Modulation of biotransformation and antioxidant enzymes by selected natural compounds

Univerzita Karlova Farmaceutická fakulta v Hradci Králové Katedra biochemických věd Kandidát: Mgr. Kateřina LNĚNIČKOVÁ Školitel: prof. Ing. Barbora SZOTÁKOVÁ, Ph.D. Název disertační práce: MODULACE BIOTRANSFORMAČNÍCH A ANTIOXIDAČNÍCH ENZYMŮ VYBRANÝMI PŘÍRODNÍMI LÁTKAMI V posledních desetiletích významně stoupl zájem o různé doplňky stravy, které obsahují léčivé byliny, rostlinné extrakty či izolované účinné látky. Jejich spotřeba celosvětově roste, a protože jsou obecně považovány za bezpečné, jsou mnohdy konzumovány v nepřiměřeně velkých dávkách. Po vstupu do organismu jsou tyto látky, stejně jako jiná xenobiotika, modifikovány především prostřednictvím biotransformačních enzymů a současně mohou tyto enzymy ovlivňovat. Případná modulace aktivity (indukce nebo inhibice) biotransformačních enzymů může významně ovlivnit farmakokinetiku současně podávaných léčiv. Pro bezpečné užívání přírodních látek je tedy nezbytná znalost jejich možného působení na biotransformační enzymy. Cílem předkládané disertační práce bylo studovat účinky extraktů vybraných rostlin a jejich obsahových látek s biologickým účinkem na aktivitu a expresi biotransformačních a antioxidačních enzymů. Zaměřili jsme se na studium účinků extraktů a obsahových látek z brusinky velkoplodé (Vaccinium macrocarpon, Ericaceae), zeleného čaje...Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Kateřina LNĚNIČKOVÁ Supervisor: prof. Ing. Barbora SZOTÁKOVÁ, Ph.D. Title of Doctoral Thesis: MODULATION OF BIOTRANSFORMATION AND ANTIOXIDANT ENZYMES BY SELECTED NATURAL COMPOUNDS Public interest in various dietary supplements containing herbs, herbal extracts or isolated active compounds has increased significantly over past decades. Consumption of these supplements increases worldwide and they are often consumed in unreasonably high doses, as they are generally considered as safe. Upon the intake to organism, these compounds are, as other xenobiotics, modified mostly by xenobiotic-metabolizing enzymes and they could influence these enzymes at the same time. Potential modulation of xenobiotic-metabolizing enzymes' activity (induction or inhibition) can seriously affect pharmacokinetics of concomitantly administered drugs. Knowledge of the possible impact of natural compounds on the xenobiotic-metabolizing enzymes is essential for their safe use. The aim of this doctoral thesis was to study the effects of selected herbal extracts and their active chemical constituents on the activity and expression of xenobiotic-metabolizing and antioxidant enzymes. We have focused on the study of effects of...Katedra biochemických vědDepartment of Biochemical SciencesFaculty of Pharmacy in Hradec KrálovéFarmaceutická fakulta v Hradci Králov

Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels

In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/or one parameter testing could produce misleading information

The use of tissue slices in xenobiotic biotransformation studies

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Lněničková Supervisor: Doc. Ing. Barbora Szotáková, Ph.D. Title of diploma thesis: The use of tissue slices in xenobiotic biotransformation studies Precision-cut tissue slices are a unique in vitro model. Maintaining the tissue architecture gives them an advantage that can not be achieved in cell suspensions. They are also less demanding for number of required laboratory animals than perfused organs. The tissue slices are used mainly in biochemical, toxicological, physiological and pharmacological studies. The aim of this project was to establish optimal conditions for preparation and incubation of precision-cut tissue slices. The liver and small intestine of laboratory rats (Rattus norvegicus, Wistar strain) were used for tissue slices preparation. Three methods of monitoring of tissue slices viability were tested - MTT assay, NR-assay and the activity of lactate dehydrogenase (LDH). Monitoring of LDH leakage into the medium provided the most accurate values and this method was used in all subsequent experiments. Viability of slices after 24 h was very low. Specific activity of glutathione S-transferase detected in the intestinal slices was low and there was a significant..

Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells

The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE) and epigallocatechin gallate (EGCG) using both proliferating (P) and differentiated (D) caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes’ activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST) activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT) activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well

Modulation of biotransformation and antioxidant enzymes by selected natural compounds

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Kateřina LNĚNIČKOVÁ Supervisor: prof. Ing. Barbora SZOTÁKOVÁ, Ph.D. Title of Doctoral Thesis: MODULATION OF BIOTRANSFORMATION AND ANTIOXIDANT ENZYMES BY SELECTED NATURAL COMPOUNDS Public interest in various dietary supplements containing herbs, herbal extracts or isolated active compounds has increased significantly over past decades. Consumption of these supplements increases worldwide and they are often consumed in unreasonably high doses, as they are generally considered as safe. Upon the intake to organism, these compounds are, as other xenobiotics, modified mostly by xenobiotic-metabolizing enzymes and they could influence these enzymes at the same time. Potential modulation of xenobiotic-metabolizing enzymes' activity (induction or inhibition) can seriously affect pharmacokinetics of concomitantly administered drugs. Knowledge of the possible impact of natural compounds on the xenobiotic-metabolizing enzymes is essential for their safe use. The aim of this doctoral thesis was to study the effects of selected herbal extracts and their active chemical constituents on the activity and expression of xenobiotic-metabolizing and antioxidant enzymes. We have focused on the study of effects of..

