doktorska disertacija
Darja Gramec (Avtor), Lucija Peterlin-Mašič (Mentor)

Povzetek

Motilci endokrinega sistema so po definiciji Svetovne zdravstvene organizacije iz leta 2002 opredeljeni kot od zunaj vnesene kemične snovi oziroma mešanice snovi, ki preko sprememb v delovanju hormonov povzročajo neželene učinke na zdravje posameznega organizma ali njegovega potomstva oziroma (sub)populacije (1). Izpostavljenost motilcem endokrinega sistema je povezana s številnimi boleznimi današnjega časa, kot so metabolni sindrom, srčno žilne bolezni, neplodnost in rak. Prisotni so povsod okoli nas, merljivi vplivi na biološke procese v telesu pa so bili dokazani že pri izredno nizkih koncentracijah, katerim smo tudi dejansko izpostavljeni. Bisfenol A, eden izmed najbolj znanih motilcev endokrinega sistema, lahko povzroči merljive spremembe v določenih celičnih funkcijah že pri koncentracijah od 1 pM-1 nM (2). Kljub obsežnim raziskavam, ki so bile v zadnjih letih opravljene na področju motilcev endokrinega sistema pa ostajajo številna vprašanja še vedno odprta in tako predstavljajo izziv za v prihodnje. V raziskavah smo se osredotočili na dve skupini potencialnih motilcev endokrinega sistema: strukturne analoge bisfenola A (BPA) in sicer bisfenol AF (BPAF), bisfenol S (BPS) in bisfenol F (BPF), ki se uporabljajo kot nadomestki BPA pri izdelavi plastičnih mas ter termalnega papirja ter na novejša bromirana zaviralca gorenja 2-etilheksil-2,3,4,5-tetrabromobenzoat (TBB) ter bis(2-etilheksil)2,3,4,5-tetrabromoftalat (TBPH). Slednja se z namenom preprečevanja gorenja dodajata izdelkom za vsakdanjo rabo, kot so na primer oblazinjeno pohištvo in elektronika. Izbrane spojine so s toksikološkega vidika izjemno zanimive, saj se množično uporabljajo in posledično smo jim ljudje vsakodnevno izpostavljeni. Kljub široki uporabi omenjenih spojin pa ostaja njihov vpliv na zdravje v veliki meri še neraziskan. V okviru doktorske naloge smo z in vitro metodami podrobno ovrednotili vpliv izbranih spojin ter njihovih metabolitov na naslednje endokrine receptorje v telesu: estrogenske (ER), androgenske (AR), tiroidne (TR), glukokortikoidne (GR), pregnanski X receptor (PXR), farnezoidni X receptor (FXR) ter na jedrna receptorja, aktivirana s peroksisomskim proliferatorjem (PPARα in PPARγ), pri čemer smo poseben poudarek namenili vplivu metabolizma na endokrino aktivnost. Metabolizem ima namreč lahko velik vpliv na samo endokrino aktivnost spojin. Za konjugate (glukuronide, sulfate,..) v večini primerov velja, da so brez aktivnosti na endokrinih receptorjih, medtem ko imajo lahko oksidativni metaboliti znatno večjo aktivnost od izhodne spojine (npr. Ipso metabolit BPA ima 500 krat večjo estrogeno aktivnost od BPA). V prvem sklopu raziskav smo podrobno proučili metabolizem izbranih bisfenolov. V članku z naslovom Differences in the glucuronidation of bisphenols F and S between two homologous human UGT enzymes, 1A9 and 1A10 smo proučevali glukuronidacijo izbranih bisfenolov tako z mikrosomskimi frakcijami kot tudi s posameznimi encimi UGT. Pokazali smo, da glukuronidacija predstavlja najpomembnejšo pot metabolizma za izbrane bisfenole, izpostavili pa smo tudi pomen ekstrahepatičnih tkiv pri glukuronidaciji bisfenolov. Zanimivo odkritje pa je, da so kljub veliki strukturni sorodnosti med izbranimi bisfenoli, različni UGT encimi odgovorni za njihovo glukuronidacijo. Jetrni encim UGT1A9 je bil najpomembnejši za glukuronidacijo BPS, UGT2B15 za BPA, črevesna UGT1A10 izooblika za BPF in UGT2A1 za BPAF (3). Pridobljene kinetične parametre glukuronidacije analogov BPA z jetrnimi