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Preparation of anticancer drugs bound in apoferritin
Fürbacherová, Pavlína ; Indra, Radek (advisor) ; Koblihová, Jitka (referee)
Cancer is one of the most serious problems, which modern medicine faces. In recent years, nanotechnologies and their use in medicine, has developed greatly. The aim is to make drug administration more effective and help to improve treatment of cancer illnesses. Incorporation of chemical substance into a nanoparticle can solve the problem with low stability of the drug, and/or it help to eliminate side effects. Nanoparticle apoferritin, which was studied in this thesis, is a form of commonly occurring protein ferritin. Its structure contains cavity, that can be used for incorporation of drug. Its chemical structure (high temperature stability and stability at wide pH range, easy manipulation by changing pH) and its biocompatibility makes apoferritin a potentionally suitable transporter. Presented thesis studied apoferritin's ability to incorporate anticancer drug cabozantinib into its structure. Cabozantinib is tyrosine kinase inhibitor which is used for treatment of thyroid cancer, renal cell carcinoma and hepatocellular carcinoma. The effect of final pH to the formation of the complex of apoferritin with cabozantinib, and stability of this complex was also studied in this thesis. Considering the results we can say that apoferritin is able to encapsulate cabozantinib into its inner structure. As we...
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The Study of metabolism of dihydromyricetin
Blecha, Tomáš ; Moserová, Michaela (advisor) ; Koblihová, Jitka (referee)
More attention has recently been paid to the chemopreventive compounds contained in food of plant origin. A large group of chemopreventive compounds are represented by flavonoids contained in many dietary supplements. One of the representatives of this group is dihydromyricetin, which is attributed to positive impact on human organism, in particular by its antioxidant, anti-inflammatory and hepatoprotective effects. Some flavonoids can act as modulators of enzymes of phase I and phase II metabolism of xenobiotics biotransformation by their inhibition or induction which can causean interference with endogenous metabolism. In first part of this present work, the effect of selected flavonoid compounds, such as biochanin A, dihydromyricetin, myricetin and their combinations on the gene expression of cytochrome P450 2E1 (CYP2E1) and sulfotransferases 1A1 (SULT1A1) in liver and small intestine of laboratory rats was studied. The small intestine was divided into three parts namely a proximal, middle and distal. At first, isolation of total RNA was made followed by conversion into cDNA using random hexamers as primers. By real-time PCR a significant decrease in gene expression of both enzymes in liver was found. The highest induction of gene expression was observed for both enzymes in proximal part of...
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Study of selected flavonoids as modulators of cytochrome P450 3A4
Skotnicová, Aneta ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
Flavonoids are secondary metabolites of plants exhibiting a wide range of biological activities. Special attention is paid to them mainly because of their antioxidant properties. Consumption of these natural substances is currently increasing especially in the form of dietary supplements. In addition, these compounds exhibit antiviral, anti-inflammatory or hepatoprotective effects and some of them also exhibit mutagenic and/or pro-oxidative properties. The study of flavonoid interactions with biotransformation enzymes such as cytochromes P450 are important because of their possible interference with the metabolism of drugs or carcinogenesis processes. In this bachelor thesis the influence of selected flavonoid compounds, dihydromyricetin and myricetin, on the expression and activity of cytochrome P450 3A4 was studied. The flavonoid premedication of rats did not resulted in induction of CYP3A4. However, dihydromyricetin and myricetin inhibit CYP3A4. IC50 were determined as 25 M for dihydromyricetin and 24 M for myricetin. Key words: Immunodetection, HPLC, inhibition, induction (In Czech)
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Function of Biotransformation Enzymes in Development of Nephropathies Caused by Aristolochic Acid
Bárta, František ; Stiborová, Marie (advisor) ; Koblihová, Jitka (referee) ; Eckschlager, Tomáš (referee)
- 6 - ABSTRACT Plant alkaloid aristolochic acid (AA) is a proven human carcinogen which causes two serious diseases: Aristolochic Acid Nephropathy (AAN) and Balkan Endemic Nephropathy (BEN). One of the characteristic features of both AAN and BEN is their close association with the development of upper urothelial carcinoma (UUC) in the renal tissue of patients. Although both nephropathies are mediated by the same compound (i.e. AA), their development differs slightly. The differences might be explained by a different exposure schedule of patients or interindividual differences in expression levels and activities of the enzymes metabolising AA in organisms. Detailed knowledge of these enzymes can contribute to the elucidation of the interindividual susceptibility to AA. In this thesis, enzymes participating in both oxidative detoxification of AAI, a major component of natural mixture of AA, and its reductive activation leading to the formation of AA-DNA adducts were studied. In a rat experimental model (Rattus norvegicus), NAD(P)H:quinone oxidoreductase 1 (NQO1) and its role in reductive bio-activation of AAI in vivo were examined utilising a specific inhibitor of this enzyme, dicoumarol. Oxidative detoxification of AAI resulting in formation of a demethylated derivative AAIa (8-hydroxyaristolochic...
