DNA comet assay: A simple screening technique for identification of some irradiated foods

DNA Comet Assay method was carried out to detect irradiation treatment of some foods like meat, spices, beans and lentils. The fresh meat of cow and duck were irradiated up to radiation doses of 3 kGy, the spices (cardamoms and cumin black) were irradiated to radiation doses of 5, 10, 15 and 20 kGy while the beans (black beans and white beans) and lentils (red and green lentils) were irradiated to 0.5 and 1 kGy. All the foods were then analyzed for radiation treatment using simple microgel electrophoresis of single cells or nuclei (DNA Comet Assay). Sedimentation, lysis and staining times were adjusted to get optimized conditions for correct and easy analysis of each food. Using these optimized conditions, it was found out that radiation damaged DNA showed comets in case of irradiated food samples, whereas in non-treated food samples, round or conical spots of stained DNA were visible. Shape, length and intensity of these comets were also radiation dose dependent. Screening of unirradiated and irradiated samples by Comet Assay was successful in the case of all the foods under consideration under the optimized conditions of assay. Therefore, for different kinds of irradiated foods studied in the present study, the DNA Comet Assay can be used as a rapid, simple and inexpensive screening test.     

Improvement of the ESR detection of irradiated food containing cellulose employing a simple extraction method

Fruit may be irradiated at rather low doses, below 1 kGy in combination treatments or for quarantine purposes. To improve the ESR detection sensitivity of irradiated fruit de Jesus et al. (Int. J. Food Sci. Technol. 34 (1999) 173.) proposed extracting the fruit pulp with 80% ethanol and measuring the residue with ESR using low power (0.25 mW) for detection of ‘cellulosic’ radicals. An improvement in ESR sensitivity using the extraction procedure could be confirmed in this paper for strawberries and papayas. In most cases, a radiation dose of 0.5 kGy could be detected in both fruits even after 2–3 weeks storage. In addition, some herbs and spices were also tested, but only for a few of them the ESR detection of the ‘cellulosic’ signal was improved by previous alcoholic extraction.

As an alternative to ESR measurements, other detection methods like DNA Comet Assay and thermoluminescence were also tested.     

Application of a rapid screening method to detect irradiated meat in Brazil

Based on the enormous potential for food irradiation in Brazil, and to ensure free consumer choice, there is a need to find a convenient and rapid method for detection of irradiated food. Since treatment with ionising radiation causes DNA fragmentation, the analysis of DNA damage might be promising. In this paper, the DNA Comet Assay was used to identify exotic meat (boar, jacaré and capybara), irradiated with ⁶⁰Co gamma rays. The applied radiation doses were 0, 1.5, 3.0 and 4.5 kGy. Analysis of the DNA migration enabled a rapid identification of the radiation treatment.     

Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma ⁶⁰Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8 kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0 kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.     

Detection of radiation treatment of beans using DNA comet assay

A simple technique of microgel electrophoresis of single cells (DNA Comet Assay) enabled a quick detection of radiation treatment of several kinds of leguminous beans (azuki, black, black eye, mung, pinto, red kidney and white beans). Each variety was exposed to radiation doses of 0.5, 1 and 5 kGy covering the permissible limits for insect disinfestation. The cells or nuclei from beans were extracted in cold PBS, embedded in agarose on microscope slides, lysed between 15 and 60 min in 2.5% SDS and electrophoresis was carried out at a voltage of 2 V/cm for 2–2.5 min. After silver staining, the slides were evaluated through an ordinary transmission microscope. In irradiated samples, fragmented DNA stretched towards the anode and the damaged cells appeared as a comet. The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. Hence, the DNA comet assay provides an inexpensive, rapid and relatively simple screening method for the detection of irradiated beans.     

ESR spectroscopy for detecting gamma-irradiated dried vegetables and estimating absorbed doses

In view of an increasing demand for food irradiation technology, the development of a reliable means of detection for the control of irradiated foods has become necessary. Various vegetable food materials (dried cabbage, carrot, chunggyungchae, garlic, onion, and green onion), which can be legally irradiated in Korea, were subjected to a detection study using ESR spectroscopy. Correlation coefficients (R²) between absorbed doses (2.5–15 kGy) and their corresponding ESR signals were identified from ESR signals. Pre-established threshold values were successfully applied to the detection of 54 coded unknown samples of dried clean vegetables (chunggyungchae, Brassica camestris var. chinensis), both non-irradiated and irradiated. The ESR signals of irradiated chunggyungchae decreased over a longer storage time, however, even after 6 months of ambient storage, these signals were still distinguishable from those of non-irradiated samples. The most successful estimates of absorbed dose (5 and 8 kGy) were obtained immediately after irradiation using a quadratic fit with average values of 4.85 and 8.65 kGy being calculated.     

