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The role of mixed function oxidases system with cytochrome P450 in metabolism of drugs and carcinogens
Mrízová, Iveta ; Stiborová, Marie (advisor) ; Levová, Kateřina (referee) ; Hansíková, Hana (referee)
6 Abstract Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activity. This antitumor agent binds to DNA and forms covalent DNA adducts. Enzymes, which are involved in its enzymatic activation, are cytochromes P450 (CYP) and peroxidases. To elucidate the effect of ellipticine on the expression and enzymatic activity of the individual components of the microsomal mixed function oxidase system in different tissues, we used rat model. Simultaneously, the effect of ellipticine and its cytotoxicity on different tumor cell lines was also investigated. Another part of the presented work was targeted on preparation of anti-peptide antibody against orphan cytochrome P450 2S1, which is highly expressed in many human tumours of the epithelial origin, for its detection in these tissues. For better understanding how CYP2S1 can contribute to the metabolism of xenobiotics, the protein was prepared by heterologous expression in E. coli. Furher, its role in metabolism of an antitumor drug ellipticine, a carcinogenic environmental pollutant benzo[a]pyrene (BaP) and its derivate BaP-7,8-dihydrodiol was examined. Utilizing a mouse model, the impact of pulmonary inflammation on the metabolism of an environmental carcinogen was...
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The use of BEAS-2B cell line for micronucleus assay in genetic toxicology
Červená, Tereza ; Rössner, Pavel (advisor) ; Rubeš, Jiří (referee)
This thesis deals with the application of BEAS-2B cell line for micronucleus assay in genetic toxicology. It is divided into two main parts: a) theoretical introduction to the analysis and search for suitable models for testing the impact of air pollution and manufactured nanoparticles, b) practical part that describes the results of micronuclei induction by polycyclic aromatic hydrocarbons (PAHs), extractable organic matter (EOM) from diesel exhaust particles obtained from emissions of three types of fuel and engineered nanoparticles. BEAS-2B cell line is a nonmalignant human model of lung epithelium which seems to be suitable for micronucleus assay. This assay is commonly used for determining the genotoxicity of various substances to wide variety of cell cultures and also in human studies. In this thesis, the following substances were tested: benzo[a]pyrene, 3-nitrobenzanthrone and 1-nitropyrene as carcinogenic PAHs commonly found in polluted air; EOMs from exhaust particles of 100 % diesel fuel, a blend of diesel fuel and 30 % of biodiesel, 100 % biodiesel and two types of engineered nanoparticles (TiO2 and Ag). The cells were treated with the compounds for 28, 48 and 72 hours. The results confirm the suitability of BEAS-2B cell line as a model for testing the genotoxicity of substances under...
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Mechanism of enzymatic activation of carcinogens and drugs by the system of cytochrome P450
Indra, Radek ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Koblihová, Jitka (referee)
13 Abstract An environmental pollutant and a human carcinogen benzo[a]pyrene (BaP) is after its activation with cytochrome P450 (CYP) able to covalently bind to DNA. In the thesis, one of the target was to investigate an influence of individual components of mixed function monooxygenase (MFO) system on metabolism of benzo[a]pyrene and generation of adducts of activated BaP with DNA. The study was particularly focused to increase our knowledge on the effect of cyt b5 on metabolism of BaP by cytochrome P450 1A1 (CYP1A1) and its potential to serve as a donor of electrons during the reaction cycle of this cytochrome P450. The effect of cyt b5 on generation of BaP metabolites and adducts of BaP with DNA was investigated. In addition the effect of two different expression systems for cytochrome P450 1A1 (prokaryotic and eukaryotic) was also studied. The influence of cyt b5 on oxidation another xenobiotic compound, a plant alkaloid ellipticine that exhibit antitumor activities, was also investigated. Its pharmacological efficiency, as well as side effects depends on its metabolic activation by cytochrome P450. CYP3A4 is very important for ellipticine activation and therefore this enzyme was used in our experiments. Furthermore, a suitability of rat as a model organism mimicking the metabolic fate of BaP...
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Role of benzo[a]pyrene in cancer development
Vaňátková, Petra ; Moserová, Michaela (advisor) ; Kubíčková, Božena (referee)
4 ABSTRACT Cancer is nowadays one of the most serious diseases. Tumor development is a multistage process in which the effect of internal and external factors lead to failure of regulatory and defense mechanisms of the organism and to the accumulation of mutations which are generated by these organisms. Chemical carcinogens and also biological and physical factors can be regarded as the main external factors. Polycyclic aromatic hydrocarbons are large group of chemical carcinogens. One of them, benzo[a]pyren is the most studied polycyclic aromatic hydrocarbon. Carcinogenic, mutagenic and teratogenic effects of benzo[a]pyrene had been shown on laboratory animals. Benzo[a]pyrene is considered as the main carcinogen in tobacco smoke and is connected with lung cancer development among smokers. Benzo[a]pyrene is metabolized in activation or detoxication pathways by enzymes of mixed function monooxygenase systeme of cytochromes P450. The most important enzymes involved in the activation of these compounds are CYP1A1 and CYP1B1 with cooperation of epoxide hydrolase. The reactive species generated in its activation pathway are able to form covalent adducts with DNA. The most important carcinogenic product of benzo[a]pyrene is benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, which can caused irreversible ganges in...
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Oxidation of benzo(a)pyrene by cytochrome P450 1A1 expressed in prokaryotic and eukaryotic systems
Kroftová, Natálie ; Stiborová, Marie (advisor) ; Kubíčková, Božena (referee)
Benzo[a]pyrene (BaP) is a human carcinogen, which is metabolized by a variety of enzyms such as cytochrome P450 (CYP) and epoxide hydrolase. The aim of this work was to study BaP metabolism in vitro by the hepatic microsomal system of rats treated with CYP inducers and by human cytochrome P450 1A1 (CYP1A1) expressed in eukaryotic and prokaryotic systems. An eukaryotic expression system consisted of microsomes isolated from insect cells, whereas a prokaryotic expression system was formed by the membrane fragments of E. coli. In the case of recombinant human CYP1A1, we investigated the influence of cytochrome b5, NADPH:cytochrome P450 reductase (CPR) and epoxide hydrolase in BaP oxidation. Isolation and purification of rabbit hepatic CPR was another aim of this work. BaP metabolites were separated by HPLC. The results found in this work demostrate the fact that hepatic microsomal systems of rats treated with an inducer of CYP1A (Sudan I), an inducer of CYP2B (phenobarbital) and an inducer of CYP3A (PCN) exhibit higher efficiency of BaP oxidation than microsomes of control rats. BaP is oxidized by human CYP1A1 expressed in the eukaryotic system to six metabolites (BaP-9,10-dihydrodiol, BaP metabolite with unknown structure, BaP-7,8-dihydrodiol, BaP-1,6-dion, BaP-3,6-dion, BaP-3-ol), whereas by human...
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Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
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Kinetics of benzo[a]pyrene destruction and identification of its products
Ryšavý, Jan ; Kolek, Emil (referee) ; Šimko, Peter (advisor)
This diploma thesis is focused on the study of conditions of benzo[a]pyrene, one of the major contaminant of foods, photodegradation under different conditions (solvents with different polarity, light sources, presence of antioxidants). In another part of the thesis, the degradation process of benzo[a]pyrene at various concentrations was studied, in order to characterise the kinetic aspects of photoinduced degradation. The attempt to identify the products of benzo[a]pyrene photodegradation was performed involving methods of gas chromatography and high performance liquid chromatography coupled with mass detectors, as well.
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