (phalanges and metatarsals) are not similar to those documented in the wild.

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Eye Abnormalities; Hindlimb; Metals, Heavy; Metamorphosis, Biological; Tretinoin; Xenopus laevis

In this study we analyzed, on an experimental model, blastemic changes occurring during the embryonic period that may later cause fetal hindlimb deformity. Experimental induction of clubfoot-like deformity in rat fetuses was performed by maternal administration of retinoic acid (RA) (120 mg/kg body weight) as a single intragastric dose on day 10 of pregnancy (previous phase of this assay). The caudal and hindlimb blastemic changes were studied by mitosis count and stereological, immunohistochemistry and AgNOR techniques in the hindlimbs of 15-day embryos and in the caudal somites of 11-day embryos. In 15-day embryos from the assay group, hypoplasia and misorientation of hindlimbs were present in 90% of the cases. The histological study showed a blastemal defect as follows: (a) reduction in mesenchymal cell activity (mitotic and AgNOR activities); (b) increase of volume of vascular lumen; (c) reduction in volume of nerve structures, and (d) reduction in the percentage of pre-rhabdomyoblastic cells. In 11-day embryos from the assay group, caudal somites showed disruption, including loss of usual morphology. Moreover, somitic AgNOR activity decreased compared to the control group. The greatest reduction in the number of black-dots per cell was in the myotome. These findings suggest that a certain pathology of the somites, very little studied to date, might be involved in clubfoot pathogenesis.

Topics: Abnormalities, Drug-Induced; Animals; Clubfoot; Embryonic and Fetal Development; Female; Hindlimb; Immunohistochemistry; Limb Deformities, Congenital; Mitosis; Nucleolus Organizer Region; Rats; Rats, Wistar; Time Factors; Tretinoin

Recently, high frequencies of malformations have been reported in amphibians across the United States. It has been suggested that the malformations may be the result of xenobiotic disruption of retinoid signaling pathways during embryogenesis and tadpole development. Therefore, a series of experiments were undertaken to examine life-stage specific effects of continuous retinoid exposure on Xenopus laevis. Continuous all-trans retinoic acid (RA) concentrations were delivered using a column saturator and a flow-through diluter system. Stage 8 embryos were exposed to RA concentrations ranging from 0.013 to 2 microgram/l. At the onset of hindlimb bud emergence (NF stage 48), a subset of tadpoles was moved to clean water, and remaining organisms were exposed continuously through metamorphosis. In addition, early limb-bud-stage tadpoles were exposed for 1 week, 2 weeks, or until tail resorption was complete, to eight concentrations of RA in the range of 0.031-3 microgram/l. RA exposure resulted in a concentration-dependent increase in mortality and dysmorphogenesis in embryos at concentrations of 0.24 microgram/l and above. However, this early embryonic exposure did not result in hindlimb abnormalities in surviving tadpoles allowed to mature in clean water. RA did not induce limb malformations in any surviving tadpole exposed during larval stages. We are confident that the concentrations used were high enough, given that the highest concentration used resulted in 100% mortality within 2 weeks of initiating the exposure. This result suggests that other aspects of growth and development, which are not externally obvious, are more sensitive to retinoids than skeletal development. From these experiments and our previous work, we conclude that it is unlikely that retinoid mimics would produce the spectrum of limb malformations which recently have been observed in amphibians collected from the field.

Topics: Abnormalities, Drug-Induced; Animals; Body Weight; Embryonic and Fetal Development; Extremities; Larva; Metamorphosis, Biological; Receptors, Retinoic Acid; Tretinoin; Water; Xenopus laevis

The response to exposure of all-trans-retinoic acid (RA) during development varies from physiologic to severe teratogenic outcomes and is dependent upon the dose and the stage of development in all species. Effects of RA-mediated teratogenesis may be due to its ability to cause apoptosis. We have recently reported the modulation of p53 in murine stem cells by RA. The aim of this study was to characterize the temporal and spatial pattern of p53 expression in Swiss Webster mouse fetuses following maternal treatment with a single oral dose of 100 mg/kg body weight of RA during organogenesis. RA treatment resulted in a decreased p53 mRNA level in fetuses 24, 48, and 72 h after maternal treatment as detected by semiquantitative reverse transcriptase polymerase chain reaction. Western blot analysis showed a decrease in p53 protein at 24 and 48 h. Immunohistochemistry revealed decreased localization of p53 in the neuroepithelium of fetuses exposed to RA in utero. RA treatment also resulted in decreased nuclear p21 and decreased expression of cytosolic as well as nuclear p27 at 72 h in the fetuses. These results demonstrated that RA-mediated teratogenesis is accompanied by a reduction in the temporal and spatial pattern of p53 gene and protein expression in addition to the disruption of the cell cycle by modulation of p21 and p27.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Blotting, Western; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Down-Regulation; Female; Gene Expression Regulation, Developmental; Genes, p53; Immunoenzyme Techniques; Maternal Exposure; Mice; Microfilament Proteins; Muscle Proteins; Organogenesis; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Teratogens; Tretinoin; Tumor Suppressor Protein p53

During formation of the secondary palate, clefting may result when critical developmental events are altered. The purpose of this study was to reduce the incidence of retinoic acid (RA)-induced cleft palate (CP) in mice by the co-administration of folic acid (FA), methionine (ME) or a combination of both.. Four groups of time-pregnant Swiss Webster mice were injected intraperitoneally with 50 mg/kg RA on gestational day (GD) 10. Likewise, 4.0 mg/kg FA and 187 mg/kg ME were administered on GD 8-11. The experiment included a control group (RA plus H2O) and three experimental groups, (RA plus therapeutic doses of FA, ME, or FA + ME). Necropsies were carried out on GD 18 and pups were analyzed for teratogenic effects.. Litters that received no therapy exhibited 100% CP with individual pups showing 76% susceptibility. Each therapy administered separately reduced the frequency of CP to approximately 6%, and the combination of FA and ME completely prevented the occurrence of RA-induced cleft palate (0%). A second experiment was conducted in which therapy levels were decreased by 25%. Litters that did not receive therapy experienced 100% clefting and individual pups exhibited CP at 86%. These therapies administered separately did not alter significantly the frequency of cleft palate. The combined doses of FA and ME, however, lowered significantly the frequency of cleft palate to 46%. Decreases in limb and tail defects with FA + ME therapy were also observed in both experiments.. Although FA and ME, at appropriate levels, can reduce individually the frequency of RA-induced cleft palate and other defects in mice, the results from the present study suggest that there is an additive interaction between the two therapeutic agents that can reduce further the teratogenic impact of RA. Further studies are needed to assess the mechanism of action of concomitant doses of FA and ME in the reduction of drug-induced birth defects.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Cleft Palate; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Female; Folic Acid; Injections, Intraperitoneal; Methionine; Mice; Pregnancy; Teratogens; Tretinoin

Abnormalities in the pontine nuclei (PN) and inferior olive are hallmarks of human retinoic acid (RA) teratogenesis. This study shows that RA exposure of the mouse at a specific embryonic stage alters morphological structures that derive from the wall of the IVth ventricle to form components of the precerebellar system (the inferior olivary nucleus and the PN). The study employs both normal and a RAREhspLacZ transgenic RA reporter mouse. It is shown that abnormalities in the PN and inferior olive result from exposure at a critical period of embryonic day 9.5 and 10.5. The abnormalities in the PN are due to a failure in their usual neurophilic migration. The compact stream of cells that leads from the anterior rhombic lip to the ventral pons is instead scattered widely over the anterior medulla. Given that the RA exposure occurs after the resolution of rhombomere identity this suggests that teratogenic RA interferes with a regulatory event that overlays this original pattern.

Topics: Abnormalities, Drug-Induced; Animals; Cell Movement; Genes, Reporter; Mice; Mice, Transgenic; Neurons; Receptors, Retinoic Acid; Rhombencephalon; Tretinoin

To investigate the morphologic changes of embryonic palatal development exposed to retinoic acid (RA) in mouse, and to detect the significance of the expression of TGFbeta1, TGFbeta3, EGF and BCL2.. The stage of palatal development was examined by light microscopy. S-P immunohistochemistry and in-situ hybridization was used to detect spatio-temporal patterns of expression of TGFbeta1, TGFbeta3, EGF and BCL2 in embryonic palate.. The fetus exposed to RA resulted in formation of small palatal shelves without contact and fusion of each other to form and intact palate. RA can regulate the embryonic palatal expression of genes involved in RA-induced cleft palate.. RA can inhibit the proliferation of MEPM cell to form small palatal shelves and induce abnormal differentiation of MEE cell causing the bi-palatal shelves no contact and fuse with each other, then induce the formation of cleft palate. RA can regulate the spatio-temporal patterns of expression of TGFbeta1, TGFbeta3 and EGF in embryonic palatal processes and the change of special expression of these genes in embryonic palatal processes are involved in RA-induced cleft palate.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Embryo, Mammalian; Epidermal Growth Factor; Female; Mice; Mice, Inbred C57BL; Palate; Transforming Growth Factor beta; Tretinoin

Prenatal exposure of rat embryos to retinoic acid induces severe malformations involving various organs. The mechanisms of this embryopathy are known only in part. This study describes the malformations of the neural crest-derived organs in this model and shows that many of them fit into the pattern of disturbed neural crest organogenic control. Pregnant rats were exposed to either all-trans retinoic acid (125 mg/kg; n=17) or vehicle ( n=10) on E10. Fetuses were recovered on E21 and external and internal malformations were sought. The craniofacial area, the trachea, parathyroids, thymus, thyroid, heart, great vessels, and adrenals were examined. In contrast with normal controls, 100% of retinoic acid animals had craniofacial, 94% anorectal, 90% limb, and 55% neural tube defects. The thymus was absent or ectopic in 76%, the parathyroids were absent or single in 88%, and the thyroid was abnormal in 41%. There were neural crest-type (outflow tract and/or pharyngeal aortic arch defects) cardiovascular malformations in 90% and the adrenals were absent in 52%. Interestingly, 9 of 11 (88%) animals with neural tube defects had absent adrenal glands. This association was significant ( p<0.01) by Fisher exact test. Among the complex mechanisms of retinoic acid teratogenesis, severe disturbances of the neural crest pathway play a leading role. The simultaneous development of neural tube defects and adrenal agenesis suggests common pathogenic pathways.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Drug Evaluation, Preclinical; Female; Fetus; Models, Animal; Neural Crest; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Tretinoin; Vitamin A

Retinoic acid (RA) alters the developmental fate of the axial skeletal anlagen. "Anteriorizations" or "posteriorizations," the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered embryonal expression domains of Hox genes after in utero RA treatment. These "homeotic" changes have been hypothesized to result from alterations of a "Hox cod" which imparts positional identity in the axial skeleton. To investigate whether such developmental alterations were specific to RA, or were a more general response to xenobiotic exposure, CD-1 pregnant mice were exposed to RA, valproic acid (VA), or bromoxynil (Br) during organogenesis. Additionally, the expression domains of two Hox genes, Hoxa7 and Hoxa10, were examined in gestation day (GD) 12.5 embryos obtained from control, RA, VA, or Br, treated gravid dams exposed on GD 6, 7, or 8. The anterior expression boundary of Hoxa7 is at the level of the C7/T1 vertebrae and that of Hoxa10 is at L6/S1. Compound-induced changes in the incidence of skeletal variants were observed. These included supernumerary cervical ribs (CSNR) lateral to C7, 8 vertebrosternal ribs, supernumerary lumbar ribs (LSNR) lateral to L1, extra presacral vertebrae, and the induction of vertebral and/or rib malformations. RA and VA administration on GD 6 caused posteriorization in the cervico-thoracic region (CSNR) while GD 8 exposure to any of the three compounds resulted in anteriorizations in the thoraco-lumbar area (LSNR and an increase in the number of presacral vertebrae). These effects occurred across regions of the axial skeleton. Analysis of gene expression demonstrated changes in the anterior boundaries of Hoxa7 expression domains in embryos treated on GD 6 and 8 with RA. VA and Br did not induce any statistically significant alterations in Hoxa7 and none of the compounds caused alterations in Hoxa10 expression domains. The studies indicate that RA GD 6 treatment-induced Hoxa7 shifts were rostral (posteriorization) while the RA-induced GD 8 anterior expression boundary shift was caudal (anteriorization), correlating with the axial skeletal changes noted. These data suggest that xenobiotic compounds such as VA and Br may induce similar axial skeletal changes by affecting different components of the developmental processes involved in the patterning of the axial skeleton.

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Developmental; Genes, Homeobox; Gestational Age; In Situ Hybridization; Litter Size; Mice; Mice, Inbred Strains; Nitriles; Pregnancy; Spine; Teratogens; Time Factors; Tretinoin; Valproic Acid

Maternal diabetes increases the risk of congenital malformations in the offspring of affected pregnancies. This increase arises from the teratogenic effect of the maternal diabetic milieu on the developing embryo, although the mechanism of this action is poorly understood. In the present study, we examined whether the vitamin A metabolite retinoic acid (RA), a common drug with well-known teratogenic properties, may interact with maternal diabetes to alter the incidence of congenital malformations in mice. Our results show that when treated with RA, embryos of diabetic mice are significantly more prone than embryos of nondiabetic mice to develop caudal regression, a defect that is highly associated with diabetic pregnancy in humans. By studying the vestigial tail (Wnt-3a(vt)) mutant, we provide evidence that Wnt-3a, a gene that controls the development of the caudal region, is directly involved in the pathogenic pathway of RA-induced caudal regression. We further show that the molecular basis of the increased susceptibility of embryos of diabetic mice to RA involves enhanced downregulation of Wnt-3a expression. This positive interaction between RA and maternal diabetes may have implications for humans in suggesting increased susceptibility to environmental teratogens during diabetic pregnancy.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Diabetes Mellitus, Experimental; Down-Regulation; Female; Genetic Predisposition to Disease; Mice; Mice, Inbred ICR; Mutation; Pregnancy; Pregnancy in Diabetics; Proteins; Teratogens; Tretinoin; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

An integral component of developmental toxicity studies is the evaluation of fetal anatomy, which consists of external, visceral, and skeletal examinations. The visceral examination includes an assessment of the fetal head which, in the rat, is typically completed after chemical fixation. Because chemical fixation requires approximately 7 days, a comprehensive visceral examination, including the head, of the fetal rodent cannot be completed at the time of cesarean section. An alternative method to chemical fixation was desired, whereby one could complete an overall visceral examination at the time of cesarean section. In addition, the method would also have to present fetal cranial structures in a manner consistent with that derived after chemical fixation.. Pregnant Sprague-Dawley rats were administered either 6-aminonicotinamide (6AN), 6 or 12 mg/kg on gestation day 13, or all-trans retinoic acid (RA) 5 or 25 mg/kg on gestation days 9 and 10, untreated dams served as a control group. On gestation day 20, fetuses were sacrificed and removed via cesarean section and one-half of the fetuses from each litter were placed in Bouin's solution (chemical fixation) and the remaining fetuses maintained under refrigeration until just before frozen tissue preparation (freeze fixation). Sectioning of the fetal head was similarly conducted after either chemical or frozen-fixation. Fetal cranial findings observed after chemical fixation were compared to those observed after frozen-fixation in the untreated control and 6AN and RA-treated groups.. The incidence and severity of the cranial findings, including effects on the eye, brain, and palate, were similarly observed, regardless of fixation method.. A frozen sectioning method for evaluation of the fetal head, yielding results comparable to those derived after chemical fixation, is described. This procedure provides a viable alternative to chemical fixation, and allows the teratologist to complete a comprehensive fetal visceral examination at the time of cesarean section.

Topics: 6-Aminonicotinamide; Abnormalities, Drug-Induced; Animals; Brain; Embryonic Structures; Female; Fixatives; Frozen Sections; Pregnancy; Rats; Rats, Sprague-Dawley; Teratogens; Tissue Fixation; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Ectromelia; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Inbred Strains; Models, Biological; Oligonucleotide Array Sequence Analysis; Research Design; Statistics as Topic; Teratogens; Transcription Factors; Tretinoin

Phocomelia, which is primarily due to a disruption in the proximodistal axis, is found in virtually all mouse embryos exposed to high doses of retinoic acid (RA) on 11 days post coitum (dpc).. To identify genes that potentially mediate the effects of retinoic acid (RA) on limb development, we have examined the expression of 9,000 clones from the IMAGE consortium by microarray analysis of RNA isolated from 11 dpc mouse forelimbs exposed to RA or vehicle for 6 hr. Eight genes that demonstrated altered expression were chosen for further study of their mRNA levels using RT-PCR. Protein levels were determined by Western blot analysis.. Of the 9,000 genes examined in the microarray, approximately 111 demonstrated altered expression (33 known genes and 78 ESTs). Of the eight known genes chosen for further study using RT-PCR, four mRNAs (PBX1a, PBX1b, IGF-Ia, and IGF-Ib) demonstrated consistent elevation ( approximately 3-fold) in their levels after RA treatment in both the forelimbs and hindlimbs as early as 3 hr after RA treatment. In addition to the two PBX1 isoforms, the mRNA level of the other two subtypes (PBX2 and PBX3) and the level of PBX1/2/3 protein were also found to be elevated in limb buds after RA treatment. Finally, we examined the expression of MEIS1, MEIS2, and MEIS3 because these proteins are necessary for PBX nuclear localization. The mRNA level of all three subtypes of MEIS were elevated approximately three- to four-fold in both the forelimbs and hindlimbs after RA treatment.. Because both PBX and MEIS (and their orthologs) are believed to be involved in the control of proximodistal axis formation in mouse and fly limbs and IGFs in the development of limbs, we suggest that increases in PBX, MEIS and IGF-1 mRNA levels may contribute to proximodistal limb reduction defects caused by teratogenic doses of RA.

Topics: Abnormalities, Drug-Induced; Animals; Ectromelia; Female; Gene Expression Regulation, Developmental; Homeodomain Proteins; Insulin-Like Growth Factor I; Limb Deformities, Congenital; Maternal-Fetal Exchange; Mice; Mice, Inbred Strains; Oligonucleotide Array Sequence Analysis; Pregnancy; Protein Isoforms; RNA, Messenger; Teratogens; Transcription Factors; Tretinoin

Previous studies in our laboratory showed a synergistic interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in regard to teratogenic effects produced in mice (M. M. Elmazar et al., 2001, TOXICOL: Appl. Pharmacol. 170, 2-9). In the present study the influence of phytol and phytanic acid (a RXR-selective ligand) on the teratogenicity of retinol and the RAR-selective ligand all-trans-retinoic acid was investigated by coadministration experiments on day 8.25 of gestation in NMRI mice. Phytol and phytanic acid, noneffective when administered alone, did not potentiate the teratogenicity induced by retinol or all-trans-retinoic acid. On the contrary, phytol and phytanic acid greatly reduced retinol-induced teratogenic effects (ear anotia, tail defects, exencephaly). The effect of phytol on all-trans-retinoic acid teratogenesis was limited (only resorptions and tail defects were reduced). Pharmacokinetic studies in nonpregnant animals revealed that phytol coadministration with retinol reduced plasma levels of retinol and retinyl esters, and drastically reduced the levels of the teratogenic retinol metabolite, all-trans-retinoic acid. Phytanic acid also reduced the oxidative metabolism and teratogenic effects of retinol. These results indicate that phytol and phytanic acid did not synergize with retinol and all-trans-retinoic acid in our mouse teratogenesis model. Instead, phytol and phytanic acid effectively blocked the teratogenic effects of retinol by drastically reducing the metabolic production of all-trans-retinoic acid. Phytol and phytanic acid may be useful for the prevention of vitamin A teratogenicity.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Chromatography, High Pressure Liquid; Drug Synergism; Female; Mice; Mice, Inbred Strains; Phytanic Acid; Phytol; Pregnancy; Prenatal Exposure Delayed Effects; Teratogens; Tretinoin; Vitamin A

Retinoic acid has been associated with a number of cardiac defects, some of which seem to be related to changes in the endocardial cushions. Studies in mice and older chick embryos have suggested that these defects may be associated with a decrease in mesenchymal cell formation within the cushion. In a previous report we showed that retinoic acid lowered the number of mesenchymal cells in a culture bioassay of mesenchyme formation and that this response was due to retinoic acid modifying the production of particulate matrix from the myocardium. In this study, we have extended these observations to the embryo by implanting a retinoic acid coated bead into the embryo and examined the effect on cardiac mesenchyme formation and in the production of the particulate matrix. In all cases the addition of retinoic acid resulted in a decrease in the number of mesenchymal cells invading the endocardial cushions. In addition retinoic acid increased the production of hLAMP-1 and fibronectin but not transferrin, confirming our earlier report. Finally, we measured the volume of the cushion and calculated the cell density of both the inferior and superior cushions. The results suggest that the superior cushion is more sensitive to retinoic acid treatment than the inferior cushion. Collectively, these results support our earlier work that suggests that the mechanism of retinoic acid cardiac abnormalities involves a disruption in the production of particulate matrix from the myocardium and a subsequent decrease in cardiac mesenchyme cells that results in a malformed cardiac cushions.

Topics: Abnormalities, Drug-Induced; Animals; Cell Count; Chick Embryo; Endocardial Cushion Defects; Endocardium; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Heart; Mesoderm; Microscopy, Fluorescence; Microscopy, Video; Transferrin; Tretinoin

The process of fetal external genitalia development might be divided into two processes. The first process accomplishes the initial outgrowth of the anlage, genital tubercle (GT). Previous analysis suggests that the distal urethral epithelium (DUE) of the GT, the Fgf8-expressing region, regulates the outgrowth of the GT. The second process eventually generates the sexually dimorphic development of the external genitalia, which is dependent on the action of steroid hormones. Several key genes, for example, RARs, RXRs, RALDH2, and CYP26, were dynamically expressed during GT development. The teratogenic dose of RA at 9.0 d.p.c. induced a drastic malformation of the urethral plate during GT formation, but did not show gross abnormalities in its outgrowth. In RA-treated embryos, Fgf8 expression was still detected in the distal GT regions. Possible regulatory roles of the FGF and RA signaling systems in external genitalia formation are discussed.

Topics: Abnormalities, Drug-Induced; Animals; Epithelium; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Genitalia, Female; Genitalia, Male; In Situ Hybridization; Male; Mice; Mice, Inbred ICR; Microscopy, Electron, Scanning; Pregnancy; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Urethra

Retinoids are involved in several physiological processes and are used in the treatment of various skin disorders. Therapy with retinoids during pregnancy may induce severe embryotoxic effects like craniofacial and cardiovascular malformations in the developing embryo. We investigated the effects of all-trans-retinoic acid (ATRA) and all-trans-retinoyl-beta-D-glucuronide (ATRAG) in an amphibian embryotoxicity assay with Ambystoma mexicanum (axolotl) as an alternative in vitro method. Embryos were exposed to various concentrations of ATRA or ATRAG for 48 h beginning with the blastula stage. Kinetic investigations in the embryonic tissue were performed during the exposure period. Both retinoids interfered with the development of the axolotl embryos. Dose-dependent effects observed included growth retardation, craniofacial and cardiovascular malformations, as well as neural tube defects. In the axolotl, ATRA induced slightly more pronounced embryotoxic effects than ATRAG. All-trans-retinal was shown to be a major endogenous retinoid in this species. Endogenous levels of all-trans-retinaldehyde were increased during exposure to both ATRA and ATRAG. The glucuronide, however, was only detected in small amounts after ATRAG exposure. The embryotoxic potential of ATRAG could be explained by deglucuronidation to ATRA.

Topics: Abnormalities, Drug-Induced; Ambystoma; Animal Testing Alternatives; Animals; Culture Techniques; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Female; Male; Tretinoin

For this study all-trans-retinoic acid was administered in pregnant white rats in their "prima gravida" pregnancy. Rats were divided in five groups. The first three groups were treated with 20 mg R.A./kg b.w. at several gestational days. The fourth group was treated with corn oil, while the fifth group remained untreated. All the animals were sacrificed during the first hours of the 21st gestational day. In the first group, three embryos, five absorptions and six compact embryonic masses were counted in litters. All the embryos presented exencephaly, combined with external anopthalmia. They also presented severe craniofacial malformations. In the second group, nine embryos and five compact embryonic masses were counted in litters. Three of the embryos presented exencephaly combined with external anopthalmia, while the six remaining presented complex craniofacial anomalies. In the third group, exencephaly was present in two embryos combined with anopthalmia, seven embryos had complex anomalies and four compact embryonic masses were counted in litters. Our results indicate the teratogenic involvement of all-trans-retinoic acid in anterior neural tube differentiation.

Topics: Abnormalities, Drug-Induced; Animals; Female; Neural Tube Defects; Pregnancy; Rats; Rats, Wistar; Tretinoin

Diverse studies on retinoic acid teratogenesis, during the recent years, indicate that the drug's analogues target on diverse cell population during differentiation in mammals. During an extended teratological protocol concerning retinoic acid influence in diverse embryonic tissue differentiation in experimental animals we studied all-trans-retinoic acid's influence on palatal development in the white rat embryo. For this purpose, six groups of white rat embryos were studied: Group 1 was treated with 100 mg/kilogram of body weight (k.b.w.) on gestational days (g.d.) 10th and 11th, Group 2 was treated with 100 mg all-trans-retinoic acid/k.b.w. on g.d. 11.5, Group 3 was treated with 50 mg all-trans retinoic acid/k.b.w. on g.d. 10th, 11th and 12th, Group 4 was treated with 50 mg all-trans-retinoic acid/k.b.w. on g.d. 11th and 12th, Group 5 was treated with 20 mg all-trans-retinoic acid/k.b.w. on g.d. 7.5, 8.5, 9.5, 10.5 and 11.5, Group 6 remained untreated. Embryonic heads aged 20 days were observed by light microscopy and scanning electron microscopy. In all treated groups clefts and malformations concerning the differentiation of palatal cell populations were observed. All our findings were compared with normal palatal morphology of untreated "control" embryos. Among the malformations, median clefts were observed, extended along only a part of the primary and all the secondary palate for group 2, the primary and secondary palate for groups 1, 3 and 5 while on group 4, an irregularity of the median palatal raphe and rugae were combined with a median incomplete cleft extended between the primary and secondary palate. Our results are discussed in relation with the international literature results.

