FAIRification of nanosafety data to improve applicability of (Q)SAR approaches: A case study on in vitro Comet assay genotoxicity data.

(Quantitative) structure-activity relationship ([Q]SAR) methodologies are widely applied to predict the (eco)toxicological effects of chemicals, and their use is envisaged in different regulatory frameworks for filling data gaps of untested substances. However, their application to the risk assessment of nanomaterials is still limited, also due to the scarcity of large and curated experimental datasets. Despite a great amount of nanosafety data having been produced over the last decade in international collaborative initiatives, their interpretation, integration and reuse has been hampered by several obstacles, such as poorly described (meta)data, non-standard terminology, lack of harmonized reporting formats and criteria. Recently, the FAIR (Findable, Accessible, Interoperable, and Reusable) principles have been established to guide the scientific community in good data management and stewardship. The EU H2020 Gov4Nano project, together with other international projects and initiatives, is addressing the challenge of improving nanosafety data FAIRness, for maximizing their availability, understanding, exchange and ultimately their reuse. These efforts are largely supported by the creation of a common Nanosafety Data Interface, which connects a row of project-specific databases applying the eNanoMapper data model. A wide variety of experimental data relating to characterization and effects of nanomaterials are stored in the database; however, the methods, protocols and parameters driving their generation are not fully mature. This article reports the progress of an ongoing case study in the Gov4nano project on the reuse of in vitro Comet genotoxicity data, focusing on the issues and challenges encountered in their FAIRification through the eNanoMapper data model. The case study is part of an iterative process in which the FAIRification of data supports the understanding of the phenomena underlying their generation and, ultimately, improves their reusability.

Silica encapsulation of ZnO nanoparticles reduces their toxicity for cumulus cell-oocyte-complex expansion.

BACKGROUND: Metal oxide nanoparticles (NPs) are increasingly used in many industrial and biomedical applications, hence their impact on occupational and public health has become a concern. In recent years, interest on the effect that exposure to NPs may exert on human reproduction has grown, however data are still scant. In the present work, we investigated whether different metal oxide NPs interfere with mouse cumulus cell-oocyte complex (COC) expansion. METHODS: Mouse COCs from pre-ovulatory follicles were cultured in vitro in the presence of various concentrations of two types of TiO2 NPs (JRC NM-103 and NM-104) and four types of ZnO NPs (JRC NM-110, NM-111, and in-house prepared uncoated and SiO2-coated NPs) and the organization of a muco-elastic extracellular matrix by cumulus cells during the process named cumulus expansion was investigated. RESULTS: We show that COC expansion was not affected by the presence of both types of TiO2 NPs at all tested doses, while ZnO NM-110 and NM-111 induced strong toxicity and inhibited COCs expansion at relatively low concentration. Medium conditioned by these NPs showed lower toxicity, suggesting that, beside ion release, inhibition of COC expansion also depends on NPs per se. To further elucidate this, we compared COC expansion in the presence of uncoated or SiO2-coated NPs. Differently from the uncoated NPs, SiO2-coated NPs underwent slower dissolution, were not internalized by the cells, and showed an overall lower toxicity. Gene expression analysis demonstrated that ZnO NPs, but not SiO2-coated ZnO NPs, affected the expression of genes fundamental for COC expansion. Dosimetry analysis revealed that the delivered-to-cell mass fractions for both NPs was very low. CONCLUSIONS: Altogether, these results suggest that chemical composition, dissolution, and cell internalization are all responsible for the adverse effects of the tested NPs and support the importance of a tailored, safer-by-design production of NPs to reduce toxicity.

Biotransformation of Silver Nanoparticles into Oro-Gastrointestinal Tract by Integrated In Vitro Testing Assay: Generation of Exposure-Dependent Physical Descriptors for Nanomaterial Grouping.

Grouping approaches of nanomaterials have the potential to facilitate high throughput and cost effective nanomaterial screening. However, an effective grouping of nanomaterials hinges on the application of suitable physicochemical descriptors to identify similarities. To address the problem, we developed an integrated testing approach coupling acellular and cellular phases, to study the full life cycle of ingested silver nanoparticles (NPs) and silver salts in the oro-gastrointestinal (OGI) tract including their impact on cellular uptake and integrity. This approach enables the derivation of exposure-dependent physical descriptors (EDPDs) upon biotransformation of undigested nanoparticles, digested nanoparticles and digested silver salts. These descriptors are identified in: size, crystallinity, chemistry of the core material, dissolution, high and low molecular weight Ag-biomolecule soluble complexes, and are compared in terms of similarities in a grouping hypothesis. Experimental results indicate that digested silver nanoparticles are neither similar to pristine nanoparticles nor completely similar to digested silver salts, due to the presence of different chemical nanoforms (silver and silver chloride nanocrystals), which were characterized in terms of their interactions with the digestive matrices. Interestingly, the cellular responses observed in the cellular phase of the integrated assay (uptake and inflammation) are also similar for the digested samples, clearly indicating a possible role of the soluble fraction of silver complexes. This study highlights the importance of quantifying exposure-related physical descriptors to advance grouping of NPs based on structural similarities.

