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Several lines of evidence have pointed towards the potential therapeutic benefit of NSAIDs in cancer therapy. In this study, we have investigated the acute bio-activation of NSAIDs and their metabolites via myeloperoxidase (MPO), a highly-expressed peroxidase enzyme in acute myeloid leukemia. As bio-activation involves the formation of reactive metabolites, we hypothesized that NSAIDs which produced reactive metabolites would be correlated with leukemia cell toxicity. We tested the enzymatic peroxidation of three NSAIDs, namely diclofenac, indomethacin, and naproxen in comparison with their hepatic metabolites, 4′- hydroxydiclofenac (4′-OHD), 5-hydroxydiclofenac (5-OHD), O-desmethyl-N-deschlorobenzoylindomethacin (DMBI), O-desmethylindomethacin (DMI) and O-desmethylnaproxen (ODN). Firstly, we used purified peroxidases in kinetic UV–vis kinetic spectrophotometry, and electron paramagnetic resonance (EPR) experiments to determine oxidation of ascorbic acid and glutathione (GSH), respectively. We then used HL-60 cells, as a model of acute myelogenous leukemia to carry out trypan blue exclusion, cellular ATP analysis, mitochondrial membrane potential (MMP) and cytofluorometric GSH assays. Our results present evidence that diclofenac, 4′-OHD, 5-OHD, DMBI and DMI demonstrated significant cytotoxic effect in the leukemic cells through oxidation by intracellular MPO. In the same vein, only diclofenac and its two metabolites caused a significant drop in the MMP and cellular ATP level; however, the cell death induced by indomethacin metabolites reflected a subtle effect on MMP or GSH content. Interestingly, only diclofenac and 4′-OHD (and not 5- OHD) caused a significant drop in HL-60 cells’ GSH content. Among diclofenac compounds, only 4′-OHD also generated GS radical and caused a significant increase in ascorbate co-oxidation rate. Lastly, even though ODN also generated GS radical and potently cooxidized ascorbate, it showed no significant cytotoxicity. These results provide evidence of a correlation between acute cytotoxicity and MPO-bioactivated NSAIDs, though this was not correlated for all compounds ( e.g. , ODN). Further studies are required to determine both the MPO-dependent and MPO-independent mechanisms of cytotoxicity. [ABSTRACT FROM AUTHOR]

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD + ) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD + adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages’ cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV–Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD + . Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV–Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product ( λ max = 326 nm) in the presence of NAD + . This adduct was confirmed to be isonicotinyl-NAD + using LC–MS analysis where the intact adduct was detected ( m / z = 769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. [ABSTRACT FROM AUTHOR]

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 μM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV–visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV–vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols. • Phenol red is redox is halogenated by myeloperoxidase by Cl− and Br−. • Phenol red oxidation by myeloperoxidase oxidizes glutathione. • Phenol red can enhance myeloperoxidase halogenation activity. • Phenol red does not appear to cause overt cytotoxicity. • Phenol red is redox active and may interfere in biological assays. [ABSTRACT FROM AUTHOR]

Fenamic acids form the substructure of many non-steroidal anti-inflammatory drugs (NSAIDs); they are amongst the most commonly prescribed drugs globally. Interactions between fenamic acid NSAIDs and peroxidases have shown that reactive metabolites can be produced, although detailed studies of these metabolites or their relative reactivity have not been studied. Adverse drug reactions may be catalyzed by peroxidase enzymes in off-target tissues. Myeloperoxidase (MPO) is a peroxidase enzyme found abundantly in neutrophils, which are recruited during inflammation and it is well known that MPO can catalyze drug oxidation. Moreover, it has been demonstrated that MPO binds to fibronectin (FN); the latter is an important multi-functional protein. The objective of this study was to examine the differential reactivity of fenamic acid NSAIDs when oxidized by MPO and the consequent effects on FN. Studies using ESR spin trapping with DMPO revealed that N-phenanthranilic acid (N-PA) produced a stable radical when oxidized by MPO/H 2 O 2 ; diphenylamine (DPA) and 3-methyldiphenylamine (3-MDA) produced ● OH (DMPO/ ● OH). In addition, the analogs showed the formation of GS ● after the addition of GSH to the MPO/H 2 O 2 system, with 3-MDA producing the most intense DMPO/GS ● spectrum. To determine the cytotoxic potential of these radical metabolites, we used HL-60 promyelocytic leukemia cells which contain MPO. 3-MDA, DPA and N-PA demonstrated MPO-catalyzed cytotoxicity in these cells, but not mefenamic acid (MFA). LC/MS analysis of the DPA product formation showed the formation of a hydroxydiphenylamine and a quinoneimine product, indicating MPO oxidation resulted in a hydroxylation reaction. The quinoneimine products appeared to react with GSH, forming a GSH conjugate. Immuno-spin trapping analysis demonstrated that MFA was the most potent in producing FN-protein free radical formation in a MPO-system. However, FN aggregation analysis by silver staining was most prominent with DPA. MFA and its analogues showed the formation of free radicals in a peroxidase-system which interferes with normal cell functioning. Future studies are needed to determine the functional impact of fenamic acid NSAID mediated damage to FN. [ABSTRACT FROM AUTHOR]

To combat tuberculosis (TB), host phagocytic cells need to survive against self-generating oxidative stress-induced necrosis. However, the effect of isoniazid (INH) in protecting cells from oxidative stress-induced necrosis has not been previously investigated. In this in vitro study, the cytotoxic effect of H 2 O 2 generation using glucose oxidase (a model of oxidative stress) was found to be abrogated by INH in a concentration-dependent manner in HL-60 cells (a human promyelocytic leukemia cell). In cells treated with glucose oxidase, both ATP and mitochondrial membrane potential were found to be decreased. However, treatment with INH demonstrated small but significant attenuation in decreasing ATP levels, and complete reversal for the decrease in mitochondrial membrane potential. Quantitative proteomics analysis identified up-regulation of 15 proteins and down-regulation of 14 proteins which all together suggest that these proteomic changes signal for increasing cellular replication, structural integrity, ATP synthesis, and inhibiting cell death. In addition, studies demonstrated that myeloperoxidase (MPO) was involved in catalyzing INH-protein adduct formation. Unexpectedly, these covalent protein adducts were correlated with INH-induced cytoprotection in HL-60 cells. Further studies are needed to determine whether the INH-protein adducts were causative in the mechanism of cytoprotection. [ABSTRACT FROM AUTHOR]

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H 2 O 2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 μM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV–Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 μM H 2 O 2 or a flux of H 2 O 2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H 2 O 2 -induced toxicity was ameliorated at high edaravone concentrations (100–200 μM). In contrast, low concentrations of edaravone (1–10 μM) exacerbated the H 2 O 2 -induced toxicity. However, the effect of edaravone at low concentration (0–10 μM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug. Image 1 • Edaravone is a substrate for myeloperoxidase. • Edaravone (≤10 μM) induced HOCl formation but attenuated at ≥ 30 μM. • The edaravone free radical metabolite was characterized, and polymers were observed. • Edaravone increased or decreased H 2 O 2 cytotoxicity depending on its concentration. [ABSTRACT FROM AUTHOR]