Hazard assessment of nanoplastics is driven by their surface-functionalization. Effects in human-derived primary endothelial cells.

During plastic waste degradation into micro/nanoplastics (MNPLs) their physicochemical characteristics including surface properties (charge, functionalization, biocorona, etc.) can change, potentially affecting their biological effects. This paper focuses on the surface functionalization of MNPLs to determine if it has a direct impact on the toxicokinetic and toxicodynamic interactions in human umbilical vein endothelial cells (HUVECs), at different exposure times. Pristine polystyrene nanoplastics (PS-NPLs), as well as their carboxylated (PS-C-NPLs) and aminated (PS-A-NPLs) forms, all around 50 nm, were used in a wide battery of toxicological assays. These assays encompassed evaluations on cell viability, cell internalization, induction of intracellular reactive oxygen species (iROS), and genotoxicity. The experiments were conducted at a concentration of 100 µg/mL, chosen to ensure a high internalization rate across all treatments while maintaining a sub-toxic concentration. Our results show that all PS-NPLs are internalized by HUVECs, but the internalization dynamic depends on the particle's functionalization. PS-NPLs and PS-C-NPLs internalization modify the morphology of the cell increasing its inner complexity/granularity. Regarding cell toxicity, only PS-A-NPLs reduced cell viability. Intracellular ROS was induced by the three different PS-NPLs but at different time points. Genotoxic damage was induced by the three PS-NPLs at short exposures (2 h), but not for PS-C-NPLs at 24 h. Overall, this study suggests that the toxicological effects of PSNPLs on HUVEC cells are surface-dependent, highlighting the relevance of using human-derived primary cells as a target.

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.

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.

Are bioplastics safe? Hazardous effects of polylactic acid (PLA) nanoplastics in Drosophila.

The expanded uses of bioplastics require understanding the potential health risks associated with their exposure. To address this issue, Drosophila melanogaster as a versatile terrestrial in vivo model was employed, and polylactic acid nanoplastics (PLA-NPLs), as a proxy for bioplastics, were tested as a material model. Effects were determined in larvae exposed for 4 days to different concentrations (25, 100, and 400 µg/mL) of 463.9 ± 129.4 nm PLA-NPLs. Transmission electron microscopy (TEM) and scanning electron microscope (SEM) approaches permitted the detection of PLA-NPLs in the midgut lumen of Drosophila larvae, interacting with symbiotic bacteria. Enzymatic vacuoles were observed as carriers, collecting PLA-NPLs and enabling the crossing of the peritrophic membrane, finally internalizing into enterocytes. Although no toxic effects were observed in egg-to-adult survival, cell uptake of PLA-NPLs causes cytological disturbances and the formation of large vacuoles. The translocation across the intestinal barrier was demonstrated by their presence in the hemolymph. PLA-NPL exposure triggered intestinal damage, oxidative stress, DNA damage, and inflammation responses, as evaluated via a wide set of marker genes. Collectively, these structural and molecular interferences caused by PLA-NPLs generated high levels of oxidative stress and DNA damage in the hemocytes of Drosophila larvae. The observed effects point out the need for further studies aiming to deepen the health risks of bioplastics before adopting their uses as a safe plastic alternative.

Alcohol-free synthesis, biological assessment, in vivo toxicological evaluation, and in silico analysis of novel silane quaternary ammonium compounds differing in structure and chain length as promising disinfectants.

Quaternary ammonium compounds (QACs) are commonly used as disinfectants for industrial, medical, and residential applications. However, adverse health outcomes have been reported. Therefore, biocompatible disinfectants must be developed to reduce these adverse effects. In this context, QACs with various alkyl chain lengths (C12-C18) were synthesized by reacting QACs with the counterion silane. The antimicrobial activities of the novel compounds against four strains of microorganisms were assessed. Several in vivo assays were conducted on Drosophila melanogaster to determine the toxicological outcomes of Si-QACs, followed by computational analyses (molecular docking, simulation, and prediction of skin sensitization). The in vivo results were combined using a cheminformatics approach to understand the descriptors responsible for the safety of Si-QAC. Si-QAC-2 was active against all tested bacteria, with minimal inhibitory concentrations ranging from 13.65 to 436.74 ppm. Drosophila exposed to Si-QAC-2 have moderate-to-low toxicological outcomes. The molecular weight, hydrophobicity/lipophilicity, and electron diffraction properties were identified as crucial descriptors for ensuring the safety of the Si-QACs. Furthermore, Si-QAC-2 exhibited good stability and notable antiviral potential with no signs of skin sensitization. Overall, Si-QAC-2 (C14) has the potential to be a novel disinfectant.

