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Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure–activity relationship and into the underlying MoA. [ABSTRACT FROM AUTHOR]

Engineered Nanomaterials (NMs), such as Superparamagnetic Iron Oxide Nanoparticles (SPIONs), offer significant benefits in a wide range of applications, including cancer diagnostic and therapeutic strategies. However, the use of NMs in biomedicine raises safety concerns due to lack of knowledge on possible biological interactions and effects. The initial basis for using SPIONs as biomedical MRI contrast enhancement agents was the idea that they are selectively taken up by macrophage cells, and not by the surrounding cancer cells. To investigate this claim, we analyzed the uptake of SPIONs into well-established cancer cell models and benchmarked this against a common macrophage cell model. In combination with fluorescent labeling of compartments and siRNA silencing of various proteins involved in common endocytic pathways, the mechanisms of internalization of SPIONs in these cell types has been ascertained utilizing reflectance confocal microscopy. Caveolar mediated endocytosis and macropinocytosis are both implicated in SPION uptake into cancer cells, whereas in macrophage cells, a clathrin-dependant route appears to predominate. Colocalization studies confirmed the eventual fate of SPIONs as accumulation in the degradative lysosomes. Dissolution of the SPIONs within the lysosomal environment has also been determined, allowing a fuller understanding of the cellular interactions, uptake, trafficking and effects of SPIONs within a variety of cancer cells and macrophages. Overall, the behavior of SPIONS in non-phagocytotic cell lines is broadly similar to that in the specialist macrophage cells, although some differences in the uptake patterns are apparent. [ABSTRACT FROM AUTHOR]

Experimental modeling to identify specific inhalation hazards for nanomaterials has in the main focused on in vivo approaches. However, these models suffer from uncertainties surrounding species-specific differences and cellular targets for biologic response. In terms of pulmonary exposure, approaches which combine ‘inhalation-like’ nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterize alterations in mRNA and miRNA within the lung. Organotypic-reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark-generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid interface (AE-ALI) system. Adverse effects were characterized using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag+) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects. [ABSTRACT FROM AUTHOR]

Toxicology and related concepts are being taught within chemistry education programs to enable students to (1) develop an awareness of toxicological principles and concepts that are otherwise absent from the chemistry curriculum for undergraduates and (2) design chemical products and processes that have reduced health and environmental hazards. This manuscript presents three model courses from different higher education institutions, presenting different approaches for introducing toxicology concepts to students. The models are presented not as prescriptive approaches, but as inspirational models for others to use to generate their own unique approaches to integrating and teaching toxicology concepts. [ABSTRACT FROM PUBLISHER]

This study investigated the role of nanoparticle size and surface chemistry on biocorona composition and its effect on uptake, toxicity and cellular responses in human umbilical vein endothelial cells (HUVEC), employing 40 and 80 nm gold nanoparticles (AuNP) with branched polyethyleneimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coatings. Proteomic analysis identified 59 hard corona proteins among the various AuNP, revealing largely surface chemistry-dependent signature adsorbomes exhibiting human serum albumin (HSA) abundance. Size distribution analysis revealed the relative instability and aggregation inducing potential of bare and corona-bound BPEI-AuNP, over LA- and PEG-AuNP. Circular dichroism analysis showed surface chemistry-dependent conformational changes of proteins binding to AuNP. Time-dependent uptake of bare, plasma corona (PC) and HSA corona-bound AuNP (HSA-AuNP) showed significant reduction in uptake with PC formation. Cell viability studies demonstrated dose-dependent toxicity of BPEI-AuNP. Transcriptional profiling studies revealed 126 genes, from 13 biological pathways, to be differentially regulated by 40 nm bare and PC-bound BPEI-AuNP (PC-BPEI-AuNP). Furthermore, PC formation relieved the toxicity of cationic BPEI-AuNP by modulating expression of genes involved in DNA damage and repair, heat shock response, mitochondrial energy metabolism, oxidative stress and antioxidant response, and ER stress and unfolded protein response cascades, which were aberrantly expressed in bare BPEI-AuNP-treated cells. NP surface chemistry is shown to play the dominant role over size in determining the biocorona composition, which in turn modulates cell uptake, and biological responses, consequently defining the potential safety and efficacy of nanoformulations. [ABSTRACT FROM AUTHOR]

Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1–50 μg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs. [ABSTRACT FROM AUTHOR]

