Seznam abstraktů 2021: SEKCE D - MONITOROVÁNÍ A TOXICITA NANOMATERIÁLŮ

Microplastics (MPs), defined as plastic particles not exceeding a size limit of 5 mm, are emerging, globally distributed micropollutants. Their presence was confirmed in various aquatic environments worldwide, including oceans and seas, lakes, rivers, and dams, even in very remote areas. Research attention has been paid mostly to the marine environment; however, MPs were also detected in water bodies serving as sources of drinking water and in raw and treated water samples obtained directly at drinking water treatment plants, supplied by both surface water and groundwater.
It is of note that the risk associated with the toxicological effects of MPs is still not fully described. Several studies investigated the impacts of MPs on living organisms or in vitro (predominantly aquatic species), and, in fact, many negative effects were recorded. However, generally, the ecotoxicological studies were performed only with a single material, mostly with larger particles and MP concentrations often greatly exceeding the values currently detected in natural environments. On the other hand, several studies documented that tissue cells may be locally exposed to elevated concentrations of MPs. For example, studies with fishes reported the ability of MPs (1 µm in diameter) to accumulate in various organs, causing toxic effects on the organism. It is also necessary to consider the fact that MPs, which are mostly hydrophobic, cannot be easily excreted from bodies. The aim of this contribution is to provide an overview of the current knowledge regarding the toxicity of MPs with the identification of important specific aspects that must be considered for future studies.

The increased use of nanomaterials in many areas of human life is accompanied by potential risk of exposure to nanoparticles (NP). Despite many nanotoxicological in vitro studies, human in vivo studies are still rare.
In our study, we repeatedly (2015-2019) monitored nanocomposite research workers and matched controls. To assess the differences in exposure to particulate matter (PM) including nanosized fractions <25-100 nm, aerosol exposure monitoring during a working shift was carried out. Two cytogenetic assays (micronucleus test using Human Pan Centromeric probes and whole-chromosome painting) and two methylation assays (global methylation analysis and gene-specific analysis using the Infinium MethylationEPIC BeadChips) were used to assess the level of DNA alterations after long-term (years) and short-term (daily) exposure to NP.
The results showed: (i) differences in the risk of exposure to NP, including chemical composition of nano-fraction, for individual working processes (welding, smelting and machining); (ii) in contrast to acute exposure, chronic exposure to NP does not affect the frequency of total micronuclei; (iii) DNA methylation pattern changed after chronic exposure, while no effect of acute NP exposure was detected; (iv) gender-related DNA damage differences were observed.
In conclusion, long-term (chronic) exposure to NP contributes to epigenetic changes followed by reduction of DNA damage. In contrast, DNA damage increases after short-term acute exposure which is not associated with a substantial adaptation of DNA methylation pattern.
Supported by the Grant Agency of the Czech Republic #18-02079S.

Air pollution is a major factor impacting public health and it is becoming a serious problem worldwide, especially in the megacities and urban areas in Europe and in Asia. One of the smallest particles in air pollution’s particulate matter are nanoparticles (NPs), of which NPs of heavy metals are especially concerning. Metal nanoparticles are used in various industries and can be generated as by-products in the pyrometallurgy of heavy metals. In the cells, NPs can cause oxidative damage, plasma membrane defects or homeostasis disruption and we are still only at the beginning of understanding their exact distribution, accumulation, toxicity and possible subsequent elimination from the organisms after relevant exposure.
In our experiment, mice were subchronically inhaling lead oxide nanoparticles in whole-body chambers for eleven weeks. The level of lead in brain tissue was then analyzed by AAS. To uncover possible degenerative changes in the brain, we employed methods such as histological analysis, TEM, SEM, immunohistochemistry, immunofluorescence, qPCR and LA-ICP-MS.
We found that inhaled lead oxide nanoparticles can enter brain tissue, where they caused pathological changes, particularly in the hippocampal region. Inclusions associated with neurodegenerative disorders, Hirano bodies, were induced in the brains of exposed mice. We also determined the alterations in the olfactory epithelium with degradation of olfactory knob and associated-cilia.
Taken together, inhaling nanoparticles of heavy metals caused significant changes in mouse brains, which were not ameliorated by the clearance period. Moreover, there seems to be a distinct pattern of changes in the brain depending on the type of lead nanoparticles.
ACKNOWLEDGEMENTS: This work was supported by Czech Science Foundation (20-02203S).