Topics: Abnormalities, Drug-Induced; Animals; Female; Microscopy, Electron, Scanning; Palate; Rats; Rats, Wistar; Tretinoin

Retinoic acid (RA) is necessary for normal differentiation of the tail bud into the secondary neural tube. Excess RA, however, is teratogenic and causes neural tube defects (NTDs). The way in which RA modulates secondary neurulation is unclear but probably involves RA-regulated downstream genes such midkine (MK), which encodes a growth factor implicated in tail bud mesenchymal-neuroepithelial conversion. Our objective was to determine whether RA-deficiency would produce similar defects and if MK is involved.. Citral, a drug that blocks endogenous RA formation, as well as a neutralizing antibody, were used to block RA activity in chick embryos. Immunohistochemistry and in situ hybridization were used to localize RA and MK in the tail bud. Competitive RT-PCR was used to examine the effects of excess RA and RA deficiency due to citral on the expression of MK mRNA.. Citral-induced NTDs displayed a morphological resemblance to those caused by excess RA. However, citral treatment did not significantly increase embryonic mortality, and RA rescue of citral-treated embryos proved unsuccessful. MK mRNA was detected in the differentiating tail bud by in situ hybridization. Competitive RT-PCR showed that excess RA decreased MK expression by 60%. Doses of citral that caused a comparable incidence of defects, however, caused only a 25% decrease.. The results show that excess RA and RA deficiency both cause defects of secondary neurulation. While excess RA decreased MK expression, RA deficiency had minimal effects. However, whether or not MK is an intermediary in the developmental phenomena regulated physiologically or pathologically by RA remains to be elucidated.

Topics: Abnormalities, Drug-Induced; Acyclic Monoterpenes; Animals; Carrier Proteins; Chick Embryo; Cytokines; Dose-Response Relationship, Drug; Immunohistochemistry; In Situ Hybridization; Keratolytic Agents; Midkine; Monoterpenes; Nerve Growth Factors; Neural Tube Defects; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tail; Teratogens; Terpenes; Time Factors; Tretinoin; Vitamin A Deficiency

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Embryo, Mammalian; Female; Humans; Mice; Pregnancy; Tretinoin

Abnormal dermatoglyphs on human volar skin have been reported in many syndromes, but little is known about the pathogenesis. Patterns of pads on rodent limb volar skin are homologous to human dermatoglyphs.. In previous studies, we showed that transplacental exposure to teratogens induced abnormal pads in mouse fetuses. Moreover, teratogens caused abnormal pad patterns at levels below those that caused skeletal malformations. In this study, we examined morphology and cytokinetics in developing abnormal pads. Pregnant mice were treated with all-trans-retinoic acid at 20 mg/kg orally at embryonal day (E) 12.5 (vaginal plug = E0). The hindlimbs of the embryos were harvested and observed under a light microscope and by scanning electron microscopy. Cell proliferation and cell death were estimated by 5-bromo-2'-deoxyuridine (BrdU) labeling, Nile blue A vital staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).. Retinoic acid induced aplasia of the fibular tarsal pad and supernumerary interdigital pads on hindlimbs. Cell proliferation was observed in the area of developing pad, but cell death was very rarely seen in either normal or abnormal pads.. Retinoic acid disturbed pad patterning as a whole rather than individual pad formation.

Topics: Abnormalities, Drug-Induced; Animals; Bromodeoxyuridine; Cell Death; Cell Survival; Embryo, Mammalian; Female; Fibula; Foot Deformities, Congenital; Hindlimb; In Situ Nick-End Labeling; Mice; Microscopy, Electron, Scanning; Pregnancy; Skin; Skin Abnormalities; Tretinoin

Within the last decade, there have been increasing reports of malformed amphibians across North America. Recently, it has been suggested that hind-limb malformations are a consequence of xenobiotic disruption of developmental pathways regulated by retinoids. To assess the validity of this hypothesis, the developmental toxicity of all-trans retinoic acid (RA) was examined in Xenopus laevis and four North American anurans, at several life stages. To determine the effects of RA on embryogenesis, mid-blastula stage embryos were exposed to 0, 6.25, 12.5, 25, or 50 ng RA/ml for 24 h. To evaluate the effects of RA on hind-limb development, early- and mid-limb bud stage tadpoles were exposed to RA concentrations of 0, 250, 500, 750, 1000, or 1250 ng RA/ml for 24 h. Mid-blastula RA exposure resulted in a concentration-dependent increase in dysmorphogenesis and mortality in the three species examined (R. clamitans, R. septentrionalis and X. laevis). RA exposure at stage 51 in X. laevis and stage 28 in R. sylvatica resulted in concentration-dependent increases in reductions and deletions of the hind limb. However, RA was ineffective at inducing hind-limb abnormalities in stages 26 and 28 of R. pipiens, stage 28 in R. clamitans, or stage 48 in X. laevis tadpoles. These results indicate that mid-blastula stage embryos are more sensitive to RA-induced dysmorphogenesis and mortality than limb-bud stage tadpoles. The significance of these findings is discussed in the context of the possible occurrence of retinoid mimics in the environment.

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Female; Hindlimb; Limb Buds; Male; Ranidae; Species Specificity; Survival Rate; Tretinoin; Xenopus laevis

Hyperhomocysteinemia is frequently associated with congenital defects of the heart and neural tube and is a suspected pathogenic factor in atherosclerosis and neoplasia. Results in the present report show homocysteine treatment disrupts normal development of avian embryos; and this effect is prevented by retinoic acid. Based on this, we hypothesize that homocysteine may exert its teratogenic effects by disrupting retinoic acid signaling during development. A reporter cell line transfected with a retinoic acid response element (RARE) linked to a lacZ reporter gene was used to identify the site of retinoid inhibition. Using this reporter cell line, we show that homocysteine inhibits the oxidation of retinal to retinoic acid with concentrations of homocysteine that are in the pathophysiological range (.05 to 0.5 mM). In contrast, homocysteine concentrations as high as 5 mM are unable to inhibit the induction of lacZ by retinoic acid. We show that cellular uptake of homocysteine is sensitive to the specific L-system transport inhibitor, bicycloheptane, and bicycloheptane blocks the inhibition of retinoic acid synthesis by homocysteine, demonstrating that this inhibition occurs intracellularly. These results suggest that homocysteine-induced congenital defects are due to the specific ability of homocysteine to inhibit conversion of retinal to retinoic acid.

Topics: Abnormalities, Drug-Induced; Animals; Biological Transport, Active; Cell Line; Chick Embryo; Genes, Reporter; Homocysteine; Hyperhomocysteinemia; Lac Operon; Receptors, Retinoic Acid; Signal Transduction; Transfection; Tretinoin

Malformations of the palate were induced in white rat embryos following maternal exposure to retinoic acid (tretinoin). Five experimental groups and the controls were treated by the following protocol: Group 1: pregnant rats received 100 mg retinoic acid (RA)/kg b.w. suspended in corn oil on gestational day (GD) 11.5; Group 2: 20 mg RA/kg b.w. from GD 8-12; Group 3: 20 mg RA/kg b.w. from GD 7.5-11.5; Group 4: 100 mg RA/kg b.w. on GD 10-11; Group 5: 100 mg RA/kg b.w. on GD 10 and 12; Group 6 received corn oil vehicle from GD 7-14.5; and Group 6: served as non-injected controls. In all retinoic acid treated groups, varying degrees of clefts with occasional attempts of fusion were noted. The severity and frequency of the malformations were dependent on dosage or gestational day of drug treatment. Our results indicate that RA, even at the lowest dose tested (20 mg/kg b.w.) severely affects the various tissues constituting the embryonic palatal shelves by altering cell interaction and possibly programmed cell death. These events would then result in lack of or inadequate differentiation with subsequent formation of aberrant craniofacial architecture.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Female; Palate; Pregnancy; Rats; Rats, Wistar; Tretinoin

Exogenous retinoic acid has been found to be teratogenic in animals and man. Craniofacial defects induced by retinoic acid have stimulated considerable research interest. The present report deals with scanning electron microscopical observations of the craniofacial region concurrent with histological examination of craniofacial dysmorphism induced in rat embryos following maternal treatment treated with varying dosages of all-trans-retinoic acid (tretinoin). Two groups of pregnant rats were treated with rat embryos exposed to retinoic acid suspended in corn oil (100 mg/kg b.w. on gestational day 11.5 and 50 mg/kg b.w. on gestational day 10, 11 and 12 respectively). A third group was treated with corn oil (vehicle) while a fourth group remained untreated. A wide spectrum of congenital abnormalities, including exophthalmos, microphthalmia and anophthalmia, maxillo-mandibular dysostosis, micrognathia of both maxilla and mandible, cleft palate, subdevelopment of ear lobe, preauricular tags and macroglossia, were observed in the offspring of retinoic acid treated animals. The abnormalities were both time and dosage dependent, and characteristic of Treacher Collins syndrome when retinoic-acid was administered on gestational day 11.5. In contrast, when retinoic acid was administered were on gestational days 10-12, the defects were similar to those seen in the first and second pharyngeal arch syndrome, as well as in the oculo-auriculo-vertebral spectrum. Whereas our data support the hypothesis that all-trans retinoic-acid disturbs growth and differentiation of several embryonic cell types essential for normal craniofacial development, its mechanism of action remains unclear.

Topics: Abnormalities, Drug-Induced; Animals; Facial Bones; Female; Male; Microscopy, Electron, Scanning; Pregnancy; Rats; Rats, Wistar; Skull; Tretinoin

The teratogenic effect of RA was found to be significantly influenced both by genetic background and by the genotype of malformation mutation Lx. The presence of the Lx mutation and BN genetic background strongly increases the teratogenic effect of RA. On the contrary, the SHR genetic background was shown to protect foetuses from RA teratogenic affliction. Recombinant inbred strain BXH2 is endowed with a specific combination of BN and SHR genes, and following RA administration it exhibits the same embryolethal effect as the BN genetic background alone. Without the Lx mutation there was no effect of RA on hind limbs in SHR/SHR or SHR/BN progeny whilst there was a significantly higher occurence of oligodactyly in SHR/BN on forelimbs as compared to SHR/SHR (92.2% vs 11.5%). In +/Lx progeny, forelimbs were significantly more afflicted with oligodactyly in SHR/BN +/Lx in comparison with both SHR/SHR and SHR/BXH2 foetuses, which indicates that BN modifiers responsible for oligodactyly were not passed to the BXH2 strain. On the contrary, hind limbs of SHR/BXH2, +/Lx progeny exhibited the highest affliction (62% of polydactyly and/or oligodactyly). In homozygous Lx/Lx progeny, polydactyly prevailed in forelimbs of SHR/BXH2 following RA administration, whilst in BN/BN progeny oligodactyly was the most frequent affliction. On the hind limbs, the highest reduction of toe number after RA treatment was connected with BN modifiers. The polymorphism of normal morphogenetic factors was shown to be responsible not only for Lx. phenotypic manifestation, but also for the variability in the response to RA teratogenic action.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Alleles; Animals; Animals, Congenic; Crosses, Genetic; Embryonic and Fetal Development; Face; Female; Forelimb; Genetic Predisposition to Disease; Genotype; Gestational Age; Hindlimb; Male; Morphogenesis; Polydactyly; Rats; Rats, Inbred BN; Rats, Inbred SHR; Rats, Mutant Strains; Syndrome; Tail; Teratogens; Toes; Tretinoin

To elucidate the underlying developmental mechanisms of persistent hyperplastic primary vitreous (PHPV) in humans, we investigated a mouse model for PHPV induced by retinoic acid. We treated C57BL/6NJcl mice at various stages of pregnancy (gestation days 7, 8, 9, 10, 11, or 12) with the teratogen retinoic acid, which affects the migration of neural crest cells. Untreated pregnant mice served as a control group. The eyes of the fetuses were examined histologically on day 18 on gestation. Developmental abnormalities of the vitreous were defined as the presence of excessive mesenchymal tissue in the vitreous cavity. The incidence of developmental abnormalities of the vitreous in all groups, except for those treated on day 12 of pregnancy, significantly exceeded that in the control group (P<0.01). The histological characteristics of the observed vitreous abnormalities in mice resembled those found in PHPV clinically. Retinoic acid-induced abnormalities in mice can serve as an experimental model for PHPV by environmental factors. Results suggest that the critical period for these retinoic acid-induced abnormalities was during days 7 to 11 of gestation, which corresponds to a critical period of 2.5 to 7 weeks of gestation for PHPV in humans.

Topics: Abnormalities, Drug-Induced; Animals; Female; Fetal Death; Gestational Age; Maternal-Fetal Exchange; Mice; Mice, Inbred C57BL; Pregnancy; Prenatal Exposure Delayed Effects; Tretinoin; Vitreous Body

To obtain a better understanding of mandibulo-facial dysostosis and hemicraniofacial microsomia in man, the authors carried out a histologic and scanning electron microscope study of the facial malformations produced in mouse embryos by retinoic acid and methyl-triazene. The administration of 400 mg/kg 13 cis-retinoic acid (RA) to pregnant C57BL mice on day 9 of gestation produced anomalies of the cephalic extremity in the embryos resembling human mandibulo-facial dysostosis. The 64 embryos collected presented hypoplasia of the branchial arches or the snout in 79% of cases, auricular anomalies in 47% and ophthalmic anomalies in 12.5%. Fourteen NMRI mice on day 10.5 of gestation were treated with 1.5 mg (0.5 mg/kg) methyl-triazene (Methyl). The 126 embryos collected had developed a very high percentage of micromandibles and anomalies of both embryonic ears (94.6% to 100%). Finally, although the facial anomalies produced by retinoic acid resemble the human mandibulo-facial dysostosis syndrome, no correlation was found between hemicraniofacial microsomia and the administration of methyl-triazene.

Topics: Abnormalities, Drug-Induced; Adolescent; Animals; Child; Facial Asymmetry; Female; Humans; Male; Mandibulofacial Dysostosis; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Pregnancy; Tretinoin; Triazenes

Topics: Abnormalities, Drug-Induced; Administration, Topical; Female; Humans; Infant, Newborn; Pregnancy; Pregnancy Trimester, First; Risk Factors; Tretinoin

Retinoic acid (RA) plays an important role in differentiation stage in which it also influences glycoconjugate metabolism. Previous work in our laboratory has shown that treatment with RA modifies glycolipid synthesis and distribution in total Xenopus embryos during development. In this study we have investigated the activity of the following anabolic enzymes involved in glycolipid biosynthesis: sialyltransferase-1 (SAT-1), GM3(beta1, 4)-N-acetylgalactosaminyltransferase (GalNAcT-1) and LacCer(beta1, 3)N-acetylglucosaminyltransferase (GlcNAcT-1). These enzymes are located at the branching point of lactosylceramide (Lc(2)) metabolism. Enzyme activities were assayed after treatment with different doses of RA added exogenously to the medium during the first 7 days of Xenopus embryo development. Our results show that RA activates GlcNAcT-1, the enzyme that drives Lc(2)to the glycolipids of the lacto-series, and SAT-1 that inserts Lc(2)in the ganglio-series pathway. These data support our previous analysis of glycolipid pattern in Xenopus embryos after RA treatment (Rizzo et al., 1995;Cell Biol Int19: 895-901) indicating a possible correlation between the distribution of glycolipids and the enzymes involved in their metabolism.

Topics: Abnormalities, Drug-Induced; Animals; Female; Glycolipids; Male; N-Acetylgalactosaminyltransferases; N-Acetylglucosaminyltransferases; Polypeptide N-acetylgalactosaminyltransferase; Sialyltransferases; Time Factors; Tretinoin; Xenopus

All-trans retinoid acid (RA) induces a stereotypic spectrum of stage-specific malformations in vertebrate conceptuses. The present work evaluated the anatomic and biochemical effects of exposure to RA in mouse embryos at a peri-implantational stage of development - gestational day (GD) 5. The RA receptors (RARs) beta and gamma, the retinoid X receptors (RXRs) alpha and beta, and the cellular retinoid acid binding proteins (CRABPs) I and II were detected by RT-PCR in both control and treated individual GD 5 decidua/embryo complexes 3 h after RA injection, indicating the presence of the mRNAs coding for the proteins that mediate the effects of RA. In contrast, the RAR alpha mRNA was detected in some but not all decidua/embryo complexes, both control and treated, suggesting that its expression is initiated at approximately GD 5, while RXR gamma mRNA was not detected. Examination of the control and RA-exposed embryos on GD 10, 12, or 17 showed that greater than 50% of the RA-exposed embryos were adversely affected, many with defects found only after serial histopathological examination. The malformations were localized primarily in the central nervous system, the branchial arches, and their derivatives. These terata included excessive folding and elevation of the neural tube floor plate, exencephaly (with detachment of the cephalic neuroepithelium and rarefied cephalic mesenchyme), persistent patency of Rathke's pouch, small trigeminal ganglia, neural diverticula (chiefly from the spinal cord), and/or various optic and otic defects. Unexpectedly, limb reduplications were not apparent in RA-exposed fetuses. Those litters examined on GD 17 had a high percentage of resorbed or malformed implantations, and the few apparently normal fetuses were developmentally delayed with respect to bone ossification. These data confirm that the development of neural- and neural crest-derived structures are severely disrupted by RA exposure prior to initial specification of the neural plate and suggest that many of the proteins that regulate RA signaling are available in early vertebrate embryos at this developmental stage.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Brain; Branchial Region; Embryo Implantation; Female; Limb Deformities, Congenital; Mice; Mice, Inbred Strains; Neural Tube Defects; Receptors, Retinoic Acid; RNA, Messenger; Teratogens; Thorax; Tretinoin; Vitamin A

To investigate the effect of the environmental pollutants, polycyclic aromatic hydrocarbons (PAHs), on retinoic acid-induced teratogenesis, all-trans-retinoic acid (RA) dissolved in corn oil (120 mg/kg) was administered orally to pregnant rats at the 11th day of gestation with and without the prior intraperitoneal treatment with 10 mg/kg 3-methylcholanthrene (3-MC) for 3 days. Dams were killed on the 20th day of pregnancy. The examinations of fetuses revealed that 3-MC barely enough to cause induction of P-450 in pregnant dams had profound embryo-toxic effects: the fetal resorption amounted to approximately 60% of total number of implantations. The fetuses survived weighed less than the control fetuses. All of RA-treated mothers had fetuses with abnormalities, and the main malformations were absence of tail (100%), caudal and sacral malformations (100%), and cleft palate (42%). Pregnant dams received both 3-MC and RA had a reduced severity of tail anomaly (33%), while the rest, 67%, had short vestigial tail. Caudal and sacral malformations were detected but at a milder degree. We did not observe cleft palate in this group. The concurrent treatment of dams with 3-MC and RA led to an increased inducibility of cytochrome P-450 and subsequently, CYP1A1 dependent enzyme activity higher than those observed after the injection of 3-MC alone. UDP-glucuronyl-transferase activity was also markedly induced in concurrent 3-MC and RA group higher than that in 3-MC alone. We suggest that the induction of P-450 and alteration of metabolic enzyme activities may play an important role in reducing the teratogenic potency of RA. However, RA-treatment did not retard the embryo-toxic effect of 3-MC but rather potentiated.

Topics: Abnormalities, Drug-Induced; Animals; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Female; Fetal Resorption; Fetus; Methylcholanthrene; Microsomes, Liver; Pregnancy; Rats; Rats, Wistar; Teratogens; Tretinoin; Vitamin A

Visceroatrial heterotaxy syndrome is characterized by abnormality of visceral laterality and complex cardiovascular anomalies usually involving both the outflow and inflow tract. Morishima et al. (1995) showed that mouse embryos treated with all-trans retinoic acid at embryonic day 6.5 (primitive streak stage) induces this syndrome.. To investigate the morphogenetic process of visceroatrial heterotaxy syndrome, we examined retinoic acid-treated mouse embryos at embryonic days 9-15 using scanning electron microscopy.. The sinoatrial connection was first distinguished for the determination of situs as early as at embryonic day 10.5. Normal visceroatrial situs was found in 57% of all treated embryos, and the rest had abnormal situs, in which right isomerism was found in 81%. In the right-isomeric mouse, the cardiac morphology was characterized by abnormal looping together with dysplasia of the inflow and outflow tract cushion; that is, the primitive right ventricle was usually deviated cranially to various degrees, the atrioventricular cushion appeared trilobed in a half of them, and unilateral ventricular hypoplasia was noted in about one-third of them after embryonic day 14.5.. An anomalous relation between the atrioventricular cushions and the interventricular septum appeared to have caused a restrictive inflow to the unilateral ventricle, leading to ventricular chamber hypoplasia on the ipsilateral side. Thus, we clarified that retinoic-acid treatment at the primitive streak stage disturbed cardiac looping and formation of atrioventricular cushion development, which secondarily influenced ventricular chamber development.

Topics: Abnormalities, Drug-Induced; Animals; Endocardial Cushion Defects; Female; Fetal Heart; Gastrula; Heart Atria; Heart Defects, Congenital; Heart Septal Defects, Ventricular; Heart Ventricles; Male; Mice; Microscopy, Electron, Scanning; Morphogenesis; Tretinoin

QUESTIONOne of my patients conceived while using a topical tretinoin preparation for acne. I know this drug is related to Accutane, which is teratogenic. How should I advise her?ANSWERAvailable evidence suggests that topical tretinoin does not increase teratogenic risk in humans.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Administration, Cutaneous; Adult; Case-Control Studies; Female; Humans; Keratolytic Agents; Pregnancy; Pregnancy Complications; Pregnancy Outcome; Pregnancy Trimester, First; Tretinoin

Topics: Abnormalities, Drug-Induced; Case-Control Studies; Dose-Response Relationship, Drug; Female; Humans; Keratolytic Agents; Pregnancy; Pregnancy Complications; Prenatal Exposure Delayed Effects; Retinoids; Tretinoin; Vitamin A

Isotretinoin is the most potent human teratogen on the market. Women for whom contraception fails may conceive during or soon after discontinuing isotretinoin therapy, making its elimination kinetics a crucial determinant of fetal safety. The steady-state pharmacokinetics of isotretinoin and its major 4-oxo metabolite were studied in 16 adult patients treated for acne who were receiving doses that ranged from 0.47 to 1.7 mg/kg daily. This is the first study of the pharmacokinetics of isotretinoin in women of childbearing age (n = 11). The clinical efficacy and tolerability of isotretinoin was investigated, and the correlation between these data and steady-state serum concentrations of isotretinoin was tested. The concentration-time data best fitted a two-compartment open model with linear elimination. There was no correlation between efficacy and tolerability of isotretinoin and steady-state serum concentrations. There was no correlation between dose of isotretinoin and steady-state concentration, due to the large variability in apparent clearance. Values for elimination half-life (t1/2) of isotretinoin and its metabolite were 29+/-40 hours and 22+/-10 hours, respectively. These data suggest a longer elimination t1/2 of the parent drug than previously reported. This is probably due to the longer sampling time used in this study (as long as 28 days). This study suggests that a greater variability exists in the safe time after discontinuation of the drug for onset of conception.

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Area Under Curve; Chromatography, High Pressure Liquid; Female; Half-Life; Humans; Isotretinoin; Keratolytic Agents; Male; Pregnancy; Teratogens; Tretinoin

(1) In animals, adapalene and tretinoin are teratogenic. (2) In humans, reports of several disturbing cases have been published after cutaneous administration. (3) Remember to warn women of child-bearing potential. Inadvertent exposure during pregnancy necessitates special monitoring.

Topics: Abnormalities, Drug-Induced; Antineoplastic Agents; Dermatologic Agents; Female; Humans; Naphthalenes; Pregnancy; Treatment Outcome; Tretinoin

Spatially-restricted expression domains of Msx 1 and Msx 2 in the developing chick face suggest that they may play a role in epithelial-mesenchymal interactions governing outgrowth of facial primordia. Retinoid application to developing chick faces reproducibly inhibits upper beak outgrowth but the lower beak is unaffected. In the normal face, high levels of Msx gene transcripts in upper and lower beak primordia correlate with regions of outgrowth. Following retinoid treatment, Msx 1 and Msx 2 transcripts are rapidly down-regulated in upper beak primordia where outgrowth is inhibited, but remain largely unchanged in lower beak primordia, where outgrowth is unaffected. Decreases in gene expression precede retinoid-induced morphological changes in the upper beak, suggesting that Msx gene products are involved in mediating the effect of retinoids on facial development.

