Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

Cloricromene effect on the enzyme activities of the tryptophan-nicotinic acid pathway in diabetic/hyperlipidemic rabbits

Since alterations of tryptophan metabolism have been reported in diabetes and atherosclerosis, it was thought of interest to investigate any role of cloricromene through the influence on the oxidative metabolism of the amino acid by using diabetic/hyperlipidemic rabbits.Male 4-month-old New Zealand white rabbits, fed a diet enriched with 1% cholesterol and 10% corn oil, were made diabetic with alloxan. During the hyperlipidemic diet, a group of rabbits was treated with cloricromene (10 mg/kg/day subcutaneously plus 1.5 mg/kg/day intravenously, for 5 weeks). The other group received saline. Normometabolic New Zealand rabbits fed standard diet, treated or not with cloricromene, were used as control.The specific activities of liver tryptophan 2,3-dioxygenase and small intestine indole 2,3-dioxygenase were not significantly changed by the drug treatment. Also the specific activities of other enzymes of the kynurenine pathway in the liver and kidneys, specifically kynurenine 3-monooxygenase, kynureninase and kynurenine-oxoglutarate transaminase, did not show any significant difference in both tissues between the two groups of rabbits. On the contrary, 3-hydroxyanthranilate 3,4-dioxygenase activity in the liver of diabetic/hyperlipidemic rabbits and control rabbits treated with cloricromene showed a slight increase in comparison with untreated animals. Conversely, the specific activity of the enzyme in kidneys was not affected by the drug treatment in diabetic/hyperlipidemic animals but was reduced in controls. Aminocarboxymuconate-semialdehyde decarboxylase specific activity remained unchanged in the liver following cloricromene treatment, instead the specific activity of the enzyme in the kidneys of the diabetic/hyperlipidemic rabbits was significantly increased by the drug, with a value more than double in comparison to untreated animals. The activity of the scavenger enzyme Cu/Zn superoxide dismutase (Cu/Zn SOD) in the small intestine was also determined and found significantly increased of about twice as much in the group of diabetic/hyperlipidemic rabbits treated with cloricromene.In conclusion, in diabetic/hyperlipidemic rabbits, cloricromene appeared to influence the enzymes involved in the last steps of tryptophan oxidative metabolism through the kynurenine pathway. This, together with the antioxidant action through the activation of Cu/Zn SOD, might deserve further investigation for evaluating any link between the observed experimental findings at the level of the kynurenine pathway and the clinical effect of the drug.     

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.     

The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes.

The metabolism of L-tryptophan by isolated liver cells prepared from control, adrenalectomized, glucocorticoid-treated, acute-diabetic, chronic-diabetic and insulin-treated chronic-diabetic rats was studied. Liver cells from adrenalectomized rats metabolized tryptophan at rates comparable with the minimum diurnal rates of controls, but different from rates determined for cells from control rats 4h later. Administration of dexamethasone phosphate increased the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) 7-8-fold, and the flux through the kynurenine pathway 3-4-fold, in cells from both control and adrenalectomized rats. Increases in flux through kynureninase (EC 3.7.1.3) and to acetyl-CoA can be explained in terms of increased substrate supply from tryptophan 2,3-dioxygenase. The metabolism of tryptophan was increased 3-fold in liver cells isolated from acutely (3 days) diabetic rats, with a 7-8-fold increase in the maximal activity of tryptophan 2,3-dioxygenase. The oxidation of tryptophan to CO2 and metabolites of the glutarate pathway increased 4-5-fold, consistent with an increase in picolinate carboxylase (EC 4.1.1.45) activity. Liver cells isolated from chronic (10 days) diabetic rats metabolized tryptophan at rates comparable with those of cells from acutely diabetic rats, but with a 50% decrease in the activity of tryptophan 2,3-dioxygenase. The proportion of flux from tryptophan 2,3-dioxygenase to acetyl-CoA, however, was increased by 50%; this was indicative of further increases in the activity of picolinate carboxylase. Administration of insulin partially reversed the effects of chronic diabetes on the activity of tryptophan 2,3-dioxygenase and flux through the kynurenine pathway, but had no effect on the increased activity of picolinate carboxylase. The role of tryptophan 2,3-dioxygenase in regulating the blood tryptophan concentration is discussed with reference to its sensitivity to the above conditions.     

