Publications by authors named "Jan Topinka"

82 Publications

The effects of age on DNA fragmentation, the condensation of chromatin and conventional semen parameters in healthy nonsmoking men exposed to traffic air pollution.

Health Sci Rep 2021 Jun 9;4(2):e260. Epub 2021 Mar 9.

Department of Genetic Toxicology and Epigenetics Institute of Experimental Medicine, Czech Academy of Science Prague Czech Republic.

Background: Numerous studies have investigated age-based declines in semen traits, but the impact of paternal age on semen parameter values remains inconclusive.

Objectives: The aim of this study was to detect an impact of age on semen quality was studied in healthy nonsmoking men exposed to traffic air pollution.

Methods: Semen samples from 150 Prague City policemen aged 23 to 63 years were examined for standard semen parameters, sperm DNA fragmentation and high DNA stainability.

Results: A significant positive correlation was found between age and %DFI ( = .359,  < .001), and negative correlations were found between age and sperm vitality ( = -.247,  < .001), the % acrosome-intact sperm ( = -.202, = .013) and the % normal sperm heads ( = -.204, = .012). A weak but significant negative correlation was found for high DNA stainability (% HDS) vs age ( = -.161, = .050). No significant correlation was detected between male age and the other investigated semen quality parameters. At ages of 23 to 30, 31 to 40, 41 to 50, and 51 to 63 years, the mean %DFI values were 12.7 ± 7.18, 14.7 ± 7.42, 19.6 ± 11.25, and 34.2 ± 15.08, respectively.

Conclusion: Our study shows a strong relationship ( < .001) between the age of men and sperm DNA fragmentation in an occupational cohort at risk of exposure to heavy traffic-related air pollution in a large city center.
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http://dx.doi.org/10.1002/hsr2.260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942397PMC
June 2021

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Nanotoxicology and Molecular Epidemiology, Institute of Experimental Medicine of the CAS, Videnska 1083, 142 20 Prague, Czech Republic.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of and was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.
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http://dx.doi.org/10.3390/ijms22010079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801947PMC
December 2020

A prolonged exposure of human lung carcinoma epithelial cells to benzo[a]pyrene induces p21-dependent epithelial-to-mesenchymal transition (EMT)-like phenotype.

Chemosphere 2021 Jan 10;263:128126. Epub 2020 Sep 10.

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Deciphering the role of the aryl hydrocarbon receptor (AhR) in lung cancer cells may help us to better understand the role of toxic AhR ligands in lung carcinogenesis, including cancer progression. We employed human lung carcinoma A549 cells to investigate their fate after continuous two-week exposure to model AhR agonists, genotoxic benzo[a]pyrene (BaP; 1 μM) and non-genotoxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM). While TCDD increased proliferative rate of A549 cells, exposure to BaP decreased cell proliferation and induced epithelial-to-mesenchymal transition (EMT)-like phenotype, which was associated with enhanced cell migration, invasion, and altered cell morphology. Although TCDD also suppressed expression of E-cadherin and activated some genes linked to EMT, it did not induce the EMT-like phenotype. The results of transcriptomic analysis, and the opposite effects of BaP and TCDD on cell proliferation, indicated that a delay in cell cycle progression, together with a slight increase of senescence (when coupled with AhR activation), favors the induction of EMT-like phenotype. The shift towards EMT-like phenotype observed after simultaneous treatment with TCDD and mitomycin C (an inhibitor of cell proliferation) confirmed the hypothesis. Since BaP decreased cell proliferative rate via induction of p21 expression, we generated the A549 cell model with reduced p21 expression and exposed it to BaP for two weeks. The p21 knockdown suppressed the BaP-mediated EMT-like phenotype in A549 cells, thus confirming that a delayed cell cycle progression, together with p21-dependent induction of senescence-related chemokine CCL2, may contribute to induction of EMT-like cell phenotype in lung cells exposed to genotoxic AhR ligands.
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http://dx.doi.org/10.1016/j.chemosphere.2020.128126DOI Listing
January 2021

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

The genotoxic effects in the leukocytes of workers handling nanocomposite materials.

Mutagenesis 2020 09;35(4):331-340

Department of Nanotoxicology and Molecular Epidemiology, Institute of Experimental Medicine of the Czech Academy of Sciences, Videnska, Prague, Czech Republic.

The extensive development of nanotechnologies and nanomaterials poses a number of questions to toxicologists about the potential health risks of exposure to nanoparticles (NP). In this study, we analysed DNA damage in the leukocytes of 20 workers who were long-term exposed (18 ± 10 years) to NP in their working environment. Blood samples were collected in September 2016, before and after a shift, to assess (i) the chronic effects of NP on DNA (pre-shift samples) and (ii) the acute effects of exposure during the shift (the difference between pre- and post-shift samples). The samples from matched controls were taken in parallel with workers before the shift. Leukocytes were isolated from heparinised blood on a Ficoll gradient. The enzyme-modified comet assay (DNA formamido-pyrimidine-glycosylase and endonuclease III) demonstrated a considerable increase of both single- and double-strand breaks in DNA (DNA-SB) and oxidised bases when compared with the controls (2.4× and 2×, respectively). Acute exposure induced a further increase of DNA-SB. The welding and smelting of nanocomposites represented a higher genotoxic risk than milling and grinding of nanocomposite surfaces. Obesity appeared to be a factor contributing to an increased risk of oxidative damage to DNA. The data also indicated a higher susceptibility of males vs. females to NP exposure. The study was repeated in September 2017. The results exhibited similar trend, but the levels of DNA damage in the exposed subjects were lower compared to previous year. This was probably associated with lower exposure to NP in consequence of changes in nanomaterial composition and working operations. The further study involving also monitoring of personal exposures to NP is necessary to identify (i) the main aerosol components responsible for genotoxic effects in workers handling nanocomposites and (ii) the primary cause of gender differences in response to NP action.
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http://dx.doi.org/10.1093/mutage/geaa016DOI Listing
September 2020

Environmental six-ring polycyclic aromatic hydrocarbons are potent inducers of the AhR-dependent signaling in human cells.

Environ Pollut 2020 Nov 3;266(Pt 2):115125. Epub 2020 Jul 3.

Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 70, 62100 Brno, Czech Republic.

