Cardiopulmonary toxicity of pulmonary exposure to occupationally relevant zinc oxide nanoparticles

Exposure to zinc oxide (ZnO) metal fumes is linked to adverse human health effects; however, the hazards of ZnO nanoparticles (ZnONPs) remain unclear. To determine pulmonary exposure to occupationally relevant ZnONPs cause cardiopulmonary injury, Sprague-Dawley rats were exposed to ZnONPs via intratracheal (IT) instillation and inhalation. The relationship between intrapulmonary zinc levels and pulmonary oxidative-inflammatory responses 72 h after ZnONP instillation was determined in bronchoalveolar lavage fluid (BALF). Instilled ZnONPs altered zinc balance and increased the levels of total cells, neutrophils, lactate dehydrogenase (LDH) and total protein in BALF and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in blood after 72 h. The ZnONPs accumulated predominantly in the lungs over 24 h, and trivial amounts of zinc were determined in the heart, liver, kidneys and blood. Furthermore, the inflammatory-oxidative responses induced by occupationally relevant levels of 1.1 and 4.9 mg/m³ of ZnONP inhalation for 2 weeks were determined in BALF and blood at 1, 7 and 30 days post-exposure. Histopathological examinations of the rat lungs and hearts were performed. Inhalation of ZnONP caused an inflammatory cytological profile. The total cell, neutrophil, LDH and total protein levels were acutely increased in the BALF, and there was an inflammatory pathology in the lungs. There were subchronic levels of white blood cells, granulocytes and 8-OHdG in the blood. Cardiac inflammation and the development of fibrosis were detected 7 days after exposure. Degeneration and necrosis of the myocardium were detected 30 days after exposure. The results demonstrate that ZnONPs cause cardiopulmonary impairments. These findings highlight the occupational health effects for ZnONP-exposed workers.     

Evidence and uptake routes for Zinc oxide nanoparticles through the gastrointestinal barrier in Xenopus laevis

The developmental toxicity of nanostructured materials, as well as their impact on the biological barriers, represents a crucial aspect to be assessed in a nanosafety policy framework. Nanosized metal oxides have been demonstrated to affect Xenopus laevis embryonic development, with nZnO specifically targeting the digestive system. To study the mechanisms of the nZnO-induced intestinal lesions, we tested two different nominally sized ZnO nanoparticles (NPs) at effective concentrations. Advanced microscopy techniques and molecular marker analyses were applied in order to describe the NP-epithelial cell interactions and the mechanisms driving NP toxicity and translocation through the intestinal barrier. We attributed the toxicity to NP-induced cell oxidative damage, the small-sized NPs being the more effective. This outcome is sustained by a marked increase in anti-oxidant genes' expression and high lipid peroxidation level in the enterocytes, where disarrangement of the cytoskeleton and cell junctions' integrity were evidenced. These events led to diffuse necrotic changes in the intestinal barrier, and trans- and paracellular NP permeation through the mucosa. The uptake routes, leading NPs to cross the intestinal barrier and reach secondary target tissues, have been documented. nZnOs embryotoxicity was confirmed to be crucially mediated by the NPs' reactivity rather than their dissolved ions. The ZnO NPs' ability to overwhelm the intestinal barrier must be taken into high consideration for a future design of safer ZnO NPs.     

Acute inhalation toxicity of cerium oxide nanoparticles in rats

The aim of the present study was to assess the acute toxic potential of cerium oxide nanoparticles (CeO₂ NPs) in rats when exposed through the head and nose inhalation route. The rats were exposed to CeO₂ NPs and the resultant effects if any, to cause cytotoxicity, oxidative stress and inflammation in the lungs were evaluated on a 24 h, 48 h and 14 day post exposure period. Our results showed a significant decrease in the cell viability, with the increase of lactate dehydogenase, total protein and alkaline phosphatase levels in the bronchoalveolar lavage fluid (BALF) of the exposed rats. Total leukocyte count and the percentage of neutrophils in BALF were elevated within 24 h of post exposure. The concentrations of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) were significantly increased in the BALF and in the blood throughout the observation period. The level of malondialdehyde was elevated with the decreased levels of intracellular reduced glutathione (GSH) in the lung after exposure. The alveolar macrophages (AMs) and neutrophils overloaded with phagocytosed CeO₂ NPs were observed along with non-phagocytosed free CeO₂ NPs that were deposited over the epithelial surfaces of the bronchi, bronchiole and alveolar regions of lungs within 24 h of post exposure and were consistent throughout the observation period. A well distributed, multifocal pulmonary microgranulomas due to impairment of clearance mechanism leading to biopersistence of CeO₂ NPs for an extended period of time were observed at the end of the 14 day post exposure period. These results suggest that acute exposure of CeO₂ NPs through inhalation route may induce cytotoxicity via oxidative stress and may lead to a chronic inflammatory response.     

