Evidence that kynurenine pathway metabolites mediate hyperbaric oxygen-induced convulsions.

Metabolism of tryptophan (TRP) through the kynurenine (KYN) pathway in brain, liver, and kidney produces intermediates including the neuroactive agonist quinolinic acid (QA) and the antagonists kynurenic acid (KA) and anthranilic acid (AA) for N-methyl D-aspartate (NMDA) receptors in the central nervous system. We hypothesized that elevated concentrations of QA, KA, or AA can moderate the convulsions that are observed during exposure of rats to hyperbaric oxygen (HBO). We found that i.p. administration of TRP or KYN (both of which cross the blood-brain barrier) had no effect on HBO-induced seizures. However, AA (administered i.p.) or gavage administration of the KYN pathway blocking drug Ro 61-8048, both of which enter the brain from the circulatory system, affect the time to first convulsion and/or coma during HBO in a manner consistent with a modulatory role for seizure activity.

Tryptophan metabolism through the kynurenine pathway in rat brain and liver slices.

We hypothesized that hyperbaric oxygen (HBO) enhances tryptophan (TRP) flux through the kynurenine (KYN) pathway because oxygen is a substrate for four pathway enzymes. Our objective was to compare the biosynthesis of KYN pathway intermediates by rat brain and liver slices with air or HBO as the gas phase. One-millimeter thick liver and brain slices were obtained from male Sprague-Dawley rats and incubated individually in chambers containing Hanks'-HEPES- buffer with (3)H-TRP (30 Ci/mmol) for 2 h (37 degrees C) in either room air or oxygen (1.2 or 5.2 atmospheres absolute [ATA] oxygen). After incubation, tissue was snap-frozen and analyzed for protein content while medium was extracted for high-performance liquid chromatography analysis. Radiolabeled nicotinamide adenine dinucleotide (NAD) was produced by brain and liver; liver (with air as the gas phase) also produced quinolinic acid (QA). HBO at 1.2 and 5.2 ATA caused increased QA and NAD from liver slices. HBO did not affect KYN metabolism in brain slices, although there was decreased production of NAD during high oxygen. We conclude that rat brain and liver contain the complete KYN pathway and that HBO enhances KYN flux in liver tissue.

Effects of oxygen on kynurenine-3-monooxygenase activity.

Kynurenine-3-monooxygenase (KM), the third enzyme in the kynurenine (KYN) pathway from tryptophan to quinolinic acid (QA), is a monooxygenase requiring oxygen, NADPH and FAD for the catalytic oxidation of L-kynurenine to 3-hydroxykynurenine and water. KM is innately low in the brain and similar in activity to indoleamine oxidase, the rate-limiting pathway enzyme. Accumulation in the CNS of QA, a known excitotoxin, is proposed to cause convulsions in several pathologies. Thus, we theorized that hyperbaric oxygen (HBO) induced convulsions arise from increased QA via oxygen K, effects on this pathway [Brown OR, Draczynska-Lusiak. Oxygen activation and inactivation of quinolinate-producing and iron-requiring 3-hydroxyanthranilic acid oxidase: a role in hyperbaric oxygen-induced convulsions? Redox Report 1995; 1: 383-385]. To complement prior studies on the effects of oxygen on pathway enzymes, in this paper we report the effects of oxygen on KM. Brain and liver KM enzyme are not known to be identical, and some systemically-produced KYN pathway intermediates can permeate the brain and might stimulate the brain pathway. Thus, KM from both brain and liver was assayed at various oxygen substrate concentrations to evaluate, in vitro, the potential effects of increases in oxygen, as would occur in mammals breathing therapeutic and convulsive HBO. In crude tissue extracts, KM was not activated during incubation in HBO up to 6 atm. The effects of oxygen as substrate on brain and liver KM activity was nearly identical: activity was nil at zero oxygen with an apparent oxygen Km of 20-22 microM. Maximum KM activity occurred at about 1000 microM oxygen and decreased slightly to plateau from 2000 to 8000 microM oxygen. This compares to approximately 30-40 microM oxygen typically reported for brain tissue of humans or rats breathing air, and an unknown but surely much lower value (perhaps below 1 microM) intracellularly at the site of KM. Thus HBO, as used therapeutically and at convulsive pressures, likely stimulates flux through the KM-catalyzed step of the KYN pathway in liver and in brain and could increase brain QA, by Km effects on brain KM, or via increased KM pathway intermediates produced systemically (in liver) and transported into the brain.

