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The changes in expression of selected drug-metabolizing enzymes in parasites after exposition to sublethal doses of drugs
Pasák, Michael ; Skálová, Lenka (advisor) ; Trejtnar, František (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michael Pasák Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: The changes in expression of selected drug-metabolizing enzymes in parasites after exposition to sublethal doses of drugs The parasite, which causes significant losses of small ruminants through a disease called haemonchosis, is the abomasal nematode Haemonchus contortus. IVM is one of the anthelmintics used in veterinary medicine to eliminate haemonchosis. The main problem with this parasite is the presence of drug resistance to almost all administered anthelmintics including IVM. It is believed that xenobiotic-metabolizing enzymes such as cytochromes P450 (CYPs) and membrane efflux transporters P- glycoproteins (P-gps), play the role in resistance in H. contortus. This study focuses on the effect of sublethal doses of IVM on expression of selected CYPs and P-gps. Females and males of susceptible ISE strain H. contortus were separated before our experiment. After incubation of nematodes with three different concentrations of IVM, RNA was isolated and the changes in expression of selected genes were analyzed using qPCR. Significant sex-differences were observed in inducibility of tested genes. Significant...
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Study of activation properties of insulin, IGF-1 and IGF-2 analogs towards the receptors for insulin and IGF-1
Chrudinová, Martina ; Selicharová, Irena (advisor) ; Šulc, Miroslav (referee) ; Skálová, Lenka (referee)
The insulin/insulin-like growth factor signaling axis is a complex system that is involved in the regulation of metabolism and body growth. It includes three related peptide hormones: insulin and two insulin-like growth factors (IGF-1 and IGF-2), and their receptors: two isoforms of insulin receptor (IR-A and IR-B), and IGF-1 receptor (IGF-1R). Whereas insulin is involved predominantly in regulation of the metabolism, IGFs participate mainly in the regulation of cell growth, proliferation and differentiation. Due to the similarities in the structures of both the hormones and the receptors, all the hormones can bind and activate all the receptors, although with different potencies. At which point and how are the "metabolic" and "mitogenic" functional outcomes of the system diversified, as well as the structural determinants responsible for the different affinities of the hormones to the individual receptors, is still not completely understood. Deregulation of the insulin/IGF system can result in many types of pathological states, mainly diabetes mellitus and cancer. Therefore, to deeply understand the functioning of the insulin/IGF system to the structural details is crucial for development of analogs with desirable properties, that are of great clinical interest. First of all, we developed a...
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Metabolism of flubendazole in human liver subcellular fractions
Holubová, Jana ; Skálová, Lenka (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jana Holubová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Metabolism of flubendazole in human liver subcellular fractions Flubendazole (FLU), a benzimidazole anthelmintic drug with a broad spectrum of activity and low toxicity has been used in veterinary as well as human medicine for a long time. Owing its mechanism of action, based on the specific binding to tubulin, FLU is now considered a promising anti-cancer agent. However, we do not have enough information about its biotransformation in human. In our project, subcellular fractions from the liver of 12 human patients (6 males and 6 females) were used to study the stereospecificity, cellular localization, coenzyme preference and possible inter-individual or sex differences in FLU reduction. In addition, the risk of FLU interaction with other drugs was evaluated. Results showed that FLU is predominantly reduced in cytosol and the NADPH coenzyme is preferred. The strict stereospecificity of FLU carbonyl reduction was proven and carbonyl reductase 1 was identified as the main enzyme of FLU reduction in the human liver. A higher reduction of FLU and a higher level of carbonyl reductase 1 protein was found in males than in...
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Modulatory effects of blueberry extract on the activities and expression of selected biotransformation enzymes in rat
Láníčková, Tereza ; Skálová, Lenka (advisor) ; Boušová, Iva (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Biochemical Sciences Candidate: Tereza Láníčková Supervisor: Prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Modulatory effects of bilberry extract on the activities and expression of selected biotransformation enzymes in rat Bilberry (Vaccinium myrtillus L., Ericacea) is one of the richest sources of anthocyanins. These bioactive polyphenolic secondary metabolites give bilberry not only a characteristic blue/black colour, but they are also responsible for many health benefits. Therefore, bilberry has potential use in the treatment and prevention of conditions associated with visual disorders, inflammation, dyslipidemia, hyperglycemia or in some other disorders associated with increased oxidative stress. The aim of this study was to evaluate the effect of bilberry extract, which was administrated for 29 and 58 days, on activity and expression of selected biotransformation enzymes in rat liver. Enzymatic activity and expression of mRNA was determined in the cytosols and microsomes of liver tissue from control and influenced rats. In this study, spectrophotometric or HPLC method was used to measure the activities of aldehyde reductase (AKR1A1), hydroxysteroid dehydrogenase (AKR1C), glutathione-S-transferase (GST),...
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New aspects of pregnane X receptor function and regulation
Hyršová, Lucie ; Trejtnar, František (advisor) ; Skálová, Lenka (referee) ; Vondráček, Jan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lucie Hyršová, MSc. First Supervisor: doc. PharmDr. František Trejtnar, CSc. Consultant: prof. PharmDr. Petr Pávek, PhD. Title of Dissertation Thesis: New aspects of pregnane X receptor function and regulation The pregnane X receptor belongs to the superfamily of nuclear receptors; it is a ligand dependent transcription factor regulating expression of its target genes. During last two decades, PXR was extensively studied as a xenosensor, i.e. the receptor, which is able to bind xenobiotics including many drugs and to regulate their metabolism by induction of the most important metabolizing enzymes of both phase I. and phase II. Induction, i.e. transcriptional stimulation of expression of the most important cytochrome P450 enzymes, by PXR ligands was described in details at many levels. Within this dissertation thesis, I am dealing with aspects of regulation via PXR, which extend the common understanding of PXR as a receptor whose exclusive function is to up-regulate drug metabolizing enzymes mediated by its agonists. Within the first, project I studied regulation of OCT1 transporter in hepatic cell models, I shown that PXR did not induce, but it rather suppressed the expression of this...
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Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta
Lukačiková, Karolína ; Vokřál, Ivan (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Karolína Lukačiková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Effect of mebendazole on the activity of selected enzymes in tapeworm Hymenolepis diminuta The resistance of parasitic helminths to anthelmintic drugs is a growing worldwide phenomenon and a concerning issue. Xenobiotic metabolizing enzymes play an important role in drug resistance development as they can lower the concentration of the anthelmintics in the parasite's body and therefore protect the parasite from the anthelmintic effect. The role of drug metabolizing enzymes in drug resistance development has been already described in the group of roundworms and flukes. Limited information is available about this topic in tapeworms. In our study we decided to test the possibility of the anthelmintic mebendazole to affect the activity of these enzymes and possibly to influence the drug resistance development in rat tapeworm (Hymenolepis diminuta). Our first goal was the isolation of adult tapeworms from the definitive host (rat, Rattus norvegicus). We used mealworm beetle (Tenebrio molitor) as an intermediate host. After the successful isolation, adult tapeworms were incubated with the mebendazole (1 and 10µM) in...
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The impact of selected prenylflavonoids on the effect of anticancer therapy in cancer cell lines
Pešková, Klára ; Matoušková, Petra (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Klára Pešková Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: The impact of selected prenylflavonoids on the effect of anticancer therapy in cancer cell lines Prenylflavonoids 6-prenylnaringenin and 8-prenylnaringenin and flavonoid naringenin are substances with anti-proliferative properties. Prenylflavonoids are present mostly in hop and in beer. Main source of naringenin are primarily citrus fruits. In this diploma thesis the effect of compounds was tested by neutral red uptake test in cell lines SW480 and SW620. These substances were tested individually and then in combination with cytostatic agent oxaliplatin. Oxaliplatin is used in treatment of cancer disease. 6-prenylnaringenin was the most effective compound in both cell lines. Statistical evaluation of the effect was carried out by Graphpad using the method One-way ANOVA in comparison with control. Combination index was determined by software program Compusyn using the method by Chou-Talalay.
