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Global Veterinaria 15 (1): 72-81, 2015 IDOSI Publications, 2015 Effect of Honey on Hydroxyl Radical Generation, Glutathione Depletion
And on Myeloperoxidase Released in the Extra-Cellular
Milieu by Activated Neutrophils
1Saad Aissat, Ha ma Benbarek, 3 Thi erry Franck, G 3 inette Deby-Dupont, ,4 idier Serteyn and Ang 1Institute of Veterinary Sciences, Ibn Khaldoun University, Tiaret 14000, Algeria.
2Faculty of Sciences of Nature and Life, Mascara University, Mascara 29000, Algeria 3Centre for Oxygen, Research and Development (CORD), Institute of Chemistry B6a, University of Liège, SartTilman, 4000 Liège 1, Belgium 4Department of Clinical Sciences, Large Animal Surgery, Faculty of Veterinary Medicine, B41, University of Liège, Sart Tilman, 4000 Liège 1, Belgium Abstract: Using Electron spin resonance we have shown that (1): nectar honey (NH) and mixed honey (MH)
at 2% and 5% (w/v)generate the hydroxyl radical (O . H) via Fenton-like reactions and noticed a partial quenching of this radical at 10% (w/v). Honeydew honey (HH) produced partial quenching at 2%, strong quenching of.
OH at 5% (w/v) and complete quenching at 10% (w/v). (2): the three honey types honey exhibited a biphasic activity on glutathione (GSH) depletion, as they lead to GSH depletion and increase reactive oxygen species (ROS) generation at low concentrations, but prevent this depletion at higher concentrations. The present study investigates also the effects of honey on the release of myeloperoxidase (MPO) by equine neutrophils (PMNs) activated with phorbol 12-myristate 13-acetate (PMA), we noticed also a biphasic effect. At low concentration the three honeys increased significantly the total MPO released in the extra-cellular milieu by activated PMNs but decreased it significantly at higher concentrations. Honey is a very complex mixture containing a number of ingredients involved in "oxidant/antioxidant physiological processes,bifunctional activities may occur at different dosages.
Key words: Honey Hydroxyl Radical Myeloperoxidase Neutrophils
acute inflammation. Similarly, the H O Escherichia coli appears to be mediated by OH via Although the basal production of ROS can be Fenton reaction [2,4]. OH reacts indiscriminately with beneficial to maintain the redox homeostasis, still the most metabolites and macromolecules, in many cases excessive production of ROS, followed by a depletion of generating other radicals in the process [1].
endogenous antioxidant enzymes, remains responsible for During the inflammatory process, phagocytosis by the disruption of the redox state balance named "oxidant PMNs is highly correlated with the respiratory burst and stress". Among these species,. OH a potent oxidizing often accompanied by neutrophils degranulation and the agent capable of reacting with a wide variety of targets extracellular release of enzymes such as MPO. MPO is over very short distances has been implicated in different also found to a much lesser extent in monocytes and some models of neutrophil-mediated tissue injury. OH cytotoxic macrophages [5]. MPO has long been considered a key effects have been shown for both prokaryotic and constituent of the neutrophil's cytotoxic armament by eukaryotic cells; bacteria, yeast and human cells [1-3]. For catalyzing the formation of hypochlorous acid, a potent example, OH production from H O oxidant that displays bactericidal activity in vitro [6].
granules is a first line of defense against bacteria during Although the generation of oxidants by MPO is beneficial Corresponding Author: Saad Aissat, Institute of Veterinary Sciences, Ibn Khaldoun University, Tiaret 14000, Algeria.
Tel: +213 046451553.
Global Veterinaria, 15 (1): 72-81, 2015 in terms of the immune response to invading pathogens, The concept "antioxidant" is usually linked to free inappropriate stimulation of oxidant formation by this radical scavenging and that ROS are essentially harmful enzyme (wrong place, wrong time, excessive levels) can and should be eliminated. However, the purpose of the result in host tissue damage [7].
