Genotoxicity Of Nanoparticles Research Articles

Biosynthesis, characterization, toxicity assessment and bio-efficacy of silver nanoparticles synthesized by Microbacterium mitrae in controlling early blight in tomato (Lycopersicon esculentum L.)

This study evaluated the biogenesis of silver nanoparticles (NPs) using Microbacterium mitrae and its possibilities against phytopathogens. Silver nanoparticles have been synthesized using a simple protocol and the synthesized NPs have been characterized using UV visible spectroscopy, particle size analyzer, transmission electron microscope (TEM). Small-sized, stable silver nanoparticles in the range of 10-25 nm have been reported. Their antimicrobial activity against three phytopathogens i.e. Alternaria solani, Thanatephorus cucumeris and Botryodiplodia theobromae, was studied. The nanoparticles showed effective antimicrobial activity against all the tested fungal pathogens under in vitro conditions. The genotoxicity of NPs was also studied using sister chromatid exchange analysis on human lymphocytes and no adverse effects were observed up to the concentration of 100μg/ml. Efficient control against early blight has been observed within cytotoxicity limits in pot study, preventing the disease outbreak in tomato plants.

Primary and Secondary Genotoxicity of Nanoparticles: Establishing a Co-Culture Protocol for Assessing Micronucleus Using Flow Cytometry.

Genotoxicity is an important endpoint to assess for understanding the risks associated with nanoparticles (NPs). Most genotoxicity studies performed on NPs have focused on primary genotoxicity analyzed by comet- or micronuclei (MN) assay using microscopic scoring. Here, we established a protocol for a more efficient version of MN assessment using flow cytometry and, importantly, both primary and secondary (inflammation-driven) genotoxicity was assessed. Human bronchial epithelial cells (HBEC-3kt) were exposed to nickel oxide (NiO) NPs directly or indirectly. The indirect exposure was done to assess secondary genotoxicity, and in this case immune cells (THP-1 derived macrophages) were exposed on inserts and the HBEC were cultured in the lower compartment. The results in monocultures showed that no increased MN formation was observed in the HBEC cells but instead a clear MN induction was noted in THP-1 cells indicating higher sensitivity. No MN formation was either observed when the HBEC were indirectly exposed, but an increase in DNA strand breaks was detected using the comet assay. Taken together, the present study emphasizes the feasibility of assessing primary and secondary genotoxicity and, furthermore, shows a clear MN induction in THP-1 monoculture following NiO NPs exposure.

ГЕНОТОКСИЧНОСТЬ МЕТАЛЛСОДЕРЖАЩИХ НАНОЧАСТИЦ

No more than 100 nm large, nanoparticles (NPs) are distinguished by unusual mechanical, electrical, optical, thermal and magnetic properties. They are used in biology, pharmacology, medicine, chemistry, physics, materials technology and engineering industry. Nanoparticles penetrate in organism through the skin, GIT and airways. Their genotoxicity depends on size, form, mode of action and composition. Two principal mechanisms of NPs genotoxicity are primary and secondary. The primary mechanism is realized in direct interaction with the genome; the secondary mechanism is achieved through mediation of the active forms of oxygen. NPs can modulate the epigenome by altering the gene functions and do not change the DNA sequence directly. Consequences of prolonged exposure to low NPs doses on the human organism still remain uninvestigated.

Nanoparticles: Weighing the Pros and Cons from an Eco-genotoxicological Perspective.

