4-hydroxyretinoic-acid and 4-oxoretinoic-acid

Retinoid signaling is essential for development of vertebrate embryos, and its action is mainly through retinoic acid (RA) binding to its RA receptors and retinoid-X receptors, while the critical concentration and localization of RA in embryos are determined by the presence and activity of retinal dehydrogenases (for RA synthesis) and cytochrome P450 RAs (Cyp26s) (for degradation of RA). Previously, we identified a novel cyp26 gene (cyp26d1) in zebrafish that is expressed in hindbrain during early development. Using reverse-phase HPLC analyses, we show here that zebrafish Cyp26D1 expressed in 293T cells could metabolize all-trans RA, 9-cis RA, and 13-cis RA, but could not metabolize retinol or retinal. The metabolites of all-trans RA produced by Cyp26D1 were the same as that produced by Cyp26A1, which are mainly 4-hydroxy-all-trans-RA and 4-oxo-all-trans-RA. Performing mRNA microinjection into zebrafish embryos, we demonstrated that overexpression of Cyp26D1 in embryos not only caused the distance between rhombomere 5 and the first somite of the injected embryos to be shorter than control embryos but also resulted in left-right asymmetry of somitogenesis in the injected embryos. These alterations were similar to those caused by the overexpression of cyp26a1 in zebrafish embryos and to that which resulted from treating embryos with 1 microm 4-diethylamino-benzaldehyde (retinal dehydrogenase inhibitor), implying that cyp26d1 can antagonize RA activity in vivo. Together, our in vitro and in vivo results provided direct evidence that zebrafish Cyp26D1 is involved in RA metabolism.

Topics: Amino Acid Sequence; Animals; Conserved Sequence; Cytochrome P-450 Enzyme System; Embryo, Nonmammalian; Molecular Sequence Data; Protein Structure, Tertiary; Retinaldehyde; Retinoic Acid 4-Hydroxylase; Sequence Homology, Amino Acid; Substrate Specificity; Tissue Distribution; Tretinoin; Vitamin A; Zebrafish Proteins

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Topics: Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Granulocytes; Humans; Integrin alphaXbeta2; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Time Factors; Tretinoin; Tumor Cells, Cultured

Significant differences between the metabolism of retinoic acid by different tissues might be an important determinant of the effectiveness of a systemically administered inhibitor at a particular tissue site. Here the metabolism of retinoic acid has been studied in microsomal fractions from different tissues (liver, kidney, intestinal mucosa, lung, skin, brain) of the male rat to determine their relative metabolic activity. Kinetic analysis revealed major differences between the activity of different tissue microsomes. This is shown by the Vmax values for the metabolism of retinoic acid-liver (102+/-39.0 pmol (mg protein)(-1) min(-1)) was 100 times more active than the lung (1+/-0.03 pmol (mg protein)(-1) min(-1)), which was the least active. The range of Km values for microsomes from the different tissues was narrow (0.48-1.40 microM). Taking into account the mass of the tissue, the gross activity ranking for metabolism of retinoic acid was liver >> skin = kidney > brain > intestinal mucosa >> lung. It is concluded that metabolism of administered retinoic acid occurs mainly in the liver but that cellular retinoic acid levels in some other tissues (skin, kidney, brain) could be reduced (metabolized) to such an extent that higher levels might be observed after the use of inhibitors of retinoic acid metabolism.

Topics: Animals; Antineoplastic Agents; Brain; Intestinal Mucosa; Kidney; Liver; Lung; Male; Microsomes; Rats; Rats, Wistar; Skin; Tretinoin

Topical all-trans retinoic acid (RA) modulates growth and differentiation of skin and is used in the treatment of various dermatological disorders. RA is metabolized to 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, which are believed to be markedly less active than RA. 3,4-didehydroretinoic acid (ddRA) is a metabolite of 3,4-didehydroretinol which is present in skin. ddRA is biologically active and acts as a morphogen. We have determined the relative biological activity of ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA as assessed by three retinoid responsive systems relevant to skin. RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA (10-100 nM) reduced epidermal transglutaminase activity in human keratinocytes to similar extents, and inhibited alpha-melanocyte-stimulating hormone-isobutylmethylxanthine-inducible tyrosinase activity in Cloudman S-91 mouse melanoma cells by 67, 39, 48, 51 and 19%, respectively, at 100 nM. Daily topical application of the retinoids to hairless mouse skin for 4 days resulted in dose-dependent changes in epidermal thickness and global histological score. The relative potencies of RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, as calculated by parallel line assay, were 1.0, 0.60, 0.34, 0.29 and 0.18, respectively, for epidermal hyperplasia and 1.0, 0.78, 0.23, 0.14 and 0.08, respectively, for global histological score. Interestingly, the compounds exhibited a similar rank order of potency with respect to induction of cellular retinoic acid binding protein-II mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Animals; Carrier Proteins; Cells, Cultured; Epidermis; Humans; Keratinocytes; Male; Melanoma, Experimental; Mice; Mice, Hairless; Monophenol Monooxygenase; Receptors, Retinoic Acid; RNA, Messenger; Skin; Transglutaminases; Tretinoin; Tumor Cells, Cultured

Incubation of [3H]retinoic acid in the presence of hamster liver 10000g supernatant produces several metabolites that are more polar than the parent compound. Two of these metabolites are identical with synthetic all-trans-4-hydroxyretinoic acid and all-trans-4-oxoretinoic acid both in ultraviolet absorption and mass spectral characteristics and in migration rates on two different reverse-phase high-pressure liquid chromatographic systems. The metabolites produced in a cell-free liver incubation reaction also migrate on a high-pressure liquid chromatography column together with metabolites isolated from a tracheal organ culture system. Both the metabolites and the synthetic standards show less biological activity than the parent all-trans-retinoic acid in a tracheal organ culture assay.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Cricetinae; Female; Liver; Male; Mass Spectrometry; Spectrophotometry, Ultraviolet; Trachea; Tretinoin; Vitamin A; Vitamin A Deficiency