Assessing the in vitro toxicity of airborne (nano)particles to the human respiratory system: from basic to advanced models.

Several studies have been conducted to address the potential adverse health risks attributed to exposure to nanoscale materials. While in vivo studies are fundamental for identifying the relationship between dose and occurrence of adverse effects, in vitro model systems provide important information regarding the mechanism(s) of action at the molecular level. With a special focus on exposure to inhaled (nano)particulate material toxicity assessment, this review provides an overview of the available human respiratory models and exposure systems for in vitro testing, advantages, limitations, and existing investigations using models of different complexity. A brief overview of the human respiratory system, pathway and fate of inhaled (nano)particles is also presented.

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.

Unveiling the Toxicity of Fine and Nano-Sized Airborne Particles Generated from Industrial Thermal Spraying Processes in Human Alveolar Epithelial Cells.

High-energy industrial processes have been associated with particle release into workplace air that can adversely affect workers' health. The present study assessed the toxicity of incidental fine (PGFP) and nanoparticles (PGNP) emitted from atmospheric plasma (APS) and high-velocity oxy-fuel (HVOF) thermal spraying. Lactate dehydrogenase (LDH) release, 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) metabolisation, intracellular reactive oxygen species (ROS) levels, cell cycle changes, histone H2AX phosphorylation (γ-H2AX) and DNA damage were evaluated in human alveolar epithelial cells at 24 h after exposure. Overall, HVOF particles were the most cytotoxic to human alveolar cells, with cell viability half-maximal inhibitory concentration (IC50) values of 20.18 µg/cm2 and 1.79 µg/cm2 for PGFP and PGNP, respectively. Only the highest tested concentration of APS-PGFP caused a slight decrease in cell viability. Particle uptake, cell cycle arrest at S + G2/M and γ-H2AX augmentation were observed after exposure to all tested particles. However, higher levels of γ-H2AX were found in cells exposed to APS-derived particles (~16%), while cells exposed to HVOF particles exhibited increased levels of oxidative damage (~17% tail intensity) and ROS (~184%). Accordingly, APS and HVOF particles seem to exert their genotoxic effects by different mechanisms, highlighting that the health risks of these process-generated particles at industrial settings should not be underestimated.

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.

Nanoparticle exposure and hazard in the ceramic industry: an overview of potential sources, toxicity and health effects.

The ceramic industry is an industrial sector of great impact in the global economy that has been benefiting from advances in materials and processing technologies. Ceramic manufacturing has a strong potential for airborne particle formation and emission, namely of ultrafine particles (UFP) and nanoparticles (NP), meaning that workers of those industries are at risk of potential exposure to these particles. At present, little is known on the impact of engineered nanoparticles (ENP) on the environment and human health and no established Occupational Exposure Limits (OEL) or specific regulations to airborne nanoparticles (ANP) exposure exist raising concerns about the possible consequences of such exposure. In this paper, we provide an overview of the current knowledge on occupational exposure to NP in the ceramic industry and their impact on human health. Possible sources and exposure scenarios, a summary of the existing methods for evaluation and monitoring of ANP in the workplace environment and proposed Nano Reference Values (NRV) for different classes of NP are presented. Case studies on occupational exposure to ANP generated at different stages of the ceramic manufacturing process are described. Finally, the toxicological potential of intentional and unintentional ANP that have been identified in the ceramic industry workplace environment is discussed based on the existing evidence from in vitro and in vivo inhalation toxicity studies.

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.

The impact of paracetamol on selected biomarkers of the mollusc species Corbicula fluminea.

The Asian clam Corbicula fluminea is an invasive bivalve that has recently spread in Europe and currently represents a large portion of the aquatic biomass in specific areas. Because of the impacts that the species may have in invaded ecosystems, increased knowledge on the physiologic features of the species life-cycle under different environmental scenarios (e.g., contamination events) is critical to understand the dynamics of the invasion and resulting ecosystem imbalance. The presence of pharmaceutical residues in the aquatic environment has recently received great attention since high levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. The present article reports toxicological biochemical effects of paracetamol to Corbicula fluminea following short- and long-term exposures. Oxidative stress parameters were specially focused namely catalase (CAT), glutathione S-transferases (GSTs), and glutathione reductase (GRed). The effect of tested substances on lipid peroxidation was also investigated. Paracetamol did not induce alterations on CAT activity, caused a significant decrease of GSTs activity following short- and long-term exposure (LOEC values of 532.78 mg L(-1) and 30.98 µg L(-1) , respectively), and was responsible for a significant and dose-dependent decrease of GRed activity in short- and long-term exposures. These results indicate that exposure to paracetamol can provoke significant alterations on the cellular redox status of C. fluminea.