Towards FAIR nanosafety data.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

A harmonized and standardized in vitro approach produces reliable results on silver nanoparticles toxicity in different cell lines.

Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results.

Use of a common European approach for nanomaterials' testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells.

In the last years, a number of in vitro studies have been performed to assess the genotoxic activity of titanium dioxide (TiO2 ). To resolve the contradictory results, in this study, we investigated the genotoxic activity of commercial TiO2 nanoparticles (NPs) and microparticles of different forms (anatase, rutile and mix of both). We evaluated micronucleus formation in stimulated lymphocytes, as well as DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells (PBMCs), a mixed population of lymphocytes and monocytes. Different responses to TiO2 exposure were obtained depending on the assay. Both TiO2 NPs and microparticles and all the crystalline forms elicited a significant increase in 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in the whole PBMC population, without a concurrent increase of micronuclei in proliferating lymphocytes. The distribution of DNA damage in PBMCs, detected by the comet assay, that measures DNA damage at level of single cells, indicated the presence of a more susceptible cell subpopulation. The measurement of side scatter signals by flow cytometry highlighted the preferential physical interaction of TiO2 particles with monocytes that also displayed higher reactive oxygen species generation, providing a mechanistic explanation for the different responses observed in genotoxicity assays with PBMCs and lymphocytes. This study confirmed the suitability of human PBMCs as multi-cell model to investigate NP-induced DNA damage, but suggested some caution in the use of stimulated lymphocytes for the assessment of NP clastogenicity.

ZnO nanoparticle tracking from uptake to genotoxic damage in human colon carcinoma cells.

Zinc Oxide (ZnO) nanoparticles are widely used both in the industry and in biomedical applications for their chemical and physical nanomaterial properties. It is therefore essential to go in depth into the cytotoxicity mechanisms and interactions between nanomaterials and cells. The aim of this work was to evaluate the dissolution of ZnO nanoparticles and their uptake, from a few minutes after treatments up to 24h. ZnO nanoparticles routes of entry into the human colon carcinoma cells (LoVo) were followed at different times by a thorough ultrastructural investigation and semiquantitative analysis. The intracellular release of Zn(2+) ions by Zinquin fluorescent dye, and phosphorylated histone H2AX (γ-H2AX) expression were evaluated. The genotoxic potential of ZnO nanoparticles was also investigated by determining the levels of 8-hydroxyl-2'-deoxyguanosine (8-oxodG). The experimental data show that ZnO nanoparticles entered LoVo cells by either passive diffusion or endocytosis or both, depending on the agglomeration state of the nanomaterial. ZnO nanoparticles coming into contact with acid pH of lysosomes altered organelles structure, resulting in the release of Zn(2+) ions. The simultaneous presence of ZnO nanoparticles and Zn(2+) ions in the LoVo cells determined the formation of reactive oxygen species at the mitochondrial and nuclear level, inducing severe DNA damage.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

Genotype-phenotype analysis of S326C OGG1 polymorphism: a risk factor for oxidative pathologies.

8-Oxoguanine DNA glycosylase (OGG) activity was measured by an in vitro assay in lymphocytes of healthy volunteers genotyped for various OGG1 polymorphisms. Only homozygous carriers of the polymorphic C326 allele showed a significantly lower OGG activity compared to the homozygous S326 genotype. The purified S326C OGG1 showed a decreased ability to complete the repair synthesis step in a base excision repair reaction reconstituted in vitro. The propensity of this variant to dimerize as well as its catalytic impairment were shown to be enhanced under oxidizing conditions. Mass spectrometry revealed that the extra cysteine of the variant protein is involved in disulfide bonds compatible with significant conformational changes and/or dimerization. We propose that the S326C OGG1 catalytic impairment and its susceptibility to dimerization and disulfide bond formation in an oxidizing environment all concur to decrease repair capacity. Consequently, the C326 homozygous carriers may be at increased risk of oxidative pathologies.

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells.

Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines.

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay.

MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh(-/-) mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity.

Role of MUTYH and MSH2 in the control of oxidative DNA damage, genetic instability, and tumorigenesis.

