tretinoin and citral

Retinoic acid, the active metabolite of vitamin A, is important for vertebrate cognition and hippocampal plasticity, but few studies have examined its role in invertebrate learning and memory, and its actions in the invertebrate central nervous system are currently unknown. Using the mollusc

Topics: Acyclic Monoterpenes; Animals; Conditioning, Operant; Lymnaea; Memory, Long-Term; Retinoids; Tretinoin

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Topics: Animals; Ecosystem; Female; Glyphosate; Isotretinoin; Methoprene; Retinaldehyde; Retinoids; Tandem Mass Spectrometry; Tretinoin; Vitamin A

Intrauterine growth restriction (IUGR) is commonly observed in human pregnancies and can result in severe clinical outcomes. IUGR is observed in Fetal Alcohol Syndrome (FAS) fetuses as a result of alcohol (ethanol) exposure during pregnancy. To further understand FAS, the severe form of Fetal Alcohol Spectrum Disorder, we performed an extensive quantitative analysis of the effects of ethanol on embryo size utilizing our Xenopus model. Ethanol-treated embryos exhibited size reduction along the anterior-posterior axis. This effect was evident primarily from the hindbrain caudally, while rostral regions appeared refractive to ethanol-induced size changes, also known as asymmetric IUGR. Interestingly, some embryo batches in addition to shortening from the hindbrain caudally also exhibited an alcohol-dependent reduction of the anterior head domain, known as symmetric IUGR. To study the connection between ethanol exposure and reduced retinoic acid levels we treated embryos with the retinaldehyde dehydrogenase inhibitors, DEAB and citral. Inhibition of retinoic acid biosynthesis recapitulated the growth defects induced by ethanol affecting mainly axial elongation from the hindbrain caudally. To study the competition between ethanol clearance and retinoic acid biosynthesis we demonstrated that, co-exposure to alcohol reduces the teratogenic effects of treatment with retinol (vitamin A), the retinoic acid precursor. These results further support the role of retinoic acid in the regulation of axial elongation.

Topics: Acyclic Monoterpenes; Animals; Benzaldehydes; Brain; Embryo, Nonmammalian; Enzyme Inhibitors; Ethanol; Fetal Alcohol Spectrum Disorders; Retinal Dehydrogenase; Signal Transduction; Tretinoin; Xenopus

Expression of the LIM homeodomain transcription factor Lhx8 is restricted to and up-regulated in the mesenchyme of the upper face prominence before lip fusion. Msx1/2 acts in early development to control cell proliferation and differentiation. Deficiency of these genes is associated with nonsyndromic cleft lip with/without cleft palate. Since retinoid is a potential patterning influence on the developing face, we have examined whether retinoic acid (RA) signaling regulated Lhx8, Msx1 and Msx2 transcription through fibroblast growth factor (FGF) signals in the maxillary prominence. Application of exogenous RA caused severe defects of the maxilla. Citral also induced a specific loss of derivatives from the maxillary prominences by blocking RA synthesis. Real-time RT-PCR and semi-quantitative RT-PCR analysis of the maxillary mesenchyme revealed that the expressions of Lhx8, Msx1 and Msx2 were significantly down-regulated by RA as well as by citral. The downregulated Lhx8 was rescued by combined treatment with FGF-8b, which indicated a downstream of RA signaling. FGF-8b induced up-regulated Lhx8 expression whereas SU5402, a pan-FGF family antagonist, down-regulated and caused defective maxillary morphogenesis and cleft lip. Our data suggest that Lhx8 is regulated by RA signaling through FGF signals and the level window of RA and FGF-8b could control the upper jaw morphogenesis.

Topics: Acyclic Monoterpenes; Animals; Cell Differentiation; Chick Embryo; Down-Regulation; Fibroblast Growth Factor 8; Homeodomain Proteins; Jaw; LIM-Homeodomain Proteins; Mesoderm; Monoterpenes; Morphogenesis; MSX1 Transcription Factor; Pyrroles; Signal Transduction; Transcription Factors; Tretinoin

The retinoic acid (RA) signaling pathway is known to play important roles during craniofacial development and skeletogenesis. However, the specific mechanism involving RA in cranial base development has not yet been clearly described. This study investigated how RA modulates endochondral bone development of the cranial base by monitoring the RA receptor RARγ, BMP4, and markers of proliferation, programmed cell death, chondrogenesis, and osteogenesis. We first examined the dynamic morphological and molecular changes in the sphenooccipital synchondrosis-forming region in the mouse embryo cranial bases at E12-E16. In vitro organ cultures employing beads soaked in RA and retinoid-signaling inhibitor citral were compared. In the RA study, the sphenooccipital synchondrosis showed reduced cartilage matrix and lower BMP4 expression while hypertrophic chondrocytes were replaced with proliferating chondrocytes. Retardation of chondrocyte hypertrophy was exhibited in citral-treated specimens, while BMP4 expression was slightly increased and programmed cell death was induced within the sphenooccipital synchondrosis. Our results demonstrate that RA modulates chondrocytes to proliferate, differentiate, or undergo programmed cell death during endochondral bone formation in the developing cranial base.

