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PubMed1989

Hydroxylamine is a vasorelaxant and a possible intermediate in the oxidative conversion of L-arginine to nitric oxide.

Our objective was to determine whether hydroxylamine is a possible intermediate in the oxidative conversion of L-arginine to nitric oxide. Vasorelaxation by hydroxylamine is known to be mediated by nitric oxide. The vasorelaxant properties of hydroxylamine were examined using rat aortic rings and an isolated rat lung perfusion model. Hydroxylamine and acetylcholine were equally effective in relaxing norepinephrine-contracted intact aortic rings, whereas only hydroxylamine relaxed aortic rings with endothelium removed. This endothelium-independent vasorelaxation by hydroxylamine indicated that the hydroxylamine-converting enzyme is not localized solely within endothelial cells. Catalase, an enzyme known to oxidize hydroxylamine to nitric oxide, was present in homogenates of intact and endothelium-denuded rings. Cyanamide, another catalase substrate and a known precursor of nitroxyl (HNO), was not a vasorelaxant of aortic rings or of isolated, hypoxia-constricted lungs. These results suggest that free nitroxyl is not an intermediate in the oxidation of hydroxylamine to nitric oxide. An overall pathway for the oxidative conversion of L-arginine through an hydroxylamine intermediate to nitric oxide is proposed.

Acetylcholine↗
PubMed2000

Hydroxylamine moiety of developmental toxicants is associated with early cell death: a structure-activity analysis.

BACKGROUND: Cellular debris, an indicator of cell death, appears in limb buds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous injection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea into the exocoelomic cavities of the embryos. This episode of early cell death appears to be central to the teratogenic action of hydroxyurea. Several chemicals that are structurally related to hydroxyurea, and that possess a terminal hydroxylamine moiety (-NHOH), also produce limb abnormalities. METHODS: To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogenesis, five structurally related hydroxylamine-bearing chemicals (hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetohydroxamic acid, and hydroxyurethane) were administered at equimolar doses to rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 micromol/embryo) injection on gestational day 12. Five additional chemicals, structurally similar to the hydroxylamine-bearing compounds, but possessing a terminal amino group (-NH(2)) (ammonium hydroxide, methylamine, urea, acetamide, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 mmol/kg or 1.30 micromol/embryo) was co-administered with the hydroxylamine-bearing compounds to determine its effect on early cell death. Embryos were harvested 4 or 8 hr after treatment and analyzed by light microscopy. RESULTS: Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compounds produced an early episode of embryonic cell death. In all cases, the antioxidant propyl gallate prevented or delayed the early episode of cell death observed after treatment with the hydroxylamine-bearing compounds. CONCLUSIONS: These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compounds involves a free radical mechanism that requires the presence of a terminal hydroxylamine group for initiation.

Abnormalities, Drug-Induced↗
PubMed2000

N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts.

Alpha-phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice. N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PBN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 microM compared with 200 microM PBN to produce a similar effect, suggesting that N-t-butyl hydroxylamine is the active form of PBN. N-Benzyl hydroxylamine and N-methyl hydroxylamine compounds unrelated to PBN were also effective in delaying senescence, suggesting the active functional group is the N-hydroxylamine. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants, as measured by the oxidation of 2', 7'-dichlorodihydrofluorescin and the increase in the GSH/GSSG ratio. The acceleration of senescence induced by hydrogen peroxide is reversed by the N-hydroxylamines. DNA damage, as determined by the level of apurinic/apyrimidinic sites, also decreased significantly following treatment with N-hydroxylamines. The N-hydroxylamines appear to be effective through mitochondria; they delay age-dependent changes in mitochondria as measured by accumulation of rhodamine-123, they prevent reduction of cytochrome C(FeIII) by superoxide radical, and they reverse an age-dependent decay of mitochondrial aconitase, suggesting they react with the superoxide radical.

Antioxidants↗
PubMed1998

Only the glutathione dependent antioxidant enzymes are inhibited by haematotoxic hydroxylamines.

