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.

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.

4-chloro-3-hydroxyanthranilate reduces local quinolinic acid synthesis, improves functional recovery, and preserves white matter after spinal cord injury.

Inflammatory processes within the central nervous system (CNS) contribute significantly to the pathogenesis of a broad range of neurologic diseases, including spinal cord injury (SCI). One mechanism by which immune activation causes neurologic symptoms and tissue injury is via the production of neurotoxins by activated macrophages and microglia. In the present study, the role of the endogenous tryptophan metabolite and neurotoxin quinolinic acid (QUIN) in secondary pathology following traumatic SCI was investigated. Adult Hartley guinea pigs were injured by lateral compression of the spinal cord at the 12th thoracic segment (T12). QUIN had accumulated at the site of injury on day 12 post-injury in proportion to the severity of functional neurologic deficits (as assessed by the cutaneus trunci muscle reflex and motor function score at 5 h post-injury). Systemic administration of the 3-hydroxyanthranilate-3,4-dioxygenase (3-HAD) inhibitor, 4-chloro-3-hydroxyanthranilate (4Cl-3HAA; approximately 100 mg/kg every 12 h, beginning 5 h after injury) attenuated local QUIN production and reduced QUIN accumulation at the site of injury by approximately 50% at day 12, without enhanced accumulations of the neuroprotective metabolite kynurenic acid (KYNA). The severity of secondary functional deficits was also reduced by 4Cl-3HAA. In toluidine blue-stained spinal cord sections, the area of surviving intact white matter at the injury site was increased by approximately 100% in the 4Cl-3HAA-treated group. Sparing of both axons and myelin contributed to this increase. These results support the conclusion that QUIN accumulations at the site of injury contribute to secondary functional deficits and tissue damage following SCI.

The mechanism of inactivation of 3-hydroxyanthranilate-3,4-dioxygenase by 4-chloro-3-hydroxyanthranilate.

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-heme Fe(II) dependent enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to quinolinate, an intermediate in the biosynthesis of nicotinamide adenine dinucleotide. Quinolinate has also been implicated in important neurological disorders. Here, we describe the mechanism by which 4-chloro-3-hydroxyanthranilate inhibits the HAD catalyzed reaction. Using overexpressed and purified bacterial HAD, we demonstrate that 4-chloro-3-hydroxyanthranilate functions as a mechanism-based inactivating agent. The inactivation results in the consumption of 2 +/- 0.8 equiv of oxygen and the production of superoxide. EPR analysis of the inactivation reaction demonstrated that the inhibitor stimulated the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated enzyme can be reactivated by treatment with DTT and Fe(II). High resolution ESI-FTMS analysis of the inactivated enzyme demonstrated that the inhibitor did not form an adduct with the enzyme and that four conserved cysteines were oxidized to two disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the enzyme, complexed to the inhibitor and O2, generates superoxide which subsequently dissociates, leaving the inhibitor and the oxidized iron center at the active site.

Structural studies on 3-hydroxyanthranilate-3,4-dioxygenase: the catalytic mechanism of a complex oxidation involved in NAD biosynthesis.

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate in the final enzymatic step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide. The enzyme requires Fe2+ as a cofactor and is inactivated by 4-chloro-3-hydroxyanthranilate. HAD from Ralstonia metallidurans was crystallized, and the structure was determined at 1.9 A resolution. The structures of HAD complexed with the inhibitor 4-chloro-3-hydroxyanthranilic acid and either molecular oxygen or nitric oxide were determined at 2.0 A resolution, and the structure of HAD complexed with 3-hydroxyanthranilate was determined at 3.2 A resolution. HAD is a homodimer with a subunit topology that is characteristic of the cupin barrel fold. Each monomer contains two iron binding sites. The catalytic iron is buried deep inside the beta-barrel with His51, Glu57, and His95 serving as ligands. The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by Cys125, Cys128, Cys162, and Cys165. The two iron sites are separated by 24 A. On the basis of the crystal structures of HAD, mutagenesis studies were carried out in order to elucidate the enzyme mechanism. In addition, a new mechanism for the enzyme inactivation by 4-chloro-3-hydroxyanthranilate is proposed.