Irradiated bivalve mollusks: Use of EPR spectroscopy for identification and dosimetry

High energy radiation treatment of foodstuff for microbial control and shelf-life extension is being used in many countries. However, for consumer protection and information, the European Union has adopted the Directives 1999/2/EC and 1999/3/EC to harmonize the rules concerning the treatment and trade of irradiated foods in EU countries. Among the validated methods to detect irradiated foods the EU directives also include Electron Paramagnetic Resonance (EPR/ESR) spectroscopy.We describe herein the use of EPR for identification of four species of bivalve mollusks, i.e. brown Venus shells (Callista chione), clams (Tapes semidecussatus), mussels (Mytilus galloprovincialis) and oysters (Ostrea edulis) irradiated with ⁶⁰Co γ-rays. EPR could definitely identify irradiated seashells due to the presence of long-lived free radicals, primarily CO₂⁻, CO₃³⁻, SO₂⁻ and SO₃⁻ radical anions. The presence of other organic free radicals, believed to originate from conchiolin, a scleroprotein present in the shells, was also ascertained. The use of one of these radicals as a marker for irradiation of brown Venus shells and clams can be envisaged. We also propose a dosimetric protocol for the reconstruction of the administered dose in irradiated oysters.     

Preliminary study on detection of irradiated foodstuffs from the Romanian market

In order to fulfil the European task for market survey in food irradiation the first Romanian laboratory for detection of irradiated foodstuffs was established at IRASM Irradiation Centre. In this preliminary study, a wide range of Romanian food samples (spices, vegetables and meat) gamma irradiated at IRASM have been studied using different detection methods: (1) DNA comet assay, (2) thermoluminescence (TL) and (3) electron spin resonance (ESR) for foodstuffs containing bone or cellulose. The results suggest that there is no general available detection method and there is no perfect detection method. In conclusion, in order to carry out a correct identification of radiation treatment of a food sample it is recommended to use at least two standardised detection methods.     

Identification of γ-ray irradiated medicinal herbs using pulsed photostimulated luminescence, thermoluminescence, and electron spin resonance spectroscopy

Dried herbal samples consisting of root, rhizome, cortex, fruit, peel, flower, spike, ramulus, folium, and whole plant of 20 different medicinal herbs were investigated using pulsed photostimulated luminescence (PPSL), thermoluminescence (TL), and electron spin resonance spectroscopy (ESR) to identify γ-ray irradiation treatment. Samples were irradiated at 0–50 kGy using a 60Co irradiator. PPSL measurement was applied as a rapid screening method. Control samples of 19 different herbs had photon counts less than the lower threshold value (700 counts 60 s−1). The photon counts of non-irradiated clematidis radix and irradiated evodia and gardenia fruits were between the lower and upper threshold values (700–5,000 counts 60 s−1). TL ratios, i.e., integrated areas of the first glow (TL1)/the second glow (TL2), were found to be less than 0.1 in all non-irradiated samples and higher than 0.1 in irradiated ones providing definite proof of radiation treatment. ESR spectroscopy was applied as an alternative rapid method. In most of the irradiated samples, mainly radiation-induced cellulosic, sugar, and relatively complicated carbohydrate radical ESR signals were detected. No radiation-specific ESR signal, except one intense singlet, was observed for irradiated scrophularia and scutellaria root and artemisiae argyi folium. Figure PPSL can be used as a rapid simple preliminary screening method and a combination of ESR and TL tests for a definite proof of gamma irradiation treatment of medicinal herbs.     

Identification of irradiated foods by monitoring radiolytically produced hydrocarbons

Many countries allow the treatment of foods with low doses of ionizing radiation to reduce microbial and insect infestations, inhibit maturation, and extend shelf life. Therefore, a reliable method is needed to identify irradiated foods and to determine their compliance with respect to allowable absorbed radiation dose. Several approaches for the identification of irradiated foods have been developed such as measurement of radiolytic products, chemiluminescence, and thermoluminescence, and the use of electron spin resonance spectroscopy to measure free radicals trapped in bone. A method for the determination of radiolytically produced hydrocarbons was developed in our laboratory to evaluate the utility of monitoring these compounds as indicators of food irradiation. The method involves the extraction of the radiolytic hydrocarbons from foods and their quantitation by gas chromatography. Concentrations of the radiolytically produced hydrocarbons increased linearly with radiation doses ranging from 0 to 6 kGy. The limit of detection appears to be approximately 1 kGy. The method was found to be useful for the identification of gamma-irradiated foods such as shrimp, frog legs, pork, beef, and poultry. Results of the method evaluation studies of these food matrices as well as factors affecting hydrocarbon production and determination will be presented.     

