Alena Gabelova - Academia.edu (original) (raw)

Papers by Alena Gabelova

Mutagenesis, Jan 22, 2016

Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial prod... more Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial products and biomedical applications. Despite this, the knowledge of human potential health risk as well as comprehensive biological and toxicological information is still limited. We have investigated the capacity of two frequently used metallic ENMs, nanosilver and magnetite nanoparticles (MNPs), to induce thymidine kinase (Tk (+/-)) mutations in L5178Y mouse lymphoma cells and transformed foci in Bhas 42 cells. Two types of nanosilver, spherical nanoparticles (AgNM300) and fibrous (AgNM302) nanorods/wires, and MNPs differing in surface modifications [MNPs coated with sodium oleate (SO-MNPs), MNPs coated with SO + polyethylene glycol (SO-PEG-MNPs) and MNPs coated with SO + PEG + poly(lactide-co-glycolic acid) SO-PEG-PLGA-MNPs] were included in this study. Spherical AgNM300 showed neither mutagenic nor carcinogenic potential. In contrast, silver nanorods/wires (AgNM302) increased significan...

Nanomaterials, 2022

Data suitable for assembling a physiologically-based pharmacokinetic (PBPK) model for nanoparticl... more Data suitable for assembling a physiologically-based pharmacokinetic (PBPK) model for nanoparticles (NPs) remain relatively scarce. Therefore, there is a trend in extrapolating the results of in vitro and in silico studies to in vivo nanoparticle hazard and risk assessment. To evaluate the reliability of such approach, a pharmacokinetic study was performed using the same polyethylene glycol-coated gold nanoparticles (PEG-AuNPs) in vitro and in vivo. As in vitro models, human cell lines TH1, A549, Hep G2, and 16HBE were employed. The in vivo PEG-AuNP biodistribution was assessed in rats. The internalization and exclusion of PEG-AuNPs in vitro were modeled as first-order rate processes with the partition coefficient describing the equilibrium distribution. The pharmacokinetic parameters were obtained by fitting the model to the in vitro data and subsequently used for PBPK simulation in vivo. Notable differences were observed in the internalized amount of Au in individual cell lines co...

Nanomaterials, 2021

Despite the obvious advantages of gold nanoparticles for biomedical applications, controversial a... more Despite the obvious advantages of gold nanoparticles for biomedical applications, controversial and incomplete toxicological data hamper their widespread use. Here, we present the results from an in vivo toxicity study using gold nanoparticles coated with polyethylene glycol (PEG-AuNPs). The pharmacokinetics and biodistribution of PEG-AuNPs were examined in the rat’s liver, lung, spleen, and kidney after a single i.v. injection (0.7 mg/kg) at different time intervals. PEG-AuNPs had a relatively long blood circulation time and accumulated primarily in the liver and spleen, where they remained for up to 28 days after administration. Increased cytoplasmic vacuolation in hepatocytes 24 h and 7 days after PEG-AuNPs exposure and apoptotic-like cells in white splenic pulp 24 h after administration has been detected, however, 28 days post-exposure were no longer observed. In contrast, at this time point, we identified significant changes in lipid metabolism, altered levels of liver injury m...

Beilstein Journal of Nanotechnology

The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired t... more The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired to reach high therapeutic efficiency while minimizing the side effects. Despite intensive research, the impact of the surface coating on the mechanism of nanoparticle uptake is not sufficiently understood yet. Herein, we present a mechanistic study of cellular internalization pathways of two magnetic iron oxide nanoparticles (MNPs) differing in surface chemistry into A549 cells. The MNP uptake was investigated in the presence of different inhibitors of endocytosis and monitored by spectroscopic and imaging techniques. The results revealed that the route of MNP entry into cells strongly depends on the surface chemistry of the MNPs. While serum bovine albumin-coated MNPs entered the cells via clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CavME) or lipid rafts were preferentially involved in the internalization of polyethylene glycol-coated MNPs. Our data indicate that ...

Small

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

Nature Protocols

The comet assay is a widely used test for the detection of DNA damage and repair activity. Howeve... more The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easi...

Cancers

Epigenetic dysregulation has been recognized as a critical factor contributing to the development... more Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identif...

