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Viaminate is a derivative of retinoic acid (tretinoin) used in the treatment of severe acne and other disorders of keratinization. Liver dysfunction due to viaminate therapy has not been well described and there have been no reported deaths due to viaminate-induced hepatotoxicity. Herein, we report the case of a woman who underwent treatment at the hospital for 38 days and died of hepatic failure after taking viaminate for more than 2 months. This case provides further insight into viaminate-induced hepatotoxicity., (Copyright © 2018 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Removal of cholesterol from macrophage-derived foam cells is a critical step to the prevention of atherosclerotic lesions. We have recently demonstrated the functional importance of retinoids in the regulation of the steroidogenic acute regulatory (StAR) protein that predominantly mediates the intramitochondrial transport of cholesterol in target tissues. In the present study, treatment of mouse macrophages with retinoids, particularly all-trans retinoic acid (atRA) and 9-cis RA, resulted in increases in cholesterol efflux to apolipoprotein AI (Apo-A1). Activation of the PKA pathway by a cAMP analog, (Bu)2cAMP, markedly augmented retinoid mediated cholesterol efflux. Macrophages overexpressing hormone-sensitive lipase increased the hydrolysis of cholesteryl esters and concomitantly enhanced the efficacy of retinoic acid receptor and liver X receptor (LXR) ligands on StAR and ATP-binding cassette transporter A1 (ABCA1) protein levels. RAs elevated StAR promoter activity in macrophages, and an increase in StAR levels augmented cholesterol efflux to Apo-A1, suggesting retinoid-mediated efflux of cholesterol involves enhanced oxysterol production. Further studies revealed that retinoids activate the LXR regulated genes, sterol receptor-element binding protein-1c and ABCA1. These findings provide insights into the regulatory events in which retinoid signaling effectively enhances macrophage cholesterol efflux and indicate that retinoid therapy may have important implications in limiting and/or regressing atherosclerotic cardiovascular disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Targeting tumor-specific metabolic adaptations is a promising anticancer strategy when tumor defense mechanisms are restrained. Here, we show that redox-modulating drugs including the retinoid N-(4-hydroxyphenyl)retinamide (4HPR), the synthetic triterpenoid bardoxolone (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester), arsenic trioxide (As2O3), and phenylethyl isothiocyanate (PEITC), while affecting tumor cell viability, induce sustained Ser9 phosphorylation of the multifunctional kinase glycogen synthase kinase 3β (GSK3β). The antioxidant N-acetylcysteine decreased GSK3β phosphorylation and poly(ADP-ribose) polymerase cleavage induced by 4HPR, As2O3, and PEITC, implicating oxidative stress in these effects. GSK3β phosphorylation was associated with up-regulation of antioxidant enzymes, in particular heme oxygenase-1 (HO-1), and transient elevation of intracellular glutathione (GSH) in cells surviving acute stress, before occurrence of irreversible damage and death. Genetic inactivation of GSK3β or transfection with the non-phosphorylatable GSK3β-S9A mutant inhibited HO-1 induction under redox stress, while tumor cells resistant to 4HPR exhibited increased GSK3β phosphorylation, HO-1 expression, and GSH levels. The above-listed findings are consistent with a role for sustained GSK3β phosphorylation in a signaling network activating antioxidant effector mechanisms during oxidoreductive stress. These data underlie the importance of combination regimens of antitumor redox drugs with inhibitors of survival signaling to improve control of tumor development and progression and overcome chemoresistance., (Copyright © 2014. Published by Elsevier Inc.)

Unusual amounts of retinoic acid (RA) isomers play an important role in abnormal morphological development of mammals; such as rat embryos. Each isomer of RA has a unique function in first steps of embryonic life. In the current study, a new method for preconcentration and simultaneous determination of all-trans retinoic acid, 13-cis retinoic acid, 9-cis retinoic acid and 9,13-di-cis retinoic acid in rat whole rudimentary embryo culture (RWEC) has been developed. RA isomers were extracted from samples by conjugation to appropriate amount of surface modified CdSe quantum dots (QDs) prior to HPLC/UV determination. In order to quickly release of the analytes with unchanged form, separated RA-QD conjugation were irradiated by intensive near infrared wavelength (NIR). Low energy NIR irradiation results in maintaining the primary forms of RA isomers during the release. The conjugation and release mechanisms were described and experimental parameters were investigated in detail. Under optimized conditions, the method was linear in the range of 0.040-34.600 pmol g(-1) for all-trans RA (R(2)=0.9996), 0.070-34.200 pmol g(-1) for 13-cis RA (R(2)=0.9992), 0.050-35.300 pmol g(-1) for 9,13-di-cis RA (R(2)=0.9998) and 0.050-32.900 pmol g(-1) for 9-cis RA (R(2)=0.9990). The present method can be useful for retinoic acid monitoring in clinical studies., (© 2013 Published by Elsevier B.V.)

Vitamin A is essential for multiple functions in mammals. Without vitamin A, mammals cannot grow, reproduce, or fight off disease. Because of its numerous functions in humans, biomarkers of vitamin A status are quite diverse. Assessment of liver reserves of vitamin A is considered the gold standard because the liver is the major storage organ. However, this measure is not feasible in human studies. Alternative biomarkers of status can be classified as biological, functional, histologic, and biochemical. Historically, signs of xerophthalmia were used to determine vitamin A deficiency. Before overt clinical damage to the eye, individuals who suffer from vitamin A deficiency are plagued by night blindness and longer vision-restoration times. These types of assessments require large population-based evaluations. Therefore, surrogate biochemical measures of vitamin A status, as defined by liver reserves, have been developed. Serum retinol concentrations are a common method used to evaluate vitamin A deficiency. Serum retinol concentrations are homeostatically controlled until liver reserves are dangerously low. Therefore, other biochemical methods that respond to liver reserves in the marginal category were developed. These included dose-response tests and isotope dilution assays. Dose-response tests work on the principle that apo-retinol-binding protein builds up in the liver as liver reserves become depleted. A challenge dose of vitamin A binds to this protein, and serum concentrations increase within a few hours if liver vitamin A concentrations are low. Isotope dilution assays use stable isotopes as tracers of total body reserves of vitamin A and evaluate a wide range of liver reserves. Resources available and study objectives often dictate the choice of a biomarker.

