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Our results show that both nPS are easily internalized by Caco-2 cells in a concentration-dependent manner, but no relevant toxic effects are observed under the analyzed exposure conditions.

Accumulation of plastic and its derivatives, micro- and nanoplastics (MNPLs), is a substantial environmental and ecological problem that could potentially become a serious health concern to humans.

The increasing presence of micro- and nanoplastics (MNPLs) in the environment, and their consequent accumulation in trophic niches, could pose a potential health threat to humans, especially due to their chronic ingestion. In vitro studies using human cells are considered pertinent approaches to determine potential health risks to humans. Nevertheless, most of such studies have been conducted using short exposure times and high concentrations. Since human exposure to MNPLs is supposed to be chronic, there is a lack of information regarding the potential in vitro MNPLs effects under chronic exposure conditions. To this aim, we assessed the accumulation and potential outcomes of polystyrene nanoparticles (PSNPs), as a model of MNPLs, in undifferentiated Caco-2 cells (as models of cell target in ingestion exposures) under a relevant long-term exposure scenario, consisting of eight weeks of exposure to sub-toxic PSNPs concentrations. In such exposure conditions, culture-media was changed every 2–3 days to maintain constant exposure. The different analyzed endpoints were cytotoxicity, dysregulation of stress-related genes, genotoxicity, oxidative DNA damage, and intracellular ROS levels. These are endpoints that showed to be sensitive enough in different studies. The obtained results attest that PSNPs accumulate in the cells through time, inducing changes at the ultrastructural and molecular levels. Nevertheless, minor changes in the different evaluated genotoxicity-related biomarkers were observed. This would indicate that no DNA damage or oxidative stress is observed in the human intestinal Caco-2 cells after long-term exposure to PSNPs. This is the first study dealing with the long-term effects of PSNPs on human cultured cells.

Carbon-based nanomaterials are being increasingly used, demanding strong information to support their safety in terms of human health. As ingestion is one of the most important exposure routes in humans, we have determined their potential risk by using an in vitro model simulating the human intestinal barrier and evaluated the effects of both graphene oxide (GO) and graphene nanoplatelets (GNPs). A coculture of differentiated Caco-2/HT29 cells presenting inherent intestinal epithelium characteristics (i.e. mucus secretion, brush border, tight junctions, etc.) were treated with GO or GNPs for 24 h. Different endpoints such as viability, membrane integrity, NPs localization, cytokines secretion, and genotoxic damage were evaluated to have a wide view of their potentially harmful effects. No cytotoxic effects were observed in the cells that constitute the barrier model. In the same way, no adverse effects were detected neither in the integrity of the barrier (TEER) nor in its permeability (LY). Nevertheless, a different bio-adhesion and biodistribution behavior was observed for GO and GNPs by confocal microscopy analysis, with a more relevant uptake of GNPs. No oxidative damage induction was detected, either by the DCFH-DA assay or the FPG enzyme in the comet assay. Conversely, both GO and GNPs were able to induce DNA breaks, as observed in the comet assay. Finally, low levels of anti-inflammatory cytokines were detected, suggesting a weak anti-inflammatory response. Our results show the moderate/severe risk posed by GO/GNPs exposures, given the observed genotoxic effects, suggesting that more extensive genotoxic evaluations must be done to properly assess the genotoxic hazard of these nanomaterials. Carbon-based nanomaterials are being increasingly used, due to their growing relevance in many fields. Their large surface area and their excellent electric conductivity offer many physicochemical advantages that are exploited in many industrial fields, including biomedical applications 1. Nevertheless, as it occurs with other nanomaterials, its wide use supposes an increased hazard for human health 2. One of the most recently discovered carbon-based nanomaterials is graphene. Since its first description in 2004 3 , different modifications have been done in their single-atom-thick sheet of planar sp 2-bound carbons, creating numerous graphene-based nanomaterials. According to the EU Graphene Flagship project, these materials can be classified according to three different characteristics: (i) the number of graphene layers, (ii) the average lateral size, and (iii) the carbon/oxygen ratio 4,5. Although a large amount of literature associated with graphene-based nanomaterials has been generated, only a few of them focus on toxicity aspects 2,6. Hence, this paper focuses on graphene oxide (GO) and graphene nanoplatelets (GNPs) exposure risk, two graphene-based nanomaterials that have shown great potential for biomedical and polymeric composites applications 7,8. GO is a highly-oxidized form of graphene, containing different ...