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PubMed1987

Dependence on Salmonella typhimurium enzymes of mutagenicities of nitrobenzene and its derivatives in the presence of rat-liver S9 and norharman.

Dependence on S. typhimurium enzymes of mutagenicities of nitrobenzene (NB) and o-, p-chloronitrobenzenes (o-, p-CNBs), which are only mutagenic in the presence of S9 and norharman (NOH), was investigated using a nitroreductase-deficient strain TA98NR and an esterifying enzyme-deficient strain TA98/1,8-DNP6. NB exhibited mutagenicity towards TA98 but did not towards TA98NR strain in spite of the presence of S9 in the assay system. The mutagenicity of o-CNB towards TA98NR was significantly lower than that of o-CNB towards TA98. In contrast to NB and o-CNB, synthesized phenylhydroxylamine (PHA) and o-chlorophenylhydroxylamine (o-CPHA) exhibited approximately the same mutagenicity towards both tester strains. These results indicate that the nitroreduction required for the appearance of mutagenicity of the nitrobenzene derivatives in the presence of S9 and NOH is dependent on the nitroreductase of the tester strain. In addition, the mutagenicities of PHA and p-CPHA were significantly higher towards TA98/1,8-DNP6 than towards TA98, suggesting that the esterification of their hydroxylamines produced inactivation rather than activation. From these results, it was concluded that S9 and NOH play a role in metabolic activation other than the reduction of the nitro group to hydroxylamine and subsequent esterification for the mutagenesis of NB and its derivatives.

Alkaloids↗

PubMed1982

Mono ADP-ribosylation and poly ADP-ribosylation of proteins in normal and malignant tissues.

Three subclasses of (ADPR)n protein conjugates were quantified from intact tissue; proteins carrying poly(ADPR) and two types of mono(ADPR) protein conjugates, one susceptible, the other resistant to neutral hydroxylamine. Mono(ADPR) conjugates were found in all major compartments of the liver cell although the two subfractions were unevenly distributed. Poly(ADPR) protein conjugates appear to be restricted to the nucleus. Independent changes of the subclasses in normal and malignant tissues associated with cell growth and differentiation also point to independent functions. Hydroxylamine-resistant mono(ADPR) protein conjugates of various tissues changed with the degree of terminal differentiation. Formation of poly(ADPR) proteins, on the other hand, was stimulated by treatment of cells with alkylating agents which lead to DNA-fragmentation. This points to an involvement of polyADP-ribosylation in DNA excision repair.

Adenosine Diphosphate Ribose↗

PubMed1989

Potent and selective inactivation of proteinases with N-peptidyl-O-acylhydroxylamines.

The reaction of seven N-peptidyl-O-acyl hydroxylamines with serine proteinases exhibiting different substrate specificity has been investigated. Depending on the structure of the peptidyl residue of the inhibitors, rapid and complete irreversible inactivation of the enzymes may be achieved. Enzyme-catalyzed turnover of inhibitor to products was detected during reaction of proline-specific endopeptidase with N-Boc-Ala-Pro-O-(4-nitrobenzoyl)hydroxylamine.

Endopeptidases↗

PubMed1990

Analysis of the reduction of nitroxides by flavin mononucleotide.

This article describes a simple method to prepare hydroxylamines from nitroxides by photo-activated flavin mononucleotide. The half-time of reduction varied from 2 to 38.4 s for a series of nitroxides. For most nitroxides short exposures to light (min) were sufficient to produce significant amounts of hydroxylamine; longer periods of exposure increased the yields of other products. Proxyl (2,2,5-trimethyl-5-alkylpyrrolidine-N-oxyl) nitroxides were unusually reactive with a much higher yield of products which could not be reoxidized by ferricyanide to the nitroxides. Optimum conditions for reversible reduction depend on the nitroxide and the amounts of other reducible substances such as oxygen and ferricyanide that may be present.