‘‘antioxidant defense network" is not to remove all ROS, Most of publications the higher level of MPO was but to control their levels so as to allow useful functions connected to enhanced free radicals productions.
whilst minimising oxidative damage [21]. Honey seems to However, some observations expand this view and show either reduce or activate the production of reactive that MPO-derived oxidants are critically involved in a oxygen species from neutrophils. Honey and its more subtle modulation of signaling pathways [8]. It has components are able to either stimulate or inhibit the been also suggested that MPO might be involved in the release of certain cytokines (tumor necrosis factor- , antioxidant, not pro-oxidant, activity of phenolic interleukin-1 , interleukin-6) from human monocytes and compounds in endothelial Cells under oxidative stress [9].
macrophages. Honey seems also, to either reduce or Peroxidases mediate in vitro the pro-oxidant or antioxidant activate the production of reactive oxygen species from activity of phenolic compounds, depending on the neutrophils. Similarly, human keratinocytes, fibroblasts chemical environment [10].
and endothelial cell responses (e.g., cell migration and Reduced GSH, a thiol-containing tripeptide, is a proliferation, collagen matrix production, chemotaxis) are significant contributor for maintaining the intracellular positively affected in the presence of honey. However, redox state and, as such, is an important component of the the immunomodulatory activity of honey is highly overall cellular defensive mechanisms against ROS. An complex because of the involvement of multiple important function of this intracellular antioxidant is to quantitatively variable compounds among honeys of scavenge ROS produced during normal aerobic cellular different origins [22].
respiration; if left unchecked, such ROS could oxidize and, The aim of this work was to evaluate the effect of thereby, damage nucleic acids, proteins and lipids [11].
three types of raw Algerian honey on hydroxyl radical Several studies have noted greater depletions of produced by the Fenton reaction and glutathione intracellular GSH in cancer, than in normal, cel s upon their depletion by using Electron Spin Resonance-spin exposures to polyphenols. Normal cells maintain a proper intracellular redox status with their antioxidant enzymes trapping and on MPO Released in the Extra-Cellular Milieu and their sufficient supply of reduced GSH and thus are by Activated Neutrophils (MPO-ELISA Assay). At the less susceptible to cytotoxic damage by pro-oxidant best of our knowledge the two latter studies have never polyphenols [12].
been achieved so far in the case of honey.
It is widely accepted that honey is beneficial to health, thanks to its antioxidant properties among all of its MATERIALS AND METHODS
beneficial aspects. Explanation(s) remain obscure and the mechanism by which it acts also so far remains to be elucidated. The antioxidant activity of honey has been paranitrophenyl phosphate, ethylene diamine tetra-acetic extensively studied, but there are remarkable acid (EDTA), phorbol 12-myristate 13-acetate discrepancies in the published data. The main cause and (PMA),Percollwere purchased from Sigma (Bornem, far from being negligible is that honey is a very complex Belgium), FeSO4 was purchased from Merck.Horseradish mixture containing a number of ingredients involved in peroxidase (HRP), diethylenetetraminepenta acetic acid "oxidant/antioxidant" physiological processes [13].
(DTPA) were purchased from Boehringer Mannheim, Besides the direct antimicrobial effects of honey [14], Trypan blue was from ICN Biomedicals, Inc (Ohio, USA).
research has also focused on identification of the Analytical-gradNaCl, KCl, H2O2 30%, were supplied by substances responsible for its anti-inflammatory [15-16] Merck (VWRI, Leuven, Belgium). all aqueous solutions and immunomodulatory effects [17-19]. It has been were prepared with water previously purified in a Milli-Q proposed that the antioxidant capacity of honey is due water system (Millipore, Bedford, MA, USA mainly to the phenolic compounds and flavonoids they contain and there is a high correlationship between Honey Samples: Three samples of Algerian honey,
polyphenols and honey antioxidant capacity, if possible honeydew honey (HH), mixed honey (MH1) and nectar a synergistic effect is observed on honey polyphenols honey (NH), were directly obtained from beekeepers, and the more than 181 compounds that form part of honey belonging to different geographical regions. Raw honeys used in this study were not submitted to thermal Global Veterinaria, 15 (1): 72-81, 2015 treatments or to pasteurization or any other operation Effect of honey on hydroxyl radical produced by the able to alter natural composition. Honey samples were Fenton reaction.
