20.500.12556/RUL-121062 Koncentracijska odvisnost učinka izbranih nesteroidnih protivnetnih učinkovin in piceatanola na androgenih in glukokortikoidnih receptorjih, izraženih v celični liniji MDA-kb2 magistrska naloga Concentration dependence of the effect of selected nonsteroidal anti-inflammatory drugs and piceatanol on the androgen and glucocorticoid receptors, expressed in MDA-kb2 cell line Široka uporaba nesteroidnih protivnetnih učinkovin (NSAID) je pripeljala do marsikaterih dolgoročnih in potencialno škodljivih posledic za organizme v okolju, tudi za človeka. Mnoge od teh so nepojasnjene, nekatere raziskave pa že potrjujejo, da določene zdravilne učinkovine, vključno z NSAID, delujejo kot hormonski motilci. Dosedanje in vitro in in vivo študije so pokazale, da so NSAID sposobne modulirati estrogenski (ER), progesteronski (PR) in s peroksisomskim proliferatorjem aktivirani receptor gama (PPARγ). Učinki NSAID na druge receptorje, kot sta npr. androgeni (AR) in glukokortikoidni receptor (GR), so manj raziskani. S tem namenom smo v okviru magistrske naloge preučevali vpliv izbranih NSAID na omenjena receptorja. Poleg dveh tipičnih predstavnikov NSAID, diklofenak (DIC) in njegov metabolit 4-hidroksidikofenak (4-HD), smo izbrali še paracetamol (PAR) in predstavnika naravnega izvora s protivnetnim delovanjem, to je piceatanol (PIC). Vsem je skupno protivnetno delovanje, ki je najbolj značilno za skupino NSAID. Večina predstavnikov NSAID inhibira dva izoencima ciklooksigenaze (COX), COX-1 in COX-2. Za DIC je v manjši meri značilna še inhibicija COX-3. Slednjo pripisujejo tudi PAR. Kot modelni sistem za raziskovanje smo uporabili celično linijo MDA-kb2, ki izraža funkcionalne androgene in glukokortikoidne receptorje z možnostjo razlikovanja agonističnega in antagonističnega delovanja testirane spojine. Najprej smo s testom citotoksičnosti testirali preiskovane spojine v prvotno izbranih koncentracijah, za katere smo potrdili, da je pri njih preživetje celic večje kot 80 %. Za DIC, PAR in PIC je bila prva necitotoksična koncentracija 175 μM, za 4-HD pa 250 μM. Prve necitotoksične in od njih nižje koncentracije spojin smo testirali v nadaljnjem in vitro testu. To je bil presejalni luciferazni test, ki temelji na aktivaciji transkripcije gena za encim luciferazo. Aktivacija transkripcije oziroma stopnja aktivnosti luciferaze je služil kot pokazatelj učinka preiskovanih spojin na AR oziroma GR. Rezultati testa so pokazali, da imajo vse preiskovane spojine pri določenih koncentracijah statistično značilno agonistično delovanje na AR in GR. Spojini DIC in PAR delujeta agonistično na AR pri koncentracijah 175, 100, 10, 1 in 0.1 μM, spojina 4-HD pri koncentracijah 250, 100, 10, 1 in 0.001 μM, spojina PIC pri koncentracijah 175, 100 in 1 μM. Agonistični učinek na GR sta spojini DIC in PAR izkazali pri koncentraciji 175 μM, spojina 4-HD pri 250, 100 in 10 μM ter PIC pri 175, 100 in 10μM. Vse preiskovane spojine, razen PIC, delujejo na AR tudi antagonistično, in sicer DIC pri koncentracijah 175, 10, 1 in 0.01 μM, 4-HD pri 250, 100, 10, 1 in 0001 μM, PAR pri 10, 1, 0.01 in 0.001 μM. Šibko antiglukokortikoidno delovanje smo dokazali samo za spojino DIC pri koncentracijah 175 in 100 μM ter za njegov metabolit 4-HD pri koncentracijah 250 in 100 μM. Na podlagi eksperimentalnih rezultatov lahko trdimo, da izbrane spojine delujejo kot modulatorji AR in GR. Ne smemo zanemariti dejstva, da so nekatere preiskovane spojine zmožne modulacije AR in GR tako pri terapevtskih koncentracijah, kot tudi pri nižjih koncentracijah. Slednje lahko najdemo tudi v okoljskih vodnih virih. Vsekakor pa so potrebne še podrobnejše in vitro in in vivo študije, s katerimi bi ovrednotili natančen mehanizem delovanja in potencialno škodljive učinke, ki jih imajo preiskovane spojine na androgeni in glukokortikoidni sistem. Common usage of nonsteroidal anti-inflammatory drugs (NSAID) has led to many longterm and potentially harmful effects for organisms, including human. Many of these effects remain