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Firefighters are the principal line of defense against fires, being at elevated risk of exposure to health-relevant pollutants released during fires and burning processes. Although many biomonitoring studies exist, only a limited number of human in vitro investigations in fire risk assessment are currently available. In vitro studies stand out as valuable tools to assess the toxicity mechanisms involved following exposure to fire pollutants at a cellular level. The aim of the present review was to contextualize existing in vitro studies using human cell models exposed to chemicals emitted from fire emissions and wood smoke and discuss the implications of the observed toxic outcomes on adverse health effects detected in firefighters. Most of the reported in vitro investigations focused on monocultures respiratory models and exposure to particulate matter (PM) extracts collected from fire effluents. Overall, (1) a decrease in cellular viability, (2) enhanced oxidative stress, (3) increased pro-inflammatory cytokines levels and (4) elevated cell death frequencies were noted. However, limited information remains regarding the toxicity mechanisms initiated by firefighting activities. Hence, more studies employing advanced in vitro models and exposure systems using human cell lines are urgently needed taking into consideration different routes of exposure and health-related pollutants released from fires. Data are needed to establish and define firefighters' occupational exposure limits and to propose mitigation strategies to promote beneficial human health.
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Poluentes Ocupacionais do Ar , Poluentes Ambientais , Bombeiros , Exposição Ocupacional , Humanos , Exposição Ocupacional/efeitos adversos , Fumaça/efeitos adversos , Material Particulado/toxicidade , Poluentes Ocupacionais do Ar/toxicidade , Poluentes Ocupacionais do Ar/análiseRESUMO
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.
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Nanopartículas , Sistema Respiratório , Humanos , Poeira , Exposição por Inalação/efeitos adversos , Nanopartículas/toxicidadeRESUMO
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.
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Células Epiteliais Alveolares , Dano ao DNA , Células Epiteliais Alveolares/metabolismo , Sobrevivência Celular , Células Epiteliais/metabolismo , Humanos , Estresse Oxidativo , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
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Poluentes Ocupacionais do Ar , Nanopartículas , Exposição Ocupacional , Poluentes Ocupacionais do Ar/análise , Poluentes Ocupacionais do Ar/toxicidade , Cerâmica/toxicidade , Monitoramento Ambiental , Humanos , Exposição por Inalação/efeitos adversos , Exposição por Inalação/análise , Nanopartículas/toxicidade , Exposição Ocupacional/análise , Tamanho da PartículaRESUMO
Immobilization of nanoparticles on inorganic supports has been recently developed, resulting in the creation of nanocomposites. Concerning titanium dioxide nanoparticles (TiO2 NPs1), these have already been developed in conjugation with clays, but so far there are no available toxicological studies on these nanocomposites. The present work intended to evaluate the hepatic toxicity of nanocomposites (C-TiO22), constituted by rutile TiO2 NPs immobilized in nanokaolin (NK3) clay, and its individual components. These nanomaterials were analysed by means of FE-SEM4 and DLS5 analysis for physicochemical characterization. HepG2 cells were exposed to rutile TiO2 NPs, NK clay and C-TiO2 nanocomposite, in the presence and absence of serum for different exposure periods. Possible interferences with the methodological procedures were determined for MTT,6 neutral red uptake, alamar blue (AB), LDH,7 and comet assays, for all studied nanomaterials. Results showed that MTT, AB and alkaline comet assay were suitable for toxicity analysis of the present materials after slight modifications to the protocol. Significant decreases in cell viability were observed after exposure to all studied nanomaterials. Furthermore, an increase in HepG2 DNA damage was observed after shorter periods of exposure in the absence of serum proteins and longer periods of exposure in their presence. Although the immobilization of nanoparticles in micron-sized supports could, in theory, decrease the toxicity of single nanoparticles, the selection of a suitable support is essential. The present results suggest that NK clay is not the appropriate substrate to decrease TiO2 NPs toxicity. Therefore, for future studies, it is critical to select a more appropriate substrate for the immobilization of TiO2 NPs.
