Effects of Zinc Oxide Nanoparticle Exposure on Human Glial Cells and Zebrafish Embryos.

Zinc oxide nanoparticles (ZnO NPs) are among the most widely used nanomaterials. They have multiple applications in cosmetics, textiles, paints, electronics and, recently, also in biomedicine. This extensive use of ZnO NPs notably increases the probability that both humans and wildlife are subjected to undesirable effects. Despite being among the most studied NPs from a toxicological point of view, much remains unknown about their ecotoxicological effects or how they may affect specific cell types, such as cells of the central nervous system. The main objective of this work was to investigate the effects of ZnO NPs on human glial cells and zebrafish embryo development and to explore the role of the released Zn2+ ions in these effects. The effects on cell viability on human A172 glial cells were assessed with an MTT assay and morphological analysis. The potential acute and developmental toxicity was assessed employing zebrafish (Danio rerio) embryos. To determine the role of Zn2+ ions in the in vitro and in vivo observed effects, we measured their release from ZnO NPs with flame atomic absorption spectrometry. Then, cells and zebrafish embryos were treated with a water-soluble salt (zinc sulfate) at concentrations that equal the number of Zn2+ ions released by the tested concentrations of ZnO NPs. Exposure to ZnO NPs induced morphological alterations and a significant decrease in cell viability depending on the concentration and duration of treatment, even after removing the overestimation due to NP interference. Although there were no signs of acute toxicity in zebrafish embryos, a decrease in hatching was detected after exposure to the highest ZnO NP concentrations tested. The ability of ZnO NPs to release Zn2+ ions into the medium in a concentration-dependent manner was confirmed. Zn2+ ions did not seem entirely responsible for the effects observed in the glial cells, but they were likely responsible for the decrease in zebrafish hatching rate. The results obtained in this work contribute to the knowledge of the toxicological potential of ZnO NPs.

Toxicological Aspects of Iron Oxide Nanoparticles.

Iron oxide nanoparticles (ION), with unique magnetic properties, have attracted huge scientific attention for a wide variety of uses, mostly in the biomedical field, due to their high biocompatibility, ability to cross biological membranes, appropriate surface architecture and easy conjugation with targeting ligands. Their current applications include diagnostic imaging, cell labelling, site-directed drug delivery and anticancer hyperthermia therapy. The ION surface may be modified by coating with different materials, aiming to stabilize the nanoparticles in different environments, to allow biomolecule binding favouring surface attachments with several molecules, and to prolong the recognition time by the immune system. Although the potential benefits of ION are considerable, and more and more ION are being manufactured to meet the demands of the rapidly proliferating field of nanomedicine, there is an urgent need to define their toxicological profile in order to avoid any potential health risks associated with their exposure and to reach optimal benefits of their use. The purpose of this chapter is to de-scribe the current knowledge on the ION toxicological features, addressing their structure and physicochemical characteristics, main exposure pathways and toxicokinetic aspects, interaction with cells, and their toxic effects, with special attention to those at the cellular and molecular level.

Salivary leucocytes as suitable biomatrix for the comet assay in human biomonitoring studies.

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples.

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci 2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay.

Suitability of the In Vitro Cytokinesis-Block Micronucleus Test for Genotoxicity Assessment of TiO2 Nanoparticles on SH-SY5Y Cells.

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.

Expanded usage of the Challenge-Comet assay as a DNA repair biomarker in human populations: protocols for fresh and cryopreserved blood samples, and for different challenge agents.

Deficiencies in DNA damage response and repair (DDRR) can cause serious pathological outcomes; therefore, having an ability to determine individual DDRR would enhance specificities in health risk assessment and in determining individual's response to cancer therapies. However, most methods for evaluating DDRR are not fully appropriate for population studies. The Challenge-Comet assay has gained acceptance for this purpose. The assay has traditionally used X-rays as challenge agent and isolated peripheral blood mononuclear cells (PBMC) as cell specimen. To enhance the usefulness of the assay, the objectives of this investigation were to use differently processed blood samples, to employ other challenge agents with different mechanisms of induction of DNA damage/repair, and to generate protocols for detecting different DDRR capacities. Fresh and frozen blood samples were challenged with bleomycin, methyl methanesulfonate (MMS) and ultraviolet light. Significant induction of damage after all treatments, and progressive and time-dependent DDRR were observed. No significant differences were obtained in the DDRR capacities of fresh or frozen whole blood samples as compared to PBMC, except that fresh blood samples showed higher MMS-induced DDRR capacity than PBMC. Results from this study show that the Challenge-Comet assay can be used as routine biomarker of DDRR capacity in human biomonitoring studies, and that whole blood is also a useful biomatrix for this assay. The collected data allow us to recommend different protocols for the Challenge-Comet assay which are useful for evaluating DDRR capacities in several key DNA repair pathways. Consequently, the usefulness of the Challenge-Comet assay can be greatly expanded.