Zinc oxide nanoparticles in modern sunscreens: An analysis of potential exposure and hazard

Abstract

Sunscreens containing metal oxide nanoparticles appear transparent on the skin and provide excellent protection against sunburn caused by UV radiation. While it is likely that nanoparticles remain on the surface of the skin of healthy adult humans, and thus are considered safe for use in sunscreens, there has been no comprehensive assessment of the impact on human health from exposure to the metal oxide nanoparticles destined for use in sunscreens, either in the workplace during the manufacturing process, in long-term use across a range of skin conditions, or upon release into the broader environment, either accidentally or consequent of normal sunscreen use. In this review, we focus on zinc oxide nanoparticles destined for use in modern sunscreens, and discuss the potential for human exposure and the health hazard at each stage of their manufacture and use. We highlight where there is a need for further research.     

Cerium oxide nanoparticle-induced pulmonary inflammation and alveolar macrophage functional change in rats

Abstract

The use of cerium compounds as diesel fuel catalyst results in the emission of cerium oxide nanoparticles (CeO₂) in the exhaust. This study characterized the potential effects of CeO₂ exposure on lung toxicity. Male Sprague Dawley rats were exposed to CeO₂ by a single intratracheal instillation at 0.15, 0.5, 1, 3.5 or 7 mg/kg body weight. At 1 day after exposure, CeO₂ significantly reduced NO production, but increased IL-12 production, by alveolar macrophages (AM) in response to ex vivo lipopolysacchride (LPS) challenge, and caused AM apoptosis, through activation of caspases 9 and 3. CeO₂ exposure markedly increased suppressor of cytokine signaling-1 at 1-day and elevated arginase-1 at 28-day post exposure in lung cells, while osteopontin was significantly elevated in lung tissue at both time points. CeO₂ induced inflammation, cytotoxicity, air/blood barrier damage, and phospholipidosis with enlarged AM. Thus, CeO₂ induced lung inflammation and injury in lungs which may lead to fibrosis.     

Cerium oxide nanoparticles protects against acrylamide induced toxicity in HepG2 cells through modulation of oxidative stress

Acrylamide (AA) is a toxic chemical compound found in cooked foods. Considerable evidences suggest that oxidative stress and mitochondrial dysfunction are contributed to AA toxicity. Ceric oxide (CeO₂) nanoparticles (nano-ceria) have the potential to be developed as a therapeutic for oxidative stress insults due to their catalytic antioxidant properties. In this study we investigated, whether nano-ceria exerted a protective effect against AA-induced cytotoxicity and oxidative damage. HepG2 human cancer cell lines were exposed to nano-ceria (50, 100, and 200?µM) and after 30?min, AA in the half maximal inhibitory concentration (IC50) concentration (200?µM) was added to the cells. Twenty four hours later, cellular viability, reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and cellular levels of glutathione (GSH) were assayed. AA decreased cell viability and pretreatment with nano-ceria significantly decreased AA-induced cytotoxicity. In addition, nano-ceria alleviated AA-induced ROS generation and LPO and depressed GSH level. Our results suggested that nano-ceria prevented cellular and oxidative damage induced by AA.     

