Effects of polystyrene microplastics acute exposure in the liver of swordtail fish (Xiphophorus helleri) revealed by LC-MS metabolomics.

As global pollution, microplastics pollution has aroused growing concerns. In our experiment, the effect of microplastics acute exposure on the liver of swordtail fish was investigated by using LC-MS metabolomics. Fishes treated with high concentration polystyrene microspheres (1 µm) for 72 h were divided into three concentration groups: (A) no microplastics, (B): 1 × 106 microspheres L-1, (C): 1 × 107 microspheres L-1. Metabolomic analysis indicated that exposure to microplastics caused alterations of metabolic profiles in swordtail fish, including 37 differential metabolites were identified in B vs. A, screened out ten significant metabolites, which involved 14 metabolic pathways. One hundred three differential metabolites were identified in C vs. A, screened out 16 significant metabolites, which involved 30 metabolic pathways. Six significant metabolites were overlapping in group B vs. A and C vs. A; they are 3-hydroxyanthranilic acid, l-histidine, citrulline, linoleic acid, pantothenate, and xanthine. In addition, four metabolic pathways are overlapping in group B vs. A and C vs. A; they are beta-alanine metabolism, biosynthesis of amino acids, linoleic acid metabolism, and aminoacyl-tRNA biosynthesis. These differential metabolites were involved in oxidative stress, immune function, energy metabolism, sugar metabolism, lipid metabolism, molecule transport, and weakened feed utilization, growth performance, nutrient metabolism, and animal growth. Furthermore, we found that the number of interfered amino acids and microplastics showed a dose-effect. In summary, great attention should be paid to the potential impact of microplastics on aquatic organisms.

Chromatographic measurement of 3-hydroxyanthranilate 3,4-dioxygenase activity reveals that edaravone can mitigate the formation of quinolinic acid through a direct enzyme inhibition.

Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and cerebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential applications in ALS treatment.

Kynurenine derivative 3-HAA is an agonist ligand for transcription factor YY1.

The 3-hydroxyanthranilic acid (3-HAA), a derivative of kynurenine, was reported to suppress tumor growth. However, the function of 3-HAA largely remains unclear. Here, we report that 3-hydroxyanthranilic acid (3-HAA) is lower in tumor cells, while adding exogenous 3-HAA induces apoptosis in hepatocellular carcinoma by binding YY1. This 3-HAA binding of YY1 leads to phosphorylation of YY1 at the Thr 398 by PKCζ, concomitantly enhances YY1 chromatin binding activity to increase expression of target genes. These findings demonstrate that 3-HAA is a ligand of YY1, suggesting it is a promising therapeutic candidate for HCC.

3-Hydroxyanthralinic acid metabolism controls the hepatic SREBP/lipoprotein axis, inhibits inflammasome activation in macrophages, and decreases atherosclerosis in Ldlr-/- mice.

AIMS: Atherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation. METHODS AND RESULTS: In vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1ß production by macrophages. Using Ldlr-/- mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores. CONCLUSIONS: We show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.

Changes in chemical structures of wheat straw auto-hydrolysis lignin by 3-hydroxyanthranilic acid as a laccase mediator.

3-Hydroxyanthranilic acid (3-HAA), as a potential natural laccase mediator, was shown to mediate the oxidation of non-phenolic lignin subunits. The problem of cost and toxicity of artificial mediators could be solved to some extent by a further study about the detailed changes of lignin chemistry structures in laccase 3-HAA system (LHS). In this work, wheat straw auto-hydrolysis lignin (AL) was prepared. Oxidations of AL by LHS and laccase 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) system were then investigated, respectively. Various structural changes of AL during the oxidation were characterized by different methods including phenolic hydroxyl group determination, nitrobenzene oxidation, ozonation, gel permeation chromatography, ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy and two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The changes in AL chemical structures were found in LHS, including unit removal, bond cleavage and biopolymerization. Compared to laccase ABTS system, a selective removal of guaiacyl-type lignin in LHS was observed, based on the results of nitrobenzene oxidation and 2D NMR analysis. The selective removal of guaiacyl-type lignin was due to improved aromatic ring cleavage and weaken lignin biopolymerization. The selectivity of guaiacyl-type lignin removal in LHS plays an important role, especially for improving bioconversion efficiency of laccase for guaiacyl-rich lignocellulosic biomass.