Cellular and Molecular Toxicity of Iron Oxide Nanoparticles.

Iron oxide nanoparticles (ION) have attracted much attention because of their particular physico-chemical properties, including superparamagnetism. These features make them suitable for many purposes and several interesting biomedical applications, such as to increase contrast in magnetic resonance imaging (MRI), as drug delivery systems and as hyperthermia agents. However, they have also shown to be easily accumulated in diverse tissues and induce toxicity at different levels. This chapter reviews the different cellular and molecular effects induced by ION reported from in vitro studies with human and non-human cell lines. Those effects are mainly dependent on ION type and concentration, time of exposure, presence and nature of coating, and cell type evaluated. They include decreases in viability, plasmatic membrane disruption, oxidative damage, mitochondrial alterations, cell cycle impairments, cytoskeleton disruption, cell death, and alterations in cell motility, and in cell integrity. Despite these negative effects, the numerous advantages of ION together with their promising applications in biomedicine, make it necessary to clearly define their toxicity in order to discard potential health risks and to reach optimal benefits of their use.

In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests.

Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5-300 µg ml(-1)), prepared in complete and serum-free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical-chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell-free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid-coated ION were less cytotoxic than silica-coated ION; besides, a serum-protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications.

Toxoplasma gondii IgG Serointensity Is Positively Associated With Frailty.

BACKGROUND: Persistent inflammation related to aging ("inflammaging") is exacerbated by chronic infections and contributes to frailty in older adults. We hypothesized associations between Toxoplasma gondii (T. gondii), a common parasite causing an oligosymptomatic unremitting infection, and frailty, and secondarily between T. gondii and previously reported markers of immune activation in frailty. METHODS: We analyzed available demographic, social, and clinical data in Spanish and Portuguese older adults [N = 601; age: mean (SD) 77.3 (8.0); 61% women]. Plasma T. gondii immunoglobulin G (IgG) serointensity was measured with an enzyme-linked immunosorbent assay. The Fried criteria were used to define frailty status. Validated translations of Mini-Mental State Examination, Geriatric Depression Scale, and the Charlson Comorbidity Index were used to evaluate confounders. Previously analyzed biomarkers that were significantly associated with frailty in both prior reports and the current study, and also related to T. gondii serointensity, were further accounted for in multivariable logistic models with frailty as outcome. RESULTS: In T. gondii-seropositives, there was a significant positive association between T. gondii IgG serointensity and frailty, accounting for age (p = .0002), and resisting adjustment for multiple successive confounders. Among biomarkers linked with frailty, kynurenine/tryptophan and soluble tumor necrosis factor receptor II were positively associated with T. gondii serointensity in seropositives (p < .05). Associations with other biomarkers were not significant. CONCLUSIONS: This first reported association between T. gondii and frailty is limited by a cross-sectional design and warrants replication. While certain biomarkers of inflammaging were associated with both T. gondii IgG serointensity and frailty, they did not fully mediate the T. gondii-frailty association.

Relationship between DNA damage measured by the comet-assay and cognitive function.

Recent studies exploring the relationship between DNA damage measured by the comet assay (single-cell gel electrophoresis) and cognitive function in both animal models and humans are reviewed and summarized. This manuscript provides an overview of studies exploring cognitive dysfunction related to DNA damage due to biological ageing process, cancer treatment, adverse environmental or occupational exposures, and prenatal genotoxic exposure. The review confirms the potential of comet assay to further explore the link between DNA damage, as indicative of genomic instability, and cognitive impairment in different research and clinical areas. Analysed studies support, in fact, the significant relationship between DNA damage and cognitive impairment, mainly affecting attention, working memory and executive functions. These cognitive domains are crucial to daily functioning and occupational performance, with important clinical implications. Although evidence support the relationship between DNA damage measured by the comet assay and cognitive function in different settings, further longitudinal research is needed to disentangle the temporal relationship between them over time, and to explore the potential of comet assay-detected DNA lesions to predict response to interventions.

Suitability of salivary leucocytes to assess DNA repair ability in human biomonitoring studies by the challenge-comet assay.