Detection of irradiated food: DNA fragmentation in grapefruits

Employing the simple microgel electrophoresis of single cells - ‘comet assay’ - on grapefruit seeds enabled a rapid identification of irradiated fruits. Fruits were exposed to radiation doses of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 kGy covering the range of potential commercial irradiation for insect disinfestation and quarantine purposes. Seeds were isolated, crushed, and the cells embedded in an agarose layer. After lysis of the cells, they were subjected to microgel electrophoresis for 2.5 minutes, and then stained. Fruits irradiated with 0.2 kGy and higher doses showed typical DNA fragmentation, the DNA fragments stretching or migrating out of the cells forming a tail towards the anode, giving the damaged cells an appearance of a comet. With increasing dose a longer extension of the DNA from the nucleus towards the anode is observed. Undamaged cells will appear as intact nuclei without tails. The DNA comet assay is thus a rapid and inexpensive screening technique to detect irradiated grapefruits. Suspected samples may subsequently be analysed by officially validated methods for detection of irradiated foods.     

Food irradiation—US regulatory considerations

The use of ionizing radiation in food processing has received increased interest as a means of reducing the level of foodborne pathogens. This overview discusses the regulatory issues connected with the use of this technology in the United States. Several recent changes in the FDA's review process are discussed. These include the current policy that utilizes an expedited review process for petitions seeking approval of additives and technologies intended to reduce pathogen levels in food, and the recent USDA rule that eliminates the need for a separate rulemaking process by USDA for irradiation of meat and poultry. Recently promulgated rules and pending petitions before the FDA associated with the use of ionizing radiation for the treatment of foods are also discussed along with the current FDA labeling requirements for irradiated foods and the 1999 advanced notice of proposed rule on labeling. Another issue that is presented is the current status of the approval of packaging materials intended for food contact during irradiation treatment of foods.     

Analysis of irradiated food

Foods, e.g. chicken, shrimps, frog legs, spices, different dried vegetables, potatoes and fruits are legally irradiated in many countries and are probably also exported into countries, which do not permit irradiation of any food. Therefore all countries need analytical methods to determine whether food has been irradiated or not. Up to now, two physical (ESR-spectroscopy and thermoluminescence) and two chemical methods (o-tyrosine and volatile compounds) are available for routine analysis. Several results of the application of these four mentioned methods on different foods are presented and a short outlook on other methods (chemiluminescence, DNA-changes, biological assays, viscometric method and photostimulated luminescence) will be given.     

Genotoxicity of 2-alkylcyclobutanones, markers for an irradiation treatment in fat-containing food—Part I: cyto- and genotoxic potential of 2-tetradecylcyclobutanone

Previous experiments had indicated a slight genotoxic potential both in rat and in human colon cells of a sample of 2-dodecylcyclobutanone, a compound formed by irradiation of food containing palmitic acid in its triglycerides. Up to date, there is no evidence that 2-alkylcyclobutanones occur in non-irradiated foodstuffs, consequently it is prudent to test several members of the class of 2-alkylcyclobutanones which are produced by treatment of fat-containing food with ionising radiation. In this work, 2-tetradecylcyclobutanone (derived from stearic acid) has been tested for its cytotoxic and genotoxic potential. Human colon tumor cell lines, i.e. HT 29 and HT 29 clone 19A, were employed as models for in vitro experiments for cytotoxicity and genotoxicity tests. Cytotoxicity was measured by tetrazolium salt reduction assays (MTT and WST-1) and genotoxicity by determining DNA damage using the Comet Assay. Neither cytotoxic nor genotoxic effects were induced by 2-TCB in HT 29 or HT 29 cl 19A cells at an incubation time of 30 min at 37°C, not even at the highest concentration (400 μM) tested. After prolonged incubation times (1–2 days) at higher concentrations (>50 μM) cytotoxicity did, however, appear. Studies on other 2-alkylcyclobutanones are in progress.     

电子束辐射固化环氧树脂的反应过程分析

对双酚A型环氧树脂的电子束辐射固化反应过程进行了分析.考察了引发剂、稀释剂对树脂体系辐射反应的影响,以环氧丙烷作为模型化合物,研究了环氧丙烷-碘盐体系的电子束辐射反应机理,证实了在电子束辐射下,碘盐分解产生质子酸,引发环氧树脂阳离子开环聚合的反应过程.观测环氧树脂辐射固化区域发现,电子束穿过样品时发生强烈的散射,在辐射方向以及周围一定区域内引发固化反应,固化反应从活性中心开始向体系内部层层扩展,整个固化区域由很多的层状结构组成.     

UV-mediated DNA strand breaks in corneal epithelial cells assessed using the comet assay procedure

Ultraviolet (UV)-mediated DNA damage in various tissues has been well documented. However, research on the damaging effect of UV irradiation on the DNA of corneal epithelium is scarce, even though this is of interest because the cornea is directly exposed to damaging solar (UV) radiation. In this study, we developed a corneal epithelium Comet assay model to assess the background DNA damage (as strand breaks) in cells retrieved from different layers of the porcine corneal epithelium, and to investigate the effect of UV irradiation on DNA damage in corneal epithelial cells. Results show that the background DNA strand breaks decreased significantly (P < 0.001) toward deeper layers of the epithelium. Exposure to the same intensity (0.216 J/cm2) of UVA, UVB and UVC caused a significant (P < 0.001) increase in DNA strand breaks of deeper-layer cells: mean +/- SD %DNA scores (10 gels per treatment, with 100 irradiated cells scored per gel) were 10.2% +/- 1.4% for UVA, 27.4% +/- 4.6% for UVB, and 14.7% +/- 1.8% for UVC compared with 4.2% +/- 0.5% for controls (ambient room light). This study has shown for the first time that the Comet assay for DNA strand breaks can be used successfully with corneal epithelial cells. This report will support future studies investigating environmental influences on corneal health and the assessment of possible protective strategies, and in applying DNA lesion-specific versions of the Comet assay in this corneal epithelial cell model.     

Volatile compounds and odor preferences of ground beef added with garlic and red wine,and irradiated with charcoal pack

Irradiation is the most efficient non-thermal technology for improving hygienic quality and extending the shelf-life of food products. One of the adverse effects of food irradiation, however, is off-flavor production, which significantly affects the sensory preferences for certain foods. In this study, garlic (5%, w/w) and red wine (1:1, w/w) were added to ground beef to increase the radiation sensitivity of pathogens and improve meat odor/flavor. Samples were irradiated at 0 or 5 kGy in the presence of charcoal pack. SPME-GC–MS analysis was performed to measure the changes in the volatile compounds and sensory characteristics of the samples. The amount of total volatile compounds produced from ground beef was greater when the sample was irradiated. When garlic and red wine were added to the ground beef, the amount of volatile compounds significantly increased, and the amount of volatile compounds increased even further after irradiation. However, when the samples were irradiated with charcoal pack, the amount of volatile compounds decreased significantly. Sensory evaluation indicated that charcoal pack significantly increased the odor preferences for both irradiated and non-irradiated ground beef added with garlic. These results indicated that addition of charcoal pack to ground beef could reduce off-odor problems induced by irradiation, and this effect was consistent even when certain additives such as garlic and red wine were added.     

Application of gamma irradiation for inhibition of food allergy

This study was carried out to evaluate the application of food irradiation technology as a method for reducing food allergy. Milk β-lactoglobulin, chicken egg albumin, and shrimp tropomyosin were used as model food allergens for experiments on allergenic and molecular properties by gamma irradiation. The amount of intact allergens in an irradiated solution was reduced by gamma irradiation depending upon the dose. These results showed that epitopes on the allergens were structurally altered by radiation treatment and that the irradiation technology can be applied to reduce allergenicity of allergic foods.     

干果类食品的样品前处理与分析检测方法研究进展

坚果、果脯等干果类食品含有丰富的营养成分,深受国内外广大消费者的喜爱.但这些食品在果实生产、加工、储运时会使用农药或产生霉变等,造成干果中农药、重金属、霉菌毒素或添加剂等有害成分残留,甚至超过国家限量要求,带来严重的食品安全问题.因此,加强干果类食品的质量监督具有重要的经济和社会意义.但干果类食品基质复杂,有害物质种类...     

Isotopes and radiation technology—Indian scene

India's isotope programme is today largely self-sustaining both in terms of availability of isotope products and the range of their applications in medicine, industry, hydrology, agriculture and research. Nuclear medicine is practised by over 200 medical institutions whereas 300 organisations offer radioimmunoassay service. Tracer technology, nucleonic gauging and isotope raiography are fairly well accepted by the Indian industry for troubleshooting, NDT and process control. There are three large radiation plants for sterilisation of medical products. Radiation chemical processing with both gamma and EB shows good promise. In agriculture, sixteen mutants of various crops have been produced using gamma-radiation and distributed for commerical cultivation. A strong programme of research on radiation preservation of food has finally resulted in the clearance of some irradiated foods by the Government of India.