Comparative toxicity of silicon dioxide,silver and iron oxide nanoparticles after repeated oral administration to rats

Although silicon dioxide (SiO₂), silver (Ag) and iron oxide (Fe₂O₃) nanoparticles are widely used in diverse applications from food to biomedicine, in vivo toxicities of these nanoparticles exposed via the oral route remain highly controversial. To examine the systemic toxicity of these nanoparticles, well‐dispersed nanoparticles were orally administered to Sprague–Dawley rats daily over a 13‐week period. Based on the results of an acute toxicity and a 14‐day repeated toxicity study, 975.9, 1030.5 and 1000 mg kg^–1 were selected as the highest dose of the SiO₂, Ag and Fe₂O₃ nanoparticles, respectively, for the 13‐week repeated oral toxicity study. The SiO₂ and Fe₂O₃ nanoparticles did not induce dose‐related changes in a number of parameters associated with the systemic toxicity up to 975.9 and 1000 mg kg^–1, respectively, whereas the Ag nanoparticles resulted in increases in serum alkaline phosphatase and calcium as well as lymphocyte infiltration in liver and kidney, raising the possibility of liver and kidney toxicity induced by the Ag nanoparticles. Compared with the SiO₂ and Fe₂O₃ nanoparticles showing no systemic distribution in all tissues tested, the Ag concentration in sampled blood and organs in the Ag nanoparticle‐treated group significantly increased with a positive and/or dose‐related trend, meaning that the systemic toxicity of the Ag nanoparticles, including liver and kidney toxicity, might be explained by extensive systemic distribution of Ag originating from the Ag nanoparticles. Our current results suggest that further study is required to identify that Ag detected outside the gastrointestinal tract were indeed a nanoparticle form or ionized form. Copyright © 2015 John Wiley & Sons, Ltd.     

Repeated exposure to iron oxide nanoparticles causes testicular toxicity in mice

The aim of this study was to determine whether repeated exposure to iron oxide nanoparticles (Fe₂O₃‐NPs) could be toxic to mice testis. Fe₂O₃‐NPs (25 and 50 mg/kg) were intraperitoneally administered into mice once a week for 4 weeks. Our study showed that Fe₂O₃‐NPs have the ability to cross the blood‐testis barrier to get into the testis. The findings showed that exposure resulted in the accumulation of Fe₂O₃‐NPs which was evidenced from the iron content and accumulation in the testis. Furthermore, 25 and 50 mg/kg Fe₂O₃‐NPs administration increased the reactive oxygen species, lipid peroxidation, protein carbonyl content, glutathione peroxidase activity, and nitric oxide levels with a concomitant decrease in the levels of antioxidants—superoxide dismutase, catalase, glutathione, and vitamin C. Increased expression of Bax, cleaved‐caspase‐3, and cleaved‐PARP confirms apoptosis. Serum testosterone levels increased with increased concentration of Fe₂O₃‐NPs exposure. In addition, the histopathological lesions like vacuolization, detachment, and sloughing of germ cells were also observed in response to Fe₂O₃‐NPs treatment. The data from our study entailed that testicular toxicity caused by Fe₂O₃‐NPs exposure may be associated with Fe₂O₃‐NPs accumulation leading to oxidative stress and apoptosis. Therefore, precautions should be taken in the safe use of Fe₂O₃‐NPs to avoid complications in the fertility of males. Further research will unravel the possible molecular mechanisms on testicular toxicity of Fe₂O₃‐NPs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 594–608, 2017.     

Investigation into the pulmonary inflammopathology of exposure to nickel oxide nanoparticles in mice

We investigated the effects of nickel oxide nanoparticles (NiONPs) on the pulmonary inflammopathology. NiONPs were intratracheally installed into mice, and lung injury and inflammation were evaluated between 1 and 28 days. NiONPs caused significant increases in LDH, total protein, and IL-6 and a decrease in IL-10 in the BALF and increases in 8-OHdG and caspase-3 in lung tissues at 24 h. Airway inflammation was present in a dose-dependent manner from the upper to lower airways at 24 h of exposure as analyzed by SPECT. Lung parenchyma inflammation and small airway inflammation were observed by CT after NiONP exposure. 8-OHdG in lung tissues had increased with formation of fibrosis at 28 days. Focal adhesion was the most important pathways identified at 24 h as determined by protemics, whereas glutathione metabolism was the most important identified at 28 days. Our results demonstrated the pulmonary inflammopathology caused by NiONPs based on image-to-biochemical approaches.     