Immunohistochemical visualization of newly formed quinolinate in the normal and excitotoxically lesioned rat striatum.

Intracerebral infusion of 3-hydroxyanthranilate (3HANA) rapidly increases the brain content of the endogenous excitotoxin quinolinate (QUIN). QUIN formation from 3HANA is readily prevented by coadministration of the specific 3-hydroxyanthranilate oxygenase inhibitor 4-chloro-3HANA (4-Cl-3HANA). This experimental paradigm was used to identify the cell populations which are responsible for the rapid de novo production of QUIN in the rat striatum in vivo. Rats received an intrastriatal infusion of 3HANA, alone or together with equimolar 4-Cl-3HANA, for 1 h. Striatal QUIN immunoreactivity (ir) was assessed immunohistochemically, using an antibody against protein-conjugated QUIN. This antibody displayed no significant crossreactivity with compounds structurally or functionally related to QUIN. QUIN-ir cells were detected after infusion with > or =300 microM 3HANA, but not in naïve striata or after co-infusion of 4-Cl-3HANA. Cellular staining was also abolished by preabsorption of the antibody with protein-conjugated QUIN. In the normal striatum, QUIN-ir was detected exclusively in cells of an apparent microglial morphology. When examined in the excitotoxically lesioned striatum, 3HANA-induced QUIN-ir localized exclusively to OX42-ir cells of an activated microglial/macrophage morphology. These data indicate that microglia and macrophages are the major source of QUIN in the rat striatum when hyperphysiological concentrations of 3HANA are used to drive QUIN synthesis. Comparison with earlier biochemical and immunohistochemical studies suggests that the enzyme responsible for microglial QUIN production is a distinct 3-hydroxyanthranilate oxygenase with high capacity and low affinity for 3HANA.

Anticonvulsant effects of the 3-hydroxyanthranilic acid dioxygenase inhibitor NCR-631.

The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the pro-convulsive excitotoxin quinolinic acid. In the present study, the anticonvulsant effect of the 3-HAO inhibitor NCR-631 was investigated in models of chemically- and sound-induced seizures. Administration of NCR-631 i.c.v. at a dose of 300nmol in Sprague-Dawley rats was found to prolong the latency of occurrence of pentylenetetrazole (PTZ)-induced seizures. Also systemic pre-treatment with NCR-631 s.c. in N.M.R.I. mice subjected to PTZ-induced seizures provided an increase in the latency until onset of seizures, concomitant with a reduction in the severity of the seizures. However, the anticonvulsant effect of NCR-631 was short lasting (15-30min), and only observed at a dose of 250 mg/kg. A similar dose- and time-dependent anticonvulsant effect of NCR-631 was found in seizure-prone DBA/2J mice following sound-induced convulsions. Hence, the findings show that NCR-631 has anticonvulsant properties against generalized tonic-clonic seizures of different origin, suggesting that it may constitute a useful tool to study the role of kynurenines in various convulsive states.

In situ produced 7-chlorokynurenate provides protection against quinolinate- and malonate-induced neurotoxicity in the rat striatum.

Excitotoxic mechanisms may play a critical role in the pathophysiology of several neurological and psychiatric diseases. Excitatory amino acid receptor antagonists are therefore of great therapeutic interest, but untoward side effects often prevent their clinical use. Targeting the glycine coagonist site of the (NMDA) receptor may bypass these shortcomings. The present study was designed to evaluate the neuroprotective characteristics of l-4-chlorokynurenine (4-Cl-KYN), a synthetic compound which is enzymatically converted to the selective glycine/NMDA receptor antagonist 7-chlorokynurenate (7-Cl-KYNA). Using slow (2 h) intrastriatal infusions of the excitotoxins quinolinate (QUIN; 120 nmol) or malonate (6.8 micromol) as the experimental paradigm, the neuroprotective potency of 4-Cl-KYN was first compared with that of exogenous 7-Cl-KYNA, using glutamate decarboxylase activity as a lesion marker. One hundred and thirty-five nanomoles of the prodrug 4-Cl-KYN or 27 nmol 7-Cl-KYNA, the former used in a pre- and cotreatment regimen, were required to block QUIN or, less efficiently, malonate toxicity. In separate animals, the metabolic fate of this neuroprotective dose of 4-Cl-KYN was examined in vivo. In control striata, the treatment gave rise to 170 +/- 25 pmol 7-Cl-KYNA/mg protein, approximately six times less than an infusion of 27 nmol exogenous 7-Cl-KYNA, indicating greatly superior efficacy of the focally produced antagonist. Notably, the conversion of 4-Cl-KYN to 7-Cl-KYNA increased by 82% in the presence of QUIN. 4-Cl-KYN was also metabolized to 4-chloro-3-hydroxyanthranilate, an established, powerful inhibitor of QUIN synthesis. This unique pharmacological profile and the fact that the prodrug, unlike 7-Cl-KYNA, readily penetrates the blood-brain barrier suggest that 4-Cl-KYN may be exceptionally useful as an anti-excitotoxic agent.