in črevesnimi mikrosomi smo v sodelovanju z raziskovalci Švicarskega inštituta za tehnologijo uporabili pri postavitvi farmakokinetičnega modela, s katerim smo želeli ovrednotiti farmakokinetiko BPA in njegovih analogov ter posledično primerjati smiselnost zamenjave BPA z ostalimi bisfenoli, ter rezultate predstavili v članku z naslovom Physiologically based pharmacokinetic (PBPK) modeling of the bisphenols BPA, BPS, BPF, and BPAF with new experimental metabolic parameters: Comparing the pharmacokinetic behavior of BPA with its substitutes. Zgrajen fiziološki farmakokinetični model je prvi, ki opisuje kinetiko strukturnih analogov BPA in tako predstavlja pomemben prispevek k znanosti na področju farmakokinetike bisfenolov. Z uporabo modela smo ugotovili, da zamenjava BPA s strukturnimi analogi ne pomeni nujno tudi manjšega tveganja za endokrino delovanje. Ob enaki izpostavljenosti vsem testiranim bisfenolom smo namreč z modelom napovedali, da BPS doseže najvišje koncentracije v plazmi (4). V nadaljevanju smo proučevali oksidativni metabolizem bisfenolov. Pri tem smo se osredotočili predvsem na BPS, ter rezultate predstavili v članku z naslovom Influence of metabolism on endocrine activities of bisphenol S. Poglavitno oksidativno pretvorbo pri BPS predstavlja orto hidroksilacija aromatskega obroča, katalizirana s citokromi CYP3A4, CYP2C19 in CYP2C9, medtem ko hidroksilacija na meta mestu in tvorba ipso metabolita potekata v veliko manjšem obsegu. V nasprotju z BPA, pri BPS nismo detektirali reaktivnih metabolitov, ki smo jih merili kot konjugate z glutationom (5). V drugem sklopu raziskav pa smo se osredotočili na endokrine učinke izbranih spojin ter njihovih metabolitov. Pri tem smo uporabili različne in vitro testne sisteme: test s poročevalskim genom na GH3.TRE-Luc celični liniji (tiroidna aktivnost), test s poročevalskim genom na MDA-kb2 celični liniji (androgena in glukokortikoidna aktivnost), YES/YAS test na kvasovkah Saccharomyces cerevisiae (estrogena in androgena aktivnost) ter po OECD protokolu validiran test s poročevalskim genom na Hela9903 celični liniji (estrogena aktivnost). V sodelovanju z raziskovalci iz Medicinske fakultete, Univerze v Perugii, smo preverili delovanje izbranih spojin in metabolitov še na PXR, FXR ter na PPARα in PPARγ. V izvirnem znanstvenem članku z naslovom Influence of metabolism on endocrine activities of bisphenol S, smo pokazali, da metabolizem v primeru BPS deluje kot zaščitni sistem, ki zmanjša endokrino delovanje BPS ter tudi pospeši njegovo izločanje iz telesa. BPS je namreč deloval šibko agonistično na ER (EC50 = 84 μM), orto hidroksiliran metabolit je izkazoval še šibkejše delovanje na ER (EC50 = 670 μM), BPS glukuronid pa je bil brez aktivnosti (5). V članku z naslovom Endocrine activities and adipogenic effects in murine preadipocytes of bisphenol AF and its main metabolite (poslan v objavo) smo določali endokrino aktivnost BPA, BPAF in BPAF glukuronida. BPAF je v večini primerov izkazal močnejše delovanje na testirane receptorje kot pa BPA. Estrogena aktivnost BPAF, določena z in vitro testom na Hela9903 celični liniji, je bila 10 krat višja od aktivnosti BPA (EC50[BPAF] = 0,15 μM; EC50[BPA] = 1,12 μM). Prav tako je BPAF deloval močnejše antagonistično na TR (IC50[BPAF] = 7,6 μM; IC50[BPA] = 88,2 μM) in AR (IC50[BPAF] = 2,9 μM; IC50[BPA] = 5,5 μM). BPAF glukuronid je bil v nasprotju z izhodno spojino neaktiven na ER, TR, AR, GR in FXR receptorjih. Ugotovili pa smo antagonistično aktivnost BPAF glukuronida na PXR in PPARγ receptorja v mikromolarnem območju in tako pokazali, da