The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the kinetics of repair process. Peripheral blood mononuclear cells (PBMC) are the cells most frequently employed in human biomonitoring studies using the challenge-comet assay, but having a validated alternative of non-invasive biomatrix would be highly convenient for certain population groups and circumstances. The objective of this study was to validate the use of salivary leucocytes in the challenge-comet assay. Leucocytes were isolated from saliva samples and challenged (either in fresh or after cryopreservation) with three genotoxic agents acting by different action mechanisms: bleomycin, methyl methanesulfonate, and ultraviolet radiation. Comet assay was performed just after treatment and at other three additional time points, in order to study repair kinetics. The results obtained demonstrated that saliva leucocytes were as suitable as PBMC for assessing DNA damage of different nature that was efficiently repaired over the evaluated time points, even after 5 months of cryopreservation (after a 24 h stimulation with PHA). Furthermore, a new parameter to determine the efficacy of the repair process, independent of the initial amount of damage induced, is proposed, and recommendations to perform the challenge-comet assay with salivary leucocytes depending on the type of DNA repair to be assessed are suggested. Validation studies are needed to verify whether the method is reproducible and results reliable and comparable among laboratories and studies.

Relationship between structure and cytotoxicity of vanadium and molybdenum complexes with pyridoxal derived ligands.

In this work four vanadium complexes (compounds 1, 2, 3 and 4) and one molybdenum complex (compound 5) with hydrazone ligands derived from pyridoxal were synthesized and characterized. All compounds are mononuclear species, two of them (compounds 3 and 5) are dioxide complexes and the other three (compounds 1, 2 and 4) monoxide complexes. The vanadium atom of the compound 3 is five-coordinated and all the other compounds have a six coordinated environment polyhedron. The poses for the potential intercalation of the compounds 2 and 3 with DNA were obtained by using AutoDock software. Optimizations were also performed at PM6-D3H4 semi-empirical level whereas the study of the nature of the interaction was carried out by means of the Energy Decomposition Analysis and the Non-Covalent Interaction index by using in both cases Density Functional Theory computations. The cytotoxicity in lung cancer cells (A549 cell line) of all the compounds was also evaluated. After 24 h of treatment, vanadium complexes showed high values of IC50, between 419.93 ± 22.58 and 685.88 ± 46.55 µM. After 48 h, the results showed that the compound 3 had the lowest IC50 value, 65.32 ± 9.95 µM, and the compound 2 the highest value, 375.28 ± 32.09 µM. The molybdenum complex showed the lowest IC50 value at 48 h (11.22 ± 1.34 µM). The toxicity of the compounds 3, 4 and 5 was tested in vivo, using zebrafish model, and the molybdenum complex showed higher toxic effects than the studied vanadium complexes.

Salivary Leucocytes as In Vitro Model to Evaluate Nanoparticle-Induced DNA Damage.

Metal oxide nanoparticles (NPs) have a wide variety of applications in many consumer products and biomedical practices. As a result, human exposure to these nanomaterials is highly frequent, becoming an issue of concern to public health. Recently, human salivary leucocytes have been proposed as an adequate non-invasive alternative to peripheral blood leucocytes to evaluate genotoxicity in vitro. The present study focused on proving the suitability of salivary leucocytes as a biomatrix in the comet assay for in vitro nanogenotoxicity studies, by testing some of the metal oxide NPs most frequently present in consumer products, namely, titanium dioxide (TiO2), zinc oxide (ZnO), and cerium dioxide (CeO2) NPs. Primary and oxidative DNA damage were evaluated by alkaline and hOGG1-modified comet assay, respectively. Any possible interference of the NPs with the methodological procedure or the hOGG1 activity was addressed before performing genotoxicity evaluation. Results obtained showed an increase of both primary and oxidative damage after NPs treatments. These data support the use of salivary leucocytes as a proper and sensitive biological sample for in vitro nanogenotoxicity studies, and contribute to increase the knowledge on the impact of metal oxide NPs on human health, reinforcing the need for a specific regulation of the nanomaterials use.

Genotoxicity of TiO2 Nanoparticles in Four Different Human Cell Lines (A549, HEPG2, A172 and SH-SY5Y).

