Ben J. van der Walt

Mechanisms by which clofazimine and dapsone inhibit the myeloperoxidase system. A possible correlation with their anti-inflammatory properties

The mechanisms by which two anti-leprotic drugs (clofazimine and dapsone), both with anti-inflammatory properties, inhibit myeloperoxidase (MPO)-catalysed reactions, were investigated. The disappearance of NADH fluorescence was used as an assay for its oxidation. Chloride stimulated the oxidation of NADH in the MPO-H2O2 system in a concentration-dependent manner (50-fold at 150 mM NaCl). Under these conditions Cl- is oxidized and the oxidant formed, presumably hypochlorous acid (HOCl), oxidizes NADH. Observations demonstrating the effect of the drugs on the MPO system, are: (1) Inhibition of Cl(-)-stimulated oxidation of NADH. (2) Inhibition of polypeptide modification in a model protein, thyroglobulin (TG). (3) Protection of MPO against loss of catalytic activity caused by chlorinating oxidants generated by the system. (4) Inhibition of haemoglobin oxidation. Only dapsone was active here. HPLC analyses suggested that the drugs were not significantly metabolized in the MPO-H2O2 system in the absence of Cl-. Bleaching of clofazimine was stimulated by Cl- in the MPO system, suggesting the involvement of HOCl. Clofazimine was found to be a more potent scavenger of HOCl than dapsone when the inhibition of NADH oxidation by reagent HOCl was used as an assay. This finding is also supported by HPLC analyses which indicated a greater sensitivity of HOCl for clofazimine than for dapsone. Relatively low concentrations of dapsone inhibited the oxidation of oxygenated haemoglobin (HbO2), suggesting that the drug was not metabolized to its N-hydroxylated derivative which is thought to be responsible for methaemoglobin (metHb) formation in vivo. It is proposed that the inhibitory mechanism of action of clofazimine is to scavenge chlorinating oxidants generated by the MPO-Cl(-)-H2O2 system, while dapsone converts MPO into its inactive compound II (ferryl) form. The different inhibitory mechanisms of clofazimine and dapsone towards the MPO system may contribute to the anti-inflammatory actions of the drugs.

Isoniazid-mediated irreversible inhibition of the myeloperoxidase antimicrobial system of the human neutrophil and the effect of thyronines

During aerobic myeloperoxidase-catalysed oxidation of isoniazid at pH 7.8, compound III was generated. Oxidation of isoniazid or hydrazine sulphate at pH values of 6.5 or 7.8 in a myeloperoxidase-H2O2 system caused considerable haem loss, which was associated with compound III formation. Haem loss and also compound III formation could be inhibited when 8 microM thyroxine was included in the reaction mixtures. During the reaction with isoniazid, an intense pink-coloured pigment with maximum absorbance at 500 nm was formed which could be bleached with ascorbate or hypochlorous acid. The pigment was more stable at pH 7.8 than at pH 6.5. A similar pink colour was generated when a mixture of isoniazid and thyroxine in alkaline solution was irradiated with light of wavelength greater than 300 nm. A possible product of thyroxine oxidation, 3,5-diiodotyrosine, could not protect the enzyme against isoniazid-mediated haem loss and no colour formation was observed. Haem loss was most extensive when isoniazid was oxidised in a myeloperoxidase system at pH 7.8 in the presence of 0.1 M NaCl. Thyroxine (8 microM), however, could still inhibit haem loss under these conditions. A good correlation was found between haem loss and irreversible loss of peroxidase activity.

Anti-oxidant properties of H2-receptor antagonists: Effects on myeloperoxidase-catalysed reactions and hydroxyl radical generation in a ferrous-hydrogen peroxide system

Ulcerogenesis of the gastroduodenal mucosa is caused by the digestive action of gastric juice and initially involves an inflammatory reaction with infiltration of phagocytes. The anti-inflammatory activity of many drugs have been attributed to the inhibition of the leukocyte enzyme, myeloperoxidase (MPO). In this study, the H2-antagonists in clinical use were found to be potent inhibitors of MPO-catalysed reactions (IC50 < 3 microM) under conditions resembling those in experiments with intact neutrophils. Since peak plasma concentrations of cimetidine, ranitidine and nizatidine are well within the micromolar range, after oral therapeutic dosing, our results may be of clinical relevance. The inhibitory actions of cimetidine and nizatidine were largely due to scavenging of hypochlorous acid (HOCl), a powerful chlorinating oxidant produced in the MPO-H2O2-Cl- system. In contrast to famotidine, ranitidine was also a potent scavenger of HOCl, while both drugs inhibited MPO reversibly by converting it to compound II, which is inactive in the oxidation of Cl-. The HOCl scavenging potencies of ranitidine and nizatidine were about three times higher than that of the anti-rheumatic drug, penicillamine, which had a potency similar to that of cimetidine. The rapid HOCl scavenging ability of penicillamine is thought to contribute to its anti-inflammatory effects. Using riboflavin as a probe, the H2-antagonists were found to be inhibitors of hydroxyl radical (.OH) generated in a Fe(2+)-H2O2 reaction mixture. Spectral analyses of the interaction of iron ions with the drugs and studies with chelators, suggest that the drugs were efficient chelators of Fe2+, in addition to their .OH scavenging abilities. Since the gastrointestinal tract can contain potentially reactive iron, the simultaneous presence of H2-antagonists may help to suppress iron-driven steps in tissue damage.

