Publications by authors named "Maria Dusinska"

126 Publications

Safety assessment of titanium dioxide (E171) as a food additive.

EFSA J 2021 May 6;19(5):e06585. Epub 2021 May 6.

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO nanoparticles (NPs) and data from an extended one-generation reproductive toxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO NPs < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO NPs (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico-chemical properties of TiO particles and the outcome of either or genotoxicity assays. A concern for genotoxicity of TiO particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut-off value for TiO particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.
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http://dx.doi.org/10.2903/j.efsa.2021.6585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101360PMC
May 2021

Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies.

Mutagenesis 2021 Mar 23. Epub 2021 Mar 23.

Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31008 P#38lona, Spain.

DNA damage and repair activity are often assessed in blood s#38les from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the s#38les on the day of collection without any type of storage. For instance, certain studies use repeated s#38ling of cells from the same subject or s#38les from different subjects collected at different time-points, and it is desirable to analyse all these s#38les in the same comet assay experiment. In addition, flawless comet assay analyses on frozen s#38les opens up for the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood s#38les can be cryopreserved with only minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen s#38les of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB s#38les.
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http://dx.doi.org/10.1093/mutage/geab012DOI Listing
March 2021

The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells.

Ocul Surf 2021 Apr 4;20:195-198. Epub 2021 Mar 4.

Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

Purpose: To assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva.

Methods: Impression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface.

Results: The mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls.

Conclusions: Micronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.
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http://dx.doi.org/10.1016/j.jtos.2021.02.011DOI Listing
April 2021

DNA repair gene polymorphisms and chromosomal aberrations in healthy, nonsmoking population.

DNA Repair (Amst) 2021 May 27;101:103079. Epub 2021 Feb 27.

Department of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; Faculty of Medicine and Biomedical Center in Pilsen, Charles University in Prague, 30605 Pilsen, Czech Republic; Division of Cancer Epidemiology, German Cancer Research Centre (DKFZ), 69120 Heidelberg, Germany.

Nonspecific structural chromosomal aberrations (CAs) can be found at around 1% of circulating lymphocytes from healthy individuals but the frequency may be higher after exposure to carcinogenic chemicals or radiation. The frequency of CAs has been measured in occupational monitoring and an increased frequency of CAs has also been associated with cancer risk. Alterations in DNA damage repair and telomere maintenance are thought to contribute to the formation of CAs, which include chromosome type of aberrations and chromatid type of aberrations. In the present study, we used the result of our published genome-wide association studies to extract data on 153 DNA repair genes from 866 nonsmoking persons who had no known occupational exposure to genotoxic substances. Considering an arbitrary cut-off level of P< 5 × 10, single nucleotide polymorphisms (SNPs) tagging 22 DNA repair genes were significantly associated with CAs and they remained significant at P < 0.05 when adjustment for multiple comparisons was done by the Binomial Sequential Goodness of Fit test. Nucleotide excision repair pathway genes showed most associations with 6 genes. Among the associated genes were several in which mutations manifest CA phenotype, including Fanconi anemia, WRN, BLM and genes that are important in maintaining genome stability, as well as PARP2 and mismatch repair genes. RPA2 and RPA3 may participate in telomere maintenance through the synthesis of the C strand of telomeres. Errors in NHEJ1 function may lead to translocations. The present results show associations with some genes with known CA phenotype and suggest other pathways with mechanistic rationale for the formation of CAs in healthy nonsmoking population.
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http://dx.doi.org/10.1016/j.dnarep.2021.103079DOI Listing
May 2021

Microfluidic In Vitro Platform for (Nano)Safety and (Nano)Drug Efficiency Screening.

Small 2021 Apr 18;17(15):e2006012. Epub 2021 Jan 18.

Fraunhofer Institute for Biomedical Engineering IBMT, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V., Joseph-von-Fraunhofer-Weg 1, Sulzbach, 66280, Germany.

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments.
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http://dx.doi.org/10.1002/smll.202006012DOI Listing
April 2021

Editorial for the Special Issue From Nanoinformatics to Nanomaterials Risk Assessment and Governance.

Nanomaterials (Basel) 2021 Jan 7;11(1). Epub 2021 Jan 7.

Department of Cheminformatics, NovaMechanics Ltd., Nicosia 1065, Cyprus.