Cyclic N-Oxides↗

PubMed1988

Cyclic GMP and cell death in rat cerebellar slices.

Incubated slices of young rat cerebellum were used to examine the possible relationship between the neurotoxic effects of excitatory amino acids and their ability to elicit large increases in the levels of cyclic GMP in this tissue. No cell death was detectable following exposure of the slices to the guanylate cyclase activator, nitroprusside (up to 0.3 mM), the phosphodiesterase inhibitor, isobutylmethylxanthine (0.5 mM), or to cyclic GMP (10 mM) and its dibutyryl and 8-bromo derivatives (0.5 mM). However, incubation of the slices with tbe guanylate cyclase inhibitors, N-methylhydroxylamine and hydroxylamine (0.1-1 mM), methylene blue (10-100 microM), ethacrynic acid (300 microM) and retinol (1 mM) caused a progressive destruction of the differentiating cells. The damage induced by N-methylhydroxylamine and hydroxylamine was inhibited by nitroprusside, cyclic GMP and isobutylmethylxanthine. It could also be reduced by lowering the partial pressure of oxygen, by oxygen radical scavenging enzymes and by omitting Ca2+ from the medium. Oxygen radical generating enzyme systems mimicked the pattern of toxicity of the guanylate cyclase inhibitors but their effects were not reduced by nitroprusside or omission of Ca2+. The results indicate that guanylate cyclase/cyclic GMP does not mediate amino acid neurotoxicity but, instead, may be part of a protective mechanism against oxygen free radicals.

Animals↗

PubMed1995

Characterization of 2-amino-1-benzylbenzimidazole and its metabolites using tandem mass spectrometry.

We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocycle, 2-amino-1-benzylbenzimidazole (ABB). Three major metabolites were isolated and structurally characterized, using a combination of off-line HPLC, in conjunction with both electron ionization and fast atom bombardment ionization tandem mass spectrometry. ABB was shown to be debenzylated to afford 2-aminobenzimidazole (AB), as well as N- and C-oxidized to give 1-benzyl-N2-hydroxyaminobenzimidazole (BHB) and 2-amino-1-benzyl-hydroxybenzimidazole, respectively. The possible reasons for formation of the exocyclic hydroxylamine BHB are discussed. Furthermore, ABB is proposed as a suitable model compound for investigating parameters that control formation of toxic hydroxylamines derived from amino-azaheterocycles.

Animals↗

PubMed1995

Kinetics and mechanism of general acid-catalysed thiolytic cleavage of 9-anilinoacridine.

The rates of the reactions of 2-mercaptoethanol (2-ME) with 9-anilinoacridine (9-ANA) have been studied in the buffer solutions of 2-ME, hydroxylamine, phosphate and morpholine. Both ionised and non-ionised forms of 2-ME and free hydroxylamine show nucleophilic reactivity toward protonated 9-ANA. The rate constants for general acid-catalysed thiolytic cleavage of protonated 9-ANA reveal a Brønsted plot of slope (alpha) of 0.93 which indicates that probably the rate-determining step involves proton transfer in a thermodynamically unfavourable direction. A stepwise mechanism for thiolysis has been suggested. General acid catalysis could be detected for thiolysis of non-protonated 9-ANA only in the buffer solutions of phosphate and morpholine. General acid catalysis seems to be unimportant when the nucleophile is non-ionised 2-ME which is attributed to the probable occurrence of intramolecular general acid catalysis.

Acids↗

PubMed2005

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations.

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.

Animals↗

PubMed2005

Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer Test of this new volatile medium-pH for HPLC-ESI-MS/MS.