glukuronid ni neaktiven metabolit, kot se je predvidevalo do sedaj. Rezultate testiranj endokrine aktivnosti smo nadgradili s proučevanjem njihovega vpliva na lipidno akumulacijo, saj je bilo že v predhodnih raziskavah potrjeno, da lahko BPA vpliva na adipogenezo. Ugotovili smo, da lahko tako BPAF kot tudi BPAF glukuronid značilno povečata lipidno akumulacijo ter ekspresijo številnih adipogenih markerjev. Zbrani rezultati kažejo, da imajo lahko tudi glukuronidi biološko aktivnost, kar predstavlja pomembno odkritje, hkrati pa kaže na nujnost nadaljnih raziskav na tem področju. Za izbrana zaviralca gorenja in njuna metabolita smo z uporabo in vitro testov ugotovili aktivnosti na številnih endokrinih receptorjih. V izvirnem znanstvenem članku z nasklovom New brominated flame retardants and their metabolites as activators of the pregnane X receptor smo z uporabo poročevalskega sistema na HepG2 celični liniji ugotovili, da sta tako zaviralca gorenja (TBB in TBPH) kot tudi njuna metabolita 2,3,4,5-tetrabromobenzojska kislina (TBBA) in 2-etilheksiltetrabromobenzoatni ester (TBMEPH) agonista PXR, ki je znan induktor metabolnih encimov in receptorjev. Metabolit TBMEPH je deloval močneje agonistično na PXR kot pa izhodna spojina TBPH. Vse testirane spojine so povečale izražanje gena za citokrom CYP3A4. Le-to lahko vodi do številnih neželenih interakcij na nivoju metabolizma (6). V članku z naslovom Estrogenic and androgenic activities of TBBA and TBMEPH, metabolites of novel brominated flame retardants, and selected bisphenols, using the XenoScreen XL YES/YAS assay smo z uporabo in vitro testa na kvasovkah preverjali delovanje izbranih spojin na ER in AR. Metabolita izbranih zaviralcev gorenja (TBBA in TBMEPH) sta delovala antagonistično na ER in AR, medtem ko sta bili izhodni spojini brez aktivnosti na omenjenih receptorjih. S tem smo potrdili hipotezo, da lahko metabolizem celo poveča biološko aktivnost izbranih spojin (7). V nadaljevanju pa smo z uporabo testa s poročevalskim genom za izbrana zaviralca gorenja določili še aktivnosti na GR in TR, ter rezultate objavili v članku z naslovom Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays. Ugotovili smo, da izkazujeta izbrana zaviralca gorenja (TBB in TBPH), ter njuna metabolita (TBBA in TBMEPH) antagonistično aktivnost na TR, izhodni spojini pa dodatno delujeta kot antagonista GR. Glede na njihovo široko uporabo in vsesplošno prisotnost v okolju obstaja utemeljena skrb za zdravje, predvsem pri najbolj ogroženih skupinah, kot so nosečnice in dojenčki (8). Med izvajanjem raziskav v okviru doktorske naloge smo pridobili obsežne podatke o metabolizmu in endokrinih učinkih bisfenolov ter novejših bromiranih zaviralcev gorenja. Potrdili smo, da izbrane spojine delujejo na številne endokrine receptorje v telesu in s tem odprli vprašanje, ali je s stališča varnosti njihova uporaba sploh upravičena. Ugotovili smo, da imajo lahko tudi glukuronidi endokrino aktivnost ter druge biološke učinke, kot je na primer vpliv na adipogenezo. S tem smo ovrgli dolgotrajno prepričanje, da so glukuronidi le neaktivni metaboliti, kar bo treba upoštevati tudi pri oceni tveganja za izbrane spojine. Opravljene raziskave predstavljajo dobro izhodišče pri izdelavi ocene tveganja za izbrane spojine, hkrati pa pridobljeni rezultati koristijo tudi splošni populaciji. Osveščanje o škodljivem delovanju izbranih spojin na endokrini sistem lahko namreč na dolgi rok vodi k manjši uporabi izbranih spojin pri izdelavi plastike, kot smo videli že pri BPA, pri čemer pa bo treba za njih najti ustrezne, varnejše alternative.

