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PubMed1989

Identification of 9-hydroxylamine-1,2,3,4-tetrahydroacridine as a hepatic microsomal metabolite of tacrine by high-performance liquid chromatography and electrochemistry.

Amperometric detection using a dual-electrode thin-layer cell in the series configuration can aid in the identification of unknown components in complicated samples by voltammetric characterization. This is shown by studying the metabolism of tacrine by rat hepatic microsomes using high-performance liquid chromatography with electrochemical detection. The major metabolite detected in microsomal incubations did not co-elute with any standard acridine available and was produced in too small a quantity for mass spectral characterization. Tentative identification of this metabolite as 9-hydroxylamine-1,2,3,4-tetrahydroacridine was made by electrochemical characterization. The electrochemistry of the metabolite was compared to that of the hydroxylamine produced and studied by cyclic voltammetry.

Aminoacridines↗
PubMed2000

In vivo EPR imaging by using an acyl-protected hydroxylamine to analyze intracerebral oxidative stress in rats after epileptic seizures.

EPR imaging by using an acyl-protected hydroxylamine, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP), in the head of a living rat after kainic acid (KA)-induced epileptic seizures was performed. ACP is a stable non-radical compound, but is easily deprotected with intracellular esterase to yield a hydroxylamine, which is oxidized by intracellular oxidative stress to yield an EPR-detectable nitroxide radical. From in vivo image data, the average values of EPR signal intensity from the hippocampus, striatum, and cerebral cortex were computed. There was no significant difference in cortical signal intensity between the control and KA-treated rats. The signal intensities from the hippocampus and striatum for the KA-treated rats were significantly higher than those for the control. The in vitro study showed that almost the same quantity of ACP moved into all regions of the brain of the control and KA-treated rats. These findings indicate that following a KA-induced seizure, the oxidative stress in the hippocampus and striatum is enhanced, but not so in the cerebral cortex.

Animals↗
PubMed1997

Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine.

The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites. Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen. Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins. To determine if free radicals could be involved in this process, rat red cells were incubated with DDS-NOH in the presence of the spin trap, 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis. Addition of DDS-NOH (25-50 microM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO-OH) standard. No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS-NOH-generated DMPO-OH signal to be replaced by a cysteamine thiyl radical adduct signal. DDS-NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide. Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals. These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia.

Animals↗
PubMed1993

Evidence for the structure of the active site heme P460 in hydroxylamine oxidoreductase of Nitrosomonas.

Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine to nitrite in nitrification by Nitrosomonas europaea. It has an alpha n subunit structure and eight covalently bound hemes per subunit. Seven of these have visible spectra indistinguishable from heme c. The eighth, designated as P460, has unusual visible spectroscopic features in the enzyme and in a heme-containing proteolytic fragment. Its structure has not been previously determined. Enzymatic digestions of HAO were performed, and various proteolytic fragments were purified. Mass spectrometry confirmed the presence of authentic heme c in some fragments, that is, iron protoporphyrin IX cross-linked by two thioether bonds to cysteine residues. It was possible to detect the presence of the P460 pigment in some fragments, based upon the sensitivity of this pigment to treatment of the holoenzyme with hydrogen peroxide. A proteolytic fragment produced by sequential digestion with trypsin and pronase was shown to contain heme c and a hydrogen peroxide-sensitive heme with an unusual visible spectrum. This fragment contained two covalently cross-linked peptides. Mass spectrometry and NMR indicated that the P460 heme was iron protoporphyrin IX covalently bonded by two thioether bridges to peptide, but in addition there was a new, third covalent bond between a meso heme carbon and an aromatic ring carbon on a tyrosyl residue. The new covalent bond has been tentatively assigned to the C2 carbon of the tyrosyl ring and the 5-meso heme carbon (IUPAC-IUB tetrapyrrole nomenclature), although this location requires further proof.

Amino Acid Sequence↗
PubMed1982

Resolution of multiple heme centers of hydroxylamine oxidoreductase from Nitrosomonas. 2. Mössbauer spectroscopy.

