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BACKGROUND: Similar to hypochlorous acid (HClO), hypobromous acid (HBrO) is one of the most notable reactive oxygen species (ROS). Overexpression of HBrO is linked to various diseases causing organ and tissue loss. Due to HBrO's role in the oxidation of micropollutants, real-time monitoring of HBrO in water-based systems is essential. Tetraphenylethylene (TPE)-based organic aggregation-induced emission luminophores (AIEgens) are an emerging category of fluorescent probe materials that have attracted considerable attentions. However, AIE probes are rarely applied to detect HBrO. Developing faster, more precise, and more sensitive AIE probes is thus crucial for detecting biological and environmental HBrO. RESULTS: A small molecule fluorescent probe 4-(1,2,2-triphenylvinyl)benzamidoxime (SWJT-21) was synthesized for the sensitive and selective detection of hypobromous acid (HBrO) based on aggregation-induced emission (AIE). The amidoxime unit of SWJT-21 would undergo an oxidation reaction with HBrO, leading to a structure differentiation between the probe and the product, and therefore the turn-on fluorescence by the AIE effect. The probe could recognize hypobromous acid rapidly (less than 3 s) in high aqueous phase (99 % water) with a turn-on fluorescence response. It was determined that the limit of detection for HBrO was 5.47 nM. Moreover, SWJT-21 demonstrates potential as a test strip for the detection of HBrO. SWJT-21 was also successfully used for the monitoring of HBrO in water samples and for the detection of endogenous/exogenous HBrO in living cells and zebrafish. SIGNIFICANCE: A special AIE fluorescence turn-on probe SWJT-21 based on tetraphenylethylene was designed for detecting HBrO in the environmental and biological systems. This probe has an extremely low detection limit of 5.47 nM and is able to detect HBrO in 99 % aqueous phase in less than 3 s.

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Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

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Atherosclerosis is a lipoprotein-driven disease, and there is no effective therapy to reverse atherosclerosis or existing plaques. Therefore, it is urgently necessary to create a noninvasive and reliable approach for early atherosclerosis detection to prevent initial plaque formation. Atherosclerosis is intimately associated with inflammation, which is accompanied by an excess of reactive oxygen species (ROS), leading to cells requiring more glutathione (GSH) to resist severe oxidative stress. Therefore, the GSH-hydrolyzed protein γ-glutamyl transpeptidase (GGT) and the ROS-hypobromous acid (HBrO) are potential biomarkers for predicting atherogenesis. Hence, to avoid false-positive diagnoses caused by a single biomarker, we constructed an ingenious sequence-activated double-locked TP fluorescent probe, C-HBrO-GGT, in which two sequential triggers of GGT and HBrO are meticulously designed to ensure that the probe fluoresces in response to HBrO only after GGT hydrolyzes the probe. By utilization of C-HBrO-GGT, the voltage-gated chloride channel (CLC-1)-HBrO-catalase (CAT)-GGT signaling pathway was confirmed in cellular level. Notably, the forthcoming atherosclerotic plaques were successfully predicted before the plaques could be observed via the naked eye or classical immunofluorescent staining. Collectively, this research proposed a powerful tool to indicate the precise position of mature plaques and provide early warning of atherosclerotic plaques.

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Dissolved ozone concentration measurement is crucial for ozone treatment. In the most used conventional indigo method, the ozone concentration is measured by the decrease in absorbance due to the loss of the C-C double bond of indigotrisulfonic acid. However, measurement of ozone concentration is difficult when water contains substances that react with C-C double bonds other than ozone. To address this concern, we developed a novel breakthrough method to measure ozone concentration by measuring the p-formylbenzoic acid (p-FBA) produced after the reaction of p-vinylbenzoic acid and ozone. The formation of p-FBA was almost not caused by other substances (hypochlorous acid, hypobromous acid, permanganate ion and hydrogen peroxide), and its yield to ozone was maintained at 1 in river water, treated wastewater and seawater. In addition, the experimental error is smaller with the new method than with indigo. Furthermore, the new method does not require cumbersome calibration unlike indigo method because highly pure forms of p-FBA are commercially available. p-FBA can be separated by liquid chromatography and detected with highly sensitive ultraviolet and mass spectrometric detectors, and hence easily analyzed simultaneously with other substances. Our new method contributes to extensive ozone treatment and ozonation management.

