Linking nanomaterial-induced mitochondrial dysfunction to existing adverse outcome pathways for chemicals.

The adverse outcome pathway (AOP) framework plays a crucial role in the paradigm shift of tox­icity testing towards the development and use of new approach methodologies. AOPs developed for chemicals are in theory applicable to nanomaterials (NMs). However, only initial efforts have been made to integrate information on NM-induced toxicity into existing AOPs. In a previous study, we identified AOPs in the AOP-Wiki associated with the molecular initiating events (MIEs) and key events (KEs) reported for NMs in scientific literature. In a next step, we analyzed these AOPs and found that mitochondrial toxicity plays a significant role in several of them at the molecular and cellular levels. In this study, we aimed to generate hypothesis-based AOPs related to NM-induced mitochondrial toxicity. This was achieved by integrating knowledge on NM-induced mitochondrial toxicity into all existing AOPs in the AOP-Wiki, which already includes mitochondrial toxicity as a MIE/KE. Several AOPs in the AOP-Wiki related to the lung, liver, cardiovascular and nervous system, with extensively defined KEs and key event relationships (KERs), could be utilized to develop AOPs that are relevant for NMs. However, the majority of the studies included in our literature review were of poor quality, particularly in reporting NM physicochemical characteristics, and NM-relevant mitochondrial MIEs were rarely reported. This study highlights the potential role of NM-induced mitochondrial toxicity in human-relevant adverse outcomes and identifies useful AOPs in the AOP-Wiki for the development of AOPs for NMs.

This article investigates commonalities in the toxicity pathways of chemicals and nanomaterials. Nanomaterials have been found to affect the function of mitochondria, the powerhouses within every human cell. Mitochondrial dysfunction may cause harmful effects such as cellular damage and inflammation. By linking these findings to existing adverse outcome pathways for chemicals, the research provides valuable insights for assessing the risks associated with nanomaterial exposure. This work is crucial for understanding the potential health implications of nanomaterials and can contribute to informed decision-making in regulatory and risk assessment processes without the use of animals.

Impact of a Polymer-Based Nanoparticle with Formoterol Drug as Nanocarrier System In Vitro and in an Experimental Asthmatic Model.

The implementation of nanotechnology in pulmonary delivery systems might result in better and more specific therapy. Therefore, a nano-sized drug carrier should be toxicologically inert and not induce adverse effects. We aimed to investigate the responses of a polymer nano drug carrier, a lysine poly-hydroxyethyl methacrylate nanoparticle (NP) [Lys-p(HEMA)], loaded with formoterol, both in vitro and in vivo in an ovalbumin (OVA) asthma model. The successfully synthesized nanodrug formulation showed an expectedly steady in vitro release profile. There was no sign of in vitro toxicity, and the 16HBE and THP-1 cell lines remained vital after exposure to the nanocarrier, both loaded and unloaded. In an experimental asthma model (Balb/c mice) of ovalbumin sensitization and challenge, the nanocarrier loaded and unloaded with formoterol was tested in a preventive strategy and compared to treatment with the drug in a normal formulation. The airway hyperresponsiveness (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL), both cellular and biochemical, were assessed. The application of formoterol as a regular drug and the unloaded and formoterol-loaded NP in OVA-sensitized mice followed by a saline challenge was not different from the control group. Yet, both the NP formulation and the normal drug application led to a more deteriorated lung function and increased lung inflammation in the OVA-sensitized and -challenged mice, showing that the use of the p(HEMA) nanocarrier loaded with formoterol needs more extensive testing before it can be applied in clinical settings.

Pollutant exposure and myocardial injury: Protocol and progress report for a toxicological systematic mapping review.

An increasing body of evidence identifies pollutant exposure as a risk factor for cardiovascular disease (CVD), while CVD incidence rises steadily with the aging population. Although numerous experimental studies are now available, the mechanisms through which lifetime exposure to environmental pollutants can result in CVD are not fully understood. To comprehensively describe and understand the pathways through which pollutant exposure leads to cardiotoxicity, a systematic mapping review of the available toxicological evidence is needed. This protocol outlines a step-by-step framework for conducting this review. Using the National Toxicology Program (NTP) Health Assessment and Translation (HAT) approach for conducting toxicological systematic reviews, we selected 362 out of 8111 in vitro (17%), in vivo (67%), and combined (16%) studies for 129 potential cardiotoxic environmental pollutants, including heavy metals (29%), air pollutants (16%), pesticides (27%), and other chemicals (28%). The internal validity of included studies is being assessed with HAT and SYRCLE Risk of Bias tools. Tabular templates are being used to extract key study elements regarding study setup, methodology, techniques, and (qualitative and quantitative) outcomes. Subsequent synthesis will consist of an explorative meta-analysis of possible pollutant-related cardiotoxicity. Evidence maps and interactive knowledge graphs will illustrate evidence streams, cardiotoxic effects and associated quality of evidence, helping researchers and regulators to efficiently identify pollutants of interest. The evidence will be integrated in novel Adverse Outcome Pathways to facilitate regulatory acceptance of non-animal methods for cardiotoxicity testing. The current article describes the progress of the steps made in the systematic mapping review process.