Hydroxylamine and some of its derivatives are known to cause oxidative effects both in vitro and in vivo. In the current study we investigated the effects of hydroxylamines on the enzymatic antioxidant defense system in human erythrocytes. The activity of catalase and superoxide dismutase was not significantly influenced by any of the hydroxylamines tested. However, the activity of glutathione peroxidase (GPX) and glutathione S-transferase (GST) was strongly inhibited by hydroxylamine and its O-derivatives (O-methyl and O-ethyl hydroxylamine). GPX was also inhibited by two N-derivatives of hydroxylamine (i.e. N-dimethyl and N,O-dimethyl hydroxylamine). This indicates that exposure to hydroxylamines not only changes the cellular oxidation-reduction status but also leads to inhibition of the glutathione dependent antioxidant enzymes. GST as well as GPX have cysteine residues at the active site of the enzymes. Such an accessible thiol group is generally susceptible to formation of protein-mixed disulphides or intramolecular disulphides. If these thiol groups are essential for activity this would be accompanied by an increase or decrease in the enzyme activity. In principle this is also true for glutathione reductase (GR), which in this study was only inhibited by N,O-dimethyl and N-methyl hydroxylamines. However, GR is capable to reduce these disulphides by taking up two electrons, either from its substrate NAPDH or from another reductant. Oxidation of these thiol groups in GR would thus not lead to impairment of GR activity. The fact that NODMH and NMH do decrease the GR activity can therefore only be explained by other modifications. The activity loss of GST and GPX on the other hand, is likely to involve oxidation of critical cysteine residues. The practical consequence of these findings is that the cellular prooxidant state that may arise in erythrocytes exposed to hydroxylamines can be further increased by activity loss of protective enzymes, which may decrease the average life span of the red blood cell.

Catalase↗
PubMed1997

Reactions of Hydroxylamine with Metal Porphyrins.

The reaction of hydroxylamine with a series of metal porphyrins was examined in methanol/chloroform media. The reductive nitrosylation reaction was observed for the manganese and iron porphyrins, leading to a nitrosyl complex that precipitated out of the solution in good isolatable yield (80-90%). This reaction could be used synthetically for the generation of iron and manganese porphyrin nitrosyl complexes and was particularly useful for making isotopically labeled nitrosyl complexes. On the other hand, Co(II)(TPP) and Cr(TPP)(Cl) did not react with hydroxylamine under anaerobic conditions. With trace amounts of oxygen, the reaction of Co(II)(TPP) with hydroxylamine led to the formation of a stable cobalt(III)-bis(hydroxylamine) complex. The infrared, resonance Raman, and proton NMR spectra were consistent with a cobalt(III)-bis(hydroxylamine) complex. The cyclic voltammetry and visible spectroelectrochemistry of this complex were examined. The one-electron reduction of Co(III)(TPP)(NH(2)OH)(2)(+) formed Co(II)(TPP), for which there was no evidence for the coordination of hydroxylamine. Further reduction led to Co(I)(TPP)(-), which reacted with the halogenated solvent to form a cobalt-alkyl complex. The difference in the reactivity of these four metal porphyrins with hydroxylamine correlated well with their E(1/2) values. Iron(III) and manganese(III) porphyrins were relatively easy to reduce and readily underwent the reductive nitrosylation reaction, while cobalt(II) and chromium(III) porphyrins are unreactive. The one-electron oxidation of the hydroxylamine complex with a M(III) porphyrin would be expected to oxidize the N-atom in the coordinated hydroxylamine. The oxidation of M(III)(NH(2)OH) with the loss of a proton would form M(II)(N(I)H(2)O)(+) by an internal electron transfer, which will eventually lead to M(NO). The relationship between the reductive nitrosyl reaction and the enzymatic interconversion of NO and hydroxylamine was discussed.

Journal Article↗
PubMed1976

Correlation between flash-induced oxygen evolution and fluorescence yield kinetics in the 0 to 16 mus range in Chlorella pyyrenoidosa during incubation with hydroxylamine.