Hydroxylamine oxidoreductase (HAO) isolated from Nitrosomas europaea is a complex protein of Mr 220000 with an (alpha beta)3 subunit structure. Each alpha beta subunit contains seven c-type hemes and approximately one unusual prosthetic group termed P-460. We have studied this enzyme in the oxidized and reduced states by using Mössbauer spectroscopy. In the fully reduced enzyme, approximately seven hemes per alpha beta subunit contributed to one spectrum characteristic of low-spin ferrous heme. The remainder of the iron (10-15% of the total) yielded an ill-defined absorption pattern. Carbon monoxide binds to the P-460 as shown by optical spectra. The Mössbauer spectra of reduced hydroxylamine oxidoreductase which had been exposed to CO showed a new spectral component, corresponding to one iron site, with parameters characteristic of a low-spin ferrous heme-carbonyl complex. It appears that this component is derived from the ill-defined spectrum observed in the reduced enzyme. This is the first direct evidence that the P-450 moiety amounts to at least one Fe per alpha beta subunit. Together the Mössbauer results and the optical spectra suggest that the P-460 moiety is a heme. The Mössbauer spectra of the oxidized (as isolated) enzyme suggest the presence of one or two low-spin ferric hemes which might be EPR undetectable because of either fast electronic spin relaxation or participation in a spin-coupled pair. The spectra gave no evidence for the presence of a ferrous site in oxidized HAO.

Carbon Monoxide↗
PubMed1985

Role of superoxide in the N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine by the rat liver cytochrome P-450 system.

The N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine (N-hydroxyphentermine, MPPNHOH) and the N-hydroxylation of 2-methyl-1-phenyl-2-propylamine (phentermine) by reconstituted systems that contained cytochromes P-450 purified from rat liver microsomes were demonstrated. The oxidation of MPPNHOH, but not of phentermine, could also be mediated by a superoxide and hydrogen peroxide generating system that contained xanthine and xanthine oxidase. Superoxide dismutase completely inhibited the oxidation of MPPNHOH by the xanthine/xanthine oxidase system and inhibited by 70% the oxidation mediated by a reconstituted cytochrome P-450 oxidase system. The majority of the microsomal oxidation was inhibited by an antibody raised against the major isozyme of cytochrome P-450 purified from livers of phenobarbital-pretreated rats. 2-Methyl-2-nitroso-1-phenylpropane (MPPNO) was found to be an intermediate in the overall oxidation of MPPNHOH to 2-methyl-2-nitro-1-phenylpropane (MPPNO2). Superoxide dismutase appeared to inhibit the first step, the conversion of MPPNHOH to MPPNO. These observations are accounted for by a sequence of two mechanistically distinct P-450-mediated oxidations. In the first reaction, N-hydroxylation of phentermine occurs by a normal cytochrome P-450 pathway. The formed hydroxylamine then uncouples the cytochrome P-450 system to generate superoxide and hydrogen peroxide. The superoxide oxidizes MPPNHOH to MPPNO which is then oxidized to MPPNO2, the ultimate product. This superoxide-mediated oxidation represents another pathway for N-oxidation by cytochrome P-450.

Animals↗
PubMed2004

Os(II)-nitrosyl and Os(II)-dinitrogen complexes from reactions between Os(VI)-nitrido and hydroxylamines and methoxylamines.

Reactions between the Os(VI)-nitrido salts (e.g., trans-[Os(VI)(tpy)(Cl)(2)(N)]PF(6) (tpy = 2,2':6',2"-terpyridine), cis-[Os(VI)(tpy)(Cl)(2)(N)]PF(6), and fac-[Os(VI)(tpm)(Cl)(2)(N)]PF(6) (tpm = tris(pyrazol-1-yl)methane)) and the hydroxylamines (e.g., H(2)NOH and MeHNOH) and the methoxylamines (e.g., H(2)NOMe and MeHNOMe) in dry MeOH at room temperature give three different types of products. They are Os(II)-dinitrogen (e.g., trans-, cis-, or fac-[Os(II)-N(2)]), Os(II)-nitrosyl [Os(II)-NO](+) (e.g., trans- or cis-[Os(II)-NO](+)), Os(IV)-hydroxyhydrazido (e.g., cis-[Os(IV)-N(H)N(Me)(OH)](+)), and Os(IV)-methoxyhydrazido (e.g., trans-/cis-[Os(IV)-N(H)N(H)(OMe)](+), and trans-/cis-[Os(IV)-N(H)N(Me)(OMe)](+)) adducts. The products depend in a subtle way on the electron content of the starting nitrido complexes, the nature of the hydroxylamines, the nature of the methoxylamines, and the reaction conditions. Their appearance can be rationalized by invoking the formation of a series of related Os(IV) adducts which are stable or decompose to give the final products by two different pathways. The first involves internal 2-electron transfer and extrusion of H(2)O, MeOH, or MeOMe to give [Os(II)-N(2)]. The second which gives [Os(II)-NO](+) appears to involve seven-coordinate Os(IV) intermediates based on the results of an (15)N-labeling study.