RSC Advances

Primary renal podocytes are more susceptible to MNPs exposure than primary renal mesangial cells.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety fo... more Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO 2 NPs, SiO 2 NPs, Fe 3 O 4 NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe 3 O 4 NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (> 700 μg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 μg/ml) concentration. Fe 3 O 4 NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO 2 NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Exposure to pesticides leads to complex, long-lasting adverse effects on human health, and poses ... more Exposure to pesticides leads to complex, long-lasting adverse effects on human health, and poses a substantial risk to those living in areas devoted to agriculture. Children are particularly vulnerable to the pesticide exposure, due to the developmental, dietary and physiological factors. Small body mass and typical exploratory behavior result in increased risk of intoxication. Thus, even exposure to low concentrations of pesticides, if of sufficient duration, may lead to permanent health disorders and limit their harmonious development. In this study 108 children, living in areas of an intense pesticide use and a control group (n = 92) of children from an agrotouristic area were investigated, whether DNA damage increased due to prolonged pesticide exposure. A presence of DNA breaks and oxidative damage to DNA bases, characterized as Fpg-sensitive sites, were detected by comet assay. Micronuclei (MN) formation was evaluated by cytokinesis-block MN assay. The exposure of children to pesticides resulted in increased number of MN in peripheral blood lymphocytes (P = 0.016), increased DNA strand breaks level (P = 0.002) and oxidative damage to DNA (P < 0.001). Negative correlation was demonstrated between the level of DNA strand breaks and acetylcholinesterase (AChE) activity in exposed group. In conclusion, despite just environmental pesticide exposure in the test group of children, significant biological effects were detected.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Drug-induced kidney injury is one of the most significant adverse events and dose limiting factor... more Drug-induced kidney injury is one of the most significant adverse events and dose limiting factor in chemotherapy as well a major cause of prospective drug attrition during pharmaceutical development. Moreover, kidney injury can also occur as a consequence of exposures to environmental xenobiotics such as heavy metals, fungal toxins and nanomaterials. The lack of adequate in vitro human kidney models that mimic more realistically the in vivo conditions and the absence of suitable and robust, cost-effective and predictive cell-based in vitro assays contribute to an underestimation of the kidney toxic potential of new drugs and xenobiotics. Therefore, a rapid screening system capable to detect potential nephrotoxicity at early stages of drug discovery is an urgent need. Here we provide an overview of human cell lines currently used as a surrogate in vitro kidney models in nephrotoxicity studies, including their advantages and limitations. In addition, the capacity of the single cell gel electrophoresis (SCGE)/comet assay as a potential tool in kidney toxicants screening is discussed. Despite a limited number of studies using the comet assay to evaluate the drug-induced kidney damage potential, a considerable variability in SCGE methodology (e.g. lysis, unwinding, and electrophoresis conditions) has been observed. Before the comet assay can be included in nephrotoxicity testing, a basic guideline has to be developed. To test its feasibility, additional in vitro experiments including inter-laboratory validation studies based on this guideline have to be performed.

Journal of Magnetism and Magnetic Materials

Abstract Magnetic iron oxide nanoparticles (MNPs) are one of the most promising types of nanopart... more Abstract Magnetic iron oxide nanoparticles (MNPs) are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy. They are used as contrast agents in magnetic resonance imaging and magnetite cell labelling. Furthermore, they are promising heating mediator in magnetic hyperthermia-based therapy, and can serve as nanocarriers in targeted gene and drug delivery as well. In biomedical applications, coating plays an important role in nanoparticle dispersion stability and biocompatibility. However, the impact of nanoparticle surface chemistry on cell uptake and proliferation has not been sufficiently investigated. The objective of this study is to prepare magnetic nanoparticles with inner magnetite core and hydrophilic outer shell of surfactant, protein and polymers that are commonly used in biomedical research. MNPs were characterized in-depth by various physicochemical methods. Magnetic hyperthermia, applied to find out the influence of MNPs coating on heating characteristics of the samples, did not show any correlation between layer thickness and specific adsorption rate. To evaluate the impact of surface chemistry on cell proliferation and internalization, the human lung adenocarcinoma epithelial (A549) cells were utilized. Substantial differences were determined in the amount of internalized MNPs and cell viability in dependence on surface coating. Our results indicate that the surface chemistry not only protects particles from agglomeration but also affect the interaction between cell and MNPs.