The exponential growth of nanotechnology and the industrial production have raised concerns over its impact on human and environmental health and safety (EHS). Although there has been substantial progress in the assessment of pristine nanoparticle toxicities, their EHS impacts require greater clarification. In this review, we discuss studies that have assessed nanoparticle eco-genotoxicity in different test systems and their fate in the environment as well as the considerable confounding factors that may complicate the results. We highlight key mechanisms of nanoparticle-mediated genotoxicity. Then we discuss the reliability of endpoint assays, such as the comet assay, the most favored assessment technique because of its versatility to measure low levels of DNA strand breakage, and the micronucleus assay, which is complementary to the former because of its greater ability to detect chromosomal DNA fragmentation. We also address the current recommendations on experimental design, including environmentally relevant concentrations and suitable exposure duration to avoid false-positive or -negative results. The genotoxicity of nanoparticles depends on their physicochemical features and the presence of co-pollutants. Thus, the effect of environmental processes (e.g., aggregation and agglomeration, adsorption, and transformation of nanoparticles) would account for when determining the actual genotoxicity relevant to environmental systems, and assay procedures must be standardized. Indeed, the engineered nanoparticles offer potential applications in different fields including biomedicine, environment, agriculture, and industry. Toxicological pathways and the potential risk factors related to genotoxic responses in biological organisms and environments need to be clarified before appropriate and sustainable applications of nanoparticles can be established.

Influence of the capping of biogenic silver nanoparticles on their toxicity and mechanism of action towards Sclerotinia sclerotiorum

BackgroundBiogenic nanoparticles possess a capping of biomolecules derived from the organism employed in the synthesis, which contributes to their stability and biological activity. These nanoparticles have been highlighted for the control of phytopathogens, so there is a need to understand their composition, mechanisms of action, and toxicity. This study aimed to investigate the importance of the capping and compare the effects of capped and uncapped biogenic silver nanoparticles synthesized using the filtrate of Trichoderma harzianum against the phytopathogenic fungus Sclerotinia sclerotiorum. Capping removal, investigation of the composition of the capping and physico-chemical characterization of the capped and uncapped nanoparticles were performed. The effects of the nanoparticles on S. sclerotiorum were evaluated in vitro. Cytotoxicity and genotoxicity of the nanoparticles on different cell lines and its effects on nontarget microorganisms were also investigated.ResultsThe capped and uncapped nanoparticles showed spherical morphology, with greater diameter of the uncapped ones. Functional groups of biomolecules, protein bands and the hydrolytic enzymes NAGase, β-1,3-glucanase, chitinase and acid protease from T. harzianum were detected in the capping. The capped nanoparticles showed great inhibitory potential against S. sclerotiorum, while the uncapped nanoparticles were ineffective. There was no difference in cytotoxicity comparing capped and uncapped nanoparticles, however higher genotoxicity of the uncapped nanoparticles was observed towards the cell lines. Regarding the effects on nontarget microorganisms, in the minimal inhibitory concentration assay only the capped nanoparticles inhibited microorganisms of agricultural importance, while in the molecular analysis of the soil microbiota there were major changes in the soils exposed to the uncapped nanoparticles.ConclusionsThe results suggest that the capping played an important role in controlling nanoparticle size and contributed to the biological activity of the nanoparticles against S. sclerotiorum. This study opens perspectives for investigations concerning the application of these nanoparticles for the control of phytopathogens.

Titanium dioxide nanoparticle genotoxicity: A review of recent in vivo and in vitro studies.

Titanium dioxide nanoparticles (TiO2 NPs, size <100 nm) find applications in a wide range of products including food and cosmetics. Studies have found that exposure to TiO2 NPs can cause inflammation, cytotoxicity, genotoxicity and cell apoptosis. In this article, we have reviewed the recent literature on the potential of TiO2 NPs to cause genotoxicity and summarized the results of two standard genotoxicity assays, the comet and micronucleus (MN) assays. Analysis of these peer-reviewed publications shows that the comet assay is the most common genotoxicity test, followed by MN, Ames, and chromosome aberration tests. These assays have reported positive as well as negative results, although there is inconsistency in some results that need to be confirmed further by well-designed experiments. We also discuss the possible mechanisms of TiO2 NP genotoxicity and point out areas that warrant further research.

Impact of metal oxide nanoparticles on in vitro DNA amplification.

Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (Fe2O3, ZnO, CeO2, Fe3O4, Al2O3, CuO, and TiO2) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs (p > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.

Genotoxicity of engineered nanoparticles in higher plants

Nanoparticles (NPs) are an emerging environmental threat. However, studies of NPs in different environmental components are limited. In this review, we discuss studies that have evaluated the genotoxicity of NPs in higher plants. Among the 29 studies reviewed, silver NPs were most studied (n = 7 articles), with fewer studies reporting the genotoxicity of carbon nanotubes (n = 3), titanium dioxide NPs (n = 4), and zinc oxide NPs (n = 3). Most of the genotoxicity studies were performed in the model plant systems Allium sp (n = 22), Nicotiana sp (n = 4) and Vicia sp (n = 4) using chromosome aberration (n = 22), micronucleus (n = 15) and comet assays (n = 14). Genotoxicity was observed in most of the studies; however, many studies did consider key determinants of NP toxicity such as particle characterization, dissolution, and uptake. From this review, we propose a set of guidelines that should be considered when reporting results of NP toxicity in plants.

Manganese oxide nanoparticles induce genotoxicity and DNA hypomethylation in the moss Physcomitrella patens

The genotoxicity of nanoparticles is a major concern for nano-safety appraisal in the bryophytes as they are the primary colonizers of bare land, indicators of atmospheric pollution and excellent accumulators of trace metals. The present study for the first time evince the in planta genotoxicity of MnONP in Physcomitrella patens a model plant system utilized for evolutionary developmental genetics. The induction of DNA strand breaks was confirmed by comet assay at all tested concentrations corroborated with the enhanced generation of ROS, increase in Mn dissolution, uptake and internalization.Genotoxicity is often coupled with epigenetic alterations. In the present study, global DNA methylation pattern at the level of single cells was studied by the methylation sensitive comet assay using the isochizomeric restriction endonucleases HpaII (digests unmethylated and hemimethylated DNA) and MspI (digests methylated DNA at 5′-CmCGG-3′). MnONP incited DNA hypomethylation in P. patens gametophores treated with the highest concentration of MnONP (20 μg/mL). The DNA hypomethylation incurred upon MnONP exposure was comparable with that of the DNA methylation blocker 5-azacytidine. This can be ascribed to its clastogenic potential mediated by the formation of H2O2, OH and O2¯.There are no reports on the epigenotoxicity of nanomaterials in plants utilizing the detection of DNA damage and DNA methylation. This can open up new avenues of research on the assessment of the epigenotoxic impact of environmentally relevant nanoparticles using bryophytes as model indicator plant system.

Evaluation of Genotoxicity of Nanoparticles in Mouse Models.

Owing to new and unique properties, engineered nanoparticles (NPs) likely pose different risks than their constituent chemicals and these risks need to be understood. In particular, it is important to assess genotoxicity, since genotoxicity is a precursor to carcinogenicity. Here we describe a battery of tests for the assessment of genotoxicity of NPs in vivo in mice. Mice can be exposed to NPs for various exposure durations and by any route of exposure, provided NPs are absorbed into the systemic blood circulation. The testing battery measures three well-established markers of DNA damage: oxidative DNA damage, double strand breaks (DSBs) and chromosomal damage. These markers are measured in peripheral blood cells by microscopic techniques. 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), indicative of oxidative DNA damage, and phosphorylated histone 2AX (γ-H2AX) foci, indicative of DSBs, are determined in white blood cells by immunofluorescence. Micronuclei, indicative of chromosomal damage, are examined in erythrocytes on Giemsa-stained peripheral blood smears. This testing battery can be easily integrated in general toxicology studies or studies examining carcinogenic potential of NPs.

Single-Dosed Genotoxicity Study of Gold Nanorod Core/Silver Shell Nanostructures by Pig-a, Micronucleus, and Comet Assays.