Journal Article↗
PubMed2005

A new supported reagent for the parallel synthesis of primary and secondary O-alkyl hydroxylamines through a base-catalyzed Mitsunobu reaction.

The growing field of applications of O-alkyl hydroxylamines in medicinal chemistry and chemical biology has motivated the search for a parallel synthesis. A solid-phase approach based on the alkylation by alcohols of a new supported N-hydroxyphthalimide reagent using a Mitsunobu reaction followed by methylaminolysis has been optimized. This study points out the importance of the linker and a specific base effect for the Mitsunobu reaction. A large variety of alcohols can be used to give with moderate to high yields diverse O-alkyl hydroxylamines in high purity.

Journal Article↗
PubMed2000

Synthesis of 2,3-dihydroisoxazoles from propargylic N-hydroxylamines via Zn(II)-catalyzed ring-closure reaction

A novel cyclization reaction of propargylic N-hydroxylamines to 2,3, 5-trisubstituted 2,3-dihydroisoxazoles in the presence of catalytic amounts (10 mol %) of ZnI(2) and DMAP is reported. The methodology provides a mild new approach to this useful class of substituted heterocycles that complements extant methods. The unique reactivity of the propargylic N-hydroxylamine substrates in the presence of Zn(II) and DMAP may have additional applications in other, related alkyne cyclization reactions.

Journal Article↗
PubMed1998

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics.

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-psi[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.

Crystallography, X-Ray↗
PubMed1993

Enzymatic phase II activation of the N-hydroxylamines of IQ, MeIQx and PhIP by various organs of monkeys and rats.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are mutagenic and carcinogenic heterocyclic amines produced during the ordinary cooking of meat. These compounds undergo metabolic activation via both cytochrome P450-mediated N-oxidation and phase II esterification in order to exert their genotoxicity. In the current study, we examined the in vitro phase II activation of N-hydroxy-IQ, N-hydroxy-PhIP and N-hydroxy-MeIQx by cytosolic acetyltransferase, sulfotransferase, aminoacyl-tRNA synthetase and phosphatase from a number of tissues including liver, kidney, colon and heart. These tissues were chosen for study because each is either a target organ for carcinogenicity or has displayed high levels of DNA adducts in in vivo studies with the heterocyclic amines. Cytosol from various tissues of both monkeys and rats was incubated with and without the respective cofactors, and carcinogen binding to calf thymus DNA was measured by 32P-postlabeling analysis. Our results show that all four phase II enzymes may participate in the activation of the N-hydroxylamines. However, the degree of activation depends on the substrate, tissue and animal species. For example, in both monkeys and rats, the highest acetyl CoA-enhanced binding was observed with N-hydroxy-IQ and the lowest acetyl CoA-enhanced binding was observed with N-hydroxy-MeIQx. In contrast, no significant adenosine 3'-phosphate 5'-phosphosulfate-dependent activation of N-hydroxy-IQ was observed with monkey cytosol from liver, kidney, heart or colon but the sulfotransferase-mediated activation of N-hydroxy-PhIP was at least 10 times higher in all four tissues of monkeys than in rats. Prolylation appears important in the activation of all three N-hydroxylamines by rat liver and heart cytosol, whereas in monkeys, prolylation appears important in kidney cytosol. The differences observed in the phase II activation of heterocyclic amines may have implications for DNA adduct formation, toxicity and carcinogenicity.

