Development of 3D spheroids from fish liver cells as in-vitro models to assess the effects of plastics in aquatic systems

Abstract

[eng] Plastic pollution poses a significant environmental threat, exacerbated by the accumulation of single-use items, especially in aquatic ecosystems. These plastics can degrade into mesoplastics and smaller micro- and nanoplastics, causing harm to aquatic organisms physically and chemically. Despite the new design of biodegradable and compostable plastics (BPs), these materials still require additives that could lead to toxic effects before complete degradation. This thesis investigated the toxicity of BPs, conventional plastics, and mesoplastics collected from Spanish beaches, and explored how plastic photodegradation and composting influence toxic responses in PLHC-1 cells. Additionally, it assessed the cellular and molecular responses of polystyrene micro- and nanoplastics in ZFL cells, focusing on lipid metabolism and antioxidant related pathways. Considering recent trends in environmental toxicity assessment using 3D cell culture techniques to simulate toxicity gradients and emulate cell functions under in vivo conditions, this thesis developed fish liver spheroids (PLHC-1, ZFL) as test models. They were characterized in terms of growth pattern, lipidomic signatures and gene expressions. PLHC-1 spheroids (7-day post seeding) were used to evaluate the toxicity of a mixture of plastic additives and their responses, while ZFL spheroids were characterized in terms of cyp1a gene expression in response to β-naphthoflavone. In addition, the effect of serum supplement (FBS) in the exposure medium was investigated in terms of changes of the lipidome of PLHC-1 spheroids and modulation of the toxicity of plastic additives. Regarding the toxicity of BPs, results showed that BP extracts significantly reduced the cell viability of PLHC-1 cells compared to conventional plastics and this toxicity increased after photodegradation or composting, while extracts from recycled plastics induced EROD activity and micronucleus formation. These results highlight the need to identify and further investigate the compounds causing toxicity in these plastics and to investigate the environmental consequences of uncontrolled dumping before the widespread use of BPs. Meanwhile, beach mesoplastic extracts contained chemicals that induced cytotoxicity and genotoxicity in PLHC-1 cells, with

further photodegradation increasing the overall toxicity. These results emphasize the utility of cell-based assays in assessing plastic pollution risks. Regarding the toxicity of MNPs, lipidomic analysis allowed the identification of an upregulation of ceramides (C16, C22, C24:1) induced by NP exposure (but not MPs), which was associated to the activation of ceramide-mediated apoptotic pathways. The study highlighted the potential of lipidomics for elucidating the mechanisms underlying nanoplastic toxicity. Furthermore, the analysis of fish liver spheroids showed a more mature liver cell phenotype than cell monolayers. Liver spheroids were characterized by a decrease in total membrane lipids (particularly PEs and PCs) with a concomitant increase in highly unsaturated phospholipids, indicating significant changes in cell membranes and inter- and extra-cellular interactions, additionally reflected by the higher PC/PE ratio observed in spheroids, which is closer to that of healthy liver. Concerning the responses to toxicants, fish liver cell spheroids exhibited increased cell viability and reduced generation of reactive oxygen species (ROS) production after exposure to a mixture of plastic additives, yet demonstrated increased sensitivity in lipidomic responses and detoxification gene expression compared to cells cultured in monolayers. Additionally, the presence of FBS in the exposure medium has been shown to significantly affect lipidomic responses induced by plastic additive mixture. These results further support the use of fish cell spheroids as more realistic in vitro approaches for aquatic toxicity studies, but they also emphasize the importance of optimizing and standardizing culture conditions before its wide application in toxicological studies. Overall, the current thesis describes the toxicity and molecular and subcellular mechanisms of action of plastic-derived toxicants using in vitro cell systems, emphasizes the importance of comprehensive environmental risk assessments associated with new emerging plastics, and highlights the key role of fish liver cell spheroids and lipidomics in advancing this field.