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Potential toxicity of terpenes and their effects in liver cells
Zárybnický, Tomáš ; Boušová, Iva (advisor) ; Anzenbacherová, Eva (referee) ; Trejtnar, František (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Tomáš Zárybnický Supervisor: assoc. prof. PharmDr. Iva Boušová, Ph.D. Consultant: assoc. prof. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: Potential toxicity of terpenes and their effects in liver cells The public interest in herbal products, supplements, extracts or isolated active compounds has been increasing during last decades. The aim of this doctoral thesis was to study selected compounds from the group of monoterpenes and sesquiterpenes and their interactions with liver cells: interactions with drug metabolising enzymes, their potential liver toxicity and looking for novel mechanisms of action. The literature about hepatotoxic properties of several monoterpenes and sesquiterpenes, that showed liver toxicity, was summarized first. Then, the study was aimed toward a known hepatotoxicant (R)-pulegone and its presumed metabolite (R)- menthofuran. The toxicity results in precision-cut human liver slices have shown that (R)- menthofuran was less toxic to human liver and that the reason may be inter-species differences between human and mice. Several sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, β-caryophyllene, and caryophyllene oxide) have previously...
Pistacia lenstiscus essential oil: in vitro testing of cytotoxicity and anti-inflammatory activity
Elstnerová, Marie ; Szotáková, Barbora (advisor) ; Boušová, Iva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Marie Elstnerová Supervisor: prof. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: Pistacia lentiscus essential oil: in vitro testing of cytotoxicity and anti-inflammatory activity This project, which has been initiated in cooperation with University of Sassari (Italy), is aimed to find out more details about biological activity of essential oil from Pistacia lentiscus. We want to contribute to the decision, if this essential oil can be used as a part of oral health products. The essential oil was prepared from leaves of wild occurring plants growing in North Sardinia. The cytotoxicity and anti-inflammatory activity of not only the pure essential oil but also of the lecithin nanoemulsion with P. lentiscus oil and Tween 80 nanoemulsion with P. lentiscus oil and moreover, the two most abundant terpenes in the essential oil, (−)-α-pinene and (−)-terpinen-4-ol, were tested. The cytotoxicity was measured by the colorimetric assay using tetrazolium salt WST-1 on four oral cell lines (gingival fibroblasts, periodontal ligament fibroblasts, dysplastic oral keratinocytes and primary gingival keratinocytes). The viability of human oral cells was the most diminished by lecithin nanoemulsion, pure...
Effect of monoterpene citral on the expression of detoxification enzymes in HepaRG cells
Švábová, Tereza ; Boušová, Iva (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Tereza Švábová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Consultant: Mgr. Michaela Šadibolová Title of diploma thesis: Effect of monoterpene citral on the expression of detoxification enzymes in HepaRG cells Citral is an acyclic monoterpene, which is mainly produced by higher plants. It is one of the many secondary metabolites, which serve the plant as detoxifying factors, morphoregulators and attractants for pollinators or defense substances. Besides other things, it has numerou positive effects such as antibacterial, anti-inflammatory or antitumor, but it may be toxic, especially for the liver, if used incorrectly. The main goal of this thesis was to find out the effect of citral on selected biotransformation and antioxidant enzymes in HepaRG cells. Differentiated HepaRG cells were incubated with 10 µM rifampicin, 10 µM β-naphthoflavone, 30 µM oltipraz, 10 µM citral, 30 µM citral and 100 µM citral for mRNA expression detection for 12 and 24 hours and for protein expression evaluation for 24 and 48 hours. At the mRNA level, rifampicin induced CYP3A4 at 12- and 24-h incubation, β-naphthoflavone induced CYP1A2 at 12- and 24-h incubation and aldo-ketoreductase (AKR1C) at 24-h incubation,...
Selection and validation of reference genes for relative mRNA quantification in human liver slices
Studenovská, Petra ; Boušová, Iva (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Petra Studenovská Supervisor: doc. PharmDr. Boušová Iva, Ph.D. Title of diploma thesis: Selection and validation of reference genes for relative mRNA quantification in human liver slices The use of precision-cut liver slices is often employed in vitro system in the biochemical, pharmacological and toxicological studies. This is mainly due to the preservation of the cellular tissue architecture and therefore its functions. The aim of this diploma thesis was to select and validate reference genes for the relative quantification of mRNA in human liver slices. Liver tissues obtained from 3 patients of different age, diagnosis and pharmacotherapy were used as samples. Liver slices, prepared from samples of liver tissue, were exposed to dimethyl sulfoxide (control) or known inducers of cytochrome P450, rifampicin (10 µM) and β-naphthoflavone (10 µM). Subsequently, the stability of the expression of reference genes was verified using RefFinder program, which integrates the programs of the geNorm, BestKeeper, NormFinder and comparative ΔCT method. The optimal number of reference genes that should be used to evaluate the relative gene expression of target genes was determined using the geNorm program....
Regulation of glutathione peroxidase 7 by microRNAs
Vávrová, Gabriela ; Matoušková, Petra (advisor) ; Boušová, Iva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Gabriela Vávrová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Regulation of glutathione peroxidase 7 by microRNA Glutathione peroxidases (GPxs) are considered to be key antioxidant enzymes. A total of 8 human genes encoding GPxs are known. The main task of these enzymes is protecting biomolecules from oxidative damage due to oxidative stress, which arises from the imbalance between the generation and removal of free radicals. These enzymes catalyze the reduction of free hydrogen peroxide and organic hydroperoxides to water or corresponding alcohols. This work is focused on influencing gene expression of monomeric enzyme GPx7 by microRNA. GPx7 is involved in the correct composition of proteins in the endoplasmic reticulum, in the proper course of adipogenesis, and suppresses development of cancer. Short single-stranded non-coding microRNA molecules are involved in post-transcriptional regulation of gene expression. These molecules negatively influence the translation process, ie the translation of mRNA into the protein. MicroRNAs bind to the binding region predominantly inside of the 3'UTR mRNA of the target gene. The aim of this study was to experimentally...
