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Application potential of screening in vitro toxicological assays in qualitative risk assessment of nanomaterials
Závodná, Táňa ; Topinka, Jan ; Danihelka, J.
Undeniable benefits of engineered nanomaterials might be discredited by their potential enhanced or unexpected toxicity arising from nano-specific properties and behavior. An analysis of the applicability of the traditional chemical risk assessment approach in nanomaterials revealed high levels of uncertainty in both hazard characterization and exposure assessment due to the lack of relevant validated methods and reliable data. This indicates the limited capability of the conventional risk assessment approach to ensure the safe use of nanomaterials. Based on the identified uncertainties, the control banding approach was proposed as a suitable tool for preliminary qualitative risk assessment of nanomaterials in occupational settings. Control banding categorizes hazard and exposure into levels referred to as bands. The combination of the hazard and exposure bands results in a risk band determining the necessary degree of control and regulatory measures. To decrease the number of cases where, based on the precautionary principle, unavailable experimental or field data would lead to the assignment to the highest hazard category requiring costly exposure control, screening evaluation of nanomaterial toxicity was proposed as an additional decision criterion. For this purpose, a battery of in vitro toxicological assays enabling screening evaluation of potential toxic effects of NMs was proposed. The assays evaluate endpoints covering basic toxic effects of substances (cytotoxicity, genotoxicity), as well as known nonspecific mechanisms of toxicity typical for nanomaterials (oxidative stress, inflammation). The proposed risk management strategy is intended to assist small and medium-sized enterprises to implement adequate measures to ensure employee safety.
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The role of dispersion medium on nanoparticle aggregation and size in biological systems
Červená, Tereza ; Rössnerová, Andrea ; Závodná, Táňa ; Vrbová, Kristýna ; Sikorová, Jitka ; Topinka, Jan ; Rössner ml., Pavel
The use of nanomaterials (NMs) in different areas has been rising for more than a decade. Along with this growth, there is visible development of different testing tools and approaches for measuring the actual size of nanomaterials in biological systems. Test conditions during in vitro toxicological assays are different from the standard conditions under which nanomaterials are characterized and careful evaluation of results is needed. The unique properties and range variety of NMs require the close look how the NMs behave in different dispersion medium over time. In this study we present the results of five types of well-characterized NMs (TiO2: NM-101 and NM-103, SiO2: NM-200, Ag: NM-300K and NM-302) of specific size and shape. The hydrodynamic size and Zeta potentials in suspensions were measured using a dynamic light scattering technique (DLS) (Zetasizer Nano ZS, Malvern, UK). The DLS method is suitable for spherical particles, nevertheless, all samples were measured in order to obtain a rough insight into agglomerate formation in the medium. NM300, NM302, and NM200 aggregated rapidly in the media, thus the cells would be most likely exposed to settled big aggregates then small clusters or individual particles. More stable NMs (NM100 and NM103) showed slight grow along with cultivation time or concentration corresponding to cluster formation. Cells exposed to those NMs would be in contact with small clusters and aggregates of NMs. Measured zeta potentials fluctuated around the stability limit corresponding to observed aggregation.
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Ultrafine particles and their possible role in etiology and development of neurodegenerative diseases
Topinka, Jan ; Závodná, Táňa ; Rössnerová, Andrea ; Rössner ml., Pavel
Air pollutants have been shown to cause a vast amount of different adverse health effects. These effects include impairment of many respiratory (e.g. asthma, chronic obstructive pulmonary disease) and cardiovascular (ischemic heart disease, infarction, stroke) diseases. However, in recent years, the evidence showing effects beyond the lungs and circulatory system are becoming more evident. Neurological diseases, namely Alzheimer's disease (AD) has shown to be associated with living near traffic. However, reason for this has remained unresolved until today. Our new H2020 project TUBE aims on revealing the mechanisms of action of ultrafine particles involved in neurological diseases. The TUBE consortium includes experts in areas of aerosol technology, emission research, engine and fuel research, human clinical studies, epidemiology, emission inventories, inhalation toxicology, neurotoxicology and disease mechanism studies. This enables research of resolving the effects of nanoparticles from different traffic modes for both air quality and concomitant toxic effect of these air pollutants. We will investigate adverse effects of air pollutants using cell cultures, animal exposures and volunteered human exposures as well as the material from epidemiological cohort study. These are going to be compared according to inflammatory, cytotoxic and genotoxic changes and furthermore beyond the current state of the art to neurotoxic and brain health effects. With this approach, we are aiming to a comprehensive understanding of the adverse brain effects of nanoparticles from traffic.