The toxicities of many environmental polycyclic aromatic hydrocarbons (PAHs), in particular those of high-molecular-weight PAHs (with MW higher than 300), remain poorly characterized. The objective of this study was to evaluate the ability of selected environmentally relevant PAHs with MW 302 (MW302 PAHs) to activate the aryl hydrocarbon receptor (AhR), since this represents a major toxic mode of action of PAHs. A large number of the evaluated compounds exhibited strong AhR-mediated activities, in particular in human models. The studied MW302 PAHs also significantly contributed to the overall calculated AhR activities of complex environmental mixtures, including both defined standard reference materials and collected diesel exhaust particles. The high AhR-mediated activities of representative MW302 PAHs, e.g. naphtho[1,2-k]fluoranthene, corresponded with the modulation of expression of relevant AhR target genes in a human lung cell model, or with the AhR-dependent suppression of cell cycle progression/proliferation in estrogen-sensitive cells. This was in a marked contrast with the limited genotoxicity of the same compound(s). Given the substantial levels of the AhR-activating MW302 PAHs in combustion particles, it seems important to continue to investigate the toxic modes of action of this large group of PAHs associated with airborne particulate matter.
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http://dx.doi.org/10.1016/j.envpol.2020.115125DOI Listing
November 2020

The Differential Effect of Carbon Dots on Gene Expression and DNA Methylation of Human Embryonic Lung Fibroblasts as a Function of Surface Charge and Dose.

Int J Mol Sci 2020 Jul 4;21(13). Epub 2020 Jul 4.

Department of Nanotoxicology and Molecular Epidemiology, Institute of Experimental Medicine of the Czech Academy of Sciences, 14220 Prague, Czech Republic.

This study presents a toxicological evaluation of two types of carbon dots (CD), similar in size (<10 nm) but differing in surface charge. Whole-genome mRNA and miRNA expression (RNAseq), as well as gene-specific DNA methylation changes, were analyzed in human embryonic lung fibroblasts (HEL 12469) after 4 h and 24 h exposure to concentrations of 10 and 50 µg/mL (for positive charged CD; pCD) or 10 and 100 µg/mL (for negative charged CD, nCD). The results showed a distinct response for the tested nanomaterials (NMs). The exposure to pCD induced the expression of a substantially lower number of mRNAs than those to nCD, with few commonly differentially expressed genes between the two CDs. For both CDs, the number of deregulated mRNAs increased with the dose and exposure time. The pathway analysis revealed a deregulation of processes associated with immune response, tumorigenesis and cell cycle regulation, after exposure to pCD. For nCD treatment, pathways relating to cell proliferation, apoptosis, oxidative stress, gene expression, and cycle regulation were detected. The expression of miRNAs followed a similar pattern: more pronounced changes after nCD exposure and few commonly differentially expressed miRNAs between the two CDs. For both CDs the pathway analysis based on miRNA-mRNA interactions, showed a deregulation of cancer-related pathways, immune processes and processes involved in extracellular matrix interactions. DNA methylation was not affected by exposure to any of the two CDs. In summary, although the tested CDs induced distinct responses on the level of mRNA and miRNA expression, pathway analyses revealed a potential common biological impact of both NMs independent of their surface charge.
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http://dx.doi.org/10.3390/ijms21134763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369946PMC
July 2020

Genotoxicant exposure, activation of the aryl hydrocarbon receptor, and lipid peroxidation in cultured human alveolar type II A549 cells.

Mutat Res 2020 05 6;853:503173. Epub 2020 Apr 6.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the CAS, Videnska 1083, 142 20, Prague, Czech Republic. Electronic address:

The aryl hydrocarbon receptor (AhR) transcription factor is activated by polycyclic aromatic hydrocarbons (PAH) and other ligands. Activated AhR binds to dioxin responsive elements (DRE) and initiates transcription of target genes, including the gene encoding prostaglandin endoperoxide synthase 2 (PTGS-2), which is also activated by the transcription factor NF-ĸB. PTGS-2 catalyzes the conversion of arachidonic acid (AA) into prostaglandins, thromboxanes or isoprostanes. 15-F2t-Isoprostane (IsoP), regarded as a universal marker of lipid peroxidation, is also induced by PAH exposure. We investigated the processes associated with lipid peroxidation in human alveolar basal epithelial cells (A549) exposed for 4 h or 24 h to model PAH (benzo[a]pyrene, BaP; 3-nitrobenzanthrone, 3-NBA) and organic extracts from ambient air particulate matter (EOM), collected in two seasons in a polluted locality. Both EOM induced the expression of CYP1A1 and CYP1B1; 24 h treatment significantly reduced PTGS-2 expression. IsoP levels decreased after both exposure periods, while the concentration of AA was not affected. The effects induced by BaP were similar to EOM except for increased IsoP levels after 4 h exposure and elevated AA concentration after 24 h treatment. In contrast, 3-NBA treatment did not induce CYP expression, had a weak effect on PTGS-2 expression, and, similar to BaP, induced IsoP levels after 4 h exposure and AA levels after 24 h treatment. All tested compounds induced the activity of NF-ĸB after the longer exposure period. In summary, our data suggest that EOM, and partly BaP, reduce lipid peroxidation by a mechanism that involves AhR-dependent inhibition of PTGS-2 expression. The effect of 3-NBA on IsoP levels is probably mediated by a different mechanism independent of AhR activation.
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http://dx.doi.org/10.1016/j.mrgentox.2020.503173DOI Listing
May 2020

DNA Methylation Profiles in a Group of Workers Occupationally Exposed to Nanoparticles.

Int J Mol Sci 2020 Mar 31;21(7). Epub 2020 Mar 31.

Department of Machining and Assembly, Department of Engineering Technology, Department of Material Science, Faculty of Mechanical Engineering, Technical University in Liberec, Studentska 1402/2 Liberec, Czech Republic.

The risk of exposure to nanoparticles (NPs) has rapidly increased during the last decade due to the vast use of nanomaterials (NMs) in many areas of human life. Despite this fact, human biomonitoring studies focused on the effect of NP exposure on DNA alterations are still rare. Furthermore, there are virtually no epigenetic data available. In this study, we investigated global and gene-specific DNA methylation profiles in a group of 20 long-term (mean 14.5 years) exposed, nanocomposite, research workers and in 20 controls. Both groups were sampled twice/day (pre-shift and post-shift) in September 2018. We applied Infinium Methylation Assay, using the Infinium MethylationEPIC BeadChips with more than 850,000 CpG loci, for identification of the DNA methylation pattern in the studied groups. Aerosol exposure monitoring, including two nanosized fractions, was also performed as proof of acute NP exposure. The obtained array data showed significant differences in methylation between the exposed and control groups related to long-term exposure, specifically 341 CpG loci were hypomethylated and 364 hypermethylated. The most significant CpG differences were mainly detected in genes involved in lipid metabolism, the immune system, lung functions, signaling pathways, cancer development and xenobiotic detoxification. In contrast, short-term acute NP exposure was not accompanied by DNA methylation changes. In summary, long-term (years) exposure to NP is associated with DNA epigenetic alterations.
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http://dx.doi.org/10.3390/ijms21072420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177382PMC
March 2020

Gene Expression and Epigenetic Changes in Mice Following Inhalation of Copper(II) Oxide Nanoparticles.

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

Department of Genetic Toxicology and Epigenetics, Institute of Experimental Medicine of the Czech Academy of Sciences, 14220 Prague, Czech Republic.

We investigated the transcriptomic response and epigenetic changes in the lungs of mice exposed to inhalation of copper(II) oxide nanoparticles (CuO NPs) (8 × 10 NPs/m) for periods of 3 days, 2 weeks, 6 weeks, and 3 months. A whole genome transcriptome and miRNA analysis was performed using next generation sequencing. Global DNA methylation was assessed by ELISA. The inhalation resulted in the deregulation of mRNA transcripts: we detected 170, 590, 534, and 1551 differentially expressed transcripts after 3 days, 2 weeks, 6 weeks, and 3 months of inhalation, respectively. Biological processes and pathways affected by inhalation, differed between 3 days exposure (collagen formation) and longer treatments (immune response). Periods of two weeks exposure further induced apoptotic processes, 6 weeks of inhalation affected the cell cycle, and 3 months of treatment impacted the processes related to cell adhesion. The expression of miRNA was not affected by 3 days of inhalation. Prolonged exposure periods modified miRNA levels, although the numbers were relatively low (17, 18, and 38 miRNAs, for periods of 2 weeks, 6 weeks, and 3 months, respectively). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis based on miRNA-mRNA interactions, revealed the deregulation of processes implicated in the immune response and carcinogenesis. Global DNA methylation was not significantly affected in any of the exposure periods. In summary, the inhalation of CuO NPs impacted on both mRNA and miRNA expression. A significant transcriptomic response was already observed after 3 days of exposure. The affected biological processes and pathways indicated the negative impacts on the immune system and potential role in carcinogenesis.
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http://dx.doi.org/10.3390/nano10030550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153614PMC
March 2020

Relation between personal exposure and outdoor concentrations of carcinogenic polycyclic aromatic hydrocarbons during smog episode.

Cent Eur J Public Health 2019 12;27(4):305-311

Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic.

Objectives: To our knowledge this is the first study measuring personal exposure to carcinogenic polycyclic aromatic hydrocarbons (cPAHs) bound to airborne particulate matter ≤ 2.5 µm (PM) in periods of high air pollution (smog episode) in which citizen were tracked.

Methods: Measurements were performed in industrial regions of the Czech Republic: Ostrava, Karviná, Havířov. The city of Prague served as a control. Personal monitoring was conducted by active personal monitors for 48 hours. Non-smoking city policemen from Prague, Karviná and Havířov, office workers from Ostrava city and volunteers from Ostrava-Radvanice and Bartovice participated in the study (N = 214).

Results: The average personal exposure to benzo[a]pyrene (B[a]P) was highest in Ostrava (17.2 ng/m), followed by Karviná, Havířov, Radvanice and Bartovice, and Prague (14.2, 12.0, 9.3, and 2.8 ng/m, respectively). We tested for association between the personal exposure to cPAHs and various health-related factors extracted from the questionnaires, including lifestyle factors and day-to-day activities.

Conclusions: Exposure to outdoor cPAHs, environmental tobacco smoke (ETS), commuting, and time spent indoors (in restaurants, workplace or home) were found to be the main determinants of the personal exposure. Daily cPAHs measurements in highly polluted areas are needed for evaluating the personal exposure and to avoid its underestimation resulting from stationary monitoring.
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http://dx.doi.org/10.21101/cejph.a5475DOI Listing
December 2019

The Biological Effects of Complete Gasoline Engine Emissions Exposure in a 3D Human Airway Model (MucilAir) and in Human Bronchial Epithelial Cells (BEAS-2B).

Int J Mol Sci 2019 Nov 14;20(22). Epub 2019 Nov 14.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the CAS, Videnska 1083, 142 20 Prague, Czech Republic.

The biological effects induced by complete engine emissions in a 3D model of the human airway (MucilAir) and in human bronchial epithelial cells (BEAS-2B) grown at the air-liquid interface were compared. The cells were exposed for one or five days to emissions generated by a Euro 5 direct injection spark ignition engine. The general condition of the cells was assessed by the measurement of transepithelial electrical resistance and mucin production. The cytotoxic effects were evaluated by adenylate kinase (AK) and lactate dehydrogenase (LDH) activity. Phosphorylation of histone H2AX was used to detect double-stranded DNA breaks. The expression of the selected 370 relevant genes was analyzed using next-generation sequencing. The exposure had minimal effects on integrity and AK leakage in both cell models. LDH activity and mucin production in BEAS-2B cells significantly increased after longer exposures; DNA breaks were also detected. The exposure affected and expression in MucilAir. There were no effects of this kind observed in BEAS-2B cells; in this system gene expression was rather affected by the time of treatment. The type of cell model was the most important factor modulating gene expression. In summary, the biological effects of complete emissions exposure were weak. In the specific conditions used in this study, the effects observed in BEAS-2B cells were induced by the exposure protocol rather than by emissions and thus this cell line seems to be less suitable for analyses of longer treatment than the 3D model.
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http://dx.doi.org/10.3390/ijms20225710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888625PMC
November 2019

The genotoxicity of organic extracts from particulate truck emissions produced at various engine operating modes using diesel or biodiesel (B100) fuel: A pilot study.

