The inhibition of lactoperoxidase catalytic activity through mesna (2-mercaptoethane sodium sulfonate).

Here, we show that mesna (sodium-2-mercaptoethane sulfonate), primarily used to prevent nephrotoxicity and urinary tract toxicity caused by chemotherapeutic agents such as cyclophosphamide and ifosfamide, modulates the catalytic activity of lactoperoxidase (LPO) by binding tightly to the enzyme, functioning either as a one electron substrate for LPO Compounds I and II, destabilizing Compound III. Lactoperoxidase is a hemoprotein that utilizes hydrogen peroxide (H2O2) and thiocyanate (SCN-) to produce hypothiocyanous acid (HOSCN), an antimicrobial agent also thought to be associated with carcinogenesis. Our results revealed that mesna binds stably to LPO within the SCN- binding site, dependent of the heme iron moiety, and its combination with LPO-Fe(III) is associated with a disturbance in the water molecule network in the heme cavity. At low concentrations, mesna accelerated the formation and decay of LPO compound II via its ability to serve as a one electron substrate for LPO compounds I and II. At higher concentrations, mesna also accelerated the formation of Compound II but it decays to LPO-Fe(III) directly or through the formation of an intermediate, Compound I*, that displays characteristic spectrum similar to that of LPO Compound I. Mesna inhibits LPO's halogenation activity (IC50 value of 9.08 µM) by switching the reaction from a 2e- to a 1e- pathway, allowing the enzyme to function with significant peroxidase activity (conversion of H2O2 to H2O without generation of HOSCN). Collectively, mesna interaction with LPO may serve as a potential mechanism for modulating its steady-state catalysis, impacting the regulation of local inflammatory and infectious events.

The inhibition effects of some natural products on lactoperoxidase purified from bovine milk.

In this study, inhibition profiles of some natural products, which are digoxin, L-Dopa, dopamine, isoliquiritigenin, and 1,1,2,2-tetrakis(p-hydroxyphenyl)ethane (Tetrakis), were investigated against bovine lactoperoxidase (LPO) enzyme. Digoxin, L-Dopa, and dopamine are active ingredients of some drugs, which have important functions in our body, especially in cases of heart failure. Isoliquiritigenin and tetrakis are types of natural phenolic compounds, which play an important role in cancer prevention and treatment. LPO enzyme was purified from bovine milk using sepharose-4B-l-tyrosine sulfonamide affinity column chromatography. LPO is responsible for the nonimmune biological defense system and has antibacterial activity so selection of these active substances is important. The inhibition studies are performed with the ABTS substrate. Bovine LPO enzyme was effectively inhibited by phenolic molecules. Ki values of these natural products were found as 0.20 ± 0.09, 0.22 ± 0.17, 0.49 ± 0.11, 0.49 ± 0.27, and 1.20 ± 0.25 µM, respectively. Tetrakis and digoxin exhibited noncompetitive inhibition, and other molecules showed competitive inhibition.

Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases.

Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in acquiring limiting nutrient iron and thrive in the gut.

Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase, lactoperoxidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase isoenzymes.

Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates. Acetylcholinesterase (AChE, E.C. 3.1.1.7) is intimately associated with the normal neurotransmission by catalysing the hydrolysis of acetylcholine to acetate and choline and acts in combination with butyrylcholinesterase (BChE) to remove acetylcholine from the synaptic cleft. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms, whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and in eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effects of rosmarinic acid on tumour-associated carbonic anhydrase IX and XII isoenzymes, AChE, BChE, LPO and GST enzymes were evaluated. Rosmarinic acid inhibited these enzymes with Kis in the range between micromolar to picomolar. The best inhibitory effect of rosmarinic acid was observed against both AChE and BChE.

(99m)Tc-amitrole as a novel selective imaging probe for solid tumor: In silico and preclinical pharmacological study.

Lactoperoxidase (LPO) inhibitors are very selective for solid tumor due to their high binding affinity to the LPO enzyme. A computational study was used to select top-ranked LPO inhibitor (alone and in complex with (99m)Tc) with high in silico affinity. The novel prepared (99m)Tc-amitrole complex demonstrated both in silico and in vivo high affinity toward solid tumors.(99m)Tc-amitrole was radio-synthesized with a high radiochemical yield (89.7±3.25). It showed in vitro stability for up to 6h. Its preclinical evaluation in solid tumor-bearing mice showed high retention and biological accumulation in solid tumor cells with a high Target/Non-Target (T/NT) ratio equal to 4.9 at 60min post-injection. The data described previously could recommend (99m)Tc-amitrole as potential targeting scintigraphic probe for solid tumor imaging.

