retinaldehyde and citral

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

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

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

The involvement of a series of microsomal cytochrome P450 (P450) isozymes in all-trans-retinoid metabolism, including the conversion of all-trans-retinal to all-trans-retinoic acid, was previously described. In the current study, we examined the role of seven liver microsomal P450 isozymes in the oxidation of three isomers of retinal. P450 1A1, which was not tested previously, is by far the most active in the conversion of all-trans-, 9-cis-, and 13-cis-retinal to the corresponding acids, as well as in the 4-hydroxylation of all-trans- and 13-cis retinal. In contrast, P450s 2B4 and 2C3 are the most active in the 4-hydroxylation of 9-cis-retinal, with turnover numbers approximately 7 times as great as that of P450 1A1. The inclusion of cytochrome b5 in the reconstituted enzyme system is without effect or inhibitory in most cases but stimulates the 4-hydroxylation of 9-cis-retinal by P450 2B4, giving a turnover of 3.7 nmol of product/min/nmol of this isozyme, the highest for any of the retinoid conversions we have studied. Evidence was obtained for two additional catalytic reactions not previously attributed to P450 oxygenases: the oxidation of all-trans- and 9-cis-retinal to the corresponding 4-oxo derivatives by isoform 1A2, and the oxidative cleavage of the acetyl ester of vitamin A (retinyl acetate) to all-trans-retinal, also by isoform 1A2. The physiological significance of the latter reaction, with a Km for the ester of 32 microM and a Vmax of 18 pmol/min/nmol of P450, remains to be established. We also examined the effect on P450 of citral, a terpenoid alpha, beta-unsaturated aldehyde and a known inhibitor of cytosolic retinoid dehydrogenases. Evidence was obtained that citral is an effective mechanism-based inactivator of isozyme 2B4, with a KI of 44 microM as determined by the oxidation of 1-phenylethanol to acetophenone, and by isozyme 1A2 in the oxidation of all-trans-retinal to the corresponding acid and by isozyme 2B4 in the 4-hydroxylation of all-trans-retinol and retinoic acid. Thus, citral is not suitable for use in attempts to distinguish between retinoid conversions catalyzed by dehydrogenases in the cytoplasm and by P450 cytochromes in the endoplasmic reticulum.

Topics: Acyclic Monoterpenes; Animals; Benzyl Alcohols; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Hydroxylation; Isoenzymes; Isomerism; Kinetics; Liver; Microsomes; Monoterpenes; Oxidation-Reduction; Rabbits; Retinaldehyde; Retinoids; Terpenes

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