Although there are thousands of articles and a wide range of nanoparticles (NPs), toxicologists face problems due to an incomplete understanding of nanoparticle physico-chemical properties in physiological environment. NPs, which appear to have desirable properties for example for clinical applications, do not perform as expected and results differ when obtained in water/solvents or in biological fluids. Characterization of NPs in these fluids is a crucial step in toxicology and concentration and incubation time should be also considered when investigating biological responses. NPs are well known for their large surface area and formation of protein corona in dispersion media. They interact with a range of molecules and therefore can cross biological barriers, influence immune response and be toxic. Different biodistribution of NPs can be also used for cell and tissue targeting and drug delivery.

Nanoparticles (NPs), nano-structures with size range between 1-100 nm have been applied widely in various fields of science and technology. Among those NPs, Cerium oxide nanoparticles (CeO2NPs) have a unique electronic structure and free radical scavenging property. The NPs have been suggested for antioxidant therapies against Age-related Macular Degeneration (AMD) by developing them in eye drops. In this study ocular bacterial communities were detected using metagenomics to answer the rising concern about the CeO2NPs impact on these communities. The bacteria were isolated on various selected agars to obtain pure cultures, afterward the strains were determined by a Sanger sequencing. Last, the isolated bacterial strains were exposed to various concentrations (0.01 to 10 mg/mL) of two types of CeO2NPs (naked and collyrium) in four biological buffers. In parallel, CeO2NPs sizes were analyzed by Differential Centrifugal Sedimentation (DCS). A primary result showed that ocular bacterial communities were low abundant. The majority was Staphylococcus and Streptococcus. Further, CeO2NPs did not show negative colony effects on Staphylococcus aureus and Staphylococcus epidermidis but the colony's colors were changed. The results lead to doubtful questions whether CeO2NPs have effects on the bacterial molecules including DNA, RNA and proteins. CeO2NPs sizes should also be replaced by an appropriate tool.

With the development of new technologies, the requirements for safety and health at workplaces are also increasing. One of these sectors are also industrial plants which process or use fine and ultrafine particles (<2,5 μm) during their activities or where these particles are created unintentionally as a by-product of the production processes. In the case of risk assessment of these particles, it is not only a matter of health risks associated with direct exposure to these particles but can have negative effects on mental health of employees. This could lead to stress, increase mental discomfort, reduce attention, performance, and motivation to work. The aim of the methodology is to evaluate the health and safety management system, increase its level and efficiency in operations loaded with fine and ultrafine particles. The methodology includes both the assessment of risks associated with the exposure of workers and the rate of perception of risks by employees in the field of information about the risks associated with fine and ultrafine particles. It also includes perception of the adequacy of existing safety measures to reduce worker exposure to these particles. The proposal of basic measures to reduce exposure of workers is also an integral part of methodology. The proposed methodology is in line with OECD guidelines focused on nanosafety.

Electron beam irradiation of collagen-based biomaterials and tissues is usually used for their sterilization. The interaction of electrons with collagen triggers variable changes in collagen structure that can modify its mechanical properties and the interaction with cells. The aim of the study was to irradiate decellularized porcine pericardium with a lower dose (25kGy) or a higher dose (50kGy) of electron beam irradiation, and to evaluate both materials physical - chemical properties and behavior of adipose-tissue derived stem cells (ASCs) on these materials. Both non-irradiated and irradiated tissues were analyzed for their resistance to trypsin treatment, for their mechanical properties; thermal stability of the tissues was assessed by differential scanning calorimetry and the changes in proteins secondary structure were analyzed by Infrared spectroscopy (FTIR). ASCs were seeded on the tissues and cell metabolic activity was measured by MTS assay; the cells were stained for alpha-actin, calponin, caldesmon and fibronectin; collagen structure was assessed by Second harmonic generation microscopy. The expression of genes for cell differentiation and extracellular matrix production, i.e. ACTA2, CNN1, SMTN, Col1, and FN1 were quantified by qPCR. The irradiated samples evinced multiple changes in their physical - chemical properties, which were connected probably with crosslinking/degradation processes. Modification by irradiation improved cell metabolic activity and forced ASCs to differentiate towards smooth muscle cells, thus making it interesting for some tissue engineering applications.
ACKNOWLEDGEMENTS: Supported by the Czech Health Research Council, the Ministry of Health of the Czech Republic. Project No. NV19-02-00068, and by MEYS (LM2018129 Czech-BioImaging).