Lactoperoxidase catalyzes in vitro activation of acrylonitrile to cyanide.

Acrylonitrile (ACN) is a widely used industrial chemical. Although it is a well reported animal carcinogen, its current designation to humans is "possibly carcinogenic". The present study aimed at investigating the ability of LPO enzyme system to oxidize ACN to cyanide (CN(-)) in vitro. Detection of CN(-) served as a marker for the possible generation of free radical intermediates implicated in ACN induced toxicity in the activation process. Optimum conditions for the oxidation of ACN to CN(-) were characterized with respect to pH, temperature and time of incubation as well as ACN, LPO and H(2)O(2) concentrations in incubation mixtures. Maximum reaction velocity (V(max)) and Michaelis-Menten constant (K(m)) were assessed. Addition of nitrite (NO(2)(-)) salts to the reaction mixtures significantly enhanced the rate of the reaction. Free radical scavengers (quercetin and trolox C), LPO enzyme inhibitor (resorcinol) and competitors for LPO binding (sodium azide and indomethacin) were found to reduce the rate of CN(-) production. Inclusion of the sulfhydryl compounds glutathione (GSH), NAC (N-acetylcysteine), D-penicillamine or L-cysteine enhanced the rate of ACN oxidation. The present results demonstrate the ability of LPO enzyme system to oxidize ACN to CN(-) and provide insight for the elucidation of ACN chronic toxicity.

Inhibition of lactoperoxidase-catalyzed 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and tyrosine oxidation by tyrosine-containing random amino acid copolymers.

Oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by lactoperoxidase was found to be inhibited by tyrosine-containing random amino acid copolymers but not by tyrosine. Both electrostatic effects and polymer size were found to be important by comparison of negatively and positively charged copolymers of varying lengths, with poly(Glu, Tyr)4:1 ([E 4Y 1] approximately 40) as the strongest competitive inhibitor (EC 50 approximately 20 nM). This polymer did not form dityrosine in the presence of lactoperoxidase (LPO) and peroxide. Furthermore, incubation with tert-butyl hydroperoxide, as opposed to hydrogen peroxide, resulted in a peculiar long lag phase of the reaction between the redox intermediate compound II and [E 4Y 1] approximately 40, indicating a very tight association between enzyme and inhibitor. We propose that interactions between multiple positively charged areas on the surface of LPO and the polymer are required for optimal inhibition.

Anti-thyroid drugs and thyroid hormone synthesis: effect of methimazole derivatives on peroxidase-catalyzed reactions.

Syntheses and characterization of the selenium analogue (MSeI) of anti-thyroid drug methimazole and a series of organoselenium compounds bearing N-methylimidazole pharmacophore are described. In contrast to the sulfur compound that exists predominantly in its thione form, the selenium analogue exists in a selenol form, which spontaneously oxidizes in air to produce the corresponding diselenide. The reduction of the diselenide by GSH or NaBH(4) affords the biologically active selenol, which effectively inhibits the lactoperoxidase (LPO) activity in vitro. The monoselenides having N-methylimidazole moiety are found to be much less active than the selenol, suggesting that the presence of a selenol moiety is important for the LPO inhibition. The kinetic and mechanistic studies reveal that MSeI inhibits the LPO activity by reducing the H(2)O(2), providing a novel method to reversibly inhibit the enzyme. Although MSeI strongly inhibits LPO, the enzyme's activity can be completely recovered by increasing the H(2)O(2) concentration. On the other hand, the inhibition by methimazole (MMI), the sulfur analogue, cannot be reversed by increasing the H(2)O(2) concentration, leading to a complete inactivation of the enzyme. The reversible inhibition of LPO by some of the selenium derivatives is correlated with their glutathione peroxidase (GPx) activity, and the high GPx activity of the selenium compounds as compared with their sulfur analogues suggests that the selenium derivatives may protect the thyroid gland from oxidative damage.

Inactivation of Coprinus cinereus peroxidase by 4-chloroaniline during turnover: comparison with horseradish peroxidase and bovine lactoperoxidase.