Zinc oxide nanoparticles induced gene mutation at the HGPRT locus and cell cycle arrest associated with apoptosis in V‐79 cells

In recent years, the large‐scale production of ZnO nanoparticles (NPs) for various applications is increasing exponentially and may pose serious health issues when inhaled either during occupational exposure or in consumer settings. The mechanisms underlying the toxicity of NPs have recently been studied intensively. Despite the existing studies, the mutagenicity of ZnO NPs in the eukaryotic system is still unclear. Therefore, the aim of the present study was to investigate the mutagenic potential of ZnO NPs using Chinese hamster lung fibroblast cells (V‐79) as an in‐vitro model. The study has demonstrated a significant uptake of ZnO NPs by flow cytometry with the confirmation of transmission electron microscopy. A reduction in cell viability was observed with a concomitant increase in reactive oxygen species (**P < 0.01, ***P < 0.001) after ZnO NP (1‐20 μg/mL) exposure. Excessive reactive oxygen species can induce oxidative stress, which leads to genotoxic insult, and further gene mutation. Apart from measuring the genotoxicity by Comet assay, a change of 2.84‐fold in the HGPRT gene mutant frequency was observed by the mammalian gene forward mutation assay. All the genotoxicity endpoints such as chromosomal break, DNA damage and mutagenicity were observed at 6 hours of ZnO NP exposure. Our results also showed that ZnO NPs manifested the cell cycle arrest, ultrastructural modifications and further cell death. A significant (**P < 0.01, ***P < 0.001) increase in the apoptotic cells was detected using annexin V‐fluorescein isothiocyanate/propidium iodide double staining by flow cytometry. Our findings presented here clearly stimulate the need for careful regulations of ZnO NPs.     

Different in vitro exposure regimens of murine primary macrophages to silver nanoparticles induce different fates of nanoparticles and different toxicological and functional consequences

Silver nanoparticles (Ag-NPs) are used in a variety of consumers’ goods. Their toxicological impact is currently intensely studied, mostly upon acute exposure, but their intracellular dissolution and fate is rather poorly documented. In this study, murine primary macrophages were exposed to a single high but non-lethal dose of Ag-NPs or to repeated, low doses of Ag-NPs. Cells were either collected immediately after acute exposure or after 72?h of recovery in the NP-free exposure medium. Ag intracellular content and distribution were analyzed by particle-induced X-ray emission, transmission electron microscopy coupled to energy-dispersive spectroscopy analysis and inductively coupled plasma mass spectrometry. In parallel, macrophage functionality as well as inflammatory and thiol-responses were assessed after Ag-NP exposure. We show that Ag accumulation in macrophages is similar upon acute and repeated exposure to Ag-NPs, and that Ag is partly expelled from cells during the 72?h recovery stage. However, acute exposure leads to a strong response of macrophages, characterized by reduced mitochondrial membrane potential, phagocytic capacity and nitric oxide (NO) production upon lipopolysaccharide (LPS) stimulation. Under this condition, we also show an increased release of proinflammatory cytokines as well as a decreased release of anti-inflammatory cytokines. This response is reversible since these biomarkers reach their basal level after the recovery phase; and is much less intense in repeatedly exposed cells. These results suggest that repeated exposure of macrophages to Ag-NPs, which is a more realistic exposure scenario than acute exposure, leads to significant Ag intracellular accumulation but a much less intense toxicological response.     

Health hazards of nanoparticles: understanding the toxicity mechanism of nanosized ZnO in cosmetic products

In recent years, nanoparticles are being used extensively in personal healthcare products such as cosmetics, sunscreens, soaps, and shampoos. Particularly, metal oxide nanoparticles are gaining competence as key industrial constituents, progressing toward a remarkable rise in their applications. Zinc oxide and titanium oxide nanoparticles are the most commonly employed metal oxide nanoparticles in sunscreens, ointments, foot care, and over the counter topical products. Dermal exposure to these metal oxides predominantly occurs through explicit use of cosmetic products and airway exposure to nanoparticle dusts is primarily mediated via occupational exposure. There is a compelling need to understand the toxicity effects of nanoparticles which can easily enter the cells and induce oxidative stress. Consequently, these products have become a direct source of pollution in the environment and thereby greatly impact our ecosystem. A complete understanding of the toxicity mechanism of nano-ZnO is intended to resolve whether and to what extent such nanoparticles may pose a threat to the environment and to human beings. In this review article, we have discussed the characteristics of metal oxide nanoparticles and its applications in the cosmetic industry. We have also highlighted about their toxicity effects and their impact on human health.     