Topics: Abnormalities, Drug-Induced; Animals; Beak; Chick Embryo; DNA-Binding Proteins; Down-Regulation; Facial Bones; Homeodomain Proteins; In Situ Hybridization; Microscopy, Electron, Scanning; Morphogenesis; MSX1 Transcription Factor; Transcription Factors; Tretinoin

Although it is well established that oral tretinoin produces embryofetal developmental toxicity in various laboratory animals, the toxic potential of topical tretinoin has not been clearly established.. This study of tretinoin administration to pregnant Wistar rats was conducted to determine whether topical tretinoin is associated with adverse effects on reproductive function or embryofetal growth and development and to compare outcomes with topical and oral tretinoin.. Topical and oral tretinoin (1 to 20 mg/kg and 1 to 10 mg/kg, respectively) or vehicles alone were administered on gestational days 6 through 16 and 15, respectively.. Topical tretinoin: After topical treatment, dams receiving 10 mg/kg daily or greater had severe local and systemic toxicity prompting discontinuation of tretinoin. At doses of 2.5 mg/kg or greater, dam weight gain and food consumption were significantly less than those of control dams. Offspring of dams receiving 5 mg/kg weighed significantly less, and offspring of dams receiving 2.5 mg/kg or greater had a significantly greater occurrence of supernumerary ribs compared with control offspring. Oral tretinoin: After oral treatment, in the absence of maternal toxicity, significantly more offspring of dams receiving 5 mg/kg or greater had supernumerary ribs, and offspring of the 10 mg/kg treatment group had a greater incidence of cleft palate than had control offspring.. The local and systemic maternal toxicity found in association with supernumerary ribs and low weights in the offspring at topical tretinoin doses of 2.5 and 5 mg/kg suggests that these developmental effects may be nonspecific or maternally mediated. Oral tretinoin at doses of 10 mg/kg, however, is clearly associated with embryofetal alterations in the Wistar rat.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Administration, Topical; Animals; Female; Fetal Diseases; Fetus; Keratolytic Agents; Pregnancy; Pregnancy, Animal; Rats; Rats, Wistar; Tretinoin

Embryofetal developmental toxicity associated with oral administration of vitamin A analogs has led to concern about risks from topical application.. This study was conducted to evaluate the potential developmental toxicity of tretinoin emollient cream when applied to the skin of pregnant New Zealand white rabbits during organogenesis (gestational days 7 through 19).. Twenty rabbits each were randomly assigned to a control group (group I) or to receive vehicle (group II) or tretinoin emollient cream topically at dosages of 10 (0.05 mg/kg*, group III) or 100 (0.5 mg/kg*, group IV) times that used clinically in humans. Does and fetuses were examined for tretinoin-induced toxic effects, and maternal plasma tretinoin and metabolite levels were measured.. The rate of abortion was increased significantly in does in group IV (p < or = 0.01) compared with the control group. Dosage-dependent increases in incidence and severity of skin reactions occurred in groups administered the vehicle and the two dosages of tretinoin. Does in groups III and IV had clinical and necropsy observations that were considered direct or indirect effects of tretinoin administration, persistent weight loss, and reduced feed consumption. Maternal endogenous plasma tretinoin levels were below the lower limit of quantitation of 5 ng/ml and were not significantly altered with treatment. Group IV had significantly reduced mean fetal body weight (p < or = 0.01) and a greater frequency of resorptions compared with group I. Although external, visceral, or skeletal alterations occurred at significantly greater levels in group III, they were unrelated to tretinoin administration because the fetal incidences were not dosage dependent, and the litter incidence did not significantly differ from the control group values.. Maternally toxic dosages of tretinoin were associated with an increased incidence of abortions and resorptions and reduced fetal body weight, two end points of developmental toxicity. Consistent with the absence of detectable tretinoin plasma levels, however, no changes in fetal morphology were attributable to tretinoin administration. *The milligrams per kilogram dosage refers to the amount of active ingredient (tretinoin). The 0.05 mg/kg and 0.5 mg/kg groups were treated with 0.005% and 0.05% wt/wt tretinoin emollient cream formulation. The 0.05% tretinoin emollient cream is the Renova clinical formulation. The 10 and 100 times clinical multiples refer to Renova clinical multiples and are based on a 50 kg adult patient's applying 500 mg of 0.05% tretinoin emollient cream formulation daily to yield a clinical dosage of 0.005 mg/kg.

Topics: Abnormalities, Drug-Induced; Abortion, Spontaneous; Administration, Topical; Animals; Dose-Response Relationship, Drug; Emollients; Female; Fetus; Keratolytic Agents; Obstetric Labor, Premature; Ointments; Pregnancy; Pregnancy, Animal; Rabbits; Tretinoin

A physiologically based pharmacokinetic (PBPK) model for all-trans-retinoic acid (tretinoin) was developed to provide a coherent description of tretinoin absorption, distribution metabolism, and excretion across species and routes of administration.. The goal of developing such a model is to provide a measure of internal dose that would be a biologically relevant surrogate for administered dose in assessing human teratogenic risk from topically applied tretinoin emollient cream.. The developed PBPK model included compartments for plasma, liver, gut, intestinal lumen, fat, skin, richly and slowly perfused tissues, placenta, and embryo. Tretinoin metabolism to 13-cis retinoic acid, oxidation, and glucuronidation were incorporated. Dose surrogates, including the maximum plasma concentration (Cmax) and area under the concentration-versus-time curve were calculated from the model.. The ability of the model to predict tretinoin pharmacokinetics and to extrapolate across species and routes of administration was tested and validated. Model-derived estimates of dose surrogates demonstrated that the internal exposure to retinoids after topical treatment with 0.05% tretinoin emollient cream is minimal in comparison to that for teratogenic oral doses. The ratio of areas under the curve for total active retinoids after teratogenic oral doses in monkeys versus therapeutic topical doses in human beings, for example, was greater than 450,000 to 1.. For topical application of tretinoin in human beings, detoxification via the glucuronidation pathway predominates, resulting in a much lower internal exposure to active retinoids than was inferred from total radioactivity data. The model predicts that topical application of tretinoin results in an internal exposure that is four to six orders of magnitude lower than a minimally teratogenic dose.

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Humans; Keratolytic Agents; Models, Biological; Tretinoin

Although topically applied all-trans-retinoic acid (tretinoin) undergoes minimal absorption and adds negligibly to normal endogenous levels, its safety in humans is occasionally questioned because oral ingestion of retinoids at therapeutic levels is known to entail teratogenic risks.. To assess the actual potential for developmental toxicity from treatment with topical tretinoin.. Risk assessments were conducted on four known human developmental toxicants (valproic acid, methotrexate, thalidomide, and isotretinoin) and a potential developmental toxicant (acetylsalicylic acid). The margin of safety for each chemical was calculated from the ratio of animal no-observed adverse effect levels to human lowest-observed adverse effect levels or estimated exposure doses.. The derived safety margin of more than 100 for topical tretinoin (with 2% absorption) contrasted sharply with the near unity values for valproic acid, methotrexate, thalidomide, and isotretinoin and was larger than that for acetylsalicylic acid.. These data support other epidemiologic and animal data that topical tretinoin is not a potential human developmental toxicant.

Topics: Abnormalities, Drug-Induced; Administration, Topical; Animals; Female; Humans; Isotretinoin; Keratolytic Agents; Methotrexate; Pregnancy; Risk Assessment; Teratogens; Thalidomide; Tretinoin; Valproic Acid

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Crosses, Genetic; Genetic Carrier Screening; Hindlimb; Inbreeding; Polydactyly; Rats; Rats, Inbred BN; Rats, Inbred Lew; Rats, Mutant Strains; Recombination, Genetic; Syndrome; Teratogens; Tretinoin

Inhalation of methanol by pregnant mice before gestation day nine (gd 9) produces fetal skeletal alterations, principally in the cervical region. The appearance of these defects suggests homeotic shifts in segment identity, patterning, or both. To explore this possibility, detailed morphological analyses of the effects of methanol on fetal skeletal development were done. Pregnant mice were gavaged with 0, 4.0, or 5.0 g/kg methanol (MeOH) split in two doses on gd 7, the most sensitive day for induction of skeletal alterations with methanol. Dams were killed on gd 18 and the fetuses were counted, weighed, and examined externally. Fetuses were double stained with alcian blue and alizarin red for examination of cartilaginous and ossified vertebral and rib characteristics, and in selected fetuses cervical vertebrae were disarticulated for more detailed analysis. Observations indicative of methanol-induced homeotic transformations were as follows: [tabular data: see abstract volume] Examination of disarticulated vertebrae revealed foramina and other distinguishing characteristics on vertebrae anterior to those on which they normally appear. These results demonstrate that maternal methanol exposure can alter segment patterning in the developing mouse embryo, producing posteriorization of cervical vertebrae.

Topics: Abnormalities, Drug-Induced; Animals; Cervical Vertebrae; Dose-Response Relationship, Drug; Female; Genes, Homeobox; Gestational Age; Maternal-Fetal Exchange; Methanol; Mice; Pregnancy; Tretinoin

Earlier studies with etretinate and its metabolite acitretin suggested that area under the concentration-time curve (AUC) is the most suitable pharmacokinetic correlate to etretinate-induced teratogenesis. In an attempt to test this hypothesis with respect to the embryotoxic effects of all-trans-retinoic acid (all-trans-RA), we determined the embryotoxicity and plasma pharmacokinetics of all-trans-RA and its metabolites following administration of all-trans-RA to Wistar rats on Gestational Day (GD) 9, either subcutaneously (sc; dose levels 1, 3, or 5 mg/kg body mass) or orally (po; 5 mg/kg body mass). The 5 mg/kg dose of all-trans-RA was not embryotoxic when administered orally but led to high rates of embryolethality and skeletal defects following sc treatment. Determination of retinoids by HPLC showed that all-trans-RA reached similar maximum plasma concentrations (C(max)) after both dosing regimens, but its plasma AUC was ca. threefold higher after sc injection than po administration due to the slower uptake rate of the drug and its limited detoxification via beta-glucuronidation following sc injection. Furthermore, retinoid analysis in rat tissues (liver, kidney, duodenum, and jejunum), collected 1 hr after sc or po administration of 5 mg all-trans-RA/kg body mass on GD 9, confirmed that formation of all-trans-retinoyl-beta-glucuronide was much more extensive after po than after sc administration. Finally, linear regression analysis of either C(max). or AUC values of all-trans-RA in rat plasma and fetal abnormality rates showed that AUC values are better correlated with the embryotoxic outcome than C(max) [AUC-based correlation coefficient (r) > 0.90; C(max)-based r < 0.43]. Our findings establish the relevance of the AUC of all-trans-RA, and not its C(max), as the most appropriate pharmacokinetic marker of embryonic exposure and embryotoxic potency of all-trans-RA and stress the importance of the duration of exposure as a major determinant of embryotoxic outcome for retinoids.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Area Under Curve; Bone and Bones; Drug Administration Routes; Embryo, Mammalian; Female; Fetus; Gestational Age; Injections, Subcutaneous; Keratolytic Agents; Linear Models; Maternal-Fetal Exchange; Pregnancy; Rats; Rats, Wistar; Tissue Distribution; Tretinoin

Topics: Abnormalities, Drug-Induced; Administration, Topical; Case-Control Studies; Cohort Studies; Contraindications; Female; Humans; Infant, Newborn; Pregnancy; Pregnancy Outcome; Pregnancy Trimester, First; Prospective Studies; Risk Factors; Tretinoin

This study reports a morphological, skeletal, and molecular characterization of the supernumerary limbs induced by systemic administration of all-trans retinoic acid to egg-cylinder stage mouse embryos. As initially described by Rutledge et al. (Proc. Natl. Acad. Sci. USA 91, 5436, 1994), we have found that oral administration of all-trans retinoic acid (70 mg/kg body weight) at 5.5 days postcoitum induced the formation of supernumerary limbs. Most often, these arose as a pair of extra buds located caudally and ventrally to the normal (orthotopic) hindlimb buds without duplication of the lower body axis. The resulting one or two supernumerary hindlimbs were connected to an imperfectly mirror-image-duplicated pelvic girdle. Variable truncations of the stylopodium and zeugopodium skeleton, as well as abnormal splitting of the distal skeleton, were frequently observed. The apical ectodermal ridge of the extra limb buds expressed expected growth factor genes. However, an ectopic anterior expression of Sonic hedgehog and Hoxd-13 was seen in the supernumerary buds, suggesting that these buds would incorporate potential polarizing cells of the hindlimb or genital field and generate an ectopic polarizing zone. This is consistent with the reverse orientation of most supernumerary limbs at later stages. Some of the buds did not express limb-specific markers and were thus expected to degenerate or form nonlimb structures, as observed in an adult specimen. Less frequently, extra limb buds with normal polarity were associated to a duplicated lower body axis. Retinoic acid also generated a novel type of duplication in which "twin" hindlimbs with two parallel apical ectodermal ridges and zones of polarizing activity arose on one side of the embryo.

Topics: Abnormalities, Drug-Induced; Animals; Female; Gene Expression Regulation, Developmental; Hedgehog Proteins; Hindlimb; Homeodomain Proteins; In Situ Hybridization; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Microscopy, Electron, Scanning; Pregnancy; Proteins; Trans-Activators; Transcription Factors; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Cloning, Molecular; Embryonic and Fetal Development; Exons; Female; Genomic Library; Genotype; Limb Deformities, Congenital; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Transgenic; Placenta; Pregnancy; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Signal Transduction; Tretinoin

Retinoic acid, an oxidative metabolite of vitamin A, is involved in the control of many biological processes including embryonic development. Excess as well as deficiency of retinoids were found to be teratogenic. The effects of retinoids in normal as well as abnormal development may be mediated by two members of retinoid receptors, the RAR's and RXR's, which exhibit a specific temporal and spatial expression during development. The significance of the retinoid receptors was investigated here by studying the teratogenic effects of retinoid ligands with relative selectivity for binding and transactivation of the retinoic acid receptors RAR alpha, RAR beta and RAR gamma. Pregnant NMRI mice were administered 5 or 15 mg/kg of CD 336 (Am 580) (alpha-ligand), CD 2019 (beta-ligand), CD 437 (gamma-ligand) or 37.5 mg/kg all-trans-retinoic acid in 25% Cremophor EL on day 8.25 or day 11 of gestation by gastric intubation. External, visceral and skeletal malformations were observed on day 18 of gestation. The order of teratogenic potency was: alpha-ligand > beta-ligand > gamma-ligand. In addition, these retinoids also produced a different spectrum of defects. The alpha-ligand induced the most varied defects including severe ear, mandible, and limb malformations. The beta-ligand induced defects of the urinary system and liver in greater frequency than expected from its relative potency. The gamma-ligand preferentially induced ossification deficiencies and defects of the sternebrae and vertebral body. Our results show that these three retinoids, which were previously demonstrated to exhibit retinoid-like activities in several systems, exert differing teratogenic activities, in regard to both potency and regioselectivity: we hypothesize that the relative selectivity for binding and transactivation of the three retinoic acid receptors could possibly be related to the differences of teratogenic effects observed in this study. The low potency of the gamma-ligand may lead the way to interesting new retinoids with improved therapeutic ratio.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Administration, Oral; Animals; Benzoates; Bone and Bones; Central Nervous System; Digestive System Abnormalities; Female; Mice; Naphthalenes; Pregnancy; Pregnancy Outcome; Receptors, Retinoic Acid; Retinoids; Tetrahydronaphthalenes; Tretinoin; Urogenital Abnormalities

The gene Fgf-3 is expressed in rhombomeres 5 and 6 of the hindbrain and has been functionally implicated in otic development. We describe new sites of expression of this gene in mouse embryos in the forebrain, the midbrain-hindbrain junction region, rhombomere boundaries, a cranial surface ectodermal domain that includes the otic placode, and in the most recently formed somite. In the early hindbrain, high levels of Fgf-3 transcripts are present in rhombomere 4. The surface ectodermal domain at first (day 8 1/2) extends laterally from rhombomeres 4 and 5 (prorhombomere B), in which neuroepithelial levels of expression are highest, to the second pharyngeal arch ventrally; at day 9, when the region of highest level of neuroepithelial Fgf-3 expression is in rhombomeres 5 and 6, the dorsal origin of the surface ectodermal domain is also at this level, extending obliquely to the otic placode and the second arch. The initially high level of Fgf-3 transcripts in the otic placode is downregulated as the placode invaginates to form the otic pit. Fgf-3 is a good marker for the epithelium of pharyngeal arches 2 and 3, and our in situ hybridization results confirm the dual identity of the apparently fused first and second arches in some retinoic acid-exposed embryos, and the fusion of the first arch with the maxillary region in others. Correlation between Fgf-3 expression and morphological pattern in craniofacial tissues of normal and retinoic acid-exposed embryos indicates that prorhombomere B, the second arch and the otic ectoderm represent a cranial segment whose structural integrity is maintained when hindbrain morphology and pharyngeal arch morphology are altered. Comparison of normal Fgf-3 expression domains with those of Fgf-4 and with the phenotype of Fgf-3-deficient mutant embryos suggests that there is some functional redundancy between Fgf-3 and Fgf-4 in otic induction and second arch development.

Topics: Abnormalities, Drug-Induced; Animals; Branchial Region; Craniofacial Abnormalities; Ear; Embryonic and Fetal Development; Female; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Genetic Markers; In Situ Hybridization; Mice; Mice, Inbred C57BL; Pregnancy; Proto-Oncogene Proteins; Reference Values; Rhombencephalon; Tretinoin

Retinoic acid (RA) is known to act as a signaling molecule during embryonic development, but little is known about the regulation of RA synthesis from retinol. The rate-limiting step in RA synthesis is the oxidation of retinol, a reaction that can be catalyzed by alcohol dehydrogenase (ADH). Ethanol is also a substrate for ADH, and high levels of ethanol inhibit ADH-catalyzed retinol oxidation. This has prompted us to hypothesize that ethanol-induced defects observed in fetal alcohol syndrome involve ethanol inhibition of ADH-catalyzed RA synthesis. Here, we have examined the effect of ethanol on RA levels in cultured mouse embryos by using a bioassay. Treatment with 100 mM ethanol, but no 10 mM, led to a significant decrease in RA detection in 7.5-day-old embryos. Using whole-mount in situ hybridization, we detected mRNA for class IV ADH, but not ethanol-active cytochrome P450 2E1, in 7.5- and 8.5-day-old embryos, indicating that an ADH-linked pathway exists at these stages for metabolizing retinol and ethanol. Thus, the observed ethanol-induced reduction in RA may be caused by ethanol inhibition of retinol oxidation catalyzed by class IV ADH. In our postulated mechanism for fetal alcohol syndrome, this enzyme may well play a crucial role.

Topics: Abnormalities, Drug-Induced; Alcohol Dehydrogenase; Animals; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Ethanol; Fetal Alcohol Spectrum Disorders; In Situ Hybridization; In Vitro Techniques; Mice; Mice, Inbred C57BL; Neural Crest; Oxidoreductases, N-Demethylating; RNA, Messenger; Tissue Distribution; Tretinoin

Retinoic acid (RA) is an active metabolite of vitamin A that is teratogenic when present in excess during mammalian embryogenesis. We have investigated the effect of embryonic exposure to nonphysiological levels of all-trans RA on the development of the mouse inner ear. Dysmorphogenesis of both vestibular and auditory portions of the inner ear, and abnormal formation of the surrounding capsule are produced by exposure to teratogenic levels of RA at an embryonic age of 9 days (E9). There was no observable teratogenic effect of RA when administered at earlier (i.e., E7 or E8) or later (i.e., E10) stages of otic morphogenesis. We hypothesize that exposure to high levels of RA during a critical period of early otic morphogenesis interferes with the inductive tissue interactions required for inner ear development.

Topics: Abnormalities, Drug-Induced; Animals; Carrier Proteins; Ear, Inner; Female; Gestational Age; Immunohistochemistry; Intermediate Filament Proteins; Male; Maternal-Fetal Exchange; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Neurofilament Proteins; Pregnancy; Sensory Receptor Cells; Teratogens; Tretinoin

The teratogenicity of all-trans-retinoic acid, 13-cis-retinoic acid, and retinol was investigated in pregnant Wistar rats given a single oral dose on Day 10 of gestation. External malformations showed dose-dependent increases and the order of potency was all-trans-retinoic acid > retinol > 13-cis-retinoic acid. The metabolites in maternal plasma were determined following a single oral dose on Day 10 of gestation. Equipotent teratogenic doses of all-trans-retinoic acid and 13-cis-retinoic acid had similar plasma levels of all-trans-retinoic acid; however, retinol teratogenicity could not be accounted for by circulating all-trans-retinoic acid or its metabolites. The teratogenicity and maternal pharmacokinetics of all-trans-retinoic were compared in pregnant Wistar rats when given as a single dose (50 mg/kg) and as three equal doses (16.66 mg/kg) over 6 hr. Divided doses were of slightly greater potency than the single dose but the maximum observed concentration (Cmax) and area under the plasma concentration-time curve (AUC) values for the second and third doses were greatly attenuated compared with the first dose; in consequence, both the total AUC and Cmax were reduced compared with the single dose. The altered profile could not be explained by increased formation of all-trans-retinoic acid glucuronide or increased isomerisation to 13-cis-retinoic acid. The maternal plasma levels of all-trans-retinoic acid in pregnant rabbits were reduced by a dose given 24 hr earlier. These data show that all-trans-retinoic acid in maternal plasma is a poor indicator of fetal exposure to teratogenic risk.

Topics: Abnormalities, Drug-Induced; Animals; Female; Glucuronates; Male; Pregnancy; Pregnancy, Animal; Rabbits; Rats; Rats, Wistar; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin

The vitamin A derivative retinoic acid (RA) is a powerful teratogen which can induce severe craniofacial and limb malformations if administered at certain stages of gestation. In addition this compound has been shown to affect patterning in regenerating systems. A classical example is the induction of supernumerary structures along the proximodistal axis of the regenerating amphibian limb. We have investigated the effect of RA on other regenerating systems, the amphibian lower and upper jaws, both in developing and adult animals. We report here that RA does not induce formation of extra structures either in the lower or in the upper jaw of adult newts under experimental conditions where duplications of the regenerating limb occur. However, RA selectively induces severe malformations in the upper jaw regenerate that resemble those induced in avian and mammalian embryos. Analysis of the expression of the newt retinoic acid receptors RAR alpha and delta in upper and lower jaws showed that RAR alpha was expressed at a significant level in the wound epidermis, but not in blastemal cells, whereas no RAR delta could be detected in the regenerate either by in situ hybridization or by using an anti-RAR delta antibody. Therefore, unlike in the limb, in jaws RAR delta is not up-regulated following amputation, and this difference in expression may be causally related to the different effects induced by RA on jaws and limbs. In order to establish whether retinoids affected regeneration of developing jaws in a similar fashion, their effects were studied in animals whose jaws had been amputated at different developmental stages. Under the experimental conditions used overall growth retardation and head defects were observed in the majority of embryos which had been amputated and treated with retinol palmitate (RP) between stages 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 45 was not significantly affected by the treatment, although the early phase of regeneration was slower than in controls. The different responses to retinoids of regenerating facial structures in embryos, larvae and adults will be discussed.

Topics: Abnormalities, Drug-Induced; Animals; Diterpenes; Gene Expression; In Situ Hybridization; Jaw; Mandible; Maxilla; Notophthalmus; Receptors, Retinoic Acid; Regeneration; Retinyl Esters; Teratogens; Tretinoin; Vitamin A

Retinoids elicit biological responses by activating a series of nuclear receptors. Six retinoid receptors belonging to two families are currently known: retinoic acid receptors (RAR alpha,beta,and gamma) and retinoid X receptors (RXR alpha,beta,and gamma). Stilbene retinoid analogs of retinoic acid (RA), such as (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)prope n-1- yl]benzoic acid (TTNPB, 1) and (E)-4-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)pro pen-1- yl]benzoic acid (3-methyl-TTNPB, 2), display differential RAR and RXR activities, depending on the substituent at C3 of the naphthalene ring. We report here structural modifications of the benzoate moiety of 2 that result in analogs with greater RXR selectivity as well as those with pan-agonist (activate both RAR and RXR receptors) activities, analyze the structural features that impart receptor selectivity, and describe a stereoselective method for the synthesis of these analogs. The biological activities associated with the RAR and RXR receptors were examined by testing representative examples with different receptor activation profiles for their ability to induce tissue transglutaminase (Tgase) activity in a human promyelocytic leukemia cell line (HL-60 cdm-1) and to inhibit tumor-promoter-induced ornithine decarboxylase (ODC) activity in hairless mouse skin. These results suggest that RAR agonists and RXR agonists may have different therapeutic applications. Finally, we show that RXR agonists are significantly reduced in teratogenic potency relative to RAR agonists and may therefore have significant advantages in clinical practice.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Benzoates; Female; Gene Expression; Humans; Mice; Mice, Hairless; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Stereoisomerism; Stilbenes; Structure-Activity Relationship; Substrate Specificity; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection

This study was designed to examine the developmental dose response for all-trans retinoic acid (TRA) administered at presomite stages in mouse embryos. Previous studies using hamsters [Shenefelt (1972) Teratology 5:103-118] have shown that developmental stages corresponding to those present early on gestational day (GD) 7 in mice are most sensitive to retinoid-induced teratogenesis. Our preliminary studies showed that at this treatment time, gavage dosages of 7.5 mg/kg maternal body weight administered to C57B1/6N mice, an inbred strain, resulted in severe craniofacial malformations representing the holoprosencephaly, aprosencephaly spectrum. Additionally, in an outbred mouse strain, CD-1, exencephaly was induced by dosages of 2.5 mg/kg TRA and above. Readily detectable abnormalities of the eyes, including anophthalmia and severe microphthalmia and iridial colobomata, were induced by even lower doses cf TRA in the C57B1/6N strain. Incidences of micro/anophthalamia were 6.7%, 8.1%, 12.9%, and 32.4% at 0, 0.313, 0.625, and 1.25 mg/kg, respectively. The dosages required to induce significant incidences of exencephaly (2.5 mg/kg) and severe ocular abnormalities (1.25 mg/kg) on GD 7 in mice are approximately 50-100-fold less than those that are commonly used to examine the teratogenicity of this compound at later developmental stages in this species. The trend toward an increase in the incidence of severe ocular malformations at the lowest dose examined and the fact that subtle ocular malformations were not taken into account for this study suggest that even lower dosages may be effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Cricetinae; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Pregnancy; Teratogens; Tretinoin

9-cis retinoic acid (RA) is a naturally occurring isomer of all-trans RA. While both isomers can bind with high affinity and activate RA receptors, only 9-cis RA is the specific ligand for the retinoid X receptors. 9-cis RA has also been shown to be much more potent than all-trans RA in inducing digit duplication in the chick embryo wing bud. To gain further insight into its mechanisms, here we investigated the teratogenic activity in pregnant mice of 9-cis RA and compared it with those of all-trans RA and 13-cis RA. Using frequency and severity of limb reduction defects as well as palatal clefts in the resultant fetuses as indicators, we found that orally administered 9-cis RA was one-half as potent a teratogen as all-trans RA. That 9-cis RA was intrinsically less active than all-trans RA was deduced by comparing the inhibitory activities of the two retinoids in the limb bud mesenchymal cell micromass cultures using chondrogenesis as an end-point. Since placental transfer of cis isomers of RA is generally poor, we monitored the identities and amounts of retinoids in the embryo after administration of 9-cis RA to the mother. We found that 9-cis RA undergoes extensive metabolism and isomerization during absorption resulting in a number of metabolites in the maternal circulation within 30 min after administration. Although some of these metabolites remain to be identified, the most abundant RA isomers in the plasma coeluted with 13-cis RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Animals; Cell Differentiation; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; In Vitro Techniques; Isotretinoin; Limb Buds; Maternal-Fetal Exchange; Mice; Mice, Inbred ICR; Placenta; Pregnancy; Pregnancy Outcome; Stereoisomerism; Teratogens; Tretinoin

We report on our experience in the experimental induction of Neural Tube Defects (NTD) in the foetal rat by maternal administration of retinoic acid. The teratogen diluted in olive oil was administered in a single intragastric dose (125 mg/kg body weight) to pregnant rats (n = 31) on the 10th day of gestation. Pure olive oil was given to control rats (n = 9). The foetuses were recovered by caesarian section on the 20th day and prepared for morphological investigation. We have studied 201 experimental and 82 control animals. There were NTD in 36.3% of experimental foetuses and none in the control ones. Sacral dysraphism was the most frequent defect but we also observed Arnold Chiari malformations and crowding of the bony limits by an enlarged neural axis. Other associated malformations found were: craneofacial (78.1%), caudal (80%), anorectal (31.4%), and limb defects (89.5%). This simple and inexpensive model may allow us to gain a better knowledge of the biology in the foetus with NTD.