The present pilot study was designed to elucidate the functional significance of amino acid subst... more The present pilot study was designed to elucidate the functional significance of amino acid substitution variants of DNA repair genes. Using the peripheral blood lymphocytes (PBLs) from healthy donors and cervical cancer patients, the contribution of four non-synonymous single nucleotide polymorphisms (SNPs) in three base excision repair genes (BER), XRCC1 (Arg194Trp and Arg399Gln), hOGG1 (Ser326Cys), and APE1 (Asp148Glu), to the susceptibility to ionizing radiation were evaluated. The level of initial, oxidative and residual DNA damage produced by 2 Gy was measured by the alkaline single cell gel electrophoresis (the comet assay), and the SNPs were determined by PCR-restriction fragment length polymorphism (RFLP) assay. No significant differences in the allele frequencies between cancer patients and controls for any of these four SNPs were detected. Although the initial DNA damage levels were approximately similar, significantly higher level of Fpg-sensitive sites were found in patients compared with controls (p<0.001) irrespective of genotype distribution. A trend towards increased values of EndoIII-sensitive sites was determined in PBLs from cancer patients compared with healthy women, mainly carriers of the XRCC1 and OGG1 variant alleles; however, the mean value of EndoIII-sensitive sites does not reach any significance. A substantial delay in DNA strand-break rejoining was ascertained in patients who carried APE1 Glu variant allele in comparison with healthy donors 15 and 60 minutes after irradiation (p< 0.05 and p< 0.01, respectively). In contrast, slightly higher but statistically significant level of residual DNA damage was estimated in controls (APE1Asp/Asp) compared with patients. An association between single nucleotide polymorphism (SNP) of two DNA repair genes functioning in the same biochemical pathway and susceptibility to radiation was found. In the combined genotype APE1/XRCC1 and APE1/hOGG1, a decreased level of residual DNA damage was detected in carriers of wild type APE1 genotype. In addition, a possible modulating effect of hOGG1 gene on the kinetics of strand-break rejoining was estimated. The lowest residual DNA damage level was determined in subjects with the combined APE1(Asp/Asp)/hOGG1(Ser/Cys+Cys/Cys) genotypes. Based on these preliminary data we suppose that a combination of several amino acid substitution variants of DNA repair genes involved in the same repair pathway rather than one low-penetrance SNP in a single gene may contribute to DNA repair outcomes. Larger study with more subjects is needed to verify these findings.

Cell Biology and Toxicology

The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in dia... more The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in diagnosis and disease management. However, as INPs are relatively difficult to fully degrade and excrete, their unintended accumulation in the tissue might result in adverse health effects. Herein, we provide a methylome–transcriptome framework for chronic effects of INPs, commonly used in biomedical applications, in human kidney TH-1 cells. Renal clearance is one of the most important routes of nanoparticle excretion; therefore, a detailed evaluation of nanoparticle-mediated nephrotoxicity is an important task. Integrated analysis of methylome and transcriptome changes induced by INPs (PEG-AuNPs, Fe3O4NPs, SiO2NPs, and TiO2NPs) revealed significantly deregulated genes with functional classification in immune response, DNA damage, and cancer-related pathways. Although most deregulated genes were unique to individual INPs, a relatively high proportion of them encoded the transcription factor...

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2009

Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our r... more Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole (N-MeDBC), induced several cellular events associated with tumor promotion in WB-F344 cells, an in vitro model of liver oval cells [J.

Materials Science and Engineering: C, 2017

Comprehensive characterization of nanoparticles associated with investigation of their cellular u... more Comprehensive characterization of nanoparticles associated with investigation of their cellular uptake creates the basis on which fundamental in vitro and in vivo studies can be built. In this work, a complex analysis of various surface-modified magnetite nanoparticles in biologically relevant environment is reported and the promotion of incorrect characterization into the results obtained from model biological experiments leading to false conclusions is demonstrated. Via a bottom-up approach from particle characterization by DLS towards interpretation of biological data based on cellular uptake, this work draws attention to the systematic propagation of errors stemming from inaccurate determination of input parameters for DLS, improper selection of particle size distribution, inadequate sampling, unknown colloidal behavior and the omission of fraction of particles complying with the internalization threshold. In addition, cellular uptake depending on the number of treated cells is shown. The definition of cellular uptake efficacy reflecting the size distribution of particles beside their absolute internalization is postulated.

