Is pulmonary inflammation a valid predictor of particle induced lung pathology? The case of amorphous and crystalline silicas.

Although inflammation is a normal and beneficial response, it is also a key event in the pathology of many chronic diseases, including pulmonary and systemic particle-induced disease. In addition, inflammation is now considered as the key response in standard settings for inhaled particles and a critical endpoint in OECD-based sub-acute/ chronic animal inhalation testing protocols. In this paper, we discuss that whilst the role of inflammation in lung disease is undeniable, it is when inflammation deviates from normal parameters that adversity occurs. We introduce the importance of the time course and in particular, the reversibility of inflammation in the progression towards tissue remodelling and neoplastic changes as commonly seen in rat inhalation studies. For this purpose, we used chronic inhalation studies with synthetic amorphous silicas (SAS) and reactive crystalline silica (RCS) as a source of data to describe the time-course of inflammation towards and beyond adversity. Whilst amorphous silicas induce an acute but reversible inflammatory response, only RCS induces a persistent, progressive response after cessation of exposure, resulting in fibrosis and carcinogenicity in rodents and humans. This suggests that the use of inflammation as a fixed endpoint at the cessation of exposure may not be a reliable predictor of particle-induced lung pathology. We therefore suggest extending the current OECD testing guidelines with a recovery period, that allows inflammation to resolve or progress into altered structure and function, such as fibrosis.

Talc Inhalation in Rats and Humans: A Review and Appraisal of Available Evidence.

BACKGROUND: Current information on the health effects and toxicology of talc suggests that this may lead to a specific target organ toxicity arising from repeated exposure (STOT-RE) classification. OBJECTIVE: To provide an assessment of the currently available inhalation toxicity data on talc and to put these data in the perspective of other poorly soluble low-toxicity particles. METHODS: A database of 177 articles was gathered from different sources. RESULTS: Relevant animal data sets were subjected to a quality review, and epidemiological studies on talc and lung effects published since 2016 were reviewed. CONCLUSIONS: Of nine original inhalation studies reviewed, only one study using rats and mice met the criteria that are needed to include for a reliable evaluation for STOT-RE. Together with the pulmonary effects observed in exposed talc miners, a STOT-RE 1 classification is warranted.

Applying Existing Particle Paradigms to Inhaled Microplastic Particles.

Ambient particulate pollution originating from plastic contaminates air, including indoor and urban environments. The recent discovery of ambient microplastic (MP) particles of a size capable of depositing in the thoracic region of the airway, if inhaled, has raised concern for public exposure and health impacts following lessons learned from other particle domains. Current microplastic exposure estimates are relatively low compared to total ambient particulate matter, but optimal analytical techniques and therefore data for risk and health impact assessments are lacking. In the absence of such an evidence base, this paper explores paradigms, metrics and dose-response curves developed in other particle domains as a starting point for predicting whether microplastic are of concern. Bio-persistence, presence of reactive sites and soluble toxicants are likely key properties in microplastic toxicity, but these are not measured in environmental studies and hence are challenging to interpret in exposure. Data from a MP inhalation study in rats is available but the study was conducted using conditions that do not replicate the known human health effects of PM2.5 or surrogate exposures: compromised, aged animal models are recommended to investigate potential parallels between MPs and PM2.5. One of these parallels is provided by tire wear particles (TWP), which form part of current ambient PM and are sometimes regarded as microplastic. A connection to epidemiological studies where PM filters are still available is recommended and consequently analytical advances are required. In summary, established particle domains and existing paradigms provide valuable insight and data that can be used to predict MP toxicity, and direct study design and key properties to consider in this emerging field.

Inflammation as a Key Outcome Pathway in Particle Induced Effects in the Lung.

Inflammation is considered a key event in the pathology of many chronic diseases, including pulmonary and systemic particle induced effects. In addition, inflammation is now considered as the key response in standard setting for poorly-soluble low toxicity (PSLT) particles and also the critical endpoint to screen for in OECD based sub-chronic animal inhalation testing protocols. During Particles & Health 2021, an afternoon session was dedicated to the subject and a brief summary of the most important messages are summarized in this paper. In the first part of this session, two speakers (Prof. Lison and Dr Duffin) provided state of the art insight into different aspects and sequels to (persistent) inflammation as a protective or adverse response. Most recent insights on the role of different macrophage cell types were presented as well as perspectives and data provided by inflammatory pathways in humans, such as in asthma and COPD. A brief review of the expert workshop on PSLT particles focusing on the regulatory impact of using persistent inflammation as a key outcome was provided by Kevin Driscoll. The second part of the session focused on the outcomes that are associated with inflammation in animal studies, with an emphasis by Drs. Harkema (Michigan State) and Weber (Anapath) on cell proliferation and other pathologies that need to be considered when comparing human and animal responses, such as outcomes from 14- or 28 day inhalation studies used for specific target organ toxicity classification.