Mismatch repair is the major pathway controlling genetic stability by removing mispairs caused by faulty replication and/or mismatches containing oxidized bases. Thus, inactivation of the Msh2 mismatch repair gene is associated with a mutator phenotype and increased cancer susceptibility. The base excision repair gene Mutyh is also involved in the maintenance of genomic integrity by repairing premutagenic lesions induced by oxidative DNA damage. Because evidence in bacteria suggested that Msh2 and Mutyh repair factors might have some overlapping functions, we investigated the biological consequences of their single and double inactivation in vitro and in vivo. Msh2(-/-) mouse embryo fibroblasts (MEF) showed a strong mutator phenotype at the hprt gene, whereas Mutyh inactivation was associated with a milder phenotype (2.9 x 10(-6) and 3.3 x 10(-7) mutation/cell/generation, respectively). The value of 2.7 x 10(-6) mutation/cell/generation in Msh2(-/-)Mutyh(-/-) MEFs did not differ significantly from Msh2(-/-) cells. When steady-state levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG) were measured in MEFs of different genotypes, single gene inactivation resulted in increases similar to those observed in doubly defective cells. In contrast, a synergistic accumulation of 8-oxoG was observed in several organs of Msh2(-/-)Mutyh(-/-) animals, suggesting that in vivo Msh2 and Mutyh provide separate repair functions and contribute independently to the control of oxidative DNA damage. Finally, a strong delay in lymphomagenesis was observed in Msh2(-/-)Mutyh(-/-) when compared with Msh2(-/-) animals. The immunophenotype of these tumors indicate that both genotypes develop B-cell lymphoblastic lymphomas displaying microsatellite instability. This suggests that a large fraction of the cancer-prone phenotype of Msh2(-/-) mice depends on Mutyh activity.

Replication of 2-hydroxyadenine-containing DNA and recognition by human MutSalpha.

2-Hydroxyadenine (2-OH-A), a product of DNA oxidation, is a potential source of mutations. We investigated how representative DNA polymerases from the A, B and Y families dealt with 2-OH-A in primer extension experiments. A template 2-OH-A reduced the rate of incorporation by DNA polymerase alpha (Pol alpha) and Klenow fragment (Kf(exo-)). Two Y family DNA polymerases, human polymerase eta (Pol eta) and the archeal Dpo4 polymerase were affected differently. Bypass by Pol eta was very inefficient whereas Dpo4 efficiently replicated 2-OH-A. Replication of a template 2-OH-A by both enzymes was mutagenic and caused base substitutions. Dpo4 additionally introduced single base deletions. Thermodynamic analysis showed that 2-OH-A forms stable base pairs with T, C and G, and to a lesser extent with A. Oligonucleotides containing 2-OH-A base pairs, including the preferred 2-OH-A:T, were recognized by the human MutSalpha mismatch repair (MMR). MutSalpha also recognized 2-OH-A located in a repeat sequence that mimics a frameshift intermediate.

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity.

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.

8-oxoguanine incorporation into DNA repeats in vitro and mismatch recognition by MutSalpha.

DNA 8-oxoguanine (8-oxoG) causes transversions and is also implicated in frameshifts. We previously identified the dNTP pool as a likely source of mutagenic DNA 8-oxoG and demonstrated that DNA mismatch repair prevented oxidation-related frameshifts in mononucleotide repeats. Here, we show that both Klenow fragment and DNA polymerase alpha can utilize 8-oxodGTP and incorporate the oxidized purine into model frameshift targets. Both polymerases incorporated 8-oxodGMP opposite C and A in repetitive DNA sequences and efficiently extended a terminal 8-oxoG. The human MutSalpha mismatch repair factor recognized DNA 8-oxoG efficiently in some contexts that resembled frameshift intermediates in the same C or A repeats. DNA 8-oxoG in other slipped/mispaired structures in the same repeats adopted configurations that prevented recognition by MutSalpha and by the OGG1 DNA glycosylase thereby rendering it invisible to DNA repair. These findings are consistent with a contribution of oxidative DNA damage to frameshifts. They also suggest how mismatch repair might reduce the burden of DNA 8-oxoG and prevent frameshift formation.

Structural and dynamic effects of single 7-hydro-8-oxoguanine bases located in a frameshift target DNA sequence.

DNA 7-hydro-8-oxoguanine (8-oxoG) is implicated in frameshift formation in an G(6) sequence of the HPRT gene in mismatch repair (MMR) defective cells. Using oligonucleotides based on this frameshift hotspot, we investigated how a single 8-oxoG modified the structural and dynamic properties of the G(6) tract. A 30 ns molecular dynamics (MD) simulation indicated compression of the minor groove in the immediate vicinity of the lesion. Fluorescence polarization anisotropy (FPA) and MD demonstrated that 8-oxoG increases DNA torsional rigidity and also constrains the movement of the single-stranded region at the single/double stranded DNA junction of model DNA replication template/primer. These constraints influenced the efficiency of primer extension by Klenow (exo(-)) DNA polymerase.

Fluorescence anisotropy in the frequency domain by an optical microscope.

Fluorescence anisotropy decay spectroscopy is a suitable tool for investigating the size and the shape of biological molecules. We coupled this technique to an optical microscope in order to reduce the excitation volume and to allow its application to spatially inhomogeneous samples. Phase modulated measurements of the fluorescence anisotropy decay were performed by feeding an intensity modulated linearly polarized laser beam to the epifluorescence port of a microscope. Here we report the test of the dynamic response of the microscope by comparing the lifetime and fluorescence polarization anisotropy decays obtained in cuvettes in a standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor for the objective depolarization is introduced in the best-fit functions for the data analysis of the decays, the results obtained on the two setups are comparable. Some applications are reported here on long DNA tracts as well on short DNA fragments containing structural anomalies.