As an increasing number of nanoproducts enter our daily life, their potential health risks have caused widespread concerns and have also promoted studies of nanotoxicity. Among these investigations, the genotoxicity of nanomaterials has attracted more attention. Engineered silver nanorod with gold core and silver shell (Au@Ag NR) was used in this study to investigate the possible genotoxicity and genotoxicity patterns in peripheral lymphocytes and hepatocytes introduced by released Ag+ and the nanoparticle itself by integrating the Pig-a gene, micronucleus, and comet assays. Most of the Au@Ag NR was rapidly cleared from the circulatory system of Sprague Dawley rats, and a small amount of Au@Ag NR was retained in the liver for at least 14 days. Our data confirmed that clastogenicity was the primary genotoxicity type induced by Au@Ag NR, and both NR particles containing Ag and the released Ag+ contribute to genotoxicity. Au@Ag NR was shown to be a clastogen through the introduction of increased %Tail DNA in peripheral lymphocytes (5.82%±0.25%) and hepatocytes (4.83%±0.17%) and promotion of the formation of micronuclei in hepatocytes (1.12%±0.13%) 3 days and 14 days after dosing, respectively (P < 0.05), which could be a result of both Ag+ and Ag shell of the Au@Ag NR. However, Au@Ag NR was not shown to be a mutagen, as the average RBCCD59- count was not changed significantly as compared with control. These data suggest the importance of adopting appropriate genotoxicity testing strategies in identifying the genotoxicity of nanoparticles in vivo.

Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques.

Human embryonic kidney 293T cells (HEK293T cells) before and after treatment with silver nanoparticles (AgNPs) were measured using advanced atomic force microscopy (AFM) force measurement technique, and the biomechanical property of cells was analyzed using a theoretical model. The biomechanical results showed that the factor of viscosity of untreated HEK293T cells reduced from 0.65 to 0.40 for cells exposure to 40 μg/mL of AgNPs. Comet assay indicated that significant DNA damage occurred in the treated cells, measured as tail DNA% and tail moment. Furthermore, gene expression analysis showed that for the cells treated with 40 μg/mL of AgNPs, the antiapoptosis genes Bcl2-t and Bclw were, respectively, downregulated to 0.65- and 0.66-fold of control, and that the proapoptosis gene Bid was upregulated to 1.55-fold of control, which indicates that apoptosis occurred in cells exposed to AgNPs. Interestingly, excellent negative correlations were found between the factor of viscosity and tail DNA%, and tail moment, which suggest that the biomechanical property can be correlated with genotoxicity of nanoparticles on the cells. Based on the above results, we conclude that (1) AgNPs can lead to biomechanical changes in HEK293T cells, concomitantly with biological changes including cell viability, DNA damage, and cell apoptosis; (2) the factor of viscosity can be exploited as a promising label-free biomechanical marker to assess the nanotoxicity of nanoparticles on the cells; and (3) the combination of AFM-based mechanical technique with conventional biological methods can provide more comprehensive understanding of the nanotoxicity of nanoparticles than merely by using the biological techniques.

ZnO and TiO2 Nanoparticles Genotoxicity According to their Structural and Morphological Characteristics Used for Medical Purposes

Defined by their small size, nanomaterials rapidly developed due to their physicochemical properties at nanoscale. Nanoparticles possess a high biological reactivity compared to their bulk size suggesting a high toxicity when the genetic material is involved. Nanogenotoxicity field refers to multidisciplinary sciences relevant for evaluation of genotoxic effect of various nanostructured materials. Due to their widespread use in medical area, zinc oxide and titanium dioxide nanoparticles are receiving researcher�s attention. The major objective of this work was to find a relationship between the structure and morphology of ZnO and TiO2 nanoparticles and their genotoxic potential.