Topics: Acyclic Monoterpenes; Animals; Apoptosis; Bone Morphogenetic Protein 4; Cell Differentiation; Cell Proliferation; Chondrocytes; Chondrogenesis; Integrin-Binding Sialoprotein; Ki-67 Antigen; Mice; Monoterpenes; Organ Culture Techniques; Osteogenesis; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Signal Transduction; Skull Base; Tretinoin

Embryonic stem (ES) cells differentiate spontaneously toward a neuroectodermal fate in serum-free, adherent monocultures. Here, we show that this spontaneous neural fate requires retinoic acid (RA) synthesis. We monitor ES cells containing reporter genes for markers of the early neural plate as well as the primitive streak and its progeny to determine the cell fates induced when RA signaling is perturbed. We demonstrate that the spontaneous neural commitment of mouse ES cells requires endogenous RA production from vitamin A (vitA) in the medium. Formation of neural progenitors is inhibited by removing vitA from the medium, by inhibiting the enzymes that catalyze the synthesis of RA, or by inhibiting RA receptors. We show that subnanomolar concentrations of RA restore neuroectodermal differentiation when RA synthesis is blocked. We demonstrate that a neural to mesodermal fate change occurring when RA signaling is inhibited is dependent on Nodal-, Wnt-, and fibroblast growth factor-signaling. We show that Nodal suppresses neural development in a Wnt-dependent manner and that Wnt-mediated inhibition of neural development is reversed by inhibition of Nodal signaling. Together, our results show that neural induction in ES cells requires RA at subnanomolar levels to suppress Nodal signaling and suggest that the mechanism by which Wnt signaling suppresses neural development is through facilitation of Nodal signaling.

Topics: Acyclic Monoterpenes; Animals; Cell Differentiation; Cells, Cultured; Disulfiram; Embryonic Stem Cells; Enzyme Inhibitors; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Genes, Reporter; Mesoderm; Mice; Monoterpenes; Naphthalenes; Neural Plate; Neurons; Nodal Protein; Receptors, Retinoic Acid; Signal Transduction; Time Factors; Transfection; Tretinoin; Vitamin A; Wnt Proteins

The initiation of meiosis is crucial to fertility. While extensive studies in rodents have enhanced our understanding of this process, studies in human fetal ovary are lacking.. We used RT-PCR and immunohistochemistry to investigate expression of meiotic factors in human fetal ovaries from 6 to 15 weeks post fertilization (wpf) and developed an organ culture model to study the initiation of human meiosis.. We observed the first meiotic cells at 11 wpf, when STRA8, SPO11 and DMC1 are first expressed. In culture, meiosis initiation is observed in 10 and 11 wpf ovaries and meiosis is maintained by addition of fetal calf serum. Meiosis is stimulated, compared with control, by retinoic acid (RA) (P < 0.05). No major change occurred in mRNA for CYP26B1, the RA-degrading enzyme proposed to control the timing of meiosis in mice. We did, however, observe increased mRNA levels for ALDH1A1 in human ovary when meiosis began, and evidence for a requirement to synthesize RA and thus sustain meiosis. Indeed, ALDH inhibition by citral prevented the appearance of meiotic cells. Finally, 8 wpf ovaries (and earlier stages) were unable to initiate meiosis whatever the length of culture, even in the presence of RA and serum. However, when human germ cells from 8 wpf ovaries were placed in a mouse ovarian environment, some did initiate meiosis.. Our data indicate that meiosis initiation in the human ovary relies partially on RA, but that the progression and regulation of this process appears to differ in many aspects from that described in mice.

Topics: Acyclic Monoterpenes; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Cytochrome P-450 Enzyme System; Female; Fetus; Humans; Meiosis; Mice; Monoterpenes; Ovary; Ovum; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Tretinoin

Retinoic acid (Ra) is crucial for the patterning and neuronal differentiation in the central nervous system (CNS). Ra deficiency in animals disrupts the motor activities and memory abilities. The molecular mechanisms underlying these behavior abnormalities remain largely unknown. In the current study, we treated the astrocytoma cells with citral, an inhibitor of Ra synthesis. We analyzed the differences in the protein concentrations between the treated and untreated astrocytoma cells by two-dimensional gel electrophoresis (2-DE), Imagemaster software, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In total, 39 of 46 altered protein spots with significant mascot scores were identified representing 36 proteins, that were involved in significantly altered glutamate metabolism, lipid metabolism, mitochondrial function, and oxidative stress response by Ingenuity Pathway Analysis (IPA). Altered 3-phosphoglycerate dehydrogenase (PHGDH) was also observed in western blot. These data provide some clues for explaining the behavioral changes caused by Ra deficiency, and support the hypothesis that Ra signaling is associated with some symptoms of neurodegenerative disorders and schizophrenia.

Topics: Acyclic Monoterpenes; Animals; Astrocytoma; Cell Line, Tumor; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Humans; Molecular Sequence Data; Monoterpenes; Phosphoglycerate Dehydrogenase; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tretinoin

Although some central effects of citral have been reported, cognitive effects on spatial memory have not been investigated. The evidence showed that citral can regulate the synthesis of retinoic acid (RA), which exerts a vital function in the development and maintenance of spatial memory. In this study, we applied Morris water maze to test the effect of citral on animals' spatial learning and memory. To elucidate the mechanism of this effect, we also measured the retinoic acid concentration in rats' hippocampus by high performance liquid chromatography (HPLC). Our data implied biphasic effects of citral. The low dose (0.1 mg/kg) of citral improved the spatial learning capability, and enhanced the spatial reference memory of rats, whereas the high dose (1.0 mg/kg) was like to produce the opposite effects. Meanwhile, the low dose of citral increased the hippocampal retinoic acid concentration, while the high dose decreased it. Due to the quick elimination and non-bioaccumulation in the body, effects of citral on spatial memory in this study seemed to be indirect actions. The change in hippocampal retinoic acid concentration induced by different doses of citral might be responsible for the biphasic effect of citral on spatial learning and memory.

Topics: Acyclic Monoterpenes; Animals; Chromatography, High Pressure Liquid; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Flavoring Agents; Hippocampus; Indicators and Reagents; Male; Maze Learning; Memory; Monoterpenes; Odorants; Rats; Rats, Sprague-Dawley; Tretinoin

Retinoic acid (RA) is a powerful differentiation agent. Barrett's oesophagus occurs when duodeno-gastro-oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett's oesophagus.. RA activity in tissues and cell lines treated with all-trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real-time PCR in Barrett's oesophagus cell lines with or without LCA. SE and Barrett's oesophagus biopsy specimens were exposed to 100 muM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT-PCR (CKs). Confocal microscopy assessed temporal changes in co-localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo-deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21.. RA biosynthesis was increased in Barrett's oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co-localised with CK8/18 at 24 h, which separated into CK8/18-positive glands and vimentin-positive stroma by 48 h. Citral-treated Barrett's oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation.. RA activity is increased in Barrett's oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the oesophageal phenotype can be altered independent of proliferation.

