sphingosine-1-phosphate and 2-acetyl-4(5)-tetrahydroxybutylimidazole

Caramel color is widely used in the food industry, and its many variations are generally considered to be safe. It has been known for a long time that THI (2-acetyl-4-(tetrahydroxybutyl)imidazole), a component of caramel color III, causes lymphopenia in animals through sphingosine 1-phosphate (S1P) lyase (S1PL) inhibition. However, this mechanism of action has not been fully validated because THI does not inhibit S1PL in vitro. To reconcile this situation, we examined molecular details of THI mechanism of action using "smaller" THI derivatives. We identified a bioactive derivative, A6770, which has the same lymphopenic effect as THI via S1PL inhibition. In the case of A6770 we observe this effect both in vitro and in vivo, and demonstrate that A6770 is phosphorylated and inhibits S1PL in the same way as 4-deoxypyridoxine. In addition, A6770 was detected in rat plasma following oral administration of THI, suggesting that A6770 is a key metabolic intermediate of THI.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Food Coloring Agents; Imidazoles; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Rats; Rats, Inbred Lew; Sphingosine; Structure-Activity Relationship

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.

Topics: Acetylation; Aldehyde-Lyases; Animals; Cell Nucleus; Down-Regulation; Histone Deacetylases; Histones; Imidazoles; Inflammation; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; MicroRNAs; Mitochondria; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Oligonucleotide Array Sequence Analysis; Phenotype; Protein Kinases; Regeneration; Sarcomeres; Sphingosine; Tissue Distribution

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Topics: Aldehyde-Lyases; Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Blood Vessels; Blotting, Western; Cell Movement; Extracellular Space; Female; Fingolimod Hydrochloride; Imidazoles; Immunohistochemistry; L-Selectin; Lectins, C-Type; Lysophospholipids; Male; Mice, Inbred C57BL; Microscopy, Confocal; Platelet Endothelial Cell Adhesion Molecule-1; Propylene Glycols; Rats, Inbred F344; Receptors, Lysosphingolipid; Sphingosine; Thymocytes; Thymus Gland; Time Factors; Up-Regulation

To test the role of sphingosine-1-phosphate (S1P) signaling system in the in vivo setting of resuscitation and survival after cardiac arrest.. A mouse model of potassium-induced cardiac arrest and resuscitation was used to test the importance of S1P homeostasis in resuscitation and survival. C57BL/6 and sphingosine kinase-1 knockout (SphK1-KO) female mice were arrested for 8 min then subjected to 5 minute CPR with epinephrine bolus given at 90s after the beginning of CPR. Animal survival was monitored for 4h post-resuscitation. Upregulation of tissue and circulatory S1P levels were achieved via inhibition of S1P lyase by 2-acetyl-5-tetrahydroxybutyl imidazole (THI). Plasma and heart tissue S1P and ceramide levels were quantified by targeted ESI-LC/MS/MS.. Lack of SphK1 and low tissue/circulatory S1P levels in SphK1-KO mice led to poor animal resuscitation after cardiac arrest and to impaired survival post-resuscitation. Inhibition of S1P lyase in SphK1-KO mice drastically improved animal resuscitation and survival. Improved resuscitation and survival of THI-treated SphK1-KO mice were better correlated with cardiac dihydro-S1P (DHS1P) than S1P levels. The lack of SphK1 and the inhibition of S1P lyase by THI were accompanied by modulation in cardiac S1PR1 and S1PR2 expression and by selective changes in plasma N-palmitoyl- and N-behenoyl-ceramide levels.. Our data provide evidence for the crucial role for SphK1 and S1P signaling system in resuscitation and survival after cardiac arrest, which may form the basis for development of novel therapeutic strategy to support resuscitation and long-term survival of cardiac arrest patients.

Topics: Aldehyde-Lyases; Animals; Cardiopulmonary Resuscitation; Ceramides; Chromatography, Liquid; Disease Models, Animal; Female; Gene Expression Regulation; Heart Arrest; Imidazoles; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Sphingosine; Sphingosine-1-Phosphate Receptors; Survival Rate; Tandem Mass Spectrometry

2-Acetyl-4(5)-tetrahydroxybutyl imidazole (THI) has been shown to reduce rodent peripheral blood lymphocytes through increasing lymphoid sphingosine 1-phosphate (S1P) by inhibiting S1P lyase. The objective of this study was to characterize the relationship between systemic THI exposure, splenic S1P concentrations, and lymphopenia in rats. Following the oral administration of 10 and 100 mg kg(-1) THI to male rats, THI was rapidly absorbed and reached a plasma peak level at 1 h post-dosing. Splenic S1P increased and reached the peak level at 24 h. Blood lymphocyte count decreased as the splenic S1P level increased. THI plasma concentration was linked to splenic S1P concentration using an indirect model incorporated with a four-step signal transduction model. In turn, the S1P level was directly coupled with blood lymphocyte number. The integrated model simultaneously captured the splenic S1P and blood lymphocyte responses. This pharmacokinetic-biomarker-pharmacodynamic model resolved the remarkable discrepancy between plasma THI concentration and the pharmacological response and quantitatively described the relationship of THI exposure, S1P, and lymphopenic response.

Topics: Administration, Oral; Animals; Computer Simulation; Imidazoles; Lymphocytes; Lysophospholipids; Male; Mice; Models, Biological; Rats; Rats, Sprague-Dawley; Sphingosine; Spleen; Time Factors

Topics: Aldehyde-Lyases; Animals; Cell Movement; Chemotaxis, Leukocyte; Diet; Enzyme Inhibitors; Fingolimod Hydrochloride; Food Coloring Agents; Imidazoles; Immunosuppressive Agents; Lymphoid Tissue; Lysophospholipids; Mice; Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocytes

Lymphocyte egress from the thymus and from peripheral lymphoid organs depends on sphingosine 1-phosphate (S1P) receptor-1 and is thought to occur in response to circulatory S1P. However, the existence of an S1P gradient between lymphoid organs and blood or lymph has not been established. To further define egress requirements, we addressed why treatment with the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI) induces lymphopenia. We found that S1P abundance in lymphoid tissues of mice is normally low but increases more than 100-fold after THI treatment and that this treatment inhibits the S1P-degrading enzyme S1P lyase. We conclude that lymphocyte egress is mediated by S1P gradients that are established by S1P lyase activity and that the lyase may represent a novel immunosuppressant drug target.

Topics: Aldehyde-Lyases; Animals; B-Lymphocytes; Chemotaxis, Leukocyte; Enzyme Inhibitors; Food Coloring Agents; Hematopoietic Stem Cells; Imidazoles; Immunosuppressive Agents; Lymph; Lymph Nodes; Lymphoid Tissue; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Pyridoxine; Receptors, Lysosphingolipid; RNA Interference; Sphingosine; T-Lymphocytes; Thymus Gland; Vitamin B 6