Inhibition of LPS-induced inflammatory mediators by 3-hydroxyanthranilic acid in macrophages through suppression of PI3K/NF-κB signaling pathways.

Many tryptophan metabolites have immunomodulatory effects on various immune cells. 3-Hydroxyanthranilic Acid (3-HAA) is a tryptophan metabolite reported to have anti-inflammatory activity. The mechanism of this activity is unclear. The present study examined the immunomodulatory effects and molecular mechanisms of 3-HAA on macrophages. Pretreatment of 3-HAA (0.1-10 µg mL(-1)) for 2 h markedly inhibited NO and cytokine production in LPS-stimulated Raw 264.7 cells. Moreover, translocation and activation of NF-κB by LPS in the nucleus were abrogated through the prevention of IκB degradation by 3-HAA treatment. 3-HAA significantly suppressed LPS-induced PI3K/Akt/mTOR activation, whereas MAPKs were not affected by 3-HAA treatment. Furthermore, the inhibition of mTOR by 3-HAA resulted in decreased production of inflammatory mediators and NF-κB activity. Similar results were also observed in primary peritoneal macrophages. Furthermore, 3-HAA modulated macrophage polarization. Collectively, the results suggest that 3-HAA has an immunomodulatory effect that may result from inhibition of PI3K/Akt/mTOR and NF-κB activation, thereby decreasing the production of pro-inflammatory mediators.

IDO expressing dendritic cells suppress allograft rejection of small bowel transplantation in mice by expansion of Foxp3+ regulatory T cells.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the enzyme that catalyzes the first and rate-limiting step of tryptophan catabolism, suppresses T-cell responses by tryptophan depletion and accumulation of kynurenine metabolites. IDO prevents allograft rejection in various transplantations. METHODS: Dendritic cells (DC) highly expressing IDO (IDO(+) DC) were cultured through transduction of adenovirus vectors carrying the IDO sequence. IDO(+) DC were incubated with CD4(+) CD25(-) T cells to detect T cell proliferation. The effects of IDO(+) DC and 3-Hydroxyanthranilic acid (3-HAA) were verified in an allogeneic murine small bowel transplantation (SBT) model. Foxp3(+) Treg cells of recipient mice were detected by flow cytometry and cytokines in plasma were determined by ELISA. RESULTS: IDO(+) DC effectively suppressed proliferation of CD4(+) CD25(-) T cells in vitro, and this effect could be enhanced by adding 3-HAA. In the SBT transplantation model, both 3-HAA (P < 0.05) and IDO(+) DC (P < 0.01) prolonged the survival time of transplanted mice. Mice treated with IDO(+) DC achieved longer mean survival time than 3-HAA administrated mice (11.5d vs. 18.5d). Grafts from IDO(+) DC, 3-HAA and combination treatment group showed reduced inflammation and minimal architectural distortion. IFN-γ production was significantly inhibited by IDO(+) DC and 3-HAA (P<0.05). The expression of IL-2 was slightly lower with 3-HAA or IDO(+) DC treatment. However, IL-10 was higher in 3-HAA, IDO(+) DC and combination treatment groups, while TGF-ß was elevated in all non-control groups. CONCLUSIONS: IDO(+) DC plus 3-HAA has an immunoprotective role and represents a potential strategy to suppress acute rejection and prolong survival of grafts in SBT.

Effects of methylprednisolone and 4-chloro-3-hydroxyanthranilic acid in experimental spinal cord injury in the guinea pig appear to be mediated by different and potentially complementary mechanisms.

