外文文献乙酸钠二乙酸钠和山梨酸钾食品添加剂的安全性

Contents lists available at ScienceDirect Food Chemistry journal homepage Safety assessment of sodium acetate sodium diacetate and potassium sorbate food additives Hossein Mohammadzadeh Aghdasha b c Yousef Sohrabia b c Ali Mohammadia Dariush Shanehbandia Parvin Dehghanb 1 Jafar Ezzati Nazhad Dolatabadid 1 aImmunology Research Center Tabriz University of Medical Sciences Tabriz Iran bDepartment of Food Science and Technology Faculty of Nutrition and Food Sciences Nutrition Research Center Tabriz University of Medical Sciences Tabriz Iran cStudent Research Committee Tabriz University of Medical Sciences Tabriz Iran dResearch Center for Pharmaceutical Nanotechnology Tabriz University of Medical Sciences Tabriz Iran A R T I C L E I N F O Keywords Sodium acetate Potassium sorbate Sodium diacetate Cytotoxicity Genotoxicity HUVECs Food additives A B S T R A C T Cytotoxicity and genotoxicity of sodium acetate SA sodium diacetate SDA and potassium sorbate PS was tested on Human Umbilical Vein Endothelial Cells HUVEC Cytotoxicity was investigated by MTT assay and fl ow cytometry analysis while genotoxicity was evaluated using DNA fragmentation and DAPI staining assays The growth of treated HUVECs with various concentrations of SA SDA and PS decreased in a dose and time dependent manner The IC50 of 487 71 485 82 and 659 96 M after 24 h and IC50 of 232 05 190 19 and 123 95 M after 48h of treatment were attained for SA SDA and PS respectively Flow cytometry analysis showed that early and late apoptosis percentage in treated cells was not considerable Also neither considerable DNA fragmentation nor DNA smear was observed using DAPI staining and DNA ladder assays Overall it can be concluded that the aforementioned food additives can be used as safe additives at low concentration in food industry 1 Introduction Synthetic food additives such as sodium acetate SA sodium dia cetate SDA and potassium sorbate PS are frequently used as fungi cide and bactericide in food industry Since food additives play a main role in food supplements their safety is one of the most signifi cant worldwide challenge in nutrition science Fathi Mohammadzadeh Aghdash Sohrabi Dehghan Piper Wu Zhong Shan Yesudhason Lalitha Gopal European Food Safety 2005 Grosulescu Juneja Ozdemir Shen Wu Chen Smith Daifas El Khoury Koukoutsis Grosulescu et al 2011 It can be added directly to food products without any limitation for human consumption and is used as a fl avoring agent adjuvant for control of pH in bakery and meat product Furthermore it is econom ical and widely available and generally recognized as safe Ghomi et al 2011 Liu Basu Miller Received in revised form 4 March 2018 Accepted 6 March 2018 Corresponding authors 1These authors contributed equally E mail addresses dehghan nut P Dehghan ezzatij tbzmed ac ir J Ezzati Nazhad Dolatabadi Food Chemistry 257 2018 211 215 Available online 08 March 2018 0308 8146 2018 Elsevier Ltd All rights reserved T Ravishankar Ozdemir G ren et al 2015 Various studies results showed that the antibacterial activity of these compounds is associated with their carboxyl group COOH and number of carbon atoms It has been confi rmed that shorter carbon chains such as acetate and sorbate additives has more antimicrobial activity compared to long carbon chains Cabezas Pizarro Redondo Solano Uma a Gamboa Sallam 2007 Despite the widespread use of additives in the food industry the mechanism of action and its eff ects on cell lines have not been clearly understood Therefore the purpose of this study is evaluation of cyto genotoxic eff ects of these extensively used food additives on human umbilical vein endothelial cells HUVECs The cytotoxic eff ects of SA SDA and PS on the growth death of HUVECs were evaluated by MTT assay For assessment of the genotoxicity eff ect DNA ladder and DAPI staining assays were used To determine the mechanism of cell toxicity fl ow cytometry study was performed 2 Materials and methods 2 1 Materials HUVECs line was purchased from the cell bank of Iran Pasteur Institute Cell culture plates and fl asks were obtained from the SPL Life Sciences Co Ltd Gyeonggi do Korea SA SDA and PS were purchased from sigma Aldrich Trypsin ethylenediaminetetraaceticacid EDTA solution Dulbecco s Modifi ed Eagle Medium DMEM medium catalog number 11995065 and fetal bovine serum FBS catalog number 10566016 was provided from Gibco Co Dublin Ireland The Anexin V FITC apoptosis detection kit was supplied from oncogene research products San Diego USA The other chemical materials were obtained from Sigma Aldrich and Merck Co 2 2 Cytotoxicity assay The cytotoxic eff ect of these food additives was determined by MTT 3 4 5 Dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide assay For MTT assay normal HUVECs were cultured in complete medium containing DMEM High Glucose that supplemented with 10 FBS The density of seeded cells on 96 well plates was 1 104cells cm2and at confl uency of 50 60 in the 96 well plates was treated with a diff erent concentration range 25 50 100 and 200 M of additives SA SDA PS and were incubated for 24 and 48h at 37 C MTT was added to cell culture at concentration of 2mg mL in PBS and after 4h incubation and formation of formazan crystals by oxidation of the MTT dye were dissolved in DMSO At the end spectrophotometric plate reader BioTek Instruments Inc Vermont USA was used for UV ab sorbance measurement at 570nm All experiments were repeated thrice Eskandani Hamishehkar Hamishehkar Khani Kashanian Ezzati