Mutation Research Environmental Mutagenesis and Related Subjects, 1981

Mutat Res Genet Toxicol E M, 2004

The genotoxic potential of extractable organic matter (EOM) associated with the respirable partic... more The genotoxic potential of extractable organic matter (EOM) associated with the respirable particulate matter (PM <10 μm) of atmospheric pollution has been determined in three European cities—Prague (Czech Republic, two monitoring sites, Libuš and Smı́chov), Košice (Slovak Republic) and Sofia (Bulgaria) using the alkaline single-cell gel electrophoresis (the comet assay). The EOM samples were extracted by dichloromethane from ambient airborne particles collected daily (24 h intervals) during 3-month sampling periods in winter and summer seasons. The human metabolically competent cell line Hep G2 was used as a test system and benzo[a]pyrene (BaP), a known carcinogen, was applied as a positive control (internal standard) in each electrophoretic run. Two-hour exposure of Hep G2 cells to equivalent EOM concentrations ranging from 5 to 150 μg EOM/ml resulted in a linear dose-dependent increase of DNA migration (r > 0.9, P < 0.01). A less significant dose-response (r = 0.61) was only induced by the EOM sample from the locality Prague-Libuš (PRG-LB) in the winter. Generally, a 1.5 to four-fold increase of DNA strand breaks over the background control level was determined in EOM-exposed cells. In order to compare the genotoxic potential of individual EOMs, a mathematical model was used to correct the ‘real’ data. No substantial location- or season-related differences were found in EOM genotoxicity (EOM μg/ml), except for the EOM sample from Sofia, collected in the summer. This EOM sample induced a nearly two-fold lower level of DNA damage in comparison with other EOMs.On the other hand, clear statistically significant location- and season-related differences (P < 0.001) in ambient air genotoxicity were determined when the EOM quantity per cubic meter of air (μg/m3) was taken into account. In that case, the genotoxicity of winter air pollution was six- to 10-fold higher in comparison with summer air. The air pollution genotoxicity in individual localities rose during the winter season in the order: PRG-LB < Košice < Prague-Smı́chov (PRG-SM) < Sofia, while during the summer season the highest ambient air genotoxicity was revealed in the locality Prague-Smı́chov and approximately equal air pollution genotoxicity was determined among localities Prague-Libuš, Košice and Sofia (PRG-LB ∼ Košice ∼ Sofia < PRG-SM). The greatest overall air pollution genotoxicity was determined in the locality Sofia during the winter season.In a time course study to evaluate the kinetics of DNA strand break rejoining it was shown that the level of DNA strand breaks in EOM-exposed cells has returned to near the background level within 24 h after the treatment.

Mutat Res Dna Repair, 1998

After treatment with N-methyl-N -nitro-N-nitrosoguanidine MNNG , methyl methanesulfonate MMS and ... more After treatment with N-methyl-N -nitro-N-nitrosoguanidine MNNG , methyl methanesulfonate MMS and hydrogen Ž . peroxide, the level of alkali-labile sites and single-strand breaks ssb in DNA was investigated, using the comet assay. The Ž . ability of antioxidant pre-treatment to decrease DNA damage was assessed. Results showed the following. a All Ž . single-strand ss DNA breaks detected immediately after MNNG-and MMS-treatment in hamster V79 cells had the Ž . character of alkali-labile sites while true ssb of DNA were represented only as a minor statistically significant p -0.01 Ž . fraction at the highest MMS concentration. b Most ss DNA breaks detected immediately after H O -treatment had the 2 2 Ž . character of true breaks in DNA and alkali-labile sites represented only a minor fraction. c Pre-treatment of hamster V79 and human CaCo2 cells with vitamin E significantly reduced the number of breaks induced by hydrogen peroxide, but has no effect on the level of breaks induced by MNNG or MMS. We suggest that MNNG and MMS do not induce significant Ž . oxidative damage of DNA. Most of breaks induced by hydrogen peroxide have the nature of oxidative lesions of DNA. d Ž . In contrast to the effect of vitamin E, stobadine STB decreased not only the breaks induced by hydrogen peroxide but also those induced by MNNG and MMS. The reduced level of DNA damage in STB pre-treated samples could be due to inactivation of these alkylating agents by STB. q 1998 Elsevier Science B.V. All rights reserved.