Comparative effects of oxygen on indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase of the kynurenine pathway

Indoleamine 2,3-dioxygenase (IDO) reacts with either oxygen or superoxide and tryptophan (trp) or other indoleamines while tryptophan 2,3-dioxygenase (TDO) reacts with oxygen and is specific for trp. These enzymes catalyze the rate-limiting step in the kynurenine (KYN) pathway from trp to quinolinic acid (QA) with TDO in kidney and liver and IDO in many tissues, including brain where it is low but inducible. QA, which does not cross the blood-brain barrier, is an excitotoxin found in the CNS during various pathologies and is associated with convulsions. We proposed that HBO-induced convulsions result from increased flux through the KYN pathway via oxygen stimulation of IDO. To test this, TDO and IDO of liver and brain, respectively, of Sprague Dawley rats were assayed with oxygen from 0 to 6.2 atm HBO. TDO activity was appreciable at even 30 microM oxygen and rose steeply to a maximum at 40 microM. Conversely, IDO had almost no detectable activity at or below 100 microM oxygen and maximum activity was not reached until about 1150 microM. (Plasma contains about 215 microM oxygen and capillaries about 20 microM oxygen when rats breathe air.) KYN was 60% higher in brains of HBO-convulsed rats compared to rats breathing air. While the oxygen concentration inside cells of rats breathing air or HBO is not known precisely, it is clear that the rate-limiting, IDO-catalyzed step in the brain KYN pathway (but not liver TDO) can be greatly accelerated in rats breathing HBO.     

Inflammation and serotonin deficiency in major depressive disorder: molecular docking of antidepressant and anti-inflammatory drugs to tryptophan and indoleamine 2,3-dioxygenases

The roles of the kynurenine pathway (KP) of tryptophan (Trp) degradation in serotonin deficiency in major depressive disorder (MDD) and the associated inflammatory state are considered in the present study. Using molecular docking in silico, we demonstrate binding of antidepressants to the crystal structure of tryptophan 2,3-dioxygenase (TDO) but not to indoleamine 2,3-dioxygenase (IDO). TDO is inhibited by a wide range of antidepressant drugs. The rapidly acting antidepressant ketamine does not dock to either enzyme but may act by inhibiting kynurenine monooxygenase thereby antagonising glutamatergic activation to normalise serotonin function. Antidepressants with anti-inflammatory properties are unlikely to act by direct inhibition of IDO but may inhibit IDO induction by lowering levels of proinflammatory cytokines in immune-activated patients. Of six anti-inflammatory drugs tested, only salicylate docks strongly to TDO and apart from celecoxib, the other five dock to IDO. TDO inhibition remains the major common property of antidepressants and TDO induction the most likely mechanism of defective serotonin synthesis in MDD. TDO inhibition and increased free Trp availability by salicylate may underpin the antidepressant effect of aspirin and distinguish it from other nonsteroidal anti-inflammatory drugs. The controversial findings with IDO in MDD patients with an inflammatory state can be explained by IDO induction being overridden by changes in subsequent KP enzymes influencing glutamatergic function. The pathophysiology of MDD may be underpinned by the interaction of serotonergic and glutamatergic activities.     

The metabolism of L-tryptophan by liver cells prepared from adrenalectomized and streptozotocin-diabetic rats.