Topics: Acyclic Monoterpenes; Barrett Esophagus; Cell Differentiation; Cell Proliferation; Esophagus; Humans; Lithocholic Acid; Monoterpenes; Organ Culture Techniques; Phenotype; Polymerase Chain Reaction; Prospective Studies; Tretinoin

Previous studies of knock-out mouse embryos have shown that the Wilms' tumor suppressor gene (Wt1) is indispensable for the development of kidneys, gonads, heart, adrenals and spleen. Using OPT (Optical Projection Tomography) we have found a new role for Wt1 in mouse liver development. In the absence of Wt1, the liver is reduced in size, and shows lobing abnormalities. In normal embryos, coelomic cells expressing Wt1, GATA-4, RALDH2 and RXRalpha delaminate from the surface of the liver, intermingle with the hepatoblasts and incorporate to the sinusoidal walls. Some of these cells express desmin, suggesting a contribution to the stellate cell population. Other cells, keeping high levels of RXRalpha immunoreactivity, are negative for stellate or smooth muscle cell markers. However, coelomic cells lining the liver of Wt1-null embryos show decreased or absent RALDH2 expression, the population of cells expressing high levels of RXRalpha is much reduced and the proliferation of hepatoblasts and RXRalpha-positive cells is significantly decreased. On the other hand, the expression of smooth muscle cell specific alpha-actin increases throughout the liver, suggesting an accelerated and probably anomalous differentiation of stellate cell progenitors. We describe a similar retardation of liver growth in RXRalpha-null mice as well as in chick embryos after inhibition of retinoic acid synthesis. We propose that Wt1 expression in cells delaminating from the coelomic epithelium is essential for the expansion of the progenitor population of liver stellate cells and for liver morphogenesis. Mechanistically, at least part of this effect is mediated via the retinoic acid signaling pathway.

Topics: Acyclic Monoterpenes; Aldehyde Oxidoreductases; Animals; Biomarkers; Cell Differentiation; Cell Proliferation; Chick Embryo; Embryo, Mammalian; Hepatocytes; Liver; Mice; Mice, Knockout; Models, Biological; Monoterpenes; Morphogenesis; Phenotype; Quail; Retinoid X Receptor alpha; Signal Transduction; Stomach; Tretinoin; WT1 Proteins

The conversion of retinol (ROH) to retinoic acid (RA) is crucial during development but has been not studied during blastocyst formation.. In vitro-produced bovine morulae were treated for 24 h with citral (which inhibits the synthesis of RA from ROH), citral + all trans retinoic acid (ATRA), ATRA or no additives. Citral interfered with blastocyst development, whereas exogenous RA had no effect. RA, however, reversed the effect of citral on development and stimulated cell proliferation. Neither citral nor RA changed the apoptotic index, but RA triggered an increase in the apoptotic frequency of the inner cell mass. Citral and RA reduced the necrotic index. Na/K-ATPase alpha1-subunit mRNA concentrations (analysed by real-time PCR) increased after hatching and showed dependence on retinoid activity, but no evidence was found of any retinoid effect on p53 expression. Nevertheless, the p53 mRNA concentration increased in response to proliferation in hatched blastocysts.. The preimplantation bovine embryo metabolizes endogenous ROH to RA, which participates in important cell processes. The true extent of the influence of RA is unknown, although the modulation of retinoid metabolism seems to be a means of increasing cell proliferation. This knowledge might be used to improve embryo quality and the efficiency of stem cell derivation.

Topics: Acyclic Monoterpenes; Animals; Apoptosis; Blastocyst; Cattle; Female; Monoterpenes; Morula; Necrosis; Protein Subunits; Sodium-Potassium-Exchanging ATPase; Tretinoin; Tumor Suppressor Protein p53; Vitamin A

Endogenous retinoids are important for patterning many aspects of the embryo including the branchial arches and frontonasal region of the embryonic face. The nasal placodes express retinaldehyde dehydrogenase-3 (RALDH3) and thus retinoids from the placode are a potential patterning influence on the developing face. We have carried out experiments that have used Citral, a RALDH antagonist, to address the function of retinoid signaling from the nasal pit in a whole embryo model. When Citral-soaked beads were implanted into the nasal pit of stage 20 chicken embryos, the result was a specific loss of derivatives from the lateral nasal prominences. Providing exogenous retinoic acid residue development of the beak demonstrating that most Citral-induced defects were produced by the specific blocking of RA synthesis. The mechanism of Citral effects was a specific increase in programmed cell death on the lateral (lateral nasal prominence) but not the medial side (frontonasal mass) of the nasal pit. Gene expression studies were focused on the Bone Morphogenetic Protein (BMP) pathway, which has a well-established role in programmed cell death. Unexpectedly, blocking RA synthesis decreased rather than increased Msx1, Msx2, and Bmp4 expression. We also examined cell survival genes, the most relevant of which was Fgf8, which is expressed around the nasal pit and in the frontonasal mass. We found that Fgf8 was not initially expressed along the lateral side of the nasal pit at the start of our experiments, whereas it was expressed on the medial side. Citral prevented upregulation of Fgf8 along the lateral edge and this may have contributed to the specific increase in programmed cell death in the lateral nasal prominence. Consistent with this idea, exogenous FGF8 was able to prevent cell death, rescue most of the morphological defects and was able to prevent a decrease in retinoic acid receptorbeta (Rarbeta) expression caused by Citral. Together, our results demonstrate that endogenous retinoids act upstream of FGF8 and the balance of these two factors is critical for regulating programmed cell death and morphogenesis in the face. In addition, our data suggest a novel role for endogenous retinoids from the nasal pit in controlling the precise downregulation of FGF in the center of the frontonasal mass observed during normal vertebrate development.

