|
|
Chicken antibodies as a tool of cytochrome P450 immunodetection
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Cytochrome P450 is an important enzyme which catalyzes a large amount of reactions of endogenous and exogenous metabolism. There are compounds which increase expression of this enzyme and it leads up to the carcinogen activation and cancer formation. It includes much propagated chemopreventive compounds which are consumed abundantly in dietary supplements. It is therefore important to have the methods for determination of cytochrome P450 induction. One of these methods is the cytochrome P450 immunodetection by Western blotting. The detection of proteins on the membrane is done by the specific antibodies mostly gained from mammals. In this bachelor thesis there is the evidence that the antibodies from hen egg serve as well as the mammalian antibodies. The cytochromes P450 1A1 and 1A2 was detected by these egg antibodies after rat exposure to the model chemopreventive compounds β-naphthoflavone.
|
|
|
Effects of chemopreventive compounds on cytochrome P450s
Křížková, Jitka ; Hodek, Petr (advisor) ; Helia, Otto (referee) ; Václavíková, Radka (referee)
CHARLES UNIVERSITY IN PRAGUE Faculty of Science Department of Biochemistry Effects of chemopreventive compounds on cytochrome P450s Summary of Ph.D. Thesis RNDr. Jitka Křížková Supervisor: prof. RNDr. Petr Hodek, CSc. Prague 2010 Introduction 1 Introduction According to the World Health Organization statistics, cancer is one of the leading causes of death in the human population worldwide for more than 50 years. Moreover, colorectal and gastrointestinal tract cancers are one of the main types of cancer leading to overall cancer mortality. Prevention consisting in a healthy lifestyle and a natural diet is suggested to be one of the main approaches to reduce cancer risk. In recent years, the consumption and use of dietary supplements containing concentrated chemopreventive phytochemicals increased dramatically. Flavonoids, as the most popular representatives of chemopreventive compounds, present in foods (fruits, vegetables, herbs, beverages) and dietary supplements have the potential to modulate the activity of xenobiotic-metabolizing enzymes [Hodek et al., 2002]. Among proteins interacting with flavonoids, cytochrome P450s (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens), play the most prominent role. The two members of CYP1A subfamily, CYP1A1 and CYP1A2, are involved in the...
|
|
|
Activity of cytochromes P450 1A1, 1A2 and 3A4 expressed in eukaryotic and prokaryotic systems
Indra, Radek ; Stiborová, Marie (advisor) ; Mizerovská, Jana (referee)
Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...
|
|
|
Isoflavonsynthasa: přítomnost a aktivita v bobovitých a nebobovitých rostlinách
Pičmanová, Martina ; Honys, David (advisor) ; Vaňková, Radomíra (referee)
Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of the plant secondary metabolites, isoflavonoids. These phenolic compounds, which are well-known for their multiple biological effects, are produced mostly in leguminous plants (family Fabaceae). However, at least 225 of them have also been described in 59 other families, without any knowledge of orthologues to hitherto known IFS genes from legumes (with the single exception of sugar beet - Beta vulgaris, from the family Chenopodiaceae). In view of these facts, this masters thesis has focused on two main objectives: (1) to identify isoflavone synthase genes in selected leguminous and non-leguminous plants exploiting the PCR strategy with degenerate and non-degenerate primers, and (2) to find a system for the verification of the correct function of these genes. Our methodology for the identification of IFS orthologues was successfully demonstrated in the case of two examined legumes - Phaseolus vulgaris L. and Pachyrhizus tuberosus (Lam.) Spreng, in the genomic DNA of which the complete IFS sequences have been newly identified. To design a procedure for ascertaining the correct function of these genes and others once they have been completely described, a pilot study with IFS from Pisum sativum L. (CYP93C18; GenBank number...
|
|
|
Study on potentiaion of pharmacological efficiencies of ellipticine
Vranová, Iveta ; Mrázová, Barbora (referee) ; Stiborová, Marie (advisor)
Cytotoxic chemotherapy offers tool for clinical treatment of neoplasia. One of the drugs suitable for chemotherapy is ellipticine. Ellipticine is an alkaloid, which has significant antineoplastic properties. It acts as a DNA intercalator, inhibitor of topoisomerase II and forms also covalent DNA adducts mediated by cytochrome P450 and/or peroxidases. Oxidation of ellipticine by CYP (CYP3A4, CYP1A1/2, CYP2C9, CYP1B1) provides several metabolites (7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13- hydroxyellipticine, and ellipticine N2 -oxide). Metabolites 12-hydroxyellipticine and 13- hydroxyellipticine, formed by CYP3A4, are responsible for formation of two major DNA adducts. Two carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium were proposed as a reactive species binding to DNA. The main metabolites generated by peroxidases are the ellipticine dimer and ellipticine N2 -oxide, which provide the same carbenium ions and same DNA adducts. Modern chemotherapy uses targeting for higher selectivity for malignant cells and lower cytotoxicity for normal cells. Ellipticine-conjugates and his derivates (N-(2-hydroxypropyl)methakrylamid-ellipticine conjugates, vasoactive intestinal peptide-ellipticine conjugates and human serum albumin-ellipticine conjugates) and epidermal...