Mutagenesis, Jan 22, 2016

Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial prod... more Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial products and biomedical applications. Despite this, the knowledge of human potential health risk as well as comprehensive biological and toxicological information is still limited. We have investigated the capacity of two frequently used metallic ENMs, nanosilver and magnetite nanoparticles (MNPs), to induce thymidine kinase (Tk (+/-)) mutations in L5178Y mouse lymphoma cells and transformed foci in Bhas 42 cells. Two types of nanosilver, spherical nanoparticles (AgNM300) and fibrous (AgNM302) nanorods/wires, and MNPs differing in surface modifications [MNPs coated with sodium oleate (SO-MNPs), MNPs coated with SO + polyethylene glycol (SO-PEG-MNPs) and MNPs coated with SO + PEG + poly(lactide-co-glycolic acid) SO-PEG-PLGA-MNPs] were included in this study. Spherical AgNM300 showed neither mutagenic nor carcinogenic potential. In contrast, silver nanorods/wires (AgNM302) increased significan...

Nanomaterials, 2022

Data suitable for assembling a physiologically-based pharmacokinetic (PBPK) model for nanoparticl... more Data suitable for assembling a physiologically-based pharmacokinetic (PBPK) model for nanoparticles (NPs) remain relatively scarce. Therefore, there is a trend in extrapolating the results of in vitro and in silico studies to in vivo nanoparticle hazard and risk assessment. To evaluate the reliability of such approach, a pharmacokinetic study was performed using the same polyethylene glycol-coated gold nanoparticles (PEG-AuNPs) in vitro and in vivo. As in vitro models, human cell lines TH1, A549, Hep G2, and 16HBE were employed. The in vivo PEG-AuNP biodistribution was assessed in rats. The internalization and exclusion of PEG-AuNPs in vitro were modeled as first-order rate processes with the partition coefficient describing the equilibrium distribution. The pharmacokinetic parameters were obtained by fitting the model to the in vitro data and subsequently used for PBPK simulation in vivo. Notable differences were observed in the internalized amount of Au in individual cell lines co...

Nanomaterials, 2021

Despite the obvious advantages of gold nanoparticles for biomedical applications, controversial a... more Despite the obvious advantages of gold nanoparticles for biomedical applications, controversial and incomplete toxicological data hamper their widespread use. Here, we present the results from an in vivo toxicity study using gold nanoparticles coated with polyethylene glycol (PEG-AuNPs). The pharmacokinetics and biodistribution of PEG-AuNPs were examined in the rat’s liver, lung, spleen, and kidney after a single i.v. injection (0.7 mg/kg) at different time intervals. PEG-AuNPs had a relatively long blood circulation time and accumulated primarily in the liver and spleen, where they remained for up to 28 days after administration. Increased cytoplasmic vacuolation in hepatocytes 24 h and 7 days after PEG-AuNPs exposure and apoptotic-like cells in white splenic pulp 24 h after administration has been detected, however, 28 days post-exposure were no longer observed. In contrast, at this time point, we identified significant changes in lipid metabolism, altered levels of liver injury m...

Beilstein Journal of Nanotechnology

The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired t... more The efficient entry of nanotechnology-based pharmaceuticals into target cells is highly desired to reach high therapeutic efficiency while minimizing the side effects. Despite intensive research, the impact of the surface coating on the mechanism of nanoparticle uptake is not sufficiently understood yet. Herein, we present a mechanistic study of cellular internalization pathways of two magnetic iron oxide nanoparticles (MNPs) differing in surface chemistry into A549 cells. The MNP uptake was investigated in the presence of different inhibitors of endocytosis and monitored by spectroscopic and imaging techniques. The results revealed that the route of MNP entry into cells strongly depends on the surface chemistry of the MNPs. While serum bovine albumin-coated MNPs entered the cells via clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CavME) or lipid rafts were preferentially involved in the internalization of polyethylene glycol-coated MNPs. Our data indicate that ...

Small

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

Nature Protocols

The comet assay is a widely used test for the detection of DNA damage and repair activity. Howeve... more The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easi...

Cancers

Epigenetic dysregulation has been recognized as a critical factor contributing to the development... more Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identif...