Topics: Acyclic Monoterpenes; Animals; Body Patterning; Bone and Bones; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Death; Chick Embryo; Craniofacial Abnormalities; Embryo, Mammalian; Embryo, Nonmammalian; Embryonic Structures; Face; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Homeodomain Proteins; In Situ Hybridization; In Situ Nick-End Labeling; Monoterpenes; Morphogenesis; MSX1 Transcription Factor; Nose; Receptors, Retinoic Acid; Signal Transduction; Skeleton; Transcription Factors; Tretinoin

The purpose of this study was to investigate the reciprocal neurotrophic interaction between regenerating limb blastemas and spinal cord explants from the newt Notophthalmus viridescens. Axon outgrowth was measured from spinal cord explants in vitro to assess the neurotrophic activity of early to mid-bud stage blastemas after various treatments. When retinoic acid, a vitamin A metabolite, was added to the medium, it increased both the number and length of axons extending from spinal cord explants. Spinal cord explants co-cultured with blastemas that were previously treated with citral, an inhibitor of retinoic acid synthesis, extended significantly fewer axons than control co-cultures. Blastemas, which were denervated by surgical resection of the brachial plexus 48 h before co-culture, also exhibited a significantly weaker neurotrophic activity than did control innervated blastemas. These results are consistent with a reciprocal interaction between blastema mesenchyme and nerves and suggest either a stimulatory or synergistic role for endogenous retinoic acid in the blastema-derived trophic activity.

Topics: Acyclic Monoterpenes; Animals; Denervation; Ethanol; Forelimb; Mitosis; Monoterpenes; Nerve Regeneration; Neurons; Organ Culture Techniques; Salamandridae; Spinal Cord; Tretinoin

The egg yolk of vertebrates contains carotenoids, which account for its characteristic yellow color in some species. Such plant-derived compounds, e.g. beta-carotene, serve as the natural precursors (provitamins) of vitamin A, which is indispensable for chordate development. As egg yolk also contains stored vitamin A, carotenoids have so far been solely discussed as pigments for the coloration of the offspring. Based on our recent molecular identification of the enzyme catalyzing provitamin A conversion to vitamin A, we address a possible role of provitamin A during zebrafish (Danio rerio) development. We cloned the zebrafish gene encoding the vitamin A-forming enzyme, a beta,beta-carotene-15,15'-oxygenase. Analysis of its mRNA expression revealed that it is under complex spatial and temporal control during development. Targeted gene knockdown using the morpholino antisense oligonucleotide technique indicated a vital role of the provitamin A-converting enzyme. Morpholino-injected embryos developed a morphological phenotype that included severe malformation of the eyes, the craniofacial skeleton and pectoral fins, as well as reduced pigmentation. Analyses of gene expression changes in the morphants revealed that distinct retinoic acid-dependent developmental processes are impaired, such as patterning of the hindbrain and differentiation of hindbrain neurons, differentiation of neural crest derivatives (including the craniofacial skeleton), and the establishment of the ventral retina. Our data provide strong evidence that, for several developmental processes, retinoic acid generation depends on local de novo formation of retinal from provitamin A via the carotene oxygenase, revealing an unexpected, essential role for carotenoids in embryonic development.

Topics: Acyclic Monoterpenes; Amino Acid Sequence; Animals; beta-Carotene 15,15'-Monooxygenase; Body Patterning; Cell Differentiation; Cloning, Molecular; Gene Expression Regulation, Developmental; Humans; In Situ Hybridization; Molecular Sequence Data; Monoterpenes; Morphogenesis; Neural Crest; Oligonucleotides, Antisense; Ovum; Oxygenases; Pharynx; Phenotype; Retina; Retinaldehyde; Rhombencephalon; Sequence Alignment; Tretinoin; Vitamin A; Zebrafish

During liver fibrogenesis or long term culture, hepatic stellate cells (HSCs) evolved from "quiescent" to activated phenotype called "myofibroblast-like", a transition prevented by retinoic acid (RA). Little is known about RA generation by HSCs. Our study aimed to check the ability of these cells to produce RA from retinol (Rol) and the alterations of this metabolic step by ethanol. To study this metabolic pathway, primary cultures of HSCs represent the most physiological model but technically suffer several drawbacks. To circumvent these problems, an immortalized rat HSC line (named PAV-1) has been established. We validated PAV-1 cell line as a convenient model to study retinoids metabolism by HSCs. Then, we showed that PAV-1 cells express Rol-binding proteins (RBPs), enzymes and nuclear receptors involved in RA signaling pathway. We also demonstrated in situ generation of functional all-trans-RA (ATRA), using transient transfections with a RA-sensitive reporter gene, in situ modulation of tissue transglutaminase (tTG) activity and HPLC experiments. This production was Rol dose-dependent; 4-methylpyrazole, citral, and ethanol-inhibited which argues in favor of an enzymatic process.In conclusion, we first demonstrate in situ RA generation from Rol in a newly immortalized rat HSC line, named PAV-1. Inhibition of RA production by ethanol in PAV-1 and recent data, suggesting fundamental role of RA to prevent fibrosis development in the liver, allow us to hypothesize that Rol metabolism could be a primary target for ethanol during development of hepatic fibrosis.

