Assessing the Genotoxicity of Cellulose Nanomaterials in a Co-Culture of Human Lung Epithelial Cells and Monocyte-Derived Macrophages.

Cellulose micro/nanomaterials (CMNMs) are innovative materials with a wide spectrum of industrial and biomedical applications. Although cellulose has been recognized as a safe material, the unique properties of its nanosized forms have raised concerns about their safety for human health. Genotoxicity is an endpoint that must be assessed to ensure that no carcinogenic risks are associated with exposure to nanomaterials. In this study, we evaluated the genotoxicity of two types of cellulose micro/nanofibrils (CMF and CNF) and one sample of cellulose nanocrystals (CNC), obtained from industrial bleached Eucalyptus globulus kraft pulp. For that, we exposed co-cultures of human alveolar epithelial A549 cells and THP-1 monocyte-derived macrophages to a concentration range of each CMNM and used the micronucleus (MN) and comet assays. Our results showed that only the lowest concentrations of the CMF sample were able to induce DNA strand breaks (FPG-comet assay). However, none of the three CMNMs produced significant chromosomal alterations (MN assay). These findings, together with results from previous in vitro studies using monocultures of A549 cells, indicate that the tested CNF and CNC are not genotoxic under the conditions tested, while the CMF display a low genotoxic potential.

The Effect of Nanomaterials on DNA Methylation: A Review.

DNA methylation is an epigenetic mechanism that involves the addition of a methyl group to a cytosine residue in CpG dinucleotides, which are particularly abundant in gene promoter regions. Several studies have highlighted the role that modifications of DNA methylation may have on the adverse health effects caused by exposure to environmental toxicants. One group of xenobiotics that is increasingly present in our daily lives are nanomaterials, whose unique physicochemical properties make them interesting for a large number of industrial and biomedical applications. Their widespread use has raised concerns about human exposure, and several toxicological studies have been performed, although the studies focusing on nanomaterials' effect on DNA methylation are still limited. The aim of this review is to investigate the possible impact of nanomaterials on DNA methylation. From the 70 studies found eligible for data analysis, the majority were in vitro, with about half using cell models related to the lungs. Among the in vivo studies, several animal models were used, but most were mice models. Only two studies were performed on human exposed populations. Global DNA methylation analyses was the most frequently applied approach. Although no trend towards hypo- or hyper-methylation could be observed, the importance of this epigenetic mechanism in the molecular response to nanomaterials is evident. Furthermore, methylation analysis of target genes and, particularly, the application of comprehensive DNA methylation analysis techniques, such as genome-wide sequencing, allowed identifying differentially methylated genes after nanomaterial exposure and affected molecular pathways, contributing to the understanding of their possible adverse health effects.

Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles-A Systematic Review.

Titanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).

Toxicological Assessment of Cellulose Nanomaterials: Oral Exposure.

Cellulose nanomaterials (CNMs) have emerged recently as an important group of sustainable bio-based nanomaterials (NMs) with potential applications in multiple sectors, including the food, food packaging, and biomedical fields. The widening of these applications leads to increased human oral exposure to these NMs and, potentially, to adverse health outcomes. Presently, the potential hazards regarding oral exposure to CNMs are insufficiently characterised. There is a need to understand and manage the potential adverse effects that might result from the ingestion of CNMs before products using CNMs reach commercialisation. This work reviews the potential applications of CNMs in the food and biomedical sectors along with the existing toxicological in vitro and in vivo studies, while also identifying current knowledge gaps. Relevant considerations when performing toxicological studies following oral exposure to CNMs are highlighted. An increasing number of studies have been published in the last years, overall showing that ingested CNMs are not toxic to the gastrointestinal tract (GIT), suggestive of the biocompatibility of the majority of the tested CNMs. However, in vitro and in vivo genotoxicity studies, as well as long-term carcinogenic or reproductive toxicity studies, are not yet available. These studies are needed to support a wider use of CNMs in applications that can lead to human oral ingestion, thereby promoting a safe and sustainable-by-design approach.

The Use of Human Biomonitoring to Assess Occupational Exposure to PAHs in Europe: A Comprehensive Review.

Polycyclic aromatic hydrocarbons (PAHs) are among the chemicals with proven impact on workers' health. The use of human biomonitoring (HBM) to assess occupational exposure to PAHs has become more common in recent years, but the data generated need an overall view to make them more usable by regulators and policymakers. This comprehensive review, developed under the Human Biomonitoring for Europe (HBM4EU) Initiative, was based on the literature available from 2008-2022, aiming to present and discuss the information on occupational exposure to PAHs, in order to identify the strengths and limitations of exposure and effect biomarkers and the knowledge needs for regulation in the workplace. The most frequently used exposure biomarker is urinary 1-hydroxypyrene (1-OH-PYR), a metabolite of pyrene. As effect biomarkers, those based on the measurement of oxidative stress (urinary 8-oxo-dG adducts) and genotoxicity (blood DNA strand-breaks) are the most common. Overall, a need to advance new harmonized approaches both in data and sample collection and in the use of appropriate biomarkers in occupational studies to obtain reliable and comparable data on PAH exposure in different industrial sectors, was noted. Moreover, the use of effect biomarkers can assist to identify work environments or activities of high risk, thus enabling preventive risk mitigation and management measures.