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.

Hydroxyl radicals detected via brain microdialysis in rats breathing air and during hyperbaric oxygen convulsions.

Microdialysis was done on 300-400 g, awake, male rats with microdialysis probes inserted through guide cannulas into the striatum (Bregma co-ordinates A 0.5, L 2.9, D -4.0 for guide cannulas implanted 5 days previously). Rats were exposed to hyperbaric oxygen (HBO; 6 atm absolute, 5 atm gauge pressure of oxygen with carbon dioxide absorbed by soda lime). Artificial cerebrospinal fluid (CSF) containing 5 mM sodium salicylate was perfused at 1 microl/min and collected over sequential 10 min intervals with rats breathing air, then HBO, and after decompression. Times to convulsions and duration and severity of convulsions were observed and recorded. CSF samples were analyzed for 2,3- and 2,5-dihydroxybenzoic acid (DHBA), reaction products of hydroxyl radicals with salicylate, by HPLC and compared to authentic standards. Recovery of DHBAs was 48% from fluid surrounding microdialysis probes, based on in vitro tests. The average time to the first convulsion was 21 min and rats convulsed an average of 4 times during 40 min in HBO. There were no significant differences in hydroxyl radical production by this protocol during any of the 10 min collection periods in air or HBO (average in pmoles for 10 microl of all samples: 2,3-DHBA = 7.0 +/- 2.5 and 2,5-DHBA = 11.3 +/- 4.1). The failure to detect an increase in hydroxyl radicals in HBO prior to or during convulsions appears valid since each rat served as its own control.

Effects of oxygen on 3-hydroxyanthranilate oxidase of the kynurenine pathway.

Iron containing 3-Hydroxyanthranilate oxidase (3HAO) converts 3-hydroxyanthranilate (3HAA) and dioxygen into a precursor which spontaneously converts to quinolinic acid (QA). 3HAO participates in de novo biosynthesis of NAD in mammalian kidney and liver, and it is present in low concentrations in brain where its function is controversial. However, QA increases in spinal fluid and is associated with convulsions in AIDS dementia, Huntington's disease, and CNS inflammation. QA is a known N-methyl, D-aspartate receptor agonist and excitotoxin that causes convulsions when injected into the brain. Hyperbaric oxygen (HBO) also causes convulsions and we investigated the interrelationships among the stimulating and toxic effects of oxygen and the role of iron in vitro using rat liver enzyme which is reported to be identical to brain enzyme and is more abundant. 3HAO requires dioxygen as a substrate but it was inactivated approximately 40% by 5.2 atm HBO in vitro in 15 min. The apparent Km was 2.6 x 10(-4) M for oxygen and 5 x 10(-5) M for 3HAA, and these values did not change for enzyme that was half-inactivated by HBO oxygen. Thus, oxygen-inactivation appears to be all-or-none for individual enzyme molecules. Freshly prepared enzyme was activated about 3-fold by incubation with acidic iron. Iron-staining of 3HAO, separated by gel electrophoresis after partial purification by FPLC, showed that loss of iron and loss of enzyme activity during HBO exposure were correlated. The apparent oxygen Km of 3HAO is far higher than the oxygen concentration in brain cells. Thus, 3HAO is capable of being stimulated initially in animals breathing HBO, and subsequently of being inactivated with potential significance for brain QA and convulsions.

HPLC analysis of quinolinic acid, a NAD biosynthesis intermediate, after fluorescence derivatization in an aqueous matrix.