Redox Homeostasis Disclosed in the Saltmarsh Plant Halimione portulacoides upon Short Waterborne Exposure to Inorganic Mercury.

The saltmarsh plant Halimione portulacoides was shortly exposed to realistic levels of inorganic mercury (iHg) with the aim of investigating the adaptative processes of the roots and leaves regarding redox homeostasis, physiology, and Hg accumulation. Plants were collected at a contaminated (CONT) and a reference (REF) site to address the interference of contamination backgrounds. The influence of major abiotic variables (i.e., temperature and light) was also examined. Total Hg levels, antioxidant enzymes, lipid peroxidation (LPO), and photosynthetic activity were analyzed after 2 and 4 h of exposure. A poor accumulation of Hg in the roots was noticed, and no translocation to the stems and leaves was found, but plants from the CONT site seemed more prone to iHg uptake (in winter). Despite this, antioxidant modulation in the roots and leaves was found, disclosing, in winter, higher thresholds for the induction of enzymatic antioxidants in CONT leaves compared to REF plants, denoting that the former are better prepared to cope with iHg redox pressure. Consistently, CONT leaves exposed to iHg had remarkably lower LPO levels. Exposure did not impair photosynthetic activity, pinpointing H. portulacoides' ability to cope with iHg toxicity under very-short-term exposure. Biochemical changes were noticed before enhancements in accumulation, reinforcing the relevance of these responses in precociously signaling iHg toxicity.

Differential Cell Metabolic Pathways in Gills and Liver of Fish (White Seabream Diplodus sargus) Coping with Dietary Methylmercury Exposure.

Mercury (Hg) is a dangerous and persistent trace element. Its organic and highly toxic form, methylmercury (MeHg), easily crosses biological membranes and accumulates in biota. Nevertheless, understanding the mechanisms of dietary MeHg toxicity in fish remains a challenge. A time-course experiment was conducted with juvenile white seabreams, Diplodus sargus (Linnaeus, 1758), exposed to realistic levels of MeHg in feed (8.7 µg g-1, dry weight), comprising exposure (E; 7 and 14 days) and post-exposure (PE; 28 days) periods. Total Hg levels increased with time in gills and liver during E and decreased significantly in PE (though levels of control fish were reached only for gills), with liver exhibiting higher levels (2.7 times) than gills. Nuclear magnetic resonance (NMR)-based metabolomics revealed multiple and often differential metabolic changes between fish organs. Gills exhibited protein catabolism, disturbances in cholinergic neurotransmission, and changes in osmoregulation and lipid and energy metabolism. However, dietary MeHg exposure provoked altered protein metabolism in the liver with decreased amino acids, likely for activation of defensive strategies. PE allowed for the partial recovery of both organs, even if with occurrence of oxidative stress and changes of energy metabolism. Overall, these findings support organ-specific responses according to their sensitivity to Hg exposure, pointing out that indications obtained in biomonitoring studies may depend also on the selected organ.

Multiparametric in vitro genotoxicity assessment of different variants of amorphous silica nanomaterials in rat alveolar epithelial cells.

The hazard posed to human health by inhaled amorphous silica nanomaterials (aSiO2 NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO2 NM variants covering different sizes (7, 15, and 40 nm) and surface modifications (unmodified, phosphonate-, amino- and trimethylsilyl-modified) on rat alveolar epithelial (RLE-6TN) cells. Cytotoxicity was evaluated at 24 h after exposure to the aSiO2 NM variants by the lactate dehydrogenase (LDH) release and WST-1 reduction assays, while genotoxicity was assessed using different endpoints: DNA damage (single- and double-strand breaks [SSB and DSB]) by the comet assay for all aSiO2 NM variants; cell cycle progression and γ-H2AX levels (DSB) by flow cytometry for those variants that presented higher cytotoxic and DNA damaging potential. The variants with higher surface area demonstrated a higher cytotoxic potential (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_15_Phospho). SiO2_40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm2), a decrease in G0/G1 subpopulation was accompanied by a significant increase in S and G2/M sub-populations after exposure to all tested materials except for SiO2_40 which did not affect cell cycle progression. Based on the obtained data, the tested variants can be ranked for its genotoxic DNA damage potential as follows: SiO2_7 = SiO2_40 = SiO2_15_Unmod > SiO2_15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction.