1. The metabolism of L-tryptophan by liver cells prepared from fed normal, adrenalectomized and streptozotocin-diabetic rats was studied. 2. At physiological concentrations (0.1 mM), the rate of oxidation of tryptophan by tryptophan 2,3-dioxygenase was 3-fold greater in liver cells from diabetic rats than in those from fed rats. In liver cells from diabetic rats, oxidation of tryptophan to CO2 and metabolites of the glutarate pathway was increased 7-fold. Quinolinate synthesis was decreased by 50%. These findings are consistent with an increase in picolinate carboxylase activity. 3. Rates of metabolism of 0.1 mM-tryptophan by hepatocytes from fed and adrenalectomized rats were similar. 4. In all three types of cell preparation, fluxes through tryptophan 2,3-dioxygenase with 2.5 mM-tryptophan were 7-fold greater than those obtained with 0.1 mM-tryptophan. Tryptophan 2,3-dioxygenase and kynureninase fluxes in hepatocytes from fed and adrenalectomized rats were comparable, whereas those in liver cells from diabetic rats were increased 2.5-fold and 3.3-fold respectively. Picolinate carboxylase activities of liver cells from diabetic rats were 15-fold greater than those of cells from fed rats, but rates of quinolinate synthesis were unchanged. 5. It is concluded that: (i) adrenal corticosteroids are not required for the maintenance of basal activities of the kynurenine pathway, whereas (ii) chronic insulin deficiency produces changes in both the rate of oxidation and metabolic fate of tryptophan carbon.     

Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease

The kynurenine pathway is the major route for the oxidative degradation of the amino acid tryptophan. Activity of the pathway is involved in several disease conditions, both in the periphery and the central nervous system, including cancer, inflammatory disorders, neurological conditions, psychiatric disorders and neurodegenerative diseases. Three enzymes are now known to catalyze the first and rate-limiting step in the catabolism of tryptophan along this pathway: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO, subsequently named IDO1), both of which have been extensively studied, and a third enzyme, indoleamine 2,3-dioxygenase 2 (IDO2), a relative newcomer to the kynurenine pathway field. The adjuvant chemotherapeutic agent, 1-methyl-d-tryptophan, was intially suggested to target IDO2, implying involvement of IDO2 in tumorigenesis. Subsequently this compound has been suggested to have alternative actions and the physiological and pathophysiological roles of IDO2 are unclear. Targeted genetic interventions and selective inhibitors provide approaches for investigating the biology of IDO2. This review focuses on the current knowledge of IDO2 biology and discusses tools that will assist in further characterizing the enzymes of the kynurenine pathway.     

Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure.

We investigated L-kynurenine distribution and metabolism in rats with experimental chronic renal failure of various severity, induced by unilateral nephrectomy and partial removal of contralateral kidney cortex. In animals with renal insufficiency the plasma concentration and the content of L-tryptophan in homogenates of kidney, liver, lung, intestine and spleen were significantly decreased. These changes were accompanied by increase activity of liver tryptophan 2,3-dioxygenase, the rate-limiting enzyme of kynurenine pathway in rats, while indoleamine 2,3-dioxygenase activity was unchanged. Conversely, the plasma concentration and tissue content of L-kynurenine, 3-hydroxykynurenine, and anthranilic, kynurenic, xanthurenic and quinolinic acids in the kidney, liver, lung, intestine, spleen and muscles were increased. The accumulation of L-kynurenine and the products of its degradation was proportional to the severity of renal failure and correlated with the concentration of renal insufficiency marker, creatinine. Kynurenine aminotransferase, kynureninase and 3-hydroxyanthranilate-3,4-dioxygenase activity was diminished or unchanged, while the activity of kynurenine 3-hydroxylase was significantly increased. We conclude that chronic renal failure is associated with the accumulation of L-kynurenine metabolites, which may be involved in the pathogenesis of certain uremic syndromes.     

