Introduction of the Comet assay for the evaluation of oxidative stress, antioxidant effects and environmental pollutants effects on animal cells | Vpeljava metode Comet za vrednotenje oksidativnega stresa, antioksidantnih učinkov in učinkov okoliških polutantov na živalske celice

Inglés. DNA damage within single mammalian cells can be one of the earliest signs of a whole range of health problems, including disease, diet and exposure to occupational or environmental toxins. The Comet assay is a relatively simple and inexpensive method for measuring this damage. It works by determining the number of breaks in the strands of DNA within the cell. Cells are embedded in agarose gel on a microscopic slide and washed to remove the cell membranes, soluble cell contents and histones from the nucleus. An electric field is then activated across and the loops of damaged DNA are pulled towards the anode. Epifluorescence microscopy of stained gels shows the image which gives the techique its name: a clump of undamaged DNA (the head) with the loops pulled away, forming a tail of a comet. We report herein the checking of the Comet assay for the evaluation of oxidative stress on chicken, mouse and pig blood cells, pig sperm cells and chicken hepatocytes. We intend to use the Comet assay as a quantitative method in some nutritional experiments with domestic animals including oxidative food components and antioxidants and for the evaluation of some environmental pollutants effects on animal and human cells.

Esloveno. Poškodbe DNK v posamičnih sesalčjih celicah so lahko zgodnji pokazatelji številnih težav pri zdravju, prehrani, izpostavljenosti toksičnim snovem na delovnem mestu in v okolju nasploh. Poskus Comet je relativno enostavna in poceni metoda za merjenje tovrstnih poškodb. Temelji na določanju stopnje prekinitev v verigah celične DNK. Celice vklopimo v agarozni gel na mikroskopskih objektnikih in jih speremo, da odstranimo celične membrane, topno celično vsebino in jedrne histone. Pri elektroforezi fragmenti poškodovane DNK potujejo proti anodi. Pri epifluorescentni mikroskopiji obarvanih preparatov dobimo značilno sliko, ki je tehniki dala ime – skupek nepoškodovane DNK (glava) in rep kometa, ki ga tvorijo fragmenti poškodovane DNK. V članku poročamo o preverjanju metode Comet za vrednotenje oksidativnega stresa na prašičjih, mišjih in kokošjih krvnih celicah, prašičjih spermijih in piščančjih hepatocitah. Metodo Comet nameravamo uporabiti za kvantitativno vrednotenje v prehranskih poskusih domačih živali, kjer bomo preverjali oksidativne učinke nekaterih prehranskih snovi in vplive antioksidantov, in za preučevanje vpliva nekaterih onesnaževalcev okolja na živalske in človeške celice.