stored at 4°C in the dark until further analysis. The three M) in the presence of FeSO (425µl, varieties of honey are in accordance with international 10 4 M), DTPA (25 µl, 10 -4M), H O (5 µl, 10 3 standards (CARI ABSL, Belgium, which performed the DMPO (50 µl, 100 mM) were added to each honey sample at the final concentration of 2, 5 or 10% (w/v). The reaction mixtures were immediately transferred into the flat Blood Collection and Isolation of PMNs: Blood
cell in the TM cavity of the spectrometer. ESR spectra samples were drawn from healthy horses by jugular were recorded at room temperature on a Bruker venipuncture in 9 ml vacutainer tubes with EDTA (1.6 spectrometer (Bruker ESP300E, Bruker, Kalsruhe, mg/mlblood) as anticoagulant. The horses were Germany) operating at X-band frequency (9.8 GHz) and at clinically normal; they were fed, bred and housed under non-saturating microwave power (20 mW).The identical conditions and not under medical treatment instrumental settings were the following: 100 KHz (Faculty of Veterinary Medicine, University of Liège, modulation frequency, 1.012 G modulation amplitude, 3480 Belgium). PMNs were isolated following the technique G magnetic field center and receiver gain was 2.10 .4 The of Pycock et al. [23] on a discontinuous density sweep width was 100 G and the total number of scans was gradient of Percoll formed by a 1.108 g/ml solution, 6. The hyperfine splitting constants were measured from overlaid with a 1.1087 g/ml solution. The anticoagulated the experimental spectra by means of a Bruker Win- whole blood, laid on the top of the gradient, was Simfonia program running under Microsoft Windows. The centrifuged at 400g for 20 min at 20°C. The PMNs were Fenton reaction in the absence of honey sample was collected at the interface between the 2 gradient layers considered as a positive control (CtrL) for DMPO-OH and washed in 2volumes of physiological saline solution.
adducts and the signal height of its ESR spectrum was The cell pellets were suspended in 20 mM phosphate taken as 100%.
buffer saline (PBS) at pH 7.4 containing 137 mMNaCl and 2.7 mMKCl. The cell preparation was =96% neutrophils EPR Spin Trapping Investigation of the Effect of Honey
with a viability of 97% as measured by the trypan blue on the Thiyl Radical Generation: The reaction mixtures
exclusion test. Each batch of neutrophils was obtained 25mg/mL HRP, 10mM GSH, 1 mM H O DMPO in phosphate buffer (pH 7, 5) were added to each from 60 ml blood drawn from one horse, the cells were honey sample at final concentrations of 2% and 10% and used immediately after isolation, the experiment was immediately transferred into the flat cell in the TM cavity completed within 5hours and each assay was performed of the spectrometer. ESR spectra were recorded at room in triplicate. Each experiment was repeated at least twice temperature on a Bruker spectrometer (Bruker ESP300E, with different cell batches.
Bruker, Kalsruhe, Germany) operating at X-band Before the experiments, neutrophils (10 c frequency (9.8 GHz) and at non-saturating microwave were incubated with diluted honey samples (2, 5, 10 and power (20 mW).). The instrumental conditions were as 20%) and the cell viability was estimated by the trypan follows : microwave power, 20 mW ; receiver gain,2x 104; blue exclusion test in order to exclude a cytotoxicity of the time constant, 164 ms ; time conversion, 40,96 ms ; center field, 3480± 50 G and number of scan,2. The hyperfine splitting constants were measured from the experimental Electron Spin Resonance (ESR)-spin Trapping
spectra by means of a Bruker Win-Simfonia program Experiments: Electron spin (or electron paramagnetic)
running under Microsoft Windows. The EPR signal, resonance (ESR) is a spectroscopic technique that detects corresponding to the spin adduct of DMPO/thiyl (GS ) unpaired electrons present within the sample. As such, resulted from the reaction of DMPO with the GS radicals ESR is the only accurate approach that can provide direct produced by the HRP enzymatic activity on GSH. The EPR evidence for the presence of a free radical. In addition, the signal obtained in the absence of honey was taken as the analysis of the ESR spectrum generally enables the control spectrum (CtrL).
determination of the identity of the free radical [24].