Genotoxicity of Silver Nanoparticles synthesized by Laser Ablation Method in Vivo

This research was conducted to evaluate three main methods usually used to assess cellular DNA damage in genotoxicity assays. These methods were single cell gel electrophoresis (comet assay), micronucleus formation, and DNA fragmentation assay. Nanoparticles genotoxicity is a subject of compelling immediate action as a result of wide application of nanotechnology in many sectors which in contact with humane health. The mentioned methods were used to assess the genotoxicity of silver nanoparticles in vivo. Silver nanoparticles were synthesized by laser ablation of pure silver plate submerged in double distilled water. The synthesized nanosilver was characterized with UV-Visible spectroscopy and atomic force microscope. After its characterization, silver nanoparticles were injected subcutaneously in to BALB/c mice at 200 µg/ kg BW for two different periods of time, one week and two weeks in daily manner. After the end of injection the animals were sacrificed and their bone marrow cells, lymphocytes, and spleen cells were collected. DNA damage in these cells was assessed using the three mentioned methods. Results indicated that the three types of DNA damage assessment methods were capable to detect the genotoxicity of silver nanoparticles in the treated animals. Spleen cells were the less DNA damaged cells as indicated with the three assays in the first week of injection. Lymphocytes and bone marrow cells was effected in more aggressive manner.

Multiple endpoints to evaluate pristine and remediated titanium dioxide nanoparticles genotoxicity in lung epithelial A549 cells

Titanium dioxide nanoparticles (TiO2 NP) are broadly used in a wide range of applications. Several studies have reported that TiO2 NP possess cytotoxic and genotoxic properties that could induce adverse health effects in humans. The FP7 Sanowork project was aimed to minimize occupational hazard and exposure to engineered nanomaterials (ENM), including TiO2 NP, through the surface modification in order to avoid possible adverse toxic effects for humans. In this study we investigated cytotoxicity, genotoxicity and epigenetic properties of TiO2 NP uncoated and coated with silica or citrate, as well as of the benchmark material P25. We used a panel of in vitro assays in the human lung epithelial cell line A549, in order to better understand if the remediation strategy adopted was able to counteract possible toxic effects of uncoated TiO2 NP. Our results showed that the uncoated TiO2 NP were both cytotoxic and genotoxic, and the remediation strategy adopted did not reduce the adverse effects of uncoated TiO2 NP. In particular, the presence of citrate was able to increase their cytotoxicity and genotoxicity, exerting also epigenotoxic effects, as evaluated by the marked reduction of LINE-1 methylation levels.

Study of TiO2 P25 Nanoparticles Genotoxicity on Lung, Blood, and Liver Cells in Lung Overload and Non-Overload Conditions After Repeated Respiratory Exposure in Rats.

Inhaled titanium dioxide (TiO2) nanoparticles (NPs) can have negative health effects, and have been shown to cause respiratory tract cancer in rats. Inflammation has been linked to oxidative stress, and both have been described as possible mechanisms for genotoxicity of NPs, but rarely examined side-by-side in animal studies. In the present study, a wide range of complementary endpoints have been performed to study TiO2 P25 NP-induced genotoxicity in lung overload and non-overload conditions. Additionally, lung burden, inflammation, cytotoxicity and oxidative stress have also been evaluated in order to link genotoxicity with these responses. To assess quick and delayed responses after recovery, endpoints were evaluated at two time points: 2 h and 35 days after three repeated instillations. This study confirmed the previously described lung overload threshold at approximately 200-300 cm2 of lung burden for total particle surface area lung deposition or 4.2 µl/kg for volume-based cumulative lung exposure dose, above which lung clearance is impaired and inflammation is induced. Our results went on to show that these overload doses induced delayed genotoxicity in lung, associated with persistent inflammation only at the highest dose. The lowest tested doses had no toxicity or genotoxicity effects in the lung. In blood, no lymphocyte DNA damage, erythrocytes chromosomal damage or gene mutation could be detected. Our data also demonstrated that only overload doses induced liver DNA lesions irrespective of the recovery time. Tested doses of TiO2 P25 NPs did not induce glutathione changes in lung, blood or liver at both recovery times.

Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity.

With the need to understand the potential biological impact of the plethora of nanoparticles (NPs) being manufactured for a wide range of potential human applications, due to their inevitable human exposure, research activities in the field of NP toxicology has grown exponentially over the last decade. Whilst such increased research efforts have elucidated an increasingly significant knowledge base pertaining to the potential human health hazard posed by NPs, understanding regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to adequately discriminate between the specific modes of action associated with the onset of genotoxicity. Additionally, in line with the recent European directives, there is an inherent need to move away from invasive animal testing strategies. Thus, in vitro systems are an important tool for expanding our mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their validity and specificity for this purpose due to the unique challenges presented when correlating NP behaviour in vitro and in vivo. This review therefore highlights the current state of the art in advanced in vitro systems and their specific advantages and disadvantages from a NP genotoxicity testing perspective. Key indicators will be given related to how these systems might be used or improved to enhance understanding of NP genotoxicity.

Overestimation of nanoparticles-induced DNA damage determined by the comet assay

The increasing use of engineered nanoparticles (NPs) in a wide range of commercial products raises concern about the possible risks that NPs pose to human health. Many aspects of the interaction between living cells and NPs are still unclear, and a reliable assessment of NP genotoxicity would be important. One of the most common tests used for genotoxicity is the comet assay, a sensitive method measuring DNA damage in individual cells. The assay was originally developed for soluble molecules, but it is also used in the assessment of genotoxicity of NPs. However, concerns have been raised recently about the reliability of this test in the case of NPs, but no conclusive results have been presented. Using nuclei isolated from human epithelial cells incubated with NPs, we obtained clear evidence of overestimation of NP genotoxicity by the comet assay in the case of CeO2, TiO2, SiO2, and polystyrene NPs. Removal of the NPs in the cytoplasm was effective in eliminating this genotoxicity overestimation (ex post damage) and determining the actual damage produced by the NPs during incubation with the cells (ex ante damage). This method could improve significantly the determination of NP genotoxicity in eukaryotic cells.

Genotoxicity of tungsten carbide–cobalt (WC–Co) nanoparticles in vitro: Mechanisms-of-action studies

We showed previously that tungsten carbide–cobalt (WC–Co) nanoparticles (NP) can be used as a nanoparticulate positive control in some in vitro mammalian genotoxicity assays. Here, we investigate the mechanisms of action involved in WC–Co NP genotoxicity in L5178Y mouse lymphoma cells and primary human lymphocytes, in vitro. Data from the micronucleus assay coupled with centromere staining and from the chromosome-aberration assay show the involvement of both clastogenic and aneugenic events. Experiments with the formamidopyrimidine DNA glycosylase (FPG)-modified comet assay showed a slight (non-significant) increase in FPG-sensitive sites in the L5178Y mouse lymphoma cells but not in the human lymphocytes. Electron paramagnetic resonance spin-trapping results showed the presence of hydroxyl radicals (OH) in WC–Co NP suspensions, with or without cells, but with time-dependent production in the presence of cells. However, a significant difference in OH production was observed between human lymphocytes from two different donors. Using H2O2, we showed that WC–Co NP can participate in Fenton-like reactions. Thus, OH might be produced either via intrinsic generation by WC–Co NP or through a Fenton-like reaction in the presence of cells.

Can the comet assay be used reliably to detect nanoparticle-induced genotoxicity?

The comet assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in nano-genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the "comet head", which implies that NPs may be present while the assay is being performed. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially impacts the comet assay results is unlikely. For photocatalytically active NPs such as TiO2 , on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%); consistency was even higher when excluding studies on TiO2 NPs (81%). The strong consistency between the comet and micronucleus assays for a range of different NPs-even though the two tests measure different endpoints-implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods.