Vpliv bisfenola A in njegovih izbranih analogov na izločanje citokinov v monocitni celični liniji THP-1

Žižek, Pia (author), Sollner Dolenc, Marija (mentor), Franko, Nina (comentor)

Bisfenol A (BPA) je najprepoznavnejši predstavnik bisfenolov, ki ga med drugim najdemo v številnih potrošniških izdelkih, zraku, pitni vodi ter bioloških vzorcih ljudi. Zaradi zaskrbljujočih učinkov tega motilca endokrinega sistema (MES) na zdravje ljudi je Evropska komisija omejila ali celo prepovedala njegovo uporabo pri proizvodnji in uvozu nekaterih potrošniških izdelkov, Evropska agencija za varnost hrane (EFSA) pa je znižala njegov dopusten dnevni vnos. To je povečalo uporabo njegovih številnih strukturnih analogov, ki jih je posledično že mogoče zaznati v okolju in pri ljudeh. Analogi BPA so problematični zlasti zaradi pomanjkanja podatkov o njihovih mehanizmih delovanja in učinkih. Namen naloge je bil preučiti vpliv sedmih bisfenolov BPA, BPAP, BPE, BPP, BPS-MAE, BPZ in TCBPA na metabolno aktivnost in imunomodulacijo v monocitni celični liniji THP-1, ki smo jo diferencirali v makrofage. Vpliv izbranih bisfenolov na metabolno aktivnost smo določali s testom z resazurinom in merjenjem fluorescence ter določili srednjo inhibitorno koncentracijo (IC50) posameznega bisfenola, kjer je bilo metabolno aktivnih 50 % celic. Najnižjo IC50 smo določili BPP (19,1 ± 0,3 μM), ki je torej najbolj citotoksičen. Sledili so BPAP (37,1 ± 1,2 μM), BPZ (43,5 ± 2,3 μM), TCBPA (64,4 ± 7,6 μM), BPA (122,6 ± 5,7 μM) in BPS-MAE (137,5 ± 7,0 μM), BPE pa je najmanj citotoksičen (220,2 ± 47,7 μM). Prav tako smo ugotovili, da obstaja korelacija med lipofilnostjo oz. logP in citotoksičnostjo. Nato smo s pretočno citometrijo preučevali vpliv treh koncentracij bisfenolov, 10 nM, 100 nM in 10 μM, na modulacijo citokinov makrofagov THP-1, aktiviranih z lipopolisaharidom (LPS). Bisfenoli so večinoma delovali provnetno, saj so povišali sproščanje vnetnih citokinov. Pri najnižji koncentraciji je prišlo do signifikantnega povišanja vnetnega citokina IL-1β (BPE) ter signifikantnega znižanja pleiotropnega citokina IL-6 (BPA), pri srednji pa do povišanja vnetnih citokinov IL-1β (BPE, BPP in TCBPA) in IL-6 (TCBPA). Najvišja koncentracija je povzročila tako znižanje (BPA in BPP) kot povišanje (TCBPA) vnetnega citokina IL-1β ter povišanje pleiotropnega citokina IL-6 (BPS-MAE, BPZ in TCBPA) in vnetnega citokina IL-8 (TCBPA). Zaključimo lahko, da imajo analogi BPA imunomodulatorne lastnosti ter pri koncentracijah, najdenih v telesnih tekočinah, delujejo provnetno, pri višjih, mikromolarnih koncentracijah pa opazimo tudi protivnetni učinek.

• Language: Slovenian
• Keywords: bisfenol A, analogi bisfenola A, celična linija THP-1, citotoksičnost, imunomodulacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-162623
• Publication date in RUL: 26.09.2024

Secondary language

• Language: English
• Title: The effect of bisphenol A and its selected analogues on cytokine secretion in the monocytic cell line THP-1

Abstract

Bisphenol A (BPA) is the best-known representative of bisphenols, found in many consumer products, air, drinking water, human biological samples etc. Due to the alarming effects of this endocrine disrupting chemical (EDC) on human health, the European Commission has restricted or even banned its use in the manufacture and import of some consumer products and the European Food Safety Authority (EFSA) has since lowered its tolerable daily intake. This in turn increased the use of its many structural analogues, which are already detectable in the environment and human fluids. BPA analogues are particularly problematic due to data scarcity on their action mechanisms and effects. The aim of this thesis was to study the effect of seven bisphenols, BPA, BPAP, BPE, BPP, BPS-MAE, BPZ and TCBPA, on metabolic activity and immunomodulation in the monocyte cell line THP-1, which were differentiated into macrophages. The effect of selected bisphenols on metabolic activity was determined by resazurin assay and fluorescence measurement, and the mean inhibitory concentration (IC50) of each bisphenol was determined, where 50% of the cells were metabolically active. The lowest IC50 was determined for BPP (19,1 ± 0,3 μM), which is therefore the most cytotoxic. BPAP (37,1 ± 1,2 μM), BPZ (43,5 ± 2,3 μM), TCBPA (64,4 ± 7,6 μM), BPA (122,6 ± 5,7 μM) and BPS-MAE (137,5 ± 7,0 μM) followed, with BPE being the least cytotoxic (220,2 ± 47,7 μM). We also found that there is a correlation between lipophilicity (logP) and cytotoxicity. The effect of three concentrations of bisphenols, 10 nM, 100 nM and 10 μM, on the modulation of cytokines of THP-1 macrophages activated by lipopolysaccharide (LPS) was then investigated by flow cytometry. Bisphenols were mostly pro-inflammatory, as they increased the release of inflammatory cytokines. At the lowest concentration, there was a significant decrease in the inflammatory cytokine IL-1β (BPE) and a significant increase in the pleiotropic cytokine IL-6 (BPA), while there was a significant increase of the inflammatory cytokines IL-1β (BPE, BPP and TCBPA) and IL-6 (TCBPA) at the medium concentration. The highest concentration caused both a significant increase (TCBPA) and decrease (BPA and BPP) of the inflammatory cytokine IL-1β and increases of the pleiotropic cytokine IL-6 (BPS-MAE, BPZ in TCBPA) and the inflammatory cytokine IL-8 (TCBPA). In conclusion, BPA analogues have immunomodulatory properties, with pro-inflammatory effects at the concentrations found in body fluids, and some anti-inflammatory effects at higher, micromolar concentrations.

• Keywords: bisphenol A, bisphenol A analogues, THP-1 cell line, cytotoxicity, immunomodulation