Bimodal Interventional Instrument Markers for Magnetic Particle Imaging and Magnetic Resonance Imaging-A Proof-of-Concept Study.

The purpose of this work was to develop instrument markers that are visible in both magnetic particle imaging (MPI) and magnetic resonance imaging (MRI). The instrument markers were based on two different magnetic nanoparticle types (synthesized in-house KLB and commercial Bayoxide E8706). Coatings containing one of both particle types were fabricated and measured with a magnetic particle spectrometer (MPS) to estimate their MPI performance. Coatings based on both particle types were then applied on a segment of a nonmetallic guidewire. Imaging experiments were conducted using a commercial, preclinical MPI scanner and a preclinical 1 tesla MRI system. MPI image reconstruction was performed based on system matrices measured with dried KLB and Bayoxide E8706 coatings. The bimodal markers were clearly visible in both methods. They caused circular signal voids in MRI and areas of high signal intensity in MPI. Both the signal voids as well as the areas of high signal intensity were larger than the real marker size. Images that were reconstructed with a Bayoxide E8706 system matrix did not show sufficient MPI signal. Instrument markers with bimodal visibility are essential for the perspective of monitoring cardiovascular interventions with MPI/MRI hybrid systems.

Optimised passive marker device visibility and automatic marker detection for 3-T MRI-guided endovascular interventions: a pulsatile flow phantom study.

BACKGROUND: Passive paramagnetic markers on magnetic resonance imaging (MRI)-compatible endovascular devices induce susceptibility artifacts, enabling MRI-visibility and real-time MRI-guidance. Optimised visibility is crucial for automatic detection and device tracking but depends on MRI technical parameters and marker characteristics. We assessed marker visibility and automatic detection robustness for varying MRI parameters and marker characteristics in a pulsatile flow phantom. METHODS: Guidewires with varying iron(II,III) oxide nanoparticle (IONP) concentration markers were imaged using gradient-echo (GRE) and balanced steady-state free precession (bSSFP) sequences at 3 T. Furthermore, echo time (TE), slice thickness (ST) and phase encoding direction (PED) were varied. Artifact width was measured and contrast-to-noise ratios were calculated. Marker visibility and image quality were scored by two MRI interventional radiologists. Additionally, a deep learning model for automatic marker detection was trained and the effects of the parameters on detection performance were evaluated. Two-tailed Wilcoxon signed-rank tests were used (significance level, p < 0.05). RESULTS: Medan artifact width (IQR) was larger in bSSFP compared to GRE images (12.7 mm (11.0-15.2) versus 8.4 mm (6.5-11.0)) (p < 0.001) and showed a positive relation with TE and IONP concentration. Switching PED and doubling ST had limited effect on artifact width. Image quality assessment scores were higher for GRE compared to bSSFP images. The deep learning model automatically detected the markers. However, the model performance was reduced after adjusting PED, TE, and IONP concentration. CONCLUSION: Marker visibility was sufficient and a large range of artifact sizes was generated by adjusting TE and IONP concentration. Deep learning-based marker detection was feasible but performance decreased for altered MR parameters. These factors should be considered to optimise device visibility and ensure reliable automatic marker detectability in MRI-guided endovascular interventions.

The parallels between particle induced lung overload and particle induced periprosthetic osteolysis (PPOL).

Background: When particles deposit for instance in the lung after inhalation or in the hip joint after local release from a hip implant material they can initiate a defense response. Even though these particles originate from inert materials such as polyethylene (PE) or titanium, they may cause harm when reaching high local doses and overwhelming local defense mechanisms. Main body: This paper describes the parallels between adverse outcome pathways (AOP) and particle properties in lung overload and periprosthetic osteolysis (PPOL). It is noted that in both outcomes in different organs , the macrophage and cytokine orchestrated persistent inflammation is the common driver of events, in the bone leading to loss of bone density and structure, and in the lung leading to fibrosis and cancer. Most evidence on lung overload and its AOP is derived from chronic inhalation studies in rats, and the relevance to man is questioned. In PPOL, the paradigms and metrics are based on human clinical data, with additional insights generated from in vitro and animal studies. In both organ pathologies the total volume of particle deposition has been used to set threshold values for the onset of pathological alterations. The estimated clinical threshold for PPOL of 130 mg/ml is much higher than the amount to cause lung overload in the rat (10 mg/ml),although the threshold in PPOL is not necessarily synonymous to particle overload. Conclusions: The paradigms developed in two very different areas of particle response in the human body have major similarities in their AOP. Connecting the clinical evidence in PPOL to lung overload challenges relevance of rat inhalation studies to the human lung cancer hazard. .

