Agglomeration State of Titanium-Dioxide (TiO2) Nanomaterials Influences the Dose Deposition and Cytotoxic Responses in Human Bronchial Epithelial Cells at the Air-Liquid Interface

Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO(2)), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO(2) NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO(2) NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO(2) agglomerates of different sizes at the air–liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO(2) (17 nm) and a non-nano TiO(2) (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO(2) particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO(2) agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs

Is aggregated synthetic amorphous silica toxicologically relevant?

The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective. This knowledge gap greatly affects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nano-sized particles (1-100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes

Air–Liquid Interface Exposure of Lung Epithelial Cells to Low Doses of Nanoparticles to Assess Pulmonary Adverse Effects

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals

Toxicological relevance of nanoparticle agglomerates and aggregates: A step towards a toxicologically relevant definition of nanomaterials

Introduction and Background: Nanotechnology is one of the fastest growing technologies in the current century and is applied in many aspects of our daily life as Manufactured Nanoparticles (MNPs) or as Manufactured Nanomaterials (MNMs), e.g. in cosmetics and skin care products, clothing, food additives, waste water treatment, specific drug delivery in the body etc. Growing production and use of Manufactured nanomaterials (MNMs) increased the risk of human exposures and raised the global concern over the potential adverse effects of MNMs. In real world applications and in exposure scenarios, MNMs also exist as aggregates and/or agglomerates (AA) of primary particles with one or more external dimensions in the size range of 1 to 100 nm. However, the biological behaviour/toxicity of the AA is not explored in detail. In addition, the current EU definition for the MNM also comprised of AA, but its toxicological relevance is not verified. Such a discrepancy, not only affecting the risk assessment of MNMs but also hamper the application of regulations and the development of guidelines to monitor SNPs in products and goods. Methods: The nano-silica (nSiO2) will be used as a model to evaluate the influence of AA on toxicity and for size distribution, nano-titanium dioxide (nTiO2) will be used. In in vitro studies, MNMs will be exposed to human cell lines such as pulmonary human cell lines (e.g.16 HBE), intestinal cell lines (e.g. Caco2 cells) and macrophages (derived from monocytes THP-1). At the end of exposure, end points such as markers of inflammation (ELISA), oxidative stress (glutathione content) and genotoxicity (comet assay) will be assessed. For in-vivo studies, Swiss albino male mice will be exposed to MNM at different doses and sacrificed at different time points. At the end of exposure, the cells (lung, BAL, blood) will be routinely processed for comet assay to determine the genotoxic potential and, to investigate the other biological end points such as tissue damage, translocation of nanomaterial, markers of inflammation and oxidative stress. Expected outcomes: From the results of in vitro and in vivo studies, the influence of MNM AA and size distribution on toxicity will be compared and analysed. The biological responses due to chronic MNM exposure will also be evaluated. Furthermore, a better understanding of MNM interference at the biological barriers and potential to translocate across these barriers will be established. Subsequently, this project will contribute to build knowledge on the potential risk of MNM for the environment and for human beings, such that a safer use of these materials can be implemented. Building knowledge in this area is one of the prerequisite to safer design and further improvement of the nanotechnologies.status: publishe

Genotoxicity of Titanium-di-oxide nanomaterials in-vitro and in-vivo influenced by their dispersion state

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Towards a toxicologically relevant definition of nanomaterials (TO2DENANO)

Introduction and Background: Nanotechnology is one of the fastest growing technologies in the current century and is applied in many aspects of our daily life as Manufactured Nanoparticles (MNPs) or as Manufactured Nanomaterials (MNMs), e.g. in cosmetics and skin care products, clothing, food additives, waste water treatment, specific drug delivery in the body etc. Growing production and use of Manufactured nanomaterials (MNMs) increased the risk of human exposures and raised the global concern over the potential adverse effects of MNMs. In real world applications and in exposure scenarios, MNMs also exist as aggregates and/or agglomerates (AA) of primary particles with one or more external dimensions in the size range of 1 to 100 nm. However, the biological behaviour/toxicity of the AA is not explored in detail. In addition, the current EU definition for the MNM also comprised of AA, but its toxicological relevance is not verified. Such a discrepancy, not only affecting the risk assessment of MNMs but also hamper the application of regulations and the development of guidelines to monitor SNPs in products and goods. Methods: The nano-silica (nSiO2) will be used as a model to evaluate the influence of AA on toxicity and for size distribution, nano-titanium dioxide (nTiO2) will be used. In in vitro studies, MNMs will be exposed to human cell lines such as pulmonary human cell lines (e.g.16 HBE), intestinal cell lines (e.g. Caco2 cells) and macrophages (derived from monocytes THP-1). At the end of exposure, end points such as markers of inflammation (ELISA), oxidative stress (glutathione content) and genotoxicity (comet assay) will be assessed. For in-vivo studies, Swiss albino male mice will be exposed to MNM at different doses and sacrificed at different time points. At the end of exposure, the cells (lung, BAL, blood) will be routinely processed for comet assay to determine the genotoxic potential and, to investigate the other biological end points such as tissue damage, translocation of nanomaterial, markers of inflammation and oxidative stress. Expected outcomes: From the results of in vitro and in vivo studies, the influence of MNM AA and size distribution on toxicity will be compared and analysed. The biological responses due to chronic MNM exposure will also be evaluated. Furthermore, a better understanding of MNM interference at the biological barriers and potential to translocate across these barriers will be established. Subsequently, this project will contribute to build knowledge on the potential risk of MNM for the environment and for human beings, such that a safer use of these materials can be implemented. Building knowledge in this area is one of the prerequisite to safer design and further improvement of the nanotechnologies.Invited lecture, invited by: FPS Public Health, Belgiumstatus: Published onlin

Assessing the Toxicological Relevance of Nanomaterial Agglomerates and Aggregates Using Realistic Exposure In Vitro

Low dose repeated exposures are considered more relevant/realistic in assessing the health risks of nanomaterials (NM), as human exposure such as in workplace occurs in low doses and in a repeated manner. Thus, in a three-week study, we assessed the biological effects (cell viability, cell proliferation, oxidative stress, pro-inflammatory response, and DNA damage) of titanium-di-oxide nanoparticle (TiO2 NP) agglomerates and synthetic amorphous silica (SAS) aggregates of different sizes in human bronchial epithelial (HBE), colon epithelial (Caco2), and human monocytic (THP-1) cell lines repeatedly exposed to a non-cytotoxic dose (0.76 µg/cm2). We noticed that neither of the two TiO2 NPs nor their agglomeration states induced any effects (compared to control) in any of the cell lines tested while SAS aggregates induced some significant effects only in HBE cell cultures. In a second set of experiments, HBE cell cultures were exposed repeatedly to different SAS suspensions for two weeks (first and second exposure cycle) and allowed to recover (without SAS exposure, recovery period) for a week. We observed that SAS aggregates of larger sizes (size ~2.5 µm) significantly affected the cell proliferation, IL-6, IL-8, and total glutathione at the end of both exposure cycle while their nanosized counterparts (size less than 100 nm) induced more pronounced effects only at the end of the first exposure cycle. As noticed in our previous short-term (24 h) exposure study, large aggregates of SAS did appear to be similarly potent as nano sized aggregates. This study also suggests that aggregates of SAS of size greater than 100 nm are toxicologically relevant and should be considered in risk assessment