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189 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2016, 16, 189-196
RESEARCH ARTICLE ISSN: 1871-5303 eISSN: 2212-3873
Impact Factor: 1.987
Pier Francesco Biagi1, Sara Boffola2, Riccardo Stefanelli2, Luigi Schiavulli1, Teresa Ligonzo1, Giuseppe Casamassima1, Maria Teresa Pappagallo4, Kiichiro Kawaguchi5, Hiroaki Takimoto6, Yoshio Kumazawa7, Sergio Fontana8, Flavia Laforgia8, Matteo Antonio Russo9, Thea Magrone2 and Emilio Jirillo2,3,* Department of Physics, University of Bari, Bari, Italy; 2Department of Basic Medical Sciences, Neuroscience and Sensory Organs, University of Bari, Bari, Italy; 3Fondazione San Raffaele, Ceglie Messapica, Italy; 4Blood Bank, Polyclinic Hospital of Bari, Bari, Italy; 5Iwaki Meisei University, Iwaki, Fukushima, Japan; 6Kitasato University, Sagamihara, Kawagawa, Japan; 7Vino Science, Japan, Inc., Kawasaki, Kanagawa, Japan; 8Farmalabor Srl, Canosa di Puglia, Italy; 9Consorzio, MEBIC, University San Raffaele, Rome, Italy
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Results: Our data will show that polyphenol pre-treatment reverts to normality the morphology of irradiated leukocytes in comparison to irradiated cells only. Conversely, leukocyte morphometry seems to be not affected by this treatment.
DOI: 10.2174/18715303166661610031530 24
Received: July 18, 2016 Revised: August 09, 2016 Accepted: September 28, 2016
Methods: In view of the evidence that polyphenols exert many beneficial effects on plants, animals and humans, leukocytes from human peripheral blood were pre-treated for 1 h with two polyphenol preparations from red grape before EMR exposure (1.8 GHz).
A R T I C L E H I S T O R Y
Abstract: Background: Our recent findings have demonstrated that electromagnetic radiations (EMR) (1.8 GHz radiofrequency) are able to in vitro induce morphometrical and morphological modifications of human leukocytes from normal donors.
Conclusion: Here, we demonstrate that polyphenols are also able to normalize leukocyte morphology per se altered before as well as after irradiation. Finally, a working hypothesis aimed at clarifying the protective mechanisms exerted by polyphenols on irradiated leukocytes will be illustrated.
Keywords: Electromagnetic radiations, leukocytes, morphology, morphometry, polyphenols. INTRODUCTION
Over recent years, a few studies have emphasized the in vitro effects of electromagnetic radiations (EMR) on peripheral blood leukocytes, using 1.8-2.4 GHz radiofrequencies. However, data of current literature on this issue are quite controversial. For instance, Cleary et al.  did not find any change of lymphocyte morphology following exposure to 2.45 GHz radiofrequency. Bouwens et al.  showed that low-frequency EMR did not modify inflammatory gene and protein expression in human monocytes. Similarly, de Klejin et al.  did not report any effect of low frequency EMR on Toll-like receptor signaling in human peripheral blood mononuclear cells. Koyama et al.  failed to demonstrate any effect following EMR exposure on phagocytosis and chemotaxis by human neutrophil-like HL-60 cells. Prenatal exposure to WiFi signals did not affect thymocyte development
Endocrine, Metabolic and ImmuneDisorders - Drug Targets
Morphometrical and Morphological Alterations of Human Leukocytes Exposed to 1.8 GHz Electromagnetic Radiations: In Vitro Protective Effects Induced by Polyphenols
*Address correspondence to this author at the Department of Basic Medical Sciences, Neuroscience and Sensory Organs, University of Bari, Bari, Italy, P. zza Giulio Cesare, 11 70124 Bari, Italy; Tel: +39 0805478492; E-mail: [email protected]
and peripheral T lymphocyte contingent in mice . In human volunteers exposed to 200 µT, 60 Hz magnetic fieldld for 4 h no evidence of peripheral blood leukocytes DNAA damage was reported . Conversely, Esmekaya et al.  ] documented mutagenic and morphological modifications s of human lymphocytes in vitro exposed to 1.8 GHz z radiofrequency. In particular, alterations of membrane e integrity, organelles and nucleus were more evident up to 48 8 h exposure, even if mitochondrial and chromatin changes s appeared earlier. In this framework, Aly et al.  have e reported changes of shape, shrinking and abnormalal expanding and rolling of human leukocytes exposed to 1.8 8 GHz radiofrequency. Furthermore, reactive oxygen species s (ROS) generation from human neutrophils was enhanced by y their in vitro exposure to low frequency EMR . In the e same direction, low frequency EMR increased the micro- bicidal effects of monocyte-derived macrophages via release e of nitric oxide (NO) . In addition, low frequency EMRR altered adhesive ability of peripheral blood mononuclearar cells by decreasing CD11a and increasing CD49d cellll markers . In this respect, another report has documented d an increased T lymphocyte adherence in vitro from both © 2016 Bentham Science Publishers
Volume 16, Number 3
190 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2016, Vol. 16, No. 3
Biagi et al.
blood donors and cancer patients following exposure to EMR . Quite interestingly, 900 MHz EMR were able to induce adaptive responses in human lymphocytes when cells were in the S-phase of the cell cycle . Finally, in terms of genetic damage following EMR exposure for 72 h (800 MHz continuous wave), increased levels of aneuploidy were in vitro detected in human peripheral blood lymphocytes .
leukocyte samples characterized by morphometrical and morphological abnormalities before EMR exposure. A working hypothesis on the mechanisms of action exerted by polyphenols in our test system will be discussed.
