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.

• MeSH
• Deoxyribonuclease (Pyrimidine Dimer) MeSH
• DNA-Formamidopyrimidine Glycosylase MeSH
• DNA drug effects   metabolism   MeSH
• Adult MeSH
• Comet Assay MeSH
• Leukocytes drug effects   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Mutagens MeSH
• Nanocomposites toxicity   MeSH
• Oxidative Stress MeSH
• DNA Damage * MeSH
• Occupational Exposure adverse effects   MeSH
• Escherichia coli Proteins MeSH
• Sex Factors MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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 m3 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.

• MeSH
• Gasoline analysis   toxicity   MeSH
• Biofuels analysis   toxicity   MeSH
• A549 Cells MeSH
• Chemical Fractionation methods   MeSH
• Carcinogens, Environmental analysis   toxicity   MeSH
• Comet Assay MeSH
• Humans MeSH
• Oxidation-Reduction MeSH
• Particulate Matter toxicity   MeSH
• Pilot Projects MeSH
• Polycyclic Aromatic Hydrocarbons isolation & purification   toxicity   MeSH
• DNA Damage MeSH
• Solvents MeSH
• Volatile Organic Compounds isolation & purification   toxicity   MeSH
• Cell Survival drug effects   MeSH
• Vehicle Emissions analysis   toxicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

INTRODUCTION: Rat mesenchymal stem cells (rMSCs) labeled with 1) poly-l-lysine-coated superparamagnetic iron oxide nanoparticles or 2) silica-coated cobalt-zinc-iron nanoparticles were implanted into the left brain hemisphere of rats, to assess their effects on the levels of oxidative damage to biological macromolecules in brain tissue. METHODS: Controls were implanted with unlabeled rMSCs. Animals were sacrificed 24 hours or 4 weeks after the treatment, and the implantation site along with the surrounding tissue was isolated from the brain. At the same intervals, parallel groups of animals were scanned in vivo by magnetic resonance imaging (MRI). The comet assay with enzymes of excision DNA repair (endonuclease III and formamidopyrimidine-DNA glycosylase) was used to analyze breaks and oxidative damage to DNA in the brain tissue. Oxidative damage to proteins and lipids was determined by measuring the levels of carbonyl groups and 15-F2t-isoprostane (enzyme-linked immunosorbent assay). MRI displayed implants of labeled cells as extensive hypointense areas in the brain tissue. In histological sections, the expression of glial fibrillary acidic protein and CD68 was analyzed to detect astrogliosis and inflammatory response. RESULTS: Both contrast labels caused a similar response in the T2-weighted magnetic resonance (MR) image and the signal was clearly visible within 4 weeks after implantation of rMSCs. No increase of oxidative damage to DNA, lipids, or proteins over the control values was detected in any sample of brain tissue from the treated animals. Also, immunohistochemistry did not indicate any serious tissue impairment around the graft. CONCLUSION: Both tested types of nanoparticles appear to be prospective and safe labels for tracking the transplanted cells by MR.

• MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Isoprostanes analysis   metabolism   MeSH
• Cobalt chemistry   MeSH
• Metal Nanoparticles administration & dosage   chemistry   toxicity   MeSH
• Magnetic Resonance Imaging methods   MeSH
• Mesenchymal Stem Cells chemistry   MeSH
• Brain diagnostic imaging   drug effects   metabolism   MeSH
• Silicon Dioxide chemistry   MeSH
• Rats, Inbred Lew MeSH
• Prospective Studies MeSH
• Tissue Extracts MeSH
• Mesenchymal Stem Cell Transplantation * MeSH
• Ferric Compounds chemistry   MeSH
• Iron chemistry   MeSH
• Zinc chemistry   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Nanomaterials are currently the subject of intense research due to their wide variety of potential applications in the biomedical, optical and electronic fields. We prepared and tested cobalt zinc ferrite nanoparticles (Co0.5Zn0.5Fe2O4+γ [CZF-NPs]) encapsulated by amorphous silica in order to find a safe contrast agent and magnetic label for tracking transplanted cells within an organism using magnetic resonance imaging (MRI). Rat mesenchymal stem cells (rMSCs) were labeled for 48 h with a low, medium or high dose of CZF-NPs (0.05; 0.11 or 0.55 mM); silica NPs (Si-NPs; 0.11 mM) served as a positive control. The internalization of NPs into cells was verified by transmission electron microscopy. Biological effects were analyzed at the end of exposure and after an additional 72 h of cell growth without NPs. Compared to untreated cells, Annexin V/Propidium Iodide labeling revealed no significant cytotoxicity for any group of treated cells and only a high dose of CZF-NPs slowed down cell proliferation and induced DNA damage, manifested as a significant increase of DNA-strand breaks and oxidized DNA bases. This was accompanied by high concentrations of 15-F2t-isoprostane and carbonyl groups, demonstrating oxidative injury to lipids and proteins, respectively. No harmful effects were detected in cells exposed to the low dose of CZF-NPs. Nevertheless, the labeled cells still exhibited an adequate relaxation rate for MRI in repeated experiments and ICP-MS confirmed sufficient magnetic label concentrations inside the cells. The results suggest that the silica-coated CZF-NPs, when applied at a non-toxic dose, represent a promising contrast agent for cell labeling.

