One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a ...relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.
Abstract
Applying validated in vitro assays to the study of nanoparticle toxicity is a growing trend in nanomaterial risk assessment. Precise characterisation of reference nanomaterials and a ...well-regulated in vitro testing system are required to determine the physicochemical descriptors which dictate the toxic potential of nanoparticles. The use of automated, high-throughput technologies to facilitate the identification and prioritisation of nanomaterials which could pose a risk is desirable and developments are underway. In this study, two mammalian fibroblast lines (Balb/c 3T3 and COS-1 cells) were treated with a range of concentrations of iron oxide nanomaterials manufactured for use in medical diagnostics, using an automated platform and high-content-imaging endpoints for cell viability, oxidative stress and DNA damage (double-strand breaks). At the same time, the high-throughput comet assay was employed to measure DNA strand breaks and oxidised bases. Our results show that these methods provide a fast way to determine the toxicity of coated and uncoated iron oxide nanoparticles and, furthermore, to predict the mechanism of toxicity in vitro.
► We performed a quantitative analysis of Gentiana asclepiadea aqueous and methanolic extracts. ► Mangiferin and an unidentified polar compound are the most pronounced antioxidants. ► Extract from ...haulm exhibited stronger antioxidant properties compared with flower extracts. ► Flower and haulm extracts enhanced repair of H2O2-induced DNA damage in human lymphocytes. ► Repair of DNA damage induced by nanosilver was stimulated by plant extracts in HEK 293 cells.
Exposure to high levels of different environmental pollutants is known to be associated with induction of DNA damage in humans. Thus DNA repair is of great importance in preventing mutations and contributes crucially to the prevention of cancer. In our study we have focused on quantitative analysis of Gentiana asclepiadea aqueous or methanolic extracts obtained from flower and haulm, their antioxidant potency in ABTS post-column derivatisation, and their potential ability to enhance DNA repair in human lymphocytes after hydrogen peroxide (H2O2) treatment (250μM, 5min). We also studied DNA repair in human kidney HEK 293 cells after exposure to 20nm silver nanoparticles (AgNPs) (100μg/ml, 30min) in the presence and absence of the plant extract. We have found that mangiferin along with unidentified polar compounds are the most pronounced antioxidants in the studied extracts. Extract from haulm exhibited slightly stronger antioxidant properties compared to flower extracts. However, all four extracts showed significant ability to enhance DNA repair in both cell types after H2O2 and AgNP treatments.
Among nanomaterials, silver nanoparticles (AgNPs) have the broadest and most commercial applications due to their antibacterial properties, highlighting the need for exploring their potential ...toxicity and underlying mechanisms of action. Our main aim was to investigate whether AgNPs exert toxicity by inducing oxidative damage to DNA in human kidney HEK 293 cells. In addition, we tested whether this damage could be counteracted by plant extracts containing phytochemicals such as swertiamarin, mangiferin and homoorientin with high antioxidant abilities. We show that AgNPs (20 nm) are taken up by cells and localised in vacuoles and cytoplasm. Exposure to 1, 25 or 100 µg/ml AgNPs leads to a significant dose-dependent increase in oxidised DNA base lesions (8-oxo-7,8-dihydroguanine or 8-oxoG) detected by the comet assay after incubation of nucleoids with 8-oxoG DNA glycosylase. Oxidised DNA base lesions and strand breaks caused by AgNPs were diminished by aqueous and methanolic extracts from both haulm and flower of Gentiana asclepiadea.
Abstract
Engineered nanoparticles (NPs) are widely used in different technologies but their unique properties might also cause adverse health effects. In reviewing recent in vitro and in vivo ...genotoxicity studies we discuss potential mechanisms of genotoxicity induced by NPs. Various factors that may influence genotoxic response, including physico-chemical properties and experimental conditions, are highlighted. From 4346 articles on NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in vivo). The most used assays are the comet assay (58 in vitro, 9 in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the chromosome aberrations test (10 in vitro, 1 in vivo) and the bacterial reverse mutation assay (13 studies). We describe advantages and potential problems with different methods and suggest the need for appropriate methodologies to be used for investigation of genotoxic effects of NPs, in vitro and in vivo.
► Novel repair protein 3-methyladenine DNA glycosylase (AlkD) was applied for detection of alkylation lesions specifically 3-methyladenine and 7-methylguanine using the comet assay. ► Modification of ...the comet assay with AlkD further enlarges its applications as biomarker of biologically relevant doses and repair of alkylation damage.
