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.

Current status and future challenges of genotoxicity OECD Test Guidelines for nanomaterials: a workshop report.

Genotoxicity testing for nanomaterials remains challenging as standard testing approaches require some adaptation, and further development of nano-specific OECD Test Guidelines (TGs) and Guidance Documents (GDs) are needed. However, the field of genotoxicology continues to progress and new approach methodologies (NAMs) are being developed that could provide relevant information on the range of mechanisms of genotoxic action that may be imparted by nanomaterials. There is a recognition of the need for implementation of new and/or adapted OECD TGs, new OECD GDs, and utilization of NAMs within a genotoxicity testing framework for nanomaterials. As such, the requirements to apply new experimental approaches and data for genotoxicity assessment of nanomaterials in a regulatory context is neither clear, nor used in practice. Thus, an international workshop with representatives from regulatory agencies, industry, government, and academic scientists was convened to discuss these issues. The expert discussion highlighted the current deficiencies that exist in standard testing approaches within exposure regimes, insufficient physicochemical characterization, lack of demonstration of cell or tissue uptake and internalization, and limitations in the coverage of genotoxic modes of action. Regarding the latter aspect, a consensus was reached on the importance of using NAMs to support the genotoxicity assessment of nanomaterials. Also highlighted was the need for close engagement between scientists and regulators to (i) provide clarity on the regulatory needs, (ii) improve the acceptance and use of NAM-generated data, and (iii) define how NAMs may be used as part of weight of evidence approaches for use in regulatory risk assessments.

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.

Long-term exposure to nanoplastics alters molecular and functional traits related to the carcinogenic process.

Micro/nanoplastics (MNPLs) are considered emergent pollutants widely spread over all environmental compartments. Although their potential biological effects are being intensively evaluated, many doubts remain about their potential health effects in humans. One of the most underdeveloped fields is the determination of the potential tumorigenic risk of MNPLs exposure. To shed light on this topic, we have designed a wide battery of different hallmarks of cancer applied to prone-to-transformed progress MEF cells exposed to polystyrene nanoplastics (PSNPLs) in the long term (6 months). Interestingly, most of the evaluated hallmarks of cancer are exacerbated after exposure, independently if they are associated with an early tumoral phenotype (changes in stress-related genes, or microRNA deregulation), advanced tumoral phenotype (growing independently of anchorage ability, and migration capacity), or an aggressive tumoral phenotype (invasion potential, changes in pluripotency markers, and ability to grow to form tumorspheres). This set of obtained data constitutes a relevant warning on the potential carcinogenic risk associated with long-term exposures to MNPLs, specifically that induced by the PSNPLs evaluated in this study.

Genotoxicity of Graphene-Based Materials.

Graphene-based materials (GBMs) are a broad family of novel carbon-based nanomaterials with many nanotechnology applications. The increasing market of GBMs raises concerns on their possible impact on human health. Here, we review the existing literature on the genotoxic potential of GBMs over the last ten years. A total of 50 articles including in vitro, in vivo, in silico, and human biomonitoring studies were selected. Graphene oxides were the most analyzed materials, followed by reduced graphene oxides. Most of the evaluations were performed in vitro using the comet assay (detecting DNA damage). The micronucleus assay (detecting chromosome damage) was the most used validated assay, whereas only two publications reported results on mammalian gene mutations. The same material was rarely assessed with more than one assay. Despite inhalation being the main exposure route in occupational settings, only one in vivo study used intratracheal instillation, and another one reported human biomonitoring data. Based on the studies, some GBMs have the potential to induce genetic damage, although the type of damage depends on the material. The broad variability of GBMs, cellular systems and methods used in the studies precludes the identification of physico-chemical properties that could drive the genotoxicity response to GBMs.

Nanoplastics and Arsenic Co-Exposures Exacerbate Oncogenic Biomarkers under an In Vitro Long-Term Exposure Scenario.