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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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Expression and purification of protein photo-initiated nanoprobe: tool to study clinically relevant protein-protein interactions
Knížek, Antonín ; Šulc, Miroslav (advisor) ; Koblihová, Jitka (referee)
Cytochrome b5 is a key protein in the function and regulation of the mixed function monooxygenase (MFO) system in mammalian endoplasmic reticulum and is, therefore, a clinically relevant target for biochemical studies. To study its interactions within the MFO system using photo-initiated crosslinking, we have developed cytochrome b5 mutants with methionine in several key amino acid positions within the primary amino acid sequence, such as serine 23 and leucine 41. Also, naturally presented Met in positions 96, 126 and 131 were mutated to Leu with no effect to cytochrome b5 activity. Our protein was expressed in E. coli B834 auxotrophic type with L-2-amino-5,5-azi-hexanoic acid (photo-Met) present in the cultivation medium. This methionine analogue with photolabile diazirine ring is readily incorporated in Met positions into the primary sequence of proteins by aminoacyl-tRNA synthetases. The whole expression protocol was optimized to achieve maximal percentage of photo-Met incorporation into the expressed protein sequence. Up to 93.4% incorporation of photo-Met was achieved. The expressed protein was isolated and photo-Met incorporation was established with MALDI-TOF mass spectrometry. After reconstitution with its natural interaction partners - full-length cytochrome P450 2B4 (rabbit isoform) or...
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The effect of dihydromyricetin and myricetin on selected biotransformation enzymes
Boštíková, Zdislava ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
Flavonoids are natural compounds commonly ingested in herbs and vegetables. They are believed to have a positive impact on human organism, in particular by their antioxidant, hepatoprotective and anti-cancer effects. In these days, it is possible to consume high concentrations of these compounds in form of dietary supplements. However it is not clear, whether flavonoids in such unnaturally high concentrations are still beneficial or rather harmful. It has already been proven, that flavonoids can influence the activity of biotransformation enzymes and interfere e.g. with the process of carcinogenesis and drug metabolism. For that reason it is important to investigate the impact of an increased intake of flavonoids. The aim of this thesis was to investigate the influence of dihydromyricetin (a potential drug to cure alcohol use disorder) and its structurally similar flavonoid myricetin on the activity of enzymes, cytochrome P450 2E1 (CYP2E1) and Nacetyltransferases 1 and 2 (NAT1/2). The research included the determination of the impact of a premedication by these flavonoids on the expression and activity of CYP2E1 and NAT1/2. The inhibition capacity of myricetin and dihydromyricetin towards the activity of CYP2E1 and NAT1/2 was also investigated. After the flavonoid premedication of rats the...
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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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Study on the interaction of chemopreventive compounds and food born carcinogens with cytochrome P450 enzymes
Brabencová, Eliška ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The use of food supplements containing natural chemopreventive compounds increased in recent years. Some of the most popular chemopreventive compounds are flavonoids. Due to their natural origin, flavonoids are generally accepted as safe compounds. They exert antioxidant, anti-cancer and anti-inflammatory properties. However, flavonoids should be considered as foreign compounds (xenobiotics). Flavonoids interact with many enzymes, among the most important belong cytochromes P450 (CYPs), key enzymes of the first phase of biotransformation of xenobiotics (e.g. drugs, carcinogens). CYPs catalyze reactions leading mainly to detoxification of xenobiotics. However, some CYPs are involved in the activation of carcinogens, particularly CYP1A1 and CYP1A2 activate e.g. heterocyclic amines. Flavonoids might enhance the activation of carcinogens via induction of these CYPs or stimulation of their activities and hence, increase the risk of a cancer development. The thesis is focused on the influence of flavonoids and food carcinogens on the induction and activity of CYP1A1 and CYP1A2 in liver and small intestine of rats. For the purpose of this study, the small intestine was dissected into three parts: proximal (nearest to stomach), middle and distal. Western blotting was used for the evaluation of CYP...
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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
HAA are compounds which are showing numerous carcinogenic impacts on studied animals even human cells. These carcinogenes arise during the heat processing of meat or during (cigarette) smoking. Activation of these compounds is required to their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP) especially subfamily 1A1 and 1A2. As a consequence of activation with these enzymes are created N-hydroxylamines, which weakly reacting with DNA. For better formation of DNA aducts one more activation is essential. More reactive acetate and sulphate esters arise by second activation from N- hydroxylamines. The esters are produced by sulphotranspherase (SULT) even N- acetotranspherase (NAT). When we affect these enzymes we could positive control the formation of carcinoma. Caffeic acid is considered as a strong inhibitor of one SULT subfamily (phenolic sulfotranspherase P - PST). On the other side as a good inhibitor of NAT is considered (known) quercetin. (in czech) Key words: Heterocyclic amine, biotransformation, cytochrome P450, sulfotransferase, N-acetyltransferase
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