Comparison of Fecal Calprotectin and Myeloperoxidase in Predicting Outcomes in Inflammatory Bowel Disease.

BACKGROUND: Biomarkers have been proposed as surrogate treatment targets for the management of inflammatory bowel disease (IBD); however, their relationship with IBD-related complications remains unclear. This study investigated the utility of neutrophil biomarkers fecal calprotectin (fCal) and fecal myeloperoxidase (fMPO) in predicting a complicated IBD course. METHODS: Participants with IBD were followed for 24 months to assess for a complicated IBD course (incident corticosteroid use, medication escalation for clinical disease relapse, IBD-related hospitalizations/surgeries). Clinically active IBD was defined as Harvey-Bradshaw index >4 for Crohn's disease (CD) and simple clinical colitis activity index >5 for ulcerative colitis (UC). Area under the receiver-operating-characteristics curves (AUROC) and multivariable logistic regression assessed the performance of baseline symptom indices, fCal, and fMPO in predicting a complicated disease IBD course at 24 months. RESULTS: One hundred and seventy-one participants were included (CD, n = 99; female, n = 90; median disease duration 13 years [interquartile range, 5-22]). Baseline fCal (250 µg/g; AUROC = 0.77; 95% confidence interval [CI], 0.69-0.84) and fMPO (12 µg/g; AUROC = 0.77; 95% CI, 0.70-0.84) predicted a complicated IBD course. Fecal calprotectin (adjusted OR = 7.85; 95% CI, 3.38-18.26) and fMPO (adjusted OR = 4.43; 95% CI, 2.03-9.64) were associated with this end point after adjustment for other baseline variables including clinical disease activity. C-reactive protein (CRP) was inferior to fecal biomarkers and clinical symptoms (pdifference < .05) at predicting a complicated IBD course. A combination of baseline CRP, fCal/fMPO, and clinical symptoms provided the greatest precision at identifying a complicated IBD course. CONCLUSIONS: Fecal biomarkers are independent predictors of IBD-related outcomes and are useful adjuncts to routine clinical care.

Elevated seminal plasma myeloperoxidase is associated with a decreased sperm concentration in young men.

Myeloperoxidase is a major neutrophil protein which generates oxidants that are highly reactive, and if present in seminal fluid, could be potentially damaging to spermatozoa. We recruited young males aged 18-35 years, unscreened for fertility status, for a pilot study measuring seminal plasma myeloperoxidase. On three occasions, over a 3-month period, we measured parameters of semen quality and correlated these with seminal myeloperoxidase protein and activity. After baseline measurement, participants were supplemented daily with 250 mg of vitamin C, a potent scavenger of reactive oxygen species with antiinflammatory activities. Seminal plasma from eight of the 12 participants had measurable concentrations of myeloperoxidase protein, across a broad range (15-250 ng/mL). Median myeloperoxidase protein concentrations were ~45-fold higher in semen samples with low vs. high sperm concentrations. Seminal plasma myeloperoxidase protein concentration was inversely correlated with the percentage of rapidly motile spermatozoa assessed by computer-assisted sperm analysis, and the total number of spermatozoa per ejaculate, but positively correlated with sperm maturity, measured by DNA staining ability. We measured an inverse correlation between semen vitamin C concentration and seminal plasma myeloperoxidase protein concentration, although vitamin C supplementation had no effect on semen quality. Our pilot data suggest that high concentrations of myeloperoxidase were present in the seminal plasma of many of our young participants, and that this may be associated with decreases in semen quality. A larger study is required to confirm these findings.

Substrates and products of eosinophil peroxidase.