Topics: Acyclic Monoterpenes; Animals; Biomarkers; Carboxylic Ester Hydrolases; Cell Line; Cell Nucleus; Esterification; Ethanol; Fomepizole; Liver; Male; Monoterpenes; Pyrazoles; Rats; Rats, Wistar; Signal Transduction; Terpenes; Tretinoin; Vitamin A

Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

Topics: Acyclic Monoterpenes; Aldehyde Oxidoreductases; Animals; Benzaldehydes; Cell Communication; Cytochrome P-450 Enzyme Inhibitors; Gastrula; Gene Expression Regulation, Developmental; Genes, Reporter; Genetic Engineering; Monoterpenes; Oxygenases; Promoter Regions, Genetic; Retinal Dehydrogenase; Teratogens; Terpenes; Transcriptional Activation; Transgenes; Tretinoin; Vitamin A Deficiency; Zebrafish; Zebrafish Proteins

The biological effects of vitamin A are mediated in part by retinoic acid (RA) modulation of gene transcription. In this study, we examined whether normal human mammary epithelial cells (HMECs) are biologically responsive to retinol (ROH), the metabolic precursor of RA. While both ROH and tRA resulted in time- and dose-dependent decreases in total cell number, tRA was markedly more potent. Metabolically, treatment of HMECs with physiological doses of ROH resulted in rapid uptake and subsequent production of both retinyl esters and tRA. Although a comparatively minor metabolite, tRA levels peaked at 6 h and remained above endogenous levels for up to 72 h in proportion to cellular ROH concentrations. In HMECs transfected with an RA-responsive luciferase reporter gene, treatment with 3 microM ROH resulted in an increase in luciferase activity to a level intermediate between that observed with 0.001 and 0.01 microM tRA. Citral, an RA-synthesis inhibitor, was also used to examine the biological activity of ROH. Compared to ROH alone, ROH plus citral treatment resulted in three-fold less tRA synthesis and a > 65% attentuation of RA-responsive reporter gene activity which persisted through 72 h. Citral also significantly attenuated the extent of ROH-mediated reductions in total HMEC number. Thus, treatment with physiological concentrations of ROH results in fewer total numbers of HMECs and this response is a consequence of cellular tRA synthesis which can induce RA-responsive gene expression.

Topics: Acyclic Monoterpenes; Biological Transport; Breast; Cells, Cultured; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Genes, Reporter; Humans; Kinetics; Luciferases; Monoterpenes; Terpenes; Time Factors; Transfection; Tretinoin; Vitamin A

In Xenopus, the primary neurons form in three domains either side of the midline in the posterior neurectoderm. At the late neurula stage there are approximately 120 primary sensory neurons on each side of the embryo. Co-injecting synthetic mRNA encoding retinoic acid receptor alpha (NR1B1) and retinoid X receptor beta (NR2B2) results in an increase in the number of primary neurons and this is further enhanced by the addition of retinoic acid indicating that elevated retinoid signalling promotes an increase in the number of cells undergoing primary neurogenesis. However, primary neurogenesis remains confined to the three domains that normally give rise to primary neurons indicating that not all regions of the neurectoderm respond equivalently to elevated retinoid signalling. The inhibition of retinoid signalling with a dominant negative retinoid receptor or treatment with citral, an inhibitor of retinoid metabolism, inhibits the formation of primary neurons. However, the lateral extent of the neurectoderm does not differ following these experimental manipulations suggesting that changes in primary neuron cell number, in response to changes in retinoid signalling, cannot be accounted for by significant gains or losses of neurectoderm. In addition, two lines of evidence are presented to suggest that retinoid signalling affects primary neurogenesis by acting directly on the neurectoderm. First, animal caps neuralized by noggin undergo primary neurogenesis in response to retinoid signalling and second primary neurogenesis is elevated in neural conjugates in which the ectodermal, but not the mesodermal, component has been co-injected with RAR/RXR mRNA.

Topics: Acyclic Monoterpenes; Animals; Dimerization; Ectoderm; Monoterpenes; Mutagenesis; Neurons, Afferent; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; Signal Transduction; Terpenes; Transcription Factors; Tretinoin; Xenopus

Besides its role in ocular development, retinoic acid (RA), which is a light-correlated byproduct of the phototransduction cycle, was recently shown to affect light-driven synaptic plasticity in the outer plexiform layer of the adult fish retina. Tuning by ambient light conditions of the retinal network properties is very prominent in outer plexiform layer circuits, and we therefore examined whether RA could affect cone horizontal cell physiology similar to ambient light. Performing intracellular recordings and dye injections in the dark-adapted inverted eyecup preparation of the carp, we found that RA reduced the receptive fields of horizontal cell somata and impaired gap junctional communication. This action was not observed among coupled axon terminals of horizontal cells and appeared to be stereospecific because it could only be attributed to all-trans and 13-cis RA but not to the 9-cis isomer and photoisomerized all-trans RA. Modulation of receptive field size occurred independently of the dopaminergic system. Furthermore, RA affected the light responsiveness of cone horizontal cells. Compared to the dark-adapted condition, responsiveness to intense light stimulation was enhanced but decreased when low intensities were used. Moreover, following RA treatment H2-type horizontal cells of dark-adapted retinae which do not give rise to colour-opponent light properties became colour-opponent and performed depolarizing responses to long-wavelength stimulation. In all these cases RA perfectly matched the effects of light adaptation, supporting the notion that RA acts as an endogenous neuromodulator.

Topics: Acyclic Monoterpenes; Adaptation, Physiological; Aldehyde Oxidoreductases; Alitretinoin; Animals; Dopamine; Eye Proteins; Gap Junctions; Goldfish; Light; Monoterpenes; Neuronal Plasticity; Retina; Retinal Dehydrogenase; Rhodopsin; Terpenes; 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

We have isolated the chick and mouse homologs of human aldehyde dehydrogenase 6 (ALDH6) that encode a third cytosolic retinaldehyde-specific aldehyde dehydrogenase. In both chick and mouse embryos, strong expression is observed in the sensory neuroepithelia of the head. In situ hybridization analysis in chick shows compartmentalized expression primarily in the ventral retina, olfactory epithelium, and otic vesicle; additional sites of expression include the isthmus, Rathke's pouch, posterior spinal cord interneurons, and developing limbs. Recombinant chick ALDH6 has a K(0.5) = 0.26 microm, V(max) = 48.4 nmol/min/mg and exhibits strong positive cooperativity (H = 1.9) toward all-trans-retinaldehyde; mouse ALDH6 has similar kinetic parameters. Expression constructs can confer 1000-fold increased sensitivity to retinoic acid receptor-dependent signaling from retinol in transient transfections experiments. The localization of ALDH6 to the developing sensory neuroepithelia of the eye, nose, and ear and discreet sites within the CNS suggests a role for RA signaling during primary neurogenesis at these sites.