Following flash excitation, oxygen pulses and fluorescence kinetics in the time range 0-16 mus were studied in the alga Chlorella pyrenoidosa during incubation with various concentrations of hydroxylamine. The obtained results could be explained considering four effects of hydroxylamine. 1. Hydroxylamine removes (reduces) oxidizing equivalents, generated in the water-splitting system by flash excitation. This process does not markedly affect the fluorescence yield kinetics between 0 and 16 mus following the ignition of a flash and reaches a constant rate within a few minutes, but possibly within a few seconds, after addition of hydroxylamine. In a sequence of flashes separated by dark time td, the steady-state oxygen yield in the flashes is exp(-ktd), the yield at td=0 being taken equal to 1, where k=(0.1 + beta[NH2OH])s-1, with [NH2OH] in mM and beta=0.6 mM-1, provided [NH2OH]greater than or equal to 0.5 mM. 2. An inhibition between Z, the physiological donor and the oxidized reaction center pigment P+ occurs, proceeding as exp (-kiti)where ti is the incubation time with hydroxylamine and ki=(alpha[NH2OH]) min-1, with [NH2OH] in mM and alpha=0.14 mM-1. This process not only inhibits oxygen evolution capability, but also decreases the amplitude of the fluorescence yield difference deltaphi=phi(16 mus)-phi(2 mus) induced by a flesh in the steady state. In a fraction of the reaction centers this inhibition occurs "immediately" after the addition of hydroxylamine. These observations, combined with the conslusion of Cheniae and Martin (1971, Plant Physiol. 47, 568-575) that the inhibition of the Hill reaction is related to the extraction of bound manganese indicate that the reaction between Z and P+ requires bound manganese. 3. In the inhibited centers a second donor for P+, D, connected to an entry site for the artificial electron donor hydroxylamine becomes apparent. 4. A flash-induced oxygen uptake signal was observed in the presence of hydroxylamine, which was shown to be caused by a system II reaction. The effects under (1) and (4) were reversed in the dark if hydroxylamine was removed by washing. The effects under (2) and (3) were reversed during illumination of a washed sample.

Chlorella↗
PubMed1997

In vitro haematotoxic effects of three methylated hydroxylamines.

Hydroxylamine (HYAM, HONH2) and some of its derivatives are known to cause erythrotoxic effects both in vitro and in vivo. Previous studies have shown that the primary in vitro effect of HYAM and O-ethyl hydroxylamine (OEH) is methaemoglobin formation, leading to liberation of free radicals which cause lipid peroxidation, enzyme inhibitions and glutathione depletion. By contrast, N-substituted N,O-dimethyl hydroxylamine (NODMH), primarily induces impairment of glucose 6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR). The oxidative potency of HYAM and the O-derivative was larger than the potency of the N,O-derivative. This seemed to indicate that attachment of an alkyl group to the nitrogen atom of hydroxylamine leads to decreased reactivity. To achieve a better understanding of the structure activity relationship for hydroxylamines three methylated derivatives were tested: N-methyl hydroxylamine (NMH). N-dimethyl hydroxylamine (NDMH) and O-methyl hydroxylamine (OMH). We were also interested in the erythrotoxic potency of OMH which recently entered industrial production. Methaemoglobin formation, high release of lipid peroxidation products, inhibition of NADPH methaemoglobin reductase and glutathione S-transferase (GST) and depletion of total glutathione (GT) were seen for OMH. The reducing enzymes G6PDH and GR were not impaired by OMH. These findings for OMH are consistent with the proposed mechanism for O-derivatives. Since both the effects caused by OMH and its potency are comparable to those of HYAM and OEH this indicates that possible occupational exposure to this compound may be approached similarly to HYAM and OEH. NMH only inhibited G6PDH and GR activity, which is fully in accord with the proposed mechanism for N-substituted derivatives of HYAM. However, NDMH a double N-substituted compound, caused a strikingly different scheme of reactivity inhibition of G6PDH but not of GR, severe methaemoglobin formation, only little lipid peroxidation and some impairment of NADPH methaemoglobin reductase. This study confirms that O-derivatives of HYAM are potent haemoglobin oxidators, leading to other oxidative effects. The main effect was confirmed for single N-derivatives as inhibition of the two protective enzymes G6PDH and GR. However, the results for NDMH indicate that this simple classification of O-derivatives and N-derivatives has to be extended for double N-substituted compounds which give a mixture of effects.

Cytochrome-B(5) Reductase↗
PubMed1977

Flash induced fluorescence kinetics in chloroplasts in the 20 microseconds-100 s time range in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea. Effects of hydroxylamine.