|
|
|
The effect of histone deacetylase inhibitor vaplroate on activity and expression of cytochromes P450 and peroxidases oxidizing ellipticine
Göttlicherová, Markéta ; Souček, Pavel (referee) ; Stiborová, Marie (advisor)
Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and inhibition of topoisomerase II. Ellipticine was also found to form covalent DNA adducts mediated by its enzymatic activation with cytochromes P450 (CYP) and peroxidases. The next study demonstrated increasing formation of these ellipticine-DNA adducts by histone deacetylase inhibitor valproate (VPA) in neuroblastoma cells. This phenomenon correlates with increasing cytotoxicity of ellipticine induced by this histone deacetylase inhibitor. This observation can be explained by several mechanisms. One of them can be loosening the structure of chromatine, which leads to accessing DNA for modification. Another one is the effect of VPA on activities and expression of enzymes metabolizing ellipticine. This study was aimed to test the second hypothesis. Since VPA has been shown to be metabolized by similar enzymes as ellipticine is, we have studied the effect of VPA (i) on oxidation of ellipticine by cytochromes P450 and peroxidases, (ii) on activities of the CYP enzymes, which significantly participate in oxidation of ellipticine (CYP1A, CYP3A) and (iii) on expression of enzymes oxidizing ellipticine (CYP1A1, CYP3A4, lactoperoxidase). Oxidation of ellipticine in vitro by model...
|
|
|
Mechanism of cancerogenic effect of nitroaromates pollutants in present environment
Čechová, Tereza ; Svobodová, Martina (referee) ; Stiborová, Marie (advisor)
Nitroaromatic compounds are mutagenic and carcinogenic substances present in all environmental compartments. Polycyclic aromatic hydrocarbons react with nitrogen oxides to form nitroaromatics under the conditions that might be expected in polluted air and in combustion processes (fossil fuel combustion, waste heat recovery, metal processing, etc.). Most of nitroaromatic compounds are potent mutagens in bacterial and mammalian systems. They are also carcinogens causing cancer, primarily in the liver, lungs and mammary glands. Nitrobenzanthrones (NBA) are nitroaromatic compounds which were recently found in environmental compartments, especially in the air. 3-Nitrobenzanthrone (3-NBA, 3-nitro-7H-benz [de] anthracene-7-one) is one of the polycyclic aromatic nitro compounds with high toxic effects. 3-NBA is present in environmental pollution, in diesel exhaust and was also detected in soil and in rain water. Bachelor's thesis describes the metabolism of this substance and it also studies its mutagenic and carcinogenic effects. This work also compares the mutagenic and carcinogenic effects of 3-NBA and its derivative, isomer 2-nitrobenzanthrone (2-NBA, 2-nitro-7H-benz [de] anthracene-7-one), which also occurs as a pollutant in air. (In Czech)
|
|
|
Modulation of activities and expression of enzymes metabolizing ellipticine by histone deacetylase inhibitor trichostatin A
Kopejtková, Barbora ; Kubíčková, Božena (referee) ; Stiborová, Marie (advisor)
Histone deacetylase inhibitor trichostatin A (TSA) increases cytotoxicity of antineoplastic agent ellipticine to human neuroblastoma cells. Its mechanism of action has not yet been explained. One of the possible mode of action is conformational change in chromatin, which leads to changes in DNA that is more accessible to covalent modification and intercalation. The aim of this work is to study another mode of action, which can explain this phenomenon. The question is, if TSA can increase cytotoxicity of ellipticine to human neuroblastoma cells by modulation of activities and expression of cytochromes P450 and peroxidases. These enzymes are responsible for cytotoxicity of ellipticine to human neuroblastoma cells. TSA has no effect on oxidation of ellipticine mediated by cytochromes P450 leading to metabolites responsible for formation of ellipticine-DNA adducts and detoxication metabolites. TSA increases formation of ellipticine dimer, which is a detoxication metabolite, forming during its oxidation by peroxidases. TSA has no effect on activities of CYP1A1, CYP1A2, CYP3A, which significantly participate in oxidation of ellipticine. TSA modulates expression of enzymes oxidizing ellipticin in human neuroblastoma cells. TSA in the presence of ellipticine increases expression of CYP1A1 a CYP3A4 in...
|
|
| |
|
|
The effect of atenolol on a fish organism
BOŘÍK, Adam
Pharmaceuticals and their metabolites are emerging pollutants of aquatic environment associated with our modern society. The aim of this bachelor's thesis was to get information about potential hazard levels of atenolol occurring in surface waters and its effects on juveniles of rainbow trout (Oncorhynchus mikyss) as a model organism. The effect of environmentally relevnt concentrations of atenolol was assased in vivo by using biochemical markers. Activity of enzymatic reactions EROD (CYP 1A), MROD (CYP 1A2) and PROD (CYP 2B) were tested as possible biomarkers. The experimental work aimed to verify wheter selected cytochromes P450 are suitable to evaluate the effects of fish exposure in natural conditions. The results of present experiment did not show a relationship between the enzymatic activity and exposure to beta-blocker atenolol. This fact implies that the enzimatic reaction EROD, MROD and PROD cannot be useful as biomarkers reflecting a metabolic response of fish after exposure to this xenobiotic.
|
guest ::