RSC Advances

Primary renal podocytes are more susceptible to MNPs exposure than primary renal mesangial cells.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety fo... more Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO 2 NPs, SiO 2 NPs, Fe 3 O 4 NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe 3 O 4 NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (> 700 μg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 μg/ml) concentration. Fe 3 O 4 NPs were most efficiently taken up while the lowest quantity of particles was determined in TiO 2 NPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Exposure to pesticides leads to complex, long-lasting adverse effects on human health, and poses ... more Exposure to pesticides leads to complex, long-lasting adverse effects on human health, and poses a substantial risk to those living in areas devoted to agriculture. Children are particularly vulnerable to the pesticide exposure, due to the developmental, dietary and physiological factors. Small body mass and typical exploratory behavior result in increased risk of intoxication. Thus, even exposure to low concentrations of pesticides, if of sufficient duration, may lead to permanent health disorders and limit their harmonious development. In this study 108 children, living in areas of an intense pesticide use and a control group (n = 92) of children from an agrotouristic area were investigated, whether DNA damage increased due to prolonged pesticide exposure. A presence of DNA breaks and oxidative damage to DNA bases, characterized as Fpg-sensitive sites, were detected by comet assay. Micronuclei (MN) formation was evaluated by cytokinesis-block MN assay. The exposure of children to pesticides resulted in increased number of MN in peripheral blood lymphocytes (P = 0.016), increased DNA strand breaks level (P = 0.002) and oxidative damage to DNA (P < 0.001). Negative correlation was demonstrated between the level of DNA strand breaks and acetylcholinesterase (AChE) activity in exposed group. In conclusion, despite just environmental pesticide exposure in the test group of children, significant biological effects were detected.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Drug-induced kidney injury is one of the most significant adverse events and dose limiting factor... more Drug-induced kidney injury is one of the most significant adverse events and dose limiting factor in chemotherapy as well a major cause of prospective drug attrition during pharmaceutical development. Moreover, kidney injury can also occur as a consequence of exposures to environmental xenobiotics such as heavy metals, fungal toxins and nanomaterials. The lack of adequate in vitro human kidney models that mimic more realistically the in vivo conditions and the absence of suitable and robust, cost-effective and predictive cell-based in vitro assays contribute to an underestimation of the kidney toxic potential of new drugs and xenobiotics. Therefore, a rapid screening system capable to detect potential nephrotoxicity at early stages of drug discovery is an urgent need. Here we provide an overview of human cell lines currently used as a surrogate in vitro kidney models in nephrotoxicity studies, including their advantages and limitations. In addition, the capacity of the single cell gel electrophoresis (SCGE)/comet assay as a potential tool in kidney toxicants screening is discussed. Despite a limited number of studies using the comet assay to evaluate the drug-induced kidney damage potential, a considerable variability in SCGE methodology (e.g. lysis, unwinding, and electrophoresis conditions) has been observed. Before the comet assay can be included in nephrotoxicity testing, a basic guideline has to be developed. To test its feasibility, additional in vitro experiments including inter-laboratory validation studies based on this guideline have to be performed.

Journal of Magnetism and Magnetic Materials

Abstract Magnetic iron oxide nanoparticles (MNPs) are one of the most promising types of nanopart... more Abstract Magnetic iron oxide nanoparticles (MNPs) are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy. They are used as contrast agents in magnetic resonance imaging and magnetite cell labelling. Furthermore, they are promising heating mediator in magnetic hyperthermia-based therapy, and can serve as nanocarriers in targeted gene and drug delivery as well. In biomedical applications, coating plays an important role in nanoparticle dispersion stability and biocompatibility. However, the impact of nanoparticle surface chemistry on cell uptake and proliferation has not been sufficiently investigated. The objective of this study is to prepare magnetic nanoparticles with inner magnetite core and hydrophilic outer shell of surfactant, protein and polymers that are commonly used in biomedical research. MNPs were characterized in-depth by various physicochemical methods. Magnetic hyperthermia, applied to find out the influence of MNPs coating on heating characteristics of the samples, did not show any correlation between layer thickness and specific adsorption rate. To evaluate the impact of surface chemistry on cell proliferation and internalization, the human lung adenocarcinoma epithelial (A549) cells were utilized. Substantial differences were determined in the amount of internalized MNPs and cell viability in dependence on surface coating. Our results indicate that the surface chemistry not only protects particles from agglomeration but also affect the interaction between cell and MNPs.