Quinolinic acid (2,3-pyridine dicarboxylic acid), a biological intermediate in nicotinamide adenine dinucleotide (NAD) biosynthesis in microbes and mammals and a brain excitotoxin, is not fluorescent nor electrochemically active and its detection sensitivity by UV absorption is comparatively low. Quinolinic acid was successfully derivatized in water-based samples by monodansylcadaverine, a fluorescence tag, and analysed by high-performance liquid chromatography (HPLC). No extraction procedure was needed and quinolinic acid was activated by water-soluble carbodiimide and derivatized under mild conditions. As little as 3 pmol (500 pg) of quinolinic acid in 5 microliter of artificial cerebrospinal fluid sample volume could be derivatized and detected at a signal to noise ratio of 3:1. Thus, detection on a mass basis by HPLC after fluorescence derivatization is about 300 times as sensitive as direct determination of quinolinic acid by UV absorbance (500 pg vs 150 ng). A variety of activators, fluorescent tags and reaction solvents and conditions were tested but found to be less effective.

Protection by selective amino acid solutions against doxorubicin induced growth inhibition of Escherichia coli.

1. Incubation of Escherichia coli with 0.7 mM doxorubicin in MBS-glucose medium resulted in complete growth inhibition, an inhibition that was blocked by placing specific amino acids (AA) in the medium. 2. The mechanism of protection by AA was similar to that reported previously for cells poisoned by hyperoxia and by paraquat, e.g. of 20 common AA, ten percent, ten do not and the branched-chair AA are among those required for inhibition. 3. Unlike hyperoxia and paraquot stringency which caused elevation of intracellular concentrations of guanosine tetraphosphate (ppGpp), doxorubicin inhibition did not elevate ppGpp. 4. Concentrations of ppGpp were increased by isoleucine starvation as expected, and the subsequent addition of doxorubicin did not abolish that increase; however, pretreatment with doxorubicin prevented the induction of stringency by isoleucine starvation. 5. This suggests that doxorubicin directly inhibits ppGpp synthesis or protein biosynthesis to leave tRNA loaded as is the case with chloramphenicol.

Dihydroxy-acid dehydratase, a [4Fe-4S] cluster-containing enzyme in Escherichia coli: effects of intracellular superoxide dismutase on its inactivation by oxidant stress.

Dihydroxy-acid dehydratase (DHAD) has a [4Fe-4S] cluster and is reported to be facilely inactivated by oxidant stress. To directly assess the biological effects in vivo of superoxide dismutase (SOD) on the oxidant sensitivity of DHAD, we used an Escherichia coli K-12 parent strain (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD. In the K-12 parent strain half the cellular DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 microM aerobic nitrofurantoin, or about 5 microM aerobic paraquat (PQ) and in about 1 min at 10 microM aerobic PQ. Oxygen and metabolism were required for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-free extracts did not restore DHAD activity. The Km was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respectively, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponential, aerobic growth. Thus, active site oxidative impairment of individual enzyme molecules apparently was all-or-none. DHAD activity was greatly decreased when measured in extracts made from strains that lacked both SODs unless SOD was added to cell suspensions before extracts were made. DHAD was more sensitive in strains lacking both SODs than in the parent strain to inactivation by aerobic PQ and HBO. Anaerobic (compared to aerobic) growth increased DHAD specific activity by 20% or less in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHAD was increased 60%. DHAD was partially inactivated by the oxidant stress of aerobic growth, but remained in a form detectable by DHAD antibody, and the ratio of active to inactive DHAD decreased greatly in cells lacking SOD. Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low levels of PQ.

Quantitative effects of redox-cycling chemicals on the oxidant-sensitive enzyme dihydroxy-acid dehydratase.