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.

Toxicological Profiling and Long-Term Effects of Bare, PEGylated- and Galacto-Oligosaccharide-Functionalized Mesoporous Silica Nanoparticles.

Mesoporous silica nanoparticles (MSNs) are amongst the most used nanoparticles in biomedicine. However, the potentially toxic effects of MSNs have not yet been fully evaluated, being a controversial matter in research. In this study, bare MSNs, PEGylated MSNs (MSNs-PEG), and galacto-oligosaccharide-functionalized MSNs (MSNs-GAL) are synthesized and characterized to assess their genotoxicity and transforming ability on human lung epithelial BEAS-2B cells in short- (48 h) and long-term (8 weeks) exposure scenarios. Initial short-term treatments show a dose-dependent increase in genotoxicity for MSNs-PEG-treated cells but not oxidative DNA damage for MSNs, MSNs-PEG, or for MSNs-GAL. In addition, after 8 weeks of continuous exposure, neither induced genotoxic nor oxidative DNA is observed. Nevertheless, long-term treatment with MSNs-PEG and MSNs-GAL, but not bare MSNs, induces cell transformation features, as evidenced by the cell's enhanced ability to grow independently of anchorage, to migrate, and to invade. Further, the secretome from cells treated with MSNs and MSNs-GAL, but not MSNs-PEG, shows certain tumor-promoting abilities, increasing the number and size of HeLa cell colonies formed in the indirect soft-agar assay. These results show that MSNs, specifically the functionalized ones, provoke some measurable adverse effects linked to tumorigenesis. These effects are in the order of other nanomaterials, such as carbon nanotubes or cerium dioxide nanoparticles, but they are lower than those provoked by some approved drugs, such as doxorubicin or dexamethasone.

Encounter rate and relative abundance of eight cetaceans off the central Catalan coast (Northwestern Mediterranean sea).

This study provides information on cetacean occurrence in the central Catalan coast (NE Iberian Peninsula), an area characterised by an underwater canyon system. Encounter rates (ER), relative abundances (RA) and seasonality were assessed for eight species. This information was combined with physiographic features of the seafloor to understand their influence on cetacean distribution. Data were collected along 189 surveys from January 2017 to December 2022. In total, 479 sightings of eight species of cetaceans were recorded. Striped, bottlenose and Risso's dolphins and fin whales were the most frequently sighted. ER and RA were similar or higher than in other areas of the Mediterranean Sea. Species were distributed differently according to physiographic features: bottlenose dolphins in coastal waters and Risso's dolphins and fin whales above the Garraf underwater canyon system. These results highlight the importance of the central Catalan coast, particularly the underwater canyon system, for at least four species of cetaceans.

Morphologically different hydroxyapatite nanoparticles exert differential genotoxic effects in Drosophila.

Hydroxyapatite (HAP) occurs naturally in sedimentary and metamorphic rocks and constitutes the hard structures in many organisms. Since synthetic nano-sized HAP (HAP-NPs) are used in orthopedic applications and for heavy metal remediation in aquatic and terrestrial media, both environment and humans are exposed to them. Due to the concerns about their potential hazards, the genotoxic effects that round/rod forms of HAP-NPs were investigated in Drosophila using the wing-spot and the comet assays. Furthermore, caspase activities were evaluated to examine the activation of cell death pathways. As a novelty, the expression of 36 genes involved in DNA repair was investigated, as a tool to indirectly determine DNA damage induction. Obtained sizes were 35-60 nm (roundHAP-NPs) and 45-90 nm (rodHAP-NPs) with a low Zeta-potential (-1.65 and 0.37 mV, respectively). Genotoxicity was detected in the wing-spot (round form), and in the comet assay (round and rod-like HA-NPs). In addition, increased expression of Caspases 3/7, 8, and 9 activities were observed. For both HAP forms, increased changes in the expression were observed for mismatch repair genes, while decreased expression was observed for genes involved in ATM, ATR, and cell cycle pathways. The observed changes in the repair pathways would reinforce the view that HAP-NPs have genotoxic potential, although more markedly in the round form. Thus, the environmental presence of engineered nanoparticles, including HAPs, raises concerns about potential effects on human health. It is essential that the effects of their use are carefully assessed and monitored to ensure safety and to mitigate any potential adverse effects.

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.

In Vitro Approaches to Determine the Potential Carcinogenic Risk of Environmental Pollutants.