Topics: Abnormalities, Drug-Induced; Animals; Disease Models, Animal; Female; Fetus; Neural Tube Defects; Pregnancy; Rats; Rats, Wistar; Tretinoin

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.

Topics: Abnormalities, Drug-Induced; Anticonvulsants; Child; Child, Preschool; Female; Humans; Infant; Infant, Newborn; Isotretinoin; Male; Oxidation-Reduction; Retinoids; Seizures; Tretinoin; Vitamin A

The Sonic hedgehog gene has been identified as a candidate for the signal mediating the function of the zone of polarizing activity (ZPA) during limb development in tetrapods. To better understand the early steps of development of paired fin buds in fish, we have analyzed the regulation of the zebrafish Sonic hedgehog gene (shh/vhh-1) in response to retinoic acid. Systemic administration of retinoic acid (RA) to zebrafish embryos during the initial stages of pectoral fin bud development resulted in the induction of ectopic expression of shh/vhh-1 on the anterior margin of the bud under the apical ectodermal ridge and in abnormal pectoral fin bud morphology. RA treatment also resulted in ectopic shh/vhh-1 expression in floor plate cells at the caudal end of the neural keel. These results suggest that the control of ZPA function during the initial stages of development of paired appendages has been conserved between fish and tetrapods.

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Nonmammalian; Embryonic Induction; Extremities; Gene Expression Regulation, Developmental; Hedgehog Proteins; Limb Deformities, Congenital; Notochord; Phylogeny; Protein Biosynthesis; Proteins; Trans-Activators; Tretinoin; Vertebrates; Zebrafish

This study demonstrates early embryonic metabolism of exogenous all-trans-3,4-didehydroretinol (vitamin A2) to all-trans-3,4-didehydroretinal and to all-trans-3,4-didehydroretinoic acid in Xenopus embryos during neurulation. The latter metabolite was recently shown to bind with high affinity and to activate various retinoic acid receptors. Embryos treated with all-trans-3,4-didehydroretinol during early or late gastrulation exhibited abnormalities along the anteroposterior axis. The abnormalities were primarily in the posterior regions of the embryo, with only minor defects anteriorally. Eye malformations, typical for early exposure to 9-cis- and all-trans-retinols and retinals (companion paper), were not observed. We also present evidence that all-trans-3,4-didehydroretinoic acid is present endogenously during early neurulation and is evenly distributed along the anteroposterior axis. After treatment with all-trans-3,4-didehydroretinol, embryonic levels of all-trans-3,4-didehydroretinoic acid exceeded endogenous levels of this metabolite during early and late neurulation. We hypothesize that the dysmorphogenic effects produced by treatment of Xenopus embryos with the alcohol precursor, all-trans-3,4-didehydroretinol, are the result of its embryonic conversion to its corresponding acid ligand.

Topics: Abnormalities, Drug-Induced; Animals; Chromatography, High Pressure Liquid; Embryo, Nonmammalian; Female; Phenotype; Teratogens; Tretinoin; Vitamin A; Xenopus laevis

We have previously demonstrated that stress proteins (SPs) are synthesized in tissues in which malformations are later observed following treatment with the developmental toxicant, retinoic acid (RA), on day 11 of gestation (GD 11). These proteins were not synthesized in tissues which did not present with malformations near partuition. The purpose of the present investigation was to determine if this correlation between early SP synthesis and later malformation was present at other times during gestation. CD-1 strain mice were dosed orally with corn oil or 100 mg/kg body weight RA on GD 10 or 13. Some of the mice in each group were given an intraperitoneal injection of 3H-leucine to label embryonic protein synthesis one hour after dosing with RA. These animals were sacrificed 1.5 hour later, and embryonic protein synthesis was determined by two-dimensional gel electrophoresis followed by autoradiography. Other animals in each group were sacrificed on day 17 of gestation, and fetuses were examined for the presence of malformations. Following treatment with RA on day 10 of gestation, malformations were observed in the forelimbs, the hindlimbs and the tail; heart defects were not observed. SPs of 20-25,000 and 90,000 relative molecular mass (Mr) were synthesized in the forelimb bud and tail; in addition, a second low molecular weight (20-25,000) and a 84,000 Mr SPs were synthesized in forelimb buds. No SPs were synthesized in the hindlimb bud or the heart. Following RA treatment on GD 13, cleft palate was observed in 58% of fetuses; no other malformations were found. Proteins of 34,000, 84,000 and 90,000 Mr were synthesized in craniofacial tissue; SPs were not observed in forelimb bud, hindlimb bud, heart or tail tissues at this time. Therefore, it appears that there may be a correlation between tissue-specific SP synthesis early in organogenesis and the presence of a malformation later in gestation.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Female; Fetus; Gestational Age; Heat-Shock Proteins; Male; Mice; Molecular Weight; Pregnancy; Tretinoin

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RAR alpha, beta, and gamma. The expression of RAR beta gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RAR beta could play specific roles during morphogenesis. Four isoforms of the RAR beta gene (beta 1-beta 4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RAR beta 2 isoform is dispensable for normal development. To ascertain the function of all RAR beta isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RAR beta gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1/11 RAR beta mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RAR beta null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RAR beta may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RAR beta isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

Topics: Abnormalities, Drug-Induced; Animals; Base Sequence; Embryonic and Fetal Development; Extremities; Female; Ganglia; Gene Targeting; Genes, Homeobox; Homozygote; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation; Receptors, Retinoic Acid; RNA, Messenger; Skull; Tretinoin

Embryonic exposure to the vitamin A metabolite retinoic acid (RA) causes malformations in numerous developing tissues, including the limbs, which serves as a model system of retinoic acid action. RA treatment of wild-type mouse embryos results in digit truncations and long bone reductions. These effects are mediated by products of the retinoic acid and retinoid X receptor genes (RARs and RXRs), members of the nuclear receptor family of ligand-dependent transcription factors. Mouse embryos homozygous for a mutation in the RXR alpha gene appear normal in limb development, although such embryos are phenotypically affected in other tissues. We now describe resistance to limb malformations normally induced by teratogenic RA exposure in the RXR alpha-/- background. RA treatments that cause limb defects in 100% of wild-type embryos fail to elicit malformations in RXR alpha homozygotes, implicating RXR alpha as a component in the teratogenic process in the limbs. Heterozygous embryos are intermediate in sensitivity to RA, suggesting the importance of RXR alpha gene dosage in limb teratogenesis. Expression of the RA-inducible gene RAR beta 2 was equivalent between wild-type and homozygous embryos after RA treatment. RA treatment also did not distinguish between wild-type and RXR alpha -/- embryos in the spatial expression of sonic hedgehog (Shh) and hoxd-12, two other genes implicated in limb development. However, the quantitative level of hoxd-12 expression was elevated in RXR alpha -/- embryos. These observations indicate that transcriptional processes which are inappropriately regulated in the mouse limb by exogenous RA require RXR alpha for their execution, and that specific teratogenic processes, as well as specific normal developmental processes under vitamin A control, occur through individual members of the RXR and RAR families.

Topics: Abnormalities, Drug-Induced; Animals; Gene Dosage; Gene Expression; Limb Deformities, Congenital; Mice; Mice, Transgenic; Models, Biological; Nuclear Proteins; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin

Visceral heterotaxy syndrome causes abnormal arrangement of thoracoabdominal organs and severe complex cardiac anomalies by abnormal laterality. The purpose of the present study is to analyze the incidence and pattern of heterotaxy syndrome in etretinate and all-tran retinoic acid treated pregnant DDY mice. Pregnant DDY mice were intragastrically given a single dose of 15 mg/kg of etretinate at day 6, 7 of gestation, 30 mg/kg of etretinate at day 7 of gestation and 20 mg/kg of all-trans retinoic acid at day 7 of gestation. The incidence of visceral heterotaxy was highest in the etretinate 15 mg/kg treated group on day 7 of gestation (38.5%). The major cardiovascular anomalies in heterotaxy syndrome were common atrium, common atrioventricular valve, atrioventricular septal defect, transposition of great arteries, pulmonary atresia, pulmonary artery hypoplasia and aortic arch anomalies. Atrial situs of heterotaxy syndrome were right isomerism, solitus-like, inversus-like and left atrial aplasia, but right isomerism was observed most frequently. The results suggest that retinoic acid exerts a significant effect on the determination of atrial situs during the development of mouse embryo.

Topics: Abnormalities, Drug-Induced; Animals; Blood Vessels; Female; Heart Defects, Congenital; Mice; Pregnancy; Syndrome; Tretinoin

These investigations provide data pertaining to the metabolism and disposition of exogenous 9-cis-retinoic acid and all-trans-retinoic acid during neurulation in Xenopus embryos. Each isomer elicited malformations of the heart, eye, and brain, but approximately 2-fold higher concentrations of all-trans-retinoic acid than 9-cis-retinoic acid were required to produce qualitatively and quantitatively similar dysmorphogenic effects. The dymorphogenic effects of all-trans-retinoic acid could not be attributed to the isomerization of all-trans-retinoic acid to 9-cis-retinoic acid. Evidence is provided that all-trans-retinoic acid and 9-cis-retinoic acid are both direct-acting dysmorphogens. After Xenopus embryos were exposed to all-trans-retinoic acid, elevated levels of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, and 13-cis-retinoic acid were detected in the embryos, whereas embryonic levels of 9-cis-retinoic acid were actually slightly lower than endogenous levels during early neurulation. After embryos were exposed to 9-cis-retinoic acid during neurulation, elevated levels of 4-oxo metabolites, glucuronides and 9,13-di-cis-retinoic acid were observed in the embryos. At equivalent concentrations, 4-oxo-13-cis-retinoic acid and 13-cis-retinoic acid elicited fewer severe multiple malformations than all-trans isomers 9,13-di-cis isomers, or 9-cis isomers. The dysmorphogenic effect of 9,13-di-cis-retinoic acid may be caused by its isomerization to 9-cis-retinoic acid. All-trans retinoyl-beta-glucuronide was only marginally teratogenic at the highest concentrations tested.

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Nonmammalian; Female; Tretinoin; Xenopus laevis

In chick embryos, the anterior greater portion of the neural tube develops by the folding, apposition, and fusion of the neuroectoderm. The smaller caudal portion that forms the secondary neural tube (lumbosacral and coccygeal regions) is derived from the tail bud, an aggregate of mesenchymal cells located at the caudal limit of the body. Tail bud mesenchyme, arranged in a solid cord, undergoes mesenchymal-epithelial transformation to form the secondary neural tube. Previous evidence suggests that this transformation is accompanied by modulation of cell surface glycoconjugates in the differentiating tissues. In this study, we show by lectin histochemistry and lectin blotting of proteins isolated by SDS-PAGE, that Datura stramonium agglutinin (DSA) binds preferentially to differentiating tail bud cells. This lectin is specific for beta 1-4-linked N-acetylglucosamine oligomers, such as the oligosaccharides of the poly-N-acetyllactosamine series that have been previously implicated in cell differentiation. Ultrastructural lectin cytochemistry indicates that at least some of the proteins binding DSA are localized extracellularly. The use of DSA as a teratogen resulted in embryos showing a variety of neural tube and notochord defects. We have also examined the binding of DSA to embryos that were treated with teratogenic doses of retinoic acid by sub-blastodermal injection, and find that the DSA-binding patterns are perturbed. Analysis of DSA-treated embryos using the TUNEL technique indicated that cell death was not a factor in DSA teratogenesis. This strongly suggests that the glycoconjugates of the cell surface have a role in the normal differentiation of tail bud mesenchyme into the neuroepithelium of the secondary neural tube. Perturbations of glycoconjugate activity results in defects of the secondary neural tube and associated tail bud derivatives.

Topics: Abnormalities, Drug-Induced; Animals; Cell Membrane; Chick Embryo; DNA Damage; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Glycoconjugates; Immunohistochemistry; Lectins; Membrane Proteins; Microscopy, Immunoelectron; Nervous System; Nervous System Malformations; Neural Tube Defects; Notochord; Survival; Tail; Tretinoin

The distribution of cellular retinoic acid-binding proteins I and II (CRABP I and II) during the first 6 days of chick development has been investigated using immunoblotting. Since retinoic acid (RA) is teratogenic to some parts of the embryo, stimulatory to other parts, and has no effect on others it may be that the distribution of cytoplasmic proteins such as CRABP I and II plays some role in this differential activity. Neither protein is expressed in the day 2 embryo, but from day 3 onwards both proteins are expressed and CRABP I is in considerable excess over CRABP II. Within the day 4 embryo there is some significant variation in the distribution according to tissue type. Neural tissues, neural crest derivatives, and limb buds most strongly express CRABP I whilst other tissues contain only moderate levels, and heart and epidermis do not express CRABP I at all. CRABP II has a widespread distribution, although at a lower level than CRABP I, with the exception of somites and ectoderm which do not express it at all. In the limb buds, there is a significant variation in CRABP I levels across the anteroposterior axis which suggests that these two CRABPs may have different functions during development. The relationship of these distributions in the embryo to the role of endogenous RA and the teratogenic effects of RA is discussed.

Topics: Abnormalities, Drug-Induced; Animals; Chick Embryo; Receptors, Retinoic Acid; Tretinoin

Birth defects registries have been established following the thalidomide epidemic in the early sixties. A first objective of registries is to monitor the frequency of specific malformations in order to detect as early as possible any variation caused by the introduction of a new teratogen in the environment. After 30 years not any new teratogen has been identified by this. It appears that the predictive value of alarms generated by surveillance is extremely low and that resources are insufficient to investigate most of warnings. However, registries have been useful to test specific hypotheses i.e. the effect of iatrogenic exposures. Three examples are given, showing the necessary characteristics of registries: topical treatment with tretinoin, chorionic biopsy and cancer treatment.

Topics: Abnormalities, Drug-Induced; Antineoplastic Agents; Chorionic Villi Sampling; Congenital Abnormalities; Female; Humans; Iatrogenic Disease; Infant, Newborn; Maternal-Fetal Exchange; Predictive Value of Tests; Pregnancy; Risk Factors; Tretinoin

Retinoic acid (RA) plays an important role during normal embryogenesis, however high doses of RA are teratogenic. Retinoic acid receptor-beta 2 (RAR-beta 2) mRNA and protein levels were previously demonstrated to undergo rapid elevation in susceptible tissues after treatment with teratogenic doses of RA. In this report we compared the effects of a number of retinoids, which represent a wide variety of chemical structures and which differ in their teratogenic potencies, on RAR-beta 2 mRNA levels in mouse embryos 6 hr after treatment. Retinoid treatments which result in a high incidence of limb defects elevated RAR-beta 2 mRNA levels similarly (10-14 fold in the limb buds, 4-8 fold in the head, and 2-4 fold in the remainder of the body). On the other hand, retinoid treatments which cause a low or no incidence of limb defects resulted in minor changes in RAR-beta 2 mRNA levels in each embryonic region. Therefore, a strong positive correlation was found between the elevation of RAR-beta 2 mRNA levels and the retinoids which produce limb defects. This provides further evidence that an elevation of RAR-beta 2 mRNA levels, and subsequently protein levels, is an important event involved in mediating the effects of RA during dysmorphogenesis.

Topics: Abnormalities, Drug-Induced; Animals; Benzoates; Ectromelia; Fatty Acids, Unsaturated; Female; Isomerism; Mice; Mice, Inbred ICR; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Tretinoin

This manuscript reports on the limb malformations and axial skeleton alterations found in legless fetuses and their heterozygote and wild-type littermates transplacentally exposed to all-trans-retinoic acid via a single intraperitoneal injection on Day 7, 8, 9, 9.5, 10, 10.5, or 11 of gestation. The most surprising aspect of the results was the temporal sensitivity of the legless mouse limb to exogenous retinoic acid. On Day 11, when both fore- and hindlimbs of nonmutant embryos can be made abnormal by retinoic acid and other teratogens, retinoic acid did not increase the frequency or severity of legless hindlimb defects and forelimb malformations were only slightly enhanced. On the other hand, retinoic acid administration on Day 7 exacerbated forelimb malformations in legless fetuses at a time when visible emergence of the affected structure is still 48 hr away. Heterozygote and wild-type fetuses had no limb malformations at this time point. A similar phenomenon was observed with hindlimb malformations after retinoic acid exposure on Day 8 except for a few mild limb malformations in heterozygotes at a high dose of retinoic acid. This early hypersensitivity of fore- and hindlimbs was followed by a period of reduced sensitivity (Day 8 forelimb; Day 9 hindlimb) when even very high doses (50 mg/kg) induced minimal changes in the typical legless malformation pattern. Subsequently, at the time of visible limb bud emergence (Day 9 forelimbs; Day 10 hindlimbs), sensitivity to exogenous retinoic acid was again detected. Surprisingly, the altered malformation patterns induced by retinoic acid in lgl mutants were nearly identical to those from earlier, pre-emergence exposure. A number of axial skeleton alterations were induced in legless fetuses by retinoic acid, especially after exposure on Days 7, 8, or 9. Posterior truncations were particularly noteworthy, showing a graded response in which frequency and severity of truncation were worst in lgl/lgl fetuses; heterozygotes gave an intermediate response, and wild-type fetuses were least affected. This exacerbation of the legless phenotype by exogenous retinoic acid coupled with the similarity between legless and retinoid malformations suggest that the legless mutation has altered endogenous retinoid homeostasis or a downstream retinoid-responsive gene.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Extremities; Female; Gestational Age; Limb Deformities, Congenital; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Pregnancy; Tretinoin

Retinoic acid (RA) has been implicated in vertebrate neural pattern formation. In this paper we analysed the expression patterns of the cellular retinoic acid binding proteins (CRABP-I and II) during early morphogenesis in normal and RA-treated mouse embryos by whole-mount in situ hybridization. This technique allowed a detailed analysis of the spatial and temporal changes in mRNA expression pattern. Both CRABPs were expressed in a rhombomere specific pattern; putative neural crest cells in the branchial arches expressed the CRABPs at levels corresponding to the rhombomere from which they were derived. CRABP-II, but not CRABP-I, was expressed in the neural epithelium caudal to the hindbrain. CRABP-I is strongly expressed in a fine net-like pattern which extends from the caudal diencephalon to the rostral hindbrain and remains predominantly dorsal to the lateral midline of the neural tube. This network corresponds to the pattern formed by the putative first axons of the embryonic mouse brain which are produced by the developing neurons of the mesencephalic nucleus of the trigeminal nerve. Although the expression of CRABP-I was unaffected by a teratogenic dose of RA, CRABP-II expression was increased slightly with no alteration in the normal spatial or temporal boundaries. These results support the suggestion that the CRABPs may play an important role in modulating endogenous RA levels, particularly in the developing nervous system and its neural crest derivatives. Furthermore, the limited ability of CRABP mRNA levels to respond to exogenous retinoids may be a factor in retinoid teratogenicity.

Topics: Abnormalities, Drug-Induced; Animals; Branchial Region; Embryonic and Fetal Development; Female; Fetal Proteins; Gene Expression Regulation; In Situ Hybridization; Mice; Morphogenesis; Nerve Tissue Proteins; Neural Crest; Pregnancy; Receptors, Retinoic Acid; Rhombencephalon; RNA, Messenger; Tretinoin

No embryotoxic or teratogenic effects, considered to be treatment related, were observed in rabbits after daily oral doses of 3 mg/kg of 13-cis-retinoic acid (13-cis-RA) from Day 8 to Day 11 of gestation. In contrast, treatment with 15 mg/kg/day significantly increased the rate of fetal resorptions (22%) and 13 out of 68 surviving fetuses (16%) were malformed. Pharmacokinetic studies with both dosing regimens of 13-cis-RA in pregnant rabbits showed that on Day 11 of gestation, high concentrations of parent compound, 13-cis-RA, and its major metabolite, 13-cis-4-oxoRA, existed in maternal plasma. Much lower concentrations were found for all-trans-4-oxoRA and all-trans-RA. The area under the concentration-time curve (AUC) of all-trans-RA following the 15 mg/kg/day dosing regimen of 13-cis-RA was only 1.2% that of parent compound 13-cis-RA. At this dose, embryo levels of 13-cis-RA, 13-cis-4-oxoRA, and all-trans-4-oxoRA were 2.5-, 4.7-, and 3.6-fold higher by AUC comparison (24-hr period of Day 11) compared with the dose of 3 mg/kg. However, embryo levels of all-trans-RA were virtually identical at both doses and were, in fact, somewhat lower than endogenous concentrations measured in untreated rabbit embryos. In contrast to mice, where isomerization from 13-cis- to all-trans-RA was suggested to be crucial for the teratogenic action of 13-cis-RA, we found that the teratogenic action of 13-cis-RA (15 mg/kg/day) in rabbits is characterized by increased whole embryo concentrations of 13-cis-RA, 13-cis-4-oxoRA, and all-trans-4-oxoRA, but not of all-trans-RA.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Fetus; Gestational Age; Isotretinoin; Maternal-Fetal Exchange; Placenta; Pregnancy; Rabbits; Stereoisomerism; Tretinoin

All retinoic acid receptors (RAR alpha, beta, gamma) have two isoforms, whose function is unknown. We now show that at least for RAR gamma, the isoforms are differentially distributed in the embryo. RAR gamma 1 and RAR gamma 2 are detected in the head region, whereas RAR gamma 2 is the sole isoform expressed in the tail region. Specifically, it is expressed in the chordoneural hinge, a region of the tailbud that has organizing properties. Treatment with high doses of retinoic acid (RA) reduces expression in this region. The results are discussed in terms of the known teratogenic effects of RA in the tail region.

Topics: Abnormalities, Drug-Induced; Animals; Gene Expression Regulation; Head; Organ Specificity; Receptors, Retinoic Acid; Species Specificity; Tail; Tretinoin; Vertebrates; Xenopus laevis

The evidence presented here strengthens the argument that RA-induced truncation defects of the embryonic limb, and probably other teratogenic effects, are mediated by the nuclear retinoid receptors, particularly the RAR-beta 2 isoform. Although apoptotic cell death and an increased transglutaminase (tTG) activity accompany teratogenesis, it should be emphasized that the increased levels of RAR-beta 2 may influence additional events in limb development, e.g., modulation of connective tissue differentiation and an inhibition of chondrogenesis. Further work entails screening the effects of RA on genes targeted by the receptors.