STUDY DESIGN: Blinded, placebo-controlled, parallel treatment group studies of the effects of methylprednisolone (MP) or 4-chloro-3-hydroxyanthranilate (4-Cl-3-HAA) on behavioral outcome and quinolinic acid tissue levels from experimental thoracic spinal cord injury in adult guinea pigs. OBJECTIVES: To compare the effects of treatment with high-dose MP, a corticosteroid, and 4-Cl-3-HAA, a compound that inhibits synthesis of the neurotoxin quinolinic acid (QUIN) by activated macrophages. To explore the effect of different times of treatment using these two approaches to ameliorating secondary tissue damage. SETTING: Laboratory animal studies at the University of North Carolina, Chapel Hill, NC, USA. METHODS: Standardized spinal cord injuries were produced in anesthetized guinea pigs, using lateral compression of the spinal cord. Behavioral impairment and recovery were measured by placing and toe-spread responses (motor function), cutaneus trunci muscle reflex receptive field areas and somatosensory-evoked potentials (sensory function). Tissue quinolinic acid levels were measured by gas chromatograph/mass spectrometry. RESULTS: The current experiments showed a reduction in delayed loss of motor and sensory function in the guinea pig with MP (150 mg kg(-1), intraperitoneally in split doses between 0.5 and 6 h), but no significant reduction in tissue QUIN. Improved sensory function was seen with a single dose of 60 mg kg(-1) MP intraperitoneally at 5 h after injury, but not at 10 h after injury. A single dose of 4-Cl-3-HAA at 5 h in the guinea pig did not produce the sensory and motor improvements seen in previous studies with 12 days of dosing, beginning at 5 h. CONCLUSION: These studies, together with earlier findings, indicate that both drugs can attenuate secondary pathologic damage after SCI, but through separate mechanisms. These are most likely an acute reduction by MP of oxidative processes and reduction by 4-Cl-3-HAA of QUIN synthesis.

Suppression of experimental autoimmune glomerulonephritis by tryptophan.

BACKGROUND: Indoleamine 2, 3-dioxygenase (IDO), a heme-containing dioxygenase, can catalyze tryptophan degradation and produce a local microenvironment with tryptophan depletion and tryptophan metabolites accumulation, which may suppress T cell-mediated immunity and play an important immunosuppressive role in many diseases. Previous studies suggested that tryptophan depletion is an important immunosuppressive mechanism of IDO, while recent evidence shows that tryptophan metabolites may also be useful for inducing the T cell immune tolerance. However, it remains unclear whether tryptophan catabolites play a protective role in anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN), which is a type 1 T-helper (Th1)-mediated autoimmune disease. METHODS: We examined the effect of tryptophan catabolites, 3-hydroxykynurenine acid and 3-hydroxyanthranilic acid, on renal injury in experimental autoimmune glomerulonephritis (EAG) of Wistar-Kyoto rats and explored their protective mechanism. RESULTS: Treatment by either 3-hydroxyanthranilic acid or 3-hydroxykynurenic acid attenuated the kidney disease of EAG rats, with decreased glomerular histological injury and inflammatory cell infiltration, lightened urinary protein, and improved renal function compared to phosphate buffered saline-treated EAG rats. This was associated with significantly increased apoptosis and decreased proliferation of splenic activated T cells in vivo, inducing the deviation of cytokines of antigen-special T cells from Th1 to Th2. CONCLUSIONS: Tryptophan metabolites play an important immunosuppressive role in the development of anti-GBM GN and might offer a new strategy for treating this disease.

The tryptophan metabolite 3-hydroxyanthranilic acid suppresses T cell responses by inhibiting dendritic cell activation.