Molecular evolution of bacterial indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD⁺). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD⁺, like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K_m, V_max and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD⁺) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking the kynU gene suggests its IDO has a function similar to eukaryotic enzymes.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

Synthesis of novel tryptanthrin derivatives as dual inhibitors of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are promising drug development targets due to their implications in pathologies such as cancer and neurodegenerative diseases. The search for IDO1 inhibitor has been intensely pursued but there is a paucity of potent TDO and IDO1/TDO dual inhibitors. Natural product tryptanthrin has been confirmed to bear IDO1 and/or TDO inhibitory activities. Herein, twelve novel tryptanthrin derivatives were synthesized and evaluated for the IDO1 and TDO inhibitory potency. All of the compounds were found to be IDO1/TDO dual inhibitors, in particular, compound 9a and 9b bore IDO1 inhibitory activity similar to that of INCB024360, and compound 5a and 9b had remarkable TDO inhibitory activity superior to that of the well-known TDO inhibitor LM10. This work enriches the collection of IDO1/TDO dual inhibitors and provides chemical molecules for potential development into drugs.     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.     

Mechanism of Delayed Increases in Kynurenine Pathway Metabolism in Damaged Brain Regions Following Transient Cerebral Ischemia

Abstract: Delayed increases in the levels of an endogenous N-methyl-D-aspartate receptor agonist, quinolinic acid (QUIN), have been demonstrated following transient ischemia in the gerbil and were postulated to be secondary to induction of indoleamine-2,3-dioxygenase (IDO) and other enzymes of the L-tryptophan-kynurenine pathway. In the present study, proportional increases in IDO activity and QUIN concentrations were found 4 days after 10 min of cerebral ischemia, with both responses in hippocampus > striatum > cerebral cortex > thalamus. These increases paralleled the severity of local brain injury and inflammation. IDO activity and QUIN concentrations were unchanged in the cerebellum of postischemic gerbils, which is consistent with the preservation of blood flow and resultant absence of pathology in this region. Blood QUIN and L-kynurenine concentrations were not affected by ischemia. Brain tissue QUIN levels at 4 days postischemia exceeded blood concentrations, minimizing a role for breakdown of the blood–brain barrier. Marked increases in the activity of kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase were also detected in hippocampus but not in cerebellum on day 4 of recirculation. In vivo synthesis of [¹³C₆]QUIN was demonstrated, using mass spectrometry, in hippocampus but not in cerebellum of 4-day postischemic animals 1 h after intracisternal administration of L-[¹³C₆]tryptophan. However, accumulation of QUIN was demonstrated in both cerebellum and hippocampus of control gerbils following an intracisternal injection of 3-hydroxyanthranilic acid, which verifies the availability of precursor to both regions when administered intracisternally. Notably, although IDO activity and QUIN concentrations were unchanged in the cerebellum of ischemic gerbils, both IDO activity and QUIN content were increased in cerebellum to approximately the same degree as in hippocampus, striatum, cerebral cortex, and thalamus 24 h after immune stimulation by systemic pokeweed mitogen administration, demonstrating that the cerebellum can increase IDO activity and QUIN content in response to immune activation. No changes in kynurenic acid concentrations in either hippocampus, cerebellum, or cerebrospinal fluid were observed in the postischemic gerbils compared with controls, in accordance with the unaffected activity of kynurenine aminotransferase activity. Collectively, these results support roles for IDO, kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase in accelerating the conversion of L-tryptophan and other substrates to QUIN in damaged brain regions following transient cerebral ischemia. Immunocytochemical results demonstrated the presence of macrophage infiltrates in hippocampus and other brain regions that parallel the extent of these biochemical changes. We hypothesize that increased kynurenine pathway metabolism after ischemia reflects the presence of macrophages and other reactive cell populations at sites of brain injury.     

Extension of life span of Drosophila melanogaster by the inhibitors of tryptophan-kynurenine metabolism

Upregulation of kynurenine (KYN) formation from tryptophan (TRY) was associated with aging in animal and human studies. TRY-KYN metabolism is affected by the activities of TRY 2,3-dioxygenase 2 (TDO) and AT P-binding cassette (ABC) transporter regulating TRY access to intracellular TDO. We studied the effects of TDO inhibitor, alpha-methyl tryptophan (aMT) and ABC transported inhibitor, 5-methyl tryptophan (5MT), on the life span of wild strain female Drosophila flies (Oregon-R). aMT and 5MT prolonged mean and maximum life span (by 27% and 43%, and 21% and 23%, resp.). The present results are the first observation of the extension of life span of Drosophila melanogaster by inhibitors of TRY-KYN metabolism, and in line with literature and previous studies on prolonged life span of TDO- and ABC-deficient female Drosophila mutants. Inhibition of TDO and ABC transporter activity might offer the new target for anti-aging interventions.     