We show here the role of retinoic acid receptor (RAR) beta and alpha signalling in proliferation and differentiation of endogenous adult forebrain neural progenitor cells (NPCs). RARbeta activation stimulates Sonic hedgehog signalling (Shh), and induces the proliferation of the NPCs. They can be induced to become Doublecortin (DCX) expressing migrating neuroblasts by RARalpha signalling, some of which differentiate into cholinergic neurons. The same signalling pathways cause the proliferation of embryonic forebrain NPCs. These cells express glial fibrillary acidic protein (GFAP) and are predominantly uni/bipolar, two characteristics of neuronal progenitor cells. We further show that fibroblast growth factor (FGF) signalling, induces the expression of the retinoic acid degrading enzyme cytochrome P450 (cyp) 26a1, and that one of its products, 4-oxo-RA, mimics the action of the RARalpha agonist in the differentiation of the NPCs into cholinergic neurons.

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.

Polyprenyl phosphates, as well as polyprenyl alcohols bearing different isopentenyl C(5) units, have been synthesized. The pH range of spontaneous vesicle formation of polyprenyl phosphates with or without polyprenyl alcohols was defined by fluorescence microscopy. A variety of the acyclic or monocyclic polyprenyl phosphates studied formed stable vesicles in water over a wide range of pHs, and the addition of polyprenyl alcohols allowed the vesicle formation of polyprenyl phosphates at higher pHs. Osmotic swelling of a suspension of unilamellar vesicles using the stopped-flow/light-scattering method enabled us to evaluate the water permeability of polyprenyl phosphate vesicles with or without 10 mol% of free polyprenyl alcohol. The addition of many polyprenyl alcohols to polyprenyl phosphate vesicles decreased the water permeability, and some reduced it even more efficiently than cholesterol.

Retinoic acid exerts a variety of effects on gene transcription that regulate growth, differentiation, and inflammation in normal and neoplastic skin cells. Because there is a lack of information regarding the influence of metabolic transformation of retinoids on their pharmacologic effects in skin, we have analyzed the functional activity of all-trans-, 9-cis-, and 13-cis-retinoic acid and their 4-oxo-metabolites in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts using gene and protein expression profiling techniques, including cDNA microarrays, two-dimensional gel electrophoresis, and MALDI-MS. It was previously thought that the 4-oxo-metabolites of RA are inert catabolic end-products but our results indicate instead that they display strong and isomer-specific transcriptional regulatory activity in both NHEKs and dermal fibroblasts. Microarray and proteomic analyses identified a number of novel genes/gene products that are influenced by RA treatment of NHEKs or fibroblasts, including genes for enzymes catalyzing biotransformation of retinoids, corticosteroids, and antioxidants and structural and transport proteins known to be essential for homeostasis. Our results expand current knowledge regarding retinoic acid action within skin cells and the target tissue/cell regulatory systems that are important for modulating the physiological and pharmacological effects of this important class of dermatological drugs.

A simple reversed phase high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA), and other retinoids in rat plasma. VN/14-1RA, alone or in combination with ATRA, is effective at inhibiting the proliferation of prostate and breast cancer cell lines in vitro. Aliquots of rat plasma were spiked with the retinoids followed by addition of acetonitrile for precipitation of plasma proteins. The decanted supernatant was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Analysis was accomplished by injection of an aliquot of the reconstituted sample into an HPLC system consisting of a Zorbax Rx-C18 column and a diode array detector. A mobile phase composed of ammonium acetate (0.1 M), acetic acid solution (2% (v/v)) and methanol at a flow rate of 1.0 mL/min was used for gradient elution. The recoveries for all compounds ranged from 65 to 85% regardless of the concentrations examined. The HPLC assay was linear over the range 0.10-5.0 microg/mL (CV < 10%) with a limit of quantification of 100 ng/mL for VN/14-1RA. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for VN/14-1RA after intravenous administration. The mean terminal elimination half-life (t(1/2)) was 19.0 +/- 3.2 min. This HPLC method is useful for the analysis and evaluation of the pharmacokinetics of VN/14-1RA in rats.

A simple and accurate high-performance liquid chromatography (HPLC) method was developed to measure levels of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its main metabolite N-(4-methoxyphenyl)retinamide (4-MPR) in tissue. Following ultrasonic extraction of fresh tissue in acetonitrile (ACN), 4-HPR and 4-MPR were measured by HPLC with UV absorbance detection at 340 nm, using isocratic elution with ACN, H(2)O, and acetic acid. N-(4-ethoxyphenyl)retinamide (4-EPR) was employed as an internal standard. The 4-HPR and 4-MPR recovery in bovine liver or bovine brain tissue samples spiked with known amounts of 4-HPR and 4-MPR ranged from 93 to 110%. The detection limit of the method was 50 ng/ml. The method was tested on actual samples from an athymic (nu/nu) mouse carrying a subcutaneous tumor xenograft originating from SMS-KCNR neuroblastoma cells. The tissues were harvested and analyzed following a 3 day long treatment with intraperitoneal injections of 4-HPR/Diluent-12. 4-HPR and the metabolite 4-MPR were detected and quantitated in the tested tissues including tumor, liver, and brain. This method can be used to quantify 4-HPR and 4-MPR in different tissues to determine the bioavailability of 4-HPR.

Numerous reports have indicated that the biological activity of all-trans retinoyl beta-glucuronide (RAG) is similar to that of all-trans-retinoic acid (RA), but without the toxic side effects of RA. In the present series of studies, we report new findings that support the contention that RAG can function as a nontoxic substitute for RA in a variety of clinic settings. One study on the effects of s.c. injected graded doses of RA and RAG (20-480 micromol/kg BW) into pregnant Sprague-Dawley rats showed that any differences between RAG and RA could be observed only at the highest dose levels of 360 and 420 micromol/kg BW, with RAG being much less toxic than RA. Similarly, daily topical application of RAG (0.16-1.6%) and RA (0.1-0.5%) to shaved swine dorsal skin for six mo resulted in redness and scabbing in RA-treated patches, and to a lesser extent in 1.6% RAG-treated, but not in other RAG-treated patches. Histological scores were significantly higher in the dermis and epidermis of RA-treated pigs than in RAG-treated pigs. Studies to document the pharmacokinetics of chronically administered RAG in mice indicated that, unlike RA, sustained blood levels of parent retinoid (RAG) can be achieved during at least 2 mo of daily administration. Another investigation to study the effects of RAG on the development and growth in nude mice of tumors derived from the human neuroblastoma cell line LA-N-5 showed that s.c. injection of RAG (30 micromol/kg BW) reduced tumor formation when the retinoid was first administered 3 d before tumor injection and continued daily for 30 d thereafter. In established tumors, RAG was shown to inhibit progressive tumor growth, the antitumor effects of RAG being comparable with RA. However, with RAG, as opposed to RA, there were no significant adverse physical side effects. Based on the results of these series of studies along with ample published reports over the last 15 y, we conclude that RAG may be a safe and effective alternative to RA and some other retinoids that are presently being utilized in the clinic.

