Citotoksično in genotoksično delovanje bisfenola A in njegovih analogov

Fras, Katarina (author), Žegura, Bojana (mentor), Štampar, Martina (comentor)

Bisfenoli (BP) so skupina industrijskih kemikalij, ki se uporabljajo pri proizvodnji plastike in epoksi smol. Iz njih izdelujejo najrazličnejše izdelke kot so obloge za konzerve, stekleničke za dojenčke, igrače, papir za račune in mnoge druge, ki se uporabljajo v našem vsakdanjem življenju in jih posledično zasledimo v okolju. Zaradi kopičenja podatkov o nevarnih lastnostih in škodljivih vplivih najpogostejšega izmed BP-jev, bisfenola A (BPA), predvsem motenj endokrinega sistema, je bila njegova uporaba v Evropski uniji postopoma omejena oziroma celo prepovedana, da bi zaščitili zdravje ljudi in okolje. Prepoved uporabe BPA je spodbudila razvoj domnevno varnejših analogov, za katere pa v literaturi ni veliko toksikoloških podatkov. Tako smo v magistrski nalogi proučevali citotoksično in genotoksično aktivnost BPA in njegovih analogov: bisfenol AF (BPAF), bisfenol AP (BPAP), bisfenol C (BPC), bisfenol FL (BPFL), bisfenol S (BPS) na jetrnem in vitro 3D celičnem modelu. Sferoide smo pripravili s prisilno plavajočo metodo, vpliv na živost celic sferoidov po izpostavitvi BP-jem smo merili s testom MTS. S pomočjo mikroskopije, smo spremljali površino, obliko in kompaktnost sferoidov, s testom komet pa smo preučevali genotoksičnost v časovnih izpostavitvah 24 in 96 ur. Statistično značilen vpliv na živost celic v sferoidu po 24 urah so imeli BPFL > BPAF = BPAP, po 96 urah pa BPFL > BPC = BPAP > BPA = BPAF. Mikroskopske analize so pokazale, da sta imela največji vpliv na rast in spremembo površine ter kompaktnosti sferoidov BPC in BPFL po 96-urni izpostavitvi. S testom komet smo pokazali, da sta bila po 24 urah izpostavitve najbolj genotoksična BPAF in BPS (⡥ 10 μM), sledil je BPAP ( ⡥ 40 μM). Po 96 urah izpostavitve je BPAP povzročil poškodbe DNA pri koncentraciji ⡥ 0,1 μM, pri vseh ostalih BP-jih smo poškodbe DNA zaznali pri višjih koncentracijah. Povzamemo lahko, da so za analoge BPA potrebne dodatne študije mehanizmov delovanja za pripravo ustrezne ocene tveganja, saj je toksična aktivnost za posamezne analoge, kljub podobni kemijski strukturi, različna.

• Language: Slovenian
• Keywords: bisfenoli, citotoksičnost, genotoksičnost, test MTS, test komet, mikroskopija, celična linija HepG2, in vitro 3D celični modeli, sferoidi
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [K. Fras]
• Year: 2022
• PID: 20.500.12556/RUL-138992
• UDC: 577.2
• COBISS.SI-ID: 120345091
• Publication date in RUL: 27.08.2022

Secondary language

• Language: English
• Title: Cytotoxic and genotoxic activity of bisphenol A and its analogues

Abstract

Bisphenols (BPs) are a group of industrial chemicals used in the manufacture of plastics and epoxy resin. They are found in various products, including the inner linings of cans, baby bottles, toys, thermal paper, and others used in our daily life. Therefore, BPs are present in the environment. The most widely used BP is bisphenol A (BPA), on which most studies have been conducted showing its adverse effects on human health, especially its endocrine disrupting activity. Therefore, the European Union began to restrict and eventually ban the use of BPA to protect human health and the environment. The ban has led to the development of supposedly safer BPA analogues, for which few toxicological data are available. In the master thesis, we investigated the cytotoxic and genotoxic activity of BPA and its analogues (BPAF, BPAP, BPC, BPFL, BPS) in a liver in vitro 3D cell model. In this study, we prepared spheroids by the forced floating method and measured cell viability after exposure to BPs using the MTS assay. We measured the surface area and observed the shape and compactness of the spheroids with microscopy and studied the genotoxic effects with the comet assay at 24 and 96-hour exposure. After 24-hour exposure to BPs, BPFL > BPAF = BPAP statistically significantly decreased the viability of HepG2 and after 96 hours, we observed a similar effect on cell viability in BPFL > BPC = BPAP > BPA = BPAF. BPC and BPFL had the greatest effect on morphological changes in growth, surface area, compactness, and shape of the spheroids that were evident at 96-hour exposure. Of the six BPs tested by the comet assay, BPAF and BPS (⡥ 10 μM) and BPAP (⡥ 40 μM) exhibited stronger genotoxic potential than BPA, BPFL, and BPC after 24 hours of exposure. After 96 hours of exposure, BPAP was the most genotoxic causing/inducing DNA damage at ⡥1 µM. All others BPs studied statistically significantly increased DNA damage at higher concentrations. To summarize, despite their similar chemical structure, the toxic activity of the analogues is not the same, and therefore further studies on the BPA analogues and their molecular activity are needed to provide/make accurate risk assessment.

• Keywords: bisphenols, cytotoxicity, genotoxicity, MTS assay, comet assay, microscopy, HepG2 cell line, in vitro 3D cell models, spheroids