Quantification of the importance of individual steps in the control of aromatic amino acid metabolism.

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.     

Reliable chromatographic assay for measuring of indoleamine 2,3-dioxygenase 1 (IDO1) activity in human cancer cells

The kynurenine pathway is the major tryptophan degradation routes generating bioactive compounds important in physiology and diseases. Depending on cell type it is initiated enzymatically by tryptophan-2,3-dioxygenase (TDO) or indoleamine-2,3-dioxygenase 1 and 2 (IDO1 and IDO2) to yield N-formylkynurenine as the precursor of further metabolites. Herein, we describe an accurate high-pressure liquid chromatography coupled with a diode array detector (HPLC-DAD) method to serve for IDO1 activity determination in human cancer cells cultured in vitro. Enzymatic activity was expressed as the rate of ʟ-kynurenine generation by 1 mg of proteins obtained from cancer cells. Our approach shows the limit of detection and limit of quantification at 12.9 and 43.0 nM Kyn, respectively. Applicability of this method was demonstrated in different cells (ovarian and breast cancer)exposed to various conditions and has successfully passed the validation process. This approach presents a useful model to study the role of kynurenine pathway in cancer biology.     

Extension of life span of Drosophila melanogaster by the inhibitors of tryptophan-kynurenine metabolism

Upregulation of kynurenine (KYN) formation from tryptophan (TRY) was associated with aging in animal and human studies. TRY - KYN metabolism is affected by the activities of TRY 2,3-dioxygenase 2 (TDO) and ATP-binding cassette (ABC) transporter regulating TRY access to intracellular TDO. We studied the effects of TDO inhibitor, alpha-methyl tryptophan (aMT), and ABC transported inhibitor, 5-methyl tryptophan (5MT), on the life span of wild strain female Drosophila flies (Oregon-R). aMT and 5MT prolonged mean and maximum life span (by 27% and 43%, and 21% and 23%, resp.). The present results are the first observation of the extension of life span of Drosophila melanogaster by inhibitors of TRY - KYN metabolism, and in line with literature and previous studies on prolonged life span of TDO- and ABC-deficient female Drosophila mutants. Inhibition of TDO and ABC transporter activity might offer the new target for anti-aging and anti-AAMPD interventions.     

Evolution of Vertebrate Indoleamine 2,3-Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the same reaction, the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed among life-forms, being found not only in eukaryotes but also in bacteria. In contrast, IDO has been found only in mammals and yeast to date. However, recent genome and EST projects have identified IDO homologues in non-mammals and found an IDO paralogue that is expressed in mice. In this study, we cloned the frog and fish IDO homologues and the mouse IDO paralogue, and characterized their enzymatic properties using recombinants. The IDOs of lower vertebrates and the mouse IDO paralogue had IDO activity but had 500–1000 times higher K _m values and very low enzyme efficiency compared with mammalian IDOs. It appears that L-Trp is not a true substrate for these enzymes in vivo, although their actual function is unknown. On the phylogenetic tree, these low-activity IDOs, which we have named “proto-IDOs,” formed a cluster that was distinct from the mammalian IDO cluster. The IDO and proto-IDO genes are present tandemly on the chromosomes of mammals, including the marsupial opossum, whereas only the proto-IDO gene is observed in chicken and fish genomes. These results suggest that (mammalian) IDOs arose from proto-IDOs by gene duplication that occurred before the divergence of marsupial and eutherian (placental) mammals in mammalian evolutionary history.