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PubMed1983

Amino acid-specific ADP-ribosylation.

[adenine-U-14C]ADP-ribose-agmatine and [adenine-U-14C ))ADP-ribose-histone were synthesized by an NAD:arginine ADP-ribosyltransferase from [14C]NAD and agmatine and histone, respectively. The pseudo-first order rate constants for breakdown of the two components either in 0.4 N NaOH or in 0.4 M neutral hydroxylamine were identical. Hydroxylamine treatment of [14C]ADP-ribose-agmatine or [32P]ADP-ribose-histone yielded a single radioactive product which was separated by high pressure liquid chromatography and identified as ADP-ribose-hydroxamate by the formation of a ferric chloride complex. Hydrolysis of ADP-ribose-hydroxamate with snake venom phosphodiesterase resulted in the formation of 5'-AMP, consistent with the presence of a pyrophosphate bond. Incubation of ADP-ribose-[14C]agmatine, synthesized by the ADP-ribosyltransferase from NAD and [14C]agmatine, with 0.4 M neutral hydroxylamine resulted in the release of [14C]agmatine rather than phosphoribosyl[14C]agmatine. In addition, neither NAD nor ADP-ribose reacts with hydroxylamine; i.e. there was no evidence of nucleophilic attack by hydroxylamine at the pyrophosphate bond. The ADP-ribosyl-protein linkage formed by the NAD:arginine ADP-ribosyltransferase is considerably more stable to hydroxylamine than is the ADP-ribose-glutamate bond. The presence of ADP-ribose-arginine and ADP-ribose-glutamate synthesized by the ADP-ribosyltransferase and poly(ADP-ribose) synthetase, respectively, may be the chemical basis for the "hydroxylamine-stable" and "hydroxylamine-labile" bonds described by Hilz (Hilz, H. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 1415-1425).

ADP Ribose Transferases↗
PubMed1972

Heterotrophic nitrifiction by Arthrobacter sp.

Arthrobacter sp. isolated from sewage oxidized ammonium to hydroxylamine, a bound hydroxylamine compound, a hydroxamic acid, a substance presumed to be a primary nitro compound, nitrite, and nitrate. The concentration of free hydroxylamine-nitrogen reached 15 mug/ml. The identification of hydroxylamine was verified by mass spectrometric analysis of its benzophenone oxime derivative. The bound hydroxylamine was tentatively identified as 1-nitrosoethanol on the basis of its mass spectrum, chemical reactions, and infrared and ultraviolet spectra. Hydroxylamine formation by growing cells was relatively independent of pH, but the accumulation of nitrite was strongly favored in alkaline solutions. The formation of hydroxylamine but not nitrite was regulated by the carbon to nitrogen ratio of the medium. The hydroxamic acid was the dominant product of nitrification in iron-deficient media, but hydroxylamine, nitrite, and 1-nitrosoethanol formation was favored in iron-rich solutions. Heterotrophic nitrification by Arthrobacter sp. was not inhibited by several compounds at concentrations which totally inhibited autotrophic nitrification.

Acetates↗
PubMed1997

Detoxication of tyramine by the flavin-containing monooxygenase: stereoselective formation of the trans oxime.

In the presence of pig or adult human liver microsomes, tyramine was metabolized to the corresponding trans oxime through the intermediacy of the hydroxylamine. The requisite intermediate, (4-hydroxyphenethyl)hydroxylamine, was retroreduced to tyramine or converted stereoselectively to the trans oxime in the presence of pig or adult human liver microsomes. Studies of the effect of metabolic inhibitors suggested that formation of the trans oxime and retroreduction of the hydroxylamine were largely dependent on NADPH and the flavin-containing monooxygenase (FMO) and cytochrome P450, respectively. The conclusion that FMO was predominantly responsible for trans oxime formation in human liver microsomes was based on the effect of incubation conditions on tyramine N-oxygenation and the observation that cDNA-expressed human FMO3 also N-oxygenated tyramine to give exclusively the trans oxime. The synthetic hydroxylamine and oxime metabolites of tyramine were examined for affinity to human and animal dopamine and serotonin receptors and the human dopamine transporter. For all of the receptors and for the transporter examined, the avidity of the hydroxylamine and oximes was greater than 10 microM and beyond the effective concentration for physiological relevance. The results suggested that tyramine was sequentially N-oxygenated in the presence of pig and human liver microsomes and cDNA-expressed FMO3 to the hydroxylamine and then to the di-N-hydroxylamine that was spontaneously dehydrated to the trans oxime. This may be facilitated by FMO through a nondissociative substrate-enzyme interaction. Based on the biogenic amine receptor or transporter affinity for the hydroxylamine and oxime metabolites of tyramine, N-oxygenation of tyramine by pig or human liver FMO may represent a detoxication reaction that terminates the pharmacological activity of tyramine.