Eosinophil peroxidase has been implicated in promoting oxidative tissue damage in a variety of inflammatory conditions, including asthma. It uses H(2)O(2) to oxidize chloride, bromide and thiocyanate to their respective hypohalous acids. The aim of this study was to establish which oxidants eosinophil peroxidase produces under physiological conditions. By measuring rates of H(2)O(2) utilization by the enzyme at neutral pH, we determined the catalytic rate constants for bromide and thiocyanate as 248 and 223 s(-1) and the Michaelis constants as 0.5 and 0.15 mM respectively. On the basis of these values thiocyanate is preferred 2.8-fold over bromide as a substrate for eosinophil peroxidase. Eosinophil peroxidase catalysed substantive oxidation of chloride only below pH 6.5. We found that when eosinophil peroxidase or myeloperoxidase oxidized thiocyanate, another product besides hypothiocyanite was formed; it also converted methionine into methionine sulphoxide. During the oxidation of thiocyanate, the peroxidases were present as their compound II forms. Compound II did not form when GSH was included to scavenge hypothiocyanite. We propose that the unidentified oxidant was derived from a radical species produced by the one-electron oxidation of hypothiocyanite. We conclude that at plasma concentrations of bromide (20-120 microM) and thiocyanate (20-100 microM), hypobromous acid and oxidation products of thiocyanate are produced by eosinophil peroxidase. Hypochlorous acid is likely to be produced only when substrates preferred over chloride are depleted. Thiocyanate should be considered to augment peroxidase-mediated toxicity because these enzymes can convert relatively benign hypothiocyanite into a stronger oxidant.

A kinetic analysis of the catalase activity of myeloperoxidase.

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.

Biomarkers of myeloperoxidase-derived hypochlorous acid.

Hypochlorous acid is the major strong oxidant generated by neutrophils. The heme enzyme myeloperoxidase catalyzes the production of hypochlorous acid from hydrogen peroxide and chloride. Although myeloperoxidase has been implicated in the tissue damage that occurs in numerous diseases that involve inflammatory cells, it has proven difficult to categorically demonstrate that it plays a crucial role in any pathology. This situation should soon be rectified with the advent of sensitive biomarkers for hypochlorous acid. In this review, we outline the advantages and limitations of chlorinated tyrosines, chlorohydrins, 5-chlorocytosine, protein carbonyls, antibodies that recognize HOCl-treated proteins, and glutathione sulfonamide as potential biomarkers of hypochlorous acid. Levels of 3-chlorotyrosine and 3,5-dichlorotyrosine are increased in proteins after exposure to low concentrations of hypochlorous acid and we conclude that their analysis by gas chromatography and mass spectrometry is currently the best method available for probing the involvement of oxidation by myeloperoxidase in the pathology of particular diseases. The appropriate use of other biomarkers should provide complementary information.Keywords-Free radicals, Myeloperoxidase, Neutrophil oxidant, Hypochlorous acid, Chlorotyrosine, Chlorohydrin, Oxidant biomarker

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production.

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1 x 10(6) M(-1)s(-1)), it reacts sluggishly with compound II (kII 7 M(-1)s(-1)). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 microM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 microM) unless superoxide dismutase was present (I50 5 microM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.

Comparison of mono- and dichlorinated tyrosines with carbonyls for detection of hypochlorous acid modified proteins.

Hypochlorous acid is a potent oxidant capable of oxidizing and chlorinating proteins. Based on its indiscriminant reactivity, it is proposed to play a major role in tissue damage associated with a range of inflammatory diseases. We have determined the relative tendencies for formation of protein carbonyls, chlorinated tyrosine residues, and epitopes recognized by an antibody raised against hypochlorous acid oxidized protein (HOP-1) when albumin is treated with hypochlorous acid. We have also tested the specificity of the HOP-1 antibody by measuring how effectively it recognizes proteins oxidized by hypobromous acid. 3-Chlorotyrosine, along with a new marker of hypochlorous acid dependent protein modification, 3, 5-dichlorotyrosine, was formed at the lowest doses of hypochlorous acid that were capable of generating protein carbonyls. Comparatively high doses of hypochlorous acid were needed to generate epitopes recognized by HOP-1, which were also produced by hypobromous acid. Our study demonstrates that it is advantageous to measure protein carbonyls and HOP-1 epitopes in conjunction with chlorinated tyrosines when attempting to identify the oxidants responsible for inflammatory tissue damage.

Initiation of rapid, P53-dependent growth arrest in cultured human skin fibroblasts by reactive chlorine species.