3-Hydroxyanthranilic acid accumulation following administration of the 3-hydroxyanthranilic acid 3,4-dioxygenase inhibitor NCR-631.

In the kynurenine pathway of tryptophan metabolism, 3-hydroxyanthranilic acid is the substrate for formation of the excitotoxin quinolinic acid by 3-hydroxyanthranilic acid 3, 4-dioxygenase. This study was designed to characterize the effects on 3-hydroxyanthranilic acid after treatment with the 3-hydroxyanthranilic acid 3,4-dioxygenase inhibitor 4, 6-di-bromo-3-hydroxyanthranilic acid (NCR-631) in Sprague-Dawley rats. The blood plasma and brain concentrations of 3-hydroxyanthranilic acid were found to increase rapidly in a dose-dependent manner after gavage administration of NCR-631. However, the effect was relatively transient, with a decline in 3-hydroxyanthranilic acid levels already at 1h after NCR-631 treatment. Similar increases in plasma levels of 3-hydroxyanthranilic acid were observed following either gavage or parenteral (i.v. or s.c.) administration of NCR-631 (25 mg/kg). Only a minor enhancement of the NCR-631-induced increase in plasma 3-hydroxyanthranilic acid levels was found after sub-chronic treatment (25 mg/kg by gavage; 7 days, b.i.d.), suggesting a low propensity for altered 3-hydroxyanthranilic acid 3,4-dioxygenase activity following repeated inhibition. Administration of [14C]NCR-631 suggested 20 min initial plasma half life and an oral absorption around 50%. A dose of 250 mg/kg [14C]NCR-631 given by gavage provided plasma levels of almost 2 micromol/ml and a brain concentration of approximately 16 nmol/g, when analyzed 15 min after administration. Neither acute nor sub-chronic administration of NCR-631 caused any substantial effects on quinolinic acid levels in plasma or brain. Also, the plasma levels of kynurenic acid, another neuroactive kynurenine pathway metabolite, were unaffected by acute NCR-631 treatment. Moreover, the brain levels of the major cerebral tryptophan metabolites 5-hydroxytryptamine and 5-hydroxyindoleacetic acid remained unchanged following administration of NCR-631. Although reversible inhibition of 3-hydroxyanthranilic acid 3, 4-dioxygenase with NCR-631 in normal rats is insufficient to cause substantial changes in the levels of quinolinic acid or other important tryptophan metabolites, it causes a major accumulation of the substrate 3-hydroxyanthranilic acid.

Effects of the 3-hydroxyanthranilic acid analogue NCR-631 on anoxia-, IL-1 beta- and LPS-induced hippocampal pyramidal cell loss in vitro.

The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the putative neuropathogen quinolinic acid (QUIN). In the present study, the neuroprotective effects of the 3-HANA analogue and 3-HAO inhibitor NCR-631 was investigated using organotypic cultures of rat hippocampus. An anoxic lesion was induced by exposing the cultures to 100% N2 for 150 min, resulting in a pronounced loss of pyramidal neurons, as identified using NMDA-R1 receptor subunit immunohistochemistry. NCR-631 provided a concentration-dependent protective effect against the anoxia. NCR-631 was also found to counteract the loss of pyramidal neurons in two models of neuroinflammatory-related damage; incubation with either LPS (10 ng/ml) or IL-1 beta (10 IU/ml). The findings suggest that NCR-631 has neuroprotective properties and that it may be a useful tool to study the role of kynurenines in neurodegeneration.