The peroxidase from Coprinus cinereus (CPX) catalyzed oxidative oligomerization of 4-chloroaniline (4-CA) forming several products: N-(4-chlorophenyl)-benzoquinone monoamine (dimer D), 4,4'-dichloroazobenzene (dimer E); 2-(4-chloroanilino)-N-(4-chlorophenyl)-benzoquinone (trimer F); 2-amino-5-chlorobenzoquinone-di-4-chloroanil (trimer G); 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil (tetramer H) and 2-amino-5-(-4-chlroanilino)-benzoquinone-di-4-chloroanil (tetramer 1). In the presence of 4-CA and H2O2, CPX was irreversibly inactivated within 10 min. Inactivation of CPX in the presence of H2O2 was a time-dependent, first-order process when the concentration of 4-CA was varied between 0 and 2.5 mM. The apparent dissociation constant (Ki) for CPX and 4-CA was 0.71 mM. The pseudo-first order rate constant for inactivation (k(inact)), was 1.15 x 10(-2) s(-1). Covalent incorporation of 20 mole 14C-4-CA per mole of inactivated CPX was observed. The partition ratio was about 2200 when either 4-CA or H2O2 was used as the limiting substrate. These results show that 4-CA is a metabolically activated inactivator (i.e. a suicide substrate). Unmodified heme and hydroxymethyl heme were isolated from native, 4-CA-inactivated and H2O2-incubated CPX. Inactivation resulted in significant losses in both heme contents. Analysis of tryptic peptides from 4-CA-inactivated CPX by MALDI-TOF/ MS and UV-VIS spectrophotometry suggested that trimer G and tetramer H were the major 4-CA derivatives that were covalently bound, including to a peptide (MGDAGF-SPDEVVDLLAAHSLASQEGLNSAIFR) containing the heme binding site. These studies show that heme destruction and covalent modification of the polypeptide chain are both important for the inactivation of CPX. These results were compared with similar studies on 4-CA-inactivated horseradish peroxidase (HRP) and bovine lactoperoxidase (LPO) during the oxidation of 4-CA.

Inhibition of peroxidase-catalyzed reactions by arylamines: mechanism for the anti-thyroid action of sulfamethazine.

Sulfonamide antibiotics, typified by sulfamethazine (SMZ), are widely used in veterinary practice. Sulfonamide residues in milk and meat products are of regulatory concern since SMZ is a thyroid carcinogen in rodents and sulfonamide-induced hypersensitivity reactions, including hypothyroidism, have been reported in humans. SMZ and other primary arylamines inhibited iodination reactions catalyzed by thyroid peroxidase (TPO) and the closely related lactoperoxidase (LPO). Inhibition of LPO-catalyzed triiodide ion formation by SMZ and other primary arylamines was complex as both apparent Km and Vmax values were affected, but consistent with a rapid equilibrium binding mechanism. The apparent Ki for SMZ inhibition of TPO- and LPO-catalyzed iodide ion oxidation was approximately 0.42 and 0.11 mM, respectively. The corresponding Ki values for a series of para-substituted anilines correlated with the ease of one-electron N-oxidation as measured by ionization potentials determined from semiempirical molecular orbital calculations. The aniline derivatives containing electron-donating substituents (e.g., p-CH3, p-OEt, p-Cl) were converted by LPO to colored products characteristic of one-electron oxidation. However, sulfonamides were not consumed in such reactions nor were any N-oxygenated derivatives formed in the absence of ascorbate (e.g., hydroxylamino, nitroso, nitro, azoxy). These observations suggest that the primary mechanism for sulfonamide-induced hypothyroidism is reversible inhibition of TPO-mediated thyroid hormone synthesis and not the formation and covalent binding of reactive N-oxygenated metabolites. These results are consistent with a hormonal mechanism for SMZ-induced thyroid carcinogenesis mediated by thyroid-stimulating hormone (TSH).(ABSTRACT TRUNCATED AT 250 WORDS)

Peroxidase-catalyzed displacement of tritium from regiospecifically labeled estradiol and 2-hydroxyestradiol.