Zalcitabine (ddC), lamivudine (3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir (PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a specific reversed-phase high-performance liquid chromatography (HPLC) method was developed for their analysis in rat plasma. The method involved a quantitative recovery of these drugs from rat plasma by solid-phase extraction on Oasis HLB Waters cartridges followed by optimised HPLC separation on an Atlantis dC18 column with acetic acid-hydroxylamine buffer (ionic strength 5mM, pH 7)-acetonitrile elution gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves were obtained within a 30-10,000 ng/mL plasma concentration range. Correlation coefficients (r2) greater than 0.992 were obtained by least-squares regression and limits of quantification were in 30-90 ng/mL concentration range. Quantitative parameters (accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been tested in HPLC/ESI-MS/MS and appears to be an efficient volatile buffer in the medium 5-7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one hundred times higher to that obtained for the usual acetic acid/ammonia buffer.

Acetic Acid↗

PubMed2005

Aerobic 2- and 4-nitroreduction of CB 1954 by human liver.

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) is an anti-tumour prodrug which recently entered clinical trials in combination with Escherichia coli nitroreductase in a gene-directed enzyme prodrug therapy (GDEPT) context. A Phase I trial of the prodrug, however, revealed dose-limiting hepatotoxicity (transaminitis). The aim of this study was to find out whether the prodrug undergoes reductive metabolism in human liver to cytotoxic metabolites which may contribute to this clinical toxicity. CB 1954 (2.5-250 microM) was incubated with human liver preparations (2-8 mg/mL of S9, cytosolic or microsomal proteins) in the presence of NAD(P)H (1 mM). The NADH- and NADPH-dependent formation of both 2- and 4-nitroreduction products was demonstrated, with NADPH being the preferred cofactor, by HPLC and mass spectrometry. The major metabolite formed in all three human liver preparations was the 4-hydroxylamine, a potent DNA cross-linking cytotoxin. The 2-hydroxylamine and 2-amine metabolites were also detected, both of which have also been demonstrated to be highly cytotoxic. 2-Nitroreduction was far greater in S9 compared with cytosol and was not detected in microsomal preparations. Although 2- and 4-nitroreduction of CB 1954 was inhibited under hyperoxic conditions, substantial metabolism was observed under atmospheric oxygen levels. These studies demonstrate that human liver is capable of aerobic reductive bioactivation of CB 1954 to cytotoxic metabolites in vitro, possibly involving multiple enzymes, which may account for the clinical hepatotoxicity observed.

Antineoplastic Agents↗

PubMed2000

Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta. An additional mechanism for nitroglycerin.

We investigated the involvement of calcitonin gene-related peptide (CGRP) in the vasodilatory mechanism of action of nitric oxide (NO) donors. The functional role of CGRP in NO donor-induced vasodilation of isolated rat aortic rings was determined by incubating these drugs with and without CGRP(8-37), a selective CGRP receptor antagonist. CGRP(8-37) (0.63 microM) induced rightward shifts in the vasodilatory concentration-response curves for nitroglycerin (NTG), Piloty's acid (PA), and SIN-1 (linsidomine). The EC(50) values for NTG, PA, and SIN-1 were increased by 8.3-, 5.2-, and 2.3-fold, respectively (P < 0.05). The release of CGRP from rat aorta in response to NTG and PA was measured specifically by radioimmunoassay. Thirty-minute incubations of NTG or PA with rat aorta induced 189.5 and 214.6% increases, respectively, in CGRP release when compared with the control (P < 0.05). The concentration-response curves of sodium nitroprusside (SNP), S-nitroso-acetylpenicillamine (SNAP), tetranitromethane (TNM), diethylamine NO complex (DEA-NO), and diethylenetriamine/nitric oxide adduct (DETA NONOate) were not inhibited significantly by CGRP(8-37) co-incubation (P 0.05). NO donors also were incubated with aortic strips, and NTG and PA alone induced significant formation of hydroxylamine, a NO(-) metabolite (232.4 and 364.9%, respectively, P < 0.05). These results indicate that only NTG and PA, and to a lesser extent SIN-1, stimulate the release of CGRP from the rat aorta, which subsequently contributes to the vasodilatory activity of these agents. The hydroxylamine formation suggests a possible link between NO(-) generation and CGRP release from the vascular wall.