Flash induced variations of the fluorescence yield have been studied at 2 degrees C over a long time range (at 1 microseconds and from 20 microseconds to 3 min) in chloroplasts in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) with or without addition of hydroxylamine. 1. In the presence of DCMU, a polyphasic rise is observed. A first fast rise (less than 1 microseconds) is ascribed to the transfer of the positive charge from the primary Photosystem II donor Chl to a secondary donor Y. Two hypotheses are proposed to interpret the existence of the subsequent slower increase (t1/2 approximately equal to 70 microseconds) which then follows the initial fast rise. 2. The effects or various hydroxylamine concentrations have been studied with a sufficient incubation time to inactivate the secondary donors Y and Z. This inactivation leads to a complete inhibition of the ability to emit oxygen. Under these conditions, the initial fast rise (less than 1 microseconds) is inhibited as shown by den Haan, G.A., Duysens, L. N. M. and Egberts, D. J. N. (1974) Biochim. Biophs. Acta 368, 409-421, and the oxidized Chl+ is reduced by an auxiliary donor D. The slow fluorescence rise observed after destruction of Y and Z has a similar kinetic behaviour to that observed in the presence of DCMU only and is polyphasic. In the presence of 10 mM hydroxylamine, the constant rate of the back reaction k1 between Chl+ and the primary acceptor Q- is estimated to be approx. (135 microseconds)-1 while the transfer of the positive charge from Chl+ to D has a rate constant k2 of approx. (105 microseconds)-1. 3. In the presence of hydroxylamine concentrations higher than 10 mM, there appears a rise in the 1-20 microseconds range ascribed to a direct reduction of oxidized Chl+ by hydroxylamine. 4. In chloroplasts treated with 10 mM hydroxylamine for 15 min and washed afterwards, the rate constant k3 of the back reaction between D+ and Q- is estimated to be approx. (100 ms)-1 which leads to a value of about 700 for the equilibrium constant between Chl and D. Hydroxylamine added under these conditions is able to reduce D+. The rate constant k4 of this reduction is estimated to be (350 ms)-1 in 0.1 mM hydroxylamine.

Chlorophyll↗
PubMed1998

Hydroxylamine-induced relaxation inhibited by K+ channel blockers in rat aortic rings.

Hydroxylamine, a putative endogenous nitric oxide donor, relaxed rat aorta in a concentration-dependent manner (0.01-30 microM). Removal of endothelium or pretreatment of aortic tissue with N(G)-nitro-L-arginine (L-NOARG, 100 microM) did not affect the relaxant effect of hydroxylamine but L-NOARG at 100 microM abolished the acetylcholine-induced relaxation. Methylene blue (10 microM) significantly reduced the relaxant effect of hydroxylamine in endothelium-denuded arteries. Tetrapentylammonium ions (0.3-3 microM), tetraethylammonium ions (1-3 mM) and charybdotoxin (100 nM) reduced the relaxant effect of hydroxylamine in the endothelium-denuded arteries while glibenclamide (3 microM) had no effect. Neither tetrapentylammonium nor tetraethylammonium ions affected relaxations induced by forskolin and verapamil. The effects of tetrapentylammonium ions (3 microM) and charybdotoxin (100 nM) were additive. Tetrapentylammonium ions (3 microM), tetraethylammonium ions (3 mM) and charybdotoxin (100 nM) decreased the relaxation induced by sodium nitroprusside in the endothelium-denuded arteries while glibenclamide (3 microM) had no effect. The concentration-relaxation curve for the relaxant effect of hydroxylamine was shifted to the right in the presence of high extracellular K+ (15-60 mM). Neither tetrapentylammonium ions (3 microM) nor charybdotoxin (100 nM) affected hydroxylamine-induced relaxation of the endothelium-denuded aorta precontracted with 60 mM K+. These results indicate that hydroxylamine relaxes the rat aorta partially through activation of tetrapentylammonium-, tetraethylammonium- and charybdotoxin-sensitive K+ channels and its action is comparable with that of sodium nitroprusside, an exogenous nitric oxide donor. The endothelium is not involved in the aortic response to hydroxylamine.

Adenosine Triphosphate↗
PubMed2004

Ammonium and hydroxylamine uptake and accumulation in Nitrosomonas.