unclarified, although some researches have already confirmed that some drugs, including NDAID, act as endocrine disruptors. The findings of in vitro and in vivo studies so far show, that NSAID are able to modulate estrogen receptor (ER), progesterone receptor (PR) and the peroxisome proliferator activated receptor gamma (PPARγ). Effects of NSAID on other receptors, e. g. androgen receptor (AR) and glucocorticoid receptor (GR) are less studied. With this purpose, we studied in our thesis the effects of selected NSAID on these two receptors. Along with two typical NSAID representatives, diclofenac (DIC) and its metabolite 4-hydroxydiclofenac (4-HD), we also chose paracetamol (PAR) and natural representative with anti-inflammatory effect, piceatannol (PIC). They all have antiinflammatory effect in common, although it is broadly known as a characteristic of NSAID. Most representatives of group of NSAID inhibit two isoforms of cyclooxygenase (COX), COX-1 and COX-2. Diclofenac in lesser extent also inhibits COX-3. They attribute this inhibition also to PAR. As a model system for determination their effects we used the cell line MDA-kb2, which expresses functional androgen and glucocorticoid receptor with the ability to differ agonistic and antagonistic properties of the selected substance. First, we used cytotoxicity test to confirm that selected substances in firstly chosen concentrations show cell survival rate more than 80 %. For DIC, PAR and PIC first non-cytotoxic concentration was 175 μM and for 4- HD it was 250 μM. The first non-cytotoxic concentrations and lower concentrations were used later on with in vitro assay. This was screening luciferase assay based on the activation of transcription of the gene for luciferase. Activation of transcription or level of luciferase activity was used as indicator of the effect of tested substances on AR or GR. The test results showed that all test substances have significant agonistic effect on AR and GR at certain concentrations. Agents DIC and PAR work as agonists of AR at concentrations 175, 100, 10, 1 and 0.1 μM, substance 4-HD at concentrations 250, 100, 10, 1 and 0.001 μM, substance PIC at 175, 100 and 1 μM. Agonistic effect on GR of DIC and PAR was showed at concentration175 μM, substance 4-HD showed this at 250, 100 and 10 μM, PIC at 175, 100 and 10 μM. All tested substances, except PIC, works also as antagonist of AR, namely DIC at concentrations 175, 10, 1 and 0.01 μM, 4-HD at 250, 100, 10, 1 and 0001 μM, PAR at 10,1, 0.01 and 0.001 μM. Weak anti-glucocorticoid effect was evidenced only for substances DIC at concentrations 175 and 100 μM, along with its metabolite 4-HD at concentrations 250 and 100 μM. On the basis of our experimental results, we can claim that selected substances modulate AR and GR. We must not neglect the fact that tested substances are able to modulate AR and GR at therapeutic concentrations and also at lower concentrations, which were also found in water sources in the environment. Definitely more detailed in vitro and in vivo studies are needed to evaluate exact mechanisms of action and potentially harmful effects of the selected substances on androgen and glucocorticoid system. hormonski motilci diklofenak 4-hidroksidiklofenak paracetamol piceatanol androgeni receptor glukokortikoidni receptor celična linija MDA-kb2 endocrine disruptors diclofenac 4-hydroxydiclofenac paracetamol piceatannol androgen receptor glucocorticoid receptor MDA-kb2 cell line true false false [B. Sladič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-09-29 13:17:47 2020-09-29 13:17:47 2022-09-01 04:01:22 0000-00-00 00:00:00 2015 0 Ljubljana 2015 Ljubljana XIV, 75 f. 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 615.9+615.357(043.3) 4008561 MAG_Sladic_Barbara_2015.pdf MAG_Sladic_Barbara_2015.pdf 1 0DF1B2363FE81787BD98B9530780CB75 d6394fc3099c2bea517dc63b4714ede680d49bce415322291f6f7028884a65cc 37379d16-a1ba-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=135921 Fakulteta za farmacijo 0 0 0