The increasing accumulation of plastic waste and the widespread presence of its derivatives, micro- and nanoplastics (MNPLs), call for an urgent evaluation of their potential health risks. In the environment, MNPLs coexist with other known hazardous contaminants and, thus, an interesting question arises as to whether MNPLs can act as carriers of such pollutants, modulating their uptake and their harmful effects. In this context, we have examined the interaction and joint effects of two relevant water contaminants: arsenic and polystyrene nanoplastics (PSNPLs), the latter being a model of nanoplastics. Since both agents are persistent pollutants, their potential effects have been evaluated under a chronic exposure scenario and measuring different effect biomarkers involved in the cell transformation process. Mouse embryonic fibroblasts deficient for oxidative DNA damage repair mechanisms, and showing a cell transformation status, were used as a sensitive cell model. Such cells were exposed to PSNPLs, arsenic, and a combination PSNPLs/arsenic for 12 weeks. Interestingly, a physical interaction between both pollutants was demonstrated by using TEM/EDX methodologies. Results also indicate that the continuous co-exposure enhances the DNA damage and the aggressive features of the initially transformed phenotype. Remarkably, co-exposed cells present a higher proportion of spindle-like cells within the population, an increased capacity to grow independently of anchorage, as well as enhanced migrating and invading potential when compared to cells exposed to arsenic or PSNPLs alone. This study highlights the need for further studies exploring the long-term effects of contaminants of emerging concern, such as MNPLs, and the importance of considering the behavior of mixtures as part of the hazard and human risk assessment approaches.

Long-Term Effects of Polystyrene Nanoplastics in Human Intestinal Caco-2 Cells.

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.

Polystyrene Nanoplastics as Carriers of Metals. Interactions of Polystyrene Nanoparticles with Silver Nanoparticles and Silver Nitrate, and Their Effects on Human Intestinal Caco-2 Cells.

Environmental plastic wastes are continuously degraded to their micro and nanoforms. Since in the environment they coexist with other pollutants, it has been suggested that they could act as vectors transporting different toxic trace elements, such as metals. To confirm this, we have assessed the potential interactions between nanopolystyrene, as a model of nanoplastic debris, and silver compounds (silver nanoparticles and silver nitrate), as models of metal contaminant. Using TEM-EDX methodological approaches, we have been able to demonstrate metal sorption by nanopolystyrene. Furthermore, using Caco-2 cells and confocal microscopy, we have observed the co-localization of nanopolystyrene/nanosilver in different cellular compartments, including the cell nucleus. Although the internalization of these complexes showed no exacerbated cytotoxic effects, compared to the effects of each compound alone, the silver/nanopolystyrene complexes modulate the cell's uptake of silver and slightly modify some harmful cellular effects of silver, such as the ability to induce genotoxic and oxidative DNA damage.

Ex vivo exposure to different types of graphene-based nanomaterials consistently alters human blood secretome.

The biomedical applications of graphene-based nanomaterials (GBN) have significantly grown in the last years. Many of these applications suppose their intravenous exposure and, in this way, GBN could encounter blood cells triggering an immunological response of unknown effects. Consequently, understanding the relationships between GBN and the immune system response should be a prerequisite for its adequate use in biomedicine. In the present study, we have conducted a little explored ex vivo exposure method in order to study the complexity of the secretome given by the interactions between GBN and blood cells. Blood samples from different healthy donors were exposed to three different types of GBN widely used in the biomedical field. In this sense, graphene oxide (GO), graphene nanoplatelets (GNPs), graphene nanoribbons (GNRs) and a panel of 105 proteins representatives of the blood secretome were evaluated. The results show broad changes in both the cytokines number and the expression levels, with important changes in inflammatory response markers. Furthermore, the indirect soft-agar assay was used as a tool to unravel the global functional impact of the found secretome changes. Our results indicate that the GBN-induced altered secretome can modify the natural anchorage-independent growth capacity of HeLa cells, used as a model. As a conclusion, this study describes an innovative approach to study the potential harmful effects of GBN, providing relevant data to be considered in the biomedical context when GBN are planned to be used in patients.

Interactions of polystyrene nanoplastics with in vitro models of the human intestinal barrier.

The universal presence of micro-nanoplastics (MNPLs) and its relative unknown effects on human health is a concern demanding reliable data to evaluate their safety. As ingestion is one of the main exposure routes for humans, we have assessed their hazard using two in vitro models that simulate the human intestinal barrier and its associated lymphoid system. Two different coculture models (differentiated Caco-2/HT29 intestinal cells and Caco-2/HT29 + Raji-B cells) were exposed to polystyrene nanoparticles (PSNPs) for 24 h. Endpoints such as viability, membrane integrity, NPS localization and translocation, ROS induction, and genotoxic damage were evaluated to have a comprehensive view of their potentially harmful effects. No significant cytotoxic effects were observed in any of the analyzed systems. In addition, no adverse effects were detected in the integrity or in the permeability of the barrier model. Nevertheless, confocal microscopy analysis showed that MNPLs were highly uptaken by both of the barrier model systems, and that translocation across the membrane occurred. Thus, MNPLs were detected into Raji-B cells, placed in the basolateral compartment of the insert. The internalization followed a dose-dependent pattern, as assessed by flow cytometry. Nonetheless, no genotoxic or oxidative DNA damage induction was detected in either case. Finally, no variations in the transcription of oxidative and stress genes could be detected in any of the in vitro barrier models. Our results show that MNPLs can enter and cross the epithelial barrier of the digestive system, as demonstrated when Raji-B cells were included in the model, but without exerting apparent hazardous effects.