Starved cells of Nitrosomonas europaea and further ammonia oxidizers were able to rapidly accumulate ammonium and hydroxylamine to an internal concentration of about 1 and 0.8 M, respectively. In kinetic studies, the uptake/accumulation rates for ammonium [3.1 mmol (g protein)(-1) min(-1)] and hydroxylamine [4.39 mmol (g protein)(-1) min(-1)] were determined. The uptake and accumulation process of ammonium and hydroxylamine was not coupled to ammonia or hydroxylamine oxidation and nitrite was not produced. In the presence of uncouplers the ammonium accumulation was completely inhibited, indicating an active, membrane-potential-driven transport mechanism. When the external ammonium or hydroxylamine pool was depleted, the internal ammonium and hydroxylamine was consumed within 12 h or 20 min, respectively. The binding of ammonium/ammonia was correlated with an energized membrane system, and hydroxylamine may bind to the hydroxylamine oxidoredutase.

Culture Media↗
PubMed1965

THE REDUCTION OF NITRATE, NITRITE AND HYDROXYLAMINE TO AMMONIA BY ENZYMES FROM CUCURBITA PEPO L. IN THE PRESENCE OF REDUCED BENZYL VIOLOGEN AS ELECTRON DONOR.

1. Enzyme systems from Cucurbita pepo have been shown to catalyse the reduction of nitrite and hydroxylamine to ammonia in yields about 90-100%. 2. Reduced benzyl viologen serves as an efficient electron donor for both systems. Activity of the nitrite-reductase system is directly related to degree of dye reduction when expressed in terms of the function for oxidation-reduction potentials, but appears to decrease to negligible activity below about 9% dye reduction. 3. NADH and NADPH alone produce negligible nitrite loss, but NADPH can be linked to an endogenous diaphorase system to reduce nitrite to ammonia in the presence of catalytic amounts of benzyl viologen. 4. The NADH- or NADPH-nitrate-reductase system that is also present can accept electrons from reduced benzyl viologen, but shows relationships opposite to that for the nitrite-reductase system with regard to effect of degree of dye reduction on activity. The product of nitrate reduction may be nitrite alone, or nitrite and ammonia, or ammonia alone, according only to the degree of dye reduction. 5. The relative activities of nitrite-reductase and hydroxylamine-reductase systems show different relationships with degree of dye reduction and may become reversed in magnitude when effects of degree of dye reduction are tested over a suitable range. 6. Nitrite severely inhibits the rate of reduction of hydroxylamine without affecting the yield of ammonia as a percentage of total substrate loss, but hydroxylamine has a negligible effect on the activity of the nitrite-reductase system. 7. The apparent K(m) for nitrite (1 mum) is substantially less than that for hydroxylamine, for which variable values between 0.05 and 0.9mm (mean 0.51 mm) have been observed. 8. The apparent K(m) values for reduced benzyl viologen differ for the nitrite-reductase and hydroxylamine-reductase systems: 60 and 7.5 mum respectively. 9. It is concluded that free hydroxylamine may not be an intermediate in the reduction of nitrite to ammonia by plants, and a possible mechanism for reduction of both compounds by the same enzyme system is discussed in the light of current ideas relating to other organisms.

Ammonia↗
PubMed2004

The effect of clarithromycin, fluconazole, and rifabutin on dapsone hydroxylamine formation in individuals with human immunodeficiency virus infection (AACTG 283).