The inhalation of zinc oxide engineered nanomaterials (ENMs) has been linked to cardiorespiratory dysfunction in mammalian models but the effects of aquatic ENM exposure on fish have not been fully investigated. Nano-zinc oxide (nZnO) is widely used in consumer products such as sunscreens and can make its way into aquatic ecosystems from domestic and commercial wastewater. This study examined the impact of an environmentally relevant nZnO formulation on cardiorespiratory function and energy metabolism in the white sucker (Catostomus commersonii), a freshwater teleost fish. Evidence of oxidative and cellular stress was present in gill tissue, including increases in malondialdehyde levels, heat shock protein (HSP) expression, and caspase 3/7 activity. Gill Na+/K+-ATPase activity was also higher by approximately three-fold in nZnO-treated fish, likely in response to increased epithelial permeability or structural remodeling. Despite evidence of toxicity in gill, plasma cortisol and lactate levels did not change in animals exposed to 1.0 mg L−1 nZnO. White suckers also exhibited a 35% decrease in heart rate during nZnO exposure, with no significant changes in resting oxygen consumption or tissue energy stores. Our results suggest that tissue damage or cellular stress resulting from nZnO exposure activates gill neuroepithelial cells, triggering a whole-animal hypoxic response. An increase in parasympathetic nervous signaling will decrease heart rate and may reduce energy demand, even in the face of an environmental toxicant. We have shown that acute exposure to nZnO is toxic to white suckers and that ENMs have the potential to negatively impact cardiorespiratory function in adult fish. [ABSTRACT FROM AUTHOR]

A weak aspect of the in vitro studies devoted to get information on the toxic, genotoxic and carcinogenic properties of nanomaterials is that they are usually conducted under acute-exposure and high-dose conditions. This makes difficult to extrapolate the results to human beings. To overcome this point, we have evaluated the cell transforming ability of cobalt nanoparticles (CoNPs) after long-term exposures (12 weeks) to sub-toxic doses (0.05 and 0.1 µg/mL). To get further information on whether CoNPs-induced oxidative DNA damage is relevant for CoNPs carcinogenesis, the cell lines selected for the study were the wild-type mouse embryonic fibroblast (MEF Ogg1+/+) and its isogenic Ogg1 knockout partner (MEF Ogg1−/−), unable to properly eliminate the 8-OH-dG lesions from DNA. Our initial short-term exposure experiments demonstrate that low doses of CoNPs are able to induce reactive oxygen species (ROS) and that MEF Ogg1−/− cells are more sensitive to CoNPs-induced acute toxicity and oxidative DNA damage. On the other hand, long-term exposures of MEF cells to sub-toxic doses of CoNPs were able to induce cell transformation, as indicated by the observed morphological cell changes, significant increases in the secretion of metalloproteinases (MMPs) and anchorage-independent cell growth ability, all cancer-like phenotypic hallmarks. Interestingly, such changes were significantly dependent on the cell line used, the Ogg1−/− cells being particularly sensitive. Altogether, the data presented here confirms the potential carcinogenic risk of CoNPs and points out the relevance of ROS and Ogg1 genetic background on CoNPs-associated effects. [ABSTRACT FROM AUTHOR]

Background: Carbon nanotubes (CNT) are fibre-like nanomaterials whose structural similarity to asbestos has raised concerns that they may also pose a mesothelioma hazard. The objective of this study was to examine the inflammatory potential of three CNT samples of differing length on the lungs and pleural cavity following introduction into the airspaces of mice. ResultsAspiration of the two short/tangled and one long CNT sample into the lungs of mice resulted in a length-dependent inflammatory response at 1 week, i.e., only the long CNT sample caused acute neutrophilic inflammation in bronchoalveolar lavage at 1 week and progressive thickening of the alveolar septa. The authors also report length-dependent inflammatory responses in the pleural lavage after exposure only to the long CNT. The inflammatory response in the pleural cavity to long fibres and the appearance of lesions along the chest wall and diaphragm was not present at 1 week and only evident by 6 weeks post-exposure. Conclusion: Length-dependent pathogenicity is a feature of asbestos and the results presented in this study demonstrate similar length-dependent pathogenicity of CNT in the lungs and pleural space following airspace deposition. The data support the contention that long CNT reach the pleura from the airspaces, and that they are retained at the parietal pleura and cause inflammation and lesion development. The parietal pleura is the site of origin of mesothelioma and inflammation is considered to be a process involved in asbestos carcinogenesis and so the data support the contention that CNT may pose an asbestos-like mesothelioma hazard. [ABSTRACT FROM AUTHOR]

Allergic asthma is a chronic inflammatory disorder of the airway associated with bronchial obstruction, airway hyper-reactivity (AHR), and mucus production. The epithelium may direct and propagate asthmatic-like responses. Central to this theory is the observation that viruses, air pollution, and allergens promote epithelial damage and trigger the generation of IL-25, IL-33, and TSLP via innate pathways such as TLRs and purinergic receptors. Similarly, engineered nanomaterials promote a Th2-associated pathophysiology. In this study, we tested the hypothesis that instillation of multi-walled carbon nanotubes (MWCNT) impair pulmonary function in C57Bl/6 mice due to the development of IL-33-dependent Th2-associated inflammation. MWCNT exposure resulted in elevated levels of IL-33 in the lavage fluid (likely originating from airway epithelial cells), enhanced AHR, eosinophil recruitment, and production of Th2-associated cytokines and chemokines. Moreover, these events were dependent on IL-13 signaling and the IL-33/ST2 axis, but independent of T and B cells. Finally, MWCNT exposure resulted in the recruitment of innate lymphoid cells. Collectively, our data suggest that MWCNT induce epithelial damage that results in release of IL-33, which in turn promotes innate lymphoid cell recruitment and the development of IL-13-dependent inflammatory response. [ABSTRACT FROM AUTHOR]

