Toxicity screening of air extracts representing different source sectors in the Greater Toronto and Hamilton areas: In vitro oxidative stress, pro-inflammatory response, and toxicogenomic analysis.

In the present study, the suitability and sensitivity of different in vitro toxicity endpoints were determined to evaluate and distinguish the specific contributions of polycyclic aromatic carbon (PAC) mixtures from various sites in Toronto (Canada), to pulmonary toxicity. Air samples were collected for two-month periods from April 2014 to March 2015 from one location, and from August 2016 to August 2017 from multiple locations reflecting different geographical areas in Toronto, and the Greater Toronto Area, with varying source emissions including background, traffic, urban, industrial and residential sites. Relative concentrations of PACs and their derivatives in these air samples were characterised. In vitro cytotoxicity, pro-inflammatory, and oxidative stress assays were employed to assess the acute pulmonary effects of urban-air-derived air pollutants. In addition, global transcriptional profiling was utilized to understand how these chemical mixtures exert their harmful effects. Lastly, the transcriptomic data and the chemical profiles for each site and season were used to relate the biological response back to individual constituents. Site-specific responses could not be derived; however, the Spring season was identified as the most responsive through benchmark concentration analysis. A combination of correlational analysis and principal component analysis revealed that nitrated and oxygenated polycyclic aromatic hydrocarbons (PAHs) drive the response at lower concentrations while specific PAHs drive the response at the highest concentration tested. Unsubstituted PAHs are the current targets for analysis as priority pollutants. The present study highlights the importance of by-products of PAH degradation in the assessment of risk. The study also demonstrates the usefulness of in vitro toxicity assays to derive meaningful data in support of risk assessment.

Inflammatory response in human alveolar epithelial cells after TiO2 NPs or ZnO NPs exposure: Inhibition of surfactant protein A expression as an indicator for loss of lung function.

The increasing use of metal oxide nanoparticles (MONPs) as TiO2 NPs or ZnO NPs has led to environmental release and human exposure. The respiratory system, effects on lamellar bodies and surfactant protein A (SP-A) of pneumocytes, can be importantly affected. Exposure of human alveolar epithelial cells (A549) induced differential responses; a higher persistence of TiO2 in cell surface and uptake (measured by Atomic Force Microscopy) and sustained inflammatory response (by means of TNF-α, IL-10, and IL-6 release) and ROS generation were observed, whereas ZnO showed a modest response and low numbers in cell surface. A reduction in SP-A levels at 24 h of exposure to TiO2 NPs (concentration-dependent) or ZnO NPs (the higher concentration) was also observed, reversed by blocking the inflammatory response (by the inhibition of IL-6). Loss of SP-A represents a relevant target of MONPs-induced inflammatory response that could contribute to cellular damage and loss of lung function.

In vitro cytotoxicity study of superparamagnetic iron oxide and silica nanoparticles on pneumocyte organelles.

Inhalation is the main route of nanoparticles (NP) exposure during manufacturing. Although many mechanisms of toxicity have been described, the interaction of NP with relevant pneumocytes organelles is not widely understood. Considering that the physicochemical properties of NP influence their toxicological responses, the objective of this study was to evaluate whether exposure to different NP, crystalline Fe3O4 NP and amorphous SiO2 NP could alter pneumocytes organelles in alveolar epithelial cells. To achieve this goal, cell viability, ultrastructural changes, lysosomal damage, mitochondrial membrane potential (MMP), lipid droplets (LD) formation and cytokines production were evaluated by MTT, electron microscopy, lysotracker red staining, JC-1, Oil Red staining and Milliplex® assay respectively. Both NP were observed within lamellar bodies (LB), lysosomes, and cytoplasm causing morphological changes. Exposure to SiO2 NP at 6 h induced lysosomal activation, but not Fe3O4 NP. MMP decreased and LD increased at the highest concentrations after both NP exposure. Pro-inflammatory cytokines were released only after SiO2 NP exposure at 48 h. These results indicate that SiO2 NP have a greater impact than Fe3O4 NP on organelles responsible for energy, secretion, degradation and metabolism in pneumocytes leading to the development of respiratory disorders or the exacerbation of preexisting conditions. Therefore, the established biocompatibility for amorphous NP has to be reconsidered.

Plasma protein adsorption on Fe3O4-PEG nanoparticles activates the complement system and induces an inflammatory response.

BACKGROUND: Understanding of iron oxide nanoparticles (IONP) interaction with the body milieu is crucial to guarantee their efficiency and biocompatibility in nanomedicine. Polymer coating to IONP, with polyethyleneglycol (PEG) and polyvinylpyrrolidone (PVP), is an accepted strategy to prevent toxicity and excessive protein binding. AIM: The aim of this study was to investigate the feature of IONP adsorption of complement proteins, their activation and consequent inflammatory response as a strategy to further elucidate their biocompatibility. METHODS: Three types of IONP with different surface characteristics were used: bare (IONP-bare), coated with PVP (IONP-PVP) and PEG-coated (IONP-PEG). IONPs were incubated with human plasma and adsorbed proteins were identified. BALB/c mice were intravenously exposed to IONP to evaluate complement activation and proinflammatory response. RESULTS: Protein corona fingerprinting showed that PEG surface around IONP promoted a selective adsorption of complement recognition molecules which would be responsible for the complement system activation. Furthermore, IONP-PEG activated in vitro, the complement system and induced a substantial increment of C3a and C4a anaphylatoxins while IONP-bare and IONP-PVP did not. In vivo IONP-PEG induced an increment in complement activation markers (C5a and C5b-9), and proinflammatory cytokines (IL-1ß, IL-6, TNF-α). CONCLUSION: The engineering of nanoparticles must incorporate the association between complement proteins and nanomedicines, which will regulate the immunostimulatory effects through a selective adsorption of plasma proteins and will enable a safer application of IONP in human therapy.

A comparison of the human and mouse protein corona profiles of functionalized SiO2 nanocarriers.

Nanoparticles are a promising cancer therapy for their use as drug carriers given their versatile functionalization with polyethylene glycol and proteins that can be recognized by overexpressed receptors in tumor cells. However, it has been suggested that in biological fluids, proteins cover nanoparticles, which gives the proteins a biological identity that could be responsible for unexpected biological responses: the so-called protein corona. A relevant biological event that is usually ignored in protein-corona formation is the interspecies differences in protein binding, which can be involved in the discrepancies observed in preclinical studies and the nanoparticle safety and efficiency. Hence, the aim of this study was to determine the differences between human and mouse plasma protein corona profiles in an active therapy model using silicon dioxide nanoparticles (SiO2 nanoparticles) functionalized with polyethylene glycol and transferrin. Functionalized SiO2 nanoparticles were made with a primary particle size of 25 nm and a transferrin content of 50 µg mg-1 of nanoparticles and were PEGylated with a cross-linker. The proteomic analysis by nanoliquid chromatography tandem-mass spectrometry (nanoLC-MS/MS) showed interspecies differences. The most abundant proteins found in the human protein corona profile were immunoglobulins, actin cytoplasmic 1, hemoglobin subunit beta, serotransferrin, ficolin-3, complement C3, and apolipoprotein A-1. Meanwhile, the mouse protein corona adsorbed the serine protease inhibitor A3K, serotransferrin, alpha-1-antitrypsin 1-2, hemoglobin subunit beta, and fibrinogen gamma and beta chains. These protein-corona profile differences in the functionalized SiO2 nanoparticles indicate that biological responses observed in in vivo models could not be translated to clinical use and must be considered in the interpretation of preclinical trials in order to design more efficient and safer nanomedicines.