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Human peroxidasin (PXDN) is a ubiquitous peroxidase enzyme expressed in most tissues in the body. PXDN represents an interesting therapeutic target for inhibition, as it plays a role in numerous pathologies, including cardiovascular disease, cancer and fibrosis. Like other peroxidases, PXDN generates hypohalous acids and free radical species, thereby facilitating oxidative modifications of numerous biomolecules. We have studied the inhibition of PXDN halogenation and peroxidase activity by phloroglucinol and 14 other peroxidase inhibitors. Although a number of compounds on their own potently inhibited PXDN halogenation activity, only five were effective in the presence of a peroxidase substrate with IC50 values in the low µM range. Using sequential stopped-flow spectrophotometry, we examined the mechanisms of inhibition for several compounds. Phloroglucinol was the most potent inhibitor with a nanomolar IC50 for purified PXDN and IC50 values of 0.95 µM and 1.6 µM for the inhibition of hypobromous acid (HOBr)-mediated collagen IV cross-linking in a decellularized extracellular matrix and a cell culture model. Other compounds were less effective in these models. Most interestingly, phloroglucinol was identified to irreversibly inhibit PXDN, either by mechanism-based inhibition or tight binding. Our work has highlighted phloroglucinol as a promising lead compound for the design of highly specific PXDN inhibitors and the assays used in this study provide a suitable approach for high-throughput screening of PXDN inhibitors.

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The development of ultrasensitive in situ detection techniques for monitoring hypobromous acid (HBrO) levels in the biological systems is of great significance to reveal its complex pathological and physiological effects. A simple mitochondria-targetable hydrazine-based near-infrared (NIR) fluorescent probe (Mito-NIR) for detecting HBrO in the mitochondria of live cells is presented in this paper. Probe Mito-NIR displays the ultrafast (< 5 s) response for HBrO. It can detect HBrO with high sensitivity. Additionally, it shows high selectivity towards HBrO over other biologically important substances. Finally, it can monitor the changes of endogenous/exogenous HBrO levels in the mitochondria of live cells. A simple mitochondria-targetable NIR fluorescent probe with picomolar sensitivity for HBrO was developed to specifically track mitochondrial HBrO.

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Peroxidasin (PXDN) is involved in the crosslinking of collagen IV, a major constituent of basement membranes. Disruption of basement membrane integrity as observed in genetic alterations of collagen IV or PXDN can result in developmental defects and diverse pathologies. Hence, the study of PXDN activity in (patho)physiological contexts is highly relevant. So far, measurements of PXDN activity have been reported from purified proteins, cell lysates and de-cellularized extracellular matrix. Here, for the first time we report the measurement of PXDN activity in live cells using the Amplex Red assay with a signal amplifying modification. We observe that bromide addition enhances the obtained signal, most likely due to formation of HOBr. Abrogation of signal amplification by the HOBr scavenger carnosine supports this hypothesis. Both, pharmacological inhibition as well as complementary genetic approaches confirm that the obtained signal is indeed related to PXDN activity. We validate the modified assay by investigating the effect of Brefeldin A, to inhibit the secretory pathway and thus the access of PXDN to the extracellular Amplex Red dye. Our method opens up new possibilities to investigate the activity of PXDN in (patho)physiological contexts.

