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Cooperativity of cytochrome P450 monooxygenase system in view of the modulation of drug and carcinogen metabolism
Dědič, Jan ; Hodek, Petr (advisor) ; Ječmen, Tomáš (referee)
System of mixed function oxidases and oxygenases is very much involved in metabolism of xenobiotics and endogenous compounds. System consists of several components: cytochrome P450, NADPH: cytochrome P450 oxidoreductase (CPR), cytochrome b5 and NADH: cytochrome b5 oxidoreductase. It was found, that all these components interact with each other, thus ensuring operation of the entire system. Cooperativity of the system is then dependent on many factors, notably the nature of the interactions between the components. Apparently the most frequently discussed are the interactions between CPR, cytochrome b5 and cytochrome P450. The main redox partner of cytochrome P450 is CPR that during the electron transport undergoes a significant conformational change. Cytochrome b5 may have both inhibitory and stimulatory effects on the enzymatic reaction and its mechanism of action has not been fully elucidated. Cytochrome P450 can interact among themselves to create complexes which presence may have significant influence on enzymatic reaction. However cooperativity of the system in terms of character of enzymatic reaction does not depend only on quantitative effects, such as inhibition and stimulation. It turns out, that also depends on qualitative effects, because it has been shown, that certain changes in the...
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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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Inhibitors of tyrosine kinases as anticancer drugs of a new generation
Hromek, Vlastimil ; Stiborová, Marie (advisor) ; Bárta, František (referee)
At the present time many types of treatment are used for curing of different cancer diseases. Among the most common types of such treatment belong a surgery, radiotherapy, chemotherapy, and immunotherapy. In the case of chemotherapy, there is used a wide (broad) spectrum of chemotherapeutics such as alkylating agents, platinum compounds, antimetabolites, anthracyclines and, at the present time, also inhibitors of tyrosine kinases. The bachelor thesis describes different types of tyrosine kinase inhibitors and their use in treatment of several cancers. They become popular because of their high specifity and minimal side efects. The first successful use of a tyrosine kinase inhibitor was treatment of the patients suffering from chronic myelogenous leukemia (CML) with imatinib. Vandetanib is another inhibitor of tyrosine kinases that is now used for treatment of another cancer, the medullary thyroid cancer. During treatment, vandetanib is biotransformed with cytochromes P450, which are the terminal oxidases of a mixed function oxidase (MFO) system, into the less efficient metabolites. In the practical part of the bachelor thesis we isolated enzymes, which metabolize xenobiotics, including vandetanib. Rat liver tissue was used for isolation of NADPH:cytochrome P450 reductase, which was isolated as a...
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Effect of pH on vandetanib oxidation
Fedák, Michal ; Indra, Radek (advisor) ; Otáhalová, Barbora (referee)
Vandetanib is anticancer drug used mainly for targeted therapy of medullary thyroid carcinoma. It acts as inhibitor of tyrosine kinase and shows selectivity for vascular endothelial growth factor 2 (VEGFR-2) and epidermal growth factor (EGFR). It also inhibits rearranged during transfection (RET) tyrosine kinase activity. Vandetanib is metabolized by cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs) in organism of humans as well as experimental animals. CYPs oxidize vandetanib to N-desmethylvandetanib. FMOs are responsible for the formation of vandetanib N-oxide. This bachelor thesis studies effect of pH on vandetanib oxidation by CYPs a FMOs present in rat hepatic microsomes induced by different agents. Collected data show that in majority of series, optimal pH levels for oxidation of vandetanib by CYPs and FMOs are similar to a large extend. The highest amount of N- desmethylvandetanib was observed mostly at the pH 8,5. Vandetanib N-oxide was also produced in the highest quantity at the same level of pH in majority of series. Results suggest that N-desmethylvandetanib is formed at levels of pH which do not fit in interval of pH for optimal CYP activity. This finding is apparently due to a fact that presence of vandeanib in its neutral form, which is effectively oxidized by CYP,...
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Metabolism of vandetanib by cytochrome P450 expressed in prokaryotic systems
Rodová, Marie ; Indra, Radek (advisor) ; Takácsová, Paulína (referee)
1 Abstract Recently, biologically targeted treatment by another name targeted molecular therapies have begun to be used in the treatment of cancers bearing specific molecular genetic or morphological traits. Vandetanib is an oral anticancer drug that belongs to a group of tyrosine kinases inhibitors. These inhibitors block signal pathway receptors, thereby inhibit growth, stimulate cell death and reduce the spread of cancer. Vandetanib was approved in April 2011 by the US FDA for a treatment of progressive or symptomatic medullary thyroid cancer. It is used in patients with metastatic or inoperable locally advanced cancer. The metabolism of vandetanib was studied in this thesis. Specifically, the kinetics of vandetanib oxidation to N-desmethylvandetanib by human recombinant cytochromes P450 3A4 expressed in the membrane of E. coli (Bactosomes). The effect of the presence of cytochrome b5 and the effect of the level of NADPH: cytochrome P450 reductase activity on the activity of cytochrome P450 3A4 were studied. The demethylated metabolite of vandetanib, N-desmethylvandetanib, was identified and separated by high performance liquid chromatography (HPLC). Enzyme kinetics studies indicate that vandetanib oxidation is affected by both, the level of NADPH:CYP reductase activity and the presence of cyt b5....
