Toxic effects of gunshot fumes from different ammunitions for small arms on lung cells exposed at the air liquid interface.

Concerns have been raised as to whether gunshot fumes induce prolonged reduced lung capacity or even cancer due to inhalation. Gunshot fumes from three different types of ammunition calibre 5.56 mm × 45 NATO were investigated. SS109 has a soft lead (Pb) core, while NM255 and NM229 have a harder steel core. Emissions from ammunitions were characterized with respect to particle number- and mass-size, and mass distribution, heavy metal content, and different gases. Lung epithelial cells were exposed to the fumes at the air liquid interface to elucidate cytotoxicity and genotoxicity. Irrespectively of ammunition type, the largest mass fraction of generated particulate matter (PM) had a size between 1 and 3 µm. The highest number of particles generated was in the size range of 30 nm. Fumes from NM255 and NM229 induced cytotoxic effects of which the emission from NM229 induced the highest effect. Fumes from NM229 induced a dose-related increase in DNA-damage. Significant effects were only achieved at the highest exposure level, which led to approximately 40% reduced cell viability after 24 h. The effect probably relates to the mass of emitted particles where the size may be of importance, in addition to emission of Cu and Zn. A complex mixture of chemical substances and PM may increase the toxicity of the fumes and should encourage measures to reduce exposure.

High throughput toxicity screening and intracellular detection of nanomaterials.

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety-preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods-namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

In vitro genotoxicity testing of four reference metal nanomaterials, titanium dioxide, zinc oxide, cerium oxide and silver: towards reliable hazard assessment.

There is serious concern about the potential harmful effects of certain nanomaterials (NMs), on account of their ability to penetrate cell membranes and the increased reactivity that results from their increased surface area compared with bulk chemicals. To assess the safety of NMs, reliable tests are needed. We have investigated the possible genotoxicity of four representative NMs, derived from titanium dioxide, zinc oxide, cerium oxide and silver, in two human cell lines, A549 alveolar epithelial cells and lymphoblastoid TK6 cells. A high-throughput version of the comet assay was used to measure DNA strand beaks (SBs) as well as oxidised purines (converted to breaks with the enzyme formamidopyrimidine DNA glycosylase). In parallel, cytotoxicity was measured with the alamarBlue® assay, and the ability of NM-treated cells to survive was assessed by their colony-forming efficiency. TiO2 and CeO2 NMs were only slightly cytotoxic by the alamarBlue® test, and had no long-term effect on colony-forming efficiency. However, both induced DNA damage at non-cytotoxic concentrations; the damage decreased from 3 to 24-h exposure, except in the case of CeO2-treated A549 cells. ZnO and Ag NMs affected cell survival, and induced high levels of DNA damage at cytotoxic concentrations. At lower concentrations, there was significant damage, which tended to persist over 24 h. The implication is that all four reference metal NMs tested-whether cytotoxic or not-are genotoxic. A full assessment of NM toxicity should include tests on different cell types, different times of incubation and a wide range of (especially non-cytotoxic) concentrations; a test for cell viability should be performed in parallel. Inclusion of Fpg in the comet assay allows detection of indirect genotoxic effects via oxidative stress.

Fibrous shape underlies the mutagenic and carcinogenic potential of nanosilver while surface chemistry affects the biosafety of iron oxide nanoparticles.

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 significantly the number of both gene mutations and transformed foci compared with the control (untreated) cells. Under the same treatment conditions, neither SO-MNPs nor SO-PEG-PLGA-MNPs increased the mutant frequency compared with control cells though an equivocal mutagenic effect was estimated for SO-PEG-MNPs. Although SO-MNPs and SO-PEG-MNPs did not show any carcinogenic potential, SO-PEG-PLGA-MNPs increased concentration dependently the number of transformed foci in Bhas 42 cells compared with the control cells. Our results revealed that fibrous shape underlies the mutagenic and carcinogenic potential of nanosilver while surface chemistry affects the biosafety of MNPs. Considering that both nanosilver and MNPs are prospective ENMs for biomedical applications, further toxicological evaluations are warranted to assess comprehensively the biosafety of these nanomaterials.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Biological impact assessment of nanomaterial used in nanomedicine. introduction to the NanoTEST project.