The [4Fe-4S] cluster-containing enzyme dihydroxy-acid dehydratase (DHAD) is susceptible to inactivation by dioxygen and active oxygen species including superoxide with an inactivation rate constant of 10(6) m-1 sec-1. Based on this property, DHAD was used to quantify and investigate the biological oxidant stress activity of various redox-cycling chemicals. Exponentially growing cultures of Escherichia coli were used as a sensitive source of the DHAD enzyme. The effects on DHAD of compounds with and without established redox activity, under aerobic and anaerobic (control) conditions were measured. Paraquat, juglone, nitrofurantoin, the nitrofuran related compound NF-963 which is 6,7-dihydro-3-(5-nitro-2-furyl-5H-imidazo-[2,1-b] thiazolium chloride), plumbagin, benzoquinone, duroquinone, hydralazine, and naphthalene inhibited DHAD activity and the concentrations required for 50% inhibition ranged from 3.5 microM for paraquat to 950 microM for naphthalene. Eleven other agents tested (including 4,4-dipyridyl which is a non-redox-cycling compound similar to paraquat and an extract and two compounds of plant origin) did not inhibit DHAD. The DHAD technique described is a useful means of detecting and comparing the oxidant-stress toxicity and mechanism of action of chemicals by a biological means on a quantitative scale relatable to paraquat.

Asparagine synthetase: an oxidant-sensitive enzyme in Escherichia coli.

Oxidant stress inhibits the growth of Escherichia coli, which is partially relieved by adding asparagine to the culture medium. Asparagine synthetase (AS), assayed using hydroxylamine as an amino donor, was decreased in a concentration-dependent manner by exposure of cultures to oxygen from near-anaerobic to hyperbaric oxygen (HBO) and by aerobic, but not by anaerobic, paraquat. The specific activity of AS was not decreased when cells were exposed to HBO without a carbon and energy source. HBO caused less AS inactivation in cells containing mutations in both superoxide dismutase (SOD) genes and producing no active SOD. Whether or not cells had catalase had no effect on HBO sensitivity of AS. Aerobic paraquat depressed AS less in cells lacking either catalase or superoxide dismutases. Cells which were decompressed following HBO poisoning had AS restored to normal activity whether or not chloramphenicol was present. These results indicate that asparagine synthetase is oxidant-sensitive; paraquat requires aerobic conditions and HBO requires energy metabolism for AS inactivation; and cells can repair oxidatively-damaged enzyme molecules. The failure of superoxide dismutase or catalase to protect AS suggests that its oxidant-inactivation in cells is not a simple effect of superoxide or hydrogen peroxide.

The inactivation of dihydroxy-acid dehydratase in Escherichia coli treated with hyperbaric oxygen occurs because of the destruction of its Fe-S cluster, but the enzyme remains in the cell in a form that can be reactivated.

The enzyme dihydroxy-acid dehydratase previously has been shown to be inactivated in vivo in Escherichia coli within minutes of exposure to hyperbaric O2. In this paper, we show its inactivation is due to the destruction of its catalytically active [4Fe-4S] cluster. The inactivation is not followed by an appreciable decrease in the amount of dihydroxy-acid dehydratase protein as determined by Western blots. Thus, the protein from the inactivated enzyme remains unproteolyzed in the cells. Dihydroxy-acid dehydratase activity recovers after the cells treated with hyperbaric O2 are returned to ambient oxygen. Since this recovery in activity is not accompanied by a significant increase in dihydroxy-acid dehydratase protein and is not prevented by chloramphenicol, it appears primarily to be due to reactivation of the previously inactivated enzyme. The reactivation occurs by reconstitution of the enzyme's Fe-S cluster. These results demonstrate that this enzyme can cycle between forms in which the Fe-S cluster is either present or absent. The facile ability to cycle between these two forms would be compatible with a regulatory role in addition to a catalytic role for this enzyme.

Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli.

The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m2/s at 310-400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.

Protein A of quinolinate synthetase is the site of oxygen poisoning of pyridine nucleotide coenzyme synthesis in Escherichia coli.