The generation of tryptophan (Trp) metabolites by indoleamine 2,3-dioxygenase (IDO) is an effective mechanism for T cell suppression. However, the effect of Trp metabolites on dendritic cells (DCs) remains unclear. Here, we investigated whether the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) directly inhibits DC activation and is responsible for T cell suppression. We found that 3-HAA treatment significantly reduced IL-12, IL-6, and TNF-α production in bone marrow-derived DCs (BMDCs) stimulated with LPS. Maturation markers CD40, CD80, CD86, and I-A were also significantly reduced. Moreover, treatment with 3-HAA decreased the ability of DCs to stimulate T cell activation and differentiation in vitro and in vivo. Finally, we observed that phospho-JNK and phospho-38 levels were reduced in 3-HAA-treated DC2.4 cells and BMDCs. These results suggest that the tryptophan metabolite 3-HAA suppresses T cell responses by inhibiting DC activation.

The tryptophan metabolite 3-hydroxyanthranilic acid lowers plasma lipids and decreases atherosclerosis in hypercholesterolaemic mice.

AIMS: Cardiovascular disease is the most common cause of death in the world and atherosclerosis, an inflammatory process in the vessel wall, accounts for the majority of these deaths. The tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) has been shown to inhibit inflammation in different experimental autoimmune disease models. However, the effect of 3-HAA in atherosclerosis has never been explored. METHODS AND RESULTS: In this study, we used the atherosclerosis prone Ldlr-/- mice, and cell culture experiments to evaluate the role of 3-HAA in atherosclerosis. Eight weeks treatment with 3-HAA significantly reduced the lesion size in the aorta, and modulated local and systemic inflammatory responses. 3-hydroxyanthranilic acid inhibited the uptake of oxLDL by macrophages, an initiating event in the formation of foam cells, a major cellular component of atherosclerotic lesions. Surprisingly, 3-HAA significantly affected plasma cholesterol and triglyceride levels in Ldlr-/- mice, likely due to modulation of signalling through peroxisome proliferator-activated receptors. CONCLUSION: 3-Hydroxyanthranilic acid inhibits atherosclerosis by regulating lipid metabolism and inflammation, two major components of this disease.

Indoleamine 2,3-dioxygenase and metabolites protect murine lung allografts and impair the calcium mobilization of T cells.

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan into kynurenine metabolites that suppress effector T-cell function. In this study, we investigated IDO and its metabolite, 3-hydroxyanthranilic acid (3HAA), in regulating lung allograft rejection, using a murine orthotopic lung transplant model with a major mismatch (BALB/c donor and C57BL6 recipient). IDO was overexpressed in murine donor lungs, using an established nonviral (polyethylenimine carrier)-based gene transfer approach, whereas 3HAA was delivered daily via intraperitoneal injection. Increased IDO expression or its metabolite, 3HAA, resulted in a remarkable therapeutic effect with near normal lung function and little acute rejection, approximately A1, compared with A3 in untreated allografts (grading based on International Society for Heart and Lung Transplantation guidelines). We found that a high IDO environment for 7 days in lung allografts resulted in impaired T-cell activation, the production of multiple effector cytokines (IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, IL-12, and IL-13), and the generation of effector memory T cells (CD62L(lo)CD44(hi) phenotype). In isolated murine splenocytes, we observed that IDO/3HAA impaired T-cell receptor (TCR)-mediated T-cell activation, and more importantly, a decrease of intracellular calcium, phospholipase C-γ1 phosphorylation, and mitochondrial mass was evident. This work further illustrates the potential role of a high IDO environment in lung transplantation, and that the high IDO environment directly impairs TCR activation via the disruption of calcium signaling.

Tryptophan in alcoholism treatment I: kynurenine metabolites inhibit the rat liver mitochondrial low Km aldehyde dehydrogenase activity, elevate blood acetaldehyde concentration and induce aversion to alcohol.