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Mammalian heme peroxidases are fascinating due to their unique peculiarity of oxidizing (pseudo)halides under physiologically relevant conditions. These proteins are able either to incorporate oxidized halides into substrates adjacent to the active site or to generate different oxidized (pseudo)halogenated species, which can take part in multiple (pseudo)halogenation and oxidation reactions with cell and tissue constituents. The present article reviews basic biochemical and redox mechanisms of (pseudo)halogenation activity as well as the physiological role of heme peroxidases. Thyroid peroxidase and peroxidasin are key enzymes for thyroid hormone synthesis and the formation of functional cross-links in collagen IV during basement membrane formation. Special attention is directed to the properties, enzymatic mechanisms, and resulting (pseudo)halogenated products of the immunologically relevant proteins such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase. The potential role of the (pseudo)halogenated products (hypochlorous acid, hypobromous acid, hypothiocyanite, and cyanate) of these three heme peroxidases is further discussed.

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The generation of undesired biofouling in medical and engineering applications results in a reduction in function and durability. Copying functionalities of natural enzymes to combat biofouling by artificial nanomaterials is highly attractive but still challenged by the inferior catalytic activity and specificity principally because of low densities of active sites. Here, an innovate strategy is demonstrated to stabilize high-density ultrasmall ceria clusters on zirconia for biofouling prevention. Benefiting from the unique structure, CeO2 @ZrO2 nanozyme can significantly enhance the haloperoxidase-mimicking activity in catalyzing the oxidation of bromide with H2 O2 into biocidal hypobromous acid as a result of abundant defects and surface strong acidity sites, inducing impressive antibacterial and antibiofouling capacity compared with that of pristine CeO2 . This work is expected to open a new avenue for the rational design of cluster catalysts for various targeting catalytic applications.

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Artificial nanozymes exerting enzyme functionality are recognized as promising alternatives of natural enzymes in biomimetic chemistry. Natural haloperoxidases that utilize hydrogen peroxide (H2 O2 ) to catalytically convert halide into strong biocidal hypohalous acid hold great promise for thwarting biofouling, while their practical application remains highly questionable as instability of natural enzymes and inadequate H2 O2 . Herein a semiconducting nanozyme consisting of chromium single atoms coordinated on carbon nitride (Cr-SA-CN) that performs bifunctional roles of nonsacrificial H2 O2 photosynthesis and haloperoxidase-mimicking activity for antibiofouling is constructed. Such nanozyme is capable of generating H2 O2 from water and O2 upon visible-light illumination, and then sustainably self-supplying H2 O2 for haloperoxidase-mimicking reaction in a sequential manner. This dual-activity Cr-SA-CN overcomes H2 O2 dilemma and yields hypobromous acid continuously, inducing remarkable bactericidal capability. When used as an eco-friendly coating additive, it is successfully demonstrated that Cr-SA-CN enables an inert surface against marine biofouling. Thereby, this study not only illustrates an attractive strategy for antibiofouling but also opens an avenue to construct valuable nanoplatform with multifunctionality for future applications.

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Understanding the complex relationship between active small molecules is of great significance in various physiological processes. Herein, we present the design and synthesis of a sequential responsive Lysosome-Naphthalene imide-Azido (lyso-NP-N3) reporter for probing the H2S and HOBr within organelle (lysosome) in living cells. Probe lyso-NP-N3 exhibited high selectivity and sensitivity towards H2S (LOD = 23.5 nM) and HOBr (LOD = 254 nM). Additionally, lyso-NP-N3 possessed an excellent lysosome targeting ability and was utilized to visualize the exogenous/endogenous H2S and HOBr in RAW 264.7, Hela and HepG2 cells. Facilitated by this sequentially activated mechanism, the probe was successfully applied to confirm that the reported scavenger of HOBr, N-acetyl-L-cysteine (NAC) mainly relied on its metabolite H2S to eliminate excess HOBr, thereby playing the role of cell regulation and protection. These results establish the crosstalk between H2S and HOBr in lysosome and provide a promising tool to study metabolite interactions.