Adult↗
PubMed2000

A role for bioactivation and covalent binding within epidermal keratinocytes in sulfonamide-induced cutaneous drug reactions.

Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Adult↗
PubMed2005

Identification of cytochrome P450 and arylamine N-acetyltransferase isoforms involved in sulfadiazine metabolism.

Sulfadiazine hydroxylamine has been postulated to be the mediator of the greatly increased rates of adverse reactions to sulfadiazine experienced by people with human immunodeficiency virus infection. Therefore, we investigated the in vitro human cytochrome P450 (P450) and N-arylamine acetyltransferase (detoxification) metabolism of sulfadiazine. Formation of both the hydroxylamine and 4-hydroxy sulfadiazine was NADPH-dependent in human liver microsomes (HLM). The average K(m) (+/-S.D.) and V(max) in HLM (n = 3) for hydroxylamine formation was 5.7 +/- 2.2 mM and 185 +/- 142 pmol/min/mg, respectively. Significant (p < 0.05) inhibition by selective P450 isoform inhibitor sulfaphenazole (2.1 microM; CYP2C9) indicated a role for CYP2C9 in the formation of the hydroxylamine. Hydroxylamine formation correlated strongly with tolbutamide 4-hydroxylation (CYP2C8/9) in HLM (r = 0.76, p < or = 0.004, n = 12). Fluconazole (CYP2C9/19 and CYP3A4 inhibitor at clinical concentrations) inhibited hydroxylamine formation, with one-enzyme model K(i) estimates ranging from 9 to 40 microM. Acetylation of sulfadiazine in human liver cytosol (HLC) correlated strongly with NAT2 activity as measured by sulfamethazine N-acetylation (r = 0.92, p < 0.001, n = 12). The average K(m) (+/-S.D.) and V(max) in HLC (n = 3) was 3.1 +/- 1.7 mM and 221.8 +/- 132.3 pmol/min/mg, respectively. The polymorphic acetylation of sulfadiazine may predispose slow acetylator patients to adverse reactions to sulfadiazine. On the basis of our K(i) estimates, clinical fluconazole concentrations of 25 microM would produce decreases of 40 to 70% in hepatic-mediated hydroxylamine production. Therefore, we predict that fluconazole may prove useful in the clinic as an in vivo inhibitor of sulfadiazine hydroxylamine formation to suppress adverse reactions to this drug.

Antiprotozoal Agents↗
PubMed1987

Rapid assay for microbially reducible ferric iron in aquatic sediments.

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.

Journal Article↗
PubMed1982

The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors.

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.

Cytochrome c Group↗
PubMed1995

Characterization of nitric oxide generator-induced hippocampal [3H]norepinephrine release. II. The role of calcium, reverse norepinephrine transport and cyclic 3',5'-guanosine monophosphate.

The mechanisms by which two nitrogen monoxide (NO) generators, hydroxylamine and S-nitroso-L-cysteine (NO-CYS), induce hippocampal [3H]norepinephrine ([3H]NE) release was investigated. Neither hydroxylamine- nor NO-CYS-induced release was affected by the guanylate cyclase inhibitors, methylene blue or LY 83,583. The effect of hydroxylamine was completely dependent on extracellular Ca++ and reduced by 40% in the presence of omega-conotoxin GVIA, an N-type Ca(++)-channel antagonist; however it was unaffected by Ni++, nifedipine, caffeine or thapsigargin. The stimulatory effect of hydroxylamine on hippocampal cyclic GMP formation was not significantly affected by removal of extracellular Ca++, indicating that Ca(++)-dependent release is not due to inhibition of NO formation from hydroxylamine. However, the response to NO-CYS was reduced by 35 to 50% in either nominally Ca(++)-free or 10 mM MgSO4-containing buffer. Interestingly, buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid dramatically enhanced the formation of NO from NO-CYS and potentiated the NO-CYS response. Both NO-CYS- and hydroxylamine-induced [3H]NE release was inhibited by NE transport blockers, indicating a prominent role for reverse transport. NO-CYS completely inhibited synaptosomal uptake of [3H]NE (IC50 approximately, 300 microM). NO generator-induced [3H]NE release has a glutamate-dependent component (see accompanying article). Inhibition of glutamate-evoked [3H]NE release by mazindol, an inhibitor of NE transport, suggests that the glutamate-dependent component also involves reversal of the NE transporter. These data suggest that NO produced from hydroxylamine or NO-CYS evoke both vesicular and nonvesicular release of hippocampal [3H]NE. Putative NO target molecules and the role of extracellular Ca++ are discussed.

