Springer Science and Business Media LLC

Abstract Background In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation. Methods A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed. Results We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation. Conclusion Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.

Zinc oxide nanoparticles (ZnO NPs) are used in an increasing number of products, including rubber manufacture, cosmetics, pigments, food additives, medicine, chemical fibers and electronics. However, the molecular mechanisms underlying ZnO NP nephrotoxicity remain unclear. In this study, we evaluated the potential toxicity of ZnO NPs in kidney cells in vitro and in vivo. We found that ZnO NPs were apparently engulfed by the HEK-293 human embryonic kidney cells and then induced reactive oxygen species (ROS) generation. Furthermore, exposure to ZnO NPs led to a reduction in cell viability and induction of apoptosis and autophagy. Interestingly, the ROS-induced hypoxia-inducible factor-1α (HIF-1α) signaling pathway was significantly increased following ZnO NPs exposure. Additionally, connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1), which are directly regulated by HIF-1 and are involved in the pathogenesis of kidney diseases, displayed significantly increased levels following ZnO NPs exposure in HEK-293 cells. HIF-1α knockdown resulted in significantly decreased levels of autophagy and increased cytotoxicity. Therefore, our results suggest that HIF-1α may have a protective role in adaptation to the toxicity of ZnO NPs in kidney cells. In an animal study, fluorescent ZnO NPs were clearly observed in the liver, lungs, kidneys, spleen and heart. ZnO NPs caused histopathological lesions in the kidney and increase in serum creatinine and blood urea nitrogen (BUN) which indicate possible renal possible damage. Moreover, ZnO NPs enhanced the HIF-1α signaling pathway, apoptosis and autophagy in mouse kidney tissues. ZnO NPs may cause nephrotoxicity, and the results demonstrate the importance of considering the toxicological hazards of ZnO NP production and application, especially for medicinal use.

This review focuses on outlining the toxicity of titanium dioxide (TiO2) particulates in vitro and in vivo, in order to understand their ability to detrimentally impact on human health. Evaluating the hazards associated with TiO2 particles is vital as it enables risk assessments to be conducted, by combining this information with knowledge on the likely exposure levels of humans. This review has concentrated on the toxicity of TiO2, due to the fact that the greatest number of studies by far have evaluated the toxicity of TiO2, in comparison to other metal oxide particulates. This derives from historical reasons (whereby the size dependency of particulate toxicity was first realised for TiO2) and due to its widespread application within consumer products (such as sunscreens). The pulmonary and dermal hazards of TiO2 have been a particular focus of the available studies, due to the past use of TiO2 as a (negative) control when assessing the pulmonary toxicity of particulates, and due to its incorporation within consumer products such as sunscreens. Mechanistic processes that are critical to TiO2 particulate toxicity will also be discussed and it is apparent that, in the main, the oxidant driven inflammatory, genotoxic and cytotoxic consequences associated with TiO2 exposure, are inherently linked, and are evident both in vivo and in vitro. The attributes of TiO2 that have been identified as being most likely to drive the observed toxicity include particle size (and therefore surface area), crystallinity (and photocatalytic activity), surface chemistry, and particle aggregation/agglomeration tendency. The experimental set up also influences toxicological outcomes, so that the species (or model) used, route of exposure, experiment duration, particle concentration and light conditions are all able to influence the findings of investigations. In addition, the applicability of the observed findings for particular TiO2 forms, to TiO2 particulates in general, requires consideration. At this time it is inappropriate to consider the findings for one TiO2 form as being representative for TiO2 particulates as a whole, due to the vast number of available TiO2 particulate forms and large variety of potential tissue and cell targets that may be affected by exposure. Thus emphasising that the physicochemical characteristics are fundamental to their toxicity.

We analyze the scientific basis and methodology used by the German MAK Commission in their recommendations for exposure limits and carcinogen classification of “granular biopersistent particles without known specific toxicity” (GBS). These recommendations are under review at the European Union level. We examine the scientific assumptions in an attempt to reproduce the results. MAK’s human equivalent concentrations (HECs) are based on a particle mass and on a volumetric model in which results from rat inhalation studies are translated to derive occupational exposure limits (OELs) and a carcinogen classification. We followed the methods as proposed by the MAK Commission and Pauluhn 2011. We also examined key assumptions in the metrics, such as surface area of the human lung, deposition fractions of inhaled dusts, human clearance rates; and risk of lung cancer among workers, presumed to have some potential for lung overload, the physiological condition in rats associated with an increase in lung cancer risk. The MAK recommendations on exposure limits for GBS have numerous incorrect assumptions that adversely affect the final results. The procedures to derive the respirable occupational exposure limit (OEL) could not be reproduced, a finding raising considerable scientific uncertainty about the reliability of the recommendations. Moreover, the scientific basis of using the rat model is confounded by the fact that rats and humans show different cellular responses to inhaled particles as demonstrated by bronchoalveolar lavage (BAL) studies in both species. Classifying all GBS as carcinogenic to humans based on rat inhalation studies in which lung overload leads to chronic inflammation and cancer is inappropriate. Studies of workers, who have been exposed to relevant levels of dust, have not indicated an increase in lung cancer risk. Using the methods proposed by the MAK, we were unable to reproduce the OEL for GBS recommended by the Commission, but identified substantial errors in the models. Considerable shortcomings in the use of lung surface area, clearance rates, deposition fractions; as well as using the mass and volumetric metrics as opposed to the particle surface area metric limit the scientific reliability of the proposed GBS OEL and carcinogen classification.

