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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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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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Food Supplements as Activation Modulators of Food Carcinogens, Phase II Metabolism
Bebová, Michaela ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
Heterocyclic aromatic amines (HAA) are representatives of carcinogenic and mutagenic compounds formed when muscle meat, including fish, is cooked, especially by frying and grilling. When these xenobiotic compounds enter the organism, they may be activated by metabolism into compounds with higher toxicity or carcinogenic potencial. Besides the enzymes of the phase I metabolism also the enzymes of the phase II metabolism mainly sulfotransferases (SULT) and N-acetyltransferases (NAT) may participate. Because these enzymes may be inducible their induction may be caused by other compounds present in food. Important inductors are some representatives of flavonoids - genistein, biochanin A and ß-naphtoflavone. These compounds have been proven to be able to to induce formation of sulfotransferases in vivo and in vitro conditions. An increased amount of enzymes may lead to the support of metabolic activation of procarcinogens, resulting in an increased formation of DNA aducts, causing development of carcinogenesis. For the research of induction of rat enzymes, rSULT and rNAT peptides were proposed that will be used for the production of antibodies suitable for their immunodetection.
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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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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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Specificities of nursing newborn children with partum cranial injuries
KOBLIHOVÁ, Jitka
Birth injuries of the new-born infants might be caused either by mechanical or anoxic traumas that occur during the birth. The injuries might be a result of insufficient attention or inadequate efforts of the obstetrician but might as well occur completely independently of the obstetrician´s activities. The character and extent of birth injuries are very wide and vary from the insignificant ones that are cured completely without any consequences to those that leave long-term consequences and that may even end in the death of the new-born infant. Cranial injuries of the new-born infants include: caput succedaneum (swollen scalp), cephalohaematoma, petechiae, suffusion, eye injuries, scalpel-incised wounds, parietal bones fractures, spinal cord injuries and intracranial haemorrhage. This Baccalaureate thesis surveys the knowledge of the nursing staff as far as birth cranial injuries are concerned and concentrates also at specific aspects of nursing new-born infants who suffer from caput succedaneum (swollen scalp) and cephalohaematoma. The theoretical part comprises the most important pieces of medical knowledge in nursing new-born infants with birth cranial injuries. The first goal of this thesis was to map the level of knowledge of the birth cranial injuries among the nursing staff. In compliance with this goal a hypothesis was stipulated: ?Members of the nursing staff are well-informed about birth cranial injuries.? This hypothesis was verified and confirmed. It was found out that members of the nursing staff have good knowledge about birth cranial injuries, their predispositions and prevention. A quantitative research was carried out using the questioning method (the questionnaire technique was used). The research was carried out at the Neonatal Departments of the hospitals Nemocnice České Budějovice, a.s, Nemocnice Písek, a. s. and Nemocnice Strakonice, a.s. The second goal of this thesis was to map the specific aspects of nursing new-born infants who suffer from caput succedaneum (swollen scalp) and cephalohaematoma. In compliance with this goal a research question was stipulated: ?What are the specific aspects of nursing the new-born infants who suffer from caput succedaneum and cephalohaematoma?? It was found out that nursing new-born infants with caput succedaneum and cephalohaematoma does not comprise many specific aspects. The most important thing is to nurse such infants carefully and with caution, to position their heads on the side without caput succedaneum or cephalohaematoma, to check regularly the bilirubin level, to observe carefully the cephalohaematoma or caput succedaneum (its colour, size, localization) and to watch the behaviour of the new-born, mainly whether it shows any signs of pain. The research was carried out in a qualitative way using the question method ? the depth interview technique. The research file consisted of two nurses and two midwives from the Neonatal Department of the České Budějovice Hospital, a.s. The research has revealed that the nursing staff at neonatal departments is well informed about birth cranial injuries and that the nursing methods applied at new-born infants who suffer from caput succedaneum and cephalohaematoma do not differ much from the methods used in case of physiological infants. The output of the thesis is an educative material concerning birth cranial injuries that is intended for the nursing staff at neonatal departments.
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