Estradiol and 2-hydroxyestradiol with 3H at different positions in rings A, B or D were incubated with lactoperoxidase without added H2O2 and their oxidative transformation was followed by transfer of 3H into 3H2O. With estradiol, 3H loss from different positions in the aromatic ring was almost equal and also occurred to a lesser extent from the alicyclic portion of the molecule. Glutathione had less effect on the formation of 3H2O for the aromatic ring of estradiol than from that of the catechol estrogen where it increased the yield 6-fold. The rate of 3H loss was also very much greater from tritiated 2-hydroxyestradiol than from estradiol and NADPH was inhibitory with both steroids. Conditions for the release of 3H from estradiol and 2-hydroxyestradiol by peroxidase as well as the effect of some biochemical inhibitors were also investigated. The possible contribution of peroxidative formation of 3H2O during the radiometric assay for catechol estrogen biosynthesis by tissue monooxygenases is discussed.

Vanadate as an inhibitor of plant and mammalian peroxidases.

Vanadate ions are shown to inhibit horseradish, squash, and rat intestinal peroxidases by following the reaction spectrophotometrically in a wide range of vanadate concentrations. I50 in phosphate buffer were 43, 9.4, and 535 microM, respectively. No inhibitory effect was found on cow milk lactoperoxidase and beef liver catalase. Gel filtration of peroxidases in the presence of vanadate, as carried out by radioactive 48V for horseradish peroxidases (either in aerobic or anoxic conditions) and neutron activation analysis (NAA) for squash peroxidase, demonstrated a binding of vanadium to these enzymes in stoichiometric amounts. Electron paramagnetic resonance spectra of the eluted peaks for the former peroxidase indicated that vanadium is in the +5 oxidation state, but an equilibrium between V (V) and V (IV) in the assay conditions cannot be discarded. Although the inhibitory mechanism remains obscure, some hypotheses are considered. The potential implications that the inhibitory effect of vanadium might have on plant and animal metabolism are also discussed.

Inhibition of sodium transport in frog skin and iodination of tyrosine in vitro by a peptide of renal origin.

Eluates from a Sephadex G-25 column through which dog renal homogenate was passed contained a factor that inhibited lactoperoxidase (LPO)-catalyzed iodination of tyrosine as well as the short circuit current (active absorptive sodium flux) in isolated skin of the bullfrog (Rana catesbeiana). The active fraction also had a sulfhydryl-containing moiety, the concentration of which was correlated directly with the extent of inhibition of the LPO enzyme. The fractions active in inhibition of the LPO enzyme failed to affect the Na/K-AT-Pase enzyme. Whether the same moiety of the active fraction inhibits both the LPO enzyme and SCC in frog skin is unknown and awaits additional study.

Cystine antagonism of the antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae.

Cystine reduction in Streptococcus agalactiae, resulting in sulfhydryl formation, may account for antagonism of the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide when cystine is present in excess of the amount needed for maximum growth. Accumulation of cystine by S. agalactiae and its reduction to form sulfhydryl compounds were demonstrated. The reduction of cystine appeared to occur by a couple reaction between glutathione reductase and glutathione-disulfide transhydrogenase activity, both of which were found in the supernatant fraction from cell homogenates. NADPH-specific glutathione reductase activity was found in the pellet and supernatant fractions from cell homogenates. Two sulfhydryls were formed for each mole of NADPH used during cystine reduction. The information presented offers a plausible explanation of how cystine, when present in excess of growth needs, may be reduced to generate sulfhydryl compounds which neutralize the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide on S. agalactiae.

Lactoperoxidase activity in guinea-pig milk and saliva: correlation in milk of lactoperoxidase with bactericidal activity against Escherichia coli.

The lactoperoxidase (LPO) activity in guinea-pig milk and saliva has been investigated in sows suckling normal young, and young orally infected with Escherichia coli. There was a 5-fold increase in activity in milk during the 3--4 weeks of lactation; infection of the young did not alter this. There was no comparable increase in lactoperoxidase activity of saliva during this same period, either in the infected or non-infected group. The antibacterial activity of milk from sows suckling normal young increased with the lactoperoxidase, and this bactericidal activity could be reversed by LPO inhibitors such as penicillamine and cysteine but not by addition of sufficient iron to saturate the lactoferrin. In milk from sows suckling infected young, bacteriostatic activity occurring in samples from about 14 days after infection needed iron or both iron and penicillamine (or cysteine) for reversal, indicating that both the antibody-lactoferrin system and the LPO system may be involved in the infected state.