Heart disease is a leading global cause of death. Recent research indicates that certain environmental chemicals can worsen heart problems. We're conducting a rigorous review of scientific studies to understand how these chemicals affect the heart. This will inform policymakers and promote non-animal testing methods for cardiotoxicity by providing a clear overview of the toxicological evidence. We have reviewed over 8,000 articles and focused on 362 studies about 129 chemicals, including heavy metals, air pollutants and pesticides, and their effects on the heart. The current manuscript describes the used methods and steps made in this process. The outcome of our systematic review of these 362 articles will be a comprehensive database that will aid the development of alternative testing methods for cardiotoxicity.

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 (TiO2), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to assess the safety of TiO2 NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO2 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 TiO2 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-TiO2 (17 nm) and a non-nano TiO2 (117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO2 particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO2 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.

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.

A strategy towards the generation of testable adverse outcome pathways for nanomaterials.

Manufactured nanomaterials (NMs) are increasingly used in a wide range of industrial applications leading to a constant increase in the market size of nano-enabled products. The increased production and use of NMs are raising concerns among different stakeholder groups with regard to their effects on human and environmental health. Currently, nanosafety hazard assessment is still widely performed using in vivo (animal) models, however the development of robust and reg­ulatory relevant strategies is required to prioritize and/or reduce animal testing. An adverse outcome pathway (AOP) is a structured representation of biological events that start from a molecular initiating event (MIE) leading to an adverse outcome (AO) through a series of key events (KEs). The AOP framework offers great advancement to risk assessment and regulatory safety assessments. While AOPs for chemicals have been more frequently reported, the AOP collection for NMs is limited. By using existing AOPs, we aimed to generate simple and testable strategies to predict if a given NM has the potential to induce a MIE leading to an AO through a series of KEs. Firstly, we identified potential MIEs or initial KEs reported for NMs in the literature. Then, we searched the identified MIE or initial KEs as keywords in the AOP-Wiki to find associated AOPs. Finally, using two case studies, we demonstrate how in vitro strategies can be used to test the identified MIE/KEs.

A novel TEM grid sampler for airborne particles to measure the cell culture surface dose.

The applied surface dose is a key parameter for the measurement of toxic effects of airborne particles by air liquid interface exposure of human lung cells. Besides online measurement of the deposited particle mass by quartz crystal microbalance frequently other dose metrics such as particle size distribution, surface and agglomeration state are required. These particle properties and their spatial distribution can be determined by digital processing of micrographs obtained by transmission electron microscopy (TEM). Here, we report the development and characterization of a novel holder for film coated TEM copper grids, which allows for sampling under identical geometric and ambient conditions as in a cell culture chamber. The sample holder avoids artefacts by reliable grounding of the grids and improves handling of the grids to prevent damage of the sensitive film. This sample holder is applied during exposure experiments with titanium dioxide nanoparticles. The measured dose of 0.2 µg/cm² corresponds well to the mass loading signal of the quartz crystal microbalance. Additionally, the spatial distribution of particles on the sampling surface shows a good homogeneity of deposition. This novel sampling method allows verifying other dosimetry methods and gives additional information about particle properties and homogeneity of the dose.

Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo.

BACKGROUND: The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. RESULTS: We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1ß increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. CONCLUSION: Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

LiCoO2 particles used in Li-ion batteries induce primary mutagenicity in lung cells via their capacity to generate hydroxyl radicals.