Modulatory effect of cis-nerolidol, trans-nerolidol and farnesol on selected phase 1 drug-metabolizing enzymes in human liver slices
Deingruberová, Kristýna ; Boušová, Iva (advisor) ; Skarková, Veronika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Bc. et Bc. Kristýna Deingruberová Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of diploma thesis: Modulatory effect of cis-nerolidol, trans-nerolidol and farnesol on selected phase 1 drug-metabolizing enzymes in human liver slices Sesquiterpenes are natural compounds which can be found in alternative medicines or food supplements. However, not all representatives have a beneficial effect on health. Indeed, sufficient attention should be paid to the evidence of serious toxicities and side effects. The aim of this diploma thesis was to find out, whether acyclic sesquiterpenes cis-nerolidol (cNER), trans-nerolidol (tNER) and farnesol (FAR) possess a modulatory effect on protein and gene expression of selected phase 1 drug-metabolizing enzymes - four cytochrome P450 (CYP) isoforms, namely CYP1A2, CYP2B6, CYP2C and CYP3A4, carbonyl reductase 1 (CBR1) and aldo-ketoreductase 1C3 (AKR1C3). The ultra-thin liver slices from five patients of both sexes aged 45 to 81 years were used as studied material. The liver slices were incubated with studied compounds at a concentration of 10 µM (in one patient, cNER and tNER also at 30 µM concentration) at 37 řC for 24 hours. The protein expression was determined by...
Survivin, a novel target of the Hedgehog/GLI signaling pathway in human tumor cells
Žáková, Petra ; Boušová, Iva (advisor) ; Skarková, Veronika (referee)
Survivin, an important antiapoptotic protein, is expressed in tumors, whereas in normal tissues the expression of this protein is extremely low, defining a role for survivin as a cancer gene. Survivin exhibits multifunctional activity in tumor cells. However, why survivin expression is sharply and invariably restricted to tumor tissue remains unclear. Here, we identified 11 putative consensus binding sites for GLI transcription factors in the survivin promoter and characterized the promoter activity. Inhibitors of the Hedgehog/GLI pathway, cyclopamine and GANT61, decreased the promoter activity in reporter assays. ΔNGLI2 (which lacks the repressor domain) was the most potent vector in activating the survivin promoter-reporter. Moreover, GANT61, a GLI1/2 inhibitor, repressed endogenous survivin protein and mRNA expression in most cells across a large panel of tumor cell lines. Chromatin immunoprecipitation showed GLI2 binding to the survivin promoter. The ectopic GLI2-evoked expression of endogenous survivin was observed in normal human fibroblasts. GANT61 decreased survivin level in nude mice tumors, mimicking the activity of GANT61 in cultured cells. The immunohistochemistry and double immunofluorescence of human tumors revealed a correlation between the tissue regions showing high GLI2 and...
Toxicity of pulegone and menthofuran and their effect on miRNA expression in human liver slices
Lancošová, Bibiána ; Boušová, Iva (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bibiána Lancošová Supervisor: doc. PharmDr. Boušová Iva, Ph.D Title of diploma thesis: Toxicity of pulegone and menthofuran and their effect on miRNA expression in human liver slices Monoterpenes are secondary plant metabolites, characterised by their odour, which have a positive impact on human body and are used not only in cosmetic and food industry, but also in medication. R-pulegone (PUL) and its metabolite R-menthofuran (MF) are the most represented in plants of genus Mentha (Mint), with higher doses of these substances exerting certain risk of hepatotoxicity. The aim of this thesis was to assess the concentration, at which the viability of liver cells decreases and also to observe the changes in individual microRNA expression as a result of influence of PUL, MF and paracetamol, as the reference compound. Five samples of human liver obtained from patients of both genders and at age of 57 to 73 years represented biological material for hepatotoxicity testing. The samples were stored in Euro-Collins solution and subsequently cut into precision-cut liver slices with thickness of 150- 170 µm, placed to 12-well plate and put into incubation medium with the tested substances for 24 hours. The...
Effect of selected sesquiterpenes on carbonyl reductase 1 activity in vitro
Mazurková, Veronika ; Boušová, Iva (advisor) ; Szotáková, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Veronika Mazurková Supervisor: doc. PharmDr. Iva Boušová, Ph.D. Title of rigorous thesis: Effect of selected sesquiterpenes on carbonyl reductase 1 activity in vitro Sesquiterpenes, the main components of plant essential oils, are integral parts of spices, traditional food and beverages. They are used in folk medicines, pharmaceutical industry as well as in cosmetics. Several sesquiterpenes possess interesting biological activities but they could interact with concurrently administered drugs via the modulation of activity and/or expression of drug-metabolizing enzymes. Enzyme carbonyl reductase 1 (CBR1) is one of the enzymes participating in the metabolism of a number of endogenous and xenobiotic compounds. One of the CBR1 substrates is chemotherapeutic agent doxorubicin, which is metabolized by this enzyme to the less effective metabolite doxorubicinol, which is responsible for the doxorubicin cardiotoxicity. The aim of this rigorous thesis was to find out the ability of five selected sesquiterpenes (i.e. valencene, β-caryophyllene oxide, α-humulene, cis-nerolidol and trans-nerolidol) to affect the activity of CBR1 in vitro. The effect of studied substances on the activity of this enzyme was...

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