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Study of metabolism air pollutants and mutagens 3-nitrobenzanthrone and 2-nitrobenzanthrone
Čechová, Tereza ; Stiborová, Marie (advisor) ; Martínek, Václav (referee)
Nitroaromatic compounds are mutagenic and carcinogenic substances present in environment. Most of nitroaromatic compounds are potent mutagens in bacterial and mammalian systems. They are also carcinogens causing development of tumors, primarily in the liver, lung and mammary glands. 3-Nitrobenzanthrone (3-NBA, 3-nitro-7H-benz [de] anthracene-7-one) is one of the polycyclic aromatic nitro compounds possesing high toxic effects. 3-NBA is an environmental pollutant present in diesel exhaust and was also detected in soil and in rain water. 2-Nitrobenzanthrone (2-NBA, 2-nitro-7H-benz [de] anthracene-7-one) is an isomer 3-NBA, which also occurs as a pollutant in air. Although the 2-NBA is a weakly toxic substance, its high abundance in air could exhibit a high health risk to humans. This thesis investigates the metabolism of 3-NBA and its isomeric derivate, isomer 2 NBA, under anaerobic and aerobic conditions. To study the metabolism of these compounds, microsomal systems isolated from the liver of rats pretreated with Sudanem I, -naphthoflavone, phenobarbital, ethanol and pregnenolon 16-carbonitrile (PCN), the inducers of cytochromes P450 1A, 2B, 2E1 and 3A, were used. We also used mouse models, a control mouse line (wild type WT) and mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting...
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Development and analysis of a database of reactions catalyzed by cytochrome P450 enzymes for machine learning applications
Komorníková, Natália ; Pluskal, Tomáš (advisor) ; Berka, Karel (referee)
Cytochrome P450 enzymes are hemoproteins showing extraordinary di- versity in the reactions they catalyze. We developed a database containing all the needed data to provide a comprehensive data source on reactions cat- alyzed by cytochrome P450 enzymes. This data mainly includes information about the substrates, products of characterized reactions, and the sequence of these enzymes. The database was developed by collecting data from reliable protein and reaction databases like UniProt and RHEA. The work presents an in-depth analysis of the created database of reactions catalyzed by cy- tochrome P450 enzymes. This database can be utilized for future machine learning approaches to predict the function of uncharacterized cytochrome P450s.
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Effect of selected perfumes on aromatase activity
Drejslarová, Iva ; Hodek, Petr (advisor) ; Smrček, Stanislav (referee)
Cytochromes P450 are enzymes involved in many physiological processes in the body, besides the metabolism of xenobiotics also in the biosynthesis and catabolism of endogenous substances such as hormones and steroids. Cytochromes P450 that are responsible for the transformation of endogenous compounds can be affected by xenobiotics, which interfere with the secretion, metabolism, transport or elimination of endogenous hormones by various mechanisms. These substances are also referred to as endocrine disruptors and include, for example, pesticides, insecticides, certain pharmaceuticals, or compounds contained in cosmetic products. One of their mechanisms of action is to interfere with steroid hormone biosynthesis through interactions with steroidogenic enzymes. One such enzyme is cytochrome P450 19, aromatase, which catalyses the conversion of testosterone to ß-estradiol in the final step of steroid hormone biosynthesis. Modulation of the activity of this enzyme by endocrine disruptors results in an imbalance of estrogen levels in the body, which can lead to impaired reproduction, osteoporosis, atherosclerosis, dementia, and the development of certain types of cancer. In this context, the effect of commercially available perfumes on the conversion of testosterone to ß-estradiol, which is catalyzed by...
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Modulation of cytochrome P450 activity by the anticancer drug lenvatinib
Ivančík, Martin ; Dračínská, Helena (advisor) ; Mrízová, Iveta (referee)
Lenvatinib, commercially marketed as Lenvima®, is an oral drug approved for the treatment of thyroid cancer, hepatocellular carcinoma and renal cell carcinoma that acts as a tyrosine kinase inhibitor. In vitro and in vivo studies have shown that lenvatinib is in the human body metabolised in liver and kidney by the cytochrome P450 enzyme system and aldehyde oxidase. Therefore, the aim of this bachelor thesis was to determine the effect of lenvatinib on the activity of individual isoforms of human cytochrome P450. Among the isoforms studied, those that ensure the metabolism of majority of foreign substances in the human body were selected. Measurements were performed in vitro using recombinant CYPs expressed in SupersomesTM and using marker reactions that are provided by individual cytochrome P450 isoforms. The activity of the enzyme in the reaction mixtures containing lenvatinib was compared with the activity of the enzyme in the reaction mixtures where only the solvent DMSO was added instead of lenvatinib. The concentration of lenvatinib corresponded to the concentration of the given substrate or was 10 times higher. Based on these measurements, the percentage activity of cytochrome P450 isoforms 1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2E1 and 3A4 in the presence of lenvatinib was calculated. A decrease in...
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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.
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Study on potentiaion of pharmacological efficiencies of ellipticine
Vranová, Iveta ; Stiborová, Marie (advisor) ; Mrázová, Barbora (referee)
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...
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Interaction of food additives with xenobiotic metabolising enzymes
Jandová, Eliška ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
Recently the use of various food supplements as a part of a healthy lifestyle has been very popular. Although most of them are natural products, their excessive consumption may not always be beneficial for health. Dietary supplements are usually of a flavonoid character. Flavonoid compounds are found in plants and they have beneficial effects on human health. For their antioxidant, anti-allergy and chemopreventive effects they are extensively studied. However, in recent years the negative impacts of flavonoids have been described, often caused by their excessive consumption. It has been shown that they interact with cytochrome P450, which play an important role in the biotransformation of xenobiotics. The change in the metabolism of xenobiotics (whether drugs or carcinogens) can cause serious health problems, including a tumor growth. Beside cytochrome P450, there is another possible points of intervention, cytochrome b5 (or NADH:cytochrome b5 reductase), which effects the catalytic cycle of cytochrome P450. Another point of potential danger is the elimination of xenobiotics out of the organism. There is a complex system of transporters, in which P-glycoprotein plays a very significant role. P-glycoprotein is involved in transmembrane efflux of xenobiotics, preventing the aggregation of these...
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Mechanism of cancerogenic effect of nitroaromates pollutants in present environment
Čechová, Tereza ; Stiborová, Marie (advisor) ; Svobodová, Martina (referee)
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)
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Heterologous expression and isolation of human cytochromes P450 1A1/2
Milichovský, Jan ; Martínek, Václav (advisor) ; Hodek, Petr (referee)
Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...
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