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.

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.

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.

Macrophage autophagy protects mice from cerium oxide nanoparticle-induced lung fibrosis.

BACKGROUND: Cerium (Ce) is a rare earth element, rapidly oxidizing to form CeO2, and currently used in numerous commercial applications, especially as nanoparticles (NP). The potential health effects of Ce remain uncertain, but literature indicates the development of rare earth pneumoconiosis accompanied with granuloma formation, interstitial fibrosis and inflammation. The exact underlying mechanisms are not yet completely understood, and we propose that autophagy could be an interesting target to study, particularly in macrophages. Therefore, the objective of our study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO2 NP in mice. Mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO2 NP by single oropharyngeal administration and sacrificed up to 1 month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFß1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype). RESULTS: Such pulmonary exposure to CeO2 NP induces a progressive and dose-dependent lung fibrosis in the bronchiolar and alveolar walls, together with the activation of autophagy. Blockage of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, via the modulation of macrophage polarization towards M2 phenotype. CONCLUSION: In conclusion, our findings bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as important players in the disease.

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.

Carbon nanotubes, but not spherical nanoparticles, block autophagy by a shape-related targeting of lysosomes in murine macrophages.

Nanoparticles (NPs) can be toxic, depending on their physico-chemical characteristics. Macroautophagy/autophagy could represent a potential underlying mechanism of this toxicity. We therefore set up a study aimed to characterize in depth the effects, on autophagy, of macrophage exposure to NPs, with a particular attention paid to the role of NP physico-chemical characteristics (specifically chemical composition, shape, size, length, crystal phase, and/or surface properties). We demonstrate that exposure to carbon nanotubes (CNT) but not to spherical NPs leads to the blockage of the autophagic flux. We further identified lysosomal dysfunction, in association with the downregulation of SNAPIN expression, as the underlying mechanism responsible for the CNT-induced autophagy blockade. These results identify for the first time the shape as a major determinant of the interaction of NPs with the autophagy pathway. Moreover, identifying the lysosomes and SNAPIN as primary targets of MWCNT toxicity opens new directions in the interpretation and understanding of nanomaterial toxicity.

Frozen dispersions of nanomaterials are a useful operational procedure in nanotoxicology.

The variability observed in nanoparticle (NP) dispersions can affect the reliability of the results obtained in short-term tests, and mainly in long-term experiments. In addition, obtaining a good dispersion is time-consuming and acts as a bottleneck in the development of high-throughput screening methodologies. The freezing of different aliquots from a stock dispersion would overcome such limitations, but no studies have explored the impact of freezing thawing the samples on the physico-chemical and biological properties of the nanomaterial (NM). This work aims to compare fresh-prepared and frozen MWCNT, ZnO-, Ag-, TiO2- and CeO2-NP dispersions, used as models. NP characterization (size and morphology by TEM), hydrodynamic size and zeta potential were performed. Viability comparisons were determined in BEAS-2B cells. Cellular NP uptake and induced ROS production was assessed by TEM and flow cytometry, respectively. The obtained results show no important differences between frozen and fresh NP in their physico-chemical characteristics or their biological effects. This study is the first to demonstrate that there is no scientific evidence to dismiss the use of frozen NP, opening the door to the development of short- and long-term experiments with higher consistency, accuracy and reproducibility in a much shorter time and using a simplified procedure.

Biomonitoring of humans exposed to arsenic, chromium, nickel, vanadium, and complex mixtures of metals by using the micronucleus test in lymphocytes.

Various metals have demonstrated genotoxic and carcinogenic potential via different mechanisms. Until now, biomonitoring and epidemiological studies have been carried out to assess the genotoxic risk to exposed human populations. In this sense, the use of the micronucleus assay in peripheral blood lymphocytes has proven to be a useful tool to determine increased levels of DNA damage, as a surrogate biomarker of cancer risk. Here we review those biomonitoring studies focused on people exposed to arsenic, chromium, nickel, vanadium and complex mixtures of metals. Only those studies that used the frequency of micronuclei in binucleated (BNMN) cells have been taken into consideration, although the inclusion of other biomarkers of exposure and genotoxicity are also reflected and discussed. Regarding arsenic, most of the occupational and environmental biomonitoring studies find an increase in BNMN among the exposed individuals. Thus, it seems conclusive that arsenic exposure increases the risk of exposed human populations. However, a lack of correlation between the level of exposure and the increase in BNMN is also common, and a limited number of studies evaluated the genotype as a risk modulator. As for chromium, a BNMN increase in occupationally exposed subjects and a correlation between level of exposure and effect is found consistently in the available literature. However, the quality score of the studies is only medium-low. On the other hand, the studies evaluating nickel and vanadium are scarce and lacks a correct characterization of the individual exposure, which difficult the building of clear conclusions. Finally, several studies with medium-high quality scores evaluated a more realistic scenario of exposure which takes into account a mixture of metals. Among them, those which correctly characterized and measured the exposure were able to find association with the level of BNMN. Also, several genes associated with DNA damage repair such as OGG1 and XRCC1 were found to influence the exposure effect.

