1. Do cytotoxicity and cell death cause false positive results in the in vitro comet assay?Amaya Azqueta, Helga Stopper, Bojana Žegura, Maria Dusinska, Peter Møller, 2022, izvirni znanstveni članek Povzetek: The comet assay is used to measure DNA damage induced by chemical and physical agents. High concentrations of test agents may cause cytotoxicity or cell death, which may give rise to false positive results in the comet assay. Systematic studies on genotoxins and cytotoxins (i.e. non-genotoxic poisons) have attempted to establish a threshold of cytotoxicity or cell death by which DNA damage results measured by the comet assay could be regarded as a false positive result. Thresholds of cytotoxicity/cell death range from 20% to 50% in various publications. Curiously, a survey of the latest literature on comet assay results from cell culture studies suggests that one-third of publications did not assess cytotoxicity or cell death. We recommend that it should be mandatory to include results from at least one type of assay on cytotoxicity, cell death or cell proliferation in publications on comet assay results. A combination of cytotoxicity (or cell death) and proliferation (or colony forming efficiency assay) is preferable in actively proliferating cells because it covers more mechanisms of action. Applying a general threshold of cytotoxicity/cell death to all types of agents may not be applicable; however, 25% compared to the concurrent negative control seems to be a good starting value to avoid false positive comet assay results. Further research is needed to establish a threshold value to distinguish between true and potentially false positive genotoxic effects detected by the comet assay.
Ključne besede: comet assay, cytotoxicity, genotoxicity, DNA damage, cell death
Objavljeno v DiRROS: 17.07.2024; Ogledov: 6; Prenosov: 3
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2. Pulsed low dose-rate irradiation response in isogenic HNSCC cell lines with different radiosensitivityVesna Todorović, Ajda Prevc, Martina Nikšić Žakelj, Monika Savarin, Simon Buček, Blaž Grošelj, Primož Strojan, Maja Čemažar, Gregor Serša, 2020, izvirni znanstveni članek Povzetek: . Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods. Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results. Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions. We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRSpositive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.
Ključne besede: DNA damage, isogenic cell lines, low dose irradiation, pulsed low dose-rate irradiation, radiosensitivity
Objavljeno v DiRROS: 12.07.2024; Ogledov: 40; Prenosov: 14
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3. Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα : dynophore-derived discoveryBarbara Herlah, Matej Janežič, Iza Ogris, Simona Golič Grdadolnik, Katja Kološa, Sonja Žabkar, Bojana Žegura, Andrej Perdih, 2024, izvirni znanstveni članek Povzetek: The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.
Ključne besede: topoisomerase II, catalytic inhibitors, chemotherapy, DNA damage, cancer
Objavljeno v DiRROS: 03.06.2024; Ogledov: 266; Prenosov: 124
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4. Subchronic exposure of rats to sublethal dose of microcystin-YR induces DNA damage in multiple organsMetka Filipič, Bojana Žegura, Bojan Sedmak, Irena Horvat-Žnidaršič, Aleksandra Milutinović Živin, Dušan Šuput, 2007, izvirni znanstveni članek Povzetek: Background. Microcystins (MCs) are cyclic heptapeptides that are considered tobe liver specific toxins. They are potent tumour promoters and recent studies indicate that they are also genotoxic. In this study we measured DNA damage in lymphocytes, liver, kidney (cortex and medulla), lung, spleen and brain cells of male Fisher F344 rats that were exposed to sublethal dose (every second day 10 Ugžkg b.w.č i.p) of microeysrin-YR (MCYR) for one month. Methods. At the end of exposure the animals were sacrificed, the lymphocytes were isolated from blood taken from jugular vein, liver cells were obtained byperfusion with collagenase A and the cells from other organs were isolated by incubating small tissue pieces with eollagenase A. The DNA damage in isolated cells was measured with the single cells gel electrophoresis (SCGE) also called the comet assay. Results. A significant increase of the % tail DNAin MCYR-exposed animals compared to the nonexposed control ones was observed in brain (2.5 fold), liver (2.1 fold), kidney medulla (1.9 fold), kidney cortex (1.8 fold) and lung (1.7 fold) cells, while the DNA from lymphocytes and spleen cells was not affected. Conclusion. This study demonstrated that subehronic exposure to sublethal doses of MCs can induce systemicgenotoxicity in mammals, and it affects not only the liver but also other vital organs.
Ključne besede: DNA damage, comet assay, cyanobacteria, bacterial toxins, rats, inbred F344
Objavljeno v DiRROS: 20.02.2024; Ogledov: 295; Prenosov: 71
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