• MeSH
• Staining and Labeling MeSH
• Cell Culture Techniques MeSH
• Isoprostanes metabolism   MeSH
• Protein Carbonylation drug effects   MeSH
• Cobalt chemistry   toxicity   MeSH
• Contrast Media chemistry   toxicity   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Magnetic Resonance Imaging MeSH
• Lipid Metabolism drug effects   MeSH
• Mesenchymal Stem Cells drug effects   metabolism   ultrastructure   MeSH
• Nanoparticles chemistry   toxicity   MeSH
• Silicon Dioxide chemistry   toxicity   MeSH
• DNA Damage * MeSH
• Surface Properties MeSH
• Cell Proliferation drug effects   MeSH
• Zinc Compounds chemistry   toxicity   MeSH
• Microscopy, Electron, Transmission MeSH
• Cell Survival drug effects   MeSH
• Dose-Response Relationship, Drug MeSH
• Ferric Compounds chemistry   toxicity   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Hlavní stanovisko práce: Na základě literárních údajů autoři upozorňují na význam hodnocení integrity DNA ve spermiích při vyšetřování příčin neplodnosti. Informace je doplněna vlastními experimentálními výsledky získanými kometovým testem a komerčním kitem Halosperm. Cíl: Abnormality v uspořádání chromatinu a/ nebo struktuře DNA spermií mohou výrazně sni- žovat fertilizační potenciál jedince i při normálním spermiogramu. Při identifikaci příčin mužské neplodnosti je proto v posledních letech kladen důraz i na hodnocení integrity DNA ve spermiích. Na našem pracovišti jsme se rozhodli k těmto účelům použít kometový test a komerčně dostupný test Halosperm s cílem porovnat obě metody při vyšetřování kontrolních jedinců a pacientů s poruchami plodnosti. Soubor pacientů a metody: U 32 kontrolních jedinců, 24 mužů s neplodností a 26 mužů s cévními poruchami mužských pohlavních orgánů (MPO) byla provedena konvenční analýza semene a následně vyšetřeny hladiny fragmentace DNA ve spermiích pomocí kometového test a Halospermu. Výsledky: Korelační analýza potvrdila dobrou shodu mezi výsledky obou metod (Pearsonův korelační keficient r=0,241; p<0,05), na rozdíl od kometového testu však výsledky Halospermu korelovaly i s motilitou (r=0,4365; p<0,001). Naproti tomu při hodnocení kometovým testem obě skupiny pacientů vykazovaly výrazně vyšší poškození DNA ve spermiích než kontroly (t-test, v obou případech p<0,001), zatímco s pomocí Halospermu se podařilo prokázat zvýšení hladin fragmentované DNA oproti kontrolám pouze u skupiny mužů s neplodností (p<0,001). Z hodnot kometového testu v kontrolním souboru byla stanovena hladina poškození 32 % Tail DNA jako horní mez tolerančního intervalu indikující snížený fertilizační potenciál. Závěr: Kometový test se osvědčil jako vhodná doplňková metoda ke standardnímu vyšetření kvality spermatu.