Mutat Res 2019 09 17;845:403034. Epub 2019 Mar 17.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech Republic. Electronic address:

An analysis of the toxic effects of emissions should reflect real traffic conditions. The exhaust emissions of particulate matter from diesel engines strongly depend on their operating conditions, with low-speed, low-load "urban creep" conditions, common for truck traffic in heavily congested urban areas, being one of the worst. We aimed to detect the genotoxicity of organic extracts from particulate matter in the exhaust of the diesel engine Zetor 1505 running on diesel and biodiesel (B100) fuels at characteristic modes of extended "urban creep", typical for transit truck traffic in Prague, comparing the first 5 min of idling with extended (20-80 min) idling, full load after idle, "stabilized" full load, and 30% load. The diluted exhaust was sampled with high volume samplers on glass fiber fluorocarbon coated filters. The filters were extracted with dichloromethane and DNA damage was analyzed in A549 cells using comet assay, with the inclusion of formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII) to recognize oxidized DNA bases. The cells were exposed to extractable organic matter (EOM) for 4 and 24 h at non-cytotoxic dose corresponding to 0.001 m of undiluted exhaust gas per ml cell media. At the 4 h exposure interval, all samples from B100 and diesel emissions induced DNA damage. EOM from the extended idle engine mode exerted the strongest genotoxic effect for both fuels. Twenty hours later, the cells exposed to diesel EOM exhibited a further increase of DNA strand breaks compared to the preceding interval. In contrast, DNA damage seemed to be fully repaired in cells treated with EOM derived from biodiesel B100. The preliminary results suggest that (i) diesel emissions are more genotoxic than the emissions from B100, (ii) biodiesel induced DNA lesions are repaired within 24 h.
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http://dx.doi.org/10.1016/j.mrgentox.2019.03.007DOI Listing
September 2019

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Disrupts Control of Cell Proliferation and Apoptosis in a Human Model of Adult Liver Progenitors.

Toxicol Sci 2019 12;172(2):368-384

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic.

The aryl hydrocarbon receptor (AhR) activation has been shown to alter proliferation, apoptosis, or differentiation of adult rat liver progenitors. Here, we investigated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated AhR activation on a human model of bipotent liver progenitors, undifferentiated HepaRG cells. We used both intact undifferentiated HepaRG cells, and the cells with silenced Hippo pathway effectors, yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which play key role(s) in tissue-specific progenitor cell self-renewal and expansion, such as in liver, cardiac, or respiratory progenitors. TCDD induced cell proliferation in confluent undifferentiated HepaRG cells; however, following YAP, and, in particular, double YAP/TAZ knockdown, TCDD promoted induction of apoptosis. These results suggested that, unlike in mature hepatocytes, or hepatocyte-like cells, activation of the AhR may sensitize undifferentiated HepaRG cells to apoptotic stimuli. Induction of apoptosis in cells with silenced YAP/TAZ was associated with upregulation of death ligand TRAIL, and seemed to involve both extrinsic and mitochondrial apoptosis pathways. Global gene expression analysis further suggested that TCDD significantly altered expression of constituents and/or transcriptional targets of signaling pathways participating in control of expansion or differentiation of liver progenitors, including EGFR, Wnt/β-catenin, or tumor growth factor-β signaling pathways. TCDD significantly upregulated cytosolic proapoptotic protein BMF (Bcl-2 modifying factor) in HepaRG cells, which could be linked with an enhanced sensitivity of TCDD-treated cells to apoptosis. Our results suggest that, in addition to promotion of cell proliferation and alteration of signaling pathways controlling expansion of human adult liver progenitors, AhR ligands may also sensitize human liver progenitor cells to apoptosis.
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http://dx.doi.org/10.1093/toxsci/kfz202DOI Listing
December 2019

Toxicity of TiO, ZnO, and SiO Nanoparticles in Human Lung Cells: Safe-by-Design Development of Construction Materials.

Nanomaterials (Basel) 2019 Jul 2;9(7). Epub 2019 Jul 2.

J. Heyrovsky Institute of Physical Chemistry of the CAS, Dolejskova 3, 18223 Prague, Czech Republic.

Rapid progress in the development of highly efficient nanoparticle-based construction technologies has not always been accompanied by a corresponding understanding of their effects on human health and ecosystems. In this study, we compare the toxicological effects of pristine TiO, ZnO, SiO, and coated SiO nanoparticles, and evaluate their suitability as additives to consolidants of weathered construction materials. First, water soluble tetrazolium 1 (WST-1) and lactate dehydrogenase (LDH) assays were used to determine the viability of human alveolar A549 cells at various nanoparticle concentrations (0-250 μg mL). While the pristine TiO and coated SiO nanoparticles did not exhibit any cytotoxic effects up to the highest tested concentration, the pristine SiO and ZnO nanoparticles significantly reduced cell viability. Second, as all developed nanoparticle-modified consolidants increased the mechanical strength of weathered sandstone, the decisive criterion for the selection of the most suitable nanoparticle additive was as low toxicity as possible. We believe that this approach would be of high importance in the industry, to identify materials representing top functional properties and low toxicity, at an early stage of the product development.
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http://dx.doi.org/10.3390/nano9070968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669541PMC
July 2019

Short-term and Long-term Exposure of the MucilAir™ Model to Polycyclic Aromatic Hydrocarbons.

Altern Lab Anim 2019 Mar 30;47(1):9-18. Epub 2019 Apr 30.

1 Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.

Cells grown in monocultures are widely used to model lung tissue. As a result of these culture conditions, these cells exhibit poor morphological similarity to those present in lung tissue. MucilAir™, a 3-D model comprising human basal, goblet and ciliated cells, represents a fully differentiated respiratory epithelium that can be used as an alternative and a more realistic system. The aim of our study was to compare the effects of short-term and long-term exposure to two polycyclic aromatic hydrocarbons (PAHs) - benzo[a]pyrene (B[a]P) and 3-nitrobenzanthrone (3-NBA) - using MucilAir as a model of human lung tissue. Two concentrations (0.1 μM and 1 μM) were tested at three time points (24 hours, 7 days and 28 days). Several aspects were assessed: cytotoxicity (lactate dehydrogenase (LDH) release), integrity of the cell layer (transepithelial electrical resistance (TEER)), induction of oxidative stress (reactive oxygen species production) and changes in the expression of selected genes involved in PAH metabolism ( and ) and the antioxidant response (, , , , and ). The results showed that exposure to B[a]P caused a spike in LDH release at day 5. Exposure to 3-NBA caused a number of spikes in LDH release, starting at day 5, and a decrease in TEER after 11 days. gene expression was upregulated after the 7-day and 28-day B[a]P exposures, as well as after the 24-hour and 7-day 3-NBA exposures. and were downregulated after both 24-hour PAH treatments. was upregulated after a 1-week exposure to 3-NBA. There were no significant changes in the messenger RNA (mRNA) levels of or . These results illustrate the potential use of this 3-D lung tissue model in studying the effects of chronic exposure to PAHs.
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http://dx.doi.org/10.1177/0261192919841484DOI Listing
March 2019

The repeated cytogenetic analysis of subjects occupationally exposed to nanoparticles: a pilot study.