Very small superparamagnetic iron oxide nanoparticles: Long-term fate and metabolic processing in atherosclerotic mice

We investigated the biotransformation of very small superparamagnetic iron oxide nanoparticles (VSOP) in atherosclerotic LDLR^?/? mice. Transmission electron microscopy revealed an uptake of VSOP not only by macrophages but also by endothelial cells in liver, spleen, and atherosclerotic lesions and their accumulation in the lysosomal compartment. Using magnetic particle spectroscopy (MPS), we show that the majority of VSOP's superparamagnetic iron was degraded within 28?days. MPS spectrum shape indicated changes in the magnetic properties of VSOP during the biodegradation process. Experiments with primary murine bone marrow derived macrophages, primary murine liver sinusoidal endothelial cells, and primary human aortic endothelial cells demonstrated that loading with VSOP induced a differential response of cellular iron homeostasis mechanisms with increased levels of ferritin and iron transport proteins in macrophages and increased levels of ferritin in endothelial cells.     

The toxicity and distribution of iron oxide–zinc oxide core‐shell nanoparticles in C57BL/6 mice after repeated subcutaneous administration

Therapeutic cancer vaccines promote immune responses by delivering tumour‐specific antigens. Recently, we developed iron oxide (Fe₃O₄)–zinc oxide (ZnO) core‐shell nanoparticles (CSNPs) as carriers for antigen delivery into dendritic cells (DCs), and the CSNPs were injected subcutaneously into C57BL/6 mice to examine the systemic toxicity, tissue distribution and excretion of the CSNPs. The doses injected were 0, 4, 20 and 200 mg kg^–1 weekly for 4 weeks. No significant changes were observed after the CSNPs administration with respect to mortality, clinical observations, body weight, food intake, water consumption, urinalysis, haematology, serum biochemistry,and organ weights. A dose‐dependent increase in granulomatous inflammation was observed at the injection site of the CSNP‐treated animals, but no other histopathological lesions in other organs could be attributed to the CSNPs. The Zn concentration, which is an indicator for CSNPs, was not significantly higher in the sampled tissues, urine, or faeces after the CSNP injection. In contrast, the Zn concentration at the subcutaneous skin of the site injected with the CSNPs increased in a dose‐dependent manner, along with a macroscopic deposition of the CSNPs. The CSNP residue at the injection site resulted in a foreign body response with the appearance of macrophage infiltration, but otherwise did not show any systemic distribution or toxicity at up to 200 mg kg^–1 during this study. In conclusion, CSNPs could be used as good antigen carriers for DC‐based immunotherapy, although further study is needed to completely clear the residue of the CSNPs at the injection site. Copyright © 2015 John Wiley & Sons, Ltd.     

Nanomaterial-induced cell death in pulmonary and hepatic cells following exposure to three different metallic materials: The role of autophagy and apoptosis

Autophagy is the catabolic process involving the sequestration of the cytoplasm within double-membrane vesicles, which fuse with lysosomes to form autolysosomes in which autophagic targets are degraded. Since most endocytic routes of nanomaterial uptake converge upon the lysosome and the possibility that autophagy induction by NMs may be an attempt by the cell to self-preserve following the external challenge, this study investigated the role of autophagy following exposure to a panel of widely used metal-based NMs with high toxicity (Ag and ZnO) or low toxicity (TiO₂) in a pulmonary (A549) and hepatic (HepG2) cell line. The in vitro exposure to the Ag and ZnO NMs resulted in the induction of both apoptosis and autophagy pathways in both cell types. However, the progression of autophagy was blocked in the formation of the autolysosome, which coincided with morphologic changes in the actin cytoskeleton. This response was not observed following the exposure to low-toxicity TiO₂ NMs. Overall, the results show that high toxicity NMs can cause a dysfunction in the autophagy pathway which is associated with apoptotic cell death.     