The retinamide, N-(4-hydroxyphenyl)retinamide (4-HPR), has shown promising anti-tumor activity, but it is unclear whether this compound is hydrolyzed to all-trans retinoic acid (atRA) and if so, whether this plays any role in its chemotherapeutic activity. To address this issue, the ability of 4-hydroxybenzylretinone (4-HBR), a carbon-linked analog of 4-HPR, to support growth in vitamin A-deficient (VAD) animals and to activate an atRA-responsive gene in vivo was compared to 4-HPR and atRA. Further, the non-hydrolyzable 4-HBR analog was used to determine whether the presence of the labile amide linkage in 4-HPR is essential for the induction of apoptosis in cultured MCF-7 breast cancer cells. Studies in VAD rats showed that 4-HPR, like atRA, supports animal growth and induces CYP26B1 mRNA expression in lung whereas 4-HBR does not. Analysis of plasma from 4-HPR- and atRA-treated VAD animals revealed the presence of atRA whereas it was not detected in plasma from animals given 4-HBR. To determine whether hydrolysis to atRA is necessary for apoptosis induced by 4-HPR in MCF-7 breast cancer cells, morphological and biochemical assays for apoptosis were performed. 4-HBR, like 4-HPR, induced apoptosis in MCF-7 cells. Apoptosis was not induced even at high concentrations of atRA, showing that 4-HPR and 4-HBR act in cells via a distinct signaling pathway. These results show that although limited hydrolysis of 4-HPR occurs in vivo, the ability to liberate atRA is not required for these 4-hydroxyphenyl retinoids to induce apoptosis in MCF-7 breast cancer cells. Thus the non-hydrolyzable analog, 4-HBR, may have significant therapeutic advantage over 4-HPR because it does not liberate atRA that can contribute to the adverse side effects of drug administration in vivo.

The cellular retinoic acid (RA) binding proteins I and II (CRABPI and CRABPII), intracellular proteins which bind retinoic acid with high affinity, are involved in the actions of RA, though their exact roles are not fully understood. We have generated several genetically engineered AB1 cell lines in which both alleles of the CRABPI gene have been deleted by homologous recombination. We have used these CRABPI knockout cell lines to examine the consequences of functional loss of CRABPI on RA-induced gene expression and RA metabolism in the murine embryonic stem cell line, AB1, which undergoes differentiation in response to RA. Complete lack of CRABPI results in decreased intracellular [3H]RA concentrations under conditions in which external concentrations of [3H]RA are low (1-10nM) and in an altered distribution of [3H] polar metabolites of [3H]RA in the cell and in the medium. Fewer [3H] polar metabolites are retained within the CRABPI(-/-) cells compared to the wild-type cells. These data suggest that CRABPI functions to regulate the intracellular concentrations of retinoic acid and to maintain high levels of oxidized retinoic acid metabolites such as 4-oxoretinoic acid within cells.

Background/aims: Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level., Methods: Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining., Results: Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis., Conclusions: AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

All-trans-retinoyl-beta-D-glucuronide (RAG) is an endogenous active metabolite of all-trans-retinoic acid (ATRA). In the present study, the pharmacokinetics of RAG was examined after the administration of a single intravenous does (5, 10, or 15 micromol/kg) and of multiple daily intravenous doses (5 micromol/kg) to rats for 8 days. The plasma concentrations of RAG and ATRA were measured by a reverse-phase HPLC method. A rapid distribution phase of approximately 1 h was observed in all of the rats after single or multiple doses. Thereafter, RAG was eliminated through a first-order process, in accord with a typical two-compartment first order pharmacokinetic profile. After single intravenous doses, the AUC of RAG increased proportionally with the dose and the clearance remained unchanged within the tested doses. There was no statistical significant difference in distribution rate constants from central compartment to peripheral compartment (K(12)) and from peripheral compartment to central compartment (K(21)) between different doses. However, as the dose increased from 5 micromol/kg to 10 micromol/kg, the volume of distribution at the steady state (V(ss)) and the volume of peripheral compartment (V(p)) decreased significantly (p < 0.05) from 1.290 +/- 0.269, 0.928 +/- 0.232. L/kg to 0.961 +/- 0.149, 0.647 +/- 0.107 L/kg, respectively. V(ss) and V(p) at a dose of 15 micromol/kg (0.924 +/- 0.187, 0.698 +/- 0.165 L/kg) were not significantly different from that at 10 micromol/kg. Thus, RAG might saturate the tissue-binding sites at higher doses. ATRA was detected as a metabolite of RAG at low levels (usually < 0.05 microM) only in the first 2 h after intravenous administration. RAG clearly was not extensively hydrolyzed to ATRA in our study. After multiple daily intravenous administration of RAG, the clearance (Cl) and the elimination rate constant (K(10)) remained unchanged (p > 0.05), indicating that long-term daily administration of RAG did not induce its accelerated metabolism. However, K(12), V(p), and V(ss) declined significantly (p < 0.05) from 1.67 +/- 0.54 h(-1), 0.928 +/- 0.232 L/kg, and 1.290 +/- 0.269 L/kg to 0.96 +/- 0.48 h(-1), 0.494 +/- 0.147 L/kg, and 0.818 +/- 0.187 L/kg, respectively. Therefore, long-term daily dosing of RAG seemed to decrease its distribution profile. Although the AUC of RAG did not change significantly after multiple dosing, the AUC of ATRA after RAG dosing significantly declined (p < 0.05) from 0.032 +/- 0.019 microM x h to 0.010 +/- 0.006 microM x h. The decline in the AUC of ATRA might reflect an increase in its uptake by tissue and/or in its metabolism. Because enhanced clearance is not associated with RAG after multiple administrations, RAG could be considered as an alternate to ATRA in appropriate clinical applications., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association)

Folate receptor (FR) type beta is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-beta expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-beta expression in vitro in myeloid leukemia cells from patient marrow. FR-beta was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D(3), or transforming growth factor beta. ATRA did not induce FR-beta expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-beta expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-beta promoter-luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-beta promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)alpha, RARbeta, or RARgamma, it appears that the retinoid effect on FR-beta expression could be mediated by ligand binding to RARs alpha, beta, or gamma, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARalpha and RARgamma selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-beta expression. Thus, blocks in the RARalpha-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-beta expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-beta expression in myeloid leukemia cells refractory to retinoid differentiation therapy.