HBM4EU Chromates Study-Genotoxicity and Oxidative Stress Biomarkers in Workers Exposed to Hexavalent Chromium.

A study was conducted within the European Human Biomonitoring Initiative (HBM4EU) to characterize occupational exposure to Cr(VI). Herein we present the results of biomarkers of genotoxicity and oxidative stress, including micronucleus analysis in lymphocytes and reticulocytes, the comet assay in whole blood, and malondialdehyde and 8-oxo-2'-deoxyguanosine in urine. Workers from several Cr(VI)-related industrial activities and controls from industrial (within company) and non-industrial (outwith company) environments were included. The significantly increased genotoxicity (p = 0.03 for MN in lymphocytes and reticulocytes; p < 0.001 for comet assay data) and oxidative stress levels (p = 0.007 and p < 0.001 for MDA and 8-OHdG levels in pre-shift urine samples, respectively) that were detected in the exposed workers over the outwith company controls suggest that Cr(VI) exposure might still represent a health risk, particularly, for chrome painters and electrolytic bath platers, despite the low Cr exposure. The within-company controls displayed DNA and chromosomal damage levels that were comparable to those of the exposed group, highlighting the relevance of considering all industry workers as potentially exposed. The use of effect biomarkers proved their capacity to detect the early biological effects from low Cr(VI) exposure, and to contribute to identifying subgroups that are at higher risk. Overall, this study reinforces the need for further re-evaluation of the occupational exposure limit and better application of protection measures. However, it also raised some additional questions and unexplained inconsistencies that need follow-up studies to be clarified.

Genotoxicity of Three Micro/Nanocelluloses with Different Physicochemical Characteristics in MG-63 and V79 Cells.

(1) Background: Nanocellulose is an innovative engineered nanomaterial with an enormous potential for use in a wide array of industrial and biomedical applications and with fast growing economic value. The expanding production of nanocellulose is leading to an increased human exposure, raising concerns about their potential health effects. This study was aimed at assessing the potential toxic and genotoxic effects of different nanocelluloses in two mammalian cell lines; (2) Methods: Two micro/nanocelluloses, produced with a TEMPO oxidation pre-treatment (CNFs) and an enzymatic pre-treatment (CMFs), and cellulose nanocrystals (CNCs) were tested in osteoblastic-like human cells (MG-63) and Chinese hamster lung fibroblasts (V79) using the MTT and clonogenic assays to analyse cytotoxicity, and the micronucleus assay to test genotoxicity; (3) Results: cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNCs, but not by the MTT assay; CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells; CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells; (4) Conclusions: All nanocelluloses revealed cytotoxicity and genotoxicity, although at different concentrations, that may be related to their physicochemical differences and availability for cell uptake, and to differences in the DNA damage response of the cell model.

Analysis of the In Vitro Toxicity of Nanocelluloses in Human Lung Cells as Compared to Multi-Walled Carbon Nanotubes.

Cellulose micro/nanomaterials (CMNM), comprising cellulose microfibrils (CMF), nanofibrils (CNF), and nanocrystals (CNC), are being recognized as promising bio-nanomaterials due to their natural and renewable source, attractive properties, and potential for applications with industrial and economical value. Thus, it is crucial to investigate their potential toxicity before starting their production at a larger scale. The present study aimed at evaluating the cell internalization and in vitro cytotoxicity and genotoxicity of CMNM as compared to two multi-walled carbon nanotubes (MWCNT), NM-401 and NM-402, in A549 cells. The exposure to all studied NM, with the exception of CNC, resulted in evident cellular uptake, as analyzed by transmission electron microscopy. However, none of the CMNM induced cytotoxic effects, in contrast to the cytotoxicity observed for the MWCNT. Furthermore, no genotoxicity was observed for CNF, CNC, and NM-402 (cytokinesis-block micronucleus assay), while CMF and NM-401 were able to significantly raise micronucleus frequency. Only NM-402 was able to induce ROS formation, although it did not induce micronuclei. Thus, it is unlikely that the observed CMF and NM-401 genotoxicity is mediated by oxidative DNA damage. More studies targeting other genotoxicity endpoints and cellular and molecular events are underway to allow for a more comprehensive safety assessment of these nanocelluloses.

New "Omics" Approaches as Tools to Explore Mechanistic Nanotoxicology.

In the last years, "omics" approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed "omics" field, transcriptomics, expects to find unique profiles of differentially-expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new "omics" studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health.

Biomarkers of effect as determined in human biomonitoring studies on hexavalent chromium and cadmium in the period 2008-2020.

A number of human biomonitoring (HBM) studies have presented data on exposure to hexavalent chromium [Cr(VI)] and cadmium (Cd), but comparatively few include results on effect biomarkers. The latter are needed to identify associations between exposure and adverse outcomes (AOs) in order to assess public health implications. To support improved derivation of EU regulation and policy making, it is of great importance to identify the most reliable effect biomarkers for these heavy metals that can be used in HBM studies. In the framework of the Human Biomonitoring for Europe (HBM4EU) initiative, our study aim was to identify effect biomarkers linking Cr(VI) and Cd exposure to selected AOs including cancer, immunotoxicity, oxidative stress, and omics/epigenetics. A comprehensive PubMed search identified recent HBM studies, in which effect biomarkers were examined. Validity and applicability of the markers in HBM studies are discussed. The most frequently analysed effect biomarkers regarding Cr(VI) exposure and its association with cancer were those indicating oxidative stress (e.g., 8-hydroxy-2'-deoxyguanosine (8-OHdG), malondialdehyde (MDA), glutathione (GSH)) and DNA or chromosomal damage (comet and micronucleus assays). With respect to Cd and to some extent Cr, ß-2-microglobulin (B2-MG) and N-acetyl-ß-D-glucosaminidase (NAG) are well-established, sensitive, and the most common effect biomarkers to relate Cd or Cr exposure to renal tubular dysfunction. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule (KIM)-1 could serve as sensitive biomarkers of acute kidney injury in response to both metals, but need further investigation in HBM studies. Omics-based biomarkers, i.e., changes in the (epi-)genome, transcriptome, proteome, and metabolome associated with Cr and/or Cd exposure, are promising effect biomarkers, but more HBM data are needed to confirm their significance. The combination of established effect markers and omics biomarkers may represent the strongest approach, especially if based on knowledge of mechanistic principles. To this aim, also mechanistic data were collected to provide guidance on the use of more sensitive and specific effect biomarkers. This also led to the identification of knowledge gaps relevant to the direction of future research.

Functional effects of differentially expressed microRNAs in A549 cells exposed to MWCNT-7 or crocidolite.

Multi-walled carbon nanotubes (MWCNT) are engineered nanomaterials widely used in industrial and biomedical applications. Yet, MWCNT inhalation may induce pulmonary adverse effects, and the MWCNT-7 (Mitsui-7) has been classified as possibly carcinogenic to humans. However, its molecular mechanisms of action are poorly understood and there are no biomarkers of exposure for occupational monitoring. Several pulmonary diseases, including lung cancer, have been associated with alterations in microRNA expression that are used as biomarkers of disease progression, and differentially-expressed microRNAs (DE miRNAs) can also allow understanding the molecular effects induced by a toxicant. In this study, we identify DE miRNAs in A549 alveolar epithelial cells following 24 h exposure to MWCNT-7 or crocidolite, as well as their enriched cellular functional pathways. These indicate that both materials change cell survival, differentiation and proliferative properties under the influence of AMPK, FoxO, TGF-ß and Hippo pathways, and their metabolic activity and cell-to-cell communication. In addition, MWCNT-7 affects the actin cytoskeleton, ubiquitin mediated proteolysis, and ECM-receptor interactions; crocidolite the PI3K-Akt and mTOR pathways, endocytosis, and central carbon metabolism. Since deregulation of these pathways may be related to carcinogenesis, an interaction network of DE miRNAs and corresponding target cancer-related genes was constructed, highlighting the carcinogenic potential of Mitsui-7.

Cytotoxicity and genotoxicity of MWCNT-7 and crocidolite: assessment in alveolar epithelial cells versus their coculture with monocyte-derived macrophages.