The kynurenine pathway and neurologic disease. Therapeutic strategies.

The neurotoxic effects of QUIN have been well established. Clinical conditions have been identified where substantial elevations in CNS QUIN levels occur. There is a relationship between the severity of neurologic impairments and macrophage activation, with the magnitude of the increases in QUIN. The magnitude of QUIN increases in experimental immune activation, and macrophages in vitro, are highest in non-human primates, intermediate in gerbils and guinea pigs, and lowest in mice and rats. Macrophages in vitro are a useful screening system to evaluate potential inhibitors of the kynurenine pathway. Several models of CNS inflammation are available, including brain injury in post-ischemic gerbils and spinal cord injury in guinea pigs. 4-Chloro-3-hydroxyanthranilate is a potent inhibitor of QUIN production by macrophages and reduces QUIN accumulations in spinal cord injury. Such reductions are associated with significant neurologic improvements in the early post-injury period. The results support further investigation of QUIN as a mediator of neurologic dysfunction and damage in neurologic diseases.

Metabolism of L-tryptophan to kynurenate and quinolinate in the central nervous system: effects of 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate.

The metabolism of L-tryptophan to the neuroactive kynurenine pathway metabolites, L-kynurenine, kynurenate and quinolinate, and the effects of two inhibitors of quinolinate synthesis (6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate) were investigated by mass spectrometric assays in cultured cells and in vivo. Cell lines obtained from astrocytoma, neuroblastoma, macrophage/monocytes, lung, and liver metabolized L-[13C6]-tryptophan to L-[13C6]kynurenine and [13C6]kynurenate, particularly after indoleamine-2,3-dioxygenase induction by interferon-gamma. Kynurenine aminotransferase activity was measurable in all cell types examined but was unaffected by interferon-gamma. These results suggest that many cell types can be sources of kynurenate following immune activation. In vivo synthesis of L-[13C6]kynurenine and [13C6]kynurenate from L-[13C6]tryptophan was studied in the CSF of macaques infected with poliovirus, as a model of inflammatory neurologic disease. The effects of 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate on the synthesis of kynurenate were different. 6-Chlorotryptophan attenuated formation of L-[13C6]kynurenine and [13C6]kynurenate and was converted to 4-chlorokynurenine and 7-chlorokynurenate. It may be an effective prodrug for the delivery of 7-chlorokynurenate, which is a potent antagonist of NMDA receptors. In contrast, 4-chloro-3-hydroxyanthranilate did not reduce accumulation of L-[13C6]kynurenine and [13C6]kynurenate. 6-Chlorotryptophan and 4-chloro-3-hydroxyanthranilate are useful tools to manipulate concentrations of quinolinate and kynurenate in the animal models of neurologic disease to evaluate physiological roles of these neuroactive metabolites.

Quinolinic acid accumulation and functional deficits following experimental spinal cord injury.

Quinolinic acid (QUIN) is an excitotoxic tryptophan metabolite that is produced by activated macrophages. Accumulations of QUIN are implicated in the aetiology of a broad spectrum of human neurological diseases, particularly inflammatory conditions. To determine whether QUIN is an endogenous neurotoxin requires agents that reduce QUIN synthesis, and animal models where QUIN levels increase in association with neurological disease. Compression injury of the spinal cord of guinea pigs results in secondary neurological deficits, related to inflammation and macrophage activation. We evaluated whether 4-chloro-3-hydroxyanthranilate (4Cl-3HAA), an inhibitor of 3-hydroxyanthranilate-3,4-dioxygenase, reduces QUIN accumulations in this model and influences the progression of neurological deficits. Intraperitoneal injections of 4Cl-3HAA (100 mg/kg every 12 h) attenuated QUIN accumulations in spinal cord following injury, and reduced the severity of delayed functional deficits. Intraperitoneal injections of the macrophage toxin, silica, also reduced QUIN levels and attenuated neurological deficits. A direct subdural infusion of Cl-3HAA into the injured spinal cord (50 microM, 1 microliter/h) promptly exacerbated functional impairments, which suggests that the infusate had direct toxic effects. These studies demonstrate that guinea pigs with spinal cord injury constitute a useful model to study the mechanisms that increase central nervous system (CNS) QUIN levels in conditions of CNS inflammation, and to evaluate the neurochemical and neurological effects of agents designed to reduce the accumulations of QUIN and other potential pathogenic mediators within the CNS. The results are consistent with a contributory role for QUIN in the pathogenesis of secondary functional impairments following spinal cord injury, although the possibility that 4Cl-3HAA had additional effects independent of QUIN cannot be excluded. Further studies are required to determine whether the beneficial effects of 4Cl-3HAA are sustained. While it is unknown whether secondary inflammatory processes contribute significantly to neurological deficits in human spinal cord injury, strategies that reduce the accumulation of QUIN are worthy of consideration and evaluation as a therapeutic target.