Retinoids are strong tissue modifiers and have been used to treat severe acne, keloids and photo-aged skin. Tocoretinate (TR), ester bound retinoic acid and tocopherol, has been topically applied for skin ulcers and, more recently, for sclerotic skin diseases. To clarify the mechanism of tissue softening by retinoids and TR, we investigated their effects on the contraction of hydrated type-1 collagen gel matrices by human dermal fibroblasts and on tenascin-C expression. TR, 13-cis-retinoic acid/isotretinoin and all trans-retinoic acid significantly inhibited collagen gel matrices contraction at concentrations from 10(-4) to 10(-8) M without significant changes of the fibroblast growth. TR and the other two retinoids dose-dependently induced tenascin-C expression in the fibroblasts. Since tenascin-C is involved in cellular detachment and tissue remodeling, these results suggest that TR and other retinoids down-regulated the tensile tension of fibroblasts in collagen gel matrices by the induction of tenascin-C.

Laser desorption ionization (LDI)- and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (LDI-MS, MALDI-MS) at 337-nm laser wavelength were used to analyze retinol (vitamin A), retinoic acid, and retinal and their analogs 3-hydroxyretinol, 3-hydroxyretinoic acid, 3-hydroxyretinal, 4-oxoretinol, 4-oxoretinoic acid, 4-oxoretinal, 3,4-didehydroretinol (vitamin A(2)), 3,4-didehydroretinoic acid, 3,4-didehydroretinal, acycloretinol, acycloretinoic acid, and acycloretinal. The compounds exhibit sufficient ionizability which allows to obtain mass spectra by LDI which are similar in quality to those obtained by MALDI. Mass spectra were recorded with a linear time-of-flight (TOF) instrument or a reflectron-type (RETOF) instrument in positive-ion mode. Under the conditions of LDI-MS the compounds form abundant radical molecular ions (M+*), whereas in the MALDI mass spectra abundant protonated molecular ions ([M + H]+) are observed. Characteristic fragment ions provide additional structural information. High-performance liquid chromatography (HPLC) coupled with UV/Vis photodiode detection was used to assist in retinoid characterization. Synthesis of 3-hydroxyretinal, 4-oxoretinal, and acycloretinal was performed by oxidative cleavage of the all-trans-carotenoids of zeaxanthin, canthaxanthin, and lycopene., (Copyright 1999 Academic Press.)

Background/aims: During hepatic fibrosis, hepatic stellate cells (HSCs) transform into myofibroblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Recently, we have demonstrated that 9,13-di-cis-retinoic acid (RA), a geometric isomer identified as a stable and major metabolite of vitamin A in circulation, stimulates the synthesis of plasminogen activator (PA) and induces PA/plasmin-dependent latent transforming growth factor (TGF)-beta activation in HSC cultures, probably via induction and activation of RA receptor (RAR) alpha. The aim of the present study was to address a potential link between the loss of retinyl esters to increased formation of RA(s), which might play a role in facilitating TGF-beta-mediated liver fibrogenesis in vivo., Methods: We examined the effect of 9,13-di-cis-RA on transactivating activity of RARalpha in HeLa cells as well as its effect on PA- and TGF-beta-dependent collagen synthesis in rat and human HSC cultures. We measured the changes in 9,13-di-cis-RA levels both during activation of rat HSCs in vitro and during porcine serum-induced rat hepatic fibrosis in vivo and correlated this with RAR alpha/beta, PA, TGF-beta and type I procollagen mRNA expression in the fibrotic liver., Results: 9,13-di-cis-RA transactivated RARalpha, and provoked PA/plasmin and TGF-beta-dependent procollagen synthesis in HSCs. 9,13-di-cis-RA levels were increased both in activated HSCs in vitro and in fibrotic liver accompanying the enhanced expression of RAR alpha/beta, PA, TGF-beta and procollagen in vivo., Conclusions: These findings suggest a potential link between 9,13-di-cis RA formation and hepatic fibrosis via formation of TGF-beta in vivo, and thus provide further insight into the biologic role of retinoids during hepatic fibrogenesis.

Retinol and retinyl esters are converted with time to slowly increasing amounts of all-trans retinoic acid (RA) in cultured human keratinocytes. Exogenous RA has been shown to limit retinol oxidation and to increase retinol esterification. Because significant amounts of retinol are present in biologic systems, we examined whether RA and all-trans-retinoyl-beta-D-glucuronide (RAG) interact with retinol in exhibiting their activities on HaCaT keratinocytes maintained in a retinoid-free culture system. RA was more potent than RAG and retinol in inducing ultrastructural changes attributed to retinoids, inhibiting cell proliferation as well as enhancing keratin 19 expression. In addition, retinoids were able to induce cellular retinoic acid-binding protein II mRNA levels in the cultures, whereas early RA and late RAG activity was detected. The described biologic effects of RA and RAG were diminished by simultaneous cell exposure to retinol. HaCaT cells quickly metabolized retinol to retinyl esters and consequently to low amounts of RA. RA treatment led to an early high peak of cellular RA followed by reduction to trace amounts. Treatment with RAG resulted in constantly high cellular RAG and low RA levels. Under the combined RA and retinol treatment retinyl esters were increased and RA was reduced in HaCaT cells, whereas extracellular RA levels were similar to those obtained by RA alone. On the other hand, the combination of RAG and retinol resulted in higher extracellular RAG, similar cellular RAG, and lower cellular RA levels than those obtained by RAG alone without any change in retinyl esters. This study demonstrates that retinoid signaling by RA and RAG is attenuated by simultaneous exposure of HaCaT keratinocytes in vitro to retinol. The presence of retinol in the medium alters the rate of RA or RAG metabolism and thus cellular RA concentrations. The intensity of retinoid signal is probably dependent on cellular RA levels. The resulting "antagonism" among retinoids is consistent with the presence of an auto-regulatory mechanism in human keratinocytes offering protection against excessive accumulation of cellular RA.

Keratinocytes differentiating in vitro exhibit greater cytosolic capacity for retinoic acid synthesis from retinol or retinaldehyde as compared to nondifferentiated cells (Siegenthaler et al. 1990. Biochem. J. 268: 371-378), and increased expression of CRABP-II (Siegenthaler et al. 1988. Exp. Cell Res. 178: 114-126). Based on these observations, the content and disposition of [3H]retinoic acids were determined in intact, nondifferentiated and differentiating keratinocytes incubated with [3H]retinaldehyde or [3H]retinol. Differentiating keratinocytes contained higher levels of [3H] retinoic acids compared to undifferentiated cells when either [3H]retinaldehyde or [3H]retinol was the substrate. The largest increases in [3H]retinoic acids were achieved with [3H]retinaldehyde. Differentiation-associated increases in [3H]retinoic acids correlated with cellular content of retinoid alcohol substrates in incubations with retinaldehyde but not in incubations with retinol. Consistent with previous observations, CRABP-II was significantly increased in differentiating cells. Moreover, newly synthesized [3H]retinoic acids were retained within cells bound to CRABP-II. The results suggest that increasing cellular concentration of retinoic acids in in vitro differentiating keratinocytes is achieved by a combination of increased activity of the retinoic acid synthesis pathway and increased cellular content of CRABP-II.