Therapeutic nanoparticles (NPs) are used in nanomedicine as drug carriers or imaging agents, providing increased selectivity/specificity for diseased tissues. The first NPs in nanomedicine were developed for increasing the efficacy of known drugs displaying dose-limiting toxicity and poor bioavailability and for enhancing disease detection. Nanotechnologies have gained much interest owing to their huge potential for applications in industry and medicine. It is necessary to ensure and control the biocompatibility of the components of therapeutic NPs to guarantee that intrinsic toxicity does not overtake the benefits. In addition to monitoring their toxicity in vitro, in vivo and in silico, it is also necessary to understand their distribution in the human body, their biodegradation and excretion routes and dispersion in the environment. Therefore, a deep understanding of their interactions with living tissues and of their possible effects in the human (and animal) body is required for the safe use of nanoparticulate formulations. Obtaining this information was the main aim of the NanoTEST project, and the goals of the reports collected together in this special issue are to summarise the observations and results obtained by the participating research teams and to provide methodological tools for evaluating the biological impact of NPs.

Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

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, (3)H-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.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles.

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.

Genotoxic and mutagenic potential of nitramines.

Climate change is one of the major challenges in the world today. To reduce the amount of CO2 released into the atmosphere, CO2 at major sources, such as power plants, can be captured. Use of aqueous amine solutions is one of the most promising methods for this purpose. However, concerns have been raised regarding its impacts on human health and the environment due to the degradation products, such as nitrosamines and nitramines that may be produced during the CO2 capture process. While several toxicity studies have been performed investigating nitrosamines, little is known about the toxic potential of nitramines. In this study a preliminary screening was performed of the genotoxic and mutagenic potential of nitramines most likely produced during amine based CO2 capture; dimethylnitramine (DMA-NO2), methylnitramine (MA-NO2), ethanolnitramine (MEA-NO2), 2-methyl-2-(nitramino)-1-propanol (AMP-NO2) and piperazine nitramine (PZ-NO2), by the Bacterial Reverse Mutation (Ames) Test, the Cytokinesis Block Micronucleus (CBMN) Assay and the in vitro Single-Cell Gel Electrophoresis (Comet) Assay. MA-NO2 and MEA-NO2 showed mutagenic potential in the Ames test and a weak genotoxic response in the CBMN Assay. AMP-NO2 and PZ-NO2 significantly increased the amount of DNA strand breaks; however, the level of breaks was below background. Most previous studies on nitramines have been performed on DMA-NO2, which in this study appeared to be the least potent nitramine. Our results indicate that it is important to investigate other nitramines that are more likely to be produced during CO2 capture, to ensure that the risk is realistically evaluated.

Screening for potential hazard effects from four nitramines on human eye and skin.

Amines have potential to be used in CO2 capture and storage (CCS) technology, but as they can be released into the environment and be degraded into more toxic compounds, such as nitrosamines and nitramines, there have been concerns about their negative impact on human health. We investigated the potential toxic effects from acute exposure to dimethylnitramine (DMA-NO2), methylnitramine (MA-NO2), ethanolnitramine (MEA-NO2) and 2-methyl-2-(nitroamino)-1-propanol (AMP-NO2). The eye irritation, and skin sensitization, irritation and corrosion potential of these substances have been evaluated in vitro using the Bovine Corneal Opacity and Permeability (BCOP) assay, VITOSENS® assay, Reconstructed Human Epidermis (RHE) skin irritation test and Corrositex Skin corrosion test, respectively. Exposure to DMA-NO2 induced a mild eye irritation response, while MA-NO2, MEA-NO2 and AMP-NO2 were shown to be very severe eye irritants. MA-NO2 and MEA-NO2 were tested for skin sensitization and found to be non-sensitizers to the skin. In addition, none of the four test substances was irritant or corrosive to the skin.

Toxicological aspects for nanomaterial in humans.

Among beneficial applications of nanotechnology, nanomedicine offers perhaps the greatest potential for improving human conditions and quality of life. Engineered nanomaterials (ENMs), with their unique properties, have potential to improve therapy of many human disorders. The properties that make ENMs so useful could also lead to unintentional adverse health effects. Challenges arising from physicochemical properties of ENMs, their characterization, exposure, and hazard assessment and other key issues of ENM safety are discussed. There is still scant knowledge about ENM cellular uptake, transport across biological barriers, distribution within the body, and possible mechanisms of toxicity. The safety of ENMs should be tested to minimize possible risk before the application. However, existing toxicity tests need to be adapted to fit to the unique features related to the nanosized material and appropriate controls and reference material should be considered.

Silver nanoparticles induce premutagenic DNA oxidation that can be prevented by phytochemicals from Gentiana asclepiadea.

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.

Impact of agglomeration and different dispersions of titanium dioxide nanoparticles on the human related in vitro cytotoxicity and genotoxicity.