Topics: Abnormalities, Drug-Induced; Animals; Apoptosis; Extremities; Female; Limb Deformities, Congenital; Maternal-Fetal Exchange; Mesoderm; Mice; Pregnancy; Receptors, Retinoic Acid; Transglutaminases; Tretinoin

Treatment of late blastula/early gastrula stage Xenopus embryos with all-trans retinoic acid results in disruption of the primary body axis through effects on both mesoderm and neuroectoderm. This effect of retinoic acid, coupled with the known presence of retinoic acid in Xenopus embryos has led to the proposal that retinoic acid may be an endogenous morphogen providing positional information in early development. To further elucidate the role of retinoic acid in early Xenopus development, we have attempted to interfere with the retinoic acid signalling pathway both at the level of retinoic acid formation, by treatment with citral (3,7-dimethy-2,6-octadienal), and at the level of nuclear retinoic acid receptor function, by microinjection of v-erbA mRNA. The feasibility of this approach was demonstrated by the ability of citral treatment and v-erbA mRNA injection to reduce the teratogenic effects of exogenous retinol and retinoic acid, respectively, in early Xenopus development. Interestingly, v-erbA mRNA injection and citral treatment of gastrula stage embryos resulted in tadpoles with a similar set of developmental defects. The defects were chiefly found in tissues that received a contribution of cells from the neural crest, suggesting that at least a subset of neural crest cells may be sensitive to the endogenous level of retinoic acid. In accord with this proposal, it was found that the expression patterns of two early markers of cranial neural crest cells, Xtwi and XAP-2, were altered in embryos injected with v-erbA mRNA. These results indicate that structures in addition to the primary axis are regulated by retinoic acid signalling during early Xenopus development.

Topics: Abnormalities, Drug-Induced; Acyclic Monoterpenes; Animals; Blotting, Western; Gastrula; Gene Expression; In Situ Hybridization; Microinjections; Monoterpenes; Morphogenesis; Oncogene Proteins v-erbB; Retroviridae Proteins, Oncogenic; RNA, Messenger; Terpenes; Tretinoin; Vitamin A; Xenopus

The effects of retinoids are mediated by two types of receptors, the retinoic acid receptors (RARs) and the retinoid-X-receptors (RXRs). The physiological ligand of the RARs is all-trans-retinoic acid whereas RXRs have high affinity for 9-cis-retinoic acid, a naturally occurring retinoid isomer. RXRs are broadly expressed in embryonic and adult tissues, and they are capable of forming homodimers as well as heterodimers with RARs and other nuclear hormone receptors. The role of 9-cis-retinoic acid in regulating the activity of RXR homodimers and RXR-containing heterodimers is poorly understood in vivo. To begin to explore the function of 9-cis-retinoic acid in morphogenesis, we have examined the activity of this isomer in the chick wing. Using reverse transcriptase polymerase chain reaction analyses, we show that RXR gamma is expressed in stage 20 wing buds. Similar to all-trans-retinoic acid, the 9-cis-isomer induces pattern duplications when locally applied to chick wing buds, but the 9-cis isomer is about 25 times more potent than the all-trans form. Furthermore, applied all-trans-retinoic acid is converted to the 9-cis isomer in the wing bud. The ratio of 9-cis to all-trans-retinoic acid established in the tissue is approximately 1:25. This quantitative agreement between the degree of conversion and the 25-fold higher efficacy of the 9-cis isomer, raises the possibility that, at least in part, the effects of all-trans-retinoic acid on the wing pattern result from a conversion to the 9-cis isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Animals; Base Sequence; Bone and Bones; Chick Embryo; Dose-Response Relationship, Drug; Embryonic Induction; Isomerism; Molecular Sequence Data; Morphogenesis; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Tretinoin; Up-Regulation; Wings, Animal

We used information from the Group Health Cooperative of Puget Sound, Washington, USA, to evaluate the risk of birth defects in mothers exposed to topical tretinoin--a retinoid preparation used to treat acne--in the first trimester of pregnancy. We identified 215 women who delivered live or stillborn infants at Group Health Cooperative hospitals and who were exposed to topical tretinoin early in pregnancy, and 430 age-matched nonexposed women who delivered live or stillborn infants at the same hospitals. The prevalence of major anomalies among babies born to the exposed women was 1.9% and among babies born to the nonexposed women was 2.6%. The relative risk estimate for having a baby with a major congenital anomaly for exposed versus nonexposed women was 0.7 (95% CI 0.2-2.3). We conclude that topical tretinoin is not associated with an increased risk for major congenital disorders.

Topics: Abnormalities, Drug-Induced; Administration, Topical; Adolescent; Adult; Case-Control Studies; Drug Prescriptions; Female; Humans; Matched-Pair Analysis; Medical Records Systems, Computerized; Pregnancy; Pregnancy Outcome; Pregnancy Trimester, First; Prenatal Exposure Delayed Effects; Prevalence; Risk Factors; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Acne Vulgaris; Administration, Topical; Female; Humans; Infant, Newborn; Pregnancy; Pregnancy Complications; Tretinoin

Topics: Abnormalities, Drug-Induced; Administration, Topical; Female; Humans; Infant, Newborn; Pregnancy; Pregnancy Trimester, First; Prenatal Exposure Delayed Effects; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Female; Forelimb; Gestational Age; Hindlimb; Homozygote; Limb Deformities, Congenital; Male; Maternal-Fetal Exchange; Mice; Mice, Mutant Strains; Phenotype; Pregnancy; Tretinoin

Full primitive streak stage chick embryos, cultured in vitro for 20 h in the presence of 10(-9) to 10(-7) moles of retinoic acid (retinol, all-trans), exhibit increasing extent of malformations. RA causes caudalization, suppression of telencephalon, formation of open and enlarged neural tube in the regions of midbrain, hindbrain and spinal cord, failure of fusion of heart tubes, and gives rise to winged or diffused, and even supernumerary somites. Extreme abnormalities include failure to form the head fold and foregut. Abnormal embryos were graded according to Rao and Chauhan (Teratology 4: 191-198, 1971), and we find that the larger the severity of malformation, the smaller the size of total cell population and blastoderm area. Concomitant to caudalization, retinoic acid suppresses the cell population growth.

Topics: Abnormalities, Drug-Induced; Animals; Blastoderm; Brain; Cell Division; Chick Embryo; Culture Techniques; Heart; Morphogenesis; Neural Tube Defects; Telencephalon; Tretinoin

When single large equimolar doses (0.38-0.41 mmol/kg BW) of all-trans retinoic acid (RA), all-trans retinoyl beta-glucose (RBGL), and all-trans retinoyl beta-glucuronide (RBG) are administered orally in oil on day 8.5 of pregnancy to Sprague-Dawley rats, RA and RBGL proved highly teratogenic, whereas RBG was not. Indeed, fetuses from RBG-treated dams were 16% heavier (P < 0.01) than control fetuses. After dosing with RA and RBGL, RA appeared in large amounts within 0.5 h in the maternal plasma and within 1.0 h in the embryo. In contrast, orally administered RBG seemed to be absorbed much more slowly, to be converted very slowly to RA, and not to accumulate either as RBG or as RA in the embryo. When incubated in vitro with embryos and attached membranes, however, both all-trans RBG and all-trans RA were partially converted to 13-cis RA. The nonteratogenicity of RBG, in contrast to RA, seems to be due to a much slower rate of GI absorption, a slow rate of hydrolysis to RA, a limited passage from the maternal circulation into the embryo, and a lower inherent toxicity.

Topics: Abnormalities, Drug-Induced; Animals; Female; Fetus; Glucose; Liver; Maternal-Fetal Exchange; Pregnancy; Rats; Rats, Sprague-Dawley; Stereoisomerism; Structure-Activity Relationship; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Biotransformation; Culture Techniques; Rats; Retinoids; Tretinoin

We have compared the transcript distribution of cellular retinoic acid binding protein (CRABP) I and II genes in mouse embryos at various stages of development. Both CRABP transcripts are present in embryonic structures from the earliest stages studied and exhibit specific patterns of distribution, suggesting that the two retinoic acid (RA) binding proteins perform different functions during mouse embryogenesis. The CRABP I transcript distribution correlates well with structures known to be targets of excess retinoid-induced teratogenesis (e.g. neural crest cells and hindbrain), suggesting that cells expressing CRABP I are those that cannot tolerate high levels of RA for their normal developmental function. The embryonic structures expressing CRABP II transcripts include those structures that have been shown to be adversely affected by excess of retinoids, such as limbs and hindbrain, but CRABP II transcripts are also found in structures not known to be specifically vulnerable to raised RA levels. The CRABP II gene is coexpressed with retinoic acid receptor (RAR)-beta and cellular retinol binding protein (CRBP) I genes in a number of tissues such as the gut endoderm, hypophysis and interdigital mesenchyme, all of which are devoid of CRABP I transcripts. Interestingly, the expression of the three genes, RAR-beta, CRABP II and CRBP I, is induced by retinoic acid, which suggests a link between the synthesis of RA from retinol and the control of expression of subsets of RA-responsive genes. The transcript distribution of CRABP I and II is discussed in relation to the teratogenic effects of RA, and compared to the RA-sensitive pattern of expression of other important developmental genes.

Topics: Abnormalities, Drug-Induced; Animals; Autoradiography; Brain; Carrier Proteins; Central Nervous System; Embryonic and Fetal Development; Extremities; Gene Expression; Genes; Genes, Developmental; Mice; Molecular Probe Techniques; Morphogenesis; Receptors, Retinoic Acid; RNA, Messenger; Tretinoin

Among the findings associated with the human Retinoic Acid Embryopathy are hindbrain defects including the Arnold-Chiari malformation. The human Arnold-Chiari malformation (ACM) is a malformation complex where the cardinal feature is herniation of the caudal hindbrain into the vertebral column; it is frequently accompanied by lumbosacral myelorachischisis and hydrocephalus. Mice exposed to all-trans-retinoic acid or etretinate on day 8.25 of pregnancy, produce offspring with hindbrain herniation and caudal lumbosacral myelorachischisis in addition to a variety of other craniofacial and caudal malformations. Several experimental animals were observed to lack the caudal myelorachischisis proving that this lesion is not required to generate hindbrain herniation. We provide evidence that the cranial malformations, including hindbrain herniation, result from primary damage to the neural crest and the rhombencephalon. The vulnerability of these sites appears to be correlated with the presence of normal physiological cell death. While these experimental animals differ in many respects from the typical human Arnold-Chiari malformation, they may provide some insight into the pathogenesis of the latter.

Topics: Abnormalities, Drug-Induced; Animals; Disease Models, Animal; Ear; Exophthalmos; Female; Humans; Isotretinoin; Male; Mandible; Mice; Mice, Inbred C57BL; Neural Crest; Pregnancy; Rhombencephalon; Tretinoin

Topics: Abnormalities, Drug-Induced; Administration, Cutaneous; Ear; Female; Humans; Infant, Newborn; Tretinoin

Mesenchymal cells in the limb buds of midgestation mouse embryos suffer prominent cell death upon exposure to retinoic acid (RA), an event likely associated with the micromelic and phocomelic anomalies of the resultant fetuses. It has been suggested, but not yet shown, that cells die by an active process termed apoptosis rather than by necrotic cytolysis. In certain cell types, investigators have previously observed a specific and early effect of RA on transcriptional activation of the gene for tissue transglutaminase (tTG), an enzyme suspected to play a role in apoptosis. We report here a distinct but transient increase in tTG activity which accompanied the initiation of cell death in the mesenchymal cells located in the central core of RA-treated limb buds. We also ascertained microscopically that the cytological appearance of the affected cells was consistent with a characterization of the process of cell death as apoptosis.

Topics: Abnormalities, Drug-Induced; Animals; Apoptosis; Dose-Response Relationship, Drug; Female; Forelimb; Liver; Mice; Mice, Inbred ICR; Pregnancy; Transglutaminases; Tretinoin

Oral administration of retinol (50 mg/kg) to NMRI mice on day 11 of gestation (vaginal plug = day 0) led to the metabolic formation of high quantities of all-trans retinoic acid and all-trans-4-oxoretinoic acid, both known as potent teratogenic agents in the mouse. A 96% reduction of the area under the concentration-versus-time-curve (AUC) of metabolically generated all-trans retinoic acid in maternal plasma, and an 84% decrease in the embryonic AUC were observed when mice had been pretreated with the alcohol dehydrogenase inhibitor 4-methylpyrazole. A similar reduction was observed for the major metabolite of all-trans retinoic acid in the mouse, all-trans-4-oxoretinoic acid. However, 4-methylpyrazole pretreatment decreased the AUC of retinol by 10% in maternal plasma and 15% in embryo. Treatment with retinol alone resulted in 55.6%, 43.9% and 56.0% skeletal anomalies of the forelimbs, hindlimbs and craniofacial structures, respectively. Pretreatment with 4-methylpyrazole lowered the retinol induced skeletal defects to 31.3%, 24.0% and 31.3%, respectively, in the forelimb, hindlimb and craniofacial region. Typical retinoid-induced malformations for gestational day 11, e.g. bent or reduced zeugopod or stylopod elements, or cleft palate, were significantly reduced by 4-methylpyrazole pretreatment but were still detected in significantly higher prevalence than in control mice. These data suggest that the teratogenic activity of a single high dose of vitamin A in mouse is partially but not exclusively dependent on the metabolic activation of retinol to all-trans retinoic acid. Thus it could be hypothesized that retinol is either a proximate teratogen or a coteratogen with all-trans retinoic acid.

Topics: Abnormalities, Drug-Induced; Alcohol Dehydrogenase; Animals; Chromatography, High Pressure Liquid; Depression, Chemical; Female; Fomepizole; Male; Mice; Pregnancy; Pyrazoles; Stereoisomerism; Teratogens; Tretinoin; Vitamin A

Topics: Abnormalities, Drug-Induced; Animals; Brain; Dose-Response Relationship, Drug; Eye Abnormalities; Female; Gestational Age; Mice; Mice, Inbred C3H; Pregnancy; Tretinoin

This study examines development of rat, mouse, and human embryonic palates in submerged, serum-free organ culture. The concentration-response profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined and the mechanisms of clefting in vitro were compared to observed in vivo responses. Craniofacial regions were dissected on gestational day (GD) 12 for mice and GD 14 for rat, and cultured for 3-4 days in Bigger's BGJb medium in flasks flushed with 50% O2, 45% N2, 5% CO2. Growth and fusion of secondary palates were scored under a dissecting microscope. In serum-free control medium, mouse and rat palatal fusion occurred within the 4-day culture period. Supplementing with fetal bovine serum (FBS) in excess of 1% interfered with growth and fusion in control medium. RA significantly inhibited fusion of mouse and rat palates at 5 x 10(-9) and 1 x 10(-10) M, respectively, with RA-induced clefting related to abnormal proliferation and differentiation of medial epithelia. In contrast, glucocorticoid-induced clefting was due to concentration-dependent inhibition of shelf growth. TRI significantly inhibited fusion at 4 x 10(-5) M, and 1 x 10(-4) M DEX or HC, inhibited fusion of 19 and 42% of shelves, respectively. The response rate for DEX in the presence of 1% FBS was increased (42% unfused). TCDD clefting was due to altered medial epithelial differentiation and 1 x 10(-8) M TCDD affected 36% of CD-1 mouse, 23% of C57BL/6N mouse, and 47% of F344 rat palates. When the medium was supplemented with 1% FBS, selenium, transferrin, and additional glutamine, the response of C57BL/6N embryos increased to 75%. This rate is similar to that reported for Trowell's-type cultures with IMEM:F12 medium and 1% FBS. The increased responsiveness to DEX or TCDD in the presence of serum suggests that an unknown factor in serum may be required for full activity. Three human embryonic palatal explants (GD 52 or 53) were cultured for 3-6 days and fused during culture. The present study demonstrates that serum-free organ culture supports development of mouse, rat, and human palatal explants. The present study demonstrates the capacity of this organ culture system to model palatogenesis for several species, and to distinguish between various mechanisms of clefting as presented through selected model compounds. This model should be useful for exploring mechanisms of activity at a cellular and mole

Topics: Abnormalities, Drug-Induced; Animals; Culture Media; Glucocorticoids; Humans; Mice; Mice, Inbred C57BL; Organ Culture Techniques; Palate; Polychlorinated Dibenzodioxins; Rats; Rats, Inbred F344; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Heart; Heat-Shock Proteins; Mice; Rats; Teratogens; Tretinoin

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.

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Mammalian; Female; Fetal Death; Macaca fascicularis; Male; Pregnancy; Teratogens; Tretinoin; Weight Gain

The joint action of all-trans retinoic acid (RA) with valproic acid (VPA) was examined at malformation-inducing concentrations for Xenopus embryos. The compounds were selected for testing to evaluate malformation as an endpoint for the developmental toxicity of chemical mixtures and to help assess whether joint action types can be related to modes or mechanisms of chemical toxicity for Xenopus. Three mixtures (3:1, 1:1, and 1:3 RA:VPA) were tested in 96 hr static-renewal exposures. Three separate tests were conducted on each mixture. Positive controls were also tested (i.e., 1:0 and 0:1 solutions). Using toxic unit analysis, the joint action for induction of malformations of all types, craniofacial malformations, and microcephaly was response addition, indicating the chemicals are dissimilar and non-interactive. The results support recent joint action studies in providing evidence that similar acting chemicals might be defined as chemicals with the same biochemical/molecular mechanism of action in inducing malformations in Xenopus embryos.

Topics: Abnormalities, Drug-Induced; Animals; Drug Synergism; Embryo, Nonmammalian; Female; Male; Tretinoin; Valproic Acid; Xenopus laevis

Topics: Abnormalities, Drug-Induced; Europe; Female; Holoprosencephaly; Humans; Infant, Newborn; Pregnancy; Pregnancy Complications; Tretinoin

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Heat-Shock Proteins; Mice; Tretinoin

Previous experiments have demonstrated that oral administration of retinoic acid to ICR mice on day 10 of gestation results in an enhanced expression of postaxial polydactyly of the forelimbs. In the present investigations, the development of the limbs from the time of treatment until the appearance of the defect on day 14 were studied at 12- to 24-hr intervals with histological and vital staining technics. A diffuse cytotoxicity in the central proximal mesoderm was noted with a safranin-toluidine blue stain 12 h after treatment which then peaked 24 hr after treatment. Treated limbs stained with Nile blue sulfate showed a significant increase of necrotic mesodermal cells in the foyer preaxial primaire, foyer marginal I and foyer marginal V when compared to the respective control group. The treated postaxial ectoderm was more hyperplastic than was the control ectoderm on day 12. The treated group developed a squarish bulge on the postaxial boundary of the fifth digital ray on day 14 that was larger than those of the controls. Most of the controls had bulges that were completely necrotic. The treated group also had some completely necrotic bulges but a large number of the treated fetuses had bulges characterized as being less than 60% necrotic.

Topics: Abnormalities, Drug-Induced; Animals; Ectoderm; Female; Forelimb; Humans; Mesoderm; Mice; Mice, Inbred ICR; Morphogenesis; Pregnancy; Teratogens; Toes; Tretinoin

The present study describes the teratogenic effects of retinoic acid (RA) on the development of the chick tail bud. Chick embryos were recovered 48 hours after treatment at HH stages 11 to 16 with various dosages of RA by subblastodermal injection. At the gross level, RA treatment resulted in varying degrees of caudal regression, scoliosis, limb malformations, and open posterior neuropores among the survivors. Histological examination of tail buds from treated embryos revealed defects which included total dysplasia of caudal structures, the presence of accessory neural tube and notochord tissue, and abnormal fusions of the notochord to the neural tube and tailgut. The incidence, severity, and location of the defects were dependent on the dose of the teratogen, and the stage of development at the time of treatment. The defects resembled those induced in previous studies by treatment with sialic acid binding lectins such as wheat germ agglutinin and limulus polyphemus lectin (Griffith and Wiley, '90b).

Topics: Abnormalities, Drug-Induced; Animals; Chick Embryo; Chickens; Dose-Response Relationship, Drug; Limb Deformities, Congenital; Neural Tube Defects; Notochord; Tail; Tretinoin

After systemic treatment with retinoic acid (RA), Ambystoma opacum and A. punctatum larvae regenerated forelimbs with a wide variety of skeletal and gross anatomical abnormalities. Yet the musculatures within the RA-treated limb regenerates were normal even in instances where the cartilages were deformed beyond recognition as components of the limb skeleton. RA is known to induce reduplication of limb structures, sometimes entire segments. When the latter condition occurred in the present study, the corresponding replicates exhibited limb musculatures which were perfect down to minute details, yet of opposite bilateral symmetry. The results attest, firstly, to the independence of myogenesis and chondrogenesis during limb regeneration. Secondly, RA treatment unmasked an otherwise hidden potential within postembryonic salamander limb tissue for the morphogenesis of the contralateral musculature.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Cartilage; Forelimb; Muscles; Regeneration; Tretinoin; Urodela

Retinoic acid is required for normal growth and development, however excessive doses are teratogenic. Recently several nuclear retinoic acid receptors (RAR) have been identified and postulated to mediate the response of retinoic acid at the gene level. We wished to determine if alpha-RAR mRNA or beta-RAR mRNA levels are modulated by teratogenic doses of retinoic acid in vivo. We have found that beta-RAR mRNA levels in 9-day-gestation mouse conceptuses are increased as early as 3 h after administration of a completely teratogenic dose of retinoic acid (100 mg/kg body weight; b.w.) and reach a maximum of approximately sixfold after 6 h of treatment. Maternal liver and maternal kidney demonstrated a similar pattern of increase in beta-RAR mRNA, however this was only approximately threefold. Retinoic acid dose-response experiments demonstrated a reduced increase of beta-RAR mRNA levels with 10 mg/kg b.w. (minimally teratogenic dose), and no increase with a more-physiological dose of 1 mg/kg b.w. in the conceptuses. beta-RAR mRNA levels were elevated in 18-day-gestation fetuses to a similar extent to that observed in the 9-day-gestation conceptuses. Therefore, the twofold difference in the extent to which beta-RAR mRNA levels increase does not occur because the fetuses are at a developmental stage that is sensitive to the teratogenic effects of retinoic acid. Finally, treatment with another teratogenic retinoid, etretinate, and a nonteratogenic retinoid, retinoyl beta-glucuronide, both resulted in increase in the level of beta-RAR mRNA in the conceptuses and the maternal tissues. Therefore, an increase in beta-RAR mRNA levels caused by treatment with retinoids does not necessarily commit a fetus to undergo an abnormal pattern of development characteristic of teratogenic retinoids.

Topics: Abnormalities, Drug-Induced; Animals; Carrier Proteins; Dose-Response Relationship, Drug; Embryonic and Fetal Development; Etretinate; Female; Gene Expression Regulation; Kidney; Liver; Mice; Pregnancy; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Tretinoin

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.

Topics: Abnormalities, Drug-Induced; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Base Sequence; Carrier Proteins; Chick Embryo; DNA; Gene Expression Regulation; Molecular Sequence Data; Morphogenesis; Nucleic Acid Hybridization; Receptors, Retinoic Acid; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cell Survival; Cells, Cultured; In Vitro Techniques; Rats; Retinoids; Teratogens; Tretinoin

Retinoic acid (RA) is teratogenic in many species, producing multiple malformations, including cleft palate. The effects of RA which lead to cleft palate vary depending on the stage of development exposed. After exposure of embryonic mice to RA on gestation day (GD) 10, abnormally small palatal shelves form. After exposure on GD 12 shelves of normal size form, but fail to fuse, as the medial cells proliferate and differentiate into a nasal-like epithelium. Growth factors and their receptors play an important role in regulating development, and the expression of EGF receptors, EGF, TGF-alpha, TFG-beta 1, and TGF-beta 2 has been reported in the mouse embryo. In a variety of cell types in culture, these growth factors are capable of regulating proliferation, differentiation, expression of matrix proteins, and other cellular events including epithelial-mesenchymal transformations. The present study examines immunohistochemically the expression of EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 in the control embryonic palatal shelves from GD 12 to 15 and the effects of RA treatment on GD 10 or 12 on their expression on GD 14 and 16. These growth factors were shown to have specific temporal and spatial expression in the palatal shelf. With advancing development the levels of TGF-alpha decreased while the expression of EGF increased. TGF-beta 2 localization became regional by GD 14-15, with higher levels found in epithelial cells and chondrogenic mesenchyme. TGF-beta 1 occurred in epithelial and mesenchymal cells and distribution did not change substantially with advancing development. RA exposure altered the expression of TFG-alpha, TGF-beta 1, and TGF-beta 2, but significant effects on EGF were not found. The effects on TGF-alpha and TGF-beta 1 expression were dependent on the gestational age exposed. Levels of TGF-alpha on GD 14 decreased after RA exposure on GD 10, but increased after GD 12 exposure. TGF-beta 1 expression in the mesenchyme was increased after exposure on GD 12, but was unaffected by RA on GD 10. After exposure on either day, the levels of TGF-beta 2 increased in GD 14 nasal epithelial cells. Acting in concert, growth factors could regulate events critical to formation of the secondary palate, including cessation of medial epithelial cell proliferation, synthesis of extracellular matrix proteins in the mesenchyme, programmed cell death of medial epithelial peridermal cells, and transformation of basal epithelial medial cells to mesenchymal cells.

Topics: Abnormalities, Drug-Induced; Animals; Cell Differentiation; Cleft Palate; Epidermal Growth Factor; Female; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Morphogenesis; Palate; Pregnancy; Transforming Growth Factors; Tretinoin

Urokinase-type plasminogen activator (uPA) has been implicated in cellular migration accompanying different biological phenomena including organogenesis. An increase in uPA activity was observed in mouse post-implantation embryos during the early organogenesis period. Since we have previously shown that all-trans retinoic acid (RA) prevented the induction of uPA in mouse peritoneal macrophages, we have now assessed whether teratogenic doses of this agent could also interfere with uPA activity in mouse embryo in vitro and in vivo. Post-implantation embryos (8.5 days) were incubated for up to 24 hr with micromolar concentration of RA resulting in the occurrence of malformations. No significant difference in uPA activity was found between control and treated embryos. Likewise, uPA activity was not altered in embryos explanted on day 9.5 from dams treated 24 hr before with a teratogenic dose of RA. This study indicates that the teratogenic activity of RA is not caused by an inhibition of the induction of uPA in embryos.