Ključne besede

kemikalije;zaviralci gorenja;bisfenol A;analogi;endokrini sistem;toksičnost;jetra;metabolizem;metaboliti;bioaktivacija;glukuronidacija;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [D. Gramec Skledar]
UDK: 612.43:620.266.1(043.3)
COBISS: 296871168 Povezava se bo odprla v novem oknu
Št. ogledov: 162
Št. prenosov: 0
Ocena: 0 (0 glasov)
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Sekundarni jezik: Angleški jezik
Sekundarni naslov: In vitro study of metabolic and endocrine effects of selected bisphenols and novel brominated flame retardants
Sekundarni povzetek: Endocrine disruptors are defined, according to World Health Organization (2002), as exogenous substances or mixtures that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations (1). Exposure to endocrine disruptors is associated with numerous diseases of our time, such as metabolic syndrome, cardiovascular disease, infertility and cancer. Their use is widespread and it is apparent that they can interfere with biological processes in our body at very low, environmentally relevant concentrations. Bisphenol A, for example, can cause measurable changes in certain cellular functions at concentrations of 1pM-1nM (2). Extensive research was performed in recent years in the area of endocrine disruptors. However, many questions remain open and therefore pose a challenge for the future. In this doctoral thesis, two groups of potential endocrine disruptors were evaluated: structural analogs of bisphenol A (BPA), like bisphenol AF (BPAF), bisphenol S (BPS) and bisphenol F (BPF), which are used as BPA substitutes in the manufacture of plastic and thermal paper and novel brominated flame retardants 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), and bis (2-ethylhexyil) 2,3,4,5-tetrabromophtalate (TBPH). Flame retardants are added to everyday products like electronic devices and furniture, to prevent fires. Selected compounds are extremely interesting from a toxicological point of view, because the general population is exposed daily to low concentrations of these chemicals. However, their impact on human health is still largely unknown. The main goal of this doctoral thesis was the evaluation of endocrine activities of selected compounds and their metabolites on the following endocrine receptors in the body: estrogen (ER), androgen (AR), thyroid (TR), glucocorticoid (GR), pregnane X receptor (PXR), farnesoid X receptor (FXR), and peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ), with particular emphasis placed on the effects of metabolism on endocrine activity. Metabolism can have a major influence on the endocrine activity of compounds. Conjugates (glucuronides, sulphates, ...) are usually without activity on endocrine receptors, while oxidative metabolites can have a significantly higher activity from the parent compound (e.g Ipso metabolite of BPA has 500 times greater estrogen activity than BPA). In the first section, the metabolism of selected bisphenols was evaluated. In an article entitled Differences in glucuronidation of bisphenols F and S between two homologous human UGT enzymes, 1A9 and 1A10, glucuronidation of selected bisphenols, both with microsomal fractions as well as with individual UGT enzymes, was studied. We showed that glucuronidation represents the most important metabolic pathway for selected bisphenols, and we highlighted the importance of non-hepatic tissues in the glucuronidation of bisphenols. Despite the great structural similarity between the selected bisphenols, various UGT enzymes are responsible for their glucuronidation. Liver UGT1A9 was the most important for glucuronidation of BPS, UGT2B15 for BPA, intestinal UGT1A10 isoform for BPF and UGT2A1 for BPAF (3). Kinetics parameters of bisphenols glucuronidation with hepatic and intestinal microsomes, that were previously determined in in vitro studies, were further used in collaboration with researchers of the Swiss Institute of Technology, for the establishment of a pharmacokinetic model. With the model we wanted to evaluate the pharmacokinetics of BPA and its analogs, and consequently compare the impact of replacing BPA with other bisphenols. The results of this work are presented in an article entitled Physiologically based pharmacokinetic (PBPK) modeling of bisphenols BPA, BPS, BPF and BPAF with new experimental metabolic parameters: Comparing the pharmacokinetic behavior of BPA with its substitutes. The built-in physiological pharmacokinetic model is the first that describes the kinetics of structural analogs of BPA, and thus represents an important contribution in the field of pharmacokinetics. With the model we found that the replacement of BPA with structural analogs does not necessarily lower the risk of endocrine activity (4). Furthermore, the oxidative metabolism of bisphenols was studied. We focused mainly on BPS oxidative metabolism and the results are collected in an article entitled Influence of metabolism on endocrine activities of bisphenol S. The major oxidative metabolic pathway of BPS is the ortho hydroxylation of the aromatic ring, catalysed by cytochromes CYP3A4, CYP2C19 and CYP2C9. Hydroxylation at meta position and the formation of ipso metabolites were minor oxidative transformations. In contrast to BPA, reactive metabolites of BPS, that were tracked as glutathione conjugates, were not detected (5). The second part of our research included evaluation of