Animals↗

PubMed1977

Effects of bleaching and regeneration on the purple membrane structure of Halobaterium halobium.

Sequential bleaching in the presence of hydroxylamine and subsequent regeneration of the purple membrane of Halobacterium halobium was studied by concomitant monitoring of its absorption and circular dichroic spectra in order to ascertain its effects on protein interaction(s) (which may result in possible excitonic interaction between the retinal chromophores), chromophore-apoprotein interaction(s), and protein conformational stability in the membrane. It was concluded that (a) although experimental results are consistent with an exciton mechanism for the interaction between retinal pi - pi* (NV(1)) transition movements in the purple membrane, no evidence for such a mechanism for interaction between retinaloxime transition moments is apparent in the case of the bleached membrane; (b) the bacteriorhodopsin molecules organized in clusters of three in the membrane appear to bleach simultaneously; (c) the retinaloxime produced on bleaching the purple membrane in the presence of hydroxylamine is strongly optically active, because of dissymmetry-inducing and/or -selecting constraints on the chromophore by a component of the membrane (most likely the apoprotein), and when the membrane is regenerated by the addition of retinal, these constraints are lost; and (d) evidence from ultraviolet absorption and circular dichroic spectra suggests that the membrane apoprotein undergoes appreciable conformational changes involving tertiary structure on bleaching with no significant secondary structure involvement. These results are compared with recently reported results from this laboratory on the effects of bleaching on the bovine rod outer segment disk membrane structure.

Circular Dichroism↗

PubMed2001

A convenient preparation of aldonohydroxamic acids in water and crystal structure of L-erythronohydroxamic acid.

Hydroxamic acids derived from aldonic acids, namely aldonohydroxamic acids, have become an increasingly important class of inhibitors of enzymes involved in the metabolism of carbohydrates. We now report the straightforward preparation of various types of aldonohydroxamic acids by a new methodology involving the use of commercial 50% aqueous hydroxylamine as the source of the free base hydroxylamine that reacts directly with the corresponding aldonolactone dissolved in water. The reaction proceeds almost instantaneously in water at room temperature, yielding generally pure products in quantitative yield. To date, this methodology is probably the most facile and efficient way to synthesize aldonohydroxamic acids. We also determined by X-ray diffraction analysis the first crystal structure of a free aldonohydroxamic acid reported to date. Crystals of L-erythronohydroxamic acid belonged to the monoclinic system, space group P2(1), a=5.511(3), b=7.556(1), c=8.071(3) A, beta=109.10 degrees, and Z=2.

Crystallization↗

PubMed1999

Action of quinolinic and indolinonic aminoxyls as radical-scavenging antioxidants.

The kinetic studies on the actions of quinolinic and indolinonic aminoxyls in the oxidation of lipid peroxidation induced by free radicals were carried out to evaluate their antioxidant activity. These aminoxyls showed a similar reactivity toward peroxyl radical with alpha-tocopherol. The antioxidant efficacies of aminoxyls against oxidation of methyl linoleate in homogeneous solution were smaller than that of alpha-tocopherol. Hydroxylamine, a reduced form of aminoxyl, possessed a comparative antioxidant efficacy with alpha-tocopherol and was capable of suppressing the consumption of alpha-tocopherol. Aminoxyls showed more potent antioxidant activity than alpha-tocopherol against the oxidation of methyl linoleate micelles induced by peroxyl radical or by a combination of copper ion and hydrogen peroxide. These results suggest that quinolinic and indolinonic aminoxyls may act as potent antioxidants against lipid peroxidation, especially in the presence of a good reductant which reduces aminoxyl radicals to hydroxylamines.

Antioxidants↗

PubMed1989

Affinity separation with polyaldehyde microsphere beads.