De novo biosynthesis of pyridine nucleotide coenzymes in Escherichia coli is initiated by an enzyme complex (quinolinate synthetase) containing protein B which converts L-aspartate into iminoaspartate and protein A, which then generates quinolinate on the pathway to the coenzymes. This complex has been shown to be poisoned by hyperbaric oxygen. We performed assays made dependent on both proteins B and A versus only protein A, using cell-free extracts of hyperbaric-oxygen poisoned and aerobically grown cells. The specific activities were reduced by similar amounts of 68% and 60%, respectively, when measured in assays made dependent on enzymes B and A virus only protein A that was derived from oxygen-poisoned extract. Thus, protein A is the oxygen-sensitive component.

Lung contains an inhibitor for nicotinatemononucleotide pyrophosphorylase (carboxylating) of NAD biosynthesis.

Rat, cow and foal lung extracts contained an inhibitor for the liver NAD biosynthetic-pathway enzyme, nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19]. The inhibitor was not dialyzable, was labile at 100 degrees C, was retained by a 30,000 dalton pore size Amicon membrane and, when partially purified by precipitation at 40-100% ammonium sulfate, inhibited the enzyme stoichiometrically. Lung reportedly does not contain nicotinate-mononucleotide pyrophosphorylase or make NAD de novo. However, the inhibitor would mask detection of the enzyme in lung extracts. We detected a low nicotinatemononucleotide pyrophosphorylase-like activity (0.003 +/- 0.001 nanomoles CO2 produced from quinolinic acid per mg of extract protein) in rat lung but none in foal or cow lung.

Skeletal muscle oxidative capacity, antioxidant enzymes, and exercise training.

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.

Overproduction of superoxide dismutase does not protect Escherichia coli from stringency-induced growth inhibition by 1mM paraquat.

Two plasmid-containing Escherichia coli strains which overproduce manganese superoxide dismutase by 4- to 5-fold and iron superoxide dismutase by about 7-fold were not more resistant than parent strains to 1 mM paraquat (a known generator of superoxide) as measured by effects on growth, survival and induction of stringency. These results indicate that overproduction of superoxide dismutase does not mitigate the growth-inhibitory effects of 1 mM paraquat, including those which are expressed through induction of the stringency mechanism.

Paraquat toxicity and pyridine nucleotide coenzyme synthesis: a data correction.

The decrease in pyridine nucleotide coenzymes which occurs during poisoning of Escherichia coli by hyperbaric oxygen or paraquat is not due to impairment of nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19] as was previously proposed (Brown, O.R. et al. Biochem. Biophys. Res. Commun. 91:982-990; 1979). This was shown directly using extracts of E. coli, prepared after exposure to 1 mM paraquat or 4.2 atmospheres of oxygen. The enzyme also was not impaired in Neurospora crassa by 1 mM paraquat. A naturally-occurring, non-dialyzable inhibitor of the enzyme was found in E. coli extracts. The inhibitor caused the erroneous, low nicotinatemononucleotide pyrophosphorylase (carboxylating) activities previously reported in extracts of E. coli poisoned by paraquat.

Persistence of gluconeogenesis in Escherichia coli poisoned by oxidant stress.

The poisoning (inhibition of growth rate) of Escherichia coli by 4.2 atmospheres of hyperbaric oxygen was less when the culture energy source was glucose, fructose-6-phosphate, or glycerol, compared to pyruvate, oxaloacetate, or amino acids. This was consistent with previous indirect data which pointed to impaired gluconeogenesis in the toxicity mechanism. However, the three enzymes unique to gluconeogenesis (fructose-1, 6-diphosphatase, phosphoenolpyruvate synthase and phosphoenolpyruvate carboxyl-kinase) were not decreased in specific activity to a biologically significant extent in cell-free extract of cells poisoned by hyperbaric oxygen. Net glycogen synthesis in vivo was not decreased from glycerol, pyruvate or oxaloacetate, compared to glucose in cells exposed to oxidant stress from hyperbaric oxygen or 1 mM aerobic paraquat with cells exposed as exponentially growing cells prior to assay or as resting cells during the assay.