Because of their short lifetime, the detection of such free Effect of honey on Total MPO Released in the Extra-
radicals is very difficult in biological samples and Cellular Milieu by Neutrophils Activated with PMA
therefore, spin trap agents (nitrones) are used to form (MPO-ELISA Assay): The neutrophil suspensions
adducts with a higher lifetime. (106cells/mL) were incubated for 10 min at 37 °C in Global Veterinaria, 15 (1): 72-81, 2015 darkness with honey at final concentrations of 2, 5, 10 and complex spectra with an increased intensity of the four- 20% (w/v) for each honey sample and then activated for line ESR signal attributed to the DMPO-OH adducts (sign 30 min at 37 °C once again in darkness with PMA at the Ø spectrum 2in Fig. 1b) and a new signal (sign * spectrum final concentration of 0.8 µM. After activation, the cell 2in Fig 1b). Similar data were obtained with MH, but are suspensions were centrifuged for 10 min (450 g) and the not presented here. When, 5% of NH (w/v) were added to supernatants were collected. To measure the total MPO the complete system, there was no real modification of the released by activated neutrophils in the extra-cellular shape of the ESR signal intensity attributed to DMPO-OH milieu, an original Equine MPO ELISA assay designed by adducts and the new signal as shown in Fig. 1a spectrum Franck et al. [25] was performed, using a specific kit 3. Upon addition of 10% of NH we noticed a decrease of provided by BiopTis (Liège, Belgium). Briefly, polyclonal the height of the spectrum VsCtrL. In contrast, the lines antibodies against equine MPO were obtained in rabbit attributed to the unidentified species were enhanced and coated on 96 wells-microtiter plates. The (Fig 1a spectrum 4). The addition of HH at 2 % (w/v) supernatants, which contained MPO released by the cells, already resulted in a slight decrease of the 4-line ESR were diluted 200-fold with PBS, loaded into the wells and spectrum of DMPO-OH adduct (sign Ø in Fig 1b spectrum incubated at 4 °C overnight. After washing, a second 2), the new signal was also present. When high polyclonal antibody against equine MPO, raised in guinea concentrations of HH (5 and 10% w/v) were added to the pigs and labeled with alkaline phosphatase, was loaded mixture, the lines corresponding to DMPO-OH adducts into the wells and incubated for 2 h in darkness. After an were strongly reduced and even totally disappeared for ultimate washing, the wells were loaded with a solution of the highest concentration of 10 %. In contrast, the second paranitrophenyl phosphate for the measurement of ESR signal (*) enhanced dramatically (Fig. 1b spectra 3 phosphatase activity and incubated for 30 min at 37 °C in darkness. The absorbance (405 nm) proportional to the content of MPO in the wells was read with Multiscan EPR Spin Trapping Investigation of the Effect of Honey
Ascent (Thermo Scientific). The control was performed on the Thiyl Radical Generation: The peroxidase-
with neutrophils activated with PMA in presence of PBS catalysed oxidation system allowed investigation of the instead of honey and was taken as 100% MPO release to formation of the thiyl (GS ) radical intermediate triggered compare with the effects of honey. by the addition of H O 2 n the presence of a peroxidase (HRP). This system was designed to assess the capability Statistical Analysis: Determination the effect of honey on
of honey to protect glutathione from the oxidant attack by total MPO released by PMA-activated PMNs was done in /2HRP couple (Fig 2). Fig (2a) shows EPR triplicate. The n value of one experimental point was 5.
spectrum characteristic of the thiyl radical produced by Data are given as mean ± SD and statistical analysis was the enzymatic system HRP/GSH/H O 2in the presence of performed with Graph Padins tat 3.05 (Graph Pad Software, DMPO (control). The addition to the complete system of San Diego California, USA). A p value < 0.05 was 2% (w/v) of HH and NH resulted in an increased intensity considered as significant.
of the four-line ESR signal attributed to the thiyl radical adduct, MH being slightly active (Fig 2b).The four-line EPR spectrum was totally abolished when HH, NHand MHat final concentration of 5%was added to the reaction Effect of Various Types of Honey on Hydroxyl Radical
mixture (Fig 2c).
Produced by the Fenton Reaction: The Fenton reaction is
based on the reduction of H O n electron donated by Effect of Honey on Total MPO Released in the Extra-
metal ions to produce hydroxyl radical ( OH). Iron is used Cellular Milieu by Neutrophils Activated with PMA
as an electron donor. We used the spin trap 5,5'-dimethyl- (MPO-ELISA Assay): The MPO amount released by
1-pyrroline-N-oxide (DMPO), which reacts with OH to activated PMNs (Ctrl PBS), in which PBS was used form a relatively stable paramagnetic species, the DMPO- instead of honey, was set as 100% MPO release (Ctrl).