The inhibitory effect of acetaminophen on the myeloperoxidase-induced antimicrobial system of the polymorphonuclear leukocyte.

Acetaminophen binds via its acetamido side chain to purified myeloperoxidase in a pH-dependent manner and maximum binding occurred around pH 6. The H2O2-dependent myeloperoxidase-catalysed polymerization products of acetaminophen had excitation maxima at 304 nm and 334 nm in acid and alkaline solutions, respectively, and an intense blue fluorescence maximum at 426 nm. Acetaminophen can compete effectively with Cl- as myeloperoxidase substrate and thus HOCl formation is suppressed while HOCl, nevertheless present, can be scavenged by the drug. In this way the microbicidal action of the myeloperoxidase-H2O2-Cl- system can be seriously limited in the presence of high concentrations of acetaminophen. To study the effect of acetaminophen on peptide bond splitting in the myeloperoxidase antimicrobial system, thyroglobulin was used as a model peptide. Peptide bond splitting was inhibited at acetaminophen concentrations below the accepted toxic range for plasma values.

Activation of chlorpromazine by the myeloperoxidase system of the human neutrophil

The univalent oxidation of chlorpromazine (CPZ) by the myeloperoxidase (MPO-H2O2) system led to the formation of a cation free radical (CPZ+) which was observed optically at 527 nm. CPZ protected MPO against loss of catalytic activity when co-oxidized in a MPO-Cl(-)-H2O2 system. Due to the stability of CPZ+ either further oxidation, or reduction back to the mother compound, become important mechanisms for disappearance of the free radical. Thus, the rate of formation and decay of CPZ+ were higher in the presence of Cl- than in its absence, since the radical can also be oxidized further by hypochlorous acid (HOCl), which is formed in the MPO-Cl(-)-H2O2 system. Decay of CPZ+ can also be due to electron acceptance from ascorbic acid or oxygenated haemoglobin (HbO2), resulting in regeneration of CPZ. When CPZ+ was generated in the MPO-H2O2 system, addition of HbO2 resulted in a sudden decrease in CPZ+ absorbance at 527 nm and a concomitant formation of metHb. When HbO2 was not added, the decay of CPZ+ was much slower. CPZ (in the absence of the MPO system) also stimulated the oxidation of HbO2 in the presence of 20 microM H2O2, but this reaction was considerably slower than when CPZ+ (generated by the MPO system) was allowed to react directly with HbO2. These results suggest that HbO2 was oxidized by CPZ+. To study the effect of CPZ intermediates, thyroglobulin (TG) was used as a model polypeptide. Chlorinated oxidants formed in the MPO system (in the absence of CPZ) induced TG peptide bond splitting. In contrast, CPZ metabolites generated by the MPO system (in the absence of Cl-) induced polymerization of TG, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Apparent hydroxyl radical generation without transition metal catalysis and tyrosine nitration during oxidation of the anti-tubercular drug, isonicotinic acid hydrazide

Aromatic hydroxylation and formation of thiobarbituric acid-reactive substances occurred in a mixture of isonicotinic acid hydrazide (isoniazid) and catalase. Since these reactions were stimulated by phytic acid (a potent metal chelator), rather than inhibited, transition metal-catalysed hydroxyl radical generation was not implicated. Hydroxylation also occurred with isoniazid and phytic acid in the absence of catalase, albeit to a lesser extent. The independent effects of catalase and phytic acid are related to their abilities to catalyse isoniazid oxidation. In the presence of tyrosine, both the isoniazid/phytic acid system and authentic peroxynitrite generated dityrosine. Authentic peroxynitrite, as well as a phytic acid-mediated isoniazid oxidation product, have absorbance maxima at 302 nm. The yield of this isoniazid-derived product increased with pH and in the presence of a superoxide-generating system. A good correlation existed between absorbance at 302 nm and aromatic hydroxylation. Acid-induced decomposition of the 302 nm absorbance in the presence of superoxide dismutase led to the formation of a product absorbing in the same region as peroxynitrite-modified superoxide dismutase (350 nm at acid pH). Catalase catalysed peroxynitrite-mediated, as well as isoniazid/phytic acid-mediated tyrosine nitration, which was accompanied by Compound II formation (ferryl-catalase) in both cases. We postulate that peroxynitrite or a similar species is formed during isoniazid oxidation.

