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Measurement and toxicity of combustion generated nanoparticles
Sikorová, Jitka ; Topinka, Jan (advisor) ; Klusoň, Petr (referee) ; Bláha, Luděk (referee)
This thesis is focused on nanoparticles produced by internal combustion engines utilized in vehicles. It deals with spatial distribution of nanoparticles within urban areas, impact of alternative fuels usage on particle production and toxicity, and a particle toxicological testing methodology. Monitoring of airborne nanoparticles identified traffic as the main source of airborne nanoparticles in places with heavy traffic load (Prague), as well as in a small city with only local traffic (Čelákovice). Most particles were likely emitted during short episodes of high emissions (e.g. uphill acceleration). During the measurements, high-emission vehicles responsible for a large fraction of the air pollution were also identified. On the other hand, small non-road internal combustion engines, which are not subject to any limit on particle emissions, such as a lawn mower, were operated during the measurement and generated a large number of nanoparticles. The amount and characteristics of the particles produced by combustion depend on the combustion technology and the fuel composition. A large part of the thesis deals with alternative fuels and their effects on the quantity of produced particles and toxicity of organic matter adsorbed on the particles. Hydrotreated vegetable oil (HVO) exhibited the lowest...
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Use of transcriptomics to study mechanism of the action of complex mixtures of organic compounds occurring in the ambient air focusing on polycyclic aromatic hydrocarbons
Líbalová, Helena ; Topinka, Jan (advisor) ; Krásný, Libor (referee) ; Postlerová, Pavla (referee)
Polycyclic aromatic hydrocarbons (PAH) represent a large group of organic compounds occuring as pollutants in ambient air. Besides their genotoxic effect, some of them are known to be complete carcinogens and act via nongenotoxic and tumor promoting mechanism. Although effects of many individual compounds are well-documented, human exposure to polycyclic aromatic hydrocarbons in ambient air occurs through complex mixtures and only few studies describe the behavior of PAH in real complex mixtures. The first part of the thesis is dealing with the global gene expression changes in human embryonic lung fibroblasts (HEL) as a consequence of the effect of complex mixtures containing PAH extracted from the respirable airborne particles PM2.5. These particles were collected in 4 localities in the Czech republic (Ostrava - Bartovice, Ostrava - Poruba, Karviná, Třeboň) differing in the level of the air pollution. Gene expression changes induced by three subtoxic concentrations of organic extracts (EOM - extractable organic matter) from each locality after 24 hour incubation were examined by microarray analysis. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to interpret gene expression data. In each locality we identified several deregulated signaling pathways...
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Interactions of cells with nanoparticles for bio-medical applications
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Benson, Veronika (referee) ; Topinka, Jan (referee)
In the past decades, nanoparticles have been viewed as a potentially powerful platform for various applications in biomedical sciences. The possible application of nanoparticles varies from drug delivery agents to novel imaging platforms and surely, some application potential still remains hidden. Thus, it is necessary to broadly study their in vitro behavior in order to assess the precise theranostic potential as well as to distinguish possible threats to human health. Even though nanoparticles are getting more and more attention in current research, still only a limited amount of information is available, especially regarding interactions of ultra-small (< 5 nm) nanoparticles with biological environment and cells. The aim of the work presented herein is to provide the reader with information concerning interactions of various ultra-small nanoparticles (silicon-based, gold, nanodiamonds) with biological environment and human cells. Dose- and time-dependent influence of the various nanoparticles on behavior of different human cells (osteoblasts, monocytes, keratinocytes, mesenchymal stem cells) was established under different conditions, stressing out the importance of protein corona (a layer of proteins originating from cultivation medium attached to nanoparticles). Biocompatibility of two...
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Interactions of cells with nanoparticles for bio-medical applications
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Benson, Veronika (referee) ; Topinka, Jan (referee)
In the past decades, nanoparticles have been viewed as a potentially powerful platform for various applications in biomedical sciences. The possible application of nanoparticles varies from drug delivery agents to novel imaging platforms and surely, some application potential still remains hidden. Thus, it is necessary to broadly study their in vitro behavior in order to assess the precise theranostic potential as well as to distinguish possible threats to human health. Even though nanoparticles are getting more and more attention in current research, still only a limited amount of information is available, especially regarding interactions of ultra-small (< 5 nm) nanoparticles with biological environment and cells. The aim of the work presented herein is to provide the reader with information concerning interactions of various ultra-small nanoparticles (silicon-based, gold, nanodiamonds) with biological environment and human cells. Dose- and time-dependent influence of the various nanoparticles on behavior of different human cells (osteoblasts, monocytes, keratinocytes, mesenchymal stem cells) was established under different conditions, stressing out the importance of protein corona (a layer of proteins originating from cultivation medium attached to nanoparticles). Biocompatibility of two...