6-Chloro-D,L-tryptophan, 4-chloro-3-hydroxyanthranilate and dexamethasone attenuate quinolinic acid accumulation in brain and blood following systemic immune activation.

Accumulations of the neurotoxin quinolinic acid (QUIN) occur in the brain and blood following immune activation and are attributed to increased metabolism of L-tryptophan through the kynurenine pathway. Systemic administration of 4-chloro-3-hydroxyanthranilate (an inhibitor of 3-hydroxyanthranilate-3,4-dioxygenase), 6-chloro-D,L-tryptophan (a substrate of the kynurenine pathway) and dexamethasone (an anti-inflammatory agent) attenuated the accumulation of QUIN in the brain and blood following systemic pokeweed mitogen administration to mice. 6-Chloro-D,L-tryptophan and dexamethasone also attenuated the increases in brain and lung indoleamine-2,3-dioxygenase activity and elevations in plasma L-kynurenine levels. We conclude that QUIN formation can be modified by drugs which act at different levels of the cascade of events that link immune stimulation to increased kynurenine pathway metabolism.

4-Chloro-3-hydroxyanthranilate inhibits quinolinate production in the rat hippocampus in vivo.

Quinolinic acid (QUIN) is a potential pathogen in a variety of excitotoxic and neuroviral brain diseases. In the present study, the ability of the QUIN synthesis inhibitor 4-chloro-3-hydroxyanthranilic acid to attenuate the production of QUIN was assessed in the hippocampus of awake rats. To this end, QUIN's immediate bioprecursor 3-hydroxyanthranilic acid (30 microM) was applied through a microdialysis probe, and QUIN production was monitored hourly in the perfusate. After 3 h, 4-chloro-3-hydroxyanthranilic acid (3 microM-3 mM) was included in the perfusion medium, and dialysis was continued for another 3 h. The drug caused dose-dependent inhibition of QUIN neosynthesis, with an apparent IC50 value of 32 microM. Discontinuation of drug administration, with continued perfusion of 3-hydroxyanthranilic acid, revealed that the drug effect was reversible. Intravenous application of 4-chloro-3-hydroxyanthranilic acid (14 mg/kg) resulted in a significant decrease in extracellular QUIN, reaching a nadir of 67% of saline-treated controls after 3 h. The data indicate that both intracerebral and systemic administration of 4-chloro-3-hydroxyanthranilic acid effectively interferes with QUIN production in the rat brain. The results suggest that QUIN synthesis inhibitors such as 4-chloro-3-hydroxyanthranilic acid may become of value in brain diseases that are caused by hyperphysiological quantities of QUIN.

A mechanism of quinolinic acid formation by brain in inflammatory neurological disease. Attenuation of synthesis from L-tryptophan by 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate.