The Modulation of Phase II Drug-Metabolizing Enzymes in Proliferating and Differentiated CaCo-2 Cells by Hop-Derived Prenylflavonoids

Prenylflavonoids in the human organism exhibit various health-beneficial activities, although they may interfere with drugs via the modulation of the expression and/or activity of drug-metabolizing enzymes. As intestinal cells are exposed to the highest concentrations of prenylflavonoids, we decided to study the cytotoxicity and modulatory effects of the four main hop-derived prenylflavonoids on the activities and mRNA expression of the main drug-conjugating enzymes in human CaCo-2 cells. Proliferating CaCo-2 cells were used for these purposes as a model of colorectal cancer cells, and differentiated CaCo-2 cells were used as an enterocyte-like model. All the tested prenylflavonoids inhibited the CaCo-2 cells proliferation, with xanthohumol proving the most effective (IC50 8.5 µM). The prenylflavonoids modulated the activities and expressions of the studied enzymes to a greater extent in the differentiated, as opposed to the proliferating, CaCo-2 cells. In the differentiated cells, all the prenylflavonoids caused a marked increase in glutathione S-transferase and catechol-O-methyltransferase activities, while the activity of sulfotransferase was significantly inhibited. Moreover, the prenylflavonoids upregulated the mRNA expression of uridine diphosphate (UDP)-glucuronosyl transferase 1A6 and downregulated that of glutathione S-transferase 1A1/2

Sulforaphane Alters β-Naphthoflavone-Induced Changes in Activity and Expression of Drug-Metabolizing Enzymes in Rat Hepatocytes

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, exerts many beneficial effects on human health such as antioxidant, anti-inflammatory, and anticancer effects. The effect of SFN alone on drug-metabolizing enzymes (DMEs) has been investigated in numerous in vitro and in vivo models, but little is known about the effect of SFN in combination with cytochrome P450 (CYP) inducer. The aim of our study was to evaluate the effect of SFN on the activity and gene expression of selected DMEs in primary cultures of rat hepatocytes treated or non-treated with β-naphthoflavone (BNF), the model CYP1A inducer. In our study, SFN alone did not significantly alter the activity and expression of the studied DMEs, except for the glutathione S-transferase (GSTA1) mRNA level, which was significantly enhanced. Co-treatment of hepatocytes with SFN and BNF led to a substantial increase in sulfotransferase, aldoketoreductase 1C, carbonylreductase 1 and NAD(P)H:quinone oxidoreductase 1 activity and a marked decrease in cytochrome P450 (CYP) Cyp1a1, Cyp2b and Cyp3a4 expression in comparison to the treatment with BNF alone. Sulforaphane is able to modulate the activity and/or expression of DMEs, thus shifting the balance of carcinogen metabolism toward deactivation, which could represent an important mechanism of its chemopreventive activity

Antiproliferative Effects of Hop-derived Prenylflavonoids and Their Influence on the Efficacy of Oxaliplatine, 5-fluorouracil and Irinotecan in Human ColorectalC Cells

Beer, the most popular beverage containing hops, is also frequently consumed by cancer patients. Moreover, non-alcoholic beer, owing to its nutritional value and high content of biological active compounds, is sometimes recommended to patients by oncologists. However, the potential benefits and negatives have to date not been sufficiently evaluated. The present study was designed to examine the effects of four main hop-derived prenylflavonoids on the viability, reactive oxygen species (ROS) formation, activity of caspases, and efficiency of the chemotherapeutics 5-fluorouracil (5-FU), oxaliplatin (OxPt) and irinotecan (IRI) in colorectal cancer cell lines SW480, SW620 and CaCo-2. All the prenylflavonoids exerted substantial antiproliferative effects in all cell lines, with xanthohumol being the most effective (IC50 ranging from 3.6 to 7.3 µM). Isoxanthohumol increased ROS formation and the activity of caspases-3/7, but 6-prenylnaringenin and 8-prenylnaringenin exerted antioxidant properties. As 6-prenylnaringenin acted synergistically with IRI, its potential in combination therapy deserves further study. However, other prenylflavonoids acted antagonistically with all chemotherapeutics at least in one cell line. Therefore, consumption of beer during chemotherapy with 5-FU, OxPt and IRI should be avoided, as the prenylflavonoids in beer could decrease the efficacy of the treatment

Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels

In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/ or one parameter testing could produce misleading information