Effects of anticholinesterase drugs on biomarkers and behavior of pumpkinseed, Lepomis gibbosus (Linnaeus, 1758).

The presence of pharmaceutical residues in the aquatic environment has recently received great attention, as potential adverse effects may arise from their presence. Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. However, other widespread anthropogenic contaminants - including pharmaceutical drugs - can exert toxic effects through ChE inhibition. Studies with aquatic species have shown that inhibition of ChE is associated with behavioral changes. Bearing this in mind, this work aimed to study the effects on individual behavior and acetylcholinesterase (AChE) activity of selected tissues of Lepomis gibbosus, after exposure to the anticholinesterasic drugs neostigmine and pyridostigmine. Results revealed that neostigmine significantly decreased the activity of AChE in the head (LOEC = 100 mg L(-1)), but not in dorsal muscle. On the other hand, pyridostigmine significantly decreased the activity of AChE in both the head (LOEC = 0.001 mg L(-1)) and dorsal muscle homogenates (LOEC = 100 mg L(-1)). The impairment of this enzymatic form was attained at ecologically relevant concentrations (in the case of pyridostigmine, for head AChE), which is an interesting finding, specially considering that these toxic effects occurred for a pharmaceutical compound. Contrarily, there were no significant differences in the behavior of L. gibbosus in any parameter, for neither drug. These results suggest that the behavioral parameters analyzed (scototaxis and lethargy) in L. gibbosus could not be regarded as suitable markers to assess the effects of drugs such as neostigmine and pyridostigmine. In contrast, the pattern of response elicited by cholinesterasic inhibition showed the usefulness of this toxicological parameter for the assessment of pharmaceuticals in the environment, namely anticholinesterasics.

Organ-Specific Metabolome Deciphering Cell Pathways to Cope with Mercury in Wild Fish (Golden Grey Mullet Chelon auratus).

Metabolomics is a powerful approach in evaluating the health status of organisms in ecotoxicological studies. However, metabolomics data reflect metabolic variations that are attributable to factors intrinsic to the environment and organism, and it is thus crucial to accurately evaluate the metabolome of the tissue/organ examined when it is exposed to no stressor. The metabolomes of the liver and gills of wild golden grey mullet (Chelon auratus) from a reference area were analyzed and compared by proton nuclear magnetic resonance (1H NMR)-based metabolomics. Both organs were characterized by amino acids, carbohydrates, osmolytes, nucleosides and their derivatives, and miscellaneous metabolites. However, similarities and differences were revealed in their metabolite profile and related to organ-specific functions. Taurine was predominant in both organs due to its involvement in osmoregulation in gills, and detoxification and antioxidant protective processes in liver. Environmental exposure to mercury (Hg) triggered multiple and often differential metabolic alterations in fish organs. Disturbances in ion-osmoregulatory processes were highlighted in the gills, whereas differential impairments between fish organs were pointed out in energy-producing metabolic pathways, protein catabolism, membrane stabilization processes, and antioxidant defense system, reflecting the induction of organ-specific adaptive and defensive strategies. Overall, a strict correlation between metabolites and organ-specific functions of fish gills and liver were discerned in this study, as well as organ-specific cytotoxicity mechanisms of Hg in fish.

Toxicity assessment of industrial engineered and airborne process-generated nanoparticles in a 3D human airway epithelial in vitro model.