Topics: Acyclic Monoterpenes; Aldehyde Dehydrogenase; Aldehyde Oxidoreductases; Amino Acid Sequence; Animals; Base Sequence; Chick Embryo; Cytosol; DNA, Complementary; Embryo, Mammalian; Humans; Kinetics; Mice; Molecular Sequence Data; Monoterpenes; Retina; Retinal Dehydrogenase; Retinaldehyde; Terpenes; Tretinoin

We had hypothesised that retinaldehyde (RAL) should be an interesting precursor for topical use.. We review our observations about its biological activities.. We performed pilot studies to explore its biological effects and tolerability in human skin and compared the effects of topical RAL to that of all-trans-retinoic acid (RA) in the mouse tail test.. The biological activities of RAL were found to be qualitatively identical to that of RA: (i) induction of cellular RA-binding protein type 2 mRNA and protein, (ii) increase in epidermal proliferation (increase in DNA synthesis, epidermal thickness, induction of 50-kD keratin mRNA and reduction in 70-kD keratin mRNA), and (iii) metaplastic effects (induction of orthokeratosis, reduction of 65-kD keratin mRNA, increase in filaggrin and loricrin mRNAs). When associated with RAL, citral (known for its capacity to inhibit the oxidation of retinol to RA in epidermis) counteracted the effects induced by RAL indicating that RAL exerts biological activities through transformation to RA. Hypothesizing that keratinocytes would metabolize 9-cis-RAL to 9-cis-RA, we compared the biological effects induced by topical 9-cis-RAL and found that hyperplastic and metaplastic responses were lower than those induced by all-trans-RAL or all-trans-RA at similar concentrations. This suggests that 9-cis-RAL has no advantage over all-trans-RAL for specific delivery of natural retinoids into the skin. As in clinical studies conducted in human skin, we also found topical RAL less irritant than RA.. These studies indicate that topical RAL has biological activity and is well tolerated.

Topics: Acyclic Monoterpenes; Administration, Topical; Animals; Dose-Response Relationship, Drug; Filaggrin Proteins; Humans; Keratins; Keratolytic Agents; Mice; Monoterpenes; Peroxidase; Retinaldehyde; Skin; Terpenes; Tretinoin

Exogenously applied retinoic acid (RA) is known to affect cartilage pattern in developing and regenerating limbs. There are, however, few reports which analyze the participation of endogenous RA in limb pattern formation. Using an organ culture system, we attempted to reduce the concentration of endogenous RA in the developing chick wing buds by the treatment with citral (3,7-dimethyl-2,6-octadienal), an inhibitor of retinoic acid formation. After this treatment, the cultured wing buds were grafted to the stumps of host embryos. These citral-treated limb buds frequently formed truncated cartilage elements and the defects were rescued by simultaneous treatment with an appropriate concentration of RA. These results suggest that endogenous RA plays a role in chick limb bud development.

Topics: Acyclic Monoterpenes; Animals; Cartilage; Cells, Cultured; Chick Embryo; Chromatography, High Pressure Liquid; Humerus; Mesoderm; Monoterpenes; Morphogenesis; Organ Culture Techniques; Radius; Terpenes; Tretinoin; Ulna; Vitamin A; Wings, Animal

This study was done to determine whether retinoic acid can be produced by excentric cleavage of beta-carotene in vivo. By using an inhibitor of retinaldehyde oxidation, citral, either retinaldehyde or beta-carotene was incorporated in a micellar solution and perfused through the upper portion of small intestine of ferrets. After 2 h perfusion of 1 microM retinaldehyde, retinoic acid rose in portal blood (+3.5 +/- 1.3 nmol/L) and was detected in the intestinal mucosa (30 +/- 2 pmol/g). When citral was added at 2 mM along with retinaldehyde, retinoic acid decreased in the portal blood and retinoic acid was not detected in the intestinal mucosa. With or without the presence of citral (2 mM), the perfusion of beta-carotene (10 microM) during 2 h caused a significant rise of retinoic acid in portal blood (+2.6 +/- 0.6 nmol/L and + 4.1 +/- 0.6 nmol/L, respectively) and in liver; moreover, significant amounts of retinoic acid were detected in the intestinal mucosa (19 +/- 3 pmol/g and 36 +/- pmol/g, respectively. This study demonstrates that after intestinal perfusion of beta-carotene in the ferret in vivo, a substantial amount of retinoic acid is formed via an excentric cleavage pathway.

Topics: Acyclic Monoterpenes; Animals; beta Carotene; Chromatography, High Pressure Liquid; Ferrets; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Liver; Male; Methylation; Monoterpenes; Retinaldehyde; Terpenes; Tretinoin; Vitamin A