The elemental changes occurring in the rat liver after exposure to PEG-coated iron oxide nanoparticles: total reflection x-ray fluorescence (TXRF) spectroscopy study

The main goal of this study was to evaluate in vivo effects of low dose of PEG-coated magnetic iron oxide nanoparticles (IONPs) on the rat liver. The IONPs was intravenously injected into rats at a dose equaled to 0.03?mg of Fe per 1?kg of an animal body weight. The elemental composition of liver tissue in rats subjected to IONPs action and controls were compared. Moreover, in order to determine the dynamics of nanoparticles (NPs) induced elemental changes, the tissues taken from animals 2?hours, 24?hours, and 7?days from IONPs injection were examined. The analysis of subtle elemental anomalies occurring as a result of IONPs action required application of highly sensitive analytical method. The total reflection X-ray fluorescence spectroscopy perfectly meets such requirements and therefore it was used in this study. The obtained results showed increasing trend of Fe level within liver occurring 2?hours from IONPs injection. One day after NPs administration, the liver Fe content presented the baseline level what suggests only the short-term accumulation of nanoparticles in the organ. The Ca, Cu, and Zn levels changed significantly as a result of NPs action. Moreover, the anomalies in their accumulation were still observed 7?days after IONPs injection. The level of Cu decreased while those of Ca and Zn increased in the liver of NPs-treated animals. The reduced liver Cu, followed by elevated serum level of this element, might be related in triggering the mechanisms responsible for Fe metabolism in the organism.     

Histopathology and analyses of inflammation intensity in the gills of mussels exposed to silver nanoparticles: role of nanoparticle size,exposure time,and uptake pathways

Environmentally induced perturbation of health parameters lead to morphological changes associated to the inflammatory response. Hematoxyline and eosin (H&;E)-stained gill filaments sections were examined for such changes and inflammation intensity was scored according to a quantitative model in order to evaluate the health status of in vivo exposed (for 3, 6, and 12?h) mussels to silver nanoparticles (Ag-NPs <50?nm and Ag-NPs <100?nm) prior and after the inhibition of two potential uptake pathways (clathrin- and caveolae-mediated endocytosis) with the aid of pharmaceutical inhibitors (amantadine and nystatin). The impacts of the nanoparticles (NPs) size, as well as their uptake routes within different time of exposure on the inflammatory response were assessed. The results showed that Ag-NPs clearly induced morphological changes associated to the inflammatory response in gill tissues (Mann–Whitney p values were <.05). It is also clear that the length of the exposure as well as the NP size highly impacted inflammation intensity (highest histopathological indices recorded with Ag-NPs <100?nm). Also, the routes of NPs entry noticed to be major factor underlying inflammatory response (significant inflammation intensity reported with Ag-NPs <50?nm after blockade of uptake routes; p?<.05). Throughout, it was concluded that inflammation intensity was related to NPs size and exposure time. Overall, uptake routes are shown to be the major factor underlying nanotoxicity.     

Glycidol induces axonopathy and aberrations of hippocampal neurogenesis affecting late-stage differentiation by exposure to rats in a framework of 28-day toxicity study

Developmental exposure to glycidol induces aberrations of late-stage neurogenesis in the hippocampal dentate gyrus of rat offspring, whereas maternal animals develop axonopathy. To investigate the possibility whether similar effects on adult neurogenesis could be induced by exposure in a framework of 28-day toxicity study, glycidol was orally administered to 5-week-old male Sprague–Dawley rats by gavage at 0, 30 or 200 mg/kg for 28 days. At 200 mg/kg, animals revealed progressively worsening gait abnormalities as well as histopathological and immunohistochemical changes suggestive of axonal injury as evidenced by generation of neurofilament-L⁺ spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells and axonal degeneration in the sciatic nerves. At the same dose, animals revealed aberrations in neurogenesis at late-stage differentiation as evidenced by decreases of both doublecortin⁺ and dihydropyrimidinase-like 3⁺ cells in the subgranular zone (SGZ) and increased reelin⁺ or calbindin-2⁺ γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein⁺ mature neurons in the dentate hilus. These effects were essentially similar to that observed in offspring after maternal exposure to glycidol. These results suggest that glycidol causes aberrations in adult neurogenesis in the SGZ at the late stage involving the process of neurite extension similar to the developmental exposure study in a standard 28-day toxicity study.