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Frequency of occurrence of selected single nucleotide polymorphisms of CYP2C8 and MDR1 in the Czech population and their influence on the effect of amiodarone
Pechandová, Kristina ; Perlík, František (advisor) ; Král, Jiří (referee) ; Anzenbacher, Pavel (referee)
Frekvence výskytu vybraných bodových polymorfismů CYP2C8 a MDR1 v české populaci a jejich vliv na působení amiodaronu Úvod: Variabilita lékové odpovědi je někdy podmíněna genetickými rozdíly v metabolismu a transportu léčiv. Interindividuální rozdíly jsou často způsobeny polymorfismy, které ovlivňují biotransformační aktivitu enzymů a expresi transportérů. V disertační práci jsme věnovali pozornost cytochromu P450 izoenzymu CYP2C8 a MDR1. Nejprve jsme popsali frekvenci výskytu vybraných variantních alel CYP2C8*2, CYP2C8*3 (2 substituce v exonu 3 a 8, CYP2C8*3G416A a CYP2C8*3A1196G), CYP2C8*4, CYP2C8 P404A u zdravé české populace a variantních alel MDR1 v exonech: 26 C3435T, 21 G2677A/T, 12 C1236T a 17 T-76A. Následně jsme sledovali vliv těchto polymorfismů na působení amiodaronu u vybraného souboru pacientů. Metody: Genotyp MDR1 a CYP2C8 jsme stanovili pomocí PCR-RFLP za využití specifických restrikčních enzymů a primerů. Frekvence genotypů MDR1jsme určili u 189 zdravých dobrovolníků a CYP2C8 u 161 zdravých osob. Do sledování jsme dále zařadili 63 pacientů užívajících amiodaron déle než dva měsíce. Jejich léčbu jsme posuzovali ze záznamů lékařské dokumentace, s využitím standardních biochemických a hematologických vyšetření a záznamů EKG. Koncentrace amiodaronu a jeho metabolitu N-...
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Modulation of activities and expression of enzymes metabolizing ellipticine by histone deacetylase inhibitor trichostatin A
Kopejtková, Barbora
Histone deacetylase inhibitor trichostatin A (TSA) increases cytotoxicity of antineoplastic agent ellipticine to human neuroblastoma cells. Its mechanism of action has not yet been explained. One of the possible mode of action is conformational change in chromatin, which leads to changes in DNA that is more accessible to covalent modification and intercalation. The aim of this work is to study another mode of action, which can explain this phenomenon. The question is, if TSA can increase cytotoxicity of ellipticine to human neuroblastoma cells by modulation of activities and expression of cytochromes P450 and peroxidases. These enzymes are responsible for cytotoxicity of ellipticine to human neuroblastoma cells. TSA has no effect on oxidation of ellipticine mediated by cytochromes P450 leading to metabolites responsible for formation of ellipticine-DNA adducts and detoxication metabolites. TSA increases formation of ellipticine dimer, which is a detoxication metabolite, forming during its oxidation by peroxidases. TSA has no effect on activities of CYP1A1, CYP1A2, CYP3A, which significantly participate in oxidation of ellipticine. TSA modulates expression of enzymes oxidizing ellipticin in human neuroblastoma cells. TSA in the presence of ellipticine increases expression of CYP1A1 a CYP3A4 in...
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Frequency of selected genetic polymorphisms of cytochrome P450 in the Czech population and the influence of CYP2C9 genotype on the hypolipidemic effect of fluvastatin
Buzková, Helena ; Perlík, František (advisor) ; Bultas, Jan (referee) ; Mičuda, Stanislav (referee)
55 Abstract Frequency of selected genetic polymorphisms of cytochrome P450 in the Czech population and the influence of CYP2C9 genotype on the hypolipidemic effect of fluvastatin Introduction: One of the main factors of genetically determined variability in response of humans to administered drugs are differences in catalytic activity of metabolizing enzymes, which are caused mainly by genetic polymorphisms in cytochrom P450 family enzymes. This thesis consists of two parts and it is presented as a commentary to the original papers. The first aim was to investigate the frequency of functionally important variant alleles of three main isoenzymes of cytochrome P450 gene: CYP2D6, CYP2C9, CYP2C19, throughout the Czech population, predict the prevalence of poor metabolizer phenotypes, and then to compare the results to the data from other populations. Secondly, we analysed the correlation between the CYP2C9 genotype and cholesterol-lowering effect of fluvastatin in human hypercholesterolemic patients. Methods: Genotypes were determined by PCR-RFLP. The presence of alleles CYP2D6*1, *6, *5, *4, *3, and gene duplication was analysed in 233 healthy volunteers, CYP2C9*1, *2 and*3 in 254 subjects and CYP2C19*1, *2 and *2 in 218 subjects. Eighty seven patients on fluvastatin therapy, and 48 patients on monotherapy...
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The effect of selected endocrine disruptors on cytochromes P450 1B1 and 3A1/2
Holecová, Jana
Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...
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