Ensuring the safe and responsible use of nanotechnologies and nanoscale materials is imperative to maximize consumer confidence and drive commercialization of nano-enabled products that underpin innovation and advances in every industrial sector [...].
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http://dx.doi.org/10.3390/nano11010121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825746PMC
January 2021

Epigenetics in Breast Cancer Therapy-New Strategies and Future Nanomedicine Perspectives.

Cancers (Basel) 2020 Dec 3;12(12). Epub 2020 Dec 3.

Cancer Research Institute, Biomedical Research Center of the Slovak Academy of Sciences, Dubravska Cesta 9, 845 05 Bratislava, Slovakia.

Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identification, allowing for patient stratification and tailored management. In combination with new-generation epi-drugs, nanomedicine can help to overcome low therapeutic efficacy in treatment-resistant tumors. This review provides an overview of ongoing clinical trials focusing on combination therapies employing epi-drugs for breast cancer treatment and summarizes the latest nano-based targeted delivery approaches for epi-drugs. Moreover, it highlights the current limitations and obstacles associated with applying these experimental strategies in the clinics.
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http://dx.doi.org/10.3390/cancers12123622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761669PMC
December 2020

An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity.

Nat Protoc 2020 12 16;15(12):3844-3878. Epub 2020 Nov 16.

Department of Pharmacology & Toxicology, School for Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, Maastricht, the Netherlands.

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.
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http://dx.doi.org/10.1038/s41596-020-0401-xDOI Listing
December 2020

Impact of genetic polymorphisms in kinetochore and spindle assembly genes on chromosomal aberration frequency in healthy humans.

Mutat Res 2020 Oct - Dec;858-860:503253. Epub 2020 Sep 15.

Department of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120, Heidelberg, Germany; Hopp Children's Cancer Center (KiTZ), Heidelberg, Germany; Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany.

Genomic instability is a characteristic of a majority of human malignancies. Chromosomal instability is a common form of genomic instability that can be caused by defects in mitotic checkpoint genes. Chromosomal aberrations in peripheral blood are also indicative of genotoxic exposure and potential cancer risk. We evaluated associations between inherited genetic variants in 33 mitotic checkpoint genes and the frequency of chromosomal aberrations (CAs) in the presence and absence of environmental genotoxic exposure. Associations with both chromosome and chromatid type of aberrations were evaluated in two cohorts of healthy individuals, namely an exposed and a reference group consisting of 607 and 866 individuals, respectively. Binary logistic and linear regression analyses were performed for the association studies. Bonferroni-corrected significant p-value was 5 × 10 for 99 tests based on the number of analyzed genes and phenotypes. In the reference group the most prominent associations were found with variants in CCNB1, a master regulator of mitosis, and in genes involved in kinetochore function, including CENPH and TEX14, whereas in the exposed group the main association was found with variants in TTK, also an important gene in kinetochore function. How the identified variants may affect the fidelity of mitotic checkpoint remains to be investigated, however, the present study suggests that genetic variation may partly explain interindividual variation in the formation of CAs.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503253DOI Listing
March 2021

Minimum Information for Reporting on the Comet Assay (MIRCA): recommendations for describing comet assay procedures and results.

Nat Protoc 2020 12 26;15(12):3817-3826. Epub 2020 Oct 26.

Department of Nutrition, University of Oslo, Oslo, Norway.

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.
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http://dx.doi.org/10.1038/s41596-020-0398-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688437PMC
December 2020

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment-A Review.

Nanomaterials (Basel) 2020 Sep 25;10(10). Epub 2020 Sep 25.

Health Effects Laboratory, NILU-Norwegian Institute for Air Research, 2007 Kjeller, Norway.

Changes in the genetic material can lead to serious human health defects, as mutations in somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic events can appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study of mechanisms leading to genotoxicity is crucially important, as well as the detection of potentially genotoxic compounds. We consider the current state of the art and describe here the main endpoints applied in standard human in vitro models as well as new advanced 3D models that are closer to the in vivo situation. We performed a literature review of in vitro studies published from 2000-2020 (August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization, organ-on-a-chip and high throughput methods.
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http://dx.doi.org/10.3390/nano10101911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601632PMC
September 2020

Improving Quality in Nanoparticle-Induced Cytotoxicity Testing by a Tiered Inter-Laboratory Comparison Study.

Nanomaterials (Basel) 2020 Jul 22;10(8). Epub 2020 Jul 22.

