doktorska disertacija
Maša Kenda (Author), Nataša Karas Kuželički (Mentor), Marija Sollner Dolenc (Co-mentor)

Abstract

Svetovna zdravstvena organizacija motilce endokrinega sistema (MES) definira kot »eksogene spojine ali zmesi, ki spremenijo funkcijo endokrinega sistema in posledično povzročajo neželene učinke pri zdravih organizmih, njihovih potomcih ali (sub)populacijah«. Povezani so s številnimi boleznimi in motnjami, kot so debelost, sladkorna bolezen, neplodnost, nepravilnosti v tiroidnem in nevroendokrinem sistemu ter razvojne nepravilnosti, spremenjeno delovanje imunskega sistema, razvoj in napredovanje rakavih obolenj zaradi učinka na povečano invazivnost celic in metastaziranje. Kljub temu pa učinki MES na celično adhezijo preko integrinov še niso dobro preučeni. Prav tako so podatki o njihovem delovanju na imunski sistem zelo heterogeni. MES na endokrini sistem delujejo preko številnih mehanizmov, pri tem pa lahko izkazujejo nemonotone učinke in učinke pri nizkih odmerkih. Testi za identifikacijo potencialnih MES temeljijo na dobro poznanih mehanizmih, vendar imajo pomanjkljivosti. V tej doktorski nalogi smo raziskali vpliv motilcev endokrinega sistema na delovanje imunskega sistema preko receptorja za aktivirano protein kinazo C 1 (RACK1) in na celične adhezijske procese preko vitronektinskega receptorja (VTNR, znan tudi kot integrin αVβ3), ter poskušali razviti in-vitro teste za identifikacijo takšnih MES. RACK1 ima pomembno vlogo pri aktivaciji imunskega odziva. Njegovo izražanje regulira ravnotežje med ravnjo glukokortikoidov in androgenov. V tej študiji smo raziskovali vpliv treh bisfenolov (BPA, BPAF in BPS) na izražanje RACK1 in na odziv prirojenega imunskega sistema na humani promielocitni celični liniji THP-1. BPA in BPAF sta znižala transkripcijsko aktivnost promotorja za RACK1, njegovo izražanje na ravni mRNA in proteina. BPS je imel obratne učinke. Kot pričakovano so učinki na izražanje RACK1 sovpadali z učinki na z lipopolisaharidom-inducirano produkcijo interlevkina 8 in tumor nekrotirajočega faktorja α. Pokazali smo vpletenost jedrnega dejavnika NF-κB, glukokortikoidnega receptorja (GR) in androgenega receptorja (AR) v regulacijo izražanja RACK1 ob izpostavitvi bisfenolom. RACK1 torej predstavlja pomembno tarčo MES in lahko služi kot biološki označevalec v presejalnih testih za imunotoksični potencial spojin. Podobni poskusi na RACK1 lahko služijo za razkritje delovanja spojin na več endokrinih receptorjev in tako pomagajo bolje osvetliti mehanizme delovanja takšnih spojin.

Keywords

motilci endokrinega sistema;presejalni testi;bisfenoli;celična adhezija;toksičnost motilcev;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FFA - Faculty of Pharmacy
Publisher: [M. Kenda]
UDC: 616.4+606-097(043.3)
COBISS: 73151747 Link will open in a new window
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Other data

Secondary language: English
Secondary title: ǂThe ǂeffect of endocrine disrupting chemicals on the function of the immune system and cell adhesion processes
Secondary abstract: World Health Organization defines endocrine disrupting chemicals (EDCs) as »exogenous substances or mixtures that alter function(s) of the endocrine system and consequently cause adverse effects in an intact organism, or its progeny, or (sub)populations«. They have been linked to numerous disorders, such as obesity, diabetes, infertility, impaired thyroid and neuroendocrine system functions, impaired development, altered function of the immune system, and cancer development and progression due to their effect on increased cell invasiveness and metastasis. However, their effects on integrinmediated cell adhesion are not yet well researched. Similarly, a very heterogenous body of data on their effect on the immune system function was produced. EDCs have various modes of action, while exerting nonmonotonic effects and effects at low doses. Tests for identification of EDCs are based on well-known mechanisms of action but have disadvantages. In this doctoral dissertation we will investigate the effect of EDCs on the immune system function through the receptor for activated kinase C 1 (RACK1), and on cell adhesion processes through the vitronectin receptor (VTNR, also known as integrin αVβ3). We will try to develop in-vitro tests for the identification of EDCs with such actions. RACK1 has an important role in immune activation and is regulated through a balance between glucocorticoid and androgen levels. In this study, we investigated the effects of three bisphenols (BPA, BPAF, BPS) on RACK1 expression and on the innate immune responses in the THP-1 human promyelocytic cell line. BPA and BPAF reduced RACK1 promoter transcriptional activity, mRNA expression, and protein levels; however, BPS had the opposite effects. As expected, these data for RACK1 expression were paralleled by lipopolysaccharide-induced interleukin-8 and tumor necrosis factor-α production. We demonstrated the involvement of nuclear factor NF-κB, glucocorticoid receptor (GR), and androgen receptor (AR) in the regulation of RACK1 expression upon exposure to bisphenols. Thus, RACK1 represents an important target of EDCs, and serves as a screening tool for immunotoxic potential. Furthermore, RACK1 can be exploited to unmask multiple molecular interactions of hormone-active substances, to better dissect out their mechanisms of action. The actions of MES on VTNR are implicated by binding sites for hormones on this receptor, which is involved in tumor angiogenesis and metastasis. VTNR-expressing human umbilical vein endothelial cells (HUVECs) were used to determine the effects of EDCs and endogenous hormones on cell adhesion to vitronectin-coated surfaces, and on VTNR activation. Cell adhesion was significantly increased for BPA, triclocarban, and triclosan (10, 100 nM; p <0.05), with similar trends for BPAF and BPS (10, 100 nM; p >0.05). These EDCs did not induce the activated conformation of VTNR, and showed nonmonotonic inverted U-shaped dose-response curves for cell adhesion. No changes in cell adhesion were seen for the endogenous hormones 5α-dihydrotestosterone, 17β-estradiol, and triiodothyronine, and for imatinib and paroxetine. These data indicate that EDC-mediated increases in HUVEC adhesion to vitronectin are not mediated through androgenic, estrogenic, or thyroid activities, nor through activation of VTNR. Although these effects of EDCs on HUVEC adhesion require further investigation of the underlying mechanism(s) of action to define their biological relevance, the low-dose effects and nonmonotonic responses revealed here define the urgent need for further risk assessment of these EDCs.
Secondary keywords: Endokrinologija;Imunski sistem;Hormonski motilci;Toksičnost;
Type (COBISS): Dissertation
Thesis comment: Univ. v Ljubljani, Fak. za farmacijo
Pages: 321 str.
ID: 15504088