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Bromide as an omnipresent matrix component in wastewater can react with ozone to form hypobromous acid (HOBr). This secondary oxidant can subsequently react with micropollutants but also with formed intermediates. Therefore, bromide and especially HOBr can highly influence the formation of transformation products (TPs). This has already been reported for the ozonation of N,N-dimethylsulfamide leading to the formation of the cancerogenic N-nitrosodimethylamine only in bromide containing waters. In this study, the influence of different bromide and ozone concentrations on the formation of TPs during the ozonation of isoproturon (ISO), metoprolol (METO) and diclofenac (DCF) were investigated. Additionally, TPs were identified, which are formed in the direct reaction of the micropollutants with HOBr with and without subsequent ozonation. The results showed that even if the reactions of ozone with the substances should be favored bromide can highly influence the formation of TPs already at low concentrations. In summary, new TPs after the reaction with HOBr (and subsequent ozonation) could be postulated for ISO, METO and DCF. This underlines that the present water matrix can have a high influence on the formation of TPs and that these mechanisms need to be investigated further.

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Hypobromous acid (HOBr), a highly reactive active species, can be formed and impact reaction processes with organic pollutants in source water during chlorination disinfection of the water containing bromide (Br-). In this study, we investigated the transformation kinetics of 10 parent polycyclic aromatic hydrocarbons (PAHs) and formation mechanisms of transformation products in the presence of Br- during chlorination. The results indicated that HOBr can accelerate the processes of electrophilic substitution (ES) and single electron transfer (ET) reactions in PAHs, and the second-order rate constants of HOBr are 102-103 times higher than those of hypochlorous acid (HOCl) with PAHs. HOBr was more conductive to induce ES reactions than HOCl. In water containing Br-, HOBr and HOCl dominate the reaction processes with PAHs, although other active bromine species may still affect reaction processes. In terms of transformation products, higher reactivity of HOBr results from faster formation of oxygenated PAHs (OPAHs) and halogenated PAHs (HPAHs) than HOCl. As an example of 3 model PAHs, anthracene transforms faster to its oxygenated products at a higher concentration, while pyrene and fluorene transform faster to halogenated products. These fundamental results were essential to understanding the transformation kinetics of PAHs and the formation of toxic disinfection by-products in the presence of Br-.

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Pool water is continuously circulated and reused after an extensive treatment including disinfection by chlorination, ozonation or UV treatment. In Germany, these methods are regulated by DIN standard 19643. Recently, the DIN standard has been extended by a new disinfection method using hypobromous acid as disinfectant formed by introducing ozone into water with naturally or artificially high bromide content during water treatment. In this study, we tested the disinfection efficacy of the ozone-bromine treatment in comparison to hypochlorous acid in a flow-through test rig using the bacterial indicator strains Escherichia coli, Enterococcus faecium, Pseudomonas aeruginosa, and Staphylococcus aureus and the viral indicators phage MS2 and phage PRD1. Furthermore, the formation of disinfection by-products and their potential toxic effects were investigated in eight pool water samples using different disinfection methods including the ozone-bromine treatment. Our results show that the efficacy of hypobromous acid, depending on its concentration and the tested organism, is comparable to that of hypochlorous acid. Hypobromous acid was effective against five of six tested indicator organisms. However, using Pseudomonas aeruginosa and drinking water as test water, both tested disinfectants (0.6 mg L-1 as Cl2 hypobromous acid as well as 0.3 mg L-1 as Cl2 hypochlorous acid) did not achieve a reduction of four log10 levels within 30 s, as required by DIN 19643. The formation of brominated disinfection by-products depends primarily on the bromide concentration of the filling water, with the treatment method having a smaller effect. The eight pool water samples did not show critical values in vitro for acute cytotoxicity or genotoxicity in the applied assays. In real pool water samples, the acute toxicological potential was not higher than for conventional disinfection methods. However, for a final assessment of toxicity, all single substance toxicities of known DBPs present in pool water treated by the ozone-bromine treatment have to be analyzed additionally.