ECAMRICERT SRL, European Center for the Sustainable Impact of Nanotechnology (ECSIN), Corso Stati Uniti 4, 35127 Padova, Italy.

The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.
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http://dx.doi.org/10.3390/nano10081430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466672PMC
July 2020

Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Small 2020 09 23;16(36):e2003303. Epub 2020 Jul 23.

TGO - Transgero Limited, Limerick, Ireland.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.
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http://dx.doi.org/10.1002/smll.202003303DOI Listing
September 2020

Embedding Ethical Impact Assessment in Nanosafety Decision Support.

Small 2020 09 23;16(36):e2002901. Epub 2020 Jul 23.

Environmental Impacts and Sustainability, NILU, Instituttveien 18, Kjeller, 2007, Norway.

Nanotechnology is a key enabling technology, which is developing fast and influences many aspects of life. Nanomaterials are already included in a broad range of products and industrial sectors. Nanosafety issues are still a matter of concern for policy makers and stakeholders, but currently, there is no platform where all stakeholders can meet and discuss these issues. A comprehensive overview of all the issues in one single dashboard presenting the output of a decision support system is also lacking. This article outlines a strategy for developing one innovative part of a modular decision support system, designed to support the work of a new Risk Governance Council (RGC) for nanomaterials which will be established through the combined efforts of the GOV4NANO, NANORIGO, and RiskGONE H2020 projects. This new module will consist of guidelines for Ethical Impact Assessment (EIA) for nanomaterials and nanoenabled products. This article offers recommendations for adapting the European Committee for Standardization (CEN) prestandard on Ethical Impact Assessment CWA (CEN Workshop Agreement) 17145-2:2017 (E), to fit into the more-encompassing decision support system for risk governance of nanomaterials within the RiskGONE project.
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http://dx.doi.org/10.1002/smll.202002901DOI Listing
September 2020

Thermodynamic Parameters at Bio-Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO, and TiO.

Chem Res Toxicol 2020 08 15;33(8):2054-2071. Epub 2020 Jul 15.

Institute of Physical Chemistry "Ilie Murgulescu" of the Romanian Academy, Bucharest 060021, Romania.

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO; NM-101), and silica (SiO; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO (NM-203) > TiO (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, Δ, for all systems. The structural stability of the protein decreased as follows: TiO (NM-101) > SiO (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA-NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00468DOI Listing
August 2020

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7 International Workshop on Genotoxicity Testing (IWGT).

Mutat Res 2020 Feb - Mar;850-851:503135. Epub 2020 Jan 15.

European Commission, Joint Research Centre (JRC), Ispra, Italy.

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7 IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503135DOI Listing
April 2020

NanoSolveIT Project: Driving nanoinformatics research to develop innovative and integrated tools for nanosafety assessment.

Comput Struct Biotechnol J 2020 7;18:583-602. Epub 2020 Mar 7.

UK Centre for Ecology and Hydrology, Library Ave, Bailrigg, Lancaster LA1 4AP, UK.

Nanotechnology has enabled the discovery of a multitude of novel materials exhibiting unique physicochemical (PChem) properties compared to their bulk analogues. These properties have led to a rapidly increasing range of commercial applications; this, however, may come at a cost, if an association to long-term health and environmental risks is discovered or even just perceived. Many nanomaterials (NMs) have not yet had their potential adverse biological effects fully assessed, due to costs and time constraints associated with the experimental assessment, frequently involving animals. Here, the available NM libraries are analyzed for their suitability for integration with novel nanoinformatics approaches and for the development of NM specific Integrated Approaches to Testing and Assessment (IATA) for human and environmental risk assessment, all within the NanoSolveIT cloud-platform. These established and well-characterized NM libraries (e.g. NanoMILE, NanoSolutions, NANoREG, NanoFASE, caLIBRAte, NanoTEST and the Nanomaterial Registry (>2000 NMs)) contain physicochemical characterization data as well as data for several relevant biological endpoints, assessed in part using harmonized Organisation for Economic Co-operation and Development (OECD) methods and test guidelines. Integration of such extensive NM information sources with the latest nanoinformatics methods will allow NanoSolveIT to model the relationships between NM structure (morphology), properties and their adverse effects and to predict the effects of other NMs for which less data is available. The project specifically addresses the needs of regulatory agencies and industry to effectively and rapidly evaluate the exposure, NM hazard and risk from nanomaterials and nano-enabled products, enabling implementation of computational 'safe-by-design' approaches to facilitate NM commercialization.
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http://dx.doi.org/10.1016/j.csbj.2020.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090366PMC
March 2020

Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model.