Topics: Abnormalities, Drug-Induced; Amiloride; Animals; Culture Techniques; Embryo Transfer; Embryo, Mammalian; Mice; Plasminogen Activators; Plasminogen Inactivators; Tretinoin; Urokinase-Type Plasminogen Activator

Treatment of pregnant Sprague-Dawley rats with etretinate or retinoic acid on pregnancy Day 8.5-9.0 resulted in craniofacial malformations in 100% of the embryos. A morphological investigation of the maxillofacial malformations was undertaken. Retinoid-exposed embryos showed a reduced skull base, flattened and elongated occiput and micrognathia. The malar bones were reduced or missing. Meckel's cartilage was delayed in differentiation as was the mandibular bone. The fusion between different facial processes was disturbed which resulted in facial and palatal clefts. Disturbance of the development of the hypophysis was combined with persisting Rathke's pouch. Aplasia of incisor and molar tooth buds was seen as was aplasia of salivary gland ducts. The facial artery was hyperplastic.

Topics: Abnormalities, Drug-Induced; Animals; Etretinate; Facial Bones; Fetal Death; Fetal Resorption; Gestational Age; Mandible; Microscopy, Electron, Scanning; Neural Tube Defects; Rats; Rats, Inbred Strains; Skull; Tooth Abnormalities; Tretinoin

Treatment of pregnant Sprague-Dawley rats with retinoic acid or etretinate pregnancy day 8.5 to 9.0 resulted in craniofacial defects in 100% of the embryos. A morphologic investigation of the malformations occurring in the ear was performed. Outer ears were missing, microtic, low-placed, and dorsally situated. External acoustic meatus was short or absent. Middle ear structures were delayed in differentiation, middle ear ossicle primordia were hypoplastic and malformed, the stapedial artery and facial nerve were hypoplastic, and their relation to the stapes was variable. In the inner ear, the otic capsule was thick, the cochlea had fewer turns and the semicircular ducts showed poor differentiation.

Topics: Abnormalities, Drug-Induced; Animals; Ear; Ear, External; Ear, Inner; Ear, Middle; Etretinate; Female; Fetal Death; Fetus; Pregnancy; Rats; Rats, Inbred Strains; Tretinoin

Time-mated Sprague-Dawley rats were administered all-trans-retinoic acid (RA) dermally on gestational days 11 through 14 at three dosage levels (25, 100, and 250 mg/kg body weight). Dams administered ethylenethiourea (ETU) dermally on gestational days 11 to 12 or RA orally on day 12 were used to indicate the strain's sensitivity to teratogenesis. The chemicals were dissolved in dimethylsulfoxide (DMSO) for dermal application or suspended in corn oil for treatment by gavage. The maternal weight gain, pup weight, number of resorptions and number of fetuses with gross malformations, and skeletal/organ-level anomalies were determined. Beginning with day 15, dams dermally treated with RA exhibited dermal lesions at the site of application, most dams showed vaginal bleeding by day 16, and approximately 20% did not survive to day 19. Relative to the DMSO control group, maternal weight gain in the dermal RA groups was decreased by approximately 50% at the lowest dose, with essentially no weight gain at the intermediate- and high-dose levels. The decrease in average fetal weight at the two higher doses was significant, whereas the resorption and malformation frequencies were not significantly increased by dermal treatment with RA. Without significantly affecting fetal weight or resorption frequency, dermal application of ETU significantly increased the frequency of skeletal anomalies, primarily tail defects. Oral administration of RA did not increase the malformation frequency nor produce significant maternal or fetotoxic effects. In summary, treatment of pregnant Sprague-Dawley rats by dermal application of RA dissolved in DMSO resulted in significant toxicity to the dam.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Administration, Topical; Animals; Behavior, Animal; Body Weight; Female; Fetal Resorption; Fetus; Pregnancy; Rats; Rats, Inbred Strains; Tretinoin

NMRI mice were treated on day 11 (day 0 = plug day) of gestation with a single oral dose of 100 mg/kg of either all-trans-etretin (acitretin) or 13-cis-etretin. For teratology studies mice were sacrificed on day 18 of gestation, and the fetuses were examined for malformations. For pharmacokinetic studies, groups of 5 mice were sacrificed after 5, 10, and 30 min and 1, 2, 4, and 8 h. The concentrations of retinoids in maternal plasma and in embryos were determined by a newly developed HPLC gradient method. All-trans-etretin induced malformations in 94% of the fetuses, mainly in fore and hind limbs and cleft palate. 13-cis-etretin did not show any teratogenic or embryo-lethal effects at the dose level used. These findings could be explained by a transplacental pharmacokinetic study. The maximum peak level and also the AUC (area under the concentration-time curve) value of all-trans-etretin in the embryos was 7-8 times higher than corresponding values for 13-cis-etretin, probably due to extensive transport of the trans-isomer and limited transport of the cis-isomer from maternal plasma to the embryos. The concentration quotient between embryo and the maternal plasma was between 0.43 and 1.10 for all-trans-etretin, and only 0.16-0.31 for 13-cis-etretin over the time period studied. An in vivo isomerization of the compounds was also observed which was more extensive for 13-cis-etretin than for all-trans-etretin. Our results indicate that the low teratogenic potency of 13-cis-etretin is due to a limited placental transfer of this compound; on the other hand, the potent teratogen all-trans-etretin is rapidly and extensively transferred to the embryo.

Topics: Abnormalities, Drug-Induced; Acitretin; Animals; Bone and Bones; Dermatologic Agents; Female; Keratolytic Agents; Maternal-Fetal Exchange; Mice; Mice, Inbred Strains; Pregnancy; Stereoisomerism; Structure-Activity Relationship; Tretinoin

Etretinate (Tegison, Tigason), a retinoid used for the treatment of skin disorders such as psoriasis, was shown to teratogenic in the human. Because of the long terminal half-life of this drug (100 days), considerable plasma levels of etretinate and its main metabolite, etretin (acitretin), were observed for up to 2 years following discontinuation of therapy. We have therefore investigated, in a newly developed animal model, the potential teratogenic risk of such persisting levels of these aromatic retinoids. Etretinate was administered by intragastric infusion throughout organogenesis in the mouse (day 8-15) via subcutaneously implanted osmotic minipumps connected to external reservoirs containing oily solutions of the drug. Dose-dependent developmental effects were found, the fetal weight decreased and the resorption rate and incidence of major malformation increased. A dose of 0.84 mg/kg/day resulted in retinoid-specific defects, in particular shortening of the limbs and cleft palate. This low dose infused resulted in mean etretinate concentrations of 6.5 ng/ml maternal plasma and 12.5 ng/g embryo (measured on days 10 and 12 of gestation). The corresponding concentrations of the metabolite etretin were 38 ng/ml plasma and 95 ng/g embryo. Our results emphasize the high teratogenic risk of relatively low, persisting concentrations of etretinate and etretin such as those observed after discontinuation of human therapy, because the area of the concentration-time curve is likely to be the decisive parameter in regard to teratogenesis.

Topics: Abnormalities, Drug-Induced; Acitretin; Animals; Embryonic and Fetal Development; Etretinate; Female; Infusion Pumps, Implantable; Maternal-Fetal Exchange; Mice; Models, Biological; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Acitretin; Animals; Biotransformation; Carbon Radioisotopes; Embryonic and Fetal Development; Female; Maternal-Fetal Exchange; Mice; Mice, Inbred Strains; Placenta; Pregnancy; Radioisotope Dilution Technique; Retinoids; Teratogens; Tretinoin

This report shows by light microscopy the appearance of secondary neurulation separated from primary neurulation and its developmental fate in the spinal cord of mice exposed to retinoic acid in utero. The embryos and fetuses were derived from pregnant mice (ICR strain) given 60, 40, or 0 mg/kg of retinoic acid in olive oil on day 8 of gestation orally and killed 1, 2, or 10 days later. Separation of the primary neural fold from the secondary neural tube was seen in 9- and 10-day-old embryos: the caudal part of the neuroepithelium of the primary neural fold was disarranged with non-closed posterior neuropore, and underneath it the secondary neural tissue extended caudally with abnormal notochord. At term, fetuses showed spina bifida, including myeloschisis, myelocele, and diplomyelia (diastematomyelia) with abnormal distribution of ganglionic cells. These cord lesions were located between the third lumbar and second coccygeal levels. The former two cord anomalies were associated with diplomyelia and split the dorsal and ventral portions of the spinal cord with an overlapping zone between the third lumbar and third sacral levels. These findings suggest that the separation from primary neurulation is due to the lesions in both primary neural folds and notochord induced by retinoic acid and that the spinal cord caudal to the third lumbar level originates from both neuroectoderm and mesenchyme-like cells while that caudal to the third sacral level originates from mesenchyme-like cells only.

Topics: Abnormalities, Drug-Induced; Animals; Female; Gestational Age; Mice; Pregnancy; Spina Bifida Occulta; Spinal Cord; Tretinoin

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.

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Ear; Female; Fetus; Isotretinoin; Macaca fascicularis; Organ Specificity; Pregnancy; Teratogens; Tretinoin

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.

Topics: Abnormalities, Drug-Induced; Animals; Female; Limb Deformities, Congenital; Mice; Mice, Inbred C57BL; Mitosis; Palate; Tretinoin

The effects of 13-cis-retinoic acid on the developing chick embryo were investigated. Fertilized eggs were injected via the yolk sac with single 50 microliters doses of either 1.5 micrograms, 15 micrograms, or 150 micrograms of 13-cis-retinoic acid in dimethyl sulfoxide on varying days of incubation (embryonic days 2, 3, 4, 5, or 6). Control embryos were given solvent alone or a mock injection. The embryos were examined on day 14 of incubation. The effects of retinoic acid on mortality and total malformations were both dose and developmental-stage responsive. The defects caused by 13-cis-retinoic acid occurred in mesenchymal tissues derived in part from the cranial neural crest ectomesenchyme. The craniofacial and cardiovascular malformations produced in the chick are analogous to those seen in animal models of retinoid teratogenesis and in human fetuses exposed to 13-cis-retinoic acid during maternal therapy for cystic acne. Following 13-cis-retinoic acid treatment, craniofacial and specific cardiovascular malformations were increased significantly compared to those in matched solvent and mock treated controls. The greatest number of malformations occurred when 13-cis-retinoic acid was given after cranial neural crest cell migration was complete. We propose that the primary effect of 13-cis-retinoic acid is on region-specific localization and differentiation of the mesenchymal subpopulation of cranial neural crest cells.

Topics: Abnormalities, Drug-Induced; Animals; Chick Embryo; Dose-Response Relationship, Drug; Face; Female; Heart Defects, Congenital; Neural Crest; Pregnancy; Pregnancy Outcome; Tretinoin

A syndrome which showed similarities to human branchial arch syndromes could be induced in Sprague-Dawley rat embryos by exposing them to retinoids prenatally. Treatment of pregnant rats with 40 mg/kg retinoic acid or 10 mg/kg etretinate on pregnancy day 8.5-9 resulted in craniofacial defects in 100% of the embryos. A scanning electron microscopic investigation of the early stages in the development of these malformations showed abnormal skull form, disorganised surface epithelium with "cell blebbing", lateral facial clefts, facial fistulas, narrowed skull-base and reduced size of the nasal and maxillary complexes. Histological examination confirmed these findings and supported the hypothesis that a main reason for this syndrome is hindrance of migration of the cranial neural crest cells to the facial processes during early craniofacial formation.

Topics: Abnormalities, Drug-Induced; Animals; Branchial Region; Facial Bones; Female; Maternal-Fetal Exchange; Models, Biological; Pregnancy; Prevalence; Rats; Skull; Syndrome; Teratogens; Tretinoin

Treatment of pregnant Sprague-Dawley rats with etretinate or retinoic acid on pregnancy days 8.5-9.0 resulted in craniofacial defects in all embryos. A morphological investigation of the malformations occurring in the nasal region is described. The external nose was deformed with a depressed nasal bridge and pronounced hump. Fusion between nasal processes was defective, resulting in fistulas and clefts. The nasal septal cartilage was reduced in size, the paranasal cartilage was missing, the parietothecal cartilage was deformed and the paraseptal cartilage was dislocated. Aberrant vessels were seen on the external nose in combination with underdevelopment of the vibrissae. The distance between the outer nasal pores was reduced after administration of retinoids.

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Mammalian; Etretinate; Female; Nose; Pregnancy; Rats; Rats, Inbred Strains; Tretinoin

All-trans-retinoic acid induced 2 types of disorganized neuroepithelium, localized and continuous, in the exencephaly of 9-day-old mouse embryos exposed to 60 or 40 mg/kg for 27 to 30 hr in utero. The localized effect appeared as a protuberance in the wall of the telencephalon and thick neural folds in the mesencephalon with the discontinuity of the apical terminal sheet. The continuous disorganization was seen from the olfactory placode to the myelencephalon with rosettes of cells and many dense bodies in the neuroepithelium. Ultrastructurally, cells in the localized disorganizations showed swelling of Golgi complexes, coated vesicles, and rough endoplasmic reticulum resulting in degeneration. The continuous disorganizations consisted of undifferentiated homogeneous cells in which the nuclei exhibited expansion of nucleolar granular portions and coagulated heterochromatin, and cytoplasm showed monosomal dispersion. In both types of disorganized neuroepithelium, junctional complexes were seen focally at the apical side or apical processes of the rosette, with few or no microfilament bundles. A layer of microfilaments at the base of the neuroepithelial cells in controls, just above the basal lamina, was not present in the monosome dispersed cytoplasm. In the neuroepithelium of controls, one phagosome was seen in the perinuclear region in 0.8% of the cells examined, whereas in the experimental neuroepithelium 2 or more phagosomes were seen in a cell, and phagocytosis occurred by pseudopods. These findings suggest that all-trans-retinoic acid induces not only cytotoxicity but also dedifferentiation in the neuroepithelial cells leading to more cell death, which activates the phagocytosis. These lesions in the neuroepithelium may be a cause of exencephaly.

Topics: Abnormalities, Drug-Induced; Animals; Brain; Cytoplasm; Cytoskeleton; Epithelial Cells; Epithelium; Female; Maternal-Fetal Exchange; Mice; Mice, Inbred ICR; Microscopy, Electron; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Adult; Female; Humans; Infant, Newborn; Male; Penis; Pregnancy; Pregnancy Complications; Psoriasis; Teratogens; Tretinoin

The concepts of sensitivity, specificity, and predictive value can be used to assess patterns of birth defects associated with human teratogens. Although sensitivity of any single defect is generally low for many known teratogens, the presence of specific defect combinations is usually predictive of the teratogen. To evaluate the patterns of birth defects associated with diabetic embryopathy, a sensitivity-specificity analysis was performed on 4929 infants with major defects ascertained by the population-based Metropolitan Atlanta Congenital Defects Program between 1968 and 1980. By reviewing hospital records, maternal insulin-dependent diabetes mellitus was confirmed in 26 infants. Patterns of defects were evaluated among infants born to mothers with insulin-dependent diabetes mellitus and compared with the rest of the Metropolitan Atlanta Congenital Defects Program case population. Multiple logistic regression analysis was used to assess defect combinations that predict for insulin-dependent diabetes mellitus. Of 26 infants, 8 had multiple defects. However, most defects and their combinations were poorly sensitive and predictive for insulin-dependent diabetes mellitus. The predictive value for insulin-dependent diabetes mellitus was greatest for the combination of vertebral and cardiovascular anomalies (6.5%). Also, several pathogenetic mechanisms were noted among patients with insulin-dependent diabetes mellitus, such as cell migration defects, cell death events, deformations, and cardiac flow lesions. The inability to find a clear-cut phenotype for diabetic embryopathy may be due to several etiologic factors and mechanisms associated with diabetic embryopathy.

Topics: Abnormalities, Drug-Induced; Congenital Abnormalities; Diabetes Mellitus, Type 1; Female; Georgia; Humans; Infant, Newborn; Pregnancy; Pregnancy in Diabetics; Rubella Syndrome, Congenital; Sensitivity and Specificity; Thalidomide; Tretinoin

TCDD is one of the most toxic man-made compounds and an extremely potent teratogen in mice. Many of its toxic symptoms resemble those seen during vitamin A deficiency. Vitamin A and its derivatives, such as alltrans-retinoic acid (RA), are also teratogenic in mice, as well as many other species. Both TCDD and RA produce cleft palate in susceptible strains of mice. However, while TCDD produces hydronephrosis, RA does not, and TCDD does not produce limb bud defects while RA does. To determine whether TCDD and RA would enhance or antagonize the teratogenic effects of the other compound, C57BL/6N dams were treated po on Gestation Day (gd) 10 or 12 with 10 ml corn oil/kg containing TCDD (0-18 micrograms/kg), RA (0-200 mg/kg), or combinations of the two chemicals. Dams were killed on gd 18 and toxicity and teratogenicity assessed. Coadministration of TCDD and RA had no effect on maternal or fetal toxicity beyond what would be expected by either compound alone. Cleft palate was induced by RA at lower doses on gd 10 than on gd 12, but by TCDD at lower doses on gd 12 than on gd 10. Sensitivity to TCDD-induced hydronephrosis was similar on both gd 10 and 12. The limb bud defects were only observed when RA was administered on gd 10, not when given on gd 12. No other soft tissue or skeletal malformations were related to administration of TCDD or RA. No effect of TCDD was observed on the incidence or severity of limb bud defects induced by RA, nor did RA influence the incidence or severity of hydronephrosis induced by TCDD. However, the incidence of cleft palate was dramatically enhanced by coadministration of the xenobiotic and vitamin. On both gd 10 and 12, the dose-response curves for cleft palate induction were parallel, suggesting some similarities in mechanism between the two compounds. However, combination treatment resulted in a synergistic response that varied with the stage of development and was tissue specific.

Topics: Abnormalities, Drug-Induced; Animals; Body Weight; Bone and Bones; Cleft Palate; Dioxins; Dose-Response Relationship, Drug; Drug Synergism; Female; Hydronephrosis; Mice; Mice, Inbred C57BL; Polychlorinated Dibenzodioxins; Pregnancy; Tretinoin

Retinoic acid, an endogenous metabolite of vitamin A (retinol), possesses striking biological activity akin to a morphogen in developing and regenerating vertebrate limbs. Systemic administration of retinoic acid (RA) to pregnant mammals during the period of limb organogenesis invariably results in dose-dependent dysmorphogenesis. In an attempt to uncover the mode of action of RA in the developing limb bud we analyzed, by HPLC methods, the levels of RA and its metabolic precursor, retinol, in embryonic mouse tissues prior to and following maternal exposure to a teratogenic dose of RA. Detectable levels of both RA and its isomer 13-cis-retinoic acid were found in the limb buds of Day 11 mouse embryos (40 +/- 2 somites). Although retinol was the major retinoid found in ethanolic extracts of either whole embryo or the limb buds, the latter is enriched in RA compared to the whole embryo. This indicated either a higher degree of retinol metabolism or a sequestration of RA in the limb bud compared to the rest of the embryo at this stage of development. A study of the time course of retinoid levels in treated embryos showed that changes occur rapidly, are stable for several hours, and then begin to return to pretreatment levels. After a maternal dose of 10 mg/kg RA, which resulted in a mild degree of limb anomalies, peak RA levels in the limb bud increased 50-fold over the endogenous level; a full 300-fold increase was found after a 100 mg/kg dose which results in 100% incidence of phocomelia. Interestingly, a dose-dependent depression in retinol levels was observed after RA treatment both in maternal plasma as well as the embryo. Studies are in progress to trace the intracellular disposition of both retinol and RA as well as any further active metabolite of RA in the limb buds and other embryonic tissues.

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Extremities; Female; Kinetics; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Morphogenesis; Pregnancy; Tretinoin; Vitamin A

Pregnant C57Bl/6J mice were treated with 100 mg/kg body weight of all-trans retinoic acid in sesame oil on day 11.0 of gestation. Among the live fetuses harvested on day 18 of gestation, 100% had mesomelic defects of the limbs as determined by gross examination and skeletal staining. Control fetuses treated with sesame oil had no observable limb malformations. Some treated and control embryos were harvested 12 hr after treatment and examined for patterns of cell death by using the supravital stain Nile blue sulphate and methylene-blue- and acid-fuchsin-stained histological sections. Retinoic-acid-induced cell death in the core of the limb was always associated with the zones of programmed cell death as seen in control embryos of comparable stages. This, in concert with previous studies demonstrating excessive cell death in regions of programmed cell death that correlated with subsequent malformations, leads us to conclude that the pathogenesis of mesomelic malformations has a primary association with the phenomenon of programmed cell death.

Topics: Abnormalities, Drug-Induced; Animals; Cell Survival; Extremities; Female; Fetal Resorption; Mice; Mice, Inbred C57BL; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Female; Humans; Infant, Newborn; Legislation, Pharmacy; Physician's Role; Physicians, Family; Pregnancy; Tretinoin; United States; United States Food and Drug Administration

Topics: Abnormalities, Drug-Induced; Female; France; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Female; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Prospective Studies; Retrospective Studies; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Female; Humans; Isotretinoin; Legislation, Drug; Pregnancy; Tretinoin; United States; United States Food and Drug Administration

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Female; Humans; Isotretinoin; Pregnancy; Pregnancy Complications; Tretinoin; United States; United States Food and Drug Administration

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Female; Humans; Isotretinoin; Pregnancy; Pregnancy Complications; Tretinoin

The transplacental pharmacokinetics of single teratogenic doses of etretinate and motretinide were compared with particular emphasis on distribution and concentrations in the exposed embryos of the free acid metabolite, etretin. The three aromatic retinoids were also tested for their direct inhibitory effect on chondrogenesis in the limb bud mesenchymal cell "micromass" culture assay. After a standard dose of 100 mg/kg administered on day 11 of gestation in NMRI mice, all three compounds were teratogenic, but they differed from each other in potency. Etretinate was most active as a teratogen, equalling the potency of our standard all-trans-retinoic acid; every exposed fetus was deformed with severe shortening of all limb bones as well as cleft palate. Etretin was less potent than etretinate, and motretinide was considerably less active as a teratogen than the other two. In the in vitro assay, only etretin suppressed chondrogenesis and this activity was equivalent to that of all-trans-retinoic acid (IC50 of 12 ng/ml). Both etretinate and motretinide (which contain an ethyl ester and ethylamide terminal group, respectively) were essentially inactive in vitro, demonstrating the fact that a free carboxylic group may be a requirement for the in vitro suppression of chondrogenesis. These differences between the results obtained in vivo and in vitro could be resolved by pharmacokinetic investigations using HPLC methods. Both etretinate and motretinide were metabolized in vivo to etretin, their likely common teratogenic metabolite. The high teratogenic potency of etretinate was probably the result of high concentrations as well as AUC values of its metabolite etretin in the embryo. On the other hand, the comparatively low teratogenicity of motretinide could be related to approximately 5 x lower embryonic peak levels as well as AUC values of etretin. A comparison of these results with those previously obtained for all-trans- and 13-cis-retinoic acids confirms the correlation between embryonic exposure and teratogenic potency in the mouse. Our results indicate that pharmacokinetic studies are essential for the interpretation of relative teratogenic potencies of retinoids as well as apparent differences between in vivo and in vitro teratogenesis. A free carboxyl group at the terminal end of the tetraene chain was necessary for high activity of the retinoids studied.

Topics: Abnormalities, Drug-Induced; Acitretin; Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Etretinate; Female; Maternal-Fetal Exchange; Mice; Mice, Inbred Strains; Molecular Structure; Placenta; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Female; Humans; Infant, Newborn; Isotretinoin; Male; New Jersey; Teratogens; Tretinoin; United States; United States Food and Drug Administration

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Female; Humans; Infant, Newborn; Isotretinoin; Male; New Jersey; Teratogens; Tretinoin; United States; United States Food and Drug Administration

Topics: Abnormalities, Drug-Induced; Female; Humans; Isotretinoin; Pregnancy; Teratogens; Tretinoin

Megadose supplements of vitamin A are under suspicion as hazards to the developing embryo after the discovery that two vitamin A-related drugs, Accutane and Tigason, are human teratogens. Retinoic acid (all-trans-RA) is a natural metabolite of vitamin A which participates in many of the known functions of vitamin A and may be the active agent in teratogenesis. In this investigation we gave a single, high oral dose of retinol (vitamin A) to pregnant mice to assess its transplacental pharmacokinetics as well as to measure the formation and distribution of its metabolites in the embryo. Retinol was estimated to be 4-fold less active than retinoic acid in the whole animal teratogenesis and 20-fold less active in the in vitro bioassay. A fully teratogenic dose, 200 mg/kg, yielded considerable quantities of retinoic acid which were transferred to the embryo with kinetics similar to that of retinol. During the first 8 hr after administration of retinol, the metabolites (including all-trans-RA, 13-cis-RA, and 4-oxo-RA) constituted almost 50% of the quantity of all retinol derivatives found in the embryo. A comparison of combined peak concentrations of the metabolites (or their AUC values) with the extent of teratogenesis associated with them individually provided sufficient evidence to implicate the metabolites themselves as mediators of retinol-induced teratogenesis. However, since both retinol and retinoic acid were present in sufficient concentrations in the embryo to act as teratogens we cannot at present rule out the possibility that they may act independently. Further experimentation will be necessary to address whether retinoic acid detected in the embryo is the product of the embryo's own metabolic capability or is transferred from the maternal circulation.

