Vpliv nanomaterialov na sprožitev fosfolipidoze in vitro na primeru izbranih celičnih linij

Kononenko, Veno (author), Drobne, Damjana (mentor), Erman, Andreja (comentor)

V doktorskem delu nas je zanimal biološki vpliv izbranih nanomaterialov, ki se lahko uporabljajo za medicinske namene. Vpliv nanomaterialov smo testirali s pasjimi ledvičnimi celicami MDCK in s človeškimi pljučnimi celicami A549 z lastnostmi alveolarnih celic tipa II. Glavni namen doktorskega dela je bil preveriti, ali lahko nanomateriali v izbranih celicah sprožijo nastanek fosfolipidoze (prekomerno znotrajcelično kopičenje fosfolipidov v lamelarnih telesih) kot enega zgodnjih znakov škodljivega vpliva nanomaterialov. Poleg tega smo želeli preveriti, ali so biološki vplivi nanomaterialov odvisni od njihove velikosti ter od njihovega raztapljanja. Na celicah MDCK, ki smo jih izpostavili necitotoksičnim koncentracijam nanodelcev ZnO, mikrodelcev ZnO in cinkove soli ZnCl2, smo zaznali poškodbe DNA le pri nanodelcih ZnO. Z merjenjem raztapljanja ZnO smo ugotovili, da sproščanje cinkovih ionov v gojišče za celice ni zadostno, da bi z njim lahko razložili škodljive vplive nanodelcev ZnO na celice. Z barvilom LipidTOX™ phospholipidosis, ki v celici obarva s fosfolipidi bogate organele, in ki se uporablja za testiranje potenciala za sprožitev fosfolipidoze, smo ugotovili, da kljub kvarnemu delovanju nanodelci ZnO ne sprožijo fosfolipidoze v celicah MDCK, kakor tudi ne v človeških pljučnih celicah A549. V celicah MDCK ni nobeden od izbranih nanomaterialov sprožil povečane količine s fosfolipidi bogatih organelov. V celicah A549 pa so nanodelci γ-Fe2O3+SiO2 sprožili povečano vsebnost s fosfolipidi bogatih organelov, povečano vsebnost fosfatidilholina in povečano vsebnost organelov, ki sodelujejo pri biogenezi lamelarnih teles. Vendar pa se je količina lamelarnih teles v izpostavljenih celicah A549 zmanjšala, povečala pa se je vsebnost avtofagnih vakuol, ki so pogosto vsebovale poškodovana lamelarna telesa, namenjena razgradnji. Rezultati na celicah A549 so pokazali, da nanodelci γ-Fe2O3+SiO2 vplivajo na metabolizem lipidov, na biogenezo lamelarnih teles in na zniževanje površinske napetosti kapljevine nad celicami. Kljub temu, da so naši rezultati pokazali, da pojav fosfolipidoze ni ustrezen indikator zgodnjih kvarnih vplivov nanomaterialov v celicah MDCK in A549, smo pokazali, da lahko nanomateriali pri necitotoksičnih koncentracijah sprožijo poškodbe DNA ter da lahko ovirajo pomembne funkcije celic.

• Language: Slovenian
• Keywords: avtofagija, celice, fosfatidilholin, fosfolipidoza, genotoksičnost, lamelarna telesa, metabolizem lipidov, nanomateriali, nanotoksikologija, pljučni surfaktant, velikost delcev
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2017
• PID: 20.500.12556/RUL-92500
• COBISS.SI-ID: 11753300
• Publication date in RUL: 05.06.2017

Secondary language

• Language: English
• Title: Influence of nanomaterials on in vitro phospholipidosis induction on selected cell lines

Abstract

We investigated the biological effects of selected nanomaterials that have potential for medical use. Effects of nanomaterials were evaluated on the canine kidney MDCK cells and on the human lung A549 cells with properties of alveolar type II cells. The main purpose of the doctoral work was to determine whether nanomaterials can induce phospholipidosis (excessive intracellular accumulation of phospholipids in lamellar bodies) as early sign of adverse effects of nanomaterials. In addition, we wanted to check if biological effects of nanomaterials depend on their size and on their dissolution. On MDCK cells that were exposed to sub-cytotoxic concentrations of ZnO nanoparticles, ZnO microparticles and to zinc salt ZnCl2, we observed increased DNA damage only in cells exposed to nanoparticles. By measuring the ZnO nanoparticle dissolution, we found that the zinc ion release in the cell culture medium is insufficient to be the main cause of ZnO nanoparticle toxicity. By staining cells with LipidTOX™ phospholipidosis dye that is used to test potential for phospholipidosis induction, we found that ZnO nanoparticles as well as all other selected nanomaterials did not increased the amount of phospholipid rich organelles in MDCK cells. In contrast, in A549 cells, we observed that silica coated maghemite nanoparticles (γ-Fe2O3+SiO2) caused increased cellular content of phospholipids rich organelles, increased content of phosphatidylcholine (PC) and increased content of organelles that are involved in lamellar body biogenesis. Despite these results, indicating intensive lamelar body biogenesis in the exposed A549 cells, the amount of lamellar bodies had been decreased. In γ-Fe2O3+SiO2 treated A549 cells, we observed an increased amount of autophagic vacuoles that often contained defective lamellar bodies destined for degradation. Results on A549 cells showed that γ-Fe2O3+SiO2 affect lipid metabolism, interfered with the lamellar body biogenesis and reduced cell capacity to lower the surface tension of hypophase. Our results show that phospholipidosis is not good indicator of nanotoxicity in MDCK and A549 cells. Nevertheless we have shown that nanomaterials can induce DNA damage and may interfere with important functions of the cell, even at non-cytotoxic concentrations.

• Keywords: autophagy, cells, genotoxicity, lamellar body, lipid metabolism, lung surfactant, nanomaterials, nanotoxicology, particle size, phosphatidylcholine, phospholipidosis