Abstract Background Several properties of multi-walled carbon nanotubes (MWCNT) have the potential to affect their bioactivity. This study examined the in vitro and in vivo outcomes of the influence of diameter, length, purification and carboxylation (in vitro testing only) of MWCNT. Methods Three original ‘as received’ MWCNT that varied in size (diameter and length) were purified and functionalized by carboxylation. The resulting MWCNT were characterized and examined for cytotoxicity and inflammasome activation in vitro using THP-1 cells and primary alveolar macrophages from C57BL/6 mice. Oropharyngeal aspiration administration was used to deliver original MWCNT and in vivo bioactivity and lung retention was examined at 1 and 7 days. Results Studies with THP-1 macrophages demonstrated that increased length or diameter corresponded with increased bioactivity as measured by inflammasome activation. Purification had little effect on the original MWCNT, and functionalization completely eliminated bioactivity. Similar results were obtained using alveolar macrophages isolated from C57BL/6 mice. The in vivo studies demonstrated that all three original MWCNT caused similar neutrophil influx at one day, but increasing length or diameter resulted in the lavaged cells to release more inflammatory cytokines (IL-6, TNF-α, and IL-1β) ex vivo. Seven-day histology revealed that, consistent with the in vitro results, increasing width or length of MWCNT caused more severe pathology with the longest MWCNT causing the most severe inflammation. In addition, the same two larger MWCNT were retained more in the lung at 7 days. Conclusions Taken together, the results indicated that in vitro and in vivo bioactivity of MWCNT increased with diameter and length. Purification had no significant modifying effect from the original MWCNT. Functionalization by carboxylation completely eliminated the bioactive potential of the MWCNT regardless of size in in vitro testing.

Using an in vitro triple cell co-culture model consisting of human epithelial cells (16HBE14o-), monocyte-derived macrophages and dendritic cells, it was recently demonstrated that macrophages and dendritic cells create a transepithelial network between the epithelial cells to capture antigens without disrupting the epithelial tightness. The expression of the different tight junction proteins in macrophages and dendritic cells, and the formation of tight junction-like structures with epithelial cells has been demonstrated. Immunofluorescent methods combined with laser scanning microscopy and quantitative real-time polymerase chain reaction were used to investigate if exposure to diesel exhaust particles (DEP) (0.5, 5, 50, 125 μg/ml), for 24 h, can modulate the expression of the tight junction mRNA/protein of occludin, in all three cell types. Only the highest dose of DEP (125 μg/ml) seemed to reduce the occludin mRNA in the cells of the defence system however not in epithelial cells, although the occludin arrangement in the latter cell type was disrupted. The transepithelial electrical resistance was reduced in epithelial cell mono-cultures but not in the triple cell co-cultures, following exposure to high DEP concentration. Cytotoxicity was not found, in either epithelial mono-cultures nor in triple cell co-cultures, after exposure to the different DEP concentrations. We concluded that high concentrations of DEP (125 μg/ml) can modulate the tight junction occludin mRNA in the cells of the defence system and that those cells play an important role maintaining the epithelial integrity following exposure to particulate antigens in lung cells.

Toxicology is about to establish itself as a leading scientific discipline in addressing potential health effects of materials on the nanosize level. Entering into a cutting-edge field, has an impact on identity-building processes within the involved academic fields. In our study, we analyzed the ways in which the entry into the field of nanosciences impacts on the formation of disciplinary identities. Using the methods of qualitative interviews with particle toxicologists in Germany, Holland, Switzerland and the USA, we could demonstrate that currently, toxicology finds itself in a transitional phase. The development of its disciplinary identity is not yet clear. Nearly all of our interview partners stressed the necessity of repositioning toxicology. However, they each suggested different approaches. While one part is already propagandizing the establishment of a new discipline – 'nanotoxicology'- others are more reserved and are demanding a clear separation of traditional and new research areas. In phases of disciplinary new-orientation, research communities do not act consistently. Rather, they establish diverse options. By expanding its disciplinary boundaries, participating in new research fields, while continuing its previous research, and only vaguely defining its topics, toxicology is feeling its way into the new fields without giving up its present self-conception. However, the toxicological research community is also discussing a new disciplinary identity. Within this, toxicology could develop from an auxiliary into a constitutive position, and take over a basic role in the cognitive, institutional and social framing of the nanosciences.