The published results on nanoparticles cytotoxicity and genotoxicity such as titanium dioxide nanoparticles (TiO(2) NPs) are inconsistent, and often conflicting and insufficient. Since different parameters may have impact on the toxicity results, there is need to lay stress on detailed characterization of NPs and the use of different testing conditions for assessment of NPs toxicity. In order to investigate whether dispersion procedures influence NP cytotoxicity and genotoxicity, we compared two protocols giving TiO(2) NP dispersions with different stability and agglomeration states. Detailed primary and secondary characteristics of both TiO(2) NP dispersions in culture media were carried out before toxicological testing; TK6 human lymphoblast cells, EUE human embryonic epithelial cells and Cos-1 monkey kidney fibroblasts were used to assess cytotoxicity (by trypan blue exclusion, proliferation activity and plating efficiency assays) and genotoxicity (by the comet assay). DNA strand breaks were detected by the alkaline comet assay. DNA oxidation lesions (especially 8-oxo-7,8-dihydroguanine, 8-oxoG) were measured with a modified comet assay including incubation with specific repair enzyme formamidopyrimidine DNA glycosylase (FPG). The TiO(2) NPs dispersion with large agglomerates (3 min sonication and no serum in stock solution) induced DNA damage in all three cell lines, while the TiO(2) NPs dispersed with agglomerates less than 200 nm (foetal serum in stock solution and sonication 15 min) had no effect on genotoxicity. An increased level of DNA oxidation lesions detected in Cos-1 and TK6 cells indicates that the leading mechanism by which TiO(2) NPs trigger genotoxicity is most likely oxidative stress. Our results show that the dispersion method used can influence the results of toxicity studies. Therefore at least two different dispersion procedures should be incorporated into assessment of cyto- and genotoxic effects of NPs. It is important, when assessing the hazard associated with NPs, to establish standard testing procedures and thorough strategies to consider the diverse conditions relevant to possible exposures.

Minimal analytical characterization of engineered nanomaterials needed for hazard assessment in biological matrices.

This paper presents the outcomes from a workshop of the European Network on the Health and Environmental Impact of Nanomaterials (NanoImpactNet). During the workshop, 45 experts in the field of safety assessment of engineered nanomaterials addressed the need to systematically study sets of engineered nanomaterials with specific metrics to generate a data set which would allow the establishment of dose-response relations. The group concluded that international cooperation and worldwide standardization of terminology, reference materials and protocols are needed to make progress in establishing lists of essential metrics. High quality data necessitates the development of harmonized study approaches and adequate reporting of data. Priority metrics can only be based on well-characterized dose-response relations derived from the systematic study of the bio-kinetics and bio-interactions of nanomaterials at both organism and (sub)-cellular levels. In addition, increased effort is needed to develop and validate analytical methods to determine these metrics in a complex matrix.

Influence of organ motion on conformal vs. intensity-modulated pelvic radiotherapy for prostate cancer.

PURPOSE: To compare an intensity-modulated radiotherapy (IMRT) planning approach for prostate pelvic RT with a conformal RT (CRT) approach taking into account the influence of organ-at-risk (OAR) motion. METHODS AND MATERIALS: A total of 20 male patients, each with one planning computed tomography scan and five to eight treatment computed tomography scans, were used for simulation of IMRT and CRT for delivery of a prescribed dose of 50 Gy to the prostate, seminal vesicles, and pelvic lymph nodes. Planning was done in Eclipse without correcting for OAR motion. Evaluation was performed using the CRT and IMRT dose matrices and the planning and treatment OAR outlines. The generalized equivalent uniform dose (gEUD) was calculated for 894 OAR volumes using a volume-effect parameter of 4, 12, and 8 for bowel, rectum and bladder, respectively. For the bowel, the gEUD was normalized to a reference volume of 200 cm(3). For each patient and each OAR, an average of the treatment gEUDs (gEUD(treat)) was calculated for CRT and IMRT. The paired t test was used to compare IMRT with CRT and gEUD(treat) with gEUD(plan). RESULTS: The mean gEUD(treat) was reduced from 43 to 40 Gy, 47 to 46 Gy, and 48 to 45 Gy with IMRT for the bowel, rectum, and bladder, respectively (p < 0.001). Differences between the gEUD(plan) and gEUD(treat) were not significant (p > 0.05) for any OAR but was >6% for the bowel in 6 of 20 patients. CONCLUSION: Intensity-modulated RT reduced the bowel, rectum, and bladder gEUDs also under influence of OAR motion. Neither CRT nor IMRT was robust against bowel motion, but IMRT was not less robust than CRT.