One important environmental/health challenge is to determine, in a feasible way, the potential carcinogenic risk associated with environmental agents/exposures. Since a significant proportion of tumors have an environmental origin, detecting the potential carcinogenic risk of environmental agents is mandatory, as regulated by national and international agencies. The challenge mainly implies finding a way of how to overcome the inefficiencies of long-term trials with rodents when thousands of agents/exposures need to be tested. To such an end, the use of in vitro cell transformation assays (CTAs) was proposed, but the existing prevalidated CTAs do not cover the complexity associated with carcinogenesis processes and present serious limitations. To overcome such limitations, we propose to use a battery of assays covering most of the hallmarks of the carcinogenesis process. For the first time, we grouped such assays as early, intermediate, or advanced biomarkers which allow for the identification of the cells in the initiation, promotion or aggressive stages of tumorigenesis. Our proposal, as a novelty, points out that using a battery containing assays from all three groups can identify if a certain agent/exposure can pose a carcinogenic risk; furthermore, it can gather mechanistic insights into the mode of the action of a specific carcinogen. This structured battery could be very useful for any type of in vitro study, containing human cell lines aiming to detect the potential carcinogenic risks of environmental agents/exposures. In fact, here, we include examples in which these approaches were successfully applied. Finally, we provide a series of advantages that, we believe, contribute to the suitability of our proposed approach for the evaluation of exposure-induced carcinogenic effects and for the development of an alternative strategy for conducting an exposure risk assessment.

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.

Insights into the potential carcinogenicity of micro- and nano-plastics.

There is a growing concern regarding the potential health effects that continuous exposure to environmental micro- and nano-plastics (MNPLs) may cause on humans. Due to their persistent nature, MNPLs may accumulate in different organs and tissues and may induce in the long term the development of cancer. The present study aimed to review the existing literature on the carcinogenic potential of MNPLs. As studies directly assessing carcinogenicity were expected to be scarce, studies dealing with indirect outcomes associated with the carcinogenic process were considered in the literature search. Of the 126 studies screened, 19 satisfied the inclusion criteria. Besides, 7 additional cross-referenced articles, identified through a careful reading of the previously selected papers, also met the inclusion criteria and, consequently, were included in the review. Most of the selected studies were performed using in vitro models whereas about 40% of the studies were done in rodents, although none of them included a 2-year carcinogenicity assay. Most of the reviewed studies pointed out the potential of MNPLs to induce inflammation and genotoxicity, the latter being recognized as a strong predictor of carcinogenicity. These, along with other important findings such as the MNPLs' ability to accumulate into cells and tissues, or their capacity to induce fibrosis, may suggest an association between MNPLs exposures and the carcinogenic potential. Nevertheless, the limited number of available studies precludes reaching clear conclusions. Therefore, this review also provides several recommendations to cover the current knowledge gaps and address the future evaluation of the MNPLs' carcinogenic risk.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

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.

Lack of mutagenicity of TiO2 nanoparticles in vitro despite cellular and nuclear uptake.

The potential genotoxicity of titanium dioxide (TiO2) nanoparticles (NPs) is a conflictive topic because both positive and negative findings have been reported. To add clarity, we have carried out a study with two cell lines (V79-4 and A549) to evaluate the effects of TiO2 NPs (NM-101), with a diameter ranging from 15 to 60 nm, at concentrations 1-75 µg/cm2. Using two different dispersion procedures, cell uptake was determined by Transmission Electron Microscopy (TEM). Mutagenicity was evaluated using the Hprt gene mutation test, while genotoxicity was determined with the comet assay, detecting both DNA breaks and oxidized DNA bases (with formamidopyrimidine glycosylase - Fpg). Cell internalization, as determined by TEM, shows TiO2 NM-101 in cytoplasmic vesicles, as well as close to and inside the nucleus. Such internalization did not depend on the state of agglomeration, nor the dispersion used. In spite of such internalization, no cytotoxicity was detected in V79-4 cells (relative growth activity and plating efficiency assays) or in A549 cells (AlamarBlue assay) after exposure lasting for 24 h. However, a significant decrease in the relative growth activity was detected at longer exposure times (48 and 72 h) and at the highest concentration 75 µg/cm2. When the modified enzyme-linked alkaline comet assay was performed on A549 cells, although no significant induction of DNA damage was detected, a positive concentration-effects relationship was observed (Spearman's correlation = 0.9, p 0.0001). Furthermore, no significant increase of DNA oxidized purine bases was observed. When the frequency of Hprt gene mutants was determined in V79-4 cells, no increase was observed in the exposed cells, relative to the unexposed cultures. Our general conclusion is that, under our experimental conditions, TiO2 NM-101 exposure does not exert mutagenic effects despite the evidence of NP uptake by V79-4 cells.