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Males-females differences in the spectrum of chromosomal aberrations in the group of nanocomposites production workers
Rössnerová, Andrea ; Pelcová, D. ; Ždímal, Vladimír ; Elzeinova, Fatima ; Margaryan, Hasmik ; Chvojková, Irena ; Topinka, Jan ; Schwarz, Jaroslav ; Ondráček, Jakub ; Koštejn, Martin ; Komarc, M. ; Vlčková, Š. ; Fenclová, Z. ; Lischková, L. ; Dvořáčková, Š. ; Rössner ml., Pavel
An increase in the use of nanomaterials (NM) has been witnessed in many areas of human life. Therefore, assessment of genotoxicity of NM and nanoparticles (NP) is one of the main objectives of genetic toxicology. Despite this fact, human cytogenetic studies following the exposure to NP are still rare. Moreover, no relevant information on possible differences in sensitivity to NP related to gender is available.\n\nIn this study we periodically (in September 2016, 2017 and 2018; pre-shift and post-shift each year) analyzed a group of workers (both genders), working long time in nanocomposites research, and matched controls. Aerosol exposure monitoring of particulate matter including nano-sized fractions was carried out during working shift. Micronucleus assay using Human Pan Centromeric probes, was applied to distinguish, besides the frequency of total MN in binucleated cells (BNC), also other types of chromosomal damage (losses and breaks). Moreover, whole-chromosome painting (WCP) for autosome #1 and both gonosomes (X and Y) were applied in third sampling period (2018) with the aim to identify the particular structural and numerical chromosomal aberrations.\n\nObtained results showed: (i) differences in the risk of exposure to NP related to individual working processes (welding, smelting and machining); (ii) differences in chemical composition of nano-fraction; (iii) no effect of chronic exposure of NP (total MN) opposite to significant effect of acute exposure; (iv) gender-related DNA damage differences (females seem to be more sensitive to chromosomal losses). Additional data from WCP suggested increased frequency of numerical aberrations in gonosomes.
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Optimalizace kultivačních podmínek lidských jaterních buněk HepG2 na 4 typech nano- a micro- vlákenných nosičů
Rössner ml., Pavel
Technologie popisuje optimalizaci počtu nasazovaných lidských hepatocytů HepG2, vhodných pro využívání v oblasti toxikologického ověřování nových léčiv či potravin, na 3D kultivační systém tvořený čtyřmi typy nano- a mikrovlákenných nosičů. Motivací pro hledání vhodných modelů testování potenciálně genotoxických účinků léčiv je nízká relevance tkáňových modelů užívaných v prvních fázích preklinického hodnocení nových látek a také redukce testování na zvířatech.
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DNA damage induced by occupational exposure to copper oxide nanoparticles
Rössner st., Pavel ; Pelcová, D. ; Elzeinová, Fatima ; Mikuška, Pavel ; Večeřa, Zbyněk ; Coufalík, Pavel ; Vlčková, Š. ; Fenclová, Z. ; Rössnerová, Andrea
Copper oxide nanoparticles (CuO NPs) have a widespread use in industry, chemistry, in production of electronic devices and as an antimicrobial agent. Although copper is an important biogenic element, CuO NPs are toxic with the ability to induce oxidative stress, apoptosis, cell cycle arrest or DNA damage. For humans, the inhalation route is the most common way of exposure to CuO NPs. In the body, CuO NPs may be either deposited in the lungs, or transported to other organs. Their presence usually causes oxidative stress or inflammatory responses, consequently leading to DNA damage. In this study, we investigated the effect of CuO NPs inhalation on DNA damage in a group of researches conducting animal exposure experiments. The subjects were exposed to various metal oxide nanoparticles, including CuO NPs, by inhalation for an average of 4.9 ± 0.4 years. The average mass concentration of Cu in the air during the experiment was 7.3 ± 3.2 ng/m3. Subjects not exposed to nanoparticles served as a control group. We applied micronucleus assay using Human Pan Centromeric probes to detect DNA damage and to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN−) micronuclei (MN) in the binucleated cells. We\ndid not find differences between both groups for either mean MN frequency (10.38 ± 2.50 vs. 11.88 ± 3.01 MN/1000 binucleated cells), or CEN+/CEN- ratio (58%/42% vs. 55%/45%), for the exposed and controls, respectively. In conclusion, inhalation of CuO NPs at this low-level exposure had no effect on chromosomal losses and/or breaks.