Mutagenesis 2019 09;34(3):253-263

Department of Machining and Assembly, Technical University in Liberec, Liberec, Czech Republic.

The application of nanomaterials has been rapidly increasing during recent years. Inhalation exposure to nanoparticles (NP) may result in negative toxic effects but there is a critical lack of human studies, especially those related to possible DNA alterations. We analyzed pre-shift and post-shift a group of nanocomposite researchers with a long-term working background (17.8 ± 10.0 years) and matched controls. The study group consisted of 73.2% males and 26.8% females. Aerosol exposure monitoring during a working shift (involving welding, smelting, machining) to assess the differences in exposure to particulate matter (PM) including nanosized fractions <25-100 nm, and their chemical analysis, was carried out. A micronucleus assay using Human Pan Centromeric probes, was applied to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN-) micronuclei (MN) in the binucleated cells. This approach allowed recognition of the types of chromosomal damage: losses and breaks. The monitoring data revealed differences in the exposure to NP related to individual working processes, and in the chemical composition of nanofraction. The cytogenetic results of this pilot study demonstrated a lack of effect of long-term (years) exposure to NP (total frequency of MN, P = 0.743), although this exposure may be responsible for DNA damage pattern changes (12% increase of chromosomal breaks-clastogenic effect). Moreover, short-term (daily shift) exposure could be a reason for the increase of chromosomal breaks in a subgroup of researchers involved in welding and smelting processes (clastogenic effect, P = 0.037). The gender and/or gender ratio of the study participants was also an important factor for the interpretation of the results. As this type of human study is unique, further research is needed to understand the effects of long-term and short-term exposure to NP.
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http://dx.doi.org/10.1093/mutage/gez016DOI Listing
September 2019

Molecular Responses in THP-1 Macrophage-Like Cells Exposed to Diverse Nanoparticles.

Nanomaterials (Basel) 2019 May 2;9(5). Epub 2019 May 2.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, 14220 Prague, Czech Republic.

In the body, engineered nanoparticles (NPs) may be recognized and processed by immune cells, among which macrophages play a crucial role. We evaluated the effects of selected NPs [NM-100 (TiO), NM-110 (ZnO), NM-200 (SiO), and NM-300 K (Ag)] on THP-1 macrophage-like cells. The cells were exposed to subcytotoxic concentrations of NPs (1-25 µg/mL) and the expression of immunologically relevant genes (VCAM1, TNFA, CXCL8, ICAM1, CD86, CD192, and IL1B) was analyzed by RT-qPCR. The expression of selected cytokines, growth factors and surface molecules was assessed by flow cytometry or ELISA. Generation of reactive oxygen species and induction of DNA breaks were also analyzed. Exposure to diverse NPs caused substantially different molecular responses. No significant effects were detected for NM-100 treatment. NM-200 induced production of IL-8, a potent attractor and activator of neutrophils, growth factors (VEGF and IGF-1) and superoxide. NM-110 triggered a proinflammatory response, characterized by the activation of transcription factor NF-κB, an enhanced production of proinflammatory cytokines (TNF-α) and chemokines (IL-8). Furthermore, the expression of cell adhesion molecules VCAM-1 and ICAM-1 and hepatocyte growth factor (HGF), as well as superoxide production and DNA breaks, were affected. NM-300 K enhanced IL-8 production and induced DNA breaks, however, it decreased the expression of chemokine receptor (CCR2) and CD86 molecule, indicating potential immunosuppressive activity. The toxicity of ZnO and Ag NPs was probably caused by their intracellular dissolution, as indicated by transmission electron microscopy imaging. The observed effects in macrophages might further influence both innate and adaptive immune responses by promoting neutrophil recruitment via IL-8 release and enhancing the adhesion and stimulation of T cells by VCAM-1 and ICAM-1 expression.
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http://dx.doi.org/10.3390/nano9050687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567235PMC
May 2019

The processes associated with lipid peroxidation in human embryonic lung fibroblasts, treated with polycyclic aromatic hydrocarbons and organic extract from particulate matter.

Mutagenesis 2019 05;34(2):153-164

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.

Polycyclic aromatic hydrocarbons (PAHs) may cause lipid peroxidation via reactive oxygen species generation. 15-F2t-isoprostane (IsoP), an oxidative stress marker, is formed from arachidonic acid (AA) by a free-radical induced oxidation. AA may also be converted to prostaglandins (PG) by prostaglandin-endoperoxide synthase (PTGS) induced by NF-κB. We treated human embryonic lung fibroblasts (HEL12469) with benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA) and extractable organic matter (EOM) from ambient air particulate matter <2.5 µm for 4 and 24 h. B[a]P and 3-NBA induced expression of PAH metabolising, but not antioxidant enzymes. The concentrations of IsoP decreased, whereas the levels of AA tended to increase. Although the activity of NF-κB was not detected, the tested compounds affected the expression of prostaglandin-endoperoxide synthase 2 (PTGS2). The levels of prostaglandin E2 (PGE2) decreased following exposure to B[a]P, whereas 3-NBA exposure tended to increase PGE2 concentration. A distinct response was observed after EOM exposure: expression of PAH-metabolising enzymes was induced, IsoP levels increased after 24-h treatment but AA concentration was not affected. The activity of NF-κB increased after both exposure periods, and a significant induction of PTGS2 expression was found following 4-h treatment. Similarly to PAHs, the EOM exposure was associated with a decrease of PGE2 levels. In summary, exposure to PAHs with low pro-oxidant potential results in a decrease of IsoP levels implying 'antioxidant' properties. For such compounds, IsoP may not be a suitable marker of lipid peroxidation.
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http://dx.doi.org/10.1093/mutage/gez004DOI Listing
May 2019

n-3 Polyunsaturated fatty acids alter benzo[a]pyrene metabolism and genotoxicity in human colon epithelial cell models.

Food Chem Toxicol 2019 Feb 17;124:374-384. Epub 2018 Dec 17.

Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno, Czech Republic. Electronic address:

Dietary carcinogens, such as benzo[a]pyrene (BaP), are suspected to contribute to colorectal cancer development. n-3 Polyunsaturated fatty acids (PUFAs) decrease colorectal cancer risk in individuals consuming diets rich in PUFAs. Here, we investigated the impact of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid on metabolism and genotoxicity of BaP in human cell models derived from the colon: HT-29 and HCT-116 cell lines. Both PUFAs reduced levels of excreted BaP metabolites, in particular BaP-tetrols and hydroxylated BaP metabolites, as well as formation of DNA adducts in HT-29 and HCT-116 cells. However, EPA appeared to be a more potent inhibitor of formation of some intracellular BaP metabolites, including BaP-7,8-dihydrodiol. EPA also reduced phosphorylation of histone H2AX (Ser139) in HT-29 cells, which indicated that it may reduce further forms of DNA damage, including DNA double strand breaks. Both PUFAs inhibited induction of CYP1 activity in colon cells determined as 7-ethoxyresorufin-O-deethylase (EROD); this was at least partly linked with inhibition of induction of CYP1A1, 1A2 and 1B1 mRNAs. The downregulation and/or inhibition of CYP1 enzymes by PUFAs could thus alter metabolism and reduce genotoxicity of BaP in human colon cells, which might contribute to known chemopreventive effects of PUFAs in colon epithelium.
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http://dx.doi.org/10.1016/j.fct.2018.12.021DOI Listing
February 2019

Inhalation of ZnO Nanoparticles: Splice Junction Expression and Alternative Splicing in Mice.

Toxicol Sci 2019 03;168(1):190-200

*Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 14220, Czech Republic.

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.
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http://dx.doi.org/10.1093/toxsci/kfy288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390655PMC
March 2019

Bulky DNA adducts, microRNA profiles, and lipid biomarkers in Norwegian tunnel finishing workers occupationally exposed to diesel exhaust.

Occup Environ Med 2019 01 13;76(1):10-16. Epub 2018 Nov 13.

Section for Toxicology and Biological Work Environment, Department of Chemical and Biological Work Environment, National Institute of Occupational Health, Oslo, Norway.

Objectives: This study aimed to assess the biological impact of occupational exposure to diesel exhaust (DE) including DE particles (DEP) from heavy-duty diesel-powered equipment in Norwegian tunnel finishing workers (TFW).

Methods: TFW (n=69) and referents (n=69) were investigated for bulky DNA adducts (by P-postlabelling) and expression of microRNAs (miRNAs) (by small RNA sequencing) in peripheral blood mononuclear cells (PBMC), as well as circulating free arachidonic acid (AA) and eicosanoid profiles in plasma (by liquid chromatography-tandem mass spectrometry).

Results: PBMC from TFW showed significantly higher levels of DNA adducts compared with referents. Levels of DNA adducts were also related to smoking habits. Seventeen miRNAs were significantly deregulated in TFW. Several of these miRNAs are related to carcinogenesis, apoptosis and antioxidant effects. Analysis of putative miRNA-gene targets revealed deregulation of pathways associated with cancer, alterations in lipid molecules, steroid biosynthesis and cell cycle. Plasma profiles showed higher levels of free AA and 15-hydroxyeicosatetraenoic acid, and lower levels of prostaglandin D and 9-hydroxyoctadecadienoic acid in TFW compared with referents.

Conclusion: Occupational exposure to DE/DEP is associated with biological alterations in TFW potentially affecting lung homoeostasis, carcinogenesis, inflammation status and the cardiovascular system. Of particular importance is the finding that tunnel finishing work is associated with an increased level of DNA adducts formation in PBMC.
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http://dx.doi.org/10.1136/oemed-2018-105445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327869PMC
January 2019

In Vitro Transformation of Human Bronchial Epithelial Cells by Diesel Exhaust Particles: Gene Expression Profiling and Early Toxic Responses.

Toxicol Sci 2018 11;166(1):51-64

Section for Toxicology and Biological Work Environment, Department of Chemical and Biological Work Environment, National Institute of Occupational Health, N-0304 Oslo, Norway.

Occupational exposure to diesel exhaust may cause lung cancer in humans. Mechanisms include DNA-damage and inflammatory responses. Here, the potential of NIST SRM2975 diesel exhaust particles (DEP) to transform human bronchial epithelial cells (HBEC3) in vitro was investigated. Long-term exposure of HBEC3 to DEP led to increased colony growth in soft agar. Several DEP-transformed cell lines were established and based on the expression of epithelial-to-mesenchymal-transition (EMT) marker genes, one of them (T2-HBEC3) was further characterized. T2-HBEC3 showed a mesenchymal/fibroblast-like morphology, reduced expression of CDH1, and induction of CDH2 and VIM. T2-HBEC3 had reduced migration potential compared with HBEC3 and little invasion capacity. Gene expression profiling showed baseline differences between HBEC3 and T2-HBEC3 linked to lung carcinogenesis. Next, to assess differences in sensitivity to DEP between parental HBEC3 and T2-HBEC3, gene expression profiling was carried out after DEP short-term exposure. Results revealed changes in genes involved in metabolism of xenobiotics and lipids, as well as inflammation. HBEC3 displayed a higher steady state of IL1B gene expression and release of IL-1β compared with T2-HBEC3. HBEC3 and T2-HBEC3 showed similar susceptibility towards DEP-induced genotoxic effects. Liquid-chromatography-tandem-mass-spectrometry was used to measure secretion of eicosanoids. Generally, major prostaglandin species were released in higher concentrations from T2-HBEC3 than from HBEC3 and several analytes were altered after DEP-exposure. In conclusion, long-term exposure to DEP-transformed human bronchial epithelial cells in vitro. Differences between HBEC3 and T2-HBEC3 regarding baseline levels and DEP-induced changes of particularly CYP1A1, IL-1β, PGE2, and PGF2α may have implications for acute inflammation and carcinogenesis.
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http://dx.doi.org/10.1093/toxsci/kfy183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204768PMC
November 2018

Adaptive changes in global gene expression profile of lung carcinoma A549 cells acutely exposed to distinct types of AhR ligands.

Toxicol Lett 2018 Aug 25;292:162-174. Epub 2018 Apr 25.

Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Exposure to persistent ligands of aryl hydrocarbon receptor (AhR) has been found to cause lung cancer in experimental animals, and lung adenocarcinomas are often associated with enhanced AhR expression and aberrant AhR activation. In order to better understand the action of toxic AhR ligands in lung epithelial cells, we performed global gene expression profiling and analyze TCDD-induced changes in A549 transcriptome, both sensitive and non-sensitive to CH223191 co-treatment. Comparison of our data with results from previously reported microarray and ChIP-seq experiments enabled us to identify candidate genes, which expression status reflects exposure of lung cancer cells to TCDD, and to predict processes, pathways (e.g. ER stress, Wnt/β-cat, IFNɣ, EGFR/Erbb1), putative TFs (e.g. STAT, AP1, E2F1, TCF4), which may be implicated in adaptive response of lung cells to TCDD-induced AhR activation. Importantly, TCDD-like expression fingerprint of selected genes was observed also in A549 cells exposed acutely to both toxic (benzo[a]pyrene, benzo[k]fluoranthene) and endogenous AhR ligands (2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester and 6-formylindolo[3,2-b]carbazole). Overall, our results suggest novel cellular candidates, which could help to improve monitoring of AhR-dependent transcriptional activity during acute exposure of lung cells to distinct types of environmental pollutants.
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http://dx.doi.org/10.1016/j.toxlet.2018.04.024DOI Listing
August 2018

Gene expression profiling in healthy newborns from diverse localities of the Czech Republic.

Environ Mol Mutagen 2018 06 30;59(5):401-415. Epub 2018 Mar 30.

Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.

Prenatal exposure to air pollution is associated with intrauterine growth restriction and low birth weight. Gene expression changes in newborns in relation to air pollution have not been sufficiently studied. We analyzed whole genome expression in cord blood leukocytes of 202 newborns from diverse localities of the Czech Republic, differing among other factors in levels of air pollution: the district of Karvina (characterized by higher concentration of air pollutants) and Ceske Budejovice (lower air pollution levels). We aimed to identify differentially expressed genes (DEGs) and pathways in relation to locality and concentration of air pollutants. We applied the linear model to identify the specific DEGs and the correlation analysis, to investigate the relationship between the concentrations of air pollutants and gene expression data. An analysis of biochemical pathways and gene set enrichment was also performed. In general, we observed modest changes of gene expression, mostly attributed to the effect of the locality. The highest number of DEGs was found in samples from the district of Karvina. A pathway analysis revealed a deregulation of processes associated with cell growth, apoptosis or cellular homeostasis, immune response-related processes or oxidative stress response. The association between concentrations of air pollutants and gene expression changes was weak, particularly for samples collected in Karvina. In summary, as we did not find a direct effect of exposure to air pollutants, we assume that the general differences in the environment, rather than actual concentrations of individual pollutants, represent a key factor affecting gene expression changes at delivery. Environ. Mol. Mutagen. 59:401-415, 2018. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22184DOI Listing
June 2018

Kinetics of ROS generation induced by polycyclic aromatic hydrocarbons and organic extracts from ambient air particulate matter in model human lung cell lines.

Mutat Res Genet Toxicol Environ Mutagen 2018 Mar 31;827:50-58. Epub 2018 Jan 31.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine CAS, Prague, 14220, Czech Republic. Electronic address:

Polycyclic aromatic hydrocarbons (PAHs) associated with particulate matter (PM) may induce oxidative damage via reactive oxygen species (ROS) generation. However, the kinetics of ROS production and the link with antioxidant response induction has not been well studied. To elucidate the differences in oxidative potential of individual PAH compounds and extractable organic matter (EOM) from PM containing various PAH mixtures, we studied ROS formation and antioxidant response [total antioxidant capacity (TAC) and expression of HMOX1 and TXNRD1] in human alveolar basal epithelial cells (A549 cells) and human embryonic lung fibroblasts (HEL12469 cells). We treated the cells with three concentrations of model PAHs (benzo[a]pyrene, B[a]P; 3-nitrobenzanthrone, 3-NBA) and EOM from PM <2.5 μm (PM2.5). ROS levels were evaluated at 8 time intervals (30 min-24 h). In both cell lines, B[a]P treatment was associated with a time-dependent decrease of ROS levels. This trend was more pronounced in HEL12469 cells and was accompanied by increased TAC. A similar response was observed upon 3-NBA treatment in HEL12469 cells. In A549 cells, however, this compound significantly increased superoxide levels. This response was accompanied by the decrease of TAC as well as HMOX1 and TXNRD1 expression. In both cell lines, a short-time exposure to EOMs tended to increase ROS levels, while a marked decrease was observed after longer treatment periods. This was accompanied by the induction of HMOX1 and TXNRD1 expression in HEL12469 cells and increased TAC in A549 cells. In summary, our data indicate that in the studied cell lines B[a]P and EOMs caused a time-dependent decrease of intracellular ROS levels, probably due to the activation of the antioxidant response. This response was not detected in A549 cells following 3-NBA treatment, which acted as a strong superoxide inducer. Pro-oxidant properties of EOMs are limited to short-time exposure periods.
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http://dx.doi.org/10.1016/j.mrgentox.2018.01.006DOI Listing
March 2018

Transcriptional response to organic compounds from diverse gasoline and biogasoline fuel emissions in human lung cells.

Toxicol In Vitro 2018 Apr 9;48:329-341. Epub 2018 Feb 9.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine AS CR, Videnska 1083, 142 20 Prague, Czech Republic. Electronic address:

Modern vehicles equipped with Gasoline Direct Injection (GDI) engine have emerged as an important source of particulate emissions potentially harmful to human health. We collected and characterized gasoline exhaust particles (GEPs) produced by neat gasoline fuel (E0) and its blends with 15% ethanol (E15), 25% n-butanol (n-But25) and 25% isobutanol (i-But25). To study the toxic effects of organic compounds extracted from GEPs, we analyzed gene expression profiles in human lung BEAS-2B cells. Despite the lowest GEP mass, n-But25 extract contained the highest concentration of polycyclic aromatic hydrocarbons (PAHs), while i-But25 extract the lowest. Gene expression analysis identified activation of the DNA damage response and other subsequent events (cell cycle arrest, modulation of extracellular matrix, cell adhesion, inhibition of cholesterol biosynthesis) following 4 h exposure to all GEP extracts. The i-But25 extract induced the most distinctive gene expression pattern particularly after 24 h exposure. Whereas E0, E15 and n-But25 extract treatments resulted in persistent stress signaling including DNA damage response, MAPK signaling, oxidative stress, metabolism of PAHs or pro-inflammatory response, i-But25 induced changes related to the metabolism of the cellular nutrients required for cell recovery. Our results indicate that i-But25 extract possessed the weakest genotoxic potency possibly due to the low PAH content.
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http://dx.doi.org/10.1016/j.tiv.2018.02.002DOI Listing
April 2018

Toxicity of surface-modified copper oxide nanoparticles in a mouse macrophage cell line: Interplay of particles, surface coating and particle dissolution.