endocrine activities of selected compounds and their metabolites. We used variety of in vitro test systems: reporter assay in GH3.TRE-Luc cell line (thyroid activity), reporter assay in MDA-kb2 cell line (androgen and glucocorticoid activity), YES/YAS assay with Saccharomyces cerevisiae (estrogen and androgen activity) and OECD validated reporter gene assay on Hela9903 cell line (estrogen activity). In collaboration with colleagues from University in Perugia, Italy, the activities of selected compounds on pregnane X receptor (PXR), farnesoid X receptor (FXR), and peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) were evaluated. In the paper entitled Influence of metabolism on endocrine activities of bisphenol S, we showed that in the case of BPS, metabolism works as a protective system that lower the endocrine activity of BPS and also accelerates its elimination from the body. BPS showed weak agonist activity on ER (EC50 = 84 μM). Ortho hydroxylated metabolite showed weaker activity on the ER than BPS (EC50 = 670 μM), while BPS glucuronide was without any activity on ER (5). The endocrine activities of BPA, BPAF and BPAF glucuronide were collected in the article entitled Endocrine activities and adipogenic effects in mouse preadipocytes of bisphenol AF and its main metabolite (sent for publication). BPAF showed more potent activity on tested receptors than BPA. The estrogenic activity of BPAF, determined by an in vitro assay using the Hela9903 cell line, was 10 times higher than the BPA activity (EC50 [BPAF] = 0.15 μM; EC50 [BPA] = 1.12 μM). BPAF was also more potent antagonist on TR (IC50 [BPAF] = 7.6 μM; IC50 [BPA] = 88.2 μM) and AR (IC50 [BPAF] = 2.9 μM; IC50 [BPA] = 5.5 μM). BPAF glucuronide, in contrast, was inactive on ER, TR, AR, GR and FXR receptors. However, weak antagonist activity of BPAF glucuronide on PXR and PPARγ receptors was determined. BPAF glucuronide is therefore not an inactive metabolite, as it was believed for a long time. The results of endocrine activities were supplemented by studying the effect of bisphenols on lipid accumulation. Both, BPAF and BPAF glucuronide, significantly increased lipid accumulation and the expression of different adipogenic markers. We showed that glucuronides can have biological activity, which is an important discovery in this field, and indicates the need for further research. Selected flame retardants and their metabolites showed activities on numerous endocrine receptors in the body. In an article entitled New brominated flame retardants and their metabolites as activators of the pregnane X receptor, the PXR agonist activities of both flame retardants (TBB and TBPH) and their metabolites 2,3,4,5-tetrabromobenzoic acid (TBBA) and the 2-ethylhexyltetrabromobenzoate ester (TBMEPH) were presented. The metabolite TBMEPH was more potent PXR agonist than TBPH. All tested compounds increased the expression of the cytochrome CYP3A4 gene, which can result in different interactions at metabolism level (6). In the article entitled Estrogenic and androgenic activities of TBBA and TBMEPH, metabolites of novel brominated flame retardants, and selected bisphenols, using the XenoScreen XL YES/YAS assay, the activities of selected flame retardants and their metabolites on the ER and AR were determined, using in vitro assay on yeasts. The metabolites of selected flame retardants (TBBA and TBMEPH) were ER and AR antagonists, while parent compounds were without activity on tested receptors. Metabolism can therefore even increase the biological activity of the selected flame retardants (7). Furthermore, using reporter gene assays, activities of selected flame retardants and their metabolites on GR and TR were determined and presented in an article entitled Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays. We found that the selected flame retardants (TBB and TBPH) and their metabolites (TBBA and TBMEPH) act as antagonists on TR, and the starting compounds further act as GR antagonists. Given their widespread use and overall presence in the environment, there is a high concern for their impact on human health, especially in the most vulnerable groups, such as pregnant women and infants (8). In the current doctoral thesis, extensive research on metabolism and endocrine effects of bisphenols and newer brominated flame retardants was conducted. We confirmed the activities of tested compounds on numerous endocrine receptors and thus raise the question about suitability of their use. We found that BPAF glucuronide can also have endocrine activity and other biological effects, like the effect on adipogenesis. We therefore refuted the long-standing belief that glucuronides are only inactive metabolites. The new knowledge about activities of glucuronides should be taken into account when performing risk assessment for selected compounds. The extensive research conducted represent a good starting point for the risk assessment of the selected compounds. Additionally, these results can be valuable for the general population. Raising awareness about the harmful effects of selected compounds on the endocrine system can in the long run lead to lower use of products containing these substances as it was already observed with BPA.
Sekundarne ključne besede: Hormonski motilci;Disertacije;
Vrsta dela (COBISS): Doktorska disertacija
Komentar na gradivo: Univ. v Ljubljani, Fak. za farmacijo
Strani: X, 283 str.
ID: 15645135