Biological effects, including oxidative stress and genotoxic damage, of polystyrene nanoparticles in different human hematopoietic cell lines.

In recent years the terms "micro-/nanoplastics" (MNPLs) have caught special attention due to the increasing levels by which humans are exposed. Among MNPLs, polystyrene nanoparticles (PSNPs) are one of the most represented MNPLs in the environment. These tiny particles may enter into the human body, translocate through human barriers, interacting with blood and lymphatic immune cells, and reaching secondary organs. By using three different human leukocytic cell lines: Raji-B (B-lymphocytes), TK6 (lymphoblasts) and THP-1 (monocytes), we pursued to determine the effects of these PSNPs on the immune cell population. With this aim, the three cell lines were exposed to spherical PSNPs of about 50 nm of diameter and cytotoxicity, cellular uptake, reactive oxygen species (ROS) production, and genotoxicity were assessed at different time-points. Results show differences in all the measured endpoints, among the selected cell lines. Thus, whilst the monocytic THP-1 cells showed the highest particle internalization, no adverse effects were observed in such cells. On the other side, although Raji-B and TK6 showed lesser PSNPs uptake, mild toxicity, ROS production and genotoxicity were detected. These results highlight the importance of the cell line selection when the biological effects of PSNPs are evaluated.

MTH1 is involved in the toxic and carcinogenic long-term effects induced by zinc oxide and cobalt nanoparticles.

The nanoparticles (NPs) exposure-related oxidative stress is considered among the main causes of the toxic effects induced by these materials. However, the importance of this mechanism has been mostly explored at short term. Previous experience with cells chronically exposed to ZnO and Co NPs hinted to the existence of an adaptative mechanism contributing to the development of oncogenic features. MTH1 is a well-described enzyme expressed exclusively in cancer cells and required to avoid the detrimental consequences of its high prooxidant microenvironment. In the present work, a significantly marked overexpression was found when MTH1 levels were monitored in long-term ZnO and Co NP-exposed cells, a fact that correlates with acquired 2.5-fold and 3.75-fold resistance to the ZnO and Co NPs treatment, respectively. The forced stable inhibition of Mth1 expression by shRNA, followed by 6 additional weeks of exposure, significantly reduced this acquired resistance and sensitized cells to the oxidizing agents H2O2 and KBrO3. When the oncogenic phenotype of Mth1 knock-down cells was evaluated, we found a decrease in several oncogenic markers, including proliferation, anchorage-independent cell growth, and migration and invasion potential. Thus, MTH1 elicits here as a relevant player in the NPs-induced toxicity and carcinogenicity. This study is the first to give a mechanistic explanation for long-term NPs exposure-derived effects. We propose MTH1 as a candidate biomarker to unravel NPs potential genotoxic and carcinogenic effects, as its expression is expected to be elevated only under exposure conditions able to induce DNA damage and the acquisition of an oncogenic phenotype.

Interactions of graphene oxide and graphene nanoplatelets with the in vitro Caco-2/HT29 model of intestinal barrier.

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.

Assessing the relevance of exposure time in differentiated Caco-2/HT29 cocultures. Effects of silver nanoparticles.

In vitro models of the intestinal barrier are being increasingly used to evaluate nanoparticles (NPs) exposure risk. Nevertheless, most of these studies have focused on short-term exposures lasting no more than 24 h of duration, which could underestimate the toxic effects of a given compound under a more realistic setting. Since the assessment of longer exposure time-points is crucial to evaluate the risk of cumulative exposure to NPs, we have analyzed the effects of AgNPs at different exposure time-points between 6 h and 4 days on the barrier model system constituted by Caco-2/HT29 cells. Our results indicate that i) the system is stable during this time frame; ii) AgNPs affect the barrier's integrity only at the highest concentration tested (100 µg/mL), and only after 96 h of exposure; iii) cellular uptake of AgNPs showed a time-dependent and concentration-dependent increase; iv) translocation through the barrier was only observed at the highest concentration and only after 96 h of exposure; v) the expression of genes involved in the barrier's structure differs depending on the exposure time analyzed. All these results reinforce our proposal of expanding exposure times beyond 24 h when performing assays for hazard assessment of NPs using in vitro models of the intestinal barrier.