BACKGROUND: Li-ion batteries (LIB) are used in most portable electronics. Among a wide variety of materials, LiCoO2 (LCO) is one of the most used for the cathode of LIB. LCO particles induce oxidative stress in mouse lungs due to their Co content, and have a strong inflammatory potential. In this study, we assessed the mutagenic potential of LCO particles in lung cells in comparison to another particulate material used in LIB, LTO (Li4Ti5O12), which has a low inflammatory potential compared to LCO particles. RESULTS: We assessed the mutagenic potential of LCO and LTO particles in vitro by performing a cytokinesis-block micronucleus (MN) assay with rat lung epithelial cells (RLE), as well as in vivo in alveolar type II epithelial (AT-II) cells. LCO particles induced MN in vitro at non-cytotoxic concentrations and in vivo at non-inflammatory doses, indicating a primary genotoxic mechanism. LTO particles did not induce MN. Electron paramagnetic resonance and terephthalate assays showed that LCO particles produce hydroxyl radicals (•OH). Catalase inhibits this •OH production. In an alkaline comet assay with the oxidative DNA damage repair enzyme human 8-oxoguanine DNA glycosylase 1, LCO particles induced DNA strand breaks and oxidative lesions. The addition of catalase reduced the frequency of MN induced by LCO particles in vitro. CONCLUSIONS: We report the mutagenic activity of LCO particles used in LIB in vitro and in vivo. Our data support the role of Co(II) ions released from these particles in their primary genotoxic activity which includes the formation of •OH by a Fenton-like reaction, oxidative DNA lesions and strand breaks, thus leading to chromosomal breaks and the formation of MN. Documenting the genotoxic potential of the other LIB particles, especially those containing Co and/or Ni, is therefore needed to guarantee a safe and sustainable development of LIB.

Is aggregated synthetic amorphous silica toxicologically relevant?

BACKGROUND: 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. RESULTS: We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR. CONCLUSION: Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.

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.

Distinct autophagy-apoptosis related pathways activated by Multi-walled (NM 400) and Single-walled carbon nanotubes (NIST-SRM2483) in human bronchial epithelial (16HBE14o-) cells.

Given the recent development in the field of particle and fibre toxicology, parallels have been drawn between Carbon nanotubes (CNTs) and asbestos. It is now established that both multi-walled (MWCNTs) and single-walled (SWCNTs) carbon nanotubes might contribute to pulmonary disease. Although multiple mechanisms might be involved in CNT induced pathogenesis, systematic understanding of the relationship between different CNT exposure (MWCNT vs SWCNT) and autophagy/ apoptosis/ necrosis, in human lung epithelial cells remains limited. In this study, we demonstrate that exposure to MWCNT (NM-400), but not SWCNT (NIST-SRM2483), leads to an autophagic response after acute exposure (24 h). MWCNT exposure was characterized by an increase in anti-apoptotic BCL2, downregulation of executor Caspase-3/7 and increase in expression of genes from the autophagy machinery. For SWCNT exposure however, we observed an overexpression of executor Caspase-3/7 and upregulation of pro-apoptotic BAX; enrichment for processes like cornification, apoptotic process, cell differentiation from proteomic analysis. These results clearly indicate a major difference in the pathways initiated by the CNTs, in vitro. While the present study design provides mechanistic understanding after an acute exposure for the tested CNTs, we believe that the information obtained here would have relevance in better understanding of CNT toxicity and pathogenesis in general.

Toxicology of silica nanoparticles: an update.

Large-scale production and use of amorphous silica nanoparticles (SiNPs) have increased the risk of human exposure to SiNPs, while their health effects remain unclear. In this review, scientific papers from 2010 to 2016 were systematically selected and sorted based on in vitro and in vivo studies: to provide an update on SiNPs toxicity and to address the knowledge gaps indicated in the review of Napierska (Part Fibre Toxicol 7:39, 2010). Toxicity of SiNPs in vitro is size, dose, and cell type dependent. SiNPs synthesized by wet route exhibited noticeably different biological effects compared to thermal route-based SiNPs. Amorphous SiNPs (particularly colloidal and stöber) induced toxicity via mechanisms similar to crystalline silica. In vivo, route of administration and physico-chemical properties of SiNPs influences the toxicokinetics. Adverse effects were mainly observed in acutely exposed animals, while no significant signs of toxicity were noted in chronically dosed animals. The correlation between in vitro and in vivo toxicity remains less well established mainly due to improper-unrealistic-dosing both in vitro and in vivo. In conclusion, notwithstanding the multiple studies published in recent years, unambiguous linking of physico-chemical properties of SiNPs types to toxicity, bioavailability, or human health effects is not yet possible.