Chemosphere 2018 Apr 29;196:482-493. Epub 2017 Dec 29.

Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. Electronic address:

The rapid dissolution of copper oxide (CuO) nanoparticles (NPs) with release of ions is thought to be one of the main factors modulating their toxicity. Here we assessed the cytotoxicity of a panel of CuO NPs (12 nm ± 4 nm) with different surface modifications, i.e., anionic sodium citrate (CIT) and sodium ascorbate (ASC), neutral polyvinylpyrrolidone (PVP), and cationic polyethylenimine (PEI), versus the pristine (uncoated) NPs, using a murine macrophage cell line (RAW264.7). Cytotoxicity, reactive oxygen species (ROS) production, and cellular uptake were assessed. The cytotoxicity results were analyzed by the benchmark dose (BMD) method and the NPs were ranked based on BMD values. The PEI-coated NPs were found to be the most cytotoxic. Despite the different properties of the coating agents, NP dissolution in cell medium was only marginally affected by surface modification. Furthermore, CuCl (used as an ion control) elicited significantly less cytotoxicity when compared to the CuO NPs. We also observed that the antioxidant, N-acetylcysteine, failed to protect against the cytotoxicity of the uncoated CuO NPs. Indeed, the toxicity of the surface-modified CuO NPs was not directly linked to particle dissolution and subsequent Cu burden in cells, nor to cellular ROS production, although CuO-ASC NPs, which were found to be the least cytotoxic, yielded lower levels of ROS in comparison to pristine NPs. Hierarchical cluster analysis suggested, instead, that the toxicity in the current in vitro model could be explained by synergistic interactions between the NPs, their dissolution, and the toxicity of the coating agents.
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http://dx.doi.org/10.1016/j.chemosphere.2017.12.182DOI Listing
April 2018

Source apportionment of aerosol particles at a European air pollution hot spot using particle number size distributions and chemical composition.

Environ Pollut 2018 Mar 22;234:145-154. Epub 2017 Nov 22.

Center for Air Resources Engineering and Science, Clarkson University, Potsdam, NY 13699-5708, USA; Department of Public Health Sciences, University of Rochester Medical Center, Rochester, NY, USA.

Ostrava in the Moravian-Silesian region (Czech Republic) is a European air pollution hot spot for airborne particulate matter (PM), polycyclic aromatic hydrocarbons (PAHs), and ultrafine particles (UFPs). Air pollution source apportionment is essential for implementation of successful abatement strategies. UFPs or nanoparticles of diameter <100 nm exhibit the highest deposition efficiency in human lungs. To permit apportionment of PM sources at the hot-spot including nanoparticles, Positive Matrix Factorization (PMF) was applied to highly time resolved particle number size distributions (NSD, 14 nm-10 μm) and PM chemical composition. Diurnal patterns, meteorological variables, gaseous pollutants, organic markers, and associations between the NSD factors and chemical composition factors were used to identify the pollution sources. The PMF on the NSD reveals two factors in the ultrafine size range: industrial UFPs (28%, number mode diameter - NMD 45 nm), industrial/fresh road traffic nanoparticles (26%, NMD 26 nm); three factors in the accumulation size range: urban background (24%, NMD 93 nm), coal burning (14%, volume mode diameter - VMD 0.5 μm), regional pollution (3%, VMD 0.8 μm) and one factor in the coarse size range: industrial coarse particles/road dust (2%, VMD 5 μm). The PMF analysis of PM revealed four factors: SIA/CC/BB (52%), road dust (18%), sinter/steel (16%), iron production (16%). The factors in the ultrafine size range resolved with NSD have a positive correlation with sinter/steel production and iron production factors resolved with chemical composition. Coal combustion factor resolved with NSD has moderate correlation with SIA/CC/BB factor. The organic markers homohopanes correlate with coal combustion and the levoglucosan correlates with urban background. The PMF applications to NSD and chemical composition datasets are complementary. PAHs in PM were found to be associated with coal combustion factor.
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http://dx.doi.org/10.1016/j.envpol.2017.10.097DOI Listing
March 2018

Adaptation of the human population to the environment: Current knowledge, clues from Czech cytogenetic and "omics" biomonitoring studies and possible mechanisms.

Mutat Res 2017 07 12;773:188-203. Epub 2017 Jul 12.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine, Czech Academy of Sciences, 14220 Prague 4, Czech Republic. Electronic address:

The human population is continually exposed to numerous harmful environmental stressors, causing negative health effects and/or deregulation of biomarker levels. However, studies reporting no or even positive impacts of some stressors on humans are also sometimes published. The main aim of this review is to provide a comprehensive overview of the last decade of Czech biomonitoring research, concerning the effect of various levels of air pollution (benzo[a]pyrene) and radiation (uranium, X-ray examination and natural radon background), on the differently exposed population groups. Because some results obtained from cytogenetic studies were opposite than hypothesized, we have searched for a meaningful interpretation in genomic/epigenetic studies. A detailed analysis of our data supported by the studies of others and current epigenetic knowledge, leads to a hypothesis of the versatile mechanism of adaptation to environmental stressors via DNA methylation settings which may even originate in prenatal development, and help to reduce the resulting DNA damage levels. This hypothesis is fully in agreement with unexpected data from our studies (e.g. lower levels of DNA damage in subjects from highly polluted regions than in controls or in subjects exposed repeatedly to a pollutant than in those without previous exposure), and is also supported by differences in DNA methylation patterns in groups from regions with various levels of pollution. In light of the adaptation hypothesis, the following points may be suggested for future research: (i) the chronic and acute exposure of study subjects should be distinguished; (ii) the exposure history should be mapped including place of residence during the life and prenatal development; (iii) changes of epigenetic markers should be monitored over time. In summary, investigation of human adaptation to the environment, one of the most important processes of survival, is a new challenge for future research in the field of human biomonitoring that may change our view on the results of biomarker analyses and potential negative health impacts of the environment.
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http://dx.doi.org/10.1016/j.mrrev.2017.07.002DOI Listing
July 2017