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The effect of cancerogenic azo dye Sudan I on expression of biotransformation enzymes
Hejduková, Žaneta ; Svášková, Dagmar (advisor) ; Dračínská, Helena (referee)
Sudan I is a widely used azo dye which has the ability to cause carcinomas in organs and tissues of experimental animals. During reactions catalyzed by microsomal monooxygenase enzyme systems or cytoplasmic biotransformation enzymes, Sudan I is oxidized to reactive metabolites that covalently bind to nucleic acids and cause their damage. Sudan I can also be metabolized by reduction, e. g. by a DT-diaphorase enzyme (NQO1). Reduction of Sudan I is considered to be a detoxification reaction. In this work, the in vivo action of Sudan I is examined in terms of its ability to induce an expression of the biotransformation enzyme DT-diaphorase in tissues of rats treated with the azo dye. The aim of this work was to quantify the degree of NQO1 induction at mRNA level. After the isolation of total RNA from organs of rats treated with Sudan I, the RNA was converted to cDNA by reverse transcription using random hexamers as primers. Using specific probes, the abundance of mRNA for the enzyme NQO1 in the organs of treated rats was quantified by "real-time" PCR, relatively to the control gene with a constant expression (β-actin). Through comparing thus determined amounts of mRNA in individual organs of treated and untreated rats, it has been found that Sudan I had caused a significant increase in the expression...
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Solubility of lipophilic model carcinogens in water environment and effect of biomolecules
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One goal was to determine solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). Another objective was to evaluate the effect of model proteins (bovine serum albumin, lysozyme) on solubility of Sudan I and ellipticine. The last aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method measuring concentration of a compound in saturated solution (In Czech) Key...
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Effect of cytochrome b5 on activity of cytochromes P450
Ličko, Vojtech ; Indra, Radek (advisor) ; Feglarová, Tereza (referee)
ABSTRACT Cytochrome b5 (CYB5) is heme protein capable of reduction of cytochromes P450 (CYP) or some other enzymes. However, his regulative capability was also observed by his apo form, i.e. in absence of heme prosthetic group in the active center. CYB5 can accept electron from cytochrome b5 reductase (CYB5R) or from cytochrome P450 reductase (CYPOR). CYPOR by itself is reduced by NADPH and is also able to forward electron to CYP independently of CYB5. CYB5R on the other hand is reduced by NADH. Efficiency of CYB5 to accept and forward an electron was studied in vitro with five different substrates - testosterone, Sudan I, aristolochic acid I (AAI), ellipticine and vandetanib. These substrates were chosen considering their characteristic reactions, which are catalyzed by their respective isoforms of CYP. The experiments with these substrates were carried out in the medium with recombinant CYPs prepared in insect cells or E. coli or in the medium with hepatic microsomes isolated from different organisms. Rats, from which the majority of these microsomes was isolated, were premedicated by different CYP inducers. The experiments were carried out in medium with NADH or NADPH in order to assess the capability of CYB5 to reduce CYP independently of CYPOR. The capability of CYB5 and CYB5R to act as a...
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Effect of cytochrome b5 on enzyme kinetics of Sudan I hydroxylation catalyzed by human cytochrome P450 1A1
Netolický, Jakub ; Martínek, Václav (advisor) ; Černá, Věra (referee)
Cytochromes P450 are the major xenobiotics converting enzymes. They are classified as mixed function monooxygenases (MFO). Isoform 1A1 is a extrahepatic form found mainly in the lung and other tissues. It is strongly induced by polycyclic aromatic hydrocarbons and their derivatives via the Ah receptor. As a marker reaction for this enzyme can be used hydroxylation of Sudan I, which has previously been widely used as a azo dye in industry, but since 1980s it is banned for coloring food and cosmetics for its negative influence on the organism. NADPH:cytochrome P450 reductase is the major electron donor for cytochrome P450 catalyzed monooxygenation reactions. Another electron carrier for cytochrome P450 catalyzed reactions is cytochrome b5. It was shown that cytochrome b5 can stimulate, inhibit or have no effect on P450 catalyzed reactions. This thesis aims to evaluate the influence of the ration between NADPH:cytochrome P450 reductase and cytochrome b5 on cytochrome P450 1A1 catalyzed Sudan I hydroxylation. The main goal is to characterize the influence of electron donor and electron transfer ratios on hydroxylation of Sudan I, and to determine the kinetic parameters KM and VMAX for selected protein ratios. Partial aims of the thesis were to characterize the recombinant proteins used in this study...
