In vitro cell-transforming potential of secondary polyethylene terephthalate and polylactic acid nanoplastics.

Continuous exposure to plastic pollutants may have serious consequences on human health. However, most toxicity assessments focus on non-environmentally relevant particles and rarely investigate long-term effects such as cancer induction. The present study assessed the carcinogenic potential of two secondary nanoplastics: polyethylene terephthalate (PET) particles generated from plastic bottles, and a biodegradable polylactic acid material, as respective examples of environmentally existing particles and new bioplastics. Pristine polystyrene nanoplastics were also included for comparison. A broad concentration range (6.25-200 µg/mL) of each nanoplastic was tested in both the initiation and promotion conditions of the regulatory assessment-accepted in vitro Bhas 42 cell transformation assay. Parallel cultures allowed confirmation of the efficient cellular internalisation of the three nanoplastics. Cell growth was enhanced by polystyrene in the initiation assay, and by PET in both conditions. Moreover, the number of transformed foci was significantly increased only by the highest PET concentration in the promotion assay, which also showed dose-dependency, indicating that nano PET can act as a non-genotoxic tumour promotor. Together, these findings support the carcinogenic risk assessment of nanoplastics and raise concerns regarding whether real-life co-exposure of PET nanoplastics and other environmental pollutants may result in synergistic transformation capacities.

Harmful effects of true-to-life nanoplastics derived from PET water bottles in human alveolar macrophages.

The increasing presence of secondary micro/nanoplastics (MNPLs) in the environment requires knowing if they represent a real health concern. To such end, an important point is to test representative MNPLs such as the denominated true-to-life MNPLs, resulting from the degradation of plastic goods in lab conditions. In this study, we have used polyethylene terephthalate (PET) NPLs resulting from the degradation of PET water bottles. Since inhalation is an important exposure route to environmental MNPLS, we have used mouse alveolar macrophages (MH-S) as a target cell, and the study focused only on the cells that have internalized them. This type of approach is novel as it may capture the realistic adverse effects of PETNPLs only in the internalized cells, thereby mitigating any biases while assessing the risk of these MNPLs. Furthermore, the study utilized a set of biomarkers including intracellular reactive oxygen species (ROS) levels, variations on the mitochondrial membrane potential values, and the macrophage polarization to M1 (pro-inflammatory response) and M2 (anti-proinflammatory response) as possible cellular effects due to PETNPLs in only the cells that internalized PETNPLs. After exposures lasting for 3 and 24 h to a range of concentrations (0, 25, 50, and 100 µg/mL) the results indicate that no toxicity was induced despite the 100% internalization observed at the highest concentration. Significant intracellular levels of ROS were observed, mainly at exposures lasting for 24 h, in an indirect concentration-effect relationship. Interestingly, a reduction in the mitochondrial membrane potential was observed, but only at exposures lasting for 24 h, but without a clear concentration-effect relationship. Finally, PETNPL exposure shows a significant polarization from M0 to M1 and M2 subtypes. Polarization to M1 (pro-inflammatory stage) was more marked and occurred at both exposure times. Polarization to M2 (anti-inflammatory stage) was only observed after exposures lasting for 24 h. Due to the relevance of the described biomarkers, our results underscore the need for further research, to better understand the health implications associated with MNPL exposure.

Titanium-doped PET nanoplastics, from opaque milk bottle degradation, as a model of environmental true-to-life nanoplastics. Hazardous effects on Drosophila.

Micro and nanoplastics (MNPLs) are emergent environmental pollutants, resulting from the degradation of plastic waste, requiring urgent information on their potential risks to human health. To determine such risks, reliable true-to-life materials are essential. In this work, we have used titanium-doped PET NPLs [PET(Ti)NPLs], obtained by grinding opaque milk polyethylene terephthalate (PET) bottles, as a true-to-life MNPLs model. These opaque PET bottles, with an average size of 112 nm, contain about 3% Ti in the form of titanium dioxide rod nanoparticles. TEM investigation confirmed the mixed Ti/PET nature of the obtained true-to-life NPLs, and the rod shape of the embedded TiO2NPs. In the in vivo Drosophila model neither PET(Ti)NPLs nor TiO2NPs reduced the survival rates, although their internalization was confirmed in different compartments of the larval body by using confocal and transmission electron microscopies. The presence of Ti in the PET(Ti)NPLs permitted to quantify its presence both in larvae (2.1 ± 2.2 µg/g of Ti) and in the resulting adults (3.4 ± 3.2 µg/g of Ti) after treatment with 500 µg/g food of PET(Ti)NPL, suggesting its potential use to track their fate in more complex organisms such as mammals. PET(Ti)NPLs, as well as TiO2NPs, altered the expression of genes driving different response pathways, inducing significant oxidative stress levels (up to 10 folds), and genotoxicity. This last result on the genotoxic effects is remarkable in the frame of the hot topic discussion on the risk that titanium compounds, used as food additives, may pose to humans.