The advanced ceramic technology has been pointed out as a potentially relevant case of occupational exposure to nanoparticles (NP). Not only when nanoscale powders are being used for production, but also in the high-temperature processing of ceramic materials there is also a high potential for NP release into the workplace environment. In vitro toxicity of engineered NP (ENP) [antimony tin oxide (Sb2O3•SnO2; ATO); zirconium oxide (ZrO2)], as well as process-generated NP (PGNP), and fine particles (PGFP), was assessed in MucilAir™ cultures at air-liquid interface (ALI). Cultures were exposed during three consecutive days to varying doses of the aerosolized NP. General cytotoxicity [lactate dehydrogenase (LDH) release, WST-1 metabolization], (oxidative) DNA damage, and the levels of pro-inflammatory mediators (IL-8 and MCP-1) were assessed. Data revealed that ENP (5.56 µg ATO/cm2 and 10.98 µg ZrO2/cm2) only caused mild cytotoxicity at early timepoints (24 h), whereas cells seemed to recover quickly since no significant changes in cytotoxicity were observed at late timepoints (72 h). No meaningful effects of the ENP were observed regarding DNA damage and cytokine levels. PGFP affected cell viability at dose levels as low as ∼9 µg/cm2, which was not seen for PGNP. However, exposure to PGNP (∼4.5 µg/cm2) caused an increase in oxidative DNA damage. These results indicated that PGFP and PGNP exhibit higher toxicity potential than ENP in mass per area unit. However, the presence of a mucociliary apparatus, as it occurs in vivo as a defense mechanism, seems to considerably attenuate the observed toxic effects. Our findings highlight the potential hazard associated with exposure to incidental NP in industrial settings.

In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures.

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air-liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.

Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM).

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

Unravelling the Potential Cytotoxic Effects of Metal Oxide Nanoparticles and Metal(Loid) Mixtures on A549 Human Cell Line.

Humans are typically exposed to environmental contaminants' mixtures that result in different toxicity than exposure to the individual counterparts. Yet, the toxicology of chemical mixtures has been overlooked. This work aims at assessing and comparing viability and cell cycle of A549 cells after exposure to single and binary mixtures of: titanium dioxide nanoparticles (TiO2NP) 0.75-75 mg/L; cerium oxide nanoparticles (CeO2NP) 0.0.75-10 µg/L; arsenic (As) 0.75-2.5 mg/L; and mercury (Hg) 5-100 mg/L. Viability was assessed through water-soluble tetrazolium (WST-1) and thiazolyl blue tetrazolium bromide (MTT) (24 h exposure) and clonogenic (seven-day exposure) assays. Cell cycle alterations were explored by flow cytometry. Viability was affected in a dose- and time-dependent manner. Prolonged exposure caused inhibition of cell proliferation even at low concentrations. Cell-cycle progression was affected by TiO2NP 75 mg/L, and As 0.75 and 2.5 µg/L, increasing the cell proportion at G0/G1 phase. Combined exposure of TiO2NP or CeO2NP mitigated As adverse effects, increasing the cell surviving factor, but cell cycle alterations were still observed. Only CeO2NP co-exposure reduced Hg toxicity, translated in a decrease of cells in Sub-G1. Toxicity was diminished for both NPs co-exposure compared to its toxicity alone, but a marked toxicity for the highest concentrations was observed for longer exposures. These findings prove that joint toxicity of contaminants must not be disregarded.

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.

Optimization of the harvesting and freezing conditions of human cell lines for DNA damage analysis by the alkaline comet assay.

The comet assay is a commonly used method for in vitro and in vivo genotoxicity assessment. This versatile assay can be performed in a wide range of tissues and cell types. Although most of the studies use samples immediately processed after collection, frozen biological samples can also be used. The present study aimed to optimize a collection and freezing protocol to minimize the DNA damage associated with these procedures in human cell line samples for comet assay analysis. This study was conducted in glial A172 and lung alveolar epithelial A549 cells. Two cell detachment methods (mechanical vs enzymatic) and two cryoprotective media [FBS + 10% DMSO vs Cell Culture Media (CCM) + 10% DMSO] were tested, and DNA damage assessed at four time points following storage at -80 °C (one, two, four and eight weeks). In both cell lines, no differences in % tail intensity were detected between fresh and frozen cells up to eight weeks, irrespective of the harvesting method and freezing medium used. However, freshly isolated A172 cells exhibited a significant lower DNA damage when resuspended in CCM + 10% DMSO, while for A549 fresh cells the preferable harvesting method was the enzymatic one since it induced less DNA damage. Although both harvesting methods and cryoprotective media tested were found suitable, our data indicate that enzymatic harvesting and cryopreservation in CCM + 10% DMSO is a preferable method for DNA integrity preservation of human cell line samples for comet assay analysis. Our data also suggest that CCM is a preferable and cost-effective alternative to FBS in cryopreservation media. This optimized protocol allows the analysis of in vitro cell samples collected and frozen at different locations, with minimal interference on the basal DNA strand break levels in samples kept frozen up to eight weeks.