Acute promyelocytic leukemia (APL) has a specific genetic rearrangement between the retinoic acid receptor (RAR)-alpha gene and the pml nuclear protein gene. All-trans retinoic acid (ATRA) induces granulocytic differentiation of APL-derived cells and is used to treat APL patients. However, ATRA interacts with normal cells with RAR throughout the entire body, and when used at high doses or over a long duration, it induces several adverse effects. The development of drugs that selectively act on APL cells may contribute to increasing the therapeutic efficacy of APL treatment as well as elucidating the mechanisms of response to ATRA. In this study, 9-cis retinoic acid alpha-tocopherol ester (9CTT) inhibited the proliferation of APL-derived NB4 and HT93 cells and induced differentiation markers, such as granulocytic maturation, nitroblue tetrazolium reduction, and CD11b expression, in these cells. The effects of 9CTT on non-APL cells, including HL-60 and U937 cells, were much weaker than those on APL cells, and tretinoin tocoferil (TT), which is an alpha-tocopherol ester of ATRA, did not induce the differentiation of APL cells as effectively as 9CTT. The differentiation-inducing effects of 9CTT were inhibited by RAR antagonists. 9CTT and TT similarly induced the transactivating activity of RARs, but were not effective on RXRs. 9CTT downregulated the expression of PML/RAR-alpha protein more effectively than TT, which suggests that it may be involved in the selectivity of 9CTT against APL cells. Interestingly, 9CTT enhanced the differentiation of APL cells induced by ATRA, 9-cis retinoic acid, and synthetic retinobenzoic acids. Combined with 1alpha,25-dihydroxyvitamin D3 (VD3), 9CTT also more than additively induced the differentiation of APL cells. Thus, 9CTT, alone or in combination with other retinoids or VD3, may be useful for the treatment of APL.

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 microm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250x4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3-100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.

A fully automated isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, has been developed using on-line solid-phase extraction and a column switching technique allowing clean-up and pre-concentration in a single step. A 500-microliter sample of serum was diluted with 750 microliters of a solution containing 20% acetonitrile and the internal standard 9,10-dimethylanthracene. About 1000 microliters of this mixture was injected on a 20 x 4.6 mm I.D. poly ether ether ketone (PEEK) pre-column with titanium frits packed with Bondapak C18, 37-53 microns, 300 A particles. Proteins and very polar compounds were washed out to waste, from the pre-column, with 0.05% trifluoroacetic acid (TFA)-acetonitrile (8.5:1.5, v/v). More than 200 aliquots of diluted serum could be injected on this pre-column before elevated back-pressure enforces replacement. Components retained on the pre-column were backflushed to the analytical column for separation and detection at 360 nm. Baseline separation was achieved using a single 250 x 4.6 mm I.D. Suplex pKb-100 column and a mobile phase containing 69:10:2:16:3 (v/v) of acetonitrile-methanol-n-butanol-2% ammonium acetate-glacial acetic acid. A total time of analysis of less than 30 min, including sample preparation, was achieved. Recoveries were in the range of 79-86%. The limit of detection was 1-7 ng/ml serum and the precision, in the concentration range 20-1000 ng/ml, was between 1.3 and 4.5% for all five compounds. The method was applied for the analysis of human serum after oral administration of 60 mg Roaccutan. The method is well suited for pharmacological studies, while the endogenous levels of some retinoic acid isomers are below the limit of quantitation.

Homoretinoic and bishomoretinoic acid have been synthesized. Fast reaction under ultrasonic activation (Wolff rearrangement, saponification) produced compounds in high yields. Only the bishomo analogue exhibits a significative activity alone and a synergistic effect with vitamin D3 on the differentiation of U937 leukemic cell line.

The retinamide-mediated apoptosis of promyelocytic cells was investigated using pHi- and DNA-sensitive fluorescent probes (BCECF and Hoechst 33342). Acidification and apoptosis were observed during prolonged (0-12 h) exposure to retinamide (1 microM), but were absent in a retinamide-resistant clone. The analysis of experiments performed by simultaneous staining with the two dyes showed that acidification and apoptosis are correlated; the half-time for acidification is about 5 h while that for apoptosis is 80% longer. On the whole, these results suggest that apoptosis is preceded by an early mechanism of acidification in human leukemia cells treated by a retinoid of clinical interest in cancer chemo prevention and therapy.

Retinoids exert various functions including anti-proliferative and anti-inflammatory effects on many cell types including keratinocytes and are widely used in skin diseases, such as psoriasis and acne. We have previously shown that human keratinocytes express low affinity immunoglobulin E receptor (FcepsilonRII/CD23) when stimulated with interleukin-4. Immunoglobulin E ligates CD23 and induces the production of nitrites (reflecting the mobilization of the nitric oxide [NO]-pathway) and tumor necrosis factor-alpha by human keratinocytes. Here, 13-cis and all-trans retinoic acid (RA) were shown to reduce the production of nitrites by immunoglobulin E-activated keratinocytes by 80% in a time- and concentration-dependent fashion. As a consequence, RA derivatives also reduced the production of tumor necrosis factor alpha by these cells by 70%. The level of inducible NO synthase activity in activated human keratinocytes was significantly decreased upon treatment of the cells with RA derivatives (inhibition by 60% of the mean inducible NO synthase activity with 13-cis RA, 2 microM). Treatment for 24 h with RA derivatives almost completely abolished transcription of inducible NO synthase-specific mRNA in activated keratinocytes. Therefore, RA derivatives downregulate tumor necrosis factor-alpha release and the NO-transduction pathway through the inhibition of inducible NO synthase transcription. Together, our data provide evidence for inhibition of the NO-pathway by 13-cis and all-trans retinoic acid on CD23-activated human keratinocytes. These data may clarify the mechanism of the anti-inflammatory activity of RA derivatives in skin diseases.

