Influence of new antimicrobial peptides of the medicinal leech Hirudo medicinalis on the functional activity of neutrophil granule proteins

BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms. AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins the main effector cells of the immune system. MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis. RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity. CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the bodys fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit if it necessary to protect against damage to the bodys own tissues.

Sitagliptin Modulates Oxidative, Nitrative and Halogenative Stress and Inflammatory Response in Rat Model of Hepatic Ischemia-Reperfusion

A possibility of repurposing sitagliptin, a well-established antidiabetic drug, for alleviating injury caused by ischemia-reperfusion (IR) is being researched. The aim of this study was to shed some light on the molecular background of the protective activity of sitagliptin during hepatic IR. The expression and/or concentration of inflammation and oxidative stress-involved factors have been determined in rat liver homogenates using quantitative RT-PCR and Luminex® xMAP® technology and markers of nitrative and halogenative stress were quantified using targeted metabolomics (LC-MS/MS). Animals (n = 36) divided into four groups were treated with sitagliptin (5 mg/kg) (S and SIR) or saline solution (C and IR), and the livers from IR and SIR were subjected to ischemia (60 min) and reperfusion (24 h). The midkine expression (by 2.2-fold) and the free 3-nitrotyrosine (by 2.5-fold) and IL-10 (by 2-fold) concentration were significantly higher and the Nox4 expression was lower (by 9.4-fold) in the IR than the C animals. As compared to IR, the SIR animals had a lower expression of interleukin-6 (by 4.2-fold) and midkine (by 2-fold), a lower concentration of 3-nitrotyrosine (by 2.5-fold) and a higher Nox4 (by 2.9-fold) and 3-bromotyrosine (by 1.4-fold). In conclusion, IR disturbs the oxidative, nitrative and halogenative balance and aggravates the inflammatory response in the liver, which can be attenuated by low doses of sitagliptin.

Physicochemical properties of lactoferrin under oxidative/halogenative stress

Lactoferrin (Lf) was discovered in the thirties of the twentieth century. Since that time a number of useful properties of Lf (antibacterial, antiviral, pro- and anti-inflammatory, etc.) have been found. That’s why Lf became a promising candidate for pharmaceuticals use. The concentration of Lf strikingly increases in inflammatory focuses due to neutrophil degranulation. At the same time, activated neutrophils starts to generate reactive oxygen and halogen species (ROS and RHS), which leads to the development of oxidative/halogenative stress. In this work, using the fluorescence analysis we found the change of the Lf structure and properties in the inflammation conditions (under oxidatives/halogenative stress). We use two forms of Lf – human Lf, excreted from human milk, and recombinant Lf, excreted from milk of transgenic goats. It was established that the amino acids of Lf (decreasing the number of tryptophanils and primary amines) and protein restructuring undergo modification under the HOCl action, while H2O2 has no influence. These changes in the molecule under the HOCl treatment result in decreasing the iron-binding capacity of Lf.

Enzymatic and bactericidal activity of myeloperoxidase in conditions of halogenative stress

Myeloperoxidase (MPO), found mainly in neutrophils, is released in inflammation. MPO produces reactive halogen species (RHS), which are bactericidal agents. However, RHS overproduction, i.e., halogenative stress, can also damage host biomolecules, and MPO itself may be targeted by RHS. Therefore, we examined the susceptibility of MPO to inactivation by its primary products (HOCl, HOBr, HOSCN) and secondary products such as taurine monochloramine (TauCl) and taurine monobromamine (TauBr). MPO was dose-dependently inhibited up to complete inactivity by treatment with HOCl or HOBr. TauBr diminished the activity but did not eliminate it. TauCl had no effect. MPO became inactivated when producing HOCl or HOBr but not HOSCN. Taurine protected MPO against inactivation when MPO was catalyzing oxidation of Cl− to HOCl, whereas taurine failed to prevent inactivation when MPO was working with Br−, either alone or in combination with Cl−. SCN− interfered with HOCl-mediated MPO inhibition. UV–vis spectra showed that heme degradation is involved in HOCl- and HOBr-mediated MPO inactivation. A negative linear correlation between the remaining chlorinating activity of HOCl- or HOBr-modified MPO and Escherichia coli survival upon incubation with MPO/H2O2/Cl− was found. This study elucidated the possibility of MPO downregulation by MPO-derived RHS, which could counteract halogenative stress.

