In vivo identification of aflatoxin-induced free radicals in rat bile

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen. We have recently detected [via electron spin resonance (ESR) spectroscopy] free radicals in vivo in rat bile following AFB1 metabolism using the spin trapping [alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN)] technique. The aim of the present study was to identify the trapped free radical intermediates from the in vivo hepatic metabolism of AFB1. Rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (1 g/kg i.p.), and bile was collected over a period of 1 h at 20 min intervals. On-line high performance liquid chromatography (HPLC) coupled to ESR was used to identify an arachidonic acid-derived radical adduct of 4-POBN in rat bile, and a methyl adduct of 4-POBN from the reaction of hydroxyl radicals with carbon-13-labeled dimethyl sulfoxide ((13)C-DMSO). The effect of metabolic inhibitors, such as desferoxamine mesylate (DFO), an iron chelator, 2-dimethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF) 525A, a cytochrome P-450 inhibitor, and gadolinium chloride (GdCl(3)), a Kupffer cell inactivator, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in radical formation as a result of DFO, SKF525A and GdCl(3) inhibition. Trapped 4-POBN radical adducts were also detected in rat bile following the in vivo metabolism of aflatoxin-M1, one of the hydroxylated metabolites of AFB1.     

Light emission from tryptophan oxidation by hypobromous acid

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N‐formylkynurenine, kynurenine, 1,2,3,3a,8,8a‐hexahydro‐3a‐hydroxypyrrolo[2,3‐b]‐indole‐2‐carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α‐amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant. Copyright © 2012 John Wiley & Sons, Ltd.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes.

Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.     

Free Radical Metabolism of Alcohols by Rat Liver Microsomes

By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN.

The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP⁺ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P₄₅₀-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P₄₅₀ where they react with alkyl alcohol molecules.

The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.     

Trace transition metal-catalyzed reactions in the microsomal metabolism of alkyl hydrazines to carbon-centered free radicals.

Radical production from alkyl hydrazines (i.e. phenelzine and benzylhydrazine) in rat liver microsomes has been proposed to occur via cytochrome P-450-catalyzed one-electron oxidation followed by beta-scission of an alkyl radical. In microsomes treated with phenelzine (2-phenylethylhydrazine), NADPH, and the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN), the 4-POBN/2-phenylethyl radical adduct was detected by electron paramagnetic resonance spectroscopy. The addition of catalase and superoxide dismutase resulted in a 28.5 and 24% decrease in radical production, respectively. The concentration of the 4-POBN/2-phenylethyl radical adduct decreased significantly in the presence of metal chelators, i.e. EDTA, diethylenetriaminepentaacetic acid (DTPA), or deferoxamine mesylate. When phenelzine was incubated with deferoxamine mesylate-washed microsomes and NADPH in Chelex-treated incubation buffer, no significant radical adduct formation was detected. Addition of iron-chelator complexes (either Fe(3+)-DTPA or Fe(3+)-EDTA) greatly stimulated production of the 4-POBN/2-phenylethyl radical adduct in this system. These results show that the 2-phenylethyl radical produced from phenelzine in a microsomal system arises via a trace transition metal-catalyzed reaction. This reaction may occur through oxidation of phenelzine by the hydroxyl radical, which has also been spin-trapped with 4-POBN in this system.     

Detection of substances with alcoholic or phenolic hydroxyl groups by generation of hydrogen peroxide with imidazole and peroxyoxalate chemiluminescence

On-line detection of substances with an alcoholic or phenolic hydroxyl group using imidazole and peroxyoxalate chemiluminescence was investigated qualitatively using a flow-injection method. The substances tested included six polyphenols, five monophenols and six sugars. After incubation at 80°C with an imidazole buffer (pH 9.5) the substances were detected by peroxyoxalate chemiluminescence. The polyphenols tested (e.g., pyrogallol, purpurogallin, and dopamine) showed the strongest light emission. The sugars with hydroxyl groups (e.g., fructose and lactose) and the monophenols (e.g., phenol, serotonin, and β-estradiol) produced only a weak light emission. Reaction of hydroxyl compounds and imidazole generated hydrogen peroxide. Imidazole served two roles, it catalysed the reaction with the hydroxyl compound and initiated peroxyoxalate chemiluminescence on-line. A novel reactor formed by packing glass beads into a flow cell (Teflon) of a chemiluminometer improved the sensitivity of light detection.     

Fatty acid radical formation in rats administered oxidized fatty acids: in vivo spin trapping investigation.