Acetyltransferases↗
PubMed1974

Promotion of seed germination by nitrate, nitrite, hydroxylamine, and ammonium salts.

Action and uptake of azides, nitrates, nitrites, hydroxylamines, and ammonium salts were measured on germination of Amaranthus albus, Lactuca sativa, Phleum pratense, Barbarea vulgaris, B. verna, and Setaria glauca seeds. Nitrate and nitrite reductase activities were measured in vivo for each of these kinds of seeds. Activities were measured in vitro for catalase, peroxidase, glycolate oxidase, and pyridine nucleotide quinone reductase on extracts of A. albus and L. sativa seeds before and after germination. The enzymic activities measured and the responsiveness of the haemproteins to inhibition by the several compounds indicate that nitrites, azides, and hydroxylamines promote seed germination by inhibition of H(2)O(2) decomposition by catalase. Ammonium salts showed pronounced promotive activity only for B. verna and B. vulgaris seeds, for which they served as metabolic substrates.The promotion of germination is thought to depend on coupling of peroxidase action to NADPH oxidation, which can regulate the pentose pathway of d-glucose 6-phosphate use. Pyridine nucleotide quinone reductase is the possible coupling enzyme. This enzyme and others required for the action are present in the seeds before imbibition of water.

Journal Article↗
PubMed1994

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite in Nitrosomonas europaea. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. The immediate acceptor of electrons from HAO is believed to be cytochrome c-554 (Cyt c-554). We have isolated a genomic DNA fragment containing the structural gene encoding HAO (hao) and a part of the gene for Cyt c-554. The nucleotide sequence of hao was determined, and its transcription was analyzed. The open reading frame (ORF) encodes amino acid sequences matching the purified peptides of HAO. A 64.28-kDa protein is encoded in this ORF, in close agreement with the empirically determined molecular mass of 63 kDa. The N terminus was located 24 amino acids from the start codon, suggesting the presence of a leader sequence. The putative eight heme-binding peptides were localized in this ORF. The gene for Cyt c-554 was located 1,200 bp downstream from the 3' end of hao. An ORF was identified in the upstream region from hao and may encode a protein of unknown function. Data bank searches did not reveal proteins with substantial similarities to HAO, but they did reveal similarities between Cyt c-554 and other c-type cytochromes.

Amino Acid Sequence↗
PubMed2006

Transcriptional analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11.

The nitrifying bacterium Nitrosomonas sp. strain ENI-11 has three copies of the gene encoding hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)) on its genome. Broad-host-range reporter plasmids containing transcriptional fusion genes between hao copies and lacZ were constructed to analyze the expression of each hydroxylamine oxidoreductase gene (hao) copy individually and quantitatively. beta-Galactosidase assays of ENI-11 harboring reporter plasmids revealed that all hao copies were transcribed in the wild-type strain. Promoter analysis of hao copies revealed that transcription of hao(3) was highest among the hao copies. Expression levels of hao(1) and hao(2) were 40% and 62% of that of hao(3) respectively. Transcription of hao(1) was negatively regulated, whereas a portion of hao(3) transcription was read through transcription from the rpsT promoter. When energy-depleted cells were incubated in the growth medium, only hao(3) expression increased. This result suggests that it is hao(3) that is responsible for recovery from energy-depleted conditions in Nitrosomonas sp. strain ENI-11.

DNA, Bacterial↗
PubMed2004

A simple and sensitive method for the determination of hydroxylamine in fresh-water samples using hypochlorite followed by gas chromatography.

A new and simple method for the determination of hydroxylamine in environmental water, such as fresh rivers and lakes using hypochlorite, followed by its gas choromatographic detection, has been developed. A glass vial filled with sample water was sealed by a butyl-rubber stopper and aluminum cap without head-space, and then sodium hypochlorite solution was injected into the vial through a syringe to convert hydroxylamine to nitrous oxide. The head-space in the glass vial was prepared with 99.9% grade N2 using a gas-tight syringe. After the glass vial was shaken for a few minutes, nitrous oxide in the gas-phase was measured by a gas chromatograph with an electron-capture detector. The dissolved nitrous oxide in the liquid-phase was calculated according to the solubility formula. The proposed method was applied to the analysis of fresh-water samples taken from Iu river and Hii river, flowing into brackish Lakes Nakaumi and Shinji, respectively.