Major statement: Based on published data, the authors highlight the importance of assessment of sperm DNA integrity in the investigation of causes of infertility. Information is complemented by our own experimental results obtained by comet assay and commercial kit Halosperm. Aim: Abnormalities in chromatin arrangement and/or structure of the sperm DNA can significantly reduce the fertilizing potential of individuals, even in cases with normal results of conventional sperm analysis. Therefore, evaluation of sperm DNA integrity is currently emphasized in identifying the causes of male infertility. For this purpose, we decided to use comet assay and a commercially available test Halosperm with the aim to compare the two methods in the investigation of control subjects and patients with fertility disorders. Patients and Methods: Conventional semen analysis was performed in 32 control subjects, 24 men with infertility and 26 men with vascular disorders of male genital organs, and then the levels of DNA fragmentation in the sperm were examined using the comet assay and Halosperm. Results: Correlation analysis confirmed good agreement between the results of both methods (Pearson coeficient r=0.241; p< 0.05); unlike the comet assay, the results of Halosperm correlated well with sperm motility (r=0.4365; p<0.001). While comet assay showed significantly higher sperm DNA damage in both groups of patients than in the controls (t-test, both p<0.001), Halosperm demonstrated increased levels of fragmented DNA over control values only in the group of men with infertility (p<0.001). From the results of comet assay in the control group, 32% of fragmented DNA per sperm was determined as the upper limit of the tolerance interval indicating a reduced fertilizing potential. Conclusion: The comet assay has proven to be a useful complementary method to the standard examination of sperm quality

• MeSH
• Chromatin pathology   MeSH
• DNA Fragmentation MeSH
• Comet Assay MeSH
• Humans MeSH
• Infertility, Male * physiopathology   MeSH
• Spermatozoa * abnormalities   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

The biological effects of several superparamagnetic iron oxide nanoparticles (SPIONs) varying in their surface coating were tested using human bone marrow mesenchymal stromal cells from two donors - hBMSCs-1 and hBMSCs-2. The measurements were performed at two intervals - after 72 h exposure to the nanoparticles and after an additional 72 h cell growth without nanoparticles. The dose of SPIONs used (15.4 μg Fe/ml) was selected as being sufficient for in vivo cell tracking using magnetic resonance imaging (MRI). Concerning cell viability and cell death, only the hBMSCs-2 seemed to be sensitive to the action of SPIONs. However, an increase of oxidative injury to lipids, proteins and DNA as a consequence of exposure to SPIONs was detected in cells from both donors. Particularly the levels of lipid peroxidation were high and increased further with time, regardless of the type of nanoparticle. Lowering intracellular label concentrations and authenticating oxidative stress levels using in vivo experiments are required to ensure the safety of SPIONs for biomedical applications.

• MeSH
• Cell Death drug effects   MeSH
• Child MeSH
• Middle Aged MeSH
• Humans MeSH
• Magnetic Resonance Imaging MeSH
• Magnetite Nanoparticles adverse effects   MeSH
• Mesenchymal Stem Cells drug effects   MeSH
• Oxidative Stress drug effects   MeSH
• Lipid Peroxidation drug effects   MeSH
• DNA Damage drug effects   MeSH
• Proteins drug effects   MeSH
• Cell Survival drug effects   MeSH
• Ferric Compounds adverse effects   MeSH
• Check Tag
• Child MeSH
• Middle Aged MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The effect of different activation protocols on DNA integrity of porcine oocytes matured in vitro was analysed using the comet assay. The oocytes from ovaries of slaughtered gilts were cultured for 48 h in modified M199 medium. They were then freed of cumulus cells and treated continuously or intermittently with a nitric oxide (NO) donor for 6 h. Standard activation with calcium ions (Ca2+) and culture without any treatment served as positive and negative controls, respectively. The activation was assessed according to the formation of pronuclei. Exposure of oocytes to Ca2+ was associated with high activation efficiency, but decreased DNA integrity. The opposite, i.e. low activation efficiency but high DNA integrity was observed after continuous exposure to NO. Intermittent action of NO increased the activation rate, while the values of DNA damage remained at low levels. Our data suggest that an increased DNA instability could be the main reason compromising the further embryonic development of oocytes activated by the standard protocol. The intermittent treatment with NO thus represents a promising step to optimization of parthenogenetic activation of pig oocytes.

• MeSH
• Cell Differentiation MeSH
• DNA genetics   metabolism   MeSH
• Cells, Cultured MeSH
• Oocytes cytology   metabolism   MeSH
• DNA Damage MeSH
• Swine MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bone marrow aspirates of 19 patients with low-risk myelodysplastic syndromes (MDS) and 14 control subjects were collected in order to assess the level of oxidative DNA damage. Glycophorin A positive and negative cells separated by miniMACS magnetic cell sorting were subjected to single cell gel electrophoresis (comet assay) combined with enzymes of base excision repair (endonuclease III and formamido-pyrimidine-glycosylase) that specifically recognize oxidized nucleotides. Compared to controls, MDS patients exhibited a significant increase of oxidative damage to DNA which could contribute to genomic instability and disease progression.