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Synthesis of epitestosterone and 4-hydroxyestrone glucuronides using recombinant human UDP-glucuronosyltransferases.
Kašparová, Michaela ; Skálová, Lenka (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Michaela Kašparová Supervisors: Prof. RNDr. Lenka Skálová, Ph.D., Erkka Järvinen, M.Sc. (Pharm.) Title of diploma thesis: Synthesis of epitestosterone and 4-hydroxyestrone glucuronides using recombinant human UDP-glucuronosyltransferases Steroid hormones constitute an important part of the human endocrine system and are involved in a variety of physiological and pathological processes. Phase I and phase II biotransformation reactions convert these lipophilic, biologically active compounds to inactive, water-soluble metabolites that are readily excretable into bile or urine. One of the most common phase II biotransformation reactions is conjugation with glucuronic acid that is enabled by the catalytic activity of UDP-glucuronosyltransferases (UGTs). This study is focused on development and optimization of an enzymatic method for producing conjugates of glucuronic acid and two naturally occurring steroid hormones, epitestosterone and 4-hydroxyestrone. Recombinant human UGT2B7 was employed as a catalyst for the production of epitestosterone 17-glucuronide and 4-hydroxyestrone 4- glucuronide while synthesis of 4-hydroxyestrone 3-glucuronide was accomplished by UGT1A10 isoform. The synthesis reactions...
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The effect of flubendazole and mebendazole on epithelial-mesenchymal transition.
Hochmalová, Martina ; Skálová, Lenka (advisor) ; Ambrož, Martin (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martina Hochmalová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Consultant: RNDr. Věra Králová, Ph.D. Title of diploma thesis: The effect of flubendazole and mebendazole on the epithelial- mesenchymal transition. Epithelial-mesenchymal transition (EMT) is a process in which a non-motile epithelial cell switches to motile mesenchymal phenotype. This phenomenon is typical for multiple biological processes including cancer metastases. It could be induced by transforming growth factor-β (TGF-β) which is produced by cancer cells and which regulates its key transcription factors including SNAIL, ZEB and TWIST. EMT involves a series of defined events mediated by microtubules, including changes in cell shape and migration into the surrounding tissue. Based on these findings we suppose that the inhibition of tubulin polymerization could prevent the EMT process therefore cell migration and also cancer metastases. In our experiments, the effect of anthelminitic drugs flubendazole (FLU) and mebendazole (MBZ) on TGF-β-induced EMT in dysplastic oral keratinocytes was tested in vitro. Expression of molecules involved in EMT process was examined on microRNA and mRNA level using RT-PCR. The protein level of EMT...
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Effect of parasite on activity of biotransformation and antioxidant enzymes in host intestine
Mládková, Zuzana ; Szotáková, Barbora (advisor) ; Skálová, Lenka (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Zuzana Mládková Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Effect of parasite on activity of biotransformation and antioxidant enzymes in host intestine Hymenolepis diminuta, known as a rat tapeworm, is commonly used in science as a model of Cestoda for studying physiology, biochemistry and drug metabolism in tapeworms. Recently, Hymenolepis diminuta has been studied for helminth-based therapy for a potential treatment of inflammatory bowel disease. The aim of this study was to determine how H. diminuta influences the activity of detoxification enzymes of the host intestine. At first 6 male rats (Wistar breed) were infected by cysticercoids of Hymenolepis diminuta previously isolated from the beetles Tenebrio molitor (intermediate host). At the same time the physiological saline solution was administered to the control group of 6 male rats. All rats were housed for 2 months in animal quarters with 12 h light/dark cycle. Thereafter, the tapeworms were removed from their intestines. Intestinal mucosa containing metabolic active enzymes was isolated. Subsequently, the subcelullar fractions were prepared and used for in vitro experiment. The activity of enzymes was...
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