3-Methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and
in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60
min. The
in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60
min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15
min of incubation, with maximum detection of lesions after 60
min’ incubation. Additionally, we tested the
in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for
in vitro and
in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.
Experiments were conducted to determine the validity of two common genotoxicity testing procedures, the comet assay and the micronucleus (MN) test, when applied to nanoparticles (NP). The comet assay ...is used to detect strand breaks (SB) induced in cellular DNA. There is a possibility of obtaining false positive results, if residual NP remain in proximity to the virtually naked DNA that results from lysis of agarose-embedded cells, and react with this DNA in ways that do not occur with chromatin in intact cells. However, data showed that if NP are deliberately present at high concentration with lysed cells, there is no change in SB with a range of NP. Only oleic acid-coated Fe
3
O
4
NP induced damage, as these particles also produced equivalent alterations in whole cells. A modification of the comet assay incorporates digestion of DNA with lesion-specific endonucleases, notably formamidopyrimidine DNA glycosylase (FPG), which detects oxidized purines. Again there is a concern regarding the presence of residual NP with DNA of lysed cells, but this time because of the risk of false negative results if NP interfere with the FPG reaction. However, it was found that incubation of cells with NP before treatment with a known 8-oxoguanine-inducing agent does not lead to any decrease in the yield of FPG-sensitive sites. Chromosomal damage is detected with the MN assay, which depends on the use of cytochalasin B (CB) to prevent cell division and accumulates binucleate cells. It is known that CB also inhibits endocytosis, and thus might prevent NP uptake. Data demonstrated that if NP are added to cells together with CB, fewer MN are induced. It is therefore necessary to treat cells with NP prior to CB in order to avoid interference and possible false negative results.
Large quantities of engineered nanoparticles (NP), such as nanosilver (AgNP), have been widely applied, leading to an increased exposure and potential health concerns. Herein, we have examined the ...ability of AgNP to induce reactive oxygen species (ROS), their role in genotoxic effects and the involvement of mitogen-activated protein kinases (MAPK). AgNP exposure induced ROS production in human epithelial embryonic cells which could be decreased by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation, induced by AgNP, was an early response but not sustained in time. Furthermore, JNK and ERK activation could be inhibited by both DPI and a free radicals scavenger N-acetyl cysteine. We also investigated the role of MAPK in the DNA damage. Using a modified comet assay for the specific detection of hOGG1 sensitive sites, we showed that AgNP induced DNA oxidation after 30-min treatment, whereas no response was observed after 2h. In conclusion, AgNP seem to induce DNA damage via a mechanism involving ROS formation. The oxidative DNA damage observed was transient, likely due to DNA repair; furthermore, higher damage was achieved upon inhibition of ERK activation by pre-treatment with U0126, suggesting a role for ERK in DNA damage repair. Activation of different MAPK might play an important role in the NP toxicity outcomes; understanding this process may be helpful for the identification of NP toxicity.
Abstract
Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of ...medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide PLGA-PEO, TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, 3H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.
The purpose of this study was to assess whether a methanol extract isolated from the flower of Gentiana asclepiadea had potential cytotoxic or genotoxic effect on COS 1 (monkey kidney) cell line. ...Five various concentrations of the extract were investigated for cytotoxicity and genotoxicity and to determine non-cytotoxic and non-genotoxic concentrations suitable for utilization in pharmacology and medicine.
Cytotoxicity was determined using the proliferation (growth activity) and the plating efficiency (colony forming ability) assays after 24 hour incubation of COS 1 cells with different concentrations of methanolic flower extract from G. asclepiadea. To assess potential genotoxicity, the comet assay or SCGE (Single-Cell Gel Electrophoresis) was used.
We found that only the highest (5 and 25 mg/ml) concentrations of the extract revealed cytotoxic and genotoxic effect. We have also determined concentrations that stimulated cell growth (0.25 mg/ml) and colony forming ability (0.25-2.5 mg/ml) and did not exhibit genotoxic effect (0.25-2.5 mg/ml).
We found out that extract of G. asclepiadea was neither cytotoxic nor genotoxic in a wide range of concentrations (0.25-2.5 mg/ml) and thus can be used to further investigate potential beneficial usage in pharmacology and medicine.
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