In the past years, several in vitro studies have addressed the pulmonary toxicity of multi-walled carbon nanotubes (MWCNT) and compared it with that caused by asbestos fibers, but their conclusions have been somewhat inconsistent and difficult to extrapolate to in vivo. Since cell coculture models were proposed to better represent the in vivo conditions than conventional monocultures, this work intended to compare the cytotoxicity and genotoxicity of MWCNT-7 (Mitsui-7) and crocidolite using A549 cells grown in a conventional monoculture or in coculture with THP-1 macrophages. Although a decrease in A549 viability was noted following exposure to a concentration range of MWCNT-7 and crocidolite, no viability change occurred in similarly exposed cocultures. Early events indicating epithelial to mesenchymal transition (EMT) were observed which could explain apoptosis resistance. The comet assay results were similar between the two models, being positive and negative for crocidolite and MWCNT-7, respectively. An increase in the micronucleus frequency was detected in the cocultured A549-treated cells with both materials, but not in the monoculture. On the other hand, exposure of A549 monocultures to MWCNT-7 induced a highly significant increase in nucleoplasmic bridges in which those were found embedded. Our overall results demonstrate that (i) both materials are cytotoxic and genotoxic, (ii) the presence of THP-1 macrophages upholds the viability of A549 cells and increases the aneugenic/clastogenic effects of both materials probably through EMT, and (iii) MWCNT-7 induces the formation of nucleoplasmic bridges in A549 cells.

Evaluating the genotoxicity of cellulose nanofibrils in a co-culture of human lung epithelial cells and monocyte-derived macrophages.

Cellulose nanofibrils (CNF) are manufactured nanofibres that hold impressive expectations in forest, food, pharmaceutical, and biomedical industries. CNF production and applications are leading to an increased human exposure and thereby it is of utmost importance to assess its safety to health. In this study, we screened the cytotoxic, immunotoxic and genotoxic effects of a CNF produced by TEMPO-mediated oxidation of an industrial bleached Eucalyptus globulus kraft pulp on a co-culture of lung epithelial alveolar (A549) cells and monocyte-derived macrophages (THP-1 cells). The results indicated that low CNF concentrations can stimulate A549 cells proliferation, whereas higher concentrations are moderately toxic. Moreover, no proinflammatory cytokine IL-1ß was detected in the co-culture medium suggesting no immunotoxicity. Although CNF treatment did not induce sizable levels of DNA damage in A549 cells, it leaded to micronuclei formation at 1.5 and 3 µg/cm2. These findings suggest that this type of CNF is genotoxic through aneugenic or clastogenic mechanisms. Noteworthy, cell overgrowth and genotoxicity, which are events relevant for cell malignant transformation, were observed at low CNF concentration levels, which are more realistic and relevant for human exposure, e.g., in occupational settings.

Conventional and novel "omics"-based approaches to the study of carbon nanotubes pulmonary toxicity.

The widespread application of carbon nanotubes (CNT) on industrial, biomedical, and consumer products can represent an emerging respiratory occupational hazard. Particularly, their similarity with the fiber-like shape of asbestos have raised a strong concern about their carcinogenic potential. Several in vitro and in vivo studies have been supporting this view by pointing to immunotoxic, cytotoxic and genotoxic effects of some CNT that may conduct to pulmonary inflammation, fibrosis, and bronchioloalveolar hyperplasia in rodents. Recently, high throughput molecular methodologies have been applied to obtain more insightful information on CNT toxicity, through the identification of the affected biological and molecular pathways. Toxicogenomic approaches are expected to identify unique gene expression profiles that, besides providing mechanistic information and guiding new research, have also the potential to be used as biomarkers for biomonitoring purposes. In this review, the potential of genomic data analysis is illustrated by gene network and gene ontology enrichment analysis of a set of 41 differentially expressed genes selected from a literature search focused on studies of C57BL/6 mice exposed to the multiwalled CNT Mitsui-7. The majority of the biological processes annotated in the network are regulatory processes and the molecular functions are related to receptor-binding signalling. Accordingly, the network-annotated pathways are cell receptor-induced pathways. A single enriched molecular function and one biological process were identified. The relevance of specific epigenomic effects triggered by CNT exposure, for example, alteration of the miRNA expression profile is also discussed in light of its use as biomarkers in occupational health studies. Environ. Mol. Mutagen. 59:334-362, 2018. © 2018 Wiley Periodicals, Inc.

PROS1 novel splice-site variant decreases protein S expression in patients from two families with thrombotic disease.

Our results prove that c.1871-14T>G is causative of type I PS deficiency, highlighting the importance of performing mRNA-based studies in order to evaluate variants pathogenicity. We evidence the increased risk of venous thromboembolism associated with this cryptic splice-site variant if present in patients with PS deficiency.

The spectrum of mutations and molecular pathogenesis of hemophilia A in 181 Portuguese patients.

Disease-causing alterations within the F8 gene were identified in 177 hemophilia A families of Portuguese origin. The spectrum of non-inversion F8 mutations in 101 families included 67 different alterations, namely: 36 missense, 8 nonsense and 4 splice site mutations, as well as 19 insertions/deletions. Thirty-four of these mutations are novel. Molecular modeling allowed prediction of the conformational changes introduced by selected amino acid substitutions and their correlation with the patients' phenotypes. The relatively frequent, population-specific, missense mutations together with de novo alterations can lead to significant differences in the spectrum of F8 mutations among different populations.