Enzymatic and bactericidal activity of monomeric and dimeric forms of myeloperoxidase

This study was carried out to compare the enzymatic and bactericidal activity of mature, dimeric myeloperoxidase (MPO) and its monomeric form. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each of them by two methods. Bactericidal activity of the MPO/Н2О2/Cl- system was tested using the Escherichia coli laboratory strain DH5a. No difference in the enzymatic and bactericidal activity between dimeric MPO and hemi-MPO was found. Both forms of the enzyme also did not differ in the resistance to HOCl, the main product of MPO. HOCl caused a dose-dependent decrease in peroxidase and chlorinating activity, and the pattern of this decrease was identical for dimeric MPO and hemi-MPO. At equal heme concentration, a somewhat higher bactericidal effect was observed for the hemi-MPO/Н2О2/Cl- system compared with the dimeric MPO/Н2О2/Cl- system. However, this is most likely not related to some specific property of hemi-MPO and can be accounted for by the higher probability of contacting between bacterial surface and hemi-MPO molecules due to their two-fold greater number relative to that of dimeric MPO molecules at the same heme concentration. By using Western-blotting with antibodies to MPO, we showed, for the first time, that the dimeric molecule of MPO could be cleaved into two monomeric subunits by HOCl, most probably due to oxidation of the disulfide bond between these subunits. This finding suggests that appearance in blood of MPO corresponding in mass to its monomer may result from the damage of dimeric MPO by reactive halogen species, especially upon their overproduction underlying oxidative/halogenative stress in inflammatory diseases.

Myeloperoxidase-Derived Halogenative Stress, a Possible Cause of Benzene Leukemogenesis

Abstract 4404 Background: Benzene, widely used in the chemical industry, is known to be myelotoxic, and to cause acute myeloid leukemia in humans. Although the mechanism has not been elucidated, benzene toxicity is considered to occur only after metabolic activation. Myeloperoxidase (MPO) is synthesized and secreted by myeloid cells catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidant derived from chloride ions and hydrogen peroxide (H2O2). HOCl plays key roles in host defense by oxidizing the cellular constituents of invading pathogens. At the same time, HOCl is also capable of damaging proteins and nucleic acids in host tissue. By damaging the DNA of host cells, MPO-induced DNA halogenation (halogenative stress) might contribute to the carcinogenesis. Objectives: Because the findings of in vivo and in vitro research so strongly implicate the involvement of reactive oxygen species (ROS) in benzene-induced toxicity, focusing on 1,2,4-benzenetriol (BT), a benzene metabolite that generates ROS by autoxidation, we designed a study to investigate the toxicity of benzene leading to leukemogenesis, specifically examining the cytotoxic effects of BT on a human myeloid cell line, a class of cells from the organ mainly affected by benzene. Methods: After exposing human myeloid cell line HL-60 to BT, we investigated the cellular effects, including apoptosis, ROS generation, DNA damage (halogenated-DNA and 8-oxo-deoxyguanosine (8-oxo-dG)), and protein damage (halogenated tyrosine). We also investigated how the cellular effects of BT were modified by H2O2 scavenger catalase, HOCl scavenger methionine, and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase-specific-inhibitor. Apoptosis was measured by Annexin V-fluorescein isothiocyanate and propidium iodide double-labeling kits. Intracellular ROS were determined flow-cytometrically using ROS-sensitive fluorescent probes. Halogenated-DNA was measured by flow cytometry after immunostaining, using a novel monoclonal antibody (mAb2D3) which recognizes the HOCl-modified 2’-deoxycytidine residue, 5-chloro-2’-deoxycytidine. 8-oxo-dG was measured by electrochemical detection after HPLC separation and halogenated tyrosines were detected immunocytochemically. Results: BT increased the levels of apoptosis and of ROS including superoxide (O2•−), H2O2, HOCl, and the hydroxyl radical (•OH). Catalase, ABAH, and methionine each inhibited the increased apoptosis caused by BT; and catalase and ABAH inhibited increases in HOCl and •OH. Indicating that HOCl was generated via the H2O2–MPO–HOCl system, this inhibition of HOCl generation by both catalase and ABAH demonstrates that, in HL-60 cell samples exposed to BT, H2O2 was certainly metabolized to HOCl by MPO. It also suggests that HOCl might trigger BT-induced apoptosis of HL-60 cells. We then investigated whether this cytotoxicity of BT was related to the induction of DNA damage. We evaluated the halogenation of DNA by HOCl, and 8-oxo-dG induction by •OH. While BT exposure increased halogenated DNA, this increase was inhibited by catalase, methionine, and ABAH. BT exposure did not, however, increase 8-oxo-dG. To confirm the induction of halogenative stress by BT, we investigated HOCl-induced protein damage in the form of halogenated tyrosines. BT exposure also increased the amount of halogenated tyrosines. Conclusions: In this study we have constructed a novel hypothesis (Figure 1): exposure to BT increases O2•− generation, possibly by autoxidation; this O2•− is chemically or enzymatically converted to H2O2; this H2O2 is metabolized to HOCl by MPO; whereupon this HOCl halogenates DNA and proteins, thus inducing myelotoxicity or leukemogenesis. The high expression of MPO from myeloid cells, along with the fact that halogenated DNA can cause gene mutation and epigenetic changes, may explain how benzene is involved in bone marrow disorders or myeloid leukemia. Previous studies of benzene toxicity have reported that MPO plays a role in the bioactivation of benzene’s phenolic metabolites. Here, we have shown for the first time that a benzene metabolite, BT, is capable of generating HOCl and consequent halogenative damage via the H2O2–MPO–HOCl system. Our findings lend strong support to the conjecture that BT-induced DNA halogenation is a primary reaction in the leukemogenesis associated with benzene. Disclosures: No relevant conflicts of interest to declare.