Nanomaterials (Basel) 2020 Mar 18;10(3). Epub 2020 Mar 18.

Health Effects Laboratory, Department for Environmental Chemistry, NILU-Norwegian Institute for Air Research, Instituttveien 18, 2007 Kjeller, Norway.

(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC 3.8 µg/cm) than in 3D cultures (EC > 30 µg/cm), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC 10.1-16.2 µg/cm). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO-NPs showed no toxicity (EC > 75 µg/cm). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.
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http://dx.doi.org/10.3390/nano10030545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153628PMC
March 2020

Effects of Titanium Dioxide Nanoparticles on the Gene Mutations in V79 Hamster Cells.

Nanomaterials (Basel) 2020 Mar 5;10(3). Epub 2020 Mar 5.

Health Effects Laboratory, Department of Environmental Chemistry, NILU-Norwegian Institute for Air Research, N-2027 Kjeller, Norway.

The genotoxicity of anatase/rutile TiO nanoparticles (TiO NPs, NM105 at 3, 15 and 75 µg/cm) was assessed with the mammalian in-vitro Hypoxanthine guanine phosphoribosyl transferase () gene mutation test in Chinese hamster lung (V79) fibroblasts after 24 h exposure. Two dispersion procedures giving different size distribution and dispersion stability were used to investigate whether the effects of TiO NPs depend on the state of agglomeration. TiO NPs were fully characterised in the previous European FP7 projects NanoTEST and NanoREG2. Uptake of TiO NPs was measured by transmission electron microscopy (TEM). TiO NPs were found in cytoplasmic vesicles, as well as close to the nucleus. The internalisation of TiO NPs did not depend on the state of agglomeration and dispersion used. The cytotoxicity of TiO NPs was measured by determining both the relative growth activity (RGA) and the plating efficiency (PE). There were no substantial effects of exposure time (24, 48 and 72 h), although a tendency to lower RGA at longer exposure was observed. No significant difference in PE values and no increases in the gene mutant frequency were found in exposed relative to unexposed cultures in spite of evidence of uptake of NPs by cells.
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http://dx.doi.org/10.3390/nano10030465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153606PMC
March 2020

The way forward for assessing the human health safety of cosmetics in the EU - Workshop proceedings.

Toxicology 2020 04 28;436:152421. Epub 2020 Feb 28.

European Commission, Joint Research Centre (JRC), Ispra, Italy.

Although the need for non-animal alternatives has been well recognised for the human health hazard assessment of chemicals in general, it has become especially pressing for cosmetic ingredients due to the full implementation of testing and marketing bans on animal testing under the European Cosmetics Regulation. This means that for the safety assessment of cosmetics, the necessary safety data for both the ingredients and the finished product can be drawn from validated (or scientifically-valid), so-called "Replacement methods". In view of the challenges for safety assessment without recourse to animal test data, the Methodology Working Group of the Scientific Committee on Consumer Safety organised a workshop in February 2019 to discuss the key issues in regard to the use of animal-free alternative methods for the safety evaluation of cosmetic ingredients. This perspective article summarises the outcomes of this workshop and reflects on the state-of-the-art and possible way forward for the safety assessment of cosmetic ingredients for which no experimental animal data exist. The use and optimisation of "New Approach Methodology" that could be useful tools in the context of the "Next Generation Risk Assessment" and the strategic framework for safety assessment of cosmetics were discussed in depth.
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http://dx.doi.org/10.1016/j.tox.2020.152421DOI Listing
April 2020

Six-week inhalation of CdO nanoparticles in mice: The effects on immune response, oxidative stress, antioxidative defense, fibrotic response, and bones.

Food Chem Toxicol 2020 Feb 9;136:110954. Epub 2019 Nov 9.

Institute of Analytical Chemistry of Czech Academy of Sciences, Veveri 97, 60200, Brno, Czech Republic.

