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Morphine alkaloid metabolism in bacteria
Zahradník, Jiří ; Kyslík, Pavel (advisor) ; Lichá, Irena (referee)
Morphine alkaloids and their derivatives are pharmaceutically important substances. Huge production and consumption of these compounds predetermines them to be significant pollutants in the environment. Some of them have been detected in surface waters. The aim of this study was to characterize effects of morphine alkaloids on the physiology of three model organisms: Agrobacterium sp. R89-1, Escherichia coli XL-1 (Blue), and Raoultella sp. kDF8, and elucidation of the mechanisms leading to toxicity. The biotransformation potential and utilization ability were characterized for model organisms. It was demonstrated that the microorganism Agrobacterium sp. R89-1 is capable of rapid biotransformation of codeine to its 14-OH derivatives. The manifestation of morphine compounds toxic effects for the strain R89-1 is the highest. In contrast, microorganism Raoultella sp. KDF8 is able to utilize codeine as a carbon and energy source. The accumulation of 14-OH-derivatives was not observed. Escherichia coli XL-1 (Blue) is not able to biotransform or utilize codeine. Α, β-unsaturated ketones (morphinone, codeinone, 14-OH-morphinone and 14-OH-codeinone) were found as a most toxic intermediates of codeine metabolism. Bacterial cell growth (strains R89-1 and KDF8) in the presence of codeine is characteristic with...
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The influence of xenobiotic metabolizing enzymes and transporters on the incidence of colorectal cancer and chemotherapy outcome
Krus, Ivona ; Souček, Pavel (advisor) ; Králíčková, Milena (referee) ; Hodis, Jiří (referee)
Introduction: Colorectal cancer (CRC) is one of the most frequent malignancies and affects approximately 5% of worldwide population. More than 75% of CRC cases represent sporadic forms. Susceptibility to nonhereditary CRC is significantly influenced by polymorphisms and mutations in low-penetrance genes. Variations in biotransformation and DNA repair genes may result in acumulation of toxins and DNA damage in cells leading to the development of cancer. Furthermore, different gene expression profiles of membrane transporters affecting the accumulation of anticancer drugs in tumour cells, e.g. ABC drug transporters, may largely influence inter-individual variability in drug response and chemotherapy outcome. The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal cancer. This study followed selected genetic alterations in xenobiotic-metabolizing enzymes (CYP1B1, GSTM1, GSTT1, GSTP1, NQO1 and EPHX1) and genes involved in response to DNA damage (CHEK2 and NBN), as potential CRC susceptibility factors. Another aim of this study was to investigate expression profile of all human ABC transporter genes to follow their prognostic and predictive potential in colorectal carcinoma. Materials and methods: The polymorphisms and other...
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Study of genetic factors modifying the risk of onset and progression of colorectal and pancreatic cancer
Mohelníková Duchoňová, Beatrice ; Souček, Pavel (advisor) ; Skálová, Lenka (referee) ; Macek, Milan (referee)
Introduction: The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal and pancreatic cancer. The first part deals with the etiological factors and the importance of polymorphisms in biotransformation enzymes and genetic alterations in the gene CHEK2 in the origin of these malignancies. In the second part, the ABC transporter genes were analyzed as potential prognostic and predictive markers of a treatment's outcome. Materials and methods: The polymorphisms and other genetic alterations were detected using real-time PCR, allelespecific PCR and PCR-RFLP methods in DNA which was extracted from the blood of patients. The frequency of polymorphisms was evaluated and their importance was assessed with regard to the available epidemiological data. Gene expressions were determined by qPCR in paired samples of tumor tissue and adjacent non-tumorous parenchyma. Results: A majority of the observed polymorphisms failed to show a relationship between their presence and the risk of any of these malignancies. CYP2A13 variant allele*7 coding inactive enzyme was found in 7 of 265 controls and in none of 235 pancreatic carcinoma patients. In contrast, GSTP1-codon 105 Val variant allele and GSTT1-null genotype were associated with an elevated...
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Biotransformation of phenols by enzymatic systems of Candida tropicalis yeast and Comamonas testosteroni bacteria
Vilímková, Lenka ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Mareš, Jaroslav (referee)
Candida tropicalis yeast and bacteria Comamonas testosteroni have been considered to be able to metabolize phenol and utilize it as the only source of carbon and energy. In our laboratory we investigated the cytoplasmic enzymes responsible for the first and second step of phenol degradation, NADPH-dependent phenol hydroxylase of both C. tropicalis and C. testosteroni and catechol 1,2-dioxygenase of C. tropicalis. The aim of our study was to isolate and partially characterize those enzymes. Phenol hydroxylase purification consisted of preparation of cytosol from C. tropicalis yeast by fraction centrifugation, chromatography and re-chromatography on a column of DEAE Sepharose, fractionation by precipitation of the enzyme with polyethylene glycol 6000 and gel permeation chromatography on a column of Sephacryl S-300. Extracellular phenol hydroxylase of C. testosteroni was purified by fraction precipitation with polyethylene glycol 6000 and by gel permeation chromatography on 4B Sepharose and Sephacryl S-300. Catechol 1,2-dioxygenase was purified using the procedure consisting of: chromatography and re- chromatography on a column of DEAE Sepharose, lyophilization of the enzyme and gel permeation chromatography on a column of Sephadex G-100. The enzyme activity was determined by two methods: use of HPLC...
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Asymmetric biotransformation of certain tetralones to tetralols
Andrejch, Jan ; Poljaková, Jitka (advisor) ; Martínek, Václav (referee)
Preparation of pure enantiomers is very important part of chemical synthesis of industrially and pharmaceutically useful compounds. In recent years traditional chemical synthesis is replaced by biotransformations. Biotransformation means the use of isolated enzymes, enzymatic systems or whole cells in catalysis of chemical reactions. In comparison with chemical synthesis it has certain advantages. In this work the activity of yeast strains Saccharomyces uvarum K8, Saccharomyces cerevisiae K12, Torulopsis azyma K43, Torulopsis sphaerica K46 and Candida tropicalis K49, K51 a K52 in catalysis of reduction 6- bromo-2-tetralone, 7-hydroxy-2-tetralone and 7-methoxy-2-tetralone was studied. Products of these reductions are used in preparation of pharmaceutical drugs. Suitable conditions for biotransformations were found and it was proved that the yeast strains Saccharomyces uvarum K8 and Candida tropicalis K52 are able to produce 2- tetralones with ee greater than 90% with conversion 100% after four-hour biotransformation. This thesis is written in Czech.
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Biotransformation of nitro aromates
Tomeček, Jaromír ; Bednařík, Karel (referee) ; Friedl, Zdeněk (advisor)
Contamination by nitroaromatic compounds, such as explosives, pesticides or other nitroaromatic compounds, is worldwide environmental problem. This text focuses on contamination by compounds from military and ammunition industry, most especially 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazinane (RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX). First goal of this work is to provide the reader with a aerobic biodegradation and transformation processes of nitroaromatic compounds named above. The second goal is to compare the toxicity of initial nitroaromates and their transformation intermediates.
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