Quinolinic acid (QUIN), kynurenic acid (KYNA) and L-kynurenine (L-KYN) are neuroactive kynurenine pathway metabolites that accumulate in inflammatory neurological diseases. These increases were attributed to the induction of indoleamine-2,3-dioxygenase (IDO), the enzyme that converts L-tryptophan into L-KYN. Direct conversion of L-tryptophan into QUIN by brain tissue occurs in conditions of CNS inflammation, but not by normal brain tissue. To investigate whether increased activity of enzymes distal to IDO may determine L-KYN conversion to QUIN, rhesus macaques were inoculated with poliovirus directly into the spinal cord, as a model of focal inflammatory neurological disease (FASEB J. 6, 2977-2989, 1992). Induction of spinal cord IDO (35.9-fold) accompanied smaller, but proportional increases in kynurenine-3-hydroxylase (2.4-fold) and kynureninase (2.3-fold) activities, which were correlated to CSF and tissue QUIN levels, as well as to measures of inflammatory lesions. 3-Hydroxyanthranilate-3,4-dioxygenase activity was unchanged. Cerebrospinal fluid KYNA levels increased in proportion to both IDO activity and L-KYN accumulation, though kynurenine aminotransferase activity was unaffected. Cerebrospinal fluid neopterin, a marker of macrophage and immune activation, accumulated in proportion to the responsive enzymes and metabolites. The cell types involved in producing QUIN were investigated in vitro. Human foetal brain cultures consisting of astrocytes and neurons converted large quantities of [13C6]L-tryptophan into L-KYN when stimulated by gamma-interferon, but very little [13C6]QUIN was formed unless macrophages (THP-1 cells) were first added to the cultures (to model a key component of brain inflammation). [13C6]L-Tryptophan was converted into [13C6]QUIN by either gamma-interferon stimulated macrophages, or following intracisternal administration into poliovirus-infected macaques. Inhibitors of the kynurenine pathway, 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilic acid, attenuated [13C6]QUIN formation by macrophages, and when co-infused with [13C6]L-tryptophan into poliovirus-infected macaques. These results suggest roles for increased activities of IDO, kynurenine-3-hydroxylase and kynureninase in accelerating the synthesis of QUIN, L-KYN and KYNA in conditions of brain inflammation. Macrophage infiltrates, and perhaps microglia, are important sources of QUIN, whereas constitutive brain cells and macrophages are sources of L-KYN. Drugs that inhibit kynurenine pathway enzymes attenuate QUIN formation in the CNS, and provide tools to examine the consequences of reduced QUIN accumulation.

4-Chloro-3-hydroxyanthranilate, 6-chlorotryptophan and norharmane attenuate quinolinic acid formation by interferon-gamma-stimulated monocytes (THP-1 cells).

Accumulation of quinolinic acid and L-kynurenine occurs in the brain and/or blood following immune activation, and may derive from L-tryptophan following induction of indoleamine 2,3-dioxygenase and other kynurenine-pathway enzymes. In the present study a survey of various cell lines derived from either brain or systemic tissues showed that, while all cells examined responded to interferon-gamma by increased conversion of L-[13C6]tryptophan into L-kynurenine (human: B-lymphocytes, neuroblastoma, glioblastoma, lung, liver, kidney; rat brain: microglia, astrocytes and oligodendrocytes), only macrophage-derived cells (peripheral-blood mononuclear cells; THP-1, U-937) and certain liver cells (SKHep1) synthesized [13C6]quinolinic acid. Tumour necrosis factor-alpha enhanced the effects of interferon-gamma in THP-1 cells. Norharmane, 6-chloro-DL-tryptophan and 4-chloro-3-hydroxyanthranilate attenuated quinolinic acid formation by THP-1 cells with IC50 values of 51 microM, 58 microM and 0.11 microM respectively. Norharmane and 6-chloro-DL-tryptophan attenuated L-kynurenine formation with IC50 values of 43 microM and 51 microM respectively, whereas 4-chloro-3-hydroxyanthranilate had no effect on L-kynurenine accumulation. The reductions in L-kynurenine and quinolinic acid formation are consistent with the reports that norharmane is an inhibitor of indoleamine 2,3-dioxygenase, 6-chloro-DL-tryptophan is metabolized through the kynurenine pathway, and 4-chloro-3-hydroxyanthranilate is an inhibitor of 3-hydroxyanthranilate 3,4-dioxygenase. These results suggest that many tissues may contribute to the production of L-kynurenine following indoleamine 2,3-dioxygenase induction and immune activation. Quinolinic acid may be directly synthesized from L-tryptophan in both macrophages and certain types of liver cells, although uptake of quinolinic acid precursors from blood may contribute to quinolinic acid synthesis in cells that cannot convert L-kynurenine into quinolinic acid.