Tretinoin tocoferil is an alpha-tocopherol ester of all-trans retinoic acid (RA) and safely used in the treatment of skin ulcer. Tretinoin tocoferil inhibited proliferation of human promyelocytic leukemia HL-60 cells and induced granulocytic differentiation of the cells, but less than RA. alpha-Tocopherol did not affect differentiation of HL-60 cells, but at high concentrations enhanced its nitroblue tetrazolium (NBT)-reducing activity and expression of surface antigen CD11b, which are markers of myelomonocytic differentiation induced by RA. Tretinoin tocoferil increased NBT reduction in HL-60 cells treated with RA. It also enhanced the differentiation of HL-60 cells induced by dimethyl sulfoxide, phorbol-12-myristate 13-acetate or 1alpha,25-dihydroxyvitamin D3 (VD3). In combination with a low concentration of VD3, it induced the NBT-reducing activity of human monoblastic U937 cells very effectively. Moreover, it enhanced the differentiation of human myelomonocytic ML-1, THP-1, P39/TSU, and P31/FUJ cells induced by VD3. In combination with VD3, it synergistically inhibited the proliferation of HL-60, U937, ML-1, THP-1, P39/TSU, and P31/FUJ cells and decreased the effective concentration of VD3 to a 10(-10) mol/L level. Because tretinoin tocoferil was reported to induce neither retinoid-related toxicity nor teratogenicity, the therapeutic advantage of the use of it in treatment of myelomonocytic leukemia is suggested.

Early events that initiate teratogenesis by Accutane or other retinoids in mammalian embryos remain unknown. It would be helpful for mechanistic considerations to know whether or not retinoids act through retinoid receptor-dependent pathways, and if they do, which of the two families of receptors (retinoic acid receptors - RARalpha, beta, gamma or retinoid X receptors - RXRalpha, beta, gamma) are more likely involved. We previously used an in vitro bioassay to demonstrate that those retinoid analogs with binding affinity and transactivational activity limited only to the RXRs have a low potential as teratogens. Here, we have extended the study to examine teratogenicity, in pregnant mice, of a number of synthetic retinoids with varying degrees of receptor selectivity. The ability of each compound to induce fetal limb and craniofacial defects after a single exposure on day 11 of gestation was assessed and compared to that of all-trans retinoic acid (RA). The highest dose selected was 100 mg/kg maternal body weight since such a regimen of all-trans RA affects virtually every exposed embryo without any indication of maternal toxicity. We found that although all RAR agonists were strong teratogens, their potencies varied over a wide magnitude. The teratogenic potencies and receptor transactivation profiles of RAR agonists were not directly correlated since compounds with similar receptor activities presented major differences in potencies. Three compounds were exclusively RXR agonists, and these were not teratogenic under our experimental conditions. Two additional compounds which turned out to be non-teratogenic were distinguished by the fact that they activated neither RARs nor RXRs. These data indicate that although RAR-dependent mechanisms are likely involved in retinoid-induced teratogenesis, there are additional factors which determine teratogenic potency. The absence of teratogenic response in the case of RXR agonists suggests that risk-benefit analyses of such receptor-selective compounds may be fruitful in further studies.

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.

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically, APL cells express PML-RAR, an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580, a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist, is a powerful inducer of granulocytic maturation in NB4, an APL-derived cell line, and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF), the compound induces granulocytic maturation, as assessed by determination of the levels of leukocyte alkaline phosphatase, CD11b, CD33, and G-CSF receptor mRNA, at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast, AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells, two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580, whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments, using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha, show that AM580 has a lower affinity than ATRA for both receptors. However, in the presence of PML-RAR, the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid, whereas, in the presence of RAR alpha, AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells.

Synthetic 4,5-didehydro GGA (geranylgeranoic acid), a potent ligand both for cellular retinoic acid-binding protein and for nuclear retinoid receptors, induced apoptosis in human hepatoma-derived cell line HuH-7 but not in primary hepatocytes, although all-trans or 9-cis retinoic acid did not induce any growth inhibition. Either exogenous transforming growth factor-alpha (TGF alpha) or epidermal growth factor(EGF) prevented the cells from apoptosis in the presence of 4,5-didehydro GGA, but hepatocyte growth factor, insulin-like growth factor-II, insulin or triiodothyronine was essentially inactive. 4,5-Didehydro GGA down-regulated the cellular levels of TGF alpha mRNA as early as 30 min after the treatment. Either anti-TGF alpha or anti-EGF receptor monoclonal antibody induced apoptosis in HuH-7 cells without using the acid. Taken together, the present study strongly suggests that 4,5-didehydro GGA induced apoptosis in HuH-7 cells through the destruction of autocrine loop consisting of TGF alpha and EGF receptor, due to the down regulation of TGF alpha gene expression.

Vitamin A is a well-established teratogen in several animal species. Case reports as well as a recent epidemiological study suggest that vitamin A intake in excess of 25,000 or 10,000 IU respectively, can result in retinoid-specific defects in the offspring. A single meal of liver contains, on the average, a 10- to 20-fold higher amount of vitamin A than what is already suspected to be teratogenic. To evaluate the risk of liver consumption during pregnancy, we have studied levels of vitamin A and a number of potentially active retinoid metabolites in plasma of ten healthy male volunteers following consumption of fried turkey liver (2 g raw weight/kg body weight). HPLC, UV spectroscopy and mass spectrometry were used for identification and quantitation of retinoids in plasma. As shown previously, vitamin A intake via liver consumption resulted in greatly increased plasma levels of 13-cis-retinoic acid (13-cis-RA) and 13-cis-4-oxo-RA, and low levels of all-trans-RA and all-trans-4-oxo-RA. In our present investigation 9-cis-RA, 9,13-di-cis-RA, and 14-hydroxy-4,14-retro-retinol (14-HRR) were identified for the first time in humans as physiological metabolites of vitamin A. 9-cis-RA is a potent teratogen as well as a high affinity ligand of retinoid receptors, and 14-HRR was previously shown to promote lymphocyte activation in vitro. The present study bears on the issue of a possible teratogenic risk of liver consumption, as active retinoids were identified in human plasma, and their levels could be related to previous human studies as well as to experimental studies in sensitive animal species.

The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes. An RAR alpha-selective ligand used with an RXR-selective ligand generated the same responses as did all-trans RA or 9-cis RA, which affect both families of receptors, suggesting an important role for RAR alpha among RAR subtypes in eliciting cellular response. Consistent with this finding, the RAR alpha antagonist, Ro 41-5253, reduced the level of the cellular responses elicited by treatment with an RAR alpha-selective ligand plus RXR-selective ligand. The coupling of the shift of RB to its hypophosphorylated form with G1/0 arrest and differentiation in response to ligands is consistent with a possible role of RB as a downstream target or effector of RAR alpha and RXR in combination.