AIMS: The aims were to provide proofs of mechanism and principle by establishing the ability of kynurenine metabolites to inhibit the liver mitochondrial low K(m) aldehyde dehydrogenase (ALDH) activity after administration and in vivo, and to induce aversion to alcohol. METHODS: Kynurenic acid (KA), 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) were administered to normal male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring blood acetaldehyde following ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. RESULTS: ALDH activity was significantly inhibited by all three metabolites by doses as small as 1 mg/kg body wt. Blood acetaldehyde accumulation after ethanol administration was strongly elevated by KA and 3-HK and to a lesser extent by 3-HAA. All three metabolites induced aversion to alcohol in rats and decreased alcohol preference in mice. CONCLUSIONS: The above kynurenine metabolites of tryptophan induce aversion to alcohol by inhibiting ALDH activity. An intellectual property covering the use of 3-HK and 3-HAA and derivatives thereof in the treatment of alcoholism by aversion awaits further development.

The tryptophan metabolite 3-hydroxyanthranilic acid plays anti-inflammatory and neuroprotective roles during inflammation: role of hemeoxygenase-1.

Tryptophan metabolism by the kynurenine pathway (KP) is important to the pathogenesis of inflammatory, infectious, and degenerative diseases. The 3-hydroxykynurenine (3-HK) branch of the KP is activated in macrophages and microglia, leading to the generation of 3-HK, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid, which are considered neurotoxic owing to their free radical-generating and N-methyl-d-aspartic acid receptor agonist activities. We investigated the role of 3-HAA in inflammatory and antioxidant gene expression and neurotoxicity in primary human fetal central nervous system cultures treated with cytokines (IL-1 with or without interferon-γ) or with Toll-like receptor ligands mimicking the proinflammatory central nervous system environment. Results were analyzed by microarray, Western blot, immunostain, enzyme-linked immunosorbent assay, and neurotoxicity assays. 3-HAA suppressed glial cytokine and chemokine expression and reduced cytokine-induced neuronal death. 3-HK also suppressed cytokine-induced neuronal death. Unexpectedly, 3-HAA was highly effective in inducing in astrocytes the expression of hemeoxygenase-1 (HO-1), an antioxidant enzyme with anti-inflammatory and cytoprotective properties. Optimal induction of HO-1 required 3-HAA and cytokines. In human microglia, 3-HAA weakly induced HO-1 and lipopolysaccharide suppressed microglial HO-1 expression. 3-HAA-mediated HO-1 expression was confirmed in cultured adult human astrocytes and in vivo after 3-HAA injection to mouse brains. Together, our results demonstrate the novel neuroprotective activity of the tryptophan metabolite 3-HAA and have implications for future therapeutic approaches for neuroinflammatory disorders.

Tryptophan metabolite 3-hydroxyanthranilic acid selectively induces activated T cell death via intracellular GSH depletion.

Tryptophan-derived metabolites, initiated by indoleamine 2,3-dioxygenase (IDO), preferentially induce activated T cell death, which is an important mechanism in IDO-mediated T cell suppression. However, the mechanism of this phenomenon remains unclear. We found that 3-hydroxyanthranilic acid (3-HAA), the most potent metabolite, selectively eliminated activated T cells, which were stimulated with the bacterial superantigen staphylococcal enterotoxin A (SEA), but not resting T cells, by inducing apoptosis. We observed 3-HAA-induced depletion of intracellular glutathione (GSH) in activated T cells. When GSH levels were maintained by addition of N-acetylcysteine (NAC) and GSH, 3-HAA-mediated T cell death was completely inhibited. This was associated with extrusion of GSH from activated T cells without increased reactive oxygen species (ROS) formation. Finally, we showed that administration of 3-HAA in mice after allogeneic bone marrow transplantation reduced acute graft-versus-host disease (GVHD) lethality by inhibition of alloreactive T cell expansion through intracellular GSH depletion. Our data suggest that direct depletion of intracellular GSH is the major mechanism of 3-HAA-mediated activated T cell death.

Suppression of T-cell response and prolongation of allograft survival in a rat model by tryptophan catabolites.

