Commet assay lab ****************************************************************************************** * ****************************************************************************************** Year of acquisition 2018-2020 Financing OP VVV CORE FACILITIES [ URL "LFHKEN-192.html "] CZ.02.1.01/0.0/0.0/16_017/0002515 Responsible Person prof. Ing. Zdeněk Fiala, CSc. Every human activity can potentially pose risks to humans and the environment under certai When these risks increase and accumulate, it is necessary to implement measures to reduce a socially acceptable standard. However, it is first necessary to characterize the potenti the identification and characterization of hazards and exposure assessment. As part of ris it is also necessary to assess the ability of the risk factor to cause damage to the genet cells, i.e., to act genotoxically. The term genotoxicity refers to substances, factors, or processes that damage the structur alter the informational content of the genome and the transfer of information, affect DNA inhibit its replication. The process of genotoxicity largely overlaps with the process of which is characterized by sudden, disordered changes in genetic information transmitted to generations of cells. Substances (factors) labeled as mutagens induce locally confined les DNA structure, which, if not removed by cellular repair systems, can be fixed into stable during DNA replication. Mutations can occur either within individual genes (gene mutations chromosome damage (chromosomal mutations). Chromosomal changes can affect either the struc chromosomes. For these reasons, mutagenicity tests can be considered genotoxicity tests. Testing carcinogenic effects on animals or analyzing carcinogenic potential through epidem studies requires high costs and usually takes years. Short-term genotoxicity tests allow f screening of several hundred thousand existing and newly produced (and used) substances, i those that signal potential carcinogenicity through their genotoxic effects. However, it i emphasize that, in addition to genotoxic mechanisms of carcinogenesis (where the first ste is induced by DNA damage through mutational changes), there are also non-genotoxic (epigen that cannot be identified by short-term genotoxicity tests. The advantage of short-term genotoxicity tests is their potential for quickly detecting th genotoxic effects and, depending on the type of test used, possibly also the mechanism of Based on the relationship between genotoxic and carcinogenic mechanisms of action, identif effect can help pinpoint substances (factors) that need attention as potential carcinogens further investigated in in vivo tests and epidemiological studies. One of the significant short-term genotoxicity tests is the "Comet assay" (CA), which is u breaks in cell nuclei. This versatile, relatively simple, and sensitive method can, depend variant, detect single and double-strand DNA breaks, alkali-labile sites (apurinic/apyrimi incomplete DNA repair, as well as oxidized purine or pyrimidine bases, cross-links, and ap DNA breaks detectable by CA can be repaired in the cell, be lethal to the cell, or be fixe resulting in a permanent mutation. DNA damage can be detected by CA in suspensions of yeas plant, and animal cells (both invertebrates and vertebrates), in both dividing and non-div Photo 1: (S) Setup for comet assay analysis (fluorescence microscope and PC with analysis example of measurements. Photo 2: Biohazard box with laminar flow featuring two workstations, allowing two students simultaneously. During the CA, cells are placed in an agarose gel. After lysing the cell and nuclear membr solution, DNA fragments migrate out of the nucleus, more precisely from the nucleoid ("hea "tail" due to electrophoresis, creating the characteristic comet image after visualization dye, which gives the method its name. The more fragmented the DNA, the larger and longer t fragmented DNA, due to its negative charge, moves towards the anode. Photo 3: Example of "comets" from a comet assay test. The CA assesses the extent of DNA damage caused by genotoxic substances present in the env occupational settings and the ability to repair damaged DNA. The method is used in in vitr epidemiological studies. Usually, a small number of cells (<50,000, some authors even ment cells) is sufficient for analysis. Its major advantage is the ability to apply/modify it f cell types and thus tissues that can be divided into individual cells. This capability all DNA damage in tissues that are the first to come into contact with carcinogens. Additional simplicity, speed, cost-effectiveness, and sensitivity in detecting even minor DNA damage As part of the CORE FACILITIES project, a laboratory for testing the genotoxic potential o substances (factors) under in vitro, in vivo, or epidemiological study conditions has been at the Faculty of Medicine in Hradec Králové. The laboratory features a biohazard box with an incubator with controlled atmosphere, a refrigerated centrifuge, a thermal block, and a microscope with a CCD camera and software that enables the measurement of selected paramet analysis. Authors: prof. Ing. Zdeněk Fiala, CSc., MUDr. Andrea Málková, Ph.D.