The present pilot study was designed to elucidate the functional significance of amino acid subst... more The present pilot study was designed to elucidate the functional significance of amino acid substitution variants of DNA repair genes. Using the peripheral blood lymphocytes (PBLs) from healthy donors and cervical cancer patients, the contribution of four non-synonymous single nucleotide polymorphisms (SNPs) in three base excision repair genes (BER), XRCC1 (Arg194Trp and Arg399Gln), hOGG1 (Ser326Cys), and APE1 (Asp148Glu), to the susceptibility to ionizing radiation were evaluated. The level of initial, oxidative and residual DNA damage produced by 2 Gy was measured by the alkaline single cell gel electrophoresis (the comet assay), and the SNPs were determined by PCR-restriction fragment length polymorphism (RFLP) assay. No significant differences in the allele frequencies between cancer patients and controls for any of these four SNPs were detected. Although the initial DNA damage levels were approximately similar, significantly higher level of Fpg-sensitive sites were found in patients compared with controls (p<0.001) irrespective of genotype distribution. A trend towards increased values of EndoIII-sensitive sites was determined in PBLs from cancer patients compared with healthy women, mainly carriers of the XRCC1 and OGG1 variant alleles; however, the mean value of EndoIII-sensitive sites does not reach any significance. A substantial delay in DNA strand-break rejoining was ascertained in patients who carried APE1 Glu variant allele in comparison with healthy donors 15 and 60 minutes after irradiation (p< 0.05 and p< 0.01, respectively). In contrast, slightly higher but statistically significant level of residual DNA damage was estimated in controls (APE1Asp/Asp) compared with patients. An association between single nucleotide polymorphism (SNP) of two DNA repair genes functioning in the same biochemical pathway and susceptibility to radiation was found. In the combined genotype APE1/XRCC1 and APE1/hOGG1, a decreased level of residual DNA damage was detected in carriers of wild type APE1 genotype. In addition, a possible modulating effect of hOGG1 gene on the kinetics of strand-break rejoining was estimated. The lowest residual DNA damage level was determined in subjects with the combined APE1(Asp/Asp)/hOGG1(Ser/Cys+Cys/Cys) genotypes. Based on these preliminary data we suppose that a combination of several amino acid substitution variants of DNA repair genes involved in the same repair pathway rather than one low-penetrance SNP in a single gene may contribute to DNA repair outcomes. Larger study with more subjects is needed to verify these findings.

Cell Biology and Toxicology

The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in dia... more The unique physicochemical properties make inorganic nanoparticles (INPs) an exciting tool in diagnosis and disease management. However, as INPs are relatively difficult to fully degrade and excrete, their unintended accumulation in the tissue might result in adverse health effects. Herein, we provide a methylome–transcriptome framework for chronic effects of INPs, commonly used in biomedical applications, in human kidney TH-1 cells. Renal clearance is one of the most important routes of nanoparticle excretion; therefore, a detailed evaluation of nanoparticle-mediated nephrotoxicity is an important task. Integrated analysis of methylome and transcriptome changes induced by INPs (PEG-AuNPs, Fe3O4NPs, SiO2NPs, and TiO2NPs) revealed significantly deregulated genes with functional classification in immune response, DNA damage, and cancer-related pathways. Although most deregulated genes were unique to individual INPs, a relatively high proportion of them encoded the transcription factor...

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2009

Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our r... more Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole (N-MeDBC), induced several cellular events associated with tumor promotion in WB-F344 cells, an in vitro model of liver oval cells [J.

Materials Science and Engineering: C, 2017

Comprehensive characterization of nanoparticles associated with investigation of their cellular u... more Comprehensive characterization of nanoparticles associated with investigation of their cellular uptake creates the basis on which fundamental in vitro and in vivo studies can be built. In this work, a complex analysis of various surface-modified magnetite nanoparticles in biologically relevant environment is reported and the promotion of incorrect characterization into the results obtained from model biological experiments leading to false conclusions is demonstrated. Via a bottom-up approach from particle characterization by DLS towards interpretation of biological data based on cellular uptake, this work draws attention to the systematic propagation of errors stemming from inaccurate determination of input parameters for DLS, improper selection of particle size distribution, inadequate sampling, unknown colloidal behavior and the omission of fraction of particles complying with the internalization threshold. In addition, cellular uptake depending on the number of treated cells is shown. The definition of cellular uptake efficacy reflecting the size distribution of particles beside their absolute internalization is postulated.