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Dose-Response Relationship, Drug; Female; Male; Mice; Mice, Inbred ICR; Pregnancy; Tretinoin; Vitamin A

Topics: Abnormalities, Drug-Induced; Female; Humans; Isotretinoin; Pregnancy; Tretinoin

Vitamin A and vitamin A derivatives have been described as etiologic factors for a number of congenital malformations. Two infants are presented with major auricular malformations including anotia and severe microtia. The infants were products of a pregnancy complicated by Accutane ingestion during the first trimester. Both infants had associated central nervous system malformations. With the increasing use of Accutane for the treatment of cystic acne in young women of child-bearing age, the dangers of teratogenesis in the head and neck area are greatly increased. This presentation will review two such cases as well as give an overview of the embryogenesis and teratogenesis of the auricle.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Acne Vulgaris; Adult; Ear, External; Female; Humans; Infant, Newborn; Isotretinoin; Male; Teratogens; Tretinoin

Recent concerns have been raised about the ability of birth defects monitoring programs to detect increases in the incidence of birth defects following the introduction of new teratogens. The authors illustrate how most monitoring programs in the United States and Europe are limited in their ability to detect new teratogens because of a combination of parameters: the small population size, the low population frequency of exposure to the new teratogen, the weakness of many suspected teratogens (measured in terms of relative risk R), the low background rate, and the etiologic heterogeneity in the measured defects. In a system that monitors 25,000 births per year, it can be shown that although a new teratogen such as thalidomide (R = 175) can lead to a significant increase in the number of observed cases in 1-2 weeks of monitoring, even strong teratogens such as valproic acid and isotretinoin (R = 20-25) require more than 20 years of monitoring to show a significant increase in the number of cases because of low exposure frequency. Also, most mild to moderate teratogens (R = 2-5) can be totally missed. To improve the ability of birth defects monitoring programs to detect new teratogens, it is suggested that surveillance systems ought to examine subsegments of the population with maximal exposure potential, classify birth defects into more etiologically homogeneous groups, and expand the sample size of the monitored population.

Topics: Abnormalities, Drug-Induced; Epidemiologic Methods; Humans; Infant, Newborn; Isotretinoin; Risk; Teratogens; Thalidomide; Tretinoin; Valproic Acid

In order to document the stage(s) at which the embryonic chick wing bud is sensitive to vitamin A teratogenesis and the kinds of defects produced by vitamin A insult to the embryonic chick wing, 1-microgram doses of retinoic acid (1 microliter RA in 90% DMSO at a concentration of 1 microgram/microliter) were locally applied to the right wing bud of chick embryos at stages 17-23 (Hamburger and Hamilton: J. Morphol., 88:49-92, '51), and the resulting limb skeleton anatomy was observed at 10 days of incubation. Local application of RA at stages 17-20 resulted in distal wing skeleton defects. There were significantly more wing skeleton defects among embryos treated at these stages with RA solution than among solvent (DMSO)-treated control embryos and than among untreated control embryos. Wings of embryos treated with RA at stages 21-23 were always normal. Scapular and vertebral defects were seen at 10 days of incubation among embryos which had been treated prior to stage 21 with both the RA solution and the solvent control. Statistical analysis and histological data suggest that scapular and vertebral defects were caused by DMSO-induced damage to somites.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Chick Embryo; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Teratogens; Tretinoin; Wings, Animal

The teratogenicity of retinoids containing either tetramethylated tetralin (Ro 13-6307 or Ro 13-2389) or tetramethylated indane (Ro 13-4306) ring system substitutions was compared to the teratogenic potency of all-trans-retinoic acid. Single oral doses, administered to Syrian Golden hamsters at 10:00 A.M. on day 8 of gestation, induced a syndrome of malformations identical to that induced by treatment with all-trans-retinoic acid. These retinoids failed to induce signs of maternal hypervitaminosis A at doses associated with a significant teratogenic response. The tetramethylated tetralin retinoids and indane retinoid were 18 and 2.4 times as embryotoxic on a molar basis, respectively, as all-trans-retinoic acid. Introduction of a supplementary ring in the side-chain restricted polyene chain flexibility and maintained the hydrophobic plane of the chain. The present results are consistent with previous studies showing that the presence of or biotransformation to a free acid congener was necessary for retinoid teratogenic activity in hamsters and that increasing conformational restriction of acidic retinoids increased teratogenic potency.

Topics: Abnormalities, Drug-Induced; Animals; Cricetinae; Female; Indans; Indenes; Mesocricetus; Naphthalenes; Pregnancy; Structure-Activity Relationship; Syndrome; Teratogens; Tetrahydronaphthalenes; Tretinoin

Although most known human teratogens often produce a combination of birth defects in an affected infant, surveillance programs aimed at detecting epidemics of birth defects usually only monitor rates of individual defects. A drawback to this approach is that an increase in the rate of infants affected with a specific combination of defects may lead to little or no increase in the rates of component defects. Using the Poisson distribution, we show that, compared with monitoring for individual defects, monitoring for combinations of two and three defects may require fewer numbers of births to detect an epidemic. In general, an increase can be detected more rapidly by monitoring the rates of defect combinations than by monitoring the rates of individual defects if most affected infants have combinations of defects rather than isolated defects. For example, in the case of Congenital rubella syndrome (CRS), monitoring for the combination of cataracts with deafness and/or patent ductus arteriosus could have led to earlier detection of an epidemic than could monitoring for cataracts alone. In contrast, in the case of thalidomide embryopathy, monitoring for reduction defects of upper limbs in combination with reduction defects of lower limbs and/or microtia/anotia would not have led to earlier detection of an epidemic than would monitoring for reduction defects of upper limbs alone. This is due mainly to the low frequency of defect combinations among affected cases. When used with regular monitoring for individual defects, surveillance of defect combinations can enhance the ability of monitoring programs to detect epidemics of birth defects.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Biometry; Female; Humans; Pregnancy; Rubella; Teratogens; Thalidomide; Tretinoin; United States

Topics: Abnormalities, Drug-Induced; Family Planning Services; Female; Humans; Isotretinoin; Pregnancy; Tretinoin

13-cis-Retinoic acid (isotretinoin) is teratogenic in man at therapeutic doses (0.5-1.5 mg/kg body wt), but only marginally teratogenic in the mouse at exceedingly high doses (greater than 100 mg/kg). On the other hand, the isomer all-trans-retinoic acid (tretinoin) is teratogenic in the mouse at dose levels which are 10 times lower than those for the 13-cis isomer. We have therefore studied whether the greatly different teratogenic potencies of these two compounds in the mouse are the result of differences in their transplacental kinetics. Following a single oral dose of 100 mg all-trans- or 13-cis-retinoic acid per kg body wt, concentrations of the parent drugs, of the C-13 isomerization products, as well as of their 4-oxo metabolites were determined in maternal plasma and embryo at two sensitive stages of organogenesis, i.e., Days 9 or 11 of gestation. All-trans-retinoic acid and its 4-oxo metabolite were transferred to the embryo to a much greater extent (embryo/maternal plasma concentration ratios, approximately 0.4) than the 13-cis-retinoic acid and its 4-oxo metabolite (embryo/maternal plasma concentration ratios, approximately 0.02). Embryo concentrations of all-trans-retinoic acid on Day 9 of gestation exceeded those on Day 11, whereas the embryo levels of 13-cis-retinoic acid were minimal at both gestational stages. The concentration of the 4-oxo metabolite of all-trans-retinoic acid was generally lower than that of the parent drug, whereas the level of the 4-oxo metabolite of the 13-cis-retinoic acid was comparable with or even higher than that of the parent compound. Concentrations of the C-13 isomerization products in maternal plasma were less than 20% of corresponding parent drug levels. However, due to the different extent of transfer of the two isomers, the concentration of all-trans-retinoic acid in the embryo exceeded that of the cis isomer even after administration of 13-cis-retinoic acid. Our results indicate that the low teratogenicity of 13-cis-retinoic acid in the mouse is the result of minimal placental transfer of this compound and of its 4-oxo metabolite, which contrast sharply with extensive placental transfer and high teratogenicity of the corresponding isomers with the all-trans configuration.

Topics: Abnormalities, Drug-Induced; Animals; Female; Isotretinoin; Maternal-Fetal Exchange; Mice; Mice, Inbred Strains; Placenta; Pregnancy; Structure-Activity Relationship; Teratogens; Tretinoin

The teratogenicity of vitamin A has been repeatedly reported in the literature and confirmed on the basis of several cases of adverse pregnancy outcome associated with maternal isotretinoin exposure. We report a case which shows a striking similarity with this syndrome, but the child was born to a mother who took a normal supplementation of vitamin A during pregnancy. The differential diagnosis is discussed.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Adult; Diagnosis, Differential; Female; Humans; Infant, Newborn; Isotretinoin; Phenotype; Pregnancy; Tretinoin; Vitamin A; Vitamins

Topics: Abnormalities, Drug-Induced; Aorta, Thoracic; Diagnosis, Differential; Ear, External; Humans; Infant, Newborn; Isotretinoin; Male; Tretinoin

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cleft Palate; Female; In Vitro Techniques; Isotretinoin; Limb Deformities, Congenital; Male; Mice; Mice, Inbred ICR; Pregnancy; Teratogens; Tretinoin

A newborn had isotretinoin embryopathy, including thymic hypoplasia. Evaluation of her immune functions demonstrated progressive attrition of T cells over the nine weeks until her death due to pneumonia. These studies strongly suggest an insult by isotretinoin to the immune system. Immune abnormalities may predispose infants with isotretinoin embryopathy to infection and contribute to their high mortality.

Topics: Abnormalities, Drug-Induced; Adult; Facial Bones; Female; Humans; Infant, Newborn; Isotretinoin; Leukocyte Count; Maternal-Fetal Exchange; Pregnancy; Skull; T-Lymphocytes; Thymus Gland; Tretinoin

In cultured fetal rat bones, cyclohexanetriones that stimulate prostaglandin synthesis inhibited retinoic acid-induced cartilage degradation in a dose-dependent manner. The inhibition by the cyclohexanetrione Ro 31-0521 was reversible, indicating that the effect was not due to cytotoxicity. Excess retinoic acid is teratogenic in rats and adversely affects the normal differentiation of various morphogenetic systems, depending on the time of administration. The following retinoic acid-induced malformations were suppressed by Ro 31-0521: malformations of long bones and of apical phalanges induced on days 13 and 15 of gestation, respectively; spina bifida and tail malformations induced on day 11 of gestation and cleft palate induced on day 15 of gestation. However, cleft palate and other head malformations including exencephaly induced by retinoic acid on day 11 of gestation were not suppressed but even increased by Ro 31-0521. At a high dose, Ro 31-0521 given alone on day 11 of gestation was embryolethal and teratogenic but was not on the tested other days, indicating that the cyclohexanetrione at specific stages and doses also interfered with normal morphogenesis like retinoic acid. Assuming that stimulation of prostaglandin synthesis is the main biological effect of the cyclohexanetriones, our findings suggest that prostaglandins may be involved in mediating retinoid action.

Topics: Abnormalities, Drug-Induced; Animals; Bone Resorption; Cartilage; Cleft Palate; Cyclohexanes; Cyclohexanones; Gestational Age; Limb Deformities, Congenital; Male; Organ Culture Techniques; Prostaglandins; Proteoglycans; Rats; Reproduction; Tretinoin

Topics: Abnormalities, Drug-Induced; Abortion, Induced; Acne Vulgaris; Ethics, Medical; Female; Humans; Isotretinoin; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Female; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Statistics as Topic; Tretinoin

Topics: Abnormalities, Drug-Induced; Humans; Isotretinoin; Tretinoin

Birth defects caused by human teratogens are an important and potentially preventable cause of perinatal morbidity and mortality. Case reports provide an initial suggestion that a specific agent may be a human teratogen and provide the basis for further study. This review discusses the importance of case reports in human teratology and provides guidance in evaluating new case reports.

Topics: Abnormalities, Drug-Induced; Animals; Antiemetics; Dicyclomine; Doxylamine; Drug Combinations; Erythromycin; Female; Humans; Infant, Newborn; Isotretinoin; Medical Records; Pregnancy; Publishing; Pyridoxine; Teratogens; Thalidomide; Tretinoin; Warfarin

Topics: Abnormalities, Drug-Induced; Etretinate; Female; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Retinoids; Skin Diseases; Teratogens; Tretinoin

Recent clinical observations strongly suggest that isotretinoin [13-cis-retinoic acid (cis RA)] is a human teratogen causing primarily heart and craniofacial malformations including ear and palatal defects. The purpose of the present study was to determine if cis RA could induce similar craniofacial malformations in mouse embryo culture. Day 8 CD-1 mouse embryos were cultured for 48 hours in rat serum in the presence or absence of various concentrations of cis RA dissolved in DMSO. DMSO by itself had no effect on embryonic development; however, cis RA at 2 X 10(-5) M (6 micrograms/ml) was clearly toxic. At 2 X 10(-6) M cis RA, growth retardation was minimal, and approximately one-third of the embryos exhibited very specific defects including a dramatic reduction in the size of the first and second visceral arches, which eventually give rise to the maxilla, mandible, and ear. Similar observations were also made with 4-oxo-13-cis RA, which is a major metabolite of cis RA in the mouse and human. These malformations would be expected to result in defects similar to those observed in the human, and preliminary observations suggest these defects are due to cis RA-induced inhibition of cranial neural crest cell migration. Using day-10 mouse embryos cultured for 48 hours in Waymouth's medium containing 50% fetal calf serum, we observed that cis RA at 2 X 10(-5) M produced a high percentage of embryos with limb defects and median cleft lip. Our results demonstrate that labeled cis RA enters the tissues of the embryo both in vivo and in vitro. Cis RA inhibited proliferation of the frontonasal mesenchyme cells in primary culture with 31% inhibition occurring at 2 X 10(-5) M cis RA.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Lip; Culture Techniques; DNA; Embryo, Mammalian; Female; Isotretinoin; Limb Deformities, Congenital; Mice; Microscopy, Electron, Scanning; Pregnancy; Proteins; Time Factors; Tretinoin

Topics: Abnormalities, Drug-Induced; Brain; Embryo, Mammalian; Female; Humans; Isotretinoin; Pregnancy; Tretinoin

Despite animal vitamin A congener teratogenicity in animal studies since 1954, striking human findings only arose in 1983 following isotretinoin (ITR) marketing for oral treatment of severe acne. By November 1985, 44 outcomes with central nervous system (CNS), cardioaortic (CV), microtia, facial palsy, micrognathia, cleft palate, and/or thymic aplasia defects, and 33 spontaneous abortions have been reported. The critical period for exposure appears to be two to five weeks postconception, although this is clinically inexact. ITR half life is less than a day, although a teratogenic metabolite, 4-oxo-isoretinoin, has a half life of several days. Seven defect outcomes and one stillbirth have been reported with another congener, etretinate (ETR), used for psoriasis. Three of these had meningomyeloceles. Half life of several months makes levels cumulative. Only one additional defect, which may have occurred by chance, is reported with use stopped before conception (4 months). Other discontinuations 1 to 6 months before conception had 11 normal outcomes and two spontaneous abortions. ITR and ETR dose ranged from 0.5 to 1.5 mg/kg. Normal outcomes are reported both with ITR and ETR, but some of these appear not to have been exposed during the critical period. Less striking defects, abortions, and normal outcomes are less well reported. Because vitamin A analogs are therapeutically important and unplanned outcome not always avoidable, further animal research is needed for better risk/benefits. Megadose vitamin A (retinol) use is widespread, but experience poorly observed. Eighteen suspicious birth defect outcomes have been reported from pregnancies with high dose exposure. Twelve had findings similar to those seen in animals and in human retinoid syndromes, e.g., CNS, CV, microtia, and clefts. Epidemiological controls are lacking to establish human teratogenicity, but based on animal studies and experience with ITR and ETR, avoiding long term megadose Vitamin A use in fertile women is warranted.

Topics: Abnormalities, Drug-Induced; Abortion, Spontaneous; Animals; Etretinate; Female; Gestational Age; Humans; Infant, Newborn; Isotretinoin; Maternal-Fetal Exchange; Pregnancy; Species Specificity; Teratogens; Tretinoin; Vitamin A

Severe congenital malformations have been associated with the inadvertant use in early pregnancy of a new dermatological drug, isotretinoin. We present proposals for the pathogenesis of this embryopathy based on the study of animal models. The characteristic malformations of the face, thymus, and great vessels were induced in mice by prenatal exposure to the drug during the early somite stages of development. From histological examination of mouse embryos it was shown that the drug directly interferes with the development of cranial neural crest cells. Subsequent deficiency of crest cell-derived mesenchyme adequately explains most of the observed malformations. Rat embryo culture studies showed that, when used at concentrations of 500 ng/ml, both isotretinoin and its main metabolite in the human, 4-oxo-isotretinoin, induce malformations similar to those seen in vivo. Since during normal repetitive dosing in the human the mean trough blood concentration of isotretinoin ranges from 132 to 196 ng/ml, while 4-oxo-isotretinoin ranges from 610 to 791 ng/ml, it is likely that the metabolite plays a major role in the induction of the isotretinoin embryopathy.

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Mammalian; Embryonic and Fetal Development; Female; Fetal Resorption; Humans; Infant, Newborn; Isotretinoin; Mice; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Neural Crest; Organ Culture Techniques; Pregnancy; Rats; Rats, Inbred Strains; Teratogens; Tretinoin

Chronokinetic synergism, a holistic and extremely sensitive experimental design, has shown in the mouse embryo that site-specific epigenetic forces differentially regulate genesis of the palate (cleft palate) and limb bud organogenesis (shortened stature). Acute exposures of 11-day pregnant mice to minimally effective doses of thymidine or ethanol followed 5 or 8 hr later by minimal exposure to retinoic acid have enabled quantitative and qualitative assay for genomic-epigenetic interactions. These site-specific morphogenetic regulations occurred during palatal genesis from the maxillary prominence of the first pharyngeal arch and during limb bud prechondrogenesis. Thymidine is presumed to induce its response by inhibition of DNA polymerase and hence by transitory cytostatic block. (Embryo size was not detectably changed). Ethanol is interpreted, guilt by associated response, indirectly to interfere with histone regulation of transcription. Two central findings have demonstrated the coordinated regulation of genomic and epigenetic positional information. First, thymidine or ethanol as epigenetic probes for limb prechondrogenesis and palatal precursor cells have activated distinctive site-specific responses. Second, responses to chronokinetic synergisms have indicated that epigenetic regulators for limb and palate dysmorphogenesis may affect distinctly different phases of the cell division cycle and hence induce differential DNA expressions. Although each of palate and limb is concurrently susceptible to epigenetic regulation, their differential intrinsic genomic capabilities appear to have been uncoupled. The putative homeostatic balance of genomic expressions in the palate precursor and the prechondrogenic limb bud cells of the 11-day mouse embryo has been characterized as epigenetically regulated, alternatively expressed, and positionally restricted. We propose that the chronokinetic synergisms have disclosed the existence of distinctive palate-determining genes and stature-determining genes.

Topics: Abnormalities, Drug-Induced; Animals; Cell Cycle; Cell Division; Cleft Palate; Embryo, Mammalian; Ethanol; Genes; Heterozygote; Limb Deformities, Congenital; Mice; Thymidine; Tretinoin

Oral administration of 40-80 mg/d of isotretinoin (13-cis-retinoic acid, Ro 4-3780, Accutane) during the first month of human pregnancy can induce severe congenital malformation. The human Accutane dysmorphic syndrome includes rudimentary external ears, absent or imperforate auditory canals, a triangular microcephalic skull, cleft palate, depressed midface, and anomalies of the brain, jaw, and heart. Children who suffer from this syndrome have large occiputs with narrowing of the frontal bone. Microphthalmia is reported in two cases; notations are made about the orbits in three cases; and the fact that infants could not follow with their eyes is noted in three cases. A decrease in muscle tone is noted in six, cleft palate is present in four, and limb reduction defects are described in two. The cardiac malformations usually include overriding aorta, interrupted or hypoplastic aortic arch, and septation defects of atria and ventricles. There are at least two cases of abnormal origin of the subclavian arteries. Oral isotretinoin in the pregnant hamster also induces similar congenital malformations. A human case of isotretinoin-induced dysmorphia is presented and compared with other affected infants and affected hamsters. The metabolic fate and pharmacokinetic parameters of isotretinoin in humans and rodents are discussed in relation to the teratogenic response. The results suggest that humans are approximately 16 times more sensitive to the teratogenic effects of oral isotretinoin than are hamsters.

Topics: Abnormalities, Drug-Induced; Animals; Cricetinae; Facial Bones; Female; Humans; Infant, Newborn; Isotretinoin; Mesocricetus; Microscopy, Electron, Scanning; Pregnancy; Skull; Species Specificity; Tretinoin

The administration of a single dose of all-trans retinoic acid on day 8 of gestation to pregnant mice, ICR strain, led to malformed fetuses in all of the litters. All-trans retinoic acid (RA) was dissolved in olive oil and given in doses of 60 or 40 mg/kg of body weight. The control mice were given vehicle alone. Examination on day 18 of gestation of the fetuses exposed to 60 mg/kg showed various malformations, such as exencephaly, exophthalmus, micrognathia, agnathia, cleft palate, cleft lower lip, spina bifida, atresia ani, tail anomalies, agenesis of the kidneys, or hydronephrosis. In the fetuses exposed to 40 mg/kg, isolated cleft palate was much more common than in those exposed to 60 mg/kg. Double-stained preparations of bone and cartilage showed cranio-facial anomalies and axial skeletal anomalies: a- or hypogenesis of palatine or maxillary bones, tympanic ring, squamosal temporal bone or otic ossicles in cartilage, and fusion of basioccipital to basisphenoid and maxilla, zygomatic and mandibular bones; a- or hypogenesis of caudal vertebrae and supernumerary thoracic and lumbar vertebrae. These results indicate that anomalies comparable to those seen in the infants of mothers treated with isotretinoin, 13-cis retinoic acid, during pregnancy can also be induced in mice and suggest that the site affected by RA may be neural crest cells, including those in the cephalic and caudal regions, and cells committed to somitic mesoderm in the trunk region.

Topics: Abnormalities, Drug-Induced; Animals; Embryo Implantation; Female; Fetus; Mice; Mice, Inbred ICR; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Autoradiography; Female; Mice; Neural Crest; Pregnancy; Tretinoin

The cyclohexanetrione Ro 31-0521, which stimulates prostaglandin synthesis, inhibited retinoic acid-induced cartilage degradation in vitro and suppressed the congenital forelimb malformations in rats treated with retinoic acid on day 13 of gestation in a dose-dependent manner.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Cartilage; Culture Techniques; Cyclohexanes; Cyclohexanones; Dose-Response Relationship, Drug; Female; Forelimb; Pregnancy; Proteoglycans; Rats; Tretinoin

Topics: Abnormalities, Drug-Induced; Child; Female; Humans; Isotretinoin; Limb Deformities, Congenital; Male; Pregnancy; Pregnancy Trimester, First; Tretinoin

Topics: Abnormalities, Drug-Induced; Female; Humans; Infant, Newborn; Isotretinoin; Limb Deformities, Congenital; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Female; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Rats; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Animals; Exostoses; Female; Humans; Isotretinoin; Lipids; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adult; Brain; Female; Heart Defects, Congenital; Humans; Hydrocephalus; Isomerism; Isotretinoin; Pregnancy; Teratogens; Tretinoin

Reproduction and teratology studies were performed with etretin, the free acid analog of the retinoid etretinate. The lowest teratogenic dose of etretin in mice, rats and rabbits was 3, 15 and 0.6 mg/kg, respectively. In all three species, the lowest dose which was embryolethal, fetolethal or reduced the survivability of the pups during lactation was 2-3 times higher than the above doses. In rats, the lowest effective dose of etretin was 3 mg/kg in both the study for fertility and general reproductive performance and the peri- and postnatal study. The main adverse effect in these two experiments was a reduced survival of the F1-generation.

Topics: Abnormalities, Drug-Induced; Acitretin; Administration, Oral; Animals; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Fetal Death; Mice; Pregnancy; Pregnancy, Animal; Rabbits; Rats; Reproduction; Teratogens; Tretinoin

Isotretinoin, a drug used for the treatment of acne, has been shown to have teratogenic effects. We report an additional case of isotretinoin teratogenicity in which the patient had agenesis of the cerebellar vermis, multiple leptomeningeal neuroglial heterotopias, hydrocephalus, and abnormalities of the corticospinal tracts. These findings are related to those reported previously.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Acne Vulgaris; Adult; Brain; Female; Humans; Infant, Newborn; Isotretinoin; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Female; Humans; Isotretinoin; Pregnancy; Risk; Tretinoin

Retinoic acid, an analogue of vitamin A, is known to be teratogenic in laboratory animals and has recently been implicated in a few clinical case reports. To study the human teratogenicity of this agent, we investigated 154 human pregnancies with fetal exposure to isotretinoin, a retinoid prescribed for severe recalcitrant cystic acne. The outcomes were 95 elective abortions, 26 infants without major malformations, 12 spontaneous abortions, and 21 malformed infants. A subset of 36 of the 154 pregnancies was observed prospectively. The outcomes in this cohort were 8 spontaneous abortions, 23 normal infants, and 5 malformed infants. Exposure to isotretinoin was associated with an unusually high relative risk for a group of selected major malformations (relative risk = 25.6; 95 per cent confidence interval, 11.4 to 57.5). Among the 21 malformed infants we found a characteristic pattern of malformation involving craniofacial, cardiac, thymic, and central nervous system structures. The malformations included microtia/anotia (15 infants), micrognathia (6), cleft palate (3), conotruncal heart defects and aortic-arch abnormalities (8), thymic defects (7), retinal or optic-nerve abnormalities (4), and central nervous system malformations (18). The pattern of malformation closely resembled that produced in animal studies of retinoid teratogenesis. It is possible that a major mechanism of isotretinoin teratogenesis is a deleterious effect on cephalic neural-crest cell activity that results in the observed craniofacial, cardiac, and thymic malformations.