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SIZE AS AN IMPORTANT FACTOR IN NANO-TiO2 TOXICITY IN MACROPHAGE-LIKE CELLS
Líbalová, Helena ; Sikorová, Jitka ; Brzicová, Táňa ; Milcová, Alena ; Vrbová, Kristýna ; Pikal, P. ; Topinka, Jan ; Rössner ml., Pavel
A set of NPs consists of 5 variants of anatase and 5 variants of rutile nanoparticles differing in their diameter (from 3 to 165 nm). TiO2 samples were characterized in the powder form and dispersed in water and cell culture media. Three cytotoxicity assays were used: MTS, WST-1, and LDH. For all nanomaterials, three independent repetitions were carried out. \n\nOverall, cytotoxicity of all NPs was low even at the highest concentration of 256 mu g/ml. The viability of cells did not decrease below 60% for WST-1 and MTS assays and 80% for the LDH assay. Besides concentration, crystalline size was identified as the most important cytotoxic factor. Clear nonlinear relationship between crystalline size and cytotoxicity was detected, higher toxicity induced NPs within the size range 20-60 nm. Increased cytotoxicity in given diameter size range would give an answer to inconsistent findings at size and cytotoxicity relationship.
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Use of the nanofiber scaffold for transfer of stem cells onto the injured ocular surface in mouse experimental model
Kössl, Jan ; Zajícová, Alena ; Heřmánková, Barbora ; Javorková, Eliška ; Boháčová, Pavla ; Holáň, Vladimír
Corneal damage is one of the most common causes of impaired vision or even blindness. When the injury is more extensive and the limbal region is involved, the natural regeneration of the cornea is not sufficient. Such damage can lead to the limbal stem cell deficiency (LSCD). The only option for LSCD treatment is transplantation of the limbal tissue or a transfer of limbal stem cells (LSCs) cultured from the healthy eye. The allogenic transplantation of the limbus or cultivated LSCs with a systemic administration of immunosuppressive drugs is needed in the case of bilateral LSCD. Nevertheless, the cell therapy is very promising approach for LSCD treatment. Transplantation of mesenchymal stem cells (MSCs) seeded on an appropriate scaffold turned out to be a suitable therapy of the LSCD. In our experimental model of LSCD we use nanofiber scaffold for MSC and LSC cultivation and for transplantation of these cells onto the chemically injured mouse eye. MSCs have immunosuppressive and immunomodulatory properties. We showed that MSCs have the ability to inhibit production of molecules associated with the inflammation and support epithelial regeneration in the damaged cornea. These inhibitory properties were confirmed in both in vitro and in vivo mouse model. Results thus showed beneficial effects of stem cell transplantation for murine corneal healing and for suppression of a local immune reaction which can impede the healing process. Such similarity of in vivo and in vitro results allows us further experiments to clarify mechanisms of MSC regenerative and healing properties after the transplantation onto the injured cornea.
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GENOTOXICITY OF NANOMATERIALS IN BEAS-2B CELLS ANALYZED BY THE IN VITRO MICRONUCLEUS ASSAY
Rössnerová, Andrea ; Červená, Tereza ; Brzicová, Táňa ; Vrbová, Kristýna ; Sikorová, Jitka ; Topinka, Jan ; Rössner ml., Pavel
The tremendous increase of the use of nanomaterials (NMs) has been witnessed during the last decade in many areas of human life including the chemical industry, cosmetics, biomedicine or food technology. The variety of NMs, their unique properties, almost ubiquitous presence and the size range of 1-100 nm raised the interest of toxicologists. The evaluation of the frequency of micronuclei (MN) as a result of the genotoxic events is a broadly utilized and well-established approach in in vitro studies for testing the risk of chemical exposure. Nevertheless, properties of the NMs give rise to the questions concerning the optimal methodological variants of the MN assay. \n\nIn our study, five types of well-characterized NMs (TiO2: NM-101 and NM-103, SiO2: NM-200, Ag: NM-300K and NM-302) of specific size, shape, or e.g. dimensions of aggregates were involved in the genotoxicity testing using four variants of protocols differing in the time of NM exposure, application of cytochalasin-B combined with simultaneous and delayed co-treatment with nanoparticles (NPs). Bronchial epithelial cells (BEAS-2B) were used in this study to fulfil these tasks. Presence of NPs was controlled by transmission electron microscopy (TEM). \n\nObtained results showed the different genotoxic potential of the various TiO2 and Ag NMs (NM-101< NM-103 and NM-300K> NM-302, respectively). Comparison of all testing strategies revealed, that the level of DNA damage can differ based on the time of exposure and the methodological approach. In general, using cytochalasin-B led most frequently to the increase of the genotoxic potential of the tested NMs.
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