BACKGROUND: Dapsone hydroxylamine formation is thought to be the cause of the high rates of adverse reactions to dapsone in human immunodeficiency virus (HIV)-infected individuals. Therefore we studied the effect of the commonly coadministered drugs fluconazole, clarithromycin, and rifabutin on hydroxylamine formation in individuals with HIV infection. METHODS: HIV-infected subjects (CD4 + > or =200 cells/mm 3 ) were enrolled in a 2-part (A or B) open-label drug interaction study. In part A, subjects (n = 12) received dapsone (100-mg tablet once daily) alone for 2 weeks and then, in a randomly assigned order, received dapsone and either fluconazole (200 mg daily), rifabutin (300 mg daily), or fluconazole plus rifabutin, each for a 2-week period. Part B (n = 11) was identical to part A except that clarithromycin (500 mg twice daily) was substituted for rifabutin. On the last study day of each 2-week period, plasma and urine were collected over ascorbic acid for 24 hours. RESULTS: In part A, fluconazole decreased the area under the plasma concentration-time curve, percent of dose excreted in 24-hour urine, and formation clearance of the hydroxylamine by 49%, 53%, and 55% (n = 12, P < .05), respectively. This inhibition of in vivo hydroxylamine formation was quantitatively consistent with that predicted from human liver microsomal experiments. Rifabutin had no effect on hydroxylamine area under the plasma concentration-time curve or percent excreted in 24-hour urine but increased formation clearance of the hydroxylamine by 92% (n = 12, P < .05). Dapsone clearance was increased by rifabutin or rifabutin plus fluconazole (67% and 38%, respectively) (n = 12, P < .05) but was unaffected by fluconazole or clarithromycin. In part B, hydroxylamine production was unaffected by clarithromycin but was affected by fluconazole in a manner identical to that in part A. CONCLUSIONS: On the basis of these data and with the assumption that the exposure to the hydroxylamine is a determinant of dapsone toxicity, we predict that coadministration of fluconazole should decrease the rate of adverse reactions to dapsone in persons with HIV infection but that rifabutin and clarithromycin will have no effect. When dapsone is given in combination with rifabutin, dapsone dosage adjustment may be necessary in light of the increase in dapsone clearance.

Adult↗
PubMed1990

Thiol-dependent passive K: Cl transport in sheep red blood cells: X. A hydroxylamine-oxidation induced K: Cl flux blocked by diethylpyrocarbonate.

Hydroxylamine, a potent oxidizing agent used to reverse carbethoxylation of histidine by diethylpyrocarbonate, activated Cl-dependent K flux (K: Cl cotransport) of low K sheep red blood cells almost sixfold. When K: Cl cotransport was already stimulated by N-ethylmaleimide, hydroxylamine caused an additional twofold activation suggesting modification of sites different from those thiol alkylated. This conclusion was supported by the finding that hydroxylamine additively augmented also the diamide-induced K: Cl flux (Lauf, P.K. 1988. J. Membrane Biol. 101: 179-188) with dithiothreitol fully reversing the diamide but not the hydroxylamine effect. Stimulation of K: Cl cotransport by hydroxylamine was completely inhibited by treatment with diethylpyrocarbonate also known to prevent K: Cl cotransport stimulation by N-ethylmaleimide, both effects being independent of the order of addition. Hence, although the effect of carbethoxy modification of K: Cl flux cannot be reversed by hydroxylamine and thus excludes histidine as the target for diethylpyrocarbonate, our finding reveals an important chemical determinant of K: Cl cotransport stimulation by both hydroxylamine oxidation and thiol group alkylation.

Animals↗
PubMed1999

Methemoglobin formation by hydroxylamine metabolites of sulfamethoxazole and dapsone: implications for differences in adverse drug reactions.

Differences in the incidence of adverse drug reactions to trimethoprim-sulfamethoxazole and dapsone may result from differences in the formation, disposition, toxicity, and/or detoxification of their hydroxylamine metabolites. In this study, we examine whether differences in the biochemical processing of sulfamethoxazole hydroxylamine (SMX-NOH) and dapsone hydroxylamine (DDS-NOH) by erythrocytes [red blood cells (RBCs)] contribute to this differential incidence. The methemoglobin (MetHgb)-forming capacity of both metabolites was compared after a 60-min incubation with washed RBCs from four healthy human volunteers. DDS-NOH was significantly more potent (P =.004) but equally efficacious with SMX-NOH in its ability to form MetHgb. The elimination of potential differences in disposition by lysing RBCs did not change the MetHgb-forming potency of either hydroxylamine. At pharmacologically relevant concentrations, greater reduction to the parent amine occurred with DDS-NOH. Maintenance of MetHgb-forming potency was dependent on recycling with glutathione, but no difference in cycling efficiency was observed between DDS-NOH and SMX-NOH. In contrast, the pharmacodynamics of hydroxylamine-induced MetHgb formation were not changed by pretreatment with the glucose 6-phosphate dehydrogenase inhibitor epiandrosterone or by compounds that alter normal antioxidant enzyme activity. Methylene blue, which stimulates NADPH-dependent MetHgb reductase activity, decreased MetHgb levels but did not alter the differential potency of these hydroxylamines. DDS-NOH was also significantly more potent when incubated with purified human hemoglobin A0. Collectively, these data suggest that the inherently greater reactivity of DDS-NOH with hemoglobin, the greater conversion of DDS-NOH to its parent amine, and potential differences in disposition of hydroxylamine metabolites may contribute to the preferential development of dapsone-induced hemotoxicity and sulfamethoxazole-induced hypersensitivity reactions.