Oxidants produced by neutrophils have been implicated in causing cancers associated with chronic inflammation. Hypochlorous acid is the most potent oxidant produced by these cells in appreciable amounts. It reacts with amines to form chloramines, which are weaker oxidants but are mutagenic. Recently, we showed that sublethal doses of hypochlorous acid increased levels of the transcription factor protein 53 (p53) and the wild-type activating fragment-1/cyclin-dependent kinase inhibitory protein-1 (WAF1/CIP1) in cultured human skin fibroblasts. WAF1/CIP1 is an important intermediate in the pathway leading to growth arrest. We now show that low doses of hypochlorous acid and physiological chloramines lead to an inhibition of both DNA synthesis and division of cultured human skin fibroblasts. Inhibition of DNA synthesis occurred within 1 h of hypochlorous acid treatment, was maintained for 24 h, and returned to a normal rate after 48 h. Cell division was inhibited by hypochlorous acid and chloramines for 48 h and returned to normal 72 h after treatment. Growth arrest was dependent on p53 because it was blocked when cells were transfected with a p53-binding oligonucleotide. We propose that reactive chlorine species will initiate WAF1/CIP1-dependent growth arrest that will counteract their mutagenic effects and minimize the possibility of the malignant transformation of cells surrounding sites of inflammation.

Mechanism of reaction of myeloperoxidase with nitrite.

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.

Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation.

Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mM), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low.

Myeloperoxidase.

This review covers recent advances in the biology of myeloperoxidase. Mechanisms of posttranslational processing and how these fail in some of the common deficiency mutants are discussed. We also review the enzymology that points to myeloperoxidase having a number of physiologic substrates in addition to chloride and the evidence that it produces hypochlorous acid in the neutrophil phagosome in sufficient quantities to be bactericidal. Evidence is accumulating that myeloperoxidase-derived oxidants modify biologic macromolecules and cell-regulatory pathways and that they play a role in atherosclerosis. Investigation of disease incidence in relation to a polymorphism in the promoter region of the gene has produced interesting associations. These links with inflammatory diseases can now be pursued further using specific biomarkers of myeloperoxidase activity.

Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase.

Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.

Current concepts of the actions of paracetamol (acetaminophen) and NSAIDs.

There is much uncertainty about the mechanism of action of paracetamol (acetaminophen). It is commonly stated that, unlike the non-steroidal anti-inflammatory drugs (NSAIDs), it is a weak inhibitor of the synthesis of prostaglandins. This conclusion is made largely from studies in which the synthesis of prostaglandins was measured in homogenized tissues. However, in several cellular systems, paracetamol is an inhibitor of the synthesis of prostaglandins with IC(50) values ranging from approximately 4 microM to 200 microM. Paracetamol is not bound significantly to plasma proteins and therefore the concentrations in plasma can be equated directly with those used in in vitro experiments. After oral doses of 1 g, the peak plasma concentrations of paracetamol are approximately 100 microM and the plasma concentrations are therefore in the range where marked inhibition of the synthesis of prostaglandins should occur in some cells. Paracetamol is metabolized by the peroxidase component of prostaglandin H synthase but the relationship of this to inhibition of the cyclooxygenase or peroxidase activities of the enzyme is unclear. Paracetamol is also metabolized by several other peroxidases, including myeloperoxidase, the enzyme in neutrophils which is responsible for the production of hypochlorous acid (HOCl). The metabolism of paracetamol by myeloperoxidase leads to the decreased total production of HOC1 by both intact neutrophils and isolated myeloperoxidase, even though the initial rate of production of HOC1 is increased. The IC(50) value, derived from inhibition of the total production of HOC1 by isolated myeloperoxidase, is 81 microM. Several NSAIDs inhibit functions of neutrophils in media containing low concentrations of protein but their effects, in contrast to that of paracetamol, are generally produced only at concentrations greater than those of the unbound drug in plasma during treatment with the NSAIDs. However, neutrophils isolated during treatment with NSAIDs, such as piroxicam, ibuprofen and indomethacin show decreased function. Paracetamol has little or no anti-inflammatory activity by itself but may potentiate the clinical activity of NSAIDs in the treatment of rheumatoid arthritis.