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Expression and activity of rat cytochromes P450 3A after exposure to benzo[a]pyrene and Sudan I
Ličko, Vojtech ; Dračínská, Helena (advisor) ; Ptáčková, Renata (referee)
The aim of this Bachelor thesis is the study of the effect of two carcinogenic compounds, benzo[a]pyrene and Sudan I, co-administered to rats individually or in combination, on the expression and the activity of important biotransformation enzymes cytochromes P450 of subfamily 3A in liver - a main organ of xenobiotic metabolism, in which the amount of CYP3A is especially high. Using the quantitative PCR method, the decrease of the gene expression of CYP3A1/2 in the livers of rats exposed to benzo[a]pyrene and Sudan I individually or in combination, was observed. Using the Western Blot method with a consecutive immunodetection, we found the decrease of the protein expression of CYP3A in the livers of rats treated with benzo[a]pyrene and Sudan I alone. Specific activity of CYP3A, determined by marker reaction of CYP3A, which is 6β-hydroxylation of testosterone, did endorse the previous results only in some of the premedicated groups of rats. It can be concluded that the exposure of rats to both studied compounds with carcinogenic potential resulted in a decrease in the expression of hepatic CYP3A in vivo. (In Czech) Keywords: cytochromes P450, benzo[a]pyrene, Sudan I, expression, enzyme activity
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Molar ratio between cytochrome b5 and its reductases affects activity of cytochrome P450 1A1 towards Sudan I
Netolický, Jakub ; Martínek, Václav (advisor) ; Dračínská, Helena (referee)
Cytochromes P450 are an evolutionary very old group of enzymes. It spread into many isoforms that can be found in animals, plants, fungi, bacteria, and some viruses. They play a major role in the first phase of the biotransformation of drugs, environmental pollutants and other xenobiotics. Also for this reason, they belong among the most researched enzymes. Cytochrome P450 for its function requires an electron donor, such as NADPH:cytochrome P450 oxidoreductase and cytochrome b5. The alternative reductase involved in this process is NADH:cytochrome b5 oxidoreductase, which is able to reduce cytochrome b5. In a eukaryotic cell, all these membrane proteins are found in the endoplasmic reticulum membrane, where they can naturally interact. This work evaluates the activity of human recombinant cytochrome P450 1A1 against the carcinogenic azo dye Sudan I, specifically it focuses on mapping the formation of major metabolites in relation to the ratio of cytochrome b5 to NADPH:cytochrome P450 oxidoreductase as well as to NADH:cytochrome b5 oxidoreductase. Keywords: cytochrome P450 1A1, NADPH:cytochrome P450 oxidoreductase, cytochrome b5, NADH:cytochrome b5 oxidoreductase, Sudan I, HPLC [In Czech]
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Solubility of lipophilic model carcinogens in water environment and effect of biomolecules
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One of our goals was to determine the solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). The second aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method utilizing determination of concentration of a compound in equilibrium with solid phase. (In Czech) Key words: solubility, UV-VIS spectroscopy, 3-nitrobenzanthrone, 2-nitrobenzanthrone, ellipticine, Sudan I
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Solubility of lipophilic model carcinogens in water environment and effect of biomolecules
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One goal was to determine solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). Another objective was to evaluate the effect of model proteins (bovine serum albumin, lysozyme) on solubility of Sudan I and ellipticine. The last aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method measuring concentration of a compound in saturated solution (In Czech) Key...
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