Indoleamine 2,3-dioxygenase is a rate-limiting enzyme involved in the conversion of tryptophan to various endogenous catabolites, such as 3-hydroxyanthranilic acid, 3-hydroxykynurenine, and L-kynurenine. This study aims to provide evidence to support the hypothesis that some of the tryptophan catabolites may exert strong immunosuppression against allograft rejection by inhibiting allogeneic T-cell response. The mixed lymphocyte proliferation assay was used as an in-vitro model and the rat cardiac allograft as an in-vivo model. We found that 3-hydroxyanthranilic acid and 3-hydroxykynurenine inhibited T-cell proliferation in a concentration-dependent manner. Both dendritic cell-stimulated T-cell proliferation and baseline T-cell proliferation (in the absence of dendritic cells) were inhibited by these tryptophan catabolites. Using the rat cardiac allograft model, we showed that a single i.v. administration of 3-hydroxyanthranilic acid+allogeneic dendritic cells seven days before cardiac grafting markedly prolonged the graft survival in the recipient (from seven days to fifteen days). Additional studies showed that this single administration of 3-hydroxyanthranilic acid wiped out a majority of specific T-cell sub-populations that were activated by allogeneic dendritic cells. This strong inhibition by 3-hydroxyanthranilic acid is believed to be an important mechanism contributing to the development of immunotolerance against the allogeneic graft. These observations offer a potential strategy for using tryptophan catabolites together with allogeneic dendritic cells to induce selective immunotolerance against allogeneic grafts in the recipients.

NO scavenging by 3-hydroxyanthranilic acid and 3-hydroxykynurenine: N-nitrosation leads via oxadiazoles to o-quinone diazides.

The tryptophan metabolites kynurenine, 3-hydroxykynurenine, anthranilic, 3-hydroxyanthranilic and 3-methoxyanthranilic acids were compared with regard to diazotation by .NO or NO+, using three different donors, nitrite at pH 5, PAPA-NONOate at pH 7.4 and NO+SbF(6)- at pH 2.0. With all three sources of NO species, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were readily nitrosated, thereby forming an intensely yellow compound. Nitrosation of the non-hydroxylated analogs did not lead to colored products within the period of observation. Competition experiments, using PAPA-NONOate as NO donor, showed that 3-hydroxyanthranilic acid is a more potent NO scavenger than N-acetylcysteine. Nitrosation of 3-hydroxykynurenine and 3-hydroxyanthranilic acid leads, presumably via a nitrosamine intermediate, to a diazonium ion, which forms an oxadiazole tautomerizing to a yellow o-quinone diazide. While the diazonium-derived quinone diazide is apparently the sole product detected directly after incubation of 3-hydroxyanthranilic acid, additional substances are formed from 3-hydroxykynurenine. Contrary to rapid carbenium ion formation from diazonium ions of non-hydroxylated anilines, nitrogen is practically not released from oxadiazoles/quinone diazides at moderate temperatures. Since carbenium ions are known to cause adduct formation with other biomolecules, and since non-hydroxylated anilines and their aminophenol analogs differ in their reactions following diazotation, these findings should be of relevance for the relative toxicity of anilines.

Effect of 3-hydroxyanthranilic acid in the immunosuppressive molecules indoleamine dioxygenase and HLA-G in macrophages.

Indoleamine 2,3-dioxygenase (IDO) and human leukocyte antigen-G (HLA-G) are two molecules involved in immune tolerance. 3-Hydroxyanthranilic acid is an IDO downstream metabolite that can produce an important immune suppression. In dendritic cells, it induces HLA-G cell surface expression and secretion to the medium. The relationship between IDO and HLA-G seems to be dependent on the cell type. In this study we analyzed the effect of the tryptophan metabolite 3-hydroxyanthranilic acid in these two proteins in monocytes and macrophages. This compound decreased IDO activity while increased HLA-G surface expression in macrophages, but not in monocytes. Also, 3-hydroxyanthranilic acid decreased HLA-G1 shedding, but not HLA-G5 secretion by macrophages. These results stress the importance of 3-hydroxyanthranilic acid as a modulator of the immune response.