The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 were examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA combed fibers. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway. [ABSTRACT FROM AUTHOR]

Surfactant protein D (SP-D) is primarily expressed in the lungs and modulates pro- and anti-inflammatory processes to toxic challenge, maintaining lung homeostasis. We investigated the interaction between NPs and SP-D and subsequent uptake by cells involved in lung immunity. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) measured NP aggregation, particle size and charge in native human SP-D (NhSP-D) and recombinant fragment SP-D (rfhSP-D). SP-D aggregated NPs, especially following the addition of calcium. Immunohistochemical analysis of A549 epithelial cells investigated the co-localization of NPs and rfhSP-D. rfhSP-D enhanced the co-localisation of NPs to epithelial A549 cells in vitro. NP uptake by alveolar macrophages (AMs) and lung dendritic cells (LDCs) from C57BL/6 and SP-D knock-out mice were compared. AMs and LDCs showed decreased uptake of NPs in SP-D deficient mice compared to wild-type mice. These data confirmed an interaction between SP-D and NPs, and subsequent enhanced NP uptake. [ABSTRACT FROM AUTHOR]

Ingested, inhaled or injected particles come into contact with biological fluids containing polymers, such as the protein fibrinogen. We studied interactions between well-characterized submicron particles or nanoparticles (NPs) and human fibrinogen. In vitro aggregation and zeta potential measurements of different sized and functionalized polystyrene, carbon black and silica NPs suspended in fibrinogen solutions were made. Particle size, surface charge and aggregation behaviour significantly changed in the presence of fibrinogen. Polymer (protein) bridging and bridge flocculation was observed. We concluded: (1) NP aggregation rate in a fibrinogen solution depended on particle surface type; (2) amine-functionalized particles aggregated more slowly in fibrinogen; and (3) particle morphology strongly influenced biologically available surface for protein attachment, but this did not correlate well with particle surface area for complex particles (calculated or measured). Interaction of particles and NPs with pro-coagulant polymers may therefore dictate the NP surface dose presentation to cells/organs and subsequent cellular effects, in and ex vivo. [ABSTRACT FROM AUTHOR]

We review the mechanisms and pathways whereby nanoparticles might cause genotoxicity. Primary and secondary mechanisms are discussed in relation to the general particle toxicology paradigm. We also discuss how we might improve genotoxicity assays for nanoparticles. In this context we describe the role of the dispersion and the protein corona, the most relevant metric, choice of controls and new endpoints for genotoxicity along with the need for a structure activity model of NP genotoxicity. [ABSTRACT FROM AUTHOR]

One of the most urgent requirements in nanotoxicology is a quantitative assessment of internalized nanomaterials in cells. We present an in vitro assay called 'max-flat' for the measurement of internalized particles in macrophages. Fluorescent polystyrene (PS) beads of diameters 1 µm, 500 nm, 200 nm, 100 nm and 20 nm were employed. Different concentrations of fibronectin (FN) coated substrates were tested to achieve a maximal cell spreading area and minimal nucleus height, hence 'max-flat'. We found this cell spreading area depends on FN concentration, and it is independent of particle concentration. An optimal condition of FN was found at 2.5×10-3 mg/ml, and it was selected for the max-flat assay to assess the internalization of PS beads. Cells under these conditions neither generate reactive oxygen species nor show noticeable differences in pro-survival/pro-apoptotic signals. Confocal images were employed for the max-flat assay and we set the interval scanning for a Z-stack as nucleus height divided by particle diameter. The max-flat assay provided a significantly higher number of internalized particles and the saturation is reached faster for nano-scale PS beads. We show how the proposed max-flat assay clearly outperform existing techniques by providing easier, more precise and far more reliable access to the number of internalized nanoparticles in macrophages. [ABSTRACT FROM AUTHOR]

Since the identification in the 1950s of deoxyribonucleic acid as the building block of life, the impact of molecular biology has been far-reaching. Understanding the processes of how DNA is replicated, transcribed into RNA and then translated into protein products has not only provided a fundamental knowledge of life but has also spawned a plethora of applications. Molecular biology has been high profile and widespread in research into the biology of disease and in drug discovery. It has additionally found application in understanding the adverse effects, or toxicity, of candidate drugs and how they interfere with biochemical and biological processes. In recent times the biggest impact of molecular biology in toxicology has been through the study of differential gene expression, largely as a result of the advent of genomics. This review seeks to describe how toxicogenomics strategies have been implemented and integrated into nonclinical studies of drug safety. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article “A rule for designing safer nanomaterials: do not interfere with the cellular redox equilibrium" by Enrico Burello, and Andrew P. Worth in the September 10, 2013 issue.