Dapsone↗
PubMed1988

Oxidation of hydroxylamines to nitroxide spin labels in living cells.

In the presence of oxygen, cells can oxidize hydroxylamines, which are the products of the reduction of nitroxides in cells, back to nitroxides. Lipid-soluble hydroxylamines are oxidized much more rapidly than water-soluble ones, and most of this oxidation is inactivated by heat or trichloroacetic acid, indicating that the principal mechanism is enzyme-linked. The rates of oxidation of some lipophilic hydroxylamines are comparable to the rates of reduction of the corresponding nitroxides. Hydroxylamines formed by reduction of aqueous soluble nitroxides are not oxidized by cells, except for slight oxidation of some pyrrolidine derivatives. The latter is due to autoxidation. The kinetics of oxidation of reduced lipid-soluble nitroxides are all first-order with respect to hydroxylamines, regardless of the position of the nitroxide group along the carbon backbone, indicating that the oxidation occurs within the membrane. The oxidation of hydroxylamines in cells in inhibited by cyanide but not by antimycin A or SKF-525A. We also describe an effective method to oxidize hydroxylamines and follow this reaction; the method is based on the use of perdeuterated [15N]Tempone.

Affinity Labels↗
PubMed2001

Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds.

Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour. The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group (ortho-position) disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix. In contrast, 4'-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group "far away" from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.

Biotransformation↗
PubMed2000

Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation.

In this study a novel hydroxylamine oxidoreductase (HAO) was purified and characterized from an anaerobic ammonium-oxidizing (Anammox) enrichment culture. The enzyme, which constituted about 9% of the protein mass in the soluble fraction of the cell extract, was able to oxidize hydroxylamine and hydrazine. When phenazine methosulfate and methylthiazolyltetrazolium bromide were used as electron acceptors, a V(max) [21 and 1.1 micromol min(-)(1) (mg of protein)(-)(1)] and K(m) (26 and 18 microM) for hydroxylamine and hydrazine were determined, respectively. The hydroxylamine oxidoreductase is a trimer and contains about 26 hemes per 183 kDa. As deduced from UV/vis spectra, hydroxylamine reduced more and different cytochromes than hydrazine. The dithionite-reduced spectrum showed an unusual 468 nm peak. Inhibition experiments with H(2)O(2) showed that hydroxylamine bound to this P-468 cytochrome, which is assumed to be the putative substrate binding site. Cyanide and hydrazine inhibited the oxidation of hydroxylamine. The amino acid sequences of several peptide fragments of HAO from Anammox showed a clear difference with the deduced amino acid sequence of HAO from the aerobic ammonia-oxidizing bacterium Nitrosomonas europaea. In EPR spectra of the Anammox HAO, two g-values (g(z)() = 2.37 and 2.42) were observed, which were not present in HAO of N. europaea.

Bacterial Proteins↗
PubMed2005

Hydroxylamines as oxygen atom nucleophiles in transition-metal-catalyzed allylic substitution.

[reaction: see text] The viability of hydroxylamines as nucleophiles in transition-metal-catalyzed allylic substitutions was examined. We have found that the oxygen atom of hydroxylamines having an N-electron-withdrawing substituent (also known as hydroxamic acids) acts as a reactive nucleophile. The palladium-catalyzed O-allylic substitution of hydroxylamines with allylic carbonate afforded the linear hydroxylamines. The selective formation of the branched hydroxylamines was observed in iridium-catalyzed reaction. Regio- and enantioselective allylic substitution of the unsymmetrical phosphates with hydroxylamines was studied by using the iridium complex of chiral pybox ligand. The aqueous-medium reaction with hydroxylamines proceeded smoothly in the presence of Ba(OH)(2).H(2)O to give the branched products with good enantioselectivities.

Allyl Compounds↗