An inducible cytochrome P-450 (P-450) catalyzing retinoic acid synthesis was purified from liver microsomes of 3-methylcholanthrene (3-MC)-treated rats, based on the activity of all-trans-retinoic acid formation from all-trans-retinal. We previously reported that the retinoic acid synthesis by microsomes was catalyzed by a cytochrome P-450-linked monooxygenase system (Tomita et al. (1993) Int. J. Biochem. 25, 1775-1784). This microsomal retinoic acid synthesis in rat liver was induced more than 8-fold by 3-MC. The purified P-450 electophoretically gave a single protein band and its minimum molecular weight was estimated to be 57.2 kDa on SDS-PAGE. The optical spectrum of the oxidized P-450 without retinal revealed it was the low-spin form, and the CO-complex exhibited a maximum peak at 447 nm. The specific activity of the reconstituted P-450-linked monooxygenase system was 29.5 nmol/min per nmol P-450 at pH 7.6 and 37 degrees C. The K(m) and Vmax values for all-trans-retinal were 11.6 microM and 38.5 nmol/min per nmol P-450, respectively. The amino-acid sequence of the N-terminal region of the P-450 was identical to that of rat P-450 1A1 (CYP 1A1). Xenobiotic activities, such as 7-ethoxycoumarin O-deethylase (7-ECOD) and 7-ethoxyresorufin O-deethylase (7-EROD) activities, of the P-450-linked monooxygenase system were specific to the P-450 1A1. The retinoic acid formation in the reconstituted monooxygenase system was specifically inhibited by alpha-naphthoflavone (alpha-NF), which is a P-450 1A1-specific inhibitor, citral, which is a retinoid analogue structurally, and an anti-rat P-450 1A1 antibody. These results further support that the purified P-450 is P-450 1A1. This paper describes that P-450 1A1 was purified and characterized as a retinoic acid synthetic P-450.

Topics: Acyclic Monoterpenes; Animals; Antibodies; Benzoflavones; Cytochrome P-450 Enzyme System; Cytochromes b5; Enzyme Induction; Immunoglobulin G; Isoenzymes; Male; Methylcholanthrene; Microsomes, Liver; Monoterpenes; Rats; Rats, Sprague-Dawley; Retinaldehyde; Sequence Analysis; Terpenes; Tretinoin; Xenobiotics

Catalysis of the oxidation of all-trans-retinol (vitamin A1) or of all-trans-retinal to all-trans-retinoic acid (all-trans-RA) by rat conceptal enzymes was investigated during organogenesis. Products of the reaction were identified and quantified with HPLC by comparing their elution times with those of authentic standard retinoids. Under the incubation and assay conditions utilized, all-trans-retinol and all-trans-retinal were converted to readily detectable quantities of all-trans-RA. Rat conceptal homogenates from gestational days 10.5, 11.5 and 12.5 each exhibited enzymatic activity for oxidation of all-trans-retinol and all-trans-retinal to all-trans-RA. Enzymatic catalysis was verified by showing that: (1) both reactions were coenzyme dependent; (2) the rates of reactions increased as concentrations of conceptal protein increased; (3) both reactions were abolished by heating the tissue homogenates (100 degrees, 5 min); and (4) both reactions exhibited substrate saturation. Under the same experimental conditions, formation of all-trans-RA from all-trans-retinol was much slower than from all-trans-retinal, suggesting that oxidation of all-trans-retinol to all-trans-retinal was the rate-limiting step for biotransformation of all-trans-retinol to all-trans-RA in embryonic tissues. When NAD or NADP were replaced by NADH or NADPH, the rate of oxidation of all-trans-retinol was reduced markedly, indicating that the reaction was catalyzed primarily by an NAD/NADP-dependent dehydrogenase(s). Carbon monoxide (CO:O2 = 90:10) did not inhibit the reaction. NAD appeared to be a more effective cofactor than NADP in catalyzing oxidation of all-trans-retinal to all-trans-RA. When NAD was omitted, formation of all-trans-RA from all-trans-retinal was reduced by approximately 55%. Replacing NAD by NADH or NADPH also reduced the conversion of all-trans-retinal to all-trans-RA by about 60%. These observations suggested at least two pathways for the generation of all-trans-RA from all-trans-retinal in embryos: oxidation catalyzed by an NAD/NADP-dependent dehydrogenase(s) and oxidation catalyzed by an oxidase(s) that did not require NAD, NADH, NADP or NADPH. Conversion of all-trans-retinol to all-trans-RA was inhibited strongly by low concentrations of citral, but not by high concentrations of sodium azide, 4-methylpyrazole, or metyrapone. Similarly, oxidation of all-trans-retinal was inhibited strongly by citral but not by metyrapone.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Acyclic Monoterpenes; Animals; Biotransformation; Embryo, Mammalian; Embryonic and Fetal Development; Enzyme Inhibitors; Gestational Age; In Vitro Techniques; Metyrapone; Monoterpenes; NAD; NADP; Rats; Rats, Sprague-Dawley; Rats, Wistar; Retinaldehyde; Terpenes; Tretinoin; Vitamin A

Endogenous retinoids are present in the embryonic mouse mandible and reach a concentration peak immediately before the formation of the dental lamina. Because exogenous retinoids alter the pattern of the dental lamina and the expression of epidermal growth factor mRNA (a transcript necessary for initiation of odontogenesis), the role of retinoic acid in the initiation of odontogenesis was studied here. Citral (3,7-dimethyl-2,6-octadienal), a known inhibitor of retinoic acid synthesis, was used to block the endogenous synthesis of retinoic acid in the mouse embryonic mandible before the formation of the dental lamina (gestational day 9). A 24-h exposure to citral totally blocked tooth formation in 7/10 mandibles. Reductions of endogenous retinoic acid concentrations were confirmed by high-performance liquid chromatography. Tooth formation was restored by simultaneous treatment with all-trans retinoic acid or 9-cis retinoic acid during the citral exposures (first 24 h of culture). Endogenous retinoic acid is necessary for the initiation of odontogenesis.