The release of polylactic acid nanoplastics (PLA-NPLs) from commercial teabags. Obtention, characterization, and hazard effects of true-to-life PLA-NPLs.

This study investigates MNPLs release from commercially available teabags and their effects on both undifferentiated monocultures of Caco-2 and HT29 and in the in vitro model of the intestinal Caco-2/HT29 barrier. Teabags were subjected to mechanical and thermodynamic forces simulating the preparation of a cup of tea. The obtained dispersions were characterized using TEM, SEM, DLS, LDV, NTA, and FTIR. Results confirmed that particles were in the nano-range, constituted by polylactic acid (PLA-NPLs), and about one million of PLA-NPLs per teabag were quantified. PLA-NPLs internalization, cytotoxicity, intracellular reactive oxygen species induction, as well as structural and functional changes in the barrier were assessed. Results show that PLA-NPLs present high uptake rates, especially in mucus-secretor cells, and bio-persisted in the tissue after 72 h of exposure. Although no significant cytotoxicity was observed after the exposure to 100 µg/mL PLA-NPLs during 48 h, a slight barrier disruption could be detected at short-time periods. The present work reveals new insights into the safety of polymer-based teabags, the behavior of true-to-life MNPLs in the human body, as well as new questions on how repeated and prolonged exposures could affect the structure and function of the human intestinal epithelium.

Effects of true-to-life PET nanoplastics using primary human nasal epithelial cells.

Since inhalation is a relevant exposure route, studies using appropriate micro/nanoplastic (MNPLs) models, representative targeted cells, and relevant biomarkers of effect are required. We have used lab-made polyethylene terephthalate (PET)NPLs obtained from PET plastic water bottles. Human primary nasal epithelial cells (HNEpCs) were used as a model of the first barrier of the respiratory system. Cell internalization and intracellular reactive oxygen species (iROS) induction, as well as the effects on mitochondria functionality and in the modulation of the autophagy pathway, were evaluated. The data indicated significant cellular uptake and increased levels of iROS. Furthermore, a loss of mitochondrial membrane potential was observed in the exposed cells. Regarding the effects on the autophagy pathway, PETNPLs exposure significantly increases LC3-II protein expression levels. PETNPLs exposure also induced significant increases in the expression of p62. This is the first study showing that true-to-life PETNPLs can alter the autophagy pathway in HNEpCs.

Titanium-doped PET nanoplastics of environmental origin as a true-to-life model of nanoplastic.

The increased presence of secondary micro/nanoplastics (MNPLs) in the environment requires urgent studies on their potentially hazardous effects on exposed organisms, including humans. In this context, it is essential to obtain representative MNPL samples for such purposes. In our study, we have obtained true-to-life NPLs resulting from the degradation, via sanding, of opaque PET bottles. Since these bottles contain titanium (TiO2NPs), the resulting MNPLs also contain embedded metal. The obtained PET(Ti)NPLs were extensively characterized from a physicochemical point of view, confirming their nanosized range and their hybrid composition. This is the first time these types of NPLs are obtained and characterized. The preliminary hazard studies show their easy internalization in different cell lines, without apparent general toxicity. The demonstration by confocal microscopy that the obtained NPLs contain Ti samples offers this material multiple advantages. Thus, they can be used in in vivo approaches to determine the fate of NPLs after exposure, escaping from the existing difficulties to follow up MNPLs in biological samples.

The potential effects of in vitro digestion on the physicochemical and biological characteristics of polystyrene nanoplastics.