In this framework, our recent report  has demonstrated morphometrical and morphological modifications of human leukocytes exposed to 1.8 GHz for different lengths of time (from 5 min up to 24 h). In particular, we used a reverberation chamber which supplies a controlled EMR intensity, as described in full details elsewhere . A large sample of leukocytes, obtained from 108 healthy blood donors, was used for separate irradiation experiments, also allowing reproducibility of data. A computerized morphometrical method was employed to measure the area size of each single leukocyte, while a parallel microscopical observation was performed, staining leukocytes with MayGrünwald-Giemsa. Modifications of leukocyte areas and marked morphological alterations of cells were noted in comparison to sham-exposed leukocytes.
MATERIALS AND METHODS
Frozen seeds from Nero di Troia Vitis vinifera grape cultivar were extracted by a percolation method in ethanol/water (70:30). Liquid chromatography with a diode array detection to define the polyphenol composition was used. Then, the extract was purified on a synthetic adsorbent brominated resin and percentage of polyphenol content (cathechins and epicatechins) was equal to 10.37. We termed this extract as poly A. The extracts were evaluated for their potential anti-oxidant effects by using the 2,2-diphenyl-1dipicrilidrazile assay which measures the ability of test agents to scavenge radicals .
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Experimental Design Twenty heparinised peripheral blood samples from healthy donors, recruited at the Blood Bank from the Polyclinic Hospital, Bari, (Italy), after giving an informed consent, were used throughout the study. Samples were treated in the following manner. Each polyphenol preparation (poly A and poly B) at different concentrations (1, 3 and 5 μg) [21, 33-35], respectively, was separately added to 1X106/ml leukocytes, and, then, mixtures were exposed to a 78±10 V/m, 1.8 GHz frequency field for 1 h by means of a reverberation chamber . After this time, samples were incubated at 37°C, 24 h, 5%CO2. Sham-exposed leukocytes in the presence or absence of polyphenols, and irradiated leukocytes in the absence of polyphenols served as controls.
Polyphenols are bioactive compounds frequently found in beverages, fruits and vegetables, in which they provide colour and flavour. Furthermore, they also contribute to plant protection from pathogens, ultra violet radiation and other sources of damage [16, 17]. Commonly, polyphenols are classified according to their chemical structures into non flavonoids as phenolic acids and phenolic amides or flavonoids, subdivided by their substituents into isoflavones, neoflavonoids, chalcones, flavones, flavonols, flavonones, flavononols, flavanols, proanthocyanidins and anthocyanidins. Numerous studies have attributed to polyphenols a wide range of biological properties, even including anti-inflammatory, anti-oxidant, cardio-protective and anti-cancer activities . Evidences for the effects of polyphenols on immune functions have been largely demonstrated, even if the knowledge of their exact mechanisms of action deserves further investigation. According to Lin et al. , some plant extracts (Bidens alba, Lycium chinense, Mentha arvensis, Plantago asiatica, Houttuynia cordata and Centella asiatica) protected human lymphocytes against hydrogen peroxide induced DNA damage. In this respect, the anti-oxidant activities by polyphenols are represented by ROS scavenging, oxidative stress protection, thiol-redox stabilization, and attenuation of membrane lipid peroxidation [24, 25]. With regard to human studies, Cao and associates  found that an augmented intake of vegetables and fruits increased the plasma anti-oxidant capacity. Other reports have clearly demonstrated that dietary polyphenols have the ability to enhance plasma antioxidant capacity, decreasing oxidative and inflammatory stress, even including DNA damage [27-30].
Grape marc from Koshu cultivar was prepared according to the method described by Tominaga and associates . Briefly, triturated grape marc and diluted with 50% water (v/v) was fermented in the presence of pre-cultured Lactobacillus plantarum (5% grape marc v/v) at 37°C for 3 days. After bacterial killing by heating, fermented grape marc (FGM) was lyophilized and obtained as a fine powder. The content of total polyphenols in FGM was colorimetrically determined and was about 2% as equivalent to gallate. Quantitative analysis of Koshu-FGM using HPLC revealed that the content of (-)-epigallocatechin gallate (EGCG) was 562 mg in 1.0 Kg. We termed this preparation as poly B.
In the light of the above concepts, the specific aims of the present work are the exploration of the in vitro effects of 1.8 GHz EMR on human leukocytes pre-treated with two different preparations of red grape polyphenols. Results will show that morphological alterations of irradiated leukocytes are abrogated by polyphenol treatment with negligible effects on the cell morphometry. The same protective effect upon cell morphology can also be observed in those
At the end of 24 h incubation, all blood samples were smeared and stained with May-Grünwald-Giemsa. For morphological analysis, blood smears were observed by an optical microscope (Olympus BH-2, Segrate, Italy) 100X immersion objective. At least 50 leukocytes per field (at least 3 fields) were read. For morphometrical assessment, blood smears were observed by an optical microscope (Leica DMRB, Milan, Italy) 100X immersion objective. A fixed number of leukocytes (50 cells per field) were acquired and, then, the size of the area occupied by each cell was determined by means of the software SPOT 5.1.
Morphometrical and Morphological Alterations
Endocrine, Metabolic & Immune Disorders - Drug Targets, 2016, Vol. 16, No. 3
human leukocytes to EMR was not univocal. In fact, some leukocyte donors exhibited normal morphological parameters before irradiation, while in other cases they were already abnormal prior to exposure to EMR. Therefore, as evidenced in the Results section, leukocytes from two different groups of donors were screened before and after EMR exposure.
Statistical Analyses Leukocyte average area (expressed in µm 2) was compared between exposed and sham exposed cells in the presence or absence of both polyphenols. The paired two tailed t test was performed using the GraphPad Prism statistical software release 5.0 for Windows Vista. Statistical significance was set at p< 0.05.
H z 1. 8