Aromatic hydroxylation during the myeloperoxidase-oxidase oxidation of hydrazines

Benzoic acid was found to be hydroxylated by a mixture of myeloperoxidase (MPO) and the mycobactericidal drug, isoniazid. Aromatic hydroxylation and formation of compound III (oxyperoxidase) were coincident during the MPO-oxidase oxidation of isoniazid which proceeded without augmentation from the reagent hydrogen peroxide. An intermediate of isoniazid reduced ferric MPO to ferrous MPO which associated with dioxygen to form compound III. Aromatic hydroxylation also occurred in a mixture of isoniazid (or phenylhydrazine) and a ferric salt. Hydroxylations in both the enzymatic and nonenzymatic reaction systems were inhibited by the iron chelator, desferal, as well as by the specific hydroxyl radical scavenger, mannitol. To distinguish between the hydroxylating intermediates in the different reaction systems, the unique properties of the natural antioxidant, phytic acid, were exploited. Phytic acid inhibited aromatic hydroxylation in the Fe(3+)-INH system, which is in accordance with its known properties as a powerful inhibitor of iron-driven reactions (.OH formation). By contrast, phytic acid stimulated hydroxylation in the enzymatic system which was accompanied by a concomitant stimulation in the rate of compound III formation. These events were, however, not directly related to each other. Phytic acid had a direct effect on the redox transformation of isoniazid by stimulating superoxide generation during auto-oxidation of the drug. In addition, phytic acid also facilitated compound III decay in the absence of isoniazid, suggesting that it may also regulate the oxygen affinity of MPO, similar to its effect on the oxygenation of haemoglobin. The data on aromatic hydroxylation in the MPO-isoniazid system do not support a role for .OH in the reaction and may fit the model for the P450 mixed oxidase system.

Cytotoxicity of myeloperoxidase-activated catechols : oxidative injury to the red blood cell

The effects of two catechols (1,2-benzenediol and nordihydroguaiaretic acid) on the myeloperoxidase-Cl(-)-H2O2 antimicrobial/cytotoxic system of the human neutrophil were investigated. To determine the cytotoxicity of myeloperoxidase-generated oxygen metabolites (mainly chlorinated oxidants such as hypochlorite) and catechol oxidation products, the well characterized erythrocyte was used as a target. At relatively low concentrations (less than 10 microM), the catechols acted as redox catalysts by stimulating the generation of chlorinated oxidants. This is visualized as a promotion of haemolysis which reached a maximum and then decreased again with increasing concentrations of the catechol. In this respect, the dicatechol, nordihydroguaiaretic acid, was more potent. At higher concentrations, the catechols competed more effectively with Cl- as electron donors and the generation of chlorinated oxidants decreased with a consequent decrease in haemolysis. Above 200 microM nordihydroguaiaretic acid, complete haemolysis occurred which might be due to high membrane concentrations of the catechol due to its high lipid solubility. In contrast, high 1,2-benzenediol concentrations did not induce haemolysis. The catechols stimulated methaemoglobin formation in a concentration-dependent fashion with 1,2-benzenediol more potent than nordihydroguaiaretic acid. There was some correlation between membrane microviscosity and haemolysis which in turn did not correlate with haemoglobin oxidation. No direct correlation existed between intracellular methaemoglobin formation and the precipitation of haemoglobin oxidation products on the membrane. Disulphide crosslinks were not involved in the covalent polymerization of haemoglobin subunits.

Characterization of the major polypeptide chains of reduced bovine thyroglobulin

The finding that reduced 19S bovine thyroglobulin showed two major, closely migrating polypeptide bands during electrophoresis in SDS-polyacrylamide gels was presented as evidence that thyroglobulin consists of two nonidentical subunits, referred to as S and F, of approximately the same size (J. Biol. Chem. 253, 1853-1858 (1978)). It was, however, not clear whether the difference in migration rates of the two subunits was due to differences in amino acid or carbohydrate composition or a small difference in size. In this study, several physical and chemical properties of purified S and F polypeptides were investigated. Three methods revealed important differences between them. It was found by cyanogen bromide degradation that S contained one fragment (Mr approximately equal to 20 000) more than F. S eluted ahead of F by column chromatography in SDS. The sedimentation coefficient of S was found to be greater than that of F. However, the two subunits were shown to have similar amino acid and carbohydrate compositions and to be indistinguishable in their interaction with SDS. The different migration rates of S and F can therefore be explained by a small difference in size which agrees with the finding (Endocrinology 108, 1285-1292 (1981)) that a proteolytic enzyme present in commercial horseradish peroxidase can convert S into F.

Interaction of methyl-xanthines with myeloperoxidase. An anti-inflammatory mechanism

1. Inhibition of myeloperoxidase (MPO)-catalyzed reactions by methyl-substituted xanthines has been investigated. 2. Except for theobromine and caffeine, all xanthines tested were potent inhibitors of the MPO-H2O2-Cl- system. 3. In contrast to methyl substitution in the 1 or 8 position of xanthine, substitution in the 3 or 7 position had a marked effect on the inhibition of MPO catalysis. 4. Two different inhibitory mechanisms were induced; scavenging of hypochlorous acid (HOCl) generated by the MPO system and accumulation of Compound II (ferryl MPO) which is inactive as a catalyst of Cl- oxidation.