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Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

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Tetrabromobisphenol S (TBBPS) is an emerging brominated flame retardant (BFR) that can cause endocrinological abnormalities in aquatic species and is neurotoxic and cytotoxic to humans. Herein, we investigated the oxidation of TBBPS by unactivated peroxymonosulfate (PMS) in aqueous solution. Results show that PMS was capable of oxidizing TBBPS without activation, and the transformation of TBBPS was pH-dependent. Interestingly, the unactivated PMS oxidation of TBBPS exhibited an autocatalytic behavior. Radical quenching experiments and electron paramagnetic resonance (EPR) analyzes ruled out the involvement of hydroxyl radical (HO•) and sulfate radical (SO4•­) as reactive species. While the generation of singlet oxygen (1O2) was confirmed in PMS solution, it was also not responsible for TBBPS oxidation. The bromine substituents are believed to be responsible for the autocatalysis observed during PMS oxidation. We propose that the initial oxidation of TBBPS by PMS resulted in the release of bromide ions (Br-) via debromination, which could be rapidly oxidized to hypobromous acid (HOBr). 3,5-Dimethyl-1H-pyrazole (DMPZ) trapping coupled with liquid chromatography-mass spectrometry (LC-MS) analysis evidenced the formation of HOBr in PMS/TBBPS system. Therefore, the presence of Br-, albeit at trace level, could significantly accelerate the oxidation of TBBPS in PMS solution via HOBr formation. The intermediate products of TBBPS were identified by solid phase extraction (SPE) coupled with high resolution-mass spectrometry (HR-MS). The oxidation of TBBPS by unactivated PMS was likely initiated through a single electron transfer mechanism, and the transformation pathways included ß-scission, debromination, and cross-coupling reactions. Further oxidation and ring-opening of the intermediates yielded three brominated disinfection byproducts (Br-DBPs), including bromoform (CHBr3), mono-, and di-bromoacetic acids (MBAA and DBAA), as quantified by gas chromatography (GC). The presence of natural organic matter (NOM) inhibited the oxidation of TBBPS and reduced the yields of Br-DBPs. Our results indicate that unactivated PMS was efficient in the abatement of TBBPS in aqueous solution due to the accelerated oxidation by bromine catalysis; however, the formation of brominated intermediate products and Br-DBPs should be scrutinized due to their potential carcinogenicity and mutagenicity.

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Kynurenic acid, a tryptophan metabolite, acts as antagonist or agonist of several receptors. Hypobromous acid (HOBr) and hypochlorous acid (HOCl) are generated by eosinophils and neutrophils. At inflammation sites, kynurenic acid may encounter HOBr and HOCl to generate products. When kynurenic acid was incubated with HOBr under neutral conditions, kynurenic acid generated a single product almost exclusively. This was identified as 3-bromokynurenic acid. Kynurenic acid reacted with HOCl, generating two products. The major product was identified as 3-chlorokynurenic acid with its oxidative decarboxylation product, 3-chloro-4-hydroxy-2(1H)-quinolinone as a by-product. Free amino acids suppressed the reactions of kynurenic acid with HOBr and HOCl. Taurine suppressed the HOCl reaction but not the HOBr reaction. An eosinophil peroxidase system containing H2O2, NaCl, and NaBr reacted with kynurenic acid, generating 3-bromokynurenic acid under mildly acidic conditions. Although a myeloperoxidase system containing H2O2 and NaCl reacted with kynurenic acid to generate 3-chlorokynurenic acid under mildly acidic conditions, the product was altered to 3-bromokynurenic acid by addition of NaBr to the system. These results suggest that 3-bromokynurenic acid and 3-chlorokynurenic acid may be generated from kynurenic acid at inflammation sites in humans, although their formation will be suppressed by coexistent amino acids.