Topics: Acyclic Monoterpenes; Animals; Chromatography, High Pressure Liquid; Dental Arch; Mandible; Mice; Monoterpenes; Odontogenesis; RNA, Messenger; Terpenes; Tooth Germ; Tretinoin; Vitamin A

Vitamin A (retinol) may play an important role in lung maturation: 1) premature delivery is simultaneously a source of vitamin A deficiency and increased risk of neonatal respiratory distress syndrome and subsequent bronchopulmonary dysplasia (BPD), due to deficit in pulmonary surfactant; 2) neonatal supplementation with retinol reduces the risk of BPD; and 3) fetal rat lung stores retinol in late gestation just before the onset of surfactant synthesis. To test the hypothesis of an implication of retinoids in the control of pulmonary surfactant synthesis, experiments were designed in the pregnant rat, aiming either at enhancing fetal lung vitamin A stores, bringing the active metabolite of vitamin A, retinoic acid (RA), or inhibiting the conversion of retinol to RA with aid of citral. Maternal administration of a single dose of 50,000 IU of retinyl palmitate on day 16 (term 22 days) increased 22 and 29%, respectively, the total phospholipid (TPL) and disaturated fraction of phosphatidylcholine (PC) in extracted fetal surfactant on day 19 but did not change surfactant protein (SP) A concentration. Chronic administration of retinyl palmitate to the mother from day 16 through 20 increased disaturated PC content on day 21 but decreased SP-A concentration. Fetal lung surfactant phospholipids were increased by chronic administration of RA and considerably reduced by citral (-31 and -35% for TPL and PC concns, respectively). RA also enhanced labeled choline incorporation into fetal lung PC on day 20. Given once on day 17, it accelerated the appearance of surfactant precursors on day 18.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetates; Acyclic Monoterpenes; Animals; Cells, Cultured; Choline; Female; Fetus; Lipid Metabolism; Lung; Maternal-Fetal Exchange; Monoterpenes; Phosphatidylcholines; Phospholipids; Pregnancy; Pulmonary Surfactants; Rats; Rats, Wistar; Retinoids; Terpenes; Tretinoin; Vitamin A

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 hypothesis that retinoic acid (RA) is produced from the excentric cleavage of beta-carotene was tested in human intestinal homogenates in vitro. Significant amounts of RA were identified by HPLC and derivatization after incubation of intestinal mucosal homogenates with retinal, beta-carotene, or beta-apocarotenals at 37 degrees C for 60 min. RA formation was inhibited, in a dose-dependent fashion, when retinal was incubated in the presence of 0.1-3.0 mM citral (3,7-dimethyl-2,6-octadienal) under identical experimental conditions. The formation of RA from both beta-carotene and beta-apocarotenals was dose and time dependent and RA was the major metabolite of both beta-apo-8'-carotenal and beta-apo-12'-carotenal after the incubation. However, citral (0.1 to 4 mM) did not inhibit the formation of beta-apocarotenals and RA from 2 microM beta-carotene (P greater than 0.05), which proves the existence of an excentric cleavage mechanism for beta-carotene conversion into retinoids. Furthermore, RA formation from both beta-apo-8'-carotenal and beta-apo-12'-carotenal in human intestinal homogenate occurred in the presence of citral, which demonstrates that RA can be produced from excentric cleavage of beta-carotene via a series of beta-apocarotenals as intermediates.

Topics: Acyclic Monoterpenes; beta Carotene; Binding, Competitive; Carotenoids; Humans; Hydrolysis; In Vitro Techniques; Intestinal Mucosa; Monoterpenes; Terpenes; Tretinoin

The food and fragrance additive citral (3,7-dimethyl-2,6-octadienal) inhibits the oxidation of retinol to retinoic acid in mouse epidermis on local application. This inhibitory property was used to test the hypothesis that oxidation to retinoic acid is rate limiting for the biological activity of vitamin A (retinol) in epithelial tissues. Citral was tested as a modulator of the biological activities of retinol and retinoic acid using two bioassays performed in Skh/hr1 (hairless) mice: (a) the ability to induce epidermal hyperplasia; (b) the ability to inhibit the induction of epidermal ornithine decarboxylase activity by tumor promoters. Citral treatment inhibited the ability of retinol, but not of retinoic acid, to induce epidermal hyperplasia. Similarly, citral treatment decreased the ability of retinol, but not of retinoic acid, to inhibit the induction of epidermal ornithine decarboxylase activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Although citral had little effect on epidermal ornithine decarboxylase activity when applied alone, it potentiated the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate. The ability of citral to inhibit retinoic acid formation from retinol and the specificity of citral for inhibition of the biological activities of retinol but not retinoic acid are evidence that oxidation to retinoic acid is obligatory for the measured biological activities of retinol. Furthermore, the ability of citral to potentiate the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate suggests that modulation of the retinol oxidation pathway by such agents may enhance susceptibility to tumor promoters.

Topics: Acyclic Monoterpenes; Animals; Epidermis; Hyperplasia; Mice; Mice, Hairless; Monoterpenes; Ornithine Decarboxylase Inhibitors; Oxidation-Reduction; Terpenes; Tetradecanoylphorbol Acetate; Tretinoin; Vitamin A

Locally applied retinol is metabolized to retinoic acid in mouse epidermis in vivo. To characterize the oxidation system we investigated the ability of soluble extracts of hairless-mouse epidermis to convert retinol and retinal into retinoic acid. The extracts oxidized retinol to retinoic acid in two steps catalysed by two NAD+-dependent enzymes that were resolved on h.p.l.c. The first enzyme catalyses the reversible oxidation of retinol to retinal and is an alcohol dehydrogenase isoenzyme. The second enzyme oxidizes retinal to retinoic acid. Retinol oxidation by epidermal extracts was inhibited by the alcohol dehydrogenase inhibitor 4-methylpyrazole and by the polyene citral. The toxicity and relatively low potency at inhibiting the epidermal alcohol dehydrogenase isoenzyme curtailed the use of 4-methylpyrazole in vivo. However, citral significantly inhibited retinoic acid formation from retinol in the epidermis in vivo. The ability to inhibit the oxidation of retinol to retinoic acid in mouse epidermis provides a potential method to resolve the roles of retinol and retinoic acid in epithelial function.

Topics: Acyclic Monoterpenes; Alcohol Dehydrogenase; Animals; Chromatography, High Pressure Liquid; Epidermis; Fomepizole; In Vitro Techniques; Mice; Monoterpenes; Oxidation-Reduction; Pyrazoles; Terpenes; Tretinoin; Vitamin A