Topics: Abnormalities, Drug-Induced; Abortion, Spontaneous; Acne Vulgaris; Adolescent; Adult; Female; Fetal Death; Heart Defects, Congenital; Humans; Infant, Newborn; Isotretinoin; Male; Pregnancy; Pregnancy Trimester, First; Prospective Studies; Retrospective Studies; Risk; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Adolescent; Cerebellum; Ear, External; Face; Female; Humans; Infant, Newborn; Isotretinoin; Male; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Brain; Ear; Heart Defects, Congenital; Humans; Isotretinoin; Syndrome; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Female; Humans; Isotretinoin; Pregnancy; Teratogens; Tretinoin

A spontaneous postaxial polydactyly, similar to type B in humans, was found in a partially inbred ICR mouse strain. The supernumerary digit could be detected grossly as early as day 14 of gestation. The incidence of polydactyly decreased with increasing gestational age. All-trans retinoic acid (RA) administered on day 10 or 11 of gestation, but not on day 9, increased the incidence of polydactyly at each gestational day examined. The day 18 levels of polydactyly were greatest after day 10 treatment. No clear dose-response relationship was observed in term fetuses following treatment on day 9, 10, or 11. RA administered on day 10 produced extra digits which were morphologically more advanced than those in the untreated controls. RA, given to the inbred C57B1/10 strain, produced low levels of polydactyly if administered on day 9, but not on day 10 or 11. F1 embryos, from reciprocal crosses between the two strains, were intermediate in response to RA. On day 14, cell death in the postaxial marginal mesoderm was apparent in all protopolydactylous embryos examined, whether treated or untreated. The supernumerary digits varied in size on day 14. The smaller digits appeared to be filled with necrotic mesodermal cells, whereas the larger digits had a necrosis-free area. The size of the extra digit on day 14 seemed to be the most important factor in the persistence of the digit until day 18.

Topics: Abnormalities, Drug-Induced; Animals; Crosses, Genetic; Dose-Response Relationship, Drug; Female; Foot Deformities, Congenital; Forelimb; Gestational Age; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Culture Techniques; Dose-Response Relationship, Drug; Female; Fetus; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Pregnancy; Toes; Tretinoin

Topics: Abnormalities, Drug-Induced; Adolescent; Embryonic and Fetal Development; Female; Gestational Age; Humans; Infant, Newborn; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Female; Humans; Infant, Newborn; Isotretinoin; Maternal-Fetal Exchange; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Adult; Female; Humans; Infant, Newborn; Isotretinoin; Male; Pregnancy; Tretinoin

Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose-dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid-induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose-dependent decrease in area of both fore- and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.

Topics: Abnormalities, Drug-Induced; Animals; Extremities; Female; Fetus; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Organ Culture Techniques; Pregnancy; Tretinoin

Two closely related retinoids, all-trans and 13-cis retinoic acids, were assessed for their relative activities as teratogens in ICR mice by monitoring the frequency with which either isomer produced discrete dysmorphogenesis of the embryonic limb and the secondary palate. A single oral dose of all-trans retinoic acid at 100 mg/kg on either day 11.5 or 12.0 of gestation (plug day = day one) was maximally effective; more than 90% of the treated embryos developed reduction defects of the limb bones and an equally high percentage also had cleft palate. The limb development was most sensitive on day 11.5 of gestation while the peak susceptibility for palatal clefts began on day 12.0. Under identical experimental conditions, treatment with 100 mg/kg 13-cis retinoic acid produced no apparent teratogenic effects. By assessing the relative incidence of readily identifiable malformations of the limb and palate associated with various doses of the two isomers, we found that 13-cis retinoic acid was four to eight times less embryopathic than all-trans retinoic acid. Since the mechanism of teratogenic action of retinoids is still far from clear, it is suggested that further studies on causative factors will be greatly assisted by the use of these two closely related retinoids, which substantially differ from each other in their teratogenic potency.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Dose-Response Relationship, Drug; Female; Isomerism; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Palate; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Cost-Benefit Analysis; Female; Humans; Infant, Newborn; Isotretinoin; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Face; Female; Fetus; Humans; Infant, Newborn; Isotretinoin; Male; Maternal-Fetal Exchange; Pregnancy; Pregnancy Complications; Skull; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Ear, External; Female; Fetus; Humans; Hydrocephalus; Infant, Newborn; Isotretinoin; Maternal-Fetal Exchange; Pregnancy; Pregnancy Complications; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Adolescent; Adult; Central Nervous System; Female; Humans; Infant, Newborn; Isotretinoin; Male; Pregnancy; Tretinoin; Triglycerides

Topics: Abnormalities, Drug-Induced; Abortion, Induced; Acne Vulgaris; Germany, West; Humans; Isotretinoin; Risk; Tretinoin

Homozygotes for the splotch (Sp) mutation in the mouse have spina bifida, whereas the heterozygotes have a white belly spot but otherwise appear normal. Spina bifida can be induced by maternal treatment with retinoic acid. Female SWV strain mice were treated intraperitoneally with retinoic acid suspended in peanut oil 8 days/12 hours after they had been mated to either Sp/+ or +/+ males. Probit analysis of the dose-response data suggests that the presence of the Sp gene causes an increased susceptibility of the embryo to the spina bifida-causing effects of retinoic acid. To study the nature of this increase litters were obtained on gestation day 9 from untreated SWV females mated as above. The mean length of the posterior neuropore (the length of the posterior neural tube that has not yet closed) was determined for each somite number between 14 and 26 and was found to be significantly greater in embryos from the Sp/+ cross. This delay of closure of the neural tube in Sp/+ cross embryos could explain the observed increase in their susceptibility to retinoic acid.

Topics: Abnormalities, Drug-Induced; Animals; Female; Mice; Mice, Mutant Strains; Neural Tube Defects; Pregnancy; Spina Bifida Occulta; Tretinoin

Topics: Abnormalities, Drug-Induced; Female; Fetus; Humans; Isotretinoin; Pregnancy; Prospective Studies; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Chick Embryo; Feathers; Limb Deformities, Congenital; Morphogenesis; Regeneration; Tretinoin; Vitamin A; Wings, Animal

Etretinate (Ro 10-9359; Tigason; 4-methoxy-2,3,6-trimethylphenyl analog of retinoic acid, ethyl ester) was evaluated for teratogenic activity in the Syrian golden hamster. Groups of pregnant hamsters were given a single oral dose of 2.8-88 mg/kg etretinate during the early primitive streak stage of gestation. No signs of maternal intoxication were observed in any of the hamsters given the retinoid and maternal body weight changes throughout gestation were not significantly different from those of the vehicle-treated group. Etretinate administration was associated with a dose-dependent increase in the incidence and severity of malformations. The average fetal body weight was significantly less in litters recovered from dams given 44 or 88 mg/kg of etretinate when compared to the average body weight of fetuses recovered from dams given an equivalent volume of the vehicle. The average crown-rump lengths also were significantly shorter in fetuses taken from the dams given 44 or 88 mg/kg etretinate as compared to the control group. The malformations induced by etretinate administration were similar to those noted following an oral dose of all-trans-retinoic acid (Willhite and Shealy, 1984). A comparison of the dose-response curves for induction of terata following treatment with etretinate or all-trans-retinoic acid revealed that etretinate was twice as potent as a teratogen in the hamster as all-trans-retinoic acid. Teratogenic activity of etretinate in the hamster was achieved at doses (mg/kg body wt) used in patients at current clinical therapeutic levels.

Topics: Abnormalities, Drug-Induced; Animals; Cricetinae; Dose-Response Relationship, Drug; Embryonic and Fetal Development; Etretinate; Female; Mesocricetus; Pregnancy; Teratogens; Tretinoin

Implanting inert carriers soaked in retinoic acid into the anterior margin of the developing limb of chicken embryos leads to orofacial malformations as well as affecting pattern formation in the limb. Using anion-exchange beads as carriers, and soaking solutions of 1-10 mg/ml retinoic acid, almost 100% of the embryos have malformations of the face. The effects on the treated limbs range from symmetrical patterns of duplicated digits (maximum number of digits being four) to truncations in which no digits were formed at all. Typically, in the malformed faces the upper beak is completely absent, no nostrils are present and the front of the face forms a scalloped rim of tissue above the mouth. By reference to normal beak development, the seven bulges of tissue that make up the rim can be identified as derivatives of the masses of tissue that normally would fuse to form the upper beak. The roof of the mouth consists of three bulges of tissue flanked by widely separated palatal shelves. The defect can thus be classified as severe bilateral clefting of the primary palate. By examining the morphology of the faces of treated embryos, the origin of the defect can be traced to failure of the frontonasal mass to enlarge. Thus, the oronasal fissures are very wide and fusion across them to form the primary palate cannot occur. The way in which retinoic acid brings about the defect is discussed in relation to possible mechanisms involved in the production of cleft palate. The parallel is noted between the associated effects of retinoic acid on beak and limb morphogenesis and the chick mutation cpp, that also affects both face and limbs.

Topics: Abnormalities, Drug-Induced; Animals; Beak; Chick Embryo; Microscopy, Electron, Scanning; Orofaciodigital Syndromes; Palate; Tretinoin

An oral dose of all-trans-retinoic acid or 13-cis-retinoic acid in the pregnant [Lak:LVG(SYR)] hamster caused a dose-dependent increase in malformations in the offspring, but an equivalent dose of all-trans-N-ethyl retinamide or 13-cis-N-ethyl retinamide failed to result in embryotoxicity. The present results show that structural modification of the retinoid skeleton can produce compounds that retain cancer chemopreventive activity but that lack the teratogenic activity common to many synthetic and naturally occurring forms of vitamin A. The results indicate that in the case of the retinoids the two kinds of activity--interference with the process of carcinogenesis and interference with embryonic development--may be divorced.

Topics: Abnormalities, Drug-Induced; Animals; Cricetinae; Embryo Implantation; Embryo, Mammalian; Female; Fetal Resorption; Fetus; Pregnancy; Structure-Activity Relationship; Teratogens; Tretinoin

Topics: Abnormalities, Drug-Induced; Chemical and Drug Induced Liver Injury; Female; Heart Defects, Congenital; Humans; Hydrocephalus; Infant, Newborn; Infant, Premature, Diseases; Isotretinoin; Liver Diseases; Tretinoin

Topics: Abnormalities, Drug-Induced; Blood Donors; Drug Labeling; Female; Gestational Age; Humans; Isotretinoin; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Female; Humans; Infant, Newborn; Isotretinoin; Maternal-Fetal Exchange; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Acne Vulgaris; Drug Administration Schedule; Drug Eruptions; Female; Humans; Isotretinoin; Mucous Membrane; Pregnancy; Tretinoin

Topics: Abnormalities, Drug-Induced; Corneal Opacity; Humans; Ileitis; Isotretinoin; Pseudotumor Cerebri; Tretinoin; Uric Acid

Swiss Webster female mice weighing 25-30 gm were injected subcutaneously on days 6-15 of gestation with the synthetic sex steroid Delalutin (17 alpha-hydroxyprogesterone caproate). Treatment was given daily in doses ranging from 42 to 833 mg/kg body weight, or 10, 100, and 200 times the human therapeutic dose. On day 18 fetuses were removed from the uterus and examined for malformations and other fetotoxic effects. Prenatal treatment with the two higher doses resulted in 8 and 13% maternal deaths, and all doses resulted in a slight increase (4-12% above control) in resorption frequency. Treatment with Delalutin did not significantly affect intrauterine growth, sex ratio, or malformation rate of the offspring. The results of the present study confirm other reports that Delalutin is not androgenic, and that it, like progesterone and certain other sex steroids, does not alter the development of nonreproductive organs.

Topics: 17 alpha-Hydroxyprogesterone Caproate; Abnormalities, Drug-Induced; Animals; Body Weight; Dose-Response Relationship, Drug; Female; Fetal Resorption; Hydroxyprogesterones; Muridae; Pregnancy; Progestins; Sex Factors; Tretinoin

Pregnant rats of CFHB strain were injected 81/2 days postcoitum with a 1% suspension of retinoic acid (RA) in arachis oil to give 20 mg RA per kg body weight. Control rats were injected with arachis oil only. After 26 hours, one uterine horn was removed from each rat and the embryos cultured in serum from untreated rats. The embryos in the other horn were allowed to continue development in vivo. After a further 48 hours the cultures were terminated and the second uterine horn removed from each rat. This provided four groups of embryos for comparison: (1) embryos from RA-treated rats, (2) cultured embryos from RA-treated rats, (3) embryos from control rats, and (4) cultured embryos from control rats. The results showed that the effects of the teratogen on the cultured embryos were similar to those on the embryos allowed to continue development for the same period in the mother. In both groups RA reduced protein synthesis, inhibited somite and limb bud formation, and caused various neural tube defects, particularly microcephaly and abnormalities in the closure of the anterior and posterior neuropores.

Topics: Abnormalities, Drug-Induced; Animals; Embryo Loss; Embryo, Mammalian; Female; In Vitro Techniques; Muridae; Neural Tube Defects; Pregnancy; Tretinoin

Local application of retinoic acid to the chick limb bud produces effects that are dose and/or stage dependent. Low doses and/or old stages tend to give normal limbs or perhaps one or two supernumerary digits of a more anterior character. Medium doses and/or intermediate stages tend to give full mirror-image supernumeraries with two or even three extra digits including particularly digits of a posterior character. High doses and/or early stages give limbs in which supernumerary digits fail to form or are lost, and in which even host skeletal elements are missing or reduced. The effects are graded over the full dose and/or stage range. Various explanations are discussed in the context of the current hypotheses of limb development. We conclude that one should not necessarily interpret the results as evidence that retinoids normally play a part in the control of development or regeneration.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Chick Embryo; Dose-Response Relationship, Drug; Tretinoin; Wings, Animal

Administration of single doses of retinoic acid to hamsters on days 7, 8, and 9 of pregnancy resulted in missing, irregular, and abnormally fused centers of ossification in the vertebrae of fetuses recovered near term. A study of early events in embryonic tissues following maternal treatment with 60 mg/kg of the teratogen on day 8 revealed a variety of changes which could be linked to the development of the bony defects. By 12 hours following treatment, the mean number of somites in teratogen-exposed embryos was significantly reduced in comparison to controls. Within 24 hours of maternal treatment, lesions were observed in the aortae of the retinoic acid-exposed embryos. The vessels were consistently damaged caudally with dissection of aortic contents into the adjacent unsegmented mesoderm. Kinking of the neural tube, notochordal irregularities, and a loss of intercellular relationships in the paraxial mesoderm accompanied the vascular lesions. By 36 hours following treatment, abnormalities were evident in the appearance of the caudal somites, and at later stages these appeared to translate into defects in the sclerotomes and subsequently, the vertebrae. The observations suggest that vascular damage plays a significant role in the induction of the vertebral defects by disrupting somitogenesis. Moreover, the results support the hypothesis that retinoic acid produces abnormalities in the vertebral skeleton by a mechanism different from that which has been suggested to operate in the induction of defects in the limb skeleton.

Topics: Abnormalities, Drug-Induced; Animals; Blood Vessels; Cricetinae; Female; Gestational Age; Mesocricetus; Microscopy, Electron, Scanning; Notochord; Pregnancy; Spine; Tretinoin

Topics: Abnormalities, Drug-Induced; Animals; Dose-Response Relationship, Drug; Extremities; Female; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Organ Culture Techniques; Pregnancy; Tretinoin

Four retinoids - retinol (vitamin A), tretinoin (retinoic acid), etretinate (the ethyl ester of trimethoxymethylphenyl retinoic acid), and isotretinoin (13-cis-retinoic acid, Accutane) - have been administered orally in humans for therapeutic purposes. A review of the available information on the clinical toxic effects of these substances indicated that, while they express similar spectra of toxicity, they also differ in the extent to which they affect various body systems. This suggests that differential efficacy of the retinoids may be related, in part, to the cutaneous sites of maximum activity.

Topics: Abnormalities, Drug-Induced; Administration, Oral; Adolescent; Adult; Child; Etretinate; Female; Humans; Infant; Isomerism; Isotretinoin; Male; Tretinoin; Vitamin A

The susceptible stages and the malformation pattern produced by excess retinoic acid were investigated in rat fetuses. Retinoic acid (120 mg/kg body weight, suspended in rape oil) was administered orally to pregnant females on one of the first 20 days of gestation. Fetuses were examined for external and, after visualization of the skeleton with alizarin red, for skeletal malformations on the 21st day of gestation. Retinoic acid was highly embryolethal when administered on days 9 and 10 of gestation (96.2 and 100% resorptions). The earliest teratogenic effect of retinoic acid was noted on the 9th day of gestation. Severe multiple defects were produced by retinoic acid administration on days 9 and 11 of gestation, but more specific malformations involving the axial skeleton, the fore- and hindlimbs, and cleft palate resulted from treatment on days 12-18 of gestation. Cycloheximide, and inhibitor of protein synthesis, reduced the incidence of limb defects induced by retinoic acid. This result indicates that the teratogenic effect of retinoic acid on limb morphogenesis may be dependent upon continuous protein synthesis and is compatible with the view that vitamin A may act like a hormone.

Topics: Abnormalities, Drug-Induced; Animals; Bone Development; Cycloheximide; Female; Gestational Age; Pregnancy; Rats; Teratogens; Tretinoin

Eight pregnant Macaca nemestrina were administered from 7.5 to 10 mg/kg retinoic acid by mouth from day 20 to 44 of gestation. All fetuses exhibited craniofacial abnormalities. The craniofacial complex was studied in detail utilizing gross photography, silver nitrate impregnation, radiography, alizarin staining, and histologic processing. Nearly all the bones of the craniofacial complex were affected; the zygomatic bone and the mandible were most severely altered. Premature fusion of the coronal suture occurred in all the specimens, and there was a clockwise rotation of the craniofacial complex on right lateral view. In general, the bones of the affected fetus were smaller and less well developed than in the control specimen. The abnormal specimens in the present investigation resembled the Treacher-Collins syndrome in humans, and may be the result of defective neural crest cell migration in the first and second branchial arches.

Topics: Abnormalities, Drug-Induced; Animals; Bone Development; Brain; Face; Female; Fetus; Gestational Age; Haplorhini; Macaca; Mandible; Neural Crest; Pregnancy; Skull; Sphenoid Bone; Tretinoin; Zygoma

Fifteen pregnant Macaca mulatta were treated with doses of 20 or 40 mg of retinoic acid between 19--45 or 17--45 days of gestation, respectively, for 4--8 consecutive days. Based on gross examination, ten malformed infants, including one stillbirth and one abortus, four normal infants, and one resorption were produced. The most critical sensitive period was between days 24--35 of gestation, and the malformations primarily involved the craniofacial skeleton. Ten treated infants and eight age-matched controls were cephalometrically analyzed using craniometric points as closely correlated as possible with those in humans in order to define the craniofacial malformations induced prenatally by retinoic acid. Although all ten animals had detectable linear and angular deviations from the controls, four had cephalometric patterns which appeared to be of similar developmental origin.

Topics: Abnormalities, Drug-Induced; Animals; Animals, Newborn; Cephalometry; Facial Bones; Female; Fetal Death; Macaca; Macaca mulatta; Monkey Diseases; Pregnancy; Radiography; Skull; Tretinoin

The teratogenic effects of retinoic acid, the alcohol-soluble acid form of vitamin A, on the craniofacial complex of 11 macaque (Macaca nemestrina) whose mothers had received the compound from days 20 to 44 are described. The fetuses ranged in gestational age from 81 to 185 days and exhibited features of the so-called retinoic acid syndrome (RAS). The syndrome includes both craniofacial defects and postcranial anomalies of the musculoskeletal and urogenital systems. The craniofacial anomalies were described with reference to gross external appearance and radiographic observations. The most frequent findings were cleft palate, malformed ears, hypertelorism, exophthalmos, hypoplasia of the bone of the mid-face and mandible, a curvature of the inferior border of the mandible, retrognathia, and distortion of the cranium. Lateral cephalograms on nine animals of the RAS sample were measured using six linear dimensions which define the cranial base, face height, palatal length, and mandibular length. The measurements were plotted relative to normal curves which describe growth of the dimensions through the macaque fetal period. For their age, the abnormal animals were small in the craniofacial region. The same measurements were then plotted relative to the size of the fetus, to investigate the possibility of a differential response of the various craniofacial areas to the teratogen. Mandibular length and anterior cranial base were the most reduced dimensions, followed by anterior and posterior face height, with palatal length the least affected. Comparison of the features of the RAS syndrome in the macaque fetus with those reported for various human mandibulofacial dysostosis syndromes yields similarities, but there are enough differences to indicate that the syndromes are not identical in the two species. The utility of the approach used, wherein several craniofacial dimensions of the abnormal are assessed relative to normal growth curves and relative to body size, is emphasized.

Topics: Abnormalities, Drug-Induced; Abnormalities, Multiple; Animals; Cephalometry; Facial Bones; Female; Fetus; Macaca nemestrina; Pregnancy; Radiography; Skull; Tretinoin

Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-unlar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.

Topics: Abnormalities, Drug-Induced; Animals; Ectromelia; Female; Forelimb; Hindlimb; Humans; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Pregnancy; Pregnancy, Animal; Time Factors; Tretinoin

Oral administration of teratogenic doses of retinoic acid to pregnant hamsters on day 10 of gestation is associated with dysmorphogenesis of the appendicular skeleton. During the 24 h following retinoic acid treatment, the developing limb bud vasculature was disorganized, with blood vessels encroaching on areas where mesenchymal condensation of the skeletal blastemata normally occurs. Large, branching marginal folds and endothelial cell vesiculations protruded into the blood vessel lumina. It is suggested that the vascular changes observed may affect the concurrent early development of the skeleton and contribute to the skeletal malformation seen in near-term fetuses.

Topics: Abnormalities, Drug-Induced; Animals; Blood Vessels; Capillaries; Cricetinae; Female; Hindlimb; Mesocricetus; Microscopy, Electron; Pregnancy; Time Factors; Tretinoin

Chondrogenesis in the fetal hamster tibia was examined 24, 36 and 48 h after maternal administration of retinoic acid at a known teratogenic dose (80 mg/kg) on day 10 of gestation. Twenty-four hours after treatment the tibial chondroblasts were more closely packed with less intervening fibrillar or granular matrix than in the controls. By 36 h post treatment, an intercellular matrix containing wide clumped fibrils unassociated with matrix granules had begun to accumulate, while the condroblasts had a relatively poorly developed Golgi apparatus and smooth, non-scalloped cell outlines. Cells frequently maintained contact chondroblasts appeared similar to controls of the same age. It is suggested that the paucity and abnormality of the extracellular matrix prevents the chondroblasts from assuming their normal spatial relations with each other, resulting in a small skeletal blastema.

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cell Differentiation; Cricetinae; Female; Hindlimb; Intercellular Junctions; Microscopy, Electron; Pregnancy; Tibia; Tretinoin; Vitamin A

Daily oral administration of 10 mg/kg retinoic acid to pregnant Macaca nemestrina monkeys on days 20 to 44 resulted in a high frequency of craniofacial and musculoskeletal malformations. Craniofacial defects including cleft palate and anomalies of the prinna were common as were ectrodactyly, kyphosis, and muscular-joint contractures. Transposition of the great vessels of the heart occurred in one animal and polycystic kidney and associated urogenital anomalies in another. Shorter treatment periods with similar or higher dosages were not teratogenic and were less fetocidal. Although only relatively long treatment courses were teratogenic, the defects that resulted were morphologically similar to those induced with retinoic acid or other vitamin A compounds in other animal orders.

Topics: Abnormalities, Drug-Induced; Animals; Female; Haplorhini; Macaca; Pregnancy; Tretinoin; Vitamin A

Rat embryos were explanted on day 8 or 9 of pregnancy and cultured for up to 48 hours in serum containing added retinol (vitamin A), retinoic acid (vitamin A acid), or absolute ethanol. They were examined morphologically and their protein content determined. Retinoic acid was more teratogenic and growth-retarding than retinol. Electron microscopy of embryos cultured for 30 minutes or one hour revealed that both forms of vitamin A brought about similar ultrastructural effects on the embryonic cells; however, the abnormally large intracellular lipid droplets observed in a previous study following exposure to retinol in vitro and retinyl palmitate in vivo were not observed in embryos exposed to retinoic acid. It is possible that the differential teratogenicity may be due to the inability of the embryonic cells to convert and store retinoic acid in a less teratogenic form.

Topics: Abnormalities, Drug-Induced; Animals; Embryo, Mammalian; Embryonic Development; Female; In Vitro Techniques; Pregnancy; Protein Biosynthesis; Rats; Tretinoin; Vitamin A

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cell Cycle; Cell Survival; Cytarabine; Extremities; Gestational Age; Limb Deformities, Congenital; Mice; Morphogenesis; Tretinoin; Vitamin A

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cell Differentiation; Cell Division; Cell Membrane; Cell Movement; Cell Survival; Collagen; Connective Tissue Cells; Cytoplasmic Granules; Extremities; Female; Humans; Humerus; Limb Deformities, Congenital; Maternal-Fetal Exchange; Mice; Pregnancy; Time Factors; Tretinoin; Ulna; Vitamin A

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Ectromelia; Female; Forelimb; Hindlimb; Limb Deformities, Congenital; Mice; Mice, Inbred DBA; Pregnancy; Teratogens; Time Factors; Tretinoin; Vitamin A