Journal Article↗
PubMedDate not supplied

Reactions of the nitroso and hydroxylamine metabolites of sulfamethoxazole with reduced glutathione. Implications for idiosyncratic toxicity.

N4-oxidation of sulfonamides has been implicated in the pathogenesis of idiosyncratic reactions to these antimicrobials. In vitro toxicity assays employing mononuclear leukocytes as target cells have shown that the toxicity of sulfamethoxazole hydroxylamine (SMX-HA) is inhibited by exogenous glutathione, suggesting that conjugation with glutathione is an important detoxification pathway. However, in these experiments, significant depletion of cellular glutathione only occurred at concentrations of SMX-HA greater than or equal to 300 microM. At concentrations of SMX-HA which produce 50% toxicity in mononuclear leukocytes (approximately 100 microM), there was not a significant loss of glutathione. SMX-HA also caused a small but significant increase in oxidized glutathione concentrations. In cell-free experiments, reduced glutathione (GSH) prevented the autooxidation of SMX-HA to nitrososulfamethoxazole (nitroso-SMX). During this process, oxidized glutathione was formed. GSH rapidly reacted with nitroso-SMX to form a labile semimercaptal conjugate. Physiologically relevant concentrations of GSH (i.e. 1 mM) favored thiolytic cleavage of the semimercaptal to form SMX-HA. Isomerization of the semimercaptal to the more stable sulfinamide occurred at low GSH concentrations. Purified glutathione transferases had no effect on the reaction of SMX-HA with GSH. Therefore, glutathione is important in protecting cells from the toxicity of SMX-HA largely by preventing its further oxidation to nitroso-SMX. Stable glutathione conjugates are likely to be formed only in small quantities under physiological conditions. Conjugation with glutathione would not be expected to be a major pathway for clearance of the hydroxylamine and nitroso metabolites of sulfonamides.

Cell-Free System↗
PubMed1995

N4-hydroxylation of sulfamethoxazole by cytochrome P450 of the cytochrome P4502C subfamily and reduction of sulfamethoxazole hydroxylamine in human and rat hepatic microsomes.

The N4-hydroxylation of sulfamethoxazole (SMX) to its hydroxylamine (SMX-HA) metabolite is the first step in the formation of reactive metabolites responsible for mediating hypersensitivity reactions associated with this compound. In rat hepatic microsomes, the NADPH-dependent oxidation of SMX to SMX-HA was increased 3-fold by pretreatment of rats with phenobarbital. Other cytochrome P450 (CYP) inducers were ineffective. The constitutive and induced SMX N-hydroxylation activities were inhibited by tolbutamide, and induction of SMX-HA activity paralleled the induction of progesterone 21-hydroxylase activity, a marker for CYP2C6. SMX N-hydroxylation in phenobarbital-treated rat hepatic microsomes was inhibited 70% by anti-CYP2C6 antisera. Thus, the N4-hydroxylation of SMX by rat hepatic microsomes was mediated by members of the CYP2C subfamily, probably CYP2C6. In a panel of human microsomes, SMX-HA formation correlated with tolbutamide hydroxylase activity (r = 0.75; p = 0.01); CYP2C9 content (r = 0.79; p < 0.01) and was inhibited 70% by 500 microM tolbutamide and 90% by 100 microM sulfaphenazole. Recombinant CYP2C9 catalyzed the N-hydroxylation of SMX. SMX-HA formation in human hepatic microsomes was therefore mediated predominantly by CYP2C9. CYP-mediated reduction of SMX-HA to SMX was markedly induced in dexamethasone and phenobarbital-treated rat hepatic microsomes, and was attributed to CYP3A and CYP2B forms. In uninduced rat and human hepatic microsomes, SMX-HA reduction was mediated predominantly by an NADH-dependent microsomal hydroxylamine reductase under aerobic conditions. Under anaerobic conditions, troleandomycin at > or = 1 microM inhibited the reduction of SMX-HA in human hepatic microsomes by 45%, whereas sulfaphenazole had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