We report in vivo evidence for fatty acid-derived free radical metabolite formation in bile of rats dosed with spin traps and oxidized polyunsaturated fatty acids (PUFA). When rats were dosed with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and oxidized PUFA, the DMPO thiyl radical adduct was formed due to a reaction between oxidized PUFA and/or its metabolites with biliary glutathione. In vitro experiments were performed to determine the conditions necessary for the elimination of radical adduct formation by ex vivo reactions. Fatty acid-derived radical adducts of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were detected in vivo in bile samples collected into a mixture of iodoacetamide, desferrioxamine, and glutathione peroxidase. Upon the administration of oxidized 13C-algal fatty acids and 4-POBN, the EPR spectrum of the radical adducts present in the bile exhibited hyperfine couplings due to 13C. Our data demonstrate that the carbon-centered radical adducts observed in in vivo experiments are unequivocally derived from oxidized PUFA. This in vivo evidence for PUFA-derived free radical formation supports the proposal that processes involving free radicals may be the molecular basis for the previously described cytotoxicity of dietary oxidized PUFA.     

Electron Spin Resonance Studies on Isolated Hepatocytes Treated with Ferrous Or Ferric Iron

Isolated rat hepatocytes incubated with iron salts in the presence of the spin trapping agent tx-4-pyridyl-l-oxide N-tert-butyl nitrone (4-POBN) generate a clear electron spin resonance signal; this signal is not detectable in the absence of exogenous iron. The hyperfine splitting constants are identical whether ferrous or ferric iron is used. The free radical trapped does not appear to be an active oxygen species but rather a carbon-centred radical, which we here ascribe to a lipodienyl radical on the basis of its hyperfine splitting features. Support to this interpretation is lent by the fact that no such radical could be generated in hepatocytes fully protected against lipid peroxidation by pretreating the donor rats with α-tocopherol.     

Prevention of hepatic ischemia-reperfusion injury by green tea extract

These experiments were designed to determine whether green tea extract (GTE), which contains polyphenolic free radical scavengers, prevents ischemia-reperfusion injury to the liver. Rats were fed a powdered diet containing 0-0.3% GTE starting 5 days before hepatic warm ischemia and reperfusion. Free radicals in bile were trapped with the spin-trapping reagent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) and measured using electron spin resonance spectroscopy. Hepatic ischemia-reperfusion increased transaminase release and caused pathological changes including focal necrosis and hepatic leukocyte infiltration in the liver. Transaminase release was diminished by over 85% and pathological changes were almost totally blocked by 0.1% dietary GTE. Ischemia-reperfusion increased 4-POBN/radical adducts in bile nearly twofold, an effect largely blocked by GTE. Epicatechin, one of the major green tea polyphenols, gave similar protection as GTE. In addition, hepatic ischemia-reperfusion activated NF-kappa B and increased TNF-alpha mRNA and protein expression. These effects were all blocked by GTE. Taken together, these results demonstrate that GTE scavenges free radicals in the liver after ischemiareoxygenation, thus preventing formation of toxic cytokines. Therefore, GTE could prove to be effective in decreasing hepatic injury in disease states where ischemia-reperfusion occurs.     

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.     

Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Melatonin and N-acetyl serotonin inhibit selectively enzymatic and non-enzymatic lipid peroxidation of rat liver microsomes

Melatonin (N-acetyl-5-methoxytryptamine) and its immediate precursor N-acetyl serotonin in the metabolism of tryptophan are free radical scavengers that have been found to protect against non-enzymatic lipid peroxidation in many experimental models. By contrast, little is known about the antioxidant ability of these indoleamines against NADPH enzymatic lipid peroxidation. The light emission produced by rat-liver microsomes, expressed as total cpm during 180 min of incubation at 37 degrees C, was two-fold greater in the presence of ascorbate (0.4mM) when compared with NADPH (0.2 mM). Maximal peaks of light emission produced by microsomes lipid peroxidized with ascorbic-Fe(2+) or NADPH and expressed as cpm were 354,208 (at 60 min) and 135,800 (at 15 min), respectively. During non-enzymatic lipid peroxidation a decrease of total chemiluminescence (inhibition of lipid peroxidation) was observed when increasing concentrations of melatonin were added to liver microsomes. The protective effect was concentration-dependent. The inhibition observed in light emission was coincident with the protection of the most PUFAs. Preincubation of microsomes with N-acetyl serotonin reduced these changes very dramatically. Thus, in the presence of both antioxidants (0.36, 0.75, 1.5 mM), light emission percent inhibition during non-enzymatic (ascorbate-Fe(2+)) lipid peroxidation of rat liver microsomes was for melatonin: 6.12, 16.20, 34.88 and for N-acetyl serotonin: 85.10, 88.48, 84.4 respectively. The incubation of rat liver microsomes in the presence of NADPH (0.36, 0.75, 1.5 mM) produce a sudden increase of chemiluminescence that gradually increased and reached a maximal value at about 15 min; however, N-acetyl serotonin reduced these changes very efficiently.     