The presence of plastic waste in our environment has continued growing and become an important environmental concern. Because of its degradation into micro- and nanoplastics (MNPLs), MNPLs are becoming environmental pollutants of special environmental/health concern. Since ingestion is one of the main exposure routes to MNPLs, the potential effects of digestion on the physicochemical/biological characteristics of polystyrene nanoplastics (PSNPLs) were determined. The results indicated a high tendency of digested PSNPLs to agglomerate and a differential presence of proteins on their surface. Interestingly, digested PSNPLs showed greater cell uptake than undigested PSNPLs in all three tested cell lines (TK6, Raji-B, and THP-1). Despite these differences in cell uptake, no differences in toxicity were observed except for high and assumed unrealistic exposures. When oxidative stress and genotoxicity induction were determined, the low effects observed after exposure to undigested PDNPLs were not observed in the digested ones. This indicated that the greater ability of digested PSNPLs to internalize was not accompanied by a greater hazard. This type of analysis should be performed with other MNPLs of varying sizes and chemical compositions.

Hazard assessment of different-sized polystyrene nanoplastics in hematopoietic human cell lines.

The environmental presence of micro/nanoplastics (MNPLs) is an environmental and human health concern. Such MNPLs can result from the physicochemical/biological degradation of plastic goods (secondary MNPLs) or can result from industrial production at that size, for different commercial purposes (primary MNPLs). Independently of their origin, the toxicological profile of MNPLs can be modulated by their size, as well as by the ability of cells/organisms to internalize them. To get more information on these topics we have determined the ability of three different sizes of polystyrene MNPLs (50, 200, and 500 nm) to produce different biological effects in three different human hematopoietic cell lines (Raji-B, THP-1, and TK6). Results show that none of the three sizes was able to induce toxicity (growth ability) in any of the tested cell types. Although transmission electron microscopy and confocal images showed cell internalization in all the cases, their quantification by flow cytometry demonstrated an important uptake by Raji-B and THP-1 cells, in comparison with TK6 cells. For the first ones, the uptake was negatively associated with the size. Interestingly, when the loss of mitochondrial membrane potential was determined, dose-related effects were observed for Raji-B and THP-1 cells, but not for TK6 cells. These effects were observed for the three different sizes. Finally, when oxidative stress induction was evaluated, no clear effects were observed for the different tested combinations. Our conclusion is that size, biological endpoint, and cell type are aspects modulating the toxicological profile of MNPLs.

Hazard Assessment of Polystyrene Nanoplastics in Primary Human Nasal Epithelial Cells, Focusing on the Autophagic Effects.

The human health risks posed by micro/nanoplastics (MNPLs), as emerging pollutants of environmental/health concern, need to be urgently addressed as part of a needed hazard assessment. The routes of MNPL exposure in humans could mainly come from oral, inhalation, or dermal means. Among them, inhalation exposure to MNPLs is the least studied area, even though their widespread presence in the air is dramatically increasing. In this context, this study focused on the potential hazard of polystyrene nanoplastics (PSNPLs with sizes 50 and 500 nm) in human primary nasal epithelial cells (HNEpCs), with the first line of cells acting as a physical and immune barrier in the respiratory system. Primarily, cellular internalization was evaluated by utilizing laboratory-labeled fluorescence PSNPLs with iDye, a commercial, pink-colored dye, using confocal microscopy, and found PSNPLs to be significantly internalized by HNEpCs. After, various cellular effects, such as the induction of intracellular reactive oxygen species (iROS), the loss of mitochondrial membrane potential (MMP), and the modulation of the autophagy pathway in the form of the accumulation of autophagosomes (LC3-II) and p62 markers (a ubiquitin involved in the clearance of cell debris), were evaluated after cell exposure. The data demonstrated significant increases in iROS, a decrease in MMP, as well as a greater accumulation of LC3-II and p62 in the presence of PSNPLs. Notably, the autophagic effects did indicate the implications of PSNPLs in defective or insufficient autophagy. This is the first study showing the autophagy pathway as a possible target for PSNPL-induced adverse effects in HNEpCs. When taken together, this study proved the cellular effects of PSNPLs in HNEpCs and adds value to the existing studies as a part of the respiratory risk assessment of MNPLs.