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Nanopartículas Metálicas/toxicidade , Nanocompostos/toxicidade , Titânio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Hep G2 , Humanos , Nanopartículas Metálicas/química , Testes de Mutagenicidade/métodos , Nanocompostos/química , Titânio/químicaRESUMO
Due to their unique electronic and optical features, gold nanoparticles (AuNP) have received a great deal of attention for application in different fields such as catalysis, electronics, and biomedicine. The large-volume manufacturing predicted for future decades and the inevitable release of these substances into the environment necessitated an assessment of potential adverse human and ecological risks due to exposure to AuNP. Accordingly, this study aimed to examine the acute and developmental toxicity attributed to a commercial suspension of Au nanorods stabilized with cetyltrimethylammonium bromide (CTAB-AuNR) using early embryonic stages of zebrafish (Danio rerio), a well-established model in ecotoxicology. Zebrafish embryos were exposed to CTAB-AuNR (0-150 µg/L) to determine for developmental assessment until 96 hr post fertilization (hpf) and lethality. Uptake of CTAB-AuNR by embryos and nanoparticles potential to induce DNA damage was also measured at 48 and 96 hpf. Analysis of the concentration-response curves with cumulative mortality at 96 hpf revealed a median lethal concentration (LC50,96h) of 110.2 µg/L. At sublethal concentrations, CTAB-AuNR suspensions were found to produce developmental abnormalities such as tail deformities, pericardial edema, decreased body length, and delayed eye, head, and tail elongation development. Further, less than 1% of the initial concentration of CTAB-AuNR present in the exposure media was internalized by zebrafish embryos prior to (48 hpf) and after hatching (96 hpf). In addition, no marked DNA damage was detected in embryos after exposure to CTAB-AuNR. Overall, CTAB-AuNR suspensions produced lethal and sublethal effects on zebrafish embryos with possible repercussions in fitness of adult stages. However, these results foresee a low risk for fish since the observed effects occurred at concentrations above the levels expected to find in the aquatic environment.
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Desenvolvimento Embrionário/efeitos dos fármacos , Ouro/toxicidade , Nanotubos/toxicidade , Peixe-Zebra/embriologia , Animais , Ensaio Cometa , Embrião não Mamífero/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Nanotubos/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Coleostephus myconis (L.) Rchb.f. (Asteraceae) is a highly disseminated plant species with ruderal and persistent growth. Owing to its advantageous agronomic properties, C. myconis might have industrial applications. However, this species needs to be comprehensively characterized before any potential use. In a previous study, the phenolic composition and antioxidant activity of different C. myconis tissues were characterized. This investigation was extended to examine the cytotoxic potential of selected plant tissues (flowers and green parts) using a HepG2 cell line by utilizing the lysosomal neutral red uptake assay or mitochondrial (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. In addition, the macronutrients content, lipophilic compounds (fatty acids, tocopherols), and amino acids were also determined. C. myconis flowers were used in the senescence stage, which was previously identified as the stage that presented maximal phenolic content and highest antioxidant activity. In contrast, stems and leaves were employed due to their high biomass proportion. Regarding cytotoxicity, mitochondrial and lysosomal damage was only significant when HepG2 cells were exposed to the highest extract concentrations (stems and leaves, 0.9 mg/ml; senescent flowers, 0.3 mg/ml). Chemically, the senescent flowers were mostly characterized by their high levels of fat, amino acids (especially threonine), oleic acid, ß-, and γ-tocopherol, while stems and leaves contained high concentrations of carbohydrates, linolenic acid, and α-tocopherol. In general, these results provide information regarding the threshold concentrations of C. myconis extracts that might be used in different applications without toxicity hazards.
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Asteraceae/química , Asteraceae/toxicidade , Citotoxinas/análise , Citotoxinas/toxicidade , Flores/química , Flores/toxicidade , Células Hep G2 , Humanos , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Folhas de Planta/química , Folhas de Planta/toxicidade , Caules de Planta/química , Caules de Planta/toxicidadeRESUMO
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.
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Bioensaio , Nanopartículas de Magnetita/toxicidade , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Nanopartículas de Magnetita/química , Neuroglia/patologia , Neurônios/patologia , Ácido Oleico/química , Reprodutibilidade dos Testes , Medição de Risco , Dióxido de Silício/química , Espectrofotometria , Fatores de TempoRESUMO
The increasing prevalence of dementia demands innovative solutions; however, existing technological products often lack tailored support for individuals living with this condition. The Living Lab approach, as a collaborative innovation method, holds promise in addressing this issue by actively involving end-users in the design and development of solutions adapted to their needs. Despite this potential, the approach still faces challenges due to its lack of recognition as a research methodology and its absence of tailored guidelines, particularly in dementia care, prompting inquiries into its effectiveness. This narrative review aims to fill this gap by identifying and analysing digital health Living Labs focusing on dementia solutions. Additionally, it proposes guidelines for enhancing their operations, ensuring sustainability, scalability, and greater impact on dementia care. Fifteen Living Labs were identified and analyzed. Based on trends, best practices, and literature, the guidelines emphasize user engagement, interdisciplinary collaboration, technological infrastructure, regulatory compliance, transparent innovation processes, impact measurement, sustainability, scalability, dissemination, and financial management. Implementing these guidelines can enhance the effectiveness and long-term impact of Living Labs in dementia care, fostering new collaborations globally.
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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.