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Reactive oxygen species (ROS) have been implicated in numerous pathological processes and their homeostasis facilitates the dynamic balance of intracellular redox states. Among ROS, hypobromous acid (HOBr) has a high similarity to hypochlorous acid (HOCl) in both chemical and physical properties, whereas it has received relatively little attention. Meanwhile, selective recognition of endogenous HOBr suffers great challenges due to the fact that the concentration of this molecule is much lower than that of HOCl. Fluorescence-based detection systems have emerged as very important tools to monitor biomolecules in living cells and organisms owing to distinct advantages, particularly the temporal and spatial sampling for in vivo imaging applications. To date, the development of HOBr-specific fluorescent probes is still proceeding quite slowly, and the research related to this area has not been systematically summarized. In this review, we are the first to review the progress made so far in fluorescent probes for selective recognition and detection of HOBr. The molecular structures, sensing mechanisms, and their successful applications of these probes as bioimaging agents are discussed here in detail. Importantly, we hope this review will call for more attention to this rising field, and that this could stimulate new future achievements.

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Peroxidasin is a heme peroxidase that oxidizes bromide to hypobromous acid (HOBr), a powerful oxidant that promotes the formation of the sulfilimine crosslink in collagen IV in basement membranes. We investigated whether HOBr released by peroxidasin leads to other oxidative modifications of proteins, particularly bromination of tyrosine residues, in peroxidasin-expressing PFHR9 cells. Using stable isotope dilution LC-MS/MS, we detected the formation of 3-bromotyrosine, a specific biomarker of HOBr-mediated protein modification. The level of 3-bromotyrosine in extracellular matrix proteins from normally cultured cells was 1.1 mmol/mol tyrosine and decreased significantly in the presence of the peroxidasin inhibitor, phloroglucinol. A negligible amount of 3-bromotyrosine was detected in peroxidasin-knockout cells. 3-Bromotyrosine formed both during cell growth in culture and in the isolated decellularized extracellular matrix when embedded peroxidasin was supplied with hydrogen peroxide and bromide. The level of 3-bromotyrosine was significantly higher in extracellular matrix than intracellular proteins, although a low amount was detected intracellularly. 3-Bromotyrosine levels increased with higher bromide concentrations and decreased in the presence of physiological concentrations of thiocyanate and urate. However, these peroxidase substrates showed moderate to minimal inhibition of collagen IV crosslinking. Our findings provide evidence that peroxidasin promotes the formation of 3-bromotyrosine in proteins. They show that HOBr produced by peroxidasin is selective for, but not limited to, the crosslinking of collagen IV. Based on our findings, the use of 3-bromotyrosine as a specific biomarker of oxidative damage by HOBr warrants further investigation in clinical conditions linked to high peroxidasin expression.

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Methotrexate is a folate antagonist cytotoxic drug employed in the therapy of cancers and rheumatoid arthritis. Hypobromous acid (HOBr) and hypochlorous acid (HOCl) are generated by eosinophils and neutrophils at inflammation sites. The administered methotrexate may encounter HOBr and HOCl, and react with them to generate products. When methotrexate was incubated with HOBr or HOCl at pH 7.4 and 37°C for 30 min, a single product was generated almost exclusively in each case, identified as 3'-bromomethotrexate for HOBr and 3'-chloromethotrexate for HOCl. When methotrexate was incubated with HOCl in the presence of NaBr, the concentration of 3'-bromomethotrexate increased with decreasing concentration of 3'-chloromethotrexate in a dose-dependent manner with NaBr, probably due to the formation of HOBr. Free amino acids suppressed the reactions of methotrexate with HOBr and HOCl. Taurine suppressed the HOCl reaction but not the HOBr reaction. These results suggest that 3'-bromomethotrexate and 3'-chloromethotrexate may be generated from methotrexate at inflammation sites in humans, although their formation will be suppressed by coexistent amino acids.

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