Mutation Research Environmental Mutagenesis and Related Subjects, 1981

Mutat Res Genet Toxicol E M, 2004

The genotoxic potential of extractable organic matter (EOM) associated with the respirable partic... more The genotoxic potential of extractable organic matter (EOM) associated with the respirable particulate matter (PM <10 μm) of atmospheric pollution has been determined in three European cities—Prague (Czech Republic, two monitoring sites, Libuš and Smı́chov), Košice (Slovak Republic) and Sofia (Bulgaria) using the alkaline single-cell gel electrophoresis (the comet assay). The EOM samples were extracted by dichloromethane from ambient airborne particles collected daily (24 h intervals) during 3-month sampling periods in winter and summer seasons. The human metabolically competent cell line Hep G2 was used as a test system and benzo[a]pyrene (BaP), a known carcinogen, was applied as a positive control (internal standard) in each electrophoretic run. Two-hour exposure of Hep G2 cells to equivalent EOM concentrations ranging from 5 to 150 μg EOM/ml resulted in a linear dose-dependent increase of DNA migration (r > 0.9, P < 0.01). A less significant dose-response (r = 0.61) was only induced by the EOM sample from the locality Prague-Libuš (PRG-LB) in the winter. Generally, a 1.5 to four-fold increase of DNA strand breaks over the background control level was determined in EOM-exposed cells. In order to compare the genotoxic potential of individual EOMs, a mathematical model was used to correct the ‘real’ data. No substantial location- or season-related differences were found in EOM genotoxicity (EOM μg/ml), except for the EOM sample from Sofia, collected in the summer. This EOM sample induced a nearly two-fold lower level of DNA damage in comparison with other EOMs.On the other hand, clear statistically significant location- and season-related differences (P < 0.001) in ambient air genotoxicity were determined when the EOM quantity per cubic meter of air (μg/m3) was taken into account. In that case, the genotoxicity of winter air pollution was six- to 10-fold higher in comparison with summer air. The air pollution genotoxicity in individual localities rose during the winter season in the order: PRG-LB < Košice < Prague-Smı́chov (PRG-SM) < Sofia, while during the summer season the highest ambient air genotoxicity was revealed in the locality Prague-Smı́chov and approximately equal air pollution genotoxicity was determined among localities Prague-Libuš, Košice and Sofia (PRG-LB ∼ Košice ∼ Sofia < PRG-SM). The greatest overall air pollution genotoxicity was determined in the locality Sofia during the winter season.In a time course study to evaluate the kinetics of DNA strand break rejoining it was shown that the level of DNA strand breaks in EOM-exposed cells has returned to near the background level within 24 h after the treatment.

Mutat Res Dna Repair, 1998

After treatment with N-methyl-N -nitro-N-nitrosoguanidine MNNG , methyl methanesulfonate MMS and ... more After treatment with N-methyl-N -nitro-N-nitrosoguanidine MNNG , methyl methanesulfonate MMS and hydrogen Ž . peroxide, the level of alkali-labile sites and single-strand breaks ssb in DNA was investigated, using the comet assay. The Ž . ability of antioxidant pre-treatment to decrease DNA damage was assessed. Results showed the following. a All Ž . single-strand ss DNA breaks detected immediately after MNNG-and MMS-treatment in hamster V79 cells had the Ž . character of alkali-labile sites while true ssb of DNA were represented only as a minor statistically significant p -0.01 Ž . fraction at the highest MMS concentration. b Most ss DNA breaks detected immediately after H O -treatment had the 2 2 Ž . character of true breaks in DNA and alkali-labile sites represented only a minor fraction. c Pre-treatment of hamster V79 and human CaCo2 cells with vitamin E significantly reduced the number of breaks induced by hydrogen peroxide, but has no effect on the level of breaks induced by MNNG or MMS. We suggest that MNNG and MMS do not induce significant Ž . oxidative damage of DNA. Most of breaks induced by hydrogen peroxide have the nature of oxidative lesions of DNA. d Ž . In contrast to the effect of vitamin E, stobadine STB decreased not only the breaks induced by hydrogen peroxide but also those induced by MNNG and MMS. The reduced level of DNA damage in STB pre-treated samples could be due to inactivation of these alkylating agents by STB. q 1998 Elsevier Science B.V. All rights reserved.