Hypochlorous acid activates the tumor suppressor protein p53 in cultured human skin fibroblasts.

The carcinogenicity associated with chronic inflammation has been attributed to neutrophils and the oxidants they produce. Neutrophils accumulate at sites of chronic inflammation, where they are stimulated to produce hydrogen peroxide which is converted to hypochlorous acid by coreleased myeloperoxidase. We report here that levels of the tumor suppressor protein p53 were increased in cultured human skin fibroblasts that had been incubated with stimulated neutrophils. The increase in p53 required the myeloperoxidase-dependent generation of hypochlorous acid and could be mimicked by exposing cells to a flux of hypochlorous acid produced by purified myeloperoxidase and a hydrogen peroxide-generating system. Levels of p53 were very sensitive to hypochlorous acid, with fluxes as low as 0.2 microM per min being effective. Levels of the p53-dependent protein WAF1/CIP1 were also elevated when fibroblasts were treated with hypochlorous acid. This result indicates that the p53 in the cells treated with hypochlorous acid was transcriptionally active. Hydrogen peroxide alone also elevated p53 and WAF1/CIP1, but the fluxes required were nearly 10-fold higher than those that were effective for hypochlorous acid. Our results implicate hypochlorous acid in the neutrophil-dependent initiation of a signal transduction pathway which could minimize the carcinogenicity of chronic inflammation.

The activation of gold complexes by cyanide produced by polymorphonuclear leukocytes. III. The formation of aurocyanide by myeloperoxidase.

There is considerable evidence that the anti-rheumatic gold complexes are activated by their conversion to aurocyanide. In order to understand the mechanism of production of aurocyanide, we investigated the involvement of myeloperoxidase in the reaction. This haem enzyme of neutrophils and monocytes uses hydrogen peroxide to oxidise chloride and thiocyanate to hypochlorous acid and hypothiocyanite, respectively. When aurothiomalate (10 microM) was incubated with thiocyanate (200 microM), hydrogen peroxide (100 microM) and myeloperoxidase (20 nM), it was transformed to a product that was spectrally identical to authentic aurocyanide. Aurothiomalate was quantitatively converted to aurocyanide in about 10 min at pH 6.0 and in 40 min at pH 7.4. Aurocyanide formation occurred after myeloperoxidase had used all the hydrogen peroxide available to produce hypothiocyanite. Thus, the cyanide must have formed from the slow decomposition of hypothiocyanite. The rate of aurocyanide production was increased in the presence of 100 mM chloride, which indicates that hypochlorous acid accelerates the formation of cyanide. Hypochlorous acid (100 to 400 microM) reacted non-enzymatically with thiocyanate (200 microM) and aurothiomalate (10 microM) to produce aurocyanide. Thus, aurocyanide is produced by two processes, involving both the formation of hypothiocyanite and hypochlorous acid. Aurocyanide is an effective inhibitor of the respiratory burst of neutrophils and monocytes and the proliferation of lymphocytes. Therefore, aurothiomalate may attenuate inflammation by acting as a pro-drug which is reliant on neutrophils and monocytes to produce hypothiocyanite. When the hypothiocyanite decays to hydrogen cyanide, the pro-drug is converted to aurocyanide which then suppresses further oxidant production by these inflammatory cells.

Thiocyanate and chloride as competing substrates for myeloperoxidase.

The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 microM. With 100 microM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 microM thiocyanate was added, and was maximal with 1mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.

Neutrophil-mediated activation of mitoxantrone to metabolites which form adducts with DNA.

In this study we have shown that phorbol ester-stimulated human neutrophils are able to oxidatively activate mitoxantrone and result in covalent incorporation of the drug into cellular DNA. The use of the myeloperoxidase inhibitor sodium azide confirmed that the activation and covalent binding of mitoxantrone to cellular DNA was due to its metabolism by the haem enzyme myeloperoxidase. Phorbol ester-stimulated neutrophils were also able to oxidatively metabolise mitoxantrone and facilitate extracellular covalent binding of the drug to calf thymus DNA. These results suggest that myeloperoxidase may contribute to the mode of action of mitoxantrone.