OH spin adduct with a characteristic four-line ESR The addition OF NH, HH and MH honey at the final spectrum (quartet with 1:2:2:1 intensities) [24]. Figure 1a concentration of 2% increased respectively the MPO and 1b spectrum 1 shows the ESR spectra obtained from release by 96,76%;64,48% and 15,53%. The addition of NH the trapping of hydroxyl radicals (•OH) by DMPO in the and HH honey at the final concentration of 5% also absence of honey samples (CtrL). The addition to the increased respectively the MPO release by 47, 59% and complete system of 2% (w/v) of NH resulted in more 28,71%, while no significant effect was observed for Global Veterinaria, 15 (1): 72-81, 2015 Fig. 1: Hydroxyl radical ( O H) by fenton reaction (Fe /D 2 and effects of honey (Fig 1a) ESR spectra obtained from the trapping of hydroxyl radicals (•OH) by DMPO in the absence of honey samples (Ctrl). (1b): same as (1a) but with addition of honey at final concentration of 2% (w/v).(1c): same as ( 1a) but with addition of honey at final concentration of 5% (w/v). ).(1): same as ( 1a) but with addition of honey at final concentration of 10% (w/v)The instrument settings are listed in materiel and methods Global Veterinaria, 15 (1): 72-81, 2015 Fig. 2: Thiyl (GS ) radical produced by the enzymatic system (HRP/GSH/H O d effects of honey.
(2a): Complete system.HRP/GSH/H O DMPO. (2b): same as (2a) but with addition of honey at final concentration of 2% (w/v).(2c): same as ( 2a) but with addition of honey at final concentration of 5% (w/v).The instrument settings are listed in materiel and methods.
MH at the same concentration. Meanwhile the addition of dilution[26]. Since the rates of hydrogen peroxide NH, HH and MH honey at the final concentration of 10% production increase with honey concentrations until the and 20% decreased respectively the MPO release by optimal dilution at which honey produces the maximal 12,7% ; 50%; 30,55% and 60.21; 59.96; 59.33. amounts of hydrogen peroxide (which is between 40 and 60%) [26],and since OH generation increases with honey dilution [27],it would thus be expected an increase of the DMPO-OH signal intensity when the concentrations of In the present work we found that NH and MH honey increased. honeys at 2% and 5% (w/v) generate OH via Fenton-like Therefore, 10 % NH (w/v) were added to the complete reactions with generation of unidentified species Fig 1a system and the shape of the ESR spectrum was spectrum 2 and 3). In1994, Mason et al. [24] ascertained monitored. In contrary, we noticed apartial quenching of that since the Fenton reaction requires hydrogen OH, but the lines attributed to the unidentified species peroxide, a substance that catalyzes hydrogen peroxide were enhanced (Fig 1a spectrum 4). formation would increase the signal. It is well established Addition of HH produced partial quenching at 2%, that honeys contain glucose oxidase enzyme which strong quenching of. OH at 5% (w/v) and complete catalyses the formation of hydrogen peroxide, after honey quenching at 10% (w/v), the signal attributed to the new Global Veterinaria, 15 (1): 72-81, 2015 species enhanced dramatically. The other radical species observed could not be unequivocally identified Fig 1b specta 2, 3 and 4). Alvarez-Suarez et al. [28] previously noticed that all Cuban honeys studied generated OH to varying extents via the Fenton reaction. While in another study carried out by the same authors, it was found on the contrary that all the studied Cuban honeys scavenged the OH generated via the Fenton reaction [29]. In a similar study Henriques et al. [30], studying three types of honey (Manuka honey, pasture honey P59 and artificial honey), reported that only pasture honey P59 was able to generate free radicals. These authors concluded that additional unidentified species might arise from secondary reactions Fig. 3: Effect of honey on Total MPO Released in the involving free radicals and the organic components of Extra-Cellular Milieu by Neutrophils Activated honey. It must be noticed that these authors used only a with PMA (MPO-ELISA Assay single dilution. Honeys were shown to cause oxidative Columns and bars represent mean ± standard damage that restricted bacterial growth and caused deviation of the results of five measurements.