• MeSH
• Bone Marrow Cells pathology   MeSH
• Financing, Organized MeSH
• Comet Assay MeSH
• Middle Aged MeSH
• Humans MeSH
• Myelodysplastic Syndromes pathology   MeSH
• Oxidative Stress MeSH
• DNA Damage MeSH
• Anemia, Refractory, with Excess of Blasts MeSH
• Anemia, Refractory MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Case-Control Studies MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH

We tested genomic instability in patients with myelodysplastic syndrome (MDS) by the comet assay and verified the suitability of this approach as a tool for analysis of ineffective hematopoiesis in refractory anemia (RA) and RA with ring sideroblasts (RARS). Erythroid and myeloid cell populations from bone marrow aspirates of 20 RA, 14 RARS and 15 control subjects were separated by differential expression of glycophorin A and subjected to comet assay. The extent of DNA migration was measured in single cells (200 cells/bone marrow fraction/subject). The results were in agreement with the concept of increased apoptosis in low-risk MDS subtypes. The RA samples had a significantly higher DNA instability than controls in glycophorin A positive cells, and the extent of DNA breakage correlated with the degree of cytopenia. Although RARS had an even higher rate of genomic instability in bone marrow cells than RA, there was no clear relationship to peripheral cytopenia. This suggests an additional DNA instability of non-apoptotic origin. Whether this increase is associated with an increased repair of oxidative damage in DNA arising due to iron deposits in ring sideroblasts remains to be formally proven. Comet assay provides a promising tool for the investigation of difference between RA and RARS pathobiology.

• MeSH
• DNA analysis   genetics   MeSH
• Adult MeSH
• Financing, Organized MeSH
• Hematopoietic Stem Cells cytology   chemistry   MeSH
• Comet Assay methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• Genomic Instability MeSH
• DNA Damage MeSH
• Anemia, Refractory genetics   physiopathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Comparative Study MeSH

DNA integrity was analyzed in the lymphocytes of 65 non-smoking city policemen during January and September 2004 using the comet assay combined with excision repair enzymes. Information about inhalation exposure was obtained by (1) stationary monitoring of PM2.5 and carcinogenic polycyclic aromatic hydrocarbons (cPAHs) during the sampling periods and (2) personal exposure monitoring of cPAHs 48h before blood sampling. The data were completed by a lifestyle questionnaire. Regardless of the season of the year, policemen working outdoors (exposed group) exhibited higher levels of DNA damage than those working indoors (controls). Within the exposed group, the levels of both unspecified and oxidative DNA damage detected in January significantly exceeded those found in September. The controls did not show analogous inter-seasonal variability. The winter levels of oxidative DNA damage positively correlated with exposure to cPAHs, probably reflecting increased oxidative stress as a result of high concentrations of PM2.5. In comparison with the wild type genotype, the carriers of at least one mutated allele, CYP1A1*2C (Ile/Val), MTHFR 2656 or MS 2656, and the EPHX1-medium phenotype appeared to be more susceptible specifically to the induction of oxidative DNA damage, while the p53 MspI mutation predisposed the carrier to a higher incidence of both breaks and oxidative lesions in DNA. In contrast, GSTM1-null and vitamin C tended rather to protect DNA integrity.

• MeSH
• Cytochrome P-450 CYP1A1 genetics   MeSH
• Adult MeSH
• Epoxide Hydrolases genetics   MeSH
• Financing, Organized MeSH
• Genotype MeSH
• Carcinogens, Environmental analysis   adverse effects   MeSH
• Comet Assay MeSH
• Humans MeSH
• Lymphocytes drug effects   MeSH
• Methylenetetrahydrofolate Reductase (NADPH2) genetics   MeSH
• Environmental Monitoring methods   MeSH
• Oxidative Stress drug effects   MeSH
• Particulate Matter analysis   adverse effects   MeSH
• Police MeSH
• Polycyclic Aromatic Hydrocarbons analysis   adverse effects   MeSH
• Polymorphism, Genetic MeSH
• DNA Damage MeSH
• Occupational Exposure MeSH
• Surveys and Questionnaires MeSH
• Seasons MeSH
• Case-Control Studies MeSH
• Check Tag
• Adult MeSH
• Humans MeSH