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Measurement and toxicity of combustion generated nanoparticles
Sikorová, Jitka ; Topinka, Jan (advisor) ; Klusoň, Petr (referee) ; Bláha, Luděk (referee)
This thesis is focused on nanoparticles produced by internal combustion engines utilized in vehicles. It deals with spatial distribution of nanoparticles within urban areas, impact of alternative fuels usage on particle production and toxicity, and a particle toxicological testing methodology. Monitoring of airborne nanoparticles identified traffic as the main source of airborne nanoparticles in places with heavy traffic load (Prague), as well as in a small city with only local traffic (Čelákovice). Most particles were likely emitted during short episodes of high emissions (e.g. uphill acceleration). During the measurements, high-emission vehicles responsible for a large fraction of the air pollution were also identified. On the other hand, small non-road internal combustion engines, which are not subject to any limit on particle emissions, such as a lawn mower, were operated during the measurement and generated a large number of nanoparticles. The amount and characteristics of the particles produced by combustion depend on the combustion technology and the fuel composition. A large part of the thesis deals with alternative fuels and their effects on the quantity of produced particles and toxicity of organic matter adsorbed on the particles. Hydrotreated vegetable oil (HVO) exhibited the lowest...
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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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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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WHOLE-GENOME EXPRESSION ANALYSIS IN THP-1 MACROPHAGE-LIKE CELLS EXPOSED TO DIVERSE NANOMATERIALS
Brzicová, Táňa ; Líbalová, Helena ; Vrbová, Kristýna ; Sikorová, Jitka ; Philimonenko, Vlada ; Kléma, J. ; Topinka, Jan ; Rössner ml., Pavel
From the perspective of the immune system, nanomaterials (NMs) represent invading agents. Macrophages are immune cells residing in all organs and tissues as the first line of defense. Interactions of macrophages with NMs can determine the fate of NMs as well as their potential toxic effects. In the present study, we compared toxicity of four different types of NMs [NM-100 (TiO2, 110 nm), NM-110 (ZnO, 20 nm), NM-200 (SiO2, 150 nm) and NM-300K (Ag, 20 nm)], towards THP-1 macrophage-like cells. Cells were incubated with non-cytotoxic concentrations (1-25 mu g/ml) of NMs for 24 hours and microarray technology was used to analyze changes in whole-genome expression. Gene expression profiling revealed a substantially different molecular response following exposure to diverse NMs. While NM-100 did not exert any significant effect on gene expression profile, all other NMs triggered a pro-inflammatory response characterized by an activation of the NF-kappa B transcription factor and induced expression of numerous chemokines and cytokines. NM-110 and NM-300K further modulated processes such as DNA damage response, oxidative and replication stress as well as cell cycle progression and proteasome function. We suppose that genotoxicity of ZnO and Ag NMs leading to DNA damage and alternatively to apoptosis in THP-1 macrophages is probably caused by the extensive intracellular dissolution of these NPs, as confirmed by TEM imaging.
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GENE EXPRESSION AND IMMUNOLOGICAL RESPONSE IN MICE EXPOSED TO ZnO NANOPARTICLES
Rössner ml., Pavel ; Vrbová, Kristýna ; Strapáčová, S. ; Rössnerová, Andrea ; Ambrož, Antonín ; Brzicová, Táňa ; Líbalová, Helena ; Javorková, Eliška ; Zajícová, Alena ; Holáň, Vladimír ; Kulich, P. ; Večeřa, Zbyněk ; Mikuška, Pavel ; Coufalík, Pavel ; Křůmal, Kamil ; Čapka, Lukáš ; Dočekal, Bohumil ; Šerý, Omar ; Machala, M. ; Topinka, Jan
We analyzed gene expression changes in the lungs and the immunological response in splenocytes of mice exposed by inhalation of ZnO nanoparticles - NP. Adult female ICR mice were treated for three days and three months, respectively. Analysis of differential expression in genes involved in oxidative stress was conducted using quantitative RT-PCR. The potential immunotoxic and immunomodulatory effects of ZnO NP were analyzed by phenotyping and cytokine production by splenocytes after three months exposure. Three days exposure resulted in down-regulation of GCLC, GSR, HMOX-1, NQO-1, NF-kB2, PTGS2 and TXNRD1 mRNA expression, three months exposure increased the expression of these genes. Three months exposure caused a significant decrease in the percentage of granulocytes in the spleen cells, and affected the production of IL-10 and IL-6 by lipopolysaccharide-stimulated leukocytes. In summary, our study revealed changes in the expression of genes involved in the oxidative stress response following acute ZnO NP exposure. Subchronic ZnO NP exposure induced immunomodulatory effects in the spleen.
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Use of transcriptomics to study mechanism of the action of complex mixtures of organic compounds occurring in the ambient air focusing on polycyclic aromatic hydrocarbons
Líbalová, Helena ; Topinka, Jan (advisor) ; Krásný, Libor (referee) ; Postlerová, Pavla (referee)
Polycyclic aromatic hydrocarbons (PAH) represent a large group of organic compounds occuring as pollutants in ambient air. Besides their genotoxic effect, some of them are known to be complete carcinogens and act via nongenotoxic and tumor promoting mechanism. Although effects of many individual compounds are well-documented, human exposure to polycyclic aromatic hydrocarbons in ambient air occurs through complex mixtures and only few studies describe the behavior of PAH in real complex mixtures. The first part of the thesis is dealing with the global gene expression changes in human embryonic lung fibroblasts (HEL) as a consequence of the effect of complex mixtures containing PAH extracted from the respirable airborne particles PM2.5. These particles were collected in 4 localities in the Czech republic (Ostrava - Bartovice, Ostrava - Poruba, Karviná, Třeboň) differing in the level of the air pollution. Gene expression changes induced by three subtoxic concentrations of organic extracts (EOM - extractable organic matter) from each locality after 24 hour incubation were examined by microarray analysis. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to interpret gene expression data. In each locality we identified several deregulated signaling pathways...
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