| 1. ObjectivesOne of the cleanest and safest methods of preserving food is high-energyionizing radiation treatment, such as high-energy gamma rays, electron beams, orx-rays that kills many viable microorganisms in meat, seafood, spices and herbs toprolong shelf life and prevent food-borne diseases. Now the irradiated food has beenin many of markets, so the technology to distinguish irradiated food from untreatedfood has been a hot field of food science.There has been many technology used to detect irradiated food, such as Gaschromatographic analysis; Photostimulated luminescence (PSL); electron spinresonance (ESR) and thermoluminescence(TL), A drawback of the above mentionedtechniques is their requirement for sophisticated and relatively expensive equipment,and sample preparation and analysis may be quite time-consuming. It's still importantto develop new methods.Since the large molecule of DNA is an easy target for ionizing radiation, changesin DNA offer potential as a detection method. A sensitive technique to detect DNAfragmentation is the microgel electrophoresis of single cells or nuclei, also called"comet assay". The Comet assay (also known as the single cell gel electrophoresisassay) is a simple, rapid and sensitive technique for analyzing and quantifying DNAdamage in individual mammalian (and to some extent prokaryotic) cells. Theprinciple behind the technique is simple. DNA is a relatively fragile molecule that ispresent in the nucleus as tightly packed super coiled loops. One nick is sufficient to uncoil the loops and to allow DNA to spread from the tightly packed core. Duringelectrophoresis the DNA fragments produced by each cell migrate toward the anode ata rate inversely related to the size of the fragments. Consequently each cell withdamage in its DNA gives the appearance of the comet. Studies on radiation biologyhave indicated that we can detect the DNA injury by SCGE in mammal cells such assperm cell, tumor cell and lymphocyte aider radiation. Recently, along with the rapidprogress of technology, new software has been exploited, which adds much newmeanings to this method.We used this technology to explore if the DNA damage in refrigerated mouseliver and muscle cells caused byγ-ray irradiated can be detected by comet assay. Tooffer a base of lab research theory that comet assay can be used to detect irradiatedmeat food.2. Materials and methods2.1 samplesSix SD mice offered by Institution of Lab Animal of Zhejiang Academy of MedicalSciences. Divide fresh mice's liver and muscle into five parts and irradiate them bydifferent dosesγ-ray. Afterwards store them at -20℃for three days and separatethem into single cells.2.2 methodsMouse's liver and muscle cells were isolated and resuspended in PBS. Then cellswere mixed with trypan blue solution and checked for viability. The following steps:slide preparation, alkaline lysis, DNA unwinding, electrophoresis and stain wereperformed. The comet lengths were measured under a confocal microscope.3. The Results3.1 DNA damage of liver cells cansed by different doses ofγ-ray irradiated3.1.1 The comet's mean tall lengths (MTL) of the three doses irradiation (1Gy, 2Gy,4Gy) groups were significantly longer than those of controls (p＜0.05 or p＜0.01). MTLincreasing significantly companion with the dose showing a dose-responserelationship(r=0.81, p＜0.01) 3.2.2 The comet's mean tail moment (MTM) of the dose irradiation (4Gy) groupswere significantly longer than those of controls (p＜0.01). MTM increasingsignificantly companion with the dose showing a dose-response relationship (r=0.69,p＜0.01)3.2 DNA damage of muscle cells caused by different doses ofγ-ray irradiated3.2.1 The comet's mean tail lengths (MTL) of the two doses irradiation (2Gy, 4Gy)groups were significantly longer than those of controls (p＜0.01). MTL increasingsignificantly companion with the dose showing a dose-response relationship (r=0.87,p＜0.01)3.2.2 The comet's mean tail moment (MTM) of the three doses irradiation (1Gy, 2Gy,4Gy) groups were significantly longer than those of controls (p＜0.05 or p＜0.01).MTM increasing significantly companion with the dose showing a dose-responserelationship (r=0.9, p＜0. 01)4. ConclusionWe make an conclusion that irradiated Mouse's liver and muscle cells will showan increased extension of the DNA from the nucleus towards the anode thusconsiderably longer comets (more fragmentation) than unirradiated cells. Unirradiatedcells will appear nearly circular or with only slight tails. So we can use Comet assayto detect the DNA damage in refrigerated mouse liver and muscle cells caused bysome doses irradiated. According to this study we know comet assay may be used intoscreening irradiated meat food. |
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