Nazhad Dolatabadi Sun et al 2017 2 4 DNA ladder assay DNA strand fragmentation as a confi rmatory test for apoptosis carried out Briefl y HUVECs were cultured in complete medium con taining DMEM and treated with SA SDA and PS for 48h Then treated cells were incubated for 10min in the lysis buff er containing 0 5 sodium dodecyl sulfate 10mM EDTA 50 mM Tris base at 37 C Chloroform isoamyl alcohol step was used for denaturing of proteins and purifying of total DNA Finally total DNA electrophoresed in 1 2 agarose gel after precipitation with isopropanol Baski Popovi Risti Gooch Hamishehkar et al 2014 Vandghanooni et al 2013 2 5 FITC labeled annexin V apoptosis assay Flow cytometry technique was used to distinguish apoptotic cell death from necrotic one in the HUVECs treated with these additives compared to untreated cells Briefl y treated and untreated cells were washed three times in PBS buff er gently and detached by trypsinization and washed three times with 500 l buff er After that the supernatant was removed and the cells were resuspended in 100 l of Annexin V binding buff er containing 5 l of Annexin V FITC Fluorescein iso thiocyanate After 15min incubation at room temperature in the dark and cell centrifugation 1000 RPM 5min supernatant was removed and 100 l of Annexin V binding buff er and 5 l propidium iodide PI staining solution were added And after 5min incubation at room temperature the cells were analyzed using Becton Dickinson FACS Calibur System San Jose USA with emission fi lters of 515 545nm for FITC green and 600nm for PI red Eskandani Hamishehkar Hao Ni Sun Vandghanooni et al 2013 2 6 Statistical analysis All results were represented as mean SD of three independent ex periments The IC50 of treated samples was calculated via Graphpad Prism 6 The one way and two way analyses of variance ANOVA were used to analyze the eff ect of SA SDA and PS 3 Results and discussion 3 1 Cytotoxic eff ects of additives on HUVECs To measure the possible eff ects of the additives on the viability of HUVECs during the experiments MTT assay was used The IC50 were 487 71 M for SA 485 82 M for SDA and 659 96 M for PS after 24h treatment while after 48 h of treatment of cells with these additives IC50 were 232 05 190 19 and 123 95 M for SA SDA and PS re spectively The results indicate that SA SDA and PS were able to de crease the growth of treated HUVECs in a dose and time dependent manner Fig 1 Our results are in accordance with the cytotoxicity study of sodium benzoate preservative which carried out by Yadav et al in 2016 Abramsson Zetterberg Yadav Kumar Das Yang et al 2014 3 2 DAPI staining assay In present study induction of apoptosis in HUVECs upon treatment with SA SDA and PS was investigated by microscopic analysis of DAPI stained cells Light and fl uorescence microscopy images of the DAPI stained cells after 48h exposure with IC50 concentration of SA SDA and PS and 7 DMSO as positive control has been shown in Fig 2 Morphology of DAPI stained cells did not show any obvious fragmen tation in the chromatin and DNA rings within the nucleus of cell s treated with the aforementioned additives and their morphology was the same as untreated normal cells When cell apoptosis occurs DNA H Mohammadzadeh Aghdash et al Food Chemistry 257 2018 211 215 212 cleavage and chromatin fragmentation should be observed Eskandani et al 2014 Hamishehkar et al 2014 Therefore it can be concluded that there was not any sign of apoptosis occurrence upon treatment of the cells with these additives 3 3 DNA ladder assay DNA fragmentation assay using agarose gel electrophoresis was performed as most reliable method to discriminate apoptotic cell death from necrotic one The agarose gel electrophoresis results did not show any signifi cant DNA fragmentation or smear upon treatment of HUVECs with IC50 concentration of SA SDA and PS Fig 3 Formation of DNA ladder can happen only due to cell apoptosis via occurrence of DNA fragmentation Since the treated cell did not show formation of DNA ladder it can be deduced that the cell growth decrease upon treatment with these additives was not owing to cell apoptosis Mamur Y zba o lu nal Saatci et al 2016 3 4 Flow cytometry annexin V PI measurement Evaluation of apoptosis with Annexin V staining has become an important biological phenomenon for studying cell toxicity Binding of Annexin V to phosphatidylserine which translocated from inner side of plasma membrane to outer side due to happening of apoptosis is used for distinguishing of apoptotic cells from necrotic cells As it is obvious in Fig 4 and Table 1 more than 4 of treated cells with SA SDA and PS after 48h were in early stage of apoptosis low percentage in Fig 1 HUVECs growth decrease in dose and time dependent manner upon treatment with various concentrations of 25 50 100 and 200 M SA SDA and PS after 24 and 48 h showed that p 0 01 Fig 2 Light and fl uorescent microscopic images of HUVEC cells stained with DAPI A1 A DNA of untreated control cells B C D DNA of cells treated with IC50 concentration of SA SDA and PS respectively after 48 h H Mohammadzadeh Aghdash et al Food Chemistry 257 2018 211 215 213 secondary stage of apoptosis and around 2 4 of treated cells were necrotic cells Based upon this theory using FITC labeled Annexin V fl ow cytometry we observed that these additives at IC50 concentration did not lead in considerable apoptosis or necrosis Bastaki Farrell Bhusari Pant Hao et al 2007 Vizzotto Porter Byrne A 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