cytotoxic degradation of DNA due to the formation of *Significant difference (*P < 0.05, **P < 0.01, OH and appear to involve some components in honey ***P<0.001) vs. CtrL. + : enhancement;-inhibition; that have not yet been identified [27,31].
ns: no significant As, the other radical species observed could not be unequivocally identified, we cannot state here if the initial studies suggested that GSH depletion was only a quenching of OH may represent an antioxidant or byproduct of oxidative stress generated during cell death, prooxidant effect of honey. Honey's therapeutic recent discoveries suggest that GSH depletion plays a properties are largely attributed to its antimicrobial and critical as regulator of apoptosis [38].
anti-oxidant activities. Bactericidal activity could be due According to Jaganathan and Mandal. [39], honey at low concentration (3% v/v) induced apoptosis of the to OH and possibly to these secondary organic radicals.
HCT-15 and HT-29 colon cancer cells by causing the Any antibacterial properties of honey will thus be a depletion of intracellular non-protein thiols and increasing balance between the opposing activities of radical the reactive oxygen species (ROS) generation.In line with generation and quenching [30].
these findings, we have also shown in the present study GSH is an intracellular antioxidant which accounts for that honey at low concentrations causes a depletion of over 90% of the intracellular non-protein thiols [32]. It is GSH and increase ROS generation (Fig 2c). commonly viewed as an universal free radical scavenger Compared to CtrL, the three honey samples at 10% and major intracellular antioxidant [33]. In a vivo studies, and 20% (w/v) decreased significantly the total MPO honey was found to maintain or enhance the level of non- released in the extra-cellular milieu by activated PMNs protein sulfhydyl substances (such as GSH) [34]. Similar observation was made by Korkmaz et al. [35]) and Very few studies reported the effect of honey on Galal et al. [36]. In agreement with what precedes,it MPO even though Medhi et al. [40] reported that Manuka appears from our results that at 5% (w/v) the three honey Honey reduced the MPO level in rats induced ulcerative types prevent GSH depletion (Fig 2b). colitis. Honey was also found to decrease the luminol- While strategies for increasing GSH levels were enhanced chemiluminescence in opsonisedzymosan- developed to increase cellular defenses and resist toxicity, stimulated whole blood and isolated leukocytes [41]. But, strategies for depleting GSH were developed to increase as was the case for GSH depletion, at the best of our the sensitivity of tumors and certain parasites to radiation, knowledge there is not available published data on the drugs or endogenous killing mechanisms [37]. Apoptosis effect of honey on the release of MPO from activated is a conserved homeostatic process critical for organ and tissue morphogenesis, development and senescence. This Surprisingly, except for MH at 5% the three honey form of programmed cell death also participates in the types at 2% and 5% increased significantly the total MPO etiology of several human diseases including cancer, released in the extra-cellular milieu by activated PMNs neurodegenerative and autoimmune disorders Although Global Veterinaria, 15 (1): 72-81, 2015 However, upon its release from neutrophils, the beneficial and adverse effects important in the enzyme becomes inactive very frequently in tissue prevention and pathogenesis of disease. The microenvironment. Therefore, both enzymatically active mechanisms by which honey affects the release of MPO MPO and enzymatically inactive MPO are present at and the GSH depletion need to be clarified. However, our inflammatory sites [42].
results herein may represent framework for further studies.
According to Papineni and Orton [43] after, Nevertheless, the immunomodulatory activity of honey is intraperitoneal administration of a single dose honey 0,1 highly complex because of the involvement of multiple ml (50% v/v) in athymic nude mice, significant quantitatively variable compounds among honeys of enhancement in MPO activity was observed within 3 hrs- different origins. probably after that honey has been over diluted by corporal fluids-with robust PMNs activation at different lymph nodes. According to Lau et al. [8] independently of his catalytic action MPO exerts leukocyte-activating The authors would like to thank Mrs Ariane Niesten functions, an event reminiscent of other PMN-derived and Jennifer Romainville for their technical assistance.
potent pro-inflammatory cytokines such as TNF- and This work was supported by Centre for Oxygen, Research and Development (CORD), University of Liège, funded by Which should attract attention in this study is that the NFSR (National Fund for Scientific Research) Belgium.
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