Due to the growing number of applications of cadmium oxide nanoparticles (CdO NPs), there is a concern about their potential deleterious effects. The objective of our study was to investigate the effect of CdO NPs on the immune response, renal and intestine oxidative stress, blood antioxidant defence, renal fibrotic response, bone density and mineral content. Six-week-old female ICR mice were exposed to CdO NPs for 6 weeks by inhalation (particle size: 9.82 nm, mass concentration: 31.7 μg CdO/m, total deposited dose: 0.195 μg CdO/g body weight). CdO NPs increased percentage of thymus CD3eCD8a cells and moderately enhanced splenocyte proliferation and production of cytokines and chemokines. CdO NPs elevated pro-fibrotic factors (TGF-β2, α-SMA and collagen I) in the kidney, and concentrations of AGEs in the intestine. The ratio of GSH and GSSG in blood was slightly reduced. Exposure to CdO NPs resulted in 10-fold higher Cd concentration in tibia bones. No differences were found in bone mass density, mineral content, bone area values, bone concentrations of Ca, P, Mg and Ca/P ratio. Our findings indicate stimulation of immune/inflammatory response, oxidative stress in the intestine, starting fibrotic response in kidneys and accumulation of CdO NPs in bones of mice.
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http://dx.doi.org/10.1016/j.fct.2019.110954DOI Listing
February 2020

Distinct pathways associated with chromosomal aberration frequency in a cohort exposed to genotoxic compounds compared to general population.

Mutagenesis 2019 12;34(4):323-330

Department of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Non-specific structural chromosomal aberrations (CAs) observed in peripheral blood lymphocytes of healthy individuals can be either chromosome-type aberrations (CSAs) or chromatid-type aberrations (CTAs) depending on the stage of cell division they are induced in and mechanism of formation. It is important to study the genetic basis of chromosomal instability as it is a marker of genotoxic exposure and a predictor of cancer risk. For that purpose, we conducted two genome-wide association studies (GWASs) on healthy individuals in the presence and absence of apparent genotoxic exposure from the Czech Republic and Slovakia. The pre-GWAS cytogenetic analysis reported the frequencies of CSA, CTA and total CA (CAtot). We performed both linear and binary logistic regression analysis with an arbitrary cut-off point of 2% for CAtot and 1% for CSA and CTA. Using the statistical threshold of 1.0 × 10-5, we identified five loci with in silico predicted functionality in the reference group and four loci in the exposed group, with no overlap between the associated regions. A meta-analysis on the two GWASs identified further four loci with moderate associations in each of the studies. From the reference group mainly loci within genes related to DNA damage response/repair were identified. Other loci identified from both the reference and exposed groups were found to be involved in the segregation of chromosomes and chromatin modification. Some of the discovered regions in each group were implicated in tumourigenesis and autism.
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http://dx.doi.org/10.1093/mutage/gez024DOI Listing
December 2019

The comet assay applied to HepG2 liver spheroids.

Mutat Res 2019 09 29;845:403033. Epub 2019 Mar 29.

Fraunhofer Institute for Biomedical Engineering IBMT, Department Bioprocessing & Bioanalytics, Joseph-von-Fraunhofer-Weg 1, 66280 Sulzbach, Germany. Electronic address:

In accordance with the 3 Rs to reduce in vivo testing, more advanced in vitro models, moving from 2D monolayer to 3D cultures, should be developed for prediction of human toxicity of industrial chemicals and environmental pollutants. In this study we compared cytotoxic and genotoxic responses induced by chemicals in 2D and 3D spheroidal cultures of the human liver cancer cell line HepG2. HepG2 spheroids were prepared by hanging drop technology. Both 3D spheroids and 2D monolayer cultures were exposed to different chemicals (colchicine, chlorpromazine hydrochloride or methyl methanesulfonate) for geno- and cytotoxicity studies. Cytotoxicity was investigated by alamarBlue assay, flow cytometry and confocal imaging. DNA damage was investigated by the comet assay with and without Fpg enzyme for detection of DNA strand breaks and oxidized or alkylated base lesions. The results from the cyto- and genotoxicity tests showed differences in sensitivity comparing the 2D and 3D HepG2 models. This study shows that human 3D spheroidal hepatocellular cultures can be successfully applied for genotoxicity testing by the comet assay and represent a promising advanced in vitro model for toxicity testing.
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http://dx.doi.org/10.1016/j.mrgentox.2019.03.006DOI Listing
September 2019

Introduction to hCOMET special issue, 'Comet assay in vitro'.

Mutat Res 2019 09 3;845:403071. Epub 2019 Jul 3.

Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Oslo, Norway.

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http://dx.doi.org/10.1016/j.mrgentox.2019.07.001DOI Listing
September 2019

Titanium dioxide nanoparticles tested for genotoxicity with the comet and micronucleus assays in vitro, ex vivo and in vivo.

Mutat Res 2019 07 2;843:57-65. Epub 2019 May 2.

NILU-Norwegian Institute for Air Research, Kjeller, Norway.

The genotoxicity of TiO nanoparticles (NPs) was assessed with the cytokinesis-block micronucleus (CBMN) assay in TK6 lymphoblastoid cells, lymphocytes from human volunteers, and bone marrow erythrocytes from rats exposed in vivo; and with the comet assay (detecting both strand breaks and oxidised purines) in human and rat peripheral blood mononuclear cells (PBMCs). NPs were dispersed using three different methods giving different size distribution and stability. On average, TiO NPs caused no increase in micronuclei in TK6 cells, rat bone marrow erythrocytes or human lymphocytes (though lymphocytes from 3 out of 13 human subjects showed significant increases). PBMCs from rats treated in vivo with a single dose of NPs dispersed by a method with low agglomeration showed an increase in strand breaks after 1 day. TiO NPs dispersed in a stable, non-agglomerated state induced DNA strand breaks at 75 μg/cm after 4 h exposure of human PBMCs and at 15 μg/cm and 75 μg/cm after 24 h exposure, but no increase in DNA oxidation was seen. Overall, NPs in an agglomerated state did not cause DNA damage. However, at the individual level, significant increases in strand breaks were seen in PBMCs from most of the volunteers. Cells from one volunteer showed positive effects in all conditions and both tests, while cells from another volunteer appeared to be completely resitant to TiO NPs. The implication is that some individuals may be more sensitive than others to effects of this nanomaterial. Differences seen in results obtained with the micronucleus and the comet assay may be due to the mechanisms underlying the genotoxic effects of TiO NPs and the different endpoints represented by the two assays.
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http://dx.doi.org/10.1016/j.mrgentox.2019.05.001DOI Listing
July 2019

Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies.

Mutat Res 2019 07 1;843:24-32. Epub 2019 May 1.

Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Sognsvannsveien 9, 0372 Oslo, Norway. Electronic address:

The comet assay (single cell gel electrophoresis) is widely used as a biomonitoring tool to assess DNA damage - strand breaks, as well as oxidised bases; it can also be adapted to measure DNA repair. It is based on the ability of breaks in the DNA to relax supercoiling, allowing DNA loops to extend from the nuclear core (nucleoid) under an electric field to form a comet-like tail. Most commonly, it is applied to white blood cells. The range of detection is between a few hundred breaks per cell and a few thousand, encompassing levels of damage that can be repaired and tolerated by human cells. Its applications include monitoring various diseases, studying the influence of nutrition on DNA stability, and investigating effects of environmental and occupational mutagens. Here we address the issue of inter-laboratory variation in comet assay results. This variation is largely due to differences in methods. Imposing a standard protocol is not practical, but users should be aware of the crucial parameters that affect performance of the assay. These include the concentration of agarose in which the cells are embedded; the duration of cell lysis, and of enzyme incubation when oxidised bases are being measured; the duration of alkaline unwinding; the duration of electrophoresis and the voltage gradient applied; and the method used to score the comets. Including reference standards in each experiment allows experimental variability to be monitored - and if variation is not extreme, results can be normalised using reference standard values. Reference standards are also essential for inter-laboratory comparison. Finally, we offer recommendations which, we believe, will limit variability and increase the usefulness of this assay in molecular epidemiology.
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http://dx.doi.org/10.1016/j.mrgentox.2019.04.007DOI Listing
July 2019

The comet assay in human biomonitoring: Technical and epidemiological perspectives.

Mutat Res 2019 07 8;843:1-2. Epub 2019 Jun 8.

Norwegian Institute for Air Research, Kjeller, Norway.

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http://dx.doi.org/10.1016/j.mrgentox.2019.06.002DOI Listing
July 2019

The comet assay in animal models: From bugs to whales - (Part 2 Vertebrates).

Mutat Res 2019 Jul - Sep;781:130-164. Epub 2019 Apr 20.

Department of Nutrition, University of Oslo, Oslo, Norway.