Titanium dioxide nanoparticles (TiO2 NPs) have a wide variety of applications in many consumer products, including as food additives, increasing the concern about the possible hazards that TiO2 NPs may pose to human health. Although most previous studies have focused on the respiratory system, ingestion must also be considered as an important exposure route. Furthermore, after inhalation or ingestion, TiO2 NPs can reach several organs, such as the liver, brain or lungs. Taking this into consideration, the present study focuses on the uptake and potential genotoxicity (micronuclei induction) of TiO2 NPs on four human cell lines of diverse origin: lung cells (A549), liver cells (HepG2), glial cells (A172) and neurons (SH-SY5Y), using flow cytometry methods. Results showed a concentration-, time- and cell-type- dependent increase in TiO2 NPs uptake but no significant induction of micronuclei in any of the tested conditions. Data obtained reinforce the importance of cell model and testing protocols choice for toxicity assessment. However, some questions remain to be answered, namely on the role of cell culture media components on the agglomeration state and mitigation of TiO2 NPs toxic effects.

Evaluation of cytotoxicity and genotoxicity induced by oleic acid-coated iron oxide nanoparticles in human astrocytes.

Iron oxide nanoparticles (ION) are gaining importance as diagnostic and therapeutic tool of central nervous system diseases. Although oleic acid-coated ION (O-ION) have been described as stable and biocompatible, their potential neurotoxicity was scarcely evaluated in human nervous cells so far. The primary aim of this work was to assess the molecular and cellular effects of O-ION on human astrocytes (A172 cells) under different experimental conditions. An extensive set of cyto- and genotoxicity tests was carried out, including lactate dehydrogenase release assay, cell cycle alterations, and cell death production, as well as comet assay, γH2AX assay, and micronucleus (MN) test, considering also iron ion release capacity and alterations in DNA repair ability. Results showed a moderate cytotoxicity related to cell cycle arrest and cell death promotion, regardless of serum presence. O-ION induced genotoxic effects, namely primary DNA damage, as detected by the comet assay and H2AX phosphorylation, but A172 cells were able to repair this particular damage because no chromosome alterations were found (confirmed by MN test results). Accordingly, no effects on the DNA repair ability were observed. The presence of serum proteins did not influence O-ION toxicity. Iron ions released from the O-ION surface seemed not to be responsible for the cytotoxic and genotoxic effects observed. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Assessment of oxidative damage induced by iron oxide nanoparticles on different nervous system cells.

Iron oxide nanoparticles (ION) have received much attention for their utility in biomedical applications, such as magnetic resonance imaging, drug delivery and hyperthermia, but concerns regarding their potential harmful effects are also growing. Even though ION may induce different toxic effects in a wide variety of cell types and animal systems, there is a notable lack of toxicological data on the human nervous system, particularly important given the increasing number of applications on this specific system. An important mechanism of nanotoxicity is reactive oxygen species (ROS) generation and oxidative stress. On this basis, the main objective of this work was to assess the oxidative potential of silica-coated (S-ION) and oleic acid-coated (O-ION) ION on human SH-SY5Y neuronal and A172 glial cells. To this aim, ability of ION to generate ROS (both in the absence and presence of cells) was determined, and consequences of oxidative potential were assessed (i) on DNA by means of the 8-oxo-7,8-dihydroguanine DNA glycosylase (OGG1)-modified comet assay, and (ii) on antioxidant reserves by analyzing ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Conditions tested included a range of concentrations, two exposure times (3 and 24 h), and absence and presence of serum in the cell culture media. Results confirmed that, even though ION were not able to produce ROS in acellular environments, ROS formation was increased in the neuronal and glial cells by ION exposure, and was parallel to induction of oxidative DNA damage and, only in the case of neuronal cells treated with S-ION, to decreases in the GSH/GSSG ratio. Present findings suggest the production of oxidative stress as a potential action mechanism leading to the previously reported cellular effects, and indicate that ION may pose a health risk to human nervous system cells by generating oxidative stress, and thus should be used with caution.

Neurotoxicity assessment of oleic acid-coated iron oxide nanoparticles in SH-SY5Y cells.

Iron oxide nanoparticles (ION) awaken a particular interest for biomedical applications due to their unique physicochemical properties, especially superparamagnetism, and ability to cross the blood-brain barrier. ION surface can be coated to improve their properties and facilitate functionalization. Still, coating may affect toxicity. The aim of this work was to evaluate the possible effects of oleic acid-coated ION (O-ION) on human neuronal cells (SH-SY5Y). A set of assays was conducted in complete and serum-free culture media for 3 and 24 h to assess O-ION cytotoxic effects - cell membrane disruption, cell cycle alteration and cell death induction -, and genotoxic effects - primary DNA damage, H2AX phosphorylation and micronuclei induction -, considering also DNA repair competence and iron ion release. Results obtained show that O-ION exhibit a moderate cytotoxicity related to cell membrane impairment, cell cycle disruption and cell death induction, especially notable in serum-free medium. Iron ion release was only observed in complete medium, indicating that cytotoxicity observed was not related to the presence of ions in the medium. However, O-ION genotoxic effects were limited to the induction of primary DNA damage, not related to double strand breaks, and this damage did not become fixed in cells in most conditions. Alterations in repair ability (DNA repair competence assay) were observed when cells where treated with O-ION before or during the challenge with H2O2, but not during the repair period. Further investigation is needed to clarify the possible role of oxidative stress and protein corona on observed O-ION toxicity.