A search for oxygen-centered free radicals in the lipoxygenase/linoleic acid system

Studies of the oxygenation of linoleic acid by soybean lipoxygenase utilizing electron spin resonance spectroscopy and oxygen uptake have been undertaken. The spin trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) was included in the lipoxygenase system to capture short-lived free radicals. Correlation of radical adduct formation rates with oxygen uptake studies indicated that the major portion of radical adduct formation occurred when the system was nearly anaerobic. Incubations containing [17O]oxygen with nuclear spin of 5/2 did not have additional ESR lines as would be expected if an oxygen-centered 4-POBN-lipid peroxyl radical adduct were formed indicating that the trapped radical must be reassigned as a carbon-centered species. To establish the presence of [17O2]oxygen in our incubations, a portion of the gas from the lipoxygenase/linoleate experiments was used to prepare the 4-POBN-superoxide radical adduct utilizing a superoxide producing microsomal/paraquat/NADPH system.     

Induction of free radicals in hepatocytes,mitochondria and microsomes of rats by ochratoxin A and its analogs

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and Oα; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe³⁺ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe³⁺. The spin adduct hyperfine splitting constants indicated the presence of α-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.     

One-electron reduction of chromate by NADPH-dependent glutathione reductase

Electron spin resonance (ESR) measurements provide evidence for the formation of Cr(V) intermediates in the enzymatic reduction of Cr(VI) by glutathione reductase (GSSG-R) in the presence of NADPH, indicating an initial single-electron transfer step in the reduction mechanism. Depending on the pH, at least two different Cr(V) species are generated which are relatively long-lived. In addition, we have detected the hydroxyl (.OH) radical formation during the GSSG-R catalyzed reduction of Cr(VI) by spin trapping, employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) as spin traps. Superoxide dismutase (SOD) causes only a minor effect on the .OH radical and Cr(V) formation, indicating that the O2- is not significantly involved in the reaction mechanism. Catalase enhances the Cr(V) formation and substantially inhibits the .OH radical formation, indicating the involvement of hydrogen peroxide (H2O2) in the reaction mechanism. Addition of H2O2 suppresses Cr(V) and enhances the .OH radical formation. Measurements involving N-ethylmaleimide show that the Cr(V) species, produced enzymatically by the reduction of Cr(VI) by GSSG-R, react with H2O2 to generate .OH radicals, which might participate in the initiation of Cr(VI) carcinogenicity.     

Acute effects of 3,4-methylenedioxymethamphetamine on brain serotonin synthesis in the dog studied by positron emission tomography

The influence of an acute dose (2 mg/kg; i.v.; infused over 10 min) of 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) on the brain serotonin synthesis in the dog was assessed using alpha [11C]methyl-L-tryptophan and positron emission tomography. The rate of serotonin synthesis measured 1 h after injection of MDMA was six times greater than the base line (before MDMA) synthesis. Five hours after the MDMA injection, serotonin synthesis was about one half that at the base line, and about one thirteenth of the synthesis at 1 h after MDMA. A large increase seen 1 h after MDMA probably relates to the large release of serotonin by MDMA and reflects an attempt of the serotonergic system to replenish released serotonin. This probably correlates with the mood changes reported by humans after MDMA intake. Decrease observed 5 h after MDMA, in part, probably relates to the inhibitory effects of the released serotonin, which could act on the activity of tryptophan hydroxylase directly or indirectly via other monoaminergic systems (e.g. dopaminergic).     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Crystal structure of tryptophan hydroxylase with bound amino acid substrate

Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O 2, and tetrahydrobiopterin (BH 4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 A resolution crystal structure of the catalytic domain (Delta1-100/Delta415-445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 A between the iron and the tryptophan Czeta3 atom that is hydroxylated. The binding of tryptophan and maybe the imidazole has caused the structural changes in the catalytic domain compared to the structure of the human TPH1 without tryptophan. The structure of chicken TPH1 is more compact, and the loops of residues Leu124-Asp139 and Ile367-Thr369 close around the active site. Similar structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of substrate analogues norleucine and thienylalanine to the PAH.BH 4 complex. In fact, the chicken TPH1.Trp.imidazole structure resembles the PAH.BH 4.thienylalanine structure more (root-mean-square deviation for Calpha atoms of 0.90 A) than the human TPH1 structure (root-mean-square deviation of 1.47 A).