The hazardous impact of true-to-life PET nanoplastics in Drosophila.

Plastic pollution is a continuously growing problem that can threaten wildlife and human beings. Environmental plastic waste is degraded into small particles termed micro/ nanoplastics (MNPLs) that, due to their small size, can be easily internalized into the exposed organisms, increasing the risks associated with their exposure. To appropriately determine the associated health risk, it is essential to obtain/test representative MNPLs' environmental samples. To such end, we have obtained NPLs resulting from sanding commercial water polyethylene terephthalate (PET) bottles. These true-to-life PETNPLs were extensively characterized, and their potential hazard impacts were explored using Drosophila melanogaster. To highlight the internalization through the digestive tract and the whole body, transmission electron microscopy (TEM) and confocal microscopy were used. In spite of the observed efficient uptake of PETNPLs into symbiotic bacteria, enterocytes, and hemocytes, the exposure failed to reduce flies' survival rates. Nevertheless, PETNPLs exposure disturbed the expression of stress, antioxidant, and DNA repair genes, as well as in those genes involved in the response to physical intestinal damage. Importantly, both oxidative stress and DNA damage induction were markedly increased as a consequence of the exposure to PETNPLs.

A new source of representative secondary PET nanoplastics. Obtention, characterization, and hazard evaluation.

Micro and nanoplastics (MNPLs) are emergent environmental pollutants requiring urgent information on their potential risks to human health. One of the problems associated with the evaluation of their undesirable effects is the lack of representative samples, matching those resulting from the environmental degradation of plastic wastes. To such end, we propose an easy method to obtain polyethylene terephthalate nanoplastics from water plastic bottles (PET-NPLs) but, in principle, applicable to any other plastic goods sources. An extensive characterization indicates that the proposed process produces uniform samples of PET-NPLs of around 100 nm, as determined by using AF4 and multi-angle and dynamic light scattering methodologies. An important point to be highlighted is that to avoid the metal contamination resulting from methods using metal blades/burrs for milling, trituration, or sanding, we propose to use diamond burrs to produce metal-free samples. To visualize the toxicological profile of the produced PET-NPLs we have evaluated their ability to be internalized by cells, their cytotoxicity, their ability to induce oxidative stress, and induce DNA damage. In this preliminary approach, we have detected their cellular uptake, but without the induction of significant biological effects. Thus, no relevant increases in toxicity, reactive oxygen species (ROS) induction, or DNA damage -as detected with the comet assay- have been observed. The use of representative samples, as produced in this study, will generate relevant data in the discussion about the potential health risks associated with MNPLs exposures.

Antagonistic in vivo interaction of polystyrene nanoplastics and silver compounds. A study using Drosophila.

Since heavy metals and micro-/nanoplastics (MNPLs) can share common environmental niches, their potential interactions could modulate their hazard impacts. The current study was planned to evaluate the potential interactions between silver compounds (silver nanoparticles or silver nitrate) and two different sizes of polystyrene nanoplastics (PSNPLs) (PS-50 and PS-500 nm), administered via ingestion to Drosophila larvae. While egg-to-adult survival was not affected by the exposure to silver compounds, PSNPLs, or their coexposures, the combined treatments succeeded to restore the delay of fly emergence induced by silver compounds. Transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) showed the ability of PSNPLs to transport silver compounds (regardless of their form) across the intestinal barrier, delivering them into the hemolymph of Drosophila larvae in a concentration exceeding that mediated by the exposure to silver compounds alone. The molecular response (gene expression) of Drosophila larvae greatly fluctuated, accordingly if exposures were administered alone or in combination. Although PSNPLs produced some oxidative stress in the hemocytes of Drosophila, especially at the highest dose (1 mM), higher levels were observed after silver exposure, regardless of its form. Interestingly, the oxidative stress of silver, especially that produced by nano­silver, drastically decreased when coexposed with PSNPLs. Similar effects were observed regarding the DNA damage induced in Drosophila hemocytes, where cotreatment decreased the genotoxicity induced by silver compounds. This antagonistic interaction could be attributed to the ability of tiny plastic specks to confine silver, avoiding its bioavailability, and diminishing their potential impacts.