Animals↗
PubMed1995

Characterization of nitric oxide generator-induced hippocampal [3H]norepinephrine release. I. The role of glutamate.

In this study we compared the effects of two nitrogen monoxide (NO) generators, hydroxylamine and S-nitroso-L-cysteine (NO-CYS), on hippocampal [3H]norepinephrine ([3H]NE) release. A 10-min incubation with hydroxylamine (3-3,000 microM) or NO-CYS (30-10,000 microM) induced a concentration-dependent increase in the basal [3H]NE efflux with EC50 values of approximately 100 microM and 1 mM, respectively. Reduced hemoglobin, a NO scavenger, blocked both hydroxylamine- and NO-CYS-evoked [3H]NE release. Long-term exposure (> or = 25 min) to 100 microM hydroxylamine, or to millimolar concentrations of NO-CYS, evoked a tetrodotoxin-insensitive [3H]NE release. However, a 10-min stimulation with either 100 microM hydroxylamine or 300 microM NO-CYS was sensitive to 0.5 microM tetrodotoxin, a voltage-sensitive sodium channel blocker. This suggested that under these conditions hydroxylamine and NO-CYS induce [3H]NE release indirectly in part, perhaps via releasing an excitatory neurotransmitter. Indeed, kynurenate, a nonselective ionotropic glutamate receptor antagonist, produced an 80% inhibition of the NO generator-evoked [3H]NE release. CGS 19755, a N-methyl-D-aspartate receptor antagonist, had no significant effect, whereas the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptor antagonists, CNQX and GYKI 52446, inhibited the hydroxylamine response by 50%. In synaptosomes, a preparation in which synaptic interactions are nonsignificant, NO-CYS induced a dose-dependent release of both [3H]NE and [3H]glutamate. These data suggest that, in hippocampal slices, NO generators evoke [3H]NE release both directly from noradrenergic terminals and indirectly via releasing glutamate.

Animals↗
PubMed1967

Genetic and biochemical studies of nitrate reduction in Aspergillus nidulans.

1. In Aspergillus nidulans nitrate and nitrite induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the three enzymes. 2. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrite reductase and hydroxylamine reductase (which are possibly a single enzyme in A. nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. 3. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. 4. Mutants at six gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. 5. Mutants are known at two gene loci, niiA and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. 6. Mutants at the niiA locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. 7. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the niiB gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. 8. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. 9. The growth and enzyme characteristics of a total of 123 mutants involving nine different genes indicate that nitrate is reduced to ammonium. Only two possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only two enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.

Aspergillus↗
PubMed1972

Mechanism of nitrification by Arthrobacter sp.

Resting cells of Arthrobacter sp. excrete as much as 60 mug of hydroxylamine-nitrogen per ml when supplied with ammonium. An organic carbon source in abundant supply is necessary for the oxidation. Resting cells oxidize hydroxylamine to nitrite and 1-nitrosoethanol, the former accumulating only when an exogenous carbon source is available. Cell-free extracts contain an enzyme catalyzing the formation of hydroxylamine from acetohydroxamic acid, a hydroxylamine-nitrite oxido-reductase, and an enzyme producing nitrite and nitrate from various primary nitro compounds. Nitrite is not produced from hydroxylamine by the extracts, but 1-nitrosoethanol is formed from hydroxylamine in the presence of acetate. 1-Nitrosoethanol is also produced from acetohydroxamic acid by these preparations. Nitrite was formed from hydroxylamine, however, by extracellular enzymes excreted by the bacterium.

Acetates↗
PubMed1998

ESR and HPLC-EC analysis of ethanol oxidation to 1-hydroxyethyl radical: rapid reduction and quantification of POBN and PBN nitroxides.