Retinol metabolism was examined in cultured human epidermal keratinocytes incubated with medium containing physiological concentrations of bovine serum albumin-free fatty acid complexes and [3H]retinol. Unsaturated 16- and 18-carbon fatty acids: (1) promoted up to a 50% increase in the steady state total cell retinoid mass which was accounted for as retinyl ester corresponding to the added fatty acid, (2) decreased the rate of endogenous retinyl ester utilization up to 80%, (3) decreased the steady state cellular concentrations of retinol, 3,4-didehydroretinol and their respective carboxylic acids up to 80%, and (4) did not alter the rate of retinoic acid metabolism. Increasing the medium retinol concentration augmented the fatty acid-stimulated increase in esters and blunted the extent of decrease in alcohols and acids. Saturated fatty acids with 14, 16, 17, and 18 carbons also reduced the rate of retinyl ester utilization and the cellular concentrations of the retinoid alcohols and acids but had little to no effect on retinyl ester synthesis. The data demonstrate that exogenous fatty acids possess the capacity to regulate the cellular concentrations of substrate retinoid alcohols, active retinoid acids, and the overall rate of retinol metabolism via substrate-mediated stimulation of retinol esterification and decreasing retinyl ester utilization. Fatty acids thus possess the potential to play a physiological role in modulating retinoid signaling in keratinocytes by regulating cellular concentrations of active retinoids.

The activity of 4-oxoretinoic acid as an inducer of gap junctional communication was investigated in C3H/10T1/2 murine fibroblasts. Two isomers of this retinoid, all-trans- and 13-cis-4-oxoretinoic acid, enhance gap junctional communication. This is accompanied by increased expression of connexin43 mRNA. Decomposition fractions of canthaxanthin were isolated by preparative high-performance liquid chromatography and shown to be active in the cell-cell communication assay. Two of the decomposition compounds were identified as all-trans- and 13-cis-4-oxoretinoic acid. Therefore, it is concluded that the biological activity of canthaxanthin regarding cell-cell communication is at least in part due to the formation of active decomposition products such as 4-oxoretinoic acid.

HuH-7 cells, a human hepatoma-derived cell line, underwent apoptosis in response to all-trans 3, 7, 11, 15-tetramethyl- 2, 4, 6, 10, 14-hexadecapentaenoic acid, or acyclic retinoid. The retinoid-induced apoptosis was verified by a characteristic step-wise fragmentation of genomic DNA and chromatin condensation. The induction of apoptosis was detected as early as 8 hrs after the addition of the retinoid and was concentration dependent (0.1-10 microM). Neither the natural retinoid all-trans retinoic acid nor 9-cis retinoic acid induced apoptosis. These data strongly indicate that the antitumor activity of the acyclic retinoid may be partly explained by the induction of apoptosis in tumor cells.

A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation.

Reversed-phase HPLC methods capable of separating several retinoic acid isomers are generally not designed for simultaneous analysis of isomers of other classes of retinoids. A reversed-phase HPLC method is presented which allows the separation of at least four retinoic acid (RA) isomers (13-cis-RA, 9,13-dicis-RA, 9-cis-RA, all-trans-RA)and of all isomers of retinoyl-beta-D-glucuronide (RAG), which have been observed in vivo (13-cis-RAG, 9-cis-RAG,all-trans-RAG). The recovery of retinoids was generally between 80 and 90%. Intra-day reproducibility (expressed as relative standard deviation) was less than or equal to 7%. As little as 0.25 ng of RA isomers and of all-trans-RAG could be detected. This method allowed the study of the metabolism of 9-cis-retinoids, where isomerization reactions play a predominant role.

The in vivo metabolism of 9-cis-retinoic acid (9-c-RA), an endogenous ligand of retinoid X receptors (RXRs), which can also bind to retinoic acid receptors (RARs), was examined in pregnant mice and rats following a single oral dose of 100 mg 9-cis-retinaldehyde (9-c-RAL)/kg body weight. 9-Cis-retinoyl-beta-D-glucuronide (9-c-RAG), a metabolite not found in vivo before, was a major metabolite of 9-c-RA in mouse plasma and was also present in all mouse tissues examined as well as in rat plasma. In both species putative oxidation products of retinoic acids and high levels of retinyl esters were found. Concentrations of retinoic acid isomers and retinoyl-beta-D-glucuronides in the mouse plasma greatly exceeded those of the rat plasma. The finding of high levels of 9-c-RAG underlines the importance of glucuronidation in the metabolism of retinoids.

The biologic activities of retinoic acid and 3,4-didehydroretinoic acid, two endogenous vitamin A derivatives in various tissues, were compared to their affinities for the nuclear retinoic acid receptors and their ability to induce transcriptional activation. Both retinoids were equipotent inducers of differentiation of F9 teratocarcinoma cells. In a morphologic assay, using reconstructed skin, retinoic acid and 3,4-didehydroretinoic acid inhibited keratinization at a concentration of 100 nM. In cultured keratinocytes, a 50% inhibition of the production of the keratinocyte transglutaminase enzyme was achieved with about 20 nM for both retinoids. The in vitro binding to the nuclear retinoic acid receptors alpha, beta, and gamma showed that retinoic acid and 3,4-didehydroretinoic acid had almost equal affinities for the receptors with Kds ranging from 3 to 47 nM. The transcriptional activation resulting from the addition of the two retinoids to cells co-transfected with alpha, beta, or gamma retinoic acid receptor expression vectors and a retinoic acid responsive element linked to the chloramphenicol acetyltransferase reporter gene was similar. Finally, it was demonstrated that retinoic acid did not metabolize to 3,4-didehydroretinoic acid, and a slow conversion of 3,4-didehydroretinoic acid into retinoic acid was not sufficient to explain the biologic effects produced by the former compound. In conclusion, the present study demonstrates that retinoic acid and 3,4-didehydroretinoic acid have the same activity in several different test systems, but their metabolism differs depending on the cell type used.