The comet assay has become one of the methods of choice for the evaluation and measurement of DNA damage. It is sensitive, quick to perform and relatively affordable for the evaluation of DNA damage and repair at the level of individual cells. The comet assay can be applied to virtually any cell type derived from different organs and tissues. Even though the comet assay is predominantly used on human cells, the application of the assay for the evaluation of DNA damage in yeast, plant and animal cells is also quite high, especially in terms of biomonitoring. The present extensive overview on the usage of the comet assay in animal models will cover both terrestrial and water environments. The first part of the review was focused on studies describing the comet assay applied in invertebrates. The second part of the review, (Part 2) will discuss the application of the comet assay in vertebrates covering cyclostomata, fishes, amphibians, reptiles, birds and mammals, in addition to chordates that are regarded as a transitional form towards vertebrates. Besides numerous vertebrate species, the assay is also performed on a range of cells, which includes blood, liver, kidney, brain, gill, bone marrow and sperm cells. These cells are readily used for the evaluation of a wide spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of vertebrate models and their role in environmental biomonitoring will also be discussed as well as the comparison of the use of the comet assay in vertebrate and human models in line with ethical principles. Although the comet assay in vertebrates is most commonly used in laboratory animals such as mice, rats and lately zebrafish, this paper will only briefly review its use regarding laboratory animal models and rather give special emphasis to the increasing usage of the assay in domestic and wildlife animals as well as in various ecotoxicological studies.
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http://dx.doi.org/10.1016/j.mrrev.2019.04.002DOI Listing
March 2020

A One Health approach to managing the applications and implications of nanotechnologies in agriculture.

Nat Nanotechnol 2019 06 5;14(6):523-531. Epub 2019 Jun 5.

GenØk Centre for Biosafety, Siva Innovation Centre, Tromsø, Norway.

The need for appropriate science and regulation to underpin nanosafety is greater than ever as ongoing advances in nanotechnology are rapidly translated into new industrial applications and nano-enabled commercial products. Nevertheless, a disconnect persists between those examining risks to human and environmental health from nanomaterials. This disconnect is not atypical in research and risk assessment and has been perpetuated in the case of engineered nanomaterials by the relatively limited overlap in human and environmental exposure pathways. The advent of agri-nanotechnologies brings both increased need and opportunity to change this status quo as it introduces significant issues of intersectionality that cannot adequately be addressed by current discipline-specific approaches alone. Here, focusing on the specific case of nanoparticles, we propose that a transdisciplinary approach, underpinned by the One Health concept, is needed to support the sustainable development of these technologies.
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http://dx.doi.org/10.1038/s41565-019-0460-8DOI Listing
June 2019

The comet assay in animal models: From bugs to whales - (Part 1 Invertebrates).

Mutat Res 2019 Jan - Mar;779:82-113. Epub 2019 Feb 16.

Department of Nutrition, University of Oslo, Oslo, Norway.

The comet assay, also called single cell gel electrophoresis, is a sensitive, rapid and low-cost technique for quantifying and analysing DNA damage and repair at the level of individual cells. The assay itself can be applied on virtually any cell type derived from different organs and tissues of eukaryotic organisms. Although it is mainly used on human cells, the assay has applications also in the evaluation of DNA damage in yeast, plant and animal cells. Therefore, the purpose of this review is to give an extensive overview on the usage of the comet assay in animal models from invertebrates to vertebrates, covering both terrestrial and water biota. The comet assay is used in a variety of invertebrate species since they are regarded as interesting subjects in ecotoxicological research due to their significance in ecosystems. Hence, the first part of the review (Part 1) will discuss the application of the comet assay in invertebrates covering protozoans, platyhelminthes, planarians, cnidarians, molluscs, annelids, arthropods and echinoderms. Besides a large number of animal species, the assay is also performed on a variety of cells, which includes haemolymph, gills, digestive gland, sperm and embryo cells. The mentioned cells have been used for the evaluation of a broad spectrum of genotoxic agents both in vitro and in vivo. Moreover, the use of invertebrate models and their role from an ecotoxicological point of view will also be discussed as well as the comparison of the use of the comet assay in invertebrate and human models. Since the comet assay is still developing, its increasing potential in assessing DNA damage in animal models is crucial especially in the field of ecotoxicology and biomonitoring at the level of different species, not only humans.
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http://dx.doi.org/10.1016/j.mrrev.2019.02.003DOI Listing
March 2020