Toxicological assessment of silica-coated iron oxide nanoparticles in human astrocytes.

Iron oxide nanoparticles (ION) have great potential for an increasing number of medical and biological applications, particularly those focused on nervous system. Although ION seem to be biocompatible and present low toxicity, it is imperative to unveil the potential risk for the nervous system associated to their exposure, especially because current data on ION effects on human nervous cells are scarce. Thus, in the present study potential toxicity associated with silica-coated ION (S-ION) exposure was evaluated on human A172 glioblastoma cells. To this aim, a complete toxicological screening testing several exposure times (3 and 24 h), nanoparticle concentrations (5-100 µg/ml), and culture media (complete and serum-free) was performed to firstly assess S-ION effects at different levels, including cytotoxicity - lactate dehydrogenase assay, analysis of cell cycle and cell death production - and genotoxicity - H2AX phosphorylation assessment, comet assay, micronucleus test and DNA repair competence assay. Results obtained showed that S-ION exhibit certain cytotoxicity, especially in serum-free medium, related to cell cycle disruption and cell death induction. However, scarce genotoxic effects and no alteration of the DNA repair process were observed. Results obtained in this work contribute to increase the knowledge on the impact of ION on the human nervous system cells.

Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing.

Cell cultures from neuronal and glial origin have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and physiology, and as neurotoxicity models to evaluate in vitro the possible effects of chemicals. But cellular heterogeneity of nervous system is considerable and these cells have been shown to respond diversely to neurotoxic insults, leading to disparate results from different studies. To shed more light on suitability of cellular models of nervous origin for neurotoxicity screening, the objective of this study was to compare the sensitivity to genetic damage induction of two nervous cell lines. To this aim, neurons (SH-SY5Y) and glial (A172) cells were treated with differently-acting genotoxic agents (bleomycin, actinomycin-D, methyl methanesulfonate, mitomycin C, and griseofulvin). After discarding cytotoxicity, genotoxicity was evaluated by a battery of assays encompassing detection of different genetic lesions. Results obtained showed that glial cells are generally more resistant to genotoxic damage induced by clastogenic agents, but more sensitive to aneugenic effects. These results highlight the need of proper design of in vitro neurotoxicology studies, especially for neurogenotoxicity screening, emphasizing the importance of employing more than one nervous cell type for testing the potential toxicity of a particular exposure.

In vitro toxicity evaluation of silica-coated iron oxide nanoparticles in human SHSY5Y neuronal cells.

Iron oxide nanoparticles (ION) have been widely used in biomedical applications, for both diagnosis and therapy, due to their unique magnetic properties. They are intensively explored in neuromedicine mostly because of their ability to cross the blood brain barrier. Hence, their potential harmful effects on neuronal cells need to be carefully assessed. The objective of this study was to evaluate the toxicity of silica-coated ION (S-ION) (10-200 µg ml-1) on human neuronal SHSY5Y cells. Alterations in the cell cycle, cell death by apoptosis or necrosis, and membrane integrity were assessed as cytotoxicity parameters. Genotoxicity was determined by a γH2AX assay, a micronucleus (MN) test, and a comet assay. Complementarily, possible effects on DNA damage repair were also analysed by means of a DNA repair competence assay. All analyses were performed in complete and serum-free cell culture media. Iron ion release from the nanoparticles was notable only in complete medium. Despite being effectively internalized by the neuronal cells, S-ION presented in general low cytotoxicity; positive results were only obtained in some assays at the highest concentrations and/or the longest exposure time tested (24 h). Genotoxicity evaluations in serum-free medium were negative for all conditions assayed; in complete medium, dose and time-dependent increase in DNA damage not related to the production of double strand breaks or chromosome loss (according to the results of the γH2AX assay and MN test), was obtained. The presence of serum slightly influenced the behaviour of S-ION; further studies to investigate the formation of a protein corona and its role in nanoparticle toxicity are necessary.