Extensive ESR spin-trapping studies have shown that ethanol is oxidized to 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. The ESR detection of POBN/HER nitroxide in bile, and formation of antibodies, which recognize HER adducts in alcoholics, suggest that HER is produced in vivo. In liver, where ethanol is primarily metabolized, only traces of PBN/HER nitroxide are documented. One limitation of the ESR spin-trapping technique, however, is that the nitroxides formed in the presence of cellular reductants can be metabolized to the corresponding ESR "silent" hydroxylamines. Ascorbate and NADPH plus liver microsomes were found to reduce rapidly both POBN/HER and PBN/HER nitroxides to their ESR "silent" hydroxylamine derivatives. An HPLC method with electrochemical detection was developed for the detection and quantification of both POBN/HER and PBN/HER nitroxides, as well as their hydroxylamines. Both the diastereomers of the POBN/HER nitroxide and hydroxylamine can be detected, as can both isomers of the PBN/HER nitroxide, and it is estimated that the sensitivity of the HPLC procedure is in the nM range when using EC detection. The hydroxylamines are stable in ethanol, while pH-dependent auto-oxidation occurs in aqueous buffers. Some of the characteristics associated with HER formation by microsomes as detected with ESR (e.g., sensitivity to SOD and catalase, increase after induction of CYP2E1) are reproduced with the HPLC method. By quantification of the POBN/HER hydroxylamine, the NADPH-dependent rates of HER formation by microsomes from pyrazole-treated rats are estimated to be about 1-1.5 nmol HER per min per mg microsomal protein. This rate is less, as compared to the two electron-dependent rate of acetaldehyde formation by these microsomes, about 10-15 nmol per min per mg protein. Thus, at first approximation, the one electron-dependent rate of ethanol oxidation is about 10% the two electron-dependent rate by isolated microsomes. The HPLC procedure can readily detect the POBN/HER and PBN/HER nitroxides and their hydroxylamine derivatives in the same sample and may be of value in detecting HER spin-trapped adducts under biological reducing conditions.

Animals↗
PubMed2001

Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabolism-dependent haptenation and T-cell proliferation in vivo.

Sulphamethoxazole has been associated with the occurrence of hypersensitivity reactions. There is controversy as to whether the immune response is metabolism-dependent or -independent. We have therefore investigated the site of antigen formation and the nature of the drug signal presented to the immune system in vivo. Male Wistar rats were dosed with sulphamethoxazole, sulphamethoxazole hydroxylamine or nitroso sulphamethoxazole. Antigen formation on cell surfaces was determined by flow cytometry using a specific anti-sulphamethoxazole antibody. Immunogenicity was determined by assessment of ex vivo T-cell proliferation. Administration of nitroso sulphamethoxazole, but not sulphamethoxazole or sulphamethoxazole hydroxylamine, resulted in antigen formation on the surface of lymphocytes, splenocytes and epidermal keratinocytes, and a strong proliferative response of splenocytes on re-stimulation with nitroso sulphamethoxazole. Rats dosed with sulphamethoxazole or sulphamethoxazole hydroxylamine did not respond to any of the test compounds. CD4+ or CD8+ depleted cells responded equally to nitroso sulphamethoxazole. The proliferative response to nitroso sulphamethoxazole was seen even after pulsing for only 5 min, and was not inhibited by glutathione. Responding cells produced IFN-gamma, but not IL-4. Haptenation of cells by sulphamethoxazole hydroxylamine was seen after depletion of glutathione by pre-treating the rats with diethyl maleate. Splenocytes from the glutathione-depleted sulphamethoxazole hydroxylamine-treated rats responded weakly to nitroso sulphamethoxazole, but not to sulphamethoxazole or sulphamethoxazole hydroxylamine. Dosing of rats with sulphamethoxazole produced a cellular response to nitroso sulphamethoxazole (but not to sulphamethoxazole or its hydroxylamine) when the animals were primed with complete Freund's adjuvant. These studies demonstrate the antigenicity of nitroso sulphamethoxazole in vivo and provide evidence for the role of drug metabolism and cell surface haptenation in the induction of a cellular immune response in the rat.

Adjuvants, Immunologic↗
PubMed1982

Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil.

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V(max)) of 0.066 and 0.003 mumol of N x min x mg of protein, respectively, at 22 degrees C. K(m) values were 15 and 42 muM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.

Journal Article↗
PubMed1990

In vitro and in vivo anti-tumor activity of L-glutamic acid gamma-monohydroxamate against L1210 leukemia and B16 melanoma.

A glutamine analogue, L-glutamic acid gamma-monohydroxamate (GAH) demonstrated complete cytotoxicity against L1210 cells in culture and marked anti-tumoral activity in vivo against L1210 leukemia and B16 melanoma. In vitro, GAH caused concentration-dependent inhibition of L1210 cell growth, with complete cell death being reached at 72 hr and at a 500 microM concentration. A minimal incubation time of 38 hr with 500 microM GAH was necessary to obtain complete cell death at 72 hr. During incubation, GAH is metabolized to hydroxylamine. Hydroxylamine acts as the active form of GAH, since the concentration-dependent inhibition of cell growth caused by hydroxylamine is the same as that observed with GAH. The cytotoxic effects of GAH and hydroxylamine on L1210 cells were not reversed or prevented by L-glutamine or L-glutamic acid and purine nucleosides but were prevented or reversed by pyruvate, 2-oxaloacetate and 2-oxoglutarate. In vivo, GAH considerably increased survival of mice bearing L1210 leukemia or a solid tumor, the B16 melanoma. Antitumor activity of GAH against L1210 leukemia and B16 melanoma was schedule-dependent. The administration of GAH 3 times daily was more effective than a twice daily treatment and the maximum ILS was observed using split-dose schedules on days 1 through 3 and 7 through 9 without noticeable toxicity. Under these conditions hydroxylamine is highly toxic, suggesting that in vivo GAH might act as an hydroxylamine releaser in the tumor cells and is not significantly metabolized in the body.

Animals↗
PubMed1989

Exchange and shuttling of electrons by nitroxide spin labels.

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.

Animals↗
PubMed1999

Taurine release modified by nitric oxide-generating compounds in the developing and adult mouse hippocampus.

The effects of the nitric oxide-generating compounds hydroxylamine, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and the nitric oxide synthase inhibitors nitroarginine and 7-nitroindazole on taurine release from hippocampal slices from adult (three-month-old) and developing (seven-day-old) mice were characterized using a superfusion system. The basal release of [3H]taurine was enhanced when the nitric oxide donors were added at the beginning of superfusion, more markedly in the adult than in the immature hippocampus. The effect of hydroxylamine was clearly concentration-dependent. Hydroxylamine also markedly enhanced the release of endogenous taurine. The K+-stimulated (50 mM) release of taurine was generally inhibited by the nitric oxide-generating compounds in both age groups. Nitric oxide is thus able to act directly at presynaptic terminals, modulating taurine release as a retrograde messenger. The N-methyl-D-aspartate-evoked taurine release was reduced by the nitric oxide donors, particularly by sodium nitroprusside, in the adult hippocampus, while hydroxylamine and S-nitroso-N-acetylpenicillamine markedly potentiated the release in developing mice. In the immature hippocampus the hydroxylamine-enhanced taurine release seems to involve a Ca2+-independent, Na+-dependent and carrier-mediated process while in adult mice only a part of the hydroxylamine-enhanced release is mediated by the same mechanism. The results show that nitric oxide-generating compounds modify the basal, K+- and N-methyl-D-aspartate-evoked releases of taurine in both adult and immature hippocampus. The enhanced N-methyl-D-aspartate receptor-evoked release may be an important mechanism protecting the immature brain against excitotoxicity.

Aging↗
PubMed1999

Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms.

Nitroxide stable radicals generally serve for probing molecular motion in membranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging. Recently nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which are depleted while attenuating oxidative damage, nitroxides can be recycled. In many cases the antioxidative activity of nitroxides is associated with switching between their oxidized and reduced forms. In the present work, superoxide radicals were generated either radiolytically or enzymatically using hypoxanthine/xanthine oxidase. Electron paramagnetic resonance (EPR) spectrometry was used to follow the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxidation. The results indicate that: a) The rate constant of superoxide reaction with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of superoxide reaction with nitroxides; b) The oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superoxide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simulation of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxides in vitro can be distinguished from that of common LMWA due to hydroxylamine oxidation by superoxide, which allows nitroxide recycling and enables its catalytic activity.

Antioxidants↗