Acute and long-term in vitro effects of zinc oxide nanoparticles.

Since most of the toxic studies of zinc oxide nanoparticles (ZnO NPs) focused on acute and high-dose exposure conditions, the aim of the present study was to fill the existing knowledge gap of long-term effects of ZnO NPs at sub-toxic doses. To overcome this point, we have evaluated the toxic, genotoxic, and carcinogenic effects of ZnO NPs under long-term treatments (12 weeks), using a sub-toxic dose (1 µg/mL) according to acute 48-h exposure. Preliminarily, oxidative stress and genotoxic/oxidative DNA damage were determined under acute exposure and high-dose conditions. To determine the role of oxidative DNA damage, a wild-type mouse embryonic fibroblast (MEF Ogg1 (+/+)) and its isogenic 8-oxo-guanine DNA glycosylase 1 (Ogg1) knockout partner (MEF Ogg1 (-/-)) cell lines were used. Although short-term exposure (24-h) experiments demonstrated that ZnO NPs were able to induce ROS, genotoxicity, and oxidative DNA damage in both cell lines, no effects were obtained under long-term exposure scenario. Thus, 1 µg/mL exposure over 12 weeks was unable to induce genotoxicity as well as cellular transformation in both cell types, as indicated by the lack of observed morphological cell changes, variations in the secretion of matrix metalloproteinases, and anchorage-independent cell growth ability, regarded as cancer-like phenotypic hallmarks. Our results indicate that short-term effects of ZnO NP exposure are not replicated under long-term and sub-toxic dose conditions. All together, the lack of genotoxic/carcinogenic effects after chronic treatments seem to indicate a reduced risk associated with ZnO NP exposure.

Oxidative DNA damage enhances the carcinogenic potential of in vitro chronic arsenic exposures.

Chronic exposure to arsenic is known to increase the incidence of cancer in humans. Our previous work demonstrated that environmentally relevant arsenic exposures generate an accelerated accumulation of pre-carcinogen 8-OH-dG DNA lesions under Ogg1-deficient backgrounds, but it remains unproved whether this observed arsenic-induced oxidative DNA damage (ODD) is certainly important in terms of cancer. Here, isogenic MEF Ogg1 (+/+) cells and MEF Ogg1 (-/-) cells-unable to properly eliminate 8-OH-dG from DNA-were exposed to 0.5, 1 and 2 µM of sodium arsenite for 40 weeks. The acquisition of an in vitro cancer-like phenotype was assessed throughout the exposure; matrix metalloproteinase (MMP) activities were measured by zymography, colony formation and promotion were evaluated by soft agar assay, and cellular invasiveness was measured by the transwell assay. Alterations in cellular morphology, growth and differentiation status were also included as complementary measures of transformation. MEF Ogg1 (-/-) cells showed a cancer-associated phenotype after 30 weeks of exposure, as indicated by morphological changes, increased proliferation, deregulated differentiation status, increased MMPs secretion, anchorage-independent cell growth and enhancement of tumor growth and invasiveness. Conversely, MEF Ogg1 (+/+) cells did not present changes in morphology or proliferation, exhibited a milder degree of gene deregulation and needed 10 weeks of additional exposure to the highest arsenite doses to show tumor enhancing effects. Thus, Ogg1 genetic background and arsenic-induced 8-OH-dG proved relevant for arsenic-mediated carcinogenic effects. To our knowledge, this is the first study directly linking ODD with arsenic carcinogenesis.

Long-term exposures to low doses of cobalt nanoparticles induce cell transformation enhanced by oxidative damage.

A weak aspect of the in vitro studies devoted to get information on the toxic, genotoxic and carcinogenic properties of nanomaterials is that they are usually conducted under acute-exposure and high-dose conditions. This makes difficult to extrapolate the results to human beings. To overcome this point, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after long-term exposures (12 weeks) to sub-toxic doses (0.05 and 0.1 µg/mL). To get further information on whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1(+/+)) and its isogenic Ogg1 knockout partner (MEF Ogg1(-)(/)(-)), unable to properly eliminate the 8-OH-dG lesions from DNA. Our initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce reactive oxygen species (ROS) and that MEF Ogg1(-)(/)(-) cells are more sensitive to CoNPs-induced acute toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorage-independent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were significantly dependent on the cell line used, the Ogg1(-)(/)(-) cells being particularly sensitive. Altogether, the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance of ROS and Ogg1 genetic background on CoNPs-associated effects.