Retinoic acid modulates proliferation and differentiation of a wide variety of normal and leukemic cells through two distinct families of transcriptional factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A stereoisomer of retinoic acid, 9-cis-retinoic acid, is a high-affinity ligand for RXR and binds efficiently to RAR. In contrast, all-trans-retinoic acid interacts 40-fold less efficiently with RXR as compared with RAR. To clarify the biologic role of retinoic acid compounds (all-trans,- 9-cis-, and 13-cis-retinoic acid) in hematopoietic cells, we studied their effects on clonal growth, differentiation, and expression of RAR-alpha and RXR-alpha genes in HL-60 cells. At very low concentrations (10(-15) to 10(-12) mmol/L), each retinoid enhanced clonal growth of HL-60 cells. These concentrations of the retinoids had no capacity to induce differentiation of leukemic cells as measured by ability either to reduce nitroblue tetrazolium and to express CD11b antigens, suggesting that retinoids at very low concentrations may stimulate proliferation of leukemic cells rather than induce their differentiation. These findings may help explain why patients with acute promyelocytic leukemia may relapse while receiving retinoic acids. With continuous therapy, retinoids are metabolized rapidly with increased urinary excretion, lowering their plasma levels to a range that may stimulate proliferation without inducing differentiation of leukemic cells. In contrast, we found that at higher concentrations (> or = 10(-11) mmol/L) each retinoid inhibited clonal growth, reduced c-myc RNA levels, and induced differentiation of HL-60 cells. 9-cis-retinoic acid was a slightly more potent inducer of differentiation than all-trans-retinoic acid; the mechanism for this increased potency and its clinical potential requires additional studies. Steady-state levels of RAR-alpha mRNA in HL-60 cells were not affected by either 9-cis- and all-trans-retinoic acid. In contrast, 9-cis-retinoic acid, but not all-trans-retinoic acid, reduced RXR-alpha mRNA accumulation in a dose-dependent manner.

Retinoidal activity of retinoic acid isomers [all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9CRA) and 13-cis-retinoic acid (13CRA)] and their oxidized derivatives [19-hydroxy and 19-oxo derivatives of ATRA (19-hydroxy-ATRA and 19-oxo-ATRA), 19-oxo derivative of 9CRA (19-oxo-9CRA), and 19-hydroxy derivative of 13CRA (19-hydroxy-13CRA)] was evaluated by means of a human promyelocytic leukemia HL-60 cell differentiation induction assay. All the compounds examined showed this activity with ED50 values of 2-30 nM, which are in accordance with their binding activity to nuclear retinoic acid receptors (RARs).

Activation of a phospholipase A2 (PLA2) is a key step in the production of precursors for the biosynthesis of lipid mediators of inflammation. Inhibition of this enzyme could result in the suppression of three important classes of inflammatory lipids, prostaglandins, leukotrienes and platelet activating factor (PAF), and offers an attractive therapeutic approach to design novel agents for the treatment of inflammation and tissue injury. In this report we describe a novel compound, BMS-181162 4(3'-carboxyphenyl)-3,7-dimethyl-9(2",6"6"-trimethyl-1"-cyclohexenyl),++ +2Z,4E,6E, 8E-nonatetraenoic acid which specifically inhibits a 14 kD human PLA2 and effectively blocks phorbol ester induced skin inflammation in mice. BMS-181162 is the first reported specific inhibitor of PLA2 and its specificity may make useful tool in the dissection of the role of PLA2 in the inflammatory process.

In clinical trials the cancer preventive drug N-(4-hydroxyphenyl)retinamide (HPR) markedly lowers plasma concentrations of retinol and retinol-binding protein (RBP). Five hours after injection of HPR (51 mumol/kg), serum concentrations of retinol and RBP were 33 and 42% lower, respectively, than control values in rats. Because the mean transit time for retinol disappearance from serum of HPR-treated rats (1.9 h) was similar to that for radiolabeled retinol in control rats in previous studies, plasma retinol turnover is apparently not accelerated by HPR treatment. To study the effects of HPR on the secretion of the retinol-RBP complex from liver, control or HPR-treated rats were injected with chylomicrons containing [3H]vitamin A and [14C]triglycerides. Both labels were rapidly cleared from plasma in the two groups. In control rats [3H]retinol concentrations began to increase in plasma after 30 min due to liver secretion of retinol bound to RBP. In HPR-treated rats, secretion was apparently inhibited because the amount of [3H]retinol bound to RBP at 4.66 h was only 2.6% of the control level. We conclude that HPR partially blocks the secretion of the retinol-RBP complex from liver and other tissues, and thus depresses plasma concentrations of vitamin A and RBP.

Inducing the expression of ICAM-1 (CD54) on the surface of epidermal keratinocytes is an important step in initiating leukocyte interaction with the epidermis. We studied the effect of keratinocyte differentiation and of drugs used to treat epidermal inflammation on the induction of this important adhesion molecule. Cell membrane expression of ICAM-1 in cultured human keratinocytes was analyzed using both immunofluorescence and FACS analysis of staining with anti-ICAM-1 monoclonal antibody and was correlated with markers of keratinocyte differentiation. Cell-surface ICAM-1 expression was induced by gamma interferon in all culture conditions, but was significantly greater (p less than 0.014) in cells grown in low-calcium medium ([Ca++] 0.03 mM), and correlated with increased staining for the basal cell keratin K5. The synthetic retinoid Etretin (Ro 10-1670) enhanced the interferon-induced ICAM-1 expression over a wide concentration range (10(-8)-10(-5) M); however, this effect was only seen in the more differentiated cells grown in 0.15 mM and 1.0 mM calcium and not in the cells grown in 0.03 mM calcium. The Etretin effects on intracellular K5 staining paralleled those on cell-surface ICAM-1. Anti-inflammatory glucocorticoids had no effect on ICAM-1 expression in cultured human keratinocytes, even at suboptimal gamma interferon doses (5 U/ml). beta-estradiol, on the other hand, mimicked the Etretin effect, increasing both IFN induction of ICAM-1 expression and K5 staining in more differentiated keratinocytes in 0.15 and 1.0 mM calcium, but not in those in 0.03 mM calcium. Both Etretin and beta-estradiol decreased staining of involucrin, a marker of terminal differentiation, supporting the proposition that in this experimental system these drugs suppress keratinocyte differentiation. The enhanced ICAM-1 induction in keratinocytes with a basal level of differentiation correlates with the in vivo effects of interferon on ICAM-1 and may be a principal determinant in the patterns of ICAM-1 seen in inflammatory skin diseases.

Conjugation reactions, such as the formation of sulfates, glucuronides and amino acid derivatives of biologically active compounds, are classically considered as detoxication processes. In essence, common nonpolar compounds are rendered less active by the formation of water-soluble metabolites, which then can readily be excreted in the urine. In the past decade, however, it has become clear that some natural metabolites become more active rather than less so by the formation of such conjugates. Several compounds in this special group will be considered in this article; namely, glucuronide and hydroxyphenylamide conjugates of retinoic acid, glutathione and cysteine conjugates of halogenated hydrocarbons, glucuronide conjugates of various xenobiotics and polyglutamates of folic acid and its inhibitors. The metabolism, biological activity and toxicity of the parent molecule is profoundly affected in complex ways by such reactions. Thus, the historical concept of conjugation reactions as general detoxication processes is no longer tenable.