The DAB-Mn++ cytochemical method revisited: validation of specificity for superoxide.

Steinbeck, Marla J.; Khan, Ahsan U.; Appel, Walter; Karnovsky, Manfred L.

doi:10.1177/41.11.8292156

Cited by 49 publications

(31 citation statements)

References 34 publications

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“…In addition, the N-chloramine assay of Witko et al (41) was performed to determine if a reaction of chloramine-T with pyridoxamine or vitamin B 6 had occurred as indicated by a decrease in the amount of chloramine-T available to oxidize KI. (42,43). A duplicate set of coverslips was coated with either dichloromethane or a solution of DNE in dichloromethane (3.6 mg/100 l) by surface evaporation at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Chlorination of Pyridinium Compounds

Daumer

Khan

Steinbeck

2000

Journal of Biological Chemistry

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Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H 2 O 2 ), hypochlorous acid/hypochlorite (HOCl/OCl ؊ ), and singlet oxygen (O 2 ( 1 ⌬g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B 6 , which share the same chemical structure and spectral properties of Pyd cross-links. Neither H 2 O 2 (125-500 M) nor O 2 ( 1 ⌬g) (10 -25 M) significantly changed the spectral properties of pyridoxamine or vitamin B 6 . Reaction of HOCl/OCl ؊ (12.5-50 M) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B 6 , which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl 2 /HOCl/OCl ؊ reacted with both pyridoxamine and vitamin B 6 . Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl 2 /HOCl/OCl ؊ on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5.0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.Inflammation associated with articular cartilage, bone, and dentin surfaces is characterized by accumulation, adhesion, and activation of neutrophils and monocytes, which results in the destruction of cartilage and the loss of bone at these sites (1-4). These effects are thought to be mediated in part by the production of reactive oxygen species (ROS), 1 a group of reactants that includes superoxide (O 2 . ), hydrogen peroxide (H 2 O 2 ), and the highly reactive species hypochlorous acid (HOCl) (5-8).ROS are produced by neutrophils and monocytes after they are recruited from the circulation to extravascular spaces. Once outside of the circulation, they adhere to extracellular matrix proteins (ECM) and undergo activation, which results in the production of ROS and release of proteolytic enzymes directly onto the matrix surface (4, 9 -13). In inflammation associated with arthritic joints, the accumulation and activation of neutrophils and monocytes and increased synovial cell formation result in the loss of synovial membrane integrity and eventually to irreversible damage and destruction of articular cartilage in the affli...

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Section: Methodsmentioning

confidence: 99%

Chlorination of Pyridinium Compounds

Daumer

Khan

Steinbeck

2000

Journal of Biological Chemistry

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“…These methods, however, give only limited information on the cellular source(s) of ROS production in situ. Therefore, we attempted to identify the source(s) of synovial oxidative stress using a cytochemical technique based on the principles described for the histochemical localization of ROS production in polymorphonuclear leukocytes (developed by Karnovsky 1993), using 3,3Ј-diaminobenzidine (DAB) and manganese ions (12). Free radicals directly react with DAB, forming an insoluble DAB polymer in a reaction catalyzed by the presence of Mn .…”

mentioning

confidence: 99%

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

Remans

Oosterhout

Smeets

et al. 2005

Arthritis & Rheumatism

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Objective. To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium.Methods. Cytochemical procedures based on the 3,3-diaminobenzidine (DAB)-Mn 2؉ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanatelabeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2,7-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested.Results. Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H 2 O 2 , since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H 2 O 2 , diphenyleneiodonium (an inhibitor of NADPH oxidase), N G -monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production.Conclusion. Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H 2 O 2 , although the oxidase(s) involved in its generation remains to be determined.

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“…Finally, macrophages and neutrophils are known sources of O 2 Ϫ and NO (2-4), hence of peroxynitrite as well. Steinbeck et al (27,55) have shown that macrophages and neutrophils indeed generate singlet oxygen in significant amounts, comparable to the amount of oxygen consumed in a respiratory burst.…”

Section: Discussionmentioning

confidence: 99%

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution

Khan

Kovacic

Kolbanovskiy

et al. 2000

Proceedings of the National Academy of Sciences

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The mechanism of decomposition of peroxynitrite (OONO ؊ ) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO ؊ was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO ؊ , after protonation at pH 7.0 to HOONO, decomposes into 1 O2 and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO ؊ solution were added to a buffer solution (final pH 7.0 -7.2), and the formation of 1 O2 and NO ؊ in high yields was observed. The 1 O2 generated was trapped as the transannular peroxide (DPAO2) of 9,10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO ؊ ) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO ؊ , the amount of singlet oxygen trapped was reduced by a factor of Ϸ2 whereas the yield of trapping of NO ؊ by methemoglobin remained unaffected. Because NO3 ؊ is known to be the ultimate decomposition product of OONO ؊ , these results suggest that the nitrate anion is not formed by a direct isomerization of OONO ؊ , but by an indirect route originating from NO ؊ .nitrosylhemoglobin ͉ diphenylanthracene endoperoxide P eroxynitrite is a potent oxidant formed by the near diffusioncontrolled reaction of nitric oxide (NO ⅐ ) and superoxide ion (O 2 Ϫ ) (1). Both nitric oxide and superoxide are produced by activated macrophages (2, 3), neutrophils (4), and endothelial cells (5, 6). There is evidence that peroxynitrite is formed in significant concentrations in vivo (7-9) and may contribute to an increased risk for cancer (10), artherosclerosis (11), stroke (12), and other diseases (13). Peroxynitrite is a stable anion in alkaline solution (pKa of 6.8); however, once protonated, it decomposes rapidly with a half-life of less than 1 s at physiological pH at 37°C (14), generating reactive species that readily react with biomolecules such as lipids (15), amino acids (16), and DNA (17). Central to the question of the biochemistry of peroxynitrite is the mechanism of decomposition and the identity of the reactive species, a subject of intense research and controversy (18-21). Speculations about the decomposition mechanisms are largely based on kinetic and thermodynamic considerations (22). It has been shown that bicarbonate ion enhances the rate of disappearance of peroxynitrite, leading to the proposal of a nitrosoperoxycarbonate anion adduct, with a distinctly different chemistry from that of OONO Ϫ (19).We reported previously that the mere acidification of an aqueous peroxynitrite solution resulted in chemiluminescence at 1,270 nm characteristic of the deactivation of singlet oxygen (23). By comparing the intensity of this emission to that of 1 O 2 generated by the reaction of hydrogen peroxide (H 2 O 2 ) with hypochlorite anion (OCl Ϫ ), which is known to be stoichiometric (24), it was...

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The DAB-Mn++ cytochemical method revisited: validation of specificity for superoxide.

Cited by 49 publications

References 34 publications

Chlorination of Pyridinium Compounds

Chlorination of Pyridinium Compounds

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution

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