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Study of inflammation using epithelial cells
Majerová, Barbora ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The human respiratory system is in constant contact with heterogeneous agents from the environment. There must be effective mechanical lung barriers and sufficient immune protection due to continuous deposition of various substances in the respiratory system. The mutual balance between the mechanisms of natural and adaptive immunity of the lungs is essential for destruction of infectious agents without initiation of inflammatory response. Overreaction of the immune system of the lungs may lead to the production of various inflammatory mediators and cytokines such as interleukins IL-1, IL-6 and IL-18. When determining the immunogenicity of a substance, it has to be exposed to lung epithelial cells, and then the concentration of cytokines produced is measured. To determine the immunogenicity of mammalian immunoglobulin G and chicken immunoglobulin Y the subsequent twenty-four hour exposure to A549 lung cancer cell line was made. Concentration measurement of cytokines IL-1 and IL-6 was performed using Luminex method, which pointed out the immunogenicity of goat immunoglobulin G and certain chicken immunoglobulins Y.
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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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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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Binding of organic dyes to proteins. Applications in practical course in biochemistry.
Hynková, Anna ; Hudeček, Jiří (advisor) ; Koblihová, Jitka (referee)
In this submitted thesis the possibility of creating a new laboratory task for advanced practical courses in biochemistry, concerning the binding of a low-molecular compound (dye) to a protein macromolecule, was experimentally verified. First intention was to modify the existing task "Dialysis kinetics" with a binding of fluorescein to a protein. However, the experiments have proved, that fluorescence measurements of this kind are not reproducible in the conditions of practical courses laboratory and absorbance measurements have low sensitivity. Therefor a whole new task was created: "Binding of bromophenol blue to serum albumin", in which the stoichiometry of this binding is studied using so-called Job plot. After the optimization of procedure a laboratory task instructions were created, which are attached to this thesis. Keywords: fluorescein, bromophenol blue, serum albumin
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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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Activation of carcinogens in gastrointestinal tract
Zawadová, Dorota ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The Bachelor Thesis deals with an activation of heterocyclic aromatic amines (HAA) which have numerous carcinogenic effects on studied animals and human cells. These carcinogens are formed during the heat processing of meat and during the smoking. However, further transformation of the compounds is required to gather their carcinogenic effect. Most of all HAA are first activated by cytochrome P450 (CYP), especially its forms 1A1 and 1A2. The products of this activation - N-hydroxylamines - are further activated in conjugation reactions. In this work, we were focused on the transformation of N-hydroxylamines to more reactive acetate esters and sulphate esters, which is catalyzed by sulphotranspherase (SULT) and N-acetyltranspherase (NAT), respectively. The affection of these enzymes can control the formation of carcinoma. For example, some dietary compounds, such as caffeic acid and quercetin, are the most common inhibitors of these enzymes: caffeic acid is considered as a strong inhibitor of phenolic sulphotranspherase (P-PST), whereas quercetin is a good inhibitor of NAT. On the other hand, some dietary compounds can also induce an opposite effect: for instance, phenol acids induce the P-PST. (in Czech) Key words: Heterocyclic aromatic amines, biotransformation, cytochrome P450,...
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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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Interaction of gut microbiota with selected flavonoids
Balatka, Štěpán ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The microflora located on the surface of and inside the human body is a very large system. The intestinal microflora of the gastrointestinal tract is a very important and still not fully explored area. The intestinal microflora consists of many types of bacteria, viruses, fungi or some single-cell eukaryotic heterotrophic organisms. The intestinal microflora is involved, for example, in the synthesis of vitamins, digestion of polysacharides or in the storing of fats. It is significantly involved in the metabolism of foreign substances and digestion of food. The activity of foreign substances can influence the composition of the intestinal microflora. It must not be forgotten that the composition of the intestinal microflora differs in separate individuals and is changeable during life. The metabolism of foreign substances in relation to the human organism is a subject of global studies. This work is devoted to the metabolism of myricetin and its influence on the digestive tract of a healthy individual and a person with celiac disease. It is also studied whether myricetin affects the biodiversity of intestinal bacteria. The collected stool samples from donor A (a person with celiac disease) and B (a healthy control) were incubated at 0, 3 and 6 hours in McDougall buffer with myricetin. They were...
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