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Effect of Genotoxic 2-Nitrofluorene and Its Metabolites on DNA In Vivo and Possibilities of Investigation of this Effect Using Electrochemical DNA Biosensors In Vitro
Stávková, Klára ; Vyskočil, Vlastimil (advisor) ; Fišer, Radovan (referee)
Presented Bachelor Thesis is targeted on the topic of hazardous 2-nitrofluorene (NF), which is known as one of the markers of the presence of nitrated polycyclic aromatic hydrocarbons. This Thesis summarizes the current knowledge about the formation of this xenobiotic, its occurrence and transport in living and working environment, and about its negative effects on living organisms, especially on their DNA. These findings obtained upon the in vivo investigations are compared in this Thesis with information obtained using electrochemical DNA biosensors, which represent very promising in vitro alternative to the study of processes proceeding in living organisms upon the interaction of their DNA with the xenobiotic. NF is mainly released to the atmosphere through the exhaust gases of diesel and gasoline engines. It is adsorbed onto solid particulate matters, through which it is spread over long distances and can enter the air passages of animals. Through an atmospheric fall-out, NF contaminates waters and soils, where it is transported together with other nutrients and food to plants and animals; by consummation of such organisms, it can finally enter the body of higher animals and the human. It has been shown that NF is a chemically stable substance. On the other hand, it is metabolized intensively...
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The Use of an Electrochemical DNA Biosensor in Detection of DNA Damage Caused by Genotoxic 2-Nitrofluorene
Stávková, Klára ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
2-Nitrofluorene is a model representative of nitrated polycyclic aromatic hydrocarbons (NPAH) which belongs to a group of mutagens and carcinogens. Interaction of DNA with genotoxic 2-nitrofluorene was monitored by an electrochemical DNA biosensor made of a glassy carbon electrode (GCE) and low molecular weight DNA from salmon sperm. Techniques used are electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square-wave voltammetry (SWV). Using the EIS technique, no damage to DNA, which would cause strand breaks in DNA, was observed, whereas using the CV technique, the intercalation of NF to the structure of DNA was observed, leading to the formation of a NF-DNA complex. The intercalation results in a reduction of electroactive sites which can be oxidized. It was verified using the SWV technique, by which a decrease of the peak heights of adenosine and guanosine was observed. Because of the dangerous effect of NF on the structure of DNA, an electroanalytical method for its determination was developed. An applicability of the method was successfully tested on a model sample of sand. For the development of the technique, differential pulse voltammetry (DPV) was used in a mixture of the Britton-Robinson buffer of pH 7.0 and ethanol in a ratio of 7:3 (v/v) and with a periodic...
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Voltammetric Determination of Genotoxic 2-Nitrofluorene at Screen-Printed Carbon Electrodes
Jelínková, Lenka ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
of the Bachelor Thesis Voltammetric behavior of genotoxic 2-nitrofluorene (NF) has been investigated using direct current voltammetry (DCV) and differential pulse voltammetry (DPV) at a screen- printed carbon electrode (SPCE). The optimum conditions have been found for its determination in a concentration range from 1×10−6 to 1×10−5 mol l−1 for DCV technique and from 1×10−7 to 1×10−5 mol l−1 for DPV technique. An optimum medium for DCV at the SPCE was a 0.1 mol l−1 acetate buffer of pH 4.6, an optimum time of stirring of the sample prior to the analysis was 120 s (the attained limit of quantification (LQ) was 8.5×10−6 mol l−1 of NF). For DPV at the SPCE, the optimum medium was an acidic component part of the Britton - Robinson buffer of pH 2.0; the attained LQs were 6.2×10−7 mol l−1 of NF for the concentration range from 1×10−6 to 1×10−5 mol l−1 (at the stirring time of the sample of 120 s) and 2.0×10−7 mol l−1 of NF for the concentration range from 1×10−7 to 1×10−6 mol l−1 (at the stirring time of the sample of 300 s).
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Effect of Genotoxic 2-Nitrofluorene and Its Metabolites on DNA In Vivo and Possibilities of Investigation of this Effect Using Electrochemical DNA Biosensors In Vitro
Stávková, Klára ; Vyskočil, Vlastimil (advisor) ; Fišer, Radovan (referee)
Presented Bachelor Thesis is targeted on the topic of hazardous 2-nitrofluorene (NF), which is known as one of the markers of the presence of nitrated polycyclic aromatic hydrocarbons. This Thesis summarizes the current knowledge about the formation of this xenobiotic, its occurrence and transport in living and working environment, and about its negative effects on living organisms, especially on their DNA. These findings obtained upon the in vivo investigations are compared in this Thesis with information obtained using electrochemical DNA biosensors, which represent very promising in vitro alternative to the study of processes proceeding in living organisms upon the interaction of their DNA with the xenobiotic. NF is mainly released to the atmosphere through the exhaust gases of diesel and gasoline engines. It is adsorbed onto solid particulate matters, through which it is spread over long distances and can enter the air passages of animals. Through an atmospheric fall-out, NF contaminates waters and soils, where it is transported together with other nutrients and food to plants and animals; by consummation of such organisms, it can finally enter the body of higher animals and the human. It has been shown that NF is a chemically stable substance. On the other hand, it is metabolized intensively...
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Voltammetric Study of the Interaction of Genotoxic 2-Nitrofluorene with DNA at a Hanging Mercury Drop Electrode
Krejčová, Zuzana ; Vyskočil, Vlastimil (advisor) ; Nesměrák, Karel (referee)
In this Diploma Thesis, an interaction of genotoxic environmental pollutant 2-nitrofluorene with a double-stranded calf thymus DNA has been studied using a hanging mercury drop electrode (HMDE) as an electrochemical sensor. Two types of DNA damage were investigated and electrochemically detected (using cyclic voltammetry and differential pulse voltammetry): (i) The DNA damage caused by the direct interaction with 2-nitrofluorene and (ii) the DNA damage caused by short-lived radicals generated by the electrochemical reduction of the nitro group in 2-nitrofluorene. For the study of direct interaction, HMDE was modified by DNA and the interaction of DNA with 2-nitrofluorene was studied, after their incubation, right at the HMDE surface (adsorptive transfer stripping technique) or the DNA was preincubated with 2-nitrofluorene and, subsequently, the interaction was studied voltammetrically (DNA titration technique). Using both detection techniques, the formation of DNA - 2-nitrofluorene complex was observed and the mutual interaction was interpreted as an intercalation between the DNA base pairs, although such interaction was not clearly confirmed by UV-visible absorption spectroscopy. An electrostatic binding of 2-nitrofluorene on DNA sugar-phosphate backbone was partially formed at low concentrations of...
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