Agarose polyaldehyde microsphere beads were prepared by encapsulating polyaldehyde microspheres of various diameters, e.g., polyacrolein or polyglutaraldehyde microspheres, within agarose beads. Amino ligands such as proteins or drugs can be bound covalently to the beads in a single step at physiological pH. The binding capacity of the beads towards various amino ligands is inversely related to the diameter of the microspheres encapsulated in the agarose matrix. Different reagents, e.g., bovine serum albumin, ethanolamine and hydroxylamine, were studied as blocking reagents of the free aldehyde groups. Blocking the remaining aldehyde groups after coupling the amino ligands to the beads is essential for increasing or retaining the reactivity of the ligands conjugated to the beads. Among the reagent studied, hydroxylamine was found to be the most suitable blocking reagent of the free aldehyde groups of beads conjugated with proteins. The extent of leakage of amino ligands bound to the agarose-polyaldehyde microsphere beads was studied as a function of the pH of aqueous solutions of the beads. At physiological pH the leakage was negligible. At acid pH, leakage of ligands containing several primary amine groups, e.g., proteins, was insignificant. However, significant leakage was detected for ligands containing a single amino group. The leakage of proteins bound to the agarose-polyaldehyde microsphere beads was found to be much less than the leakage of the same proteins bound to agarose beads through the cyanogen bromide activation method.

Aldehydes↗

PubMed1996

Identification of domains in human recombinant GABAA receptors that are photoaffinity labelled by [3H]flunitrazepam and [3H]Ro15-4513.

We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.

Affinity Labels↗

PubMed1998

Functional characterization of visual and nonvisual pigments of American chameleon (Anolis carolinensis).

Using only 11-cis 3, 4-dehydroretinal as a chromophore in the pure-cone retina, American chameleon (Anolis carolinensis) detects a wide range of color from ultraviolet (UV) to infrared. We previously characterized its visual opsin genes sws1Ac, sws2Ac, rh1Ac, rh2Ac, and LwsAc that encode SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, and LWSAc opsins, respectively, and the pineal gland-specific opsin (PAc) gene. Here we present the light absorption profiles of the visual pigments obtained by expressing these opsins and reconstituting them with 11-cis retinal using the COS1 cell cDNA expression system. The purified SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, LWSAc, and PAc pigments have the wavelengths of maximal absorption at 358, 437, 491, 495, 560, and 482 nm, respectively. SWS1Ac is the first vertebrate UV opsin whose spectral sensitivity has been directly evaluated. RH1 pigments, orthologous to the rod pigments of other vertebrates, are sensitive to hydroxylamine in the dark, exhibiting a cone pigment-like characteristic, probably reflecting their adaptation to the pure cone retina. Interestingly, the blue-sensitive SWS2Ac pigment shows an exceptionally low level of sensitivity to hydroxylamine, possessing a rod pigment-like characteristic.

Animals↗

PubMed1998

Prevention of the influence of fibrin and alpha2-macroglobulin in the continuous measurement of the thrombin potential: implications for an endpoint determination of the optical density.

We proposed the endogenous thrombin potential (ETP) as an overall function test of the coagulation system. We recently introduced a routine test which requires defibrinated plasma. In order to develop an assay in which the ETP-value can be directly obtained by measuring the optical density, we investigated two methods to inhibit fibrinogen clottability and to inactivate alpha2-macroglobulin. The first method makes use of hydroxylamine to inactivate alpha2-macroglobulin and H-Gly-Pro-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incubated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37 degrees C resulted in a reduced endlevel of the amidolytic activity on small chromogenic substrates. The second method uses a metalloprotease purified from Crotalus basiliscus to remove alpha2-macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro-OH. Herein plasma is incubated with 3.5 LM protease during 15 minutes at 37 degrees C in the presence of 1 mg/mL polymerization inhibitor. The enzymatic method results in a zero endlevel of the amidolytic activity and this would imply that measurement of the ETP is reduced to an endpoint determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculated ETP in plasmas with different degrees of anticoagulation.

Biomarkers↗