Assuntos
Células Epiteliais Alveolares , Nanoestruturas , Ratos , Humanos , Animais , Dióxido de Silício/toxicidade , Dano ao DNA , Ensaio Cometa , Nanoestruturas/toxicidadeRESUMO
Surfaces with antimicrobial properties are gaining notoriety as an efficient method to avoid surface contamination. Self-disinfecting paints are a promising strategy towards cleaner indoor environments by preventing the colonization of walls with microorganisms. However, its widespread use needs an appropriate toxicological safety evaluation due to the potential for biological disturbance associated to its biocidal activity. In this work, the cyto- and genotoxic assessment of two self-disinfecting paints containing the antimicrobial substances triclosan (TCS) and isoborneol (ISB) is performed. HaCaT and A549 cell lines models were selected for the in vitro assessment. To evaluate the cytotoxicity, tests by direct contact and on extracts obtained from leaching were performed following ISO 10993, whereas the genotoxicity was assessed by comet assay and cytokinesis-block micronucleus (CBMN) assay. The results showed low levels of cyto- and genotoxicity under the models and conditions tested, indicating that these substances have commercial potential.
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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 (Sb2O3â¢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 â¼9 µg/cm2, which was not seen for PGNP. However, exposure to PGNP (â¼4.5 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.
Assuntos
Nanopartículas , Sobrevivência Celular , Dano ao DNA , Humanos , Nanopartículas/toxicidade , Estresse Oxidativo , Tamanho da PartículaRESUMO
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.
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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.
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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.
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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.
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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.
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Células Epiteliais Alveolares , Ensaio Cometa/métodos , Criopreservação/métodos , Dano ao DNA , Neuroglia , Manejo de Espécimes/métodos , Células A549 , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Separação Celular/métodos , Crioprotetores/farmacologia , Meios de Cultura/farmacologia , Quebras de DNA , Dimetil Sulfóxido/farmacologia , Sangue Fetal , Humanos , Concentração de Íons de Hidrogênio , Neuroglia/efeitos dos fármacos , Soluções/farmacologia , Fatores de TempoRESUMO
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.
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Nanopartículas de Magnetita/toxicidade , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Estresse Oxidativo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Dano ao DNA , DNA Glicosilases/farmacologia , Relação Dose-Resposta a Droga , Glioblastoma/patologia , Glutationa/metabolismo , Humanos , Nanopartículas de Magnetita/química , Neuroblastoma/patologia , Ácido Oleico , Oxirredução , Tamanho da Partícula , Espécies Reativas de Oxigênio , Dióxido de Silício , Propriedades de SuperfícieRESUMO
Due to their unique physicochemical properties, including superparamagnetism, iron oxide nanoparticles (ION) have a number of interesting applications, especially in the biomedical field, that make them one of the most fascinating nanomaterials. They are used as contrast agents for magnetic resonance imaging, in targeted drug delivery, and for induced hyperthermia cancer treatments. Together with these valuable uses, concerns regarding the onset of unexpected adverse health effects following exposure have been also raised. Nevertheless, despite the numerous ION purposes being explored, currently available information on their potential toxicity is still scarce and controversial data have been reported. Although ION have traditionally been considered as biocompatible - mainly on the basis of viability tests results - influence of nanoparticle surface coating, size, or dose, and of other experimental factors such as treatment time or cell type, has been demonstrated to be important for ION in vitro toxicity manifestation. In vivo studies have shown distribution of ION to different tissues and organs, including brain after passing the blood-brain barrier; nevertheless results from acute toxicity, genotoxicity, immunotoxicity, neurotoxicity and reproductive toxicity investigations in different animal models do not provide a clear overview on ION safety yet, and epidemiological studies are almost inexistent. Much work has still to be done to fully understand how these nanomaterials interact with cellular systems and what, if any, potential adverse health consequences can derive from ION exposure.
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Compostos Férricos/toxicidade , Nanopartículas/toxicidade , Animais , Compostos Férricos/análise , Compostos Férricos/metabolismo , Humanos , Nanopartículas/análise , Nanopartículas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aerial parts of Medicago sativa L. have been used as food and its consumption has been associated with health benefits, one among the most important being menopausal symptoms control. This work was aimed to explore possible pharmacological effects of two new alfalfa-derived products that have recently emerged as daily beverage preparations. In exploring their potential estrogenic effects, they produced no relevant alteration in the uterus. However, lowering glucose levels until normal values without causing further hypoglycemic effect were observed, when rats were treated with 1.5 g/kg/day samples. In vivo acute toxicity was not found when the alfalfa products were tested up to 3 g/kg rat weight. Furthermore, in vitro studies were conducted to assess their possible toxic effects. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase tests were carried out on the Caco-2 cell model to determine cell viability and membrane integrity. A concentration-dependent effect was observed, with a significant decrease in cell viability after exposure to concentrations of alfalfa product up to 100 mg/mL (after 3 h of incubation) and 50 mg/mL (after 24 h of treatment). Although in vitro level, the decrease in cell viability at these still low doses may underlie some toxicity, making necessary additional studies before any recommendation of a sustained consumption of these products by humans.