Comment on Saber et al. (2019), "Commentary: the chronic inhalation study in rats for assessing lung cancer risk may be better than its reputation".

In their Commentary Saber et al. (Part Fibre Toxicol 16: 44, 2019) argue that chronic inhalation studies in rats can be used for assessing the lung cancer risk of insoluble nanomaterials. The authors make several significant errors in their interpretation and representation of the underlying science. In this Letter to the Editor we discuss these inaccuracies to correct the scientific record. When the science is recounted accurately it does not support Saber et al's statements and conclusions.

Expert workshop on the hazards and risks of poorly soluble low toxicity particles.

'Lung particle overload' refers to the impaired lung particle clearance and increased particle retention occurring with high lung doses of poorly soluble low toxicity (PSLT) particles. In rats, lung particle overload is associated with inflammation, epithelial hyperplasia, and, in extreme cases, lung cancer. While the human relevance of rat lung tumors occurring under overload has been questioned, recent regulatory decisions have considered these outcomes evidence of possible human hazard. To better understand the state-of-the-science on PSLT toxicology, an Expert Workshop was held to document agreements and differences amongst a panel of highly experienced scientists and regulators. Key outcomes included: a functional definition of PSLTs; agreement the rat is a sensitive model for PSLT inhalation toxicology; identifying lung inflammation as a critical endpoint for PSLT risk assessment; and, agreement rat lung cancer occurring only under conditions of lung particle overload does not imply a cancer hazard for humans under non-overloading exposures. Moreover, when asked - should PSLTs be considered as human lung carcinogens based on rat data alone (and no supporting data from other species), the expert consensus was: 'No. However, the experts noted the current default regulatory position on rat lung overload data alone would be the suspicion of human carcinogen hazard.' The many areas of the expert agreement provide guidance for design, interpretation, and extrapolating PSLT inhalation toxicology studies. Considering the workshop outcomes, the authors recommend guidelines for evaluation and classification of PSLT be reassessed; and, prior decisions on PSLT hazard classification be revisited to determine if they remain appropriate.

The hazards and risks of inhaled poorly soluble particles - where do we stand after 30 years of research?

BACKGROUND: In 2006, titanium dioxide and carbon black were classified by IARC as "possibly carcinogenic to humans" and in 2017 the European Chemicals Agency's (ECHA) Committee for Risk Assessment concluded titanium dioxide meets the criteria to be classified as suspected of causing cancer (category 2, through the inhalation route). These classifications were based primarily on the occurrence of lung cancer in rats exposed chronically to high concentrations of these materials, as no such responses have been observed in other animal species similarly exposed. After the EU classification of titanium dioxide, it was suggested that Poorly Soluble particles of Low Toxicity (PSLTs) can be evaluated as a group. MAIN BODY: To better understand the current state of scientific opinion, we sought perspective from several international experts on topics relevant to the classification of carbon black; titanium dioxide; and, the potential future classification of PSLTs. Areas discussed included: grouping of PSLTs; the relevance of rat lung cancer responses to high concentrations of PSLTs; and, clearance overload and implications for interpretation of inhalation toxicology studies. We found there were several areas where a large majority of experts, including ourselves, agreed. These included concerns on the grouping of PSLT and the definition of clearance overload. Regarding the extrapolation of PSLT associated lung cancer in rats there were some strongly held differences, although most experts questioned the relevance when excessive exposures which overwhelm lung clearance were required. SHORT CONCLUSION: Given the ongoing discussion on PSLT classification and safety, we believe it is important to re-activate the public debate including experts and stakeholders. Such an open discussion would serve to formally document where scientific consensus and differences exist. This could form the basis for design of future safety programs and safety assessments.

Labeling of Collagen Type I Templates with a Naturally Derived Contrast Agent for Noninvasive MR Imaging in Soft Tissue Engineering.

In vivo monitoring of tissue-engineered constructs is important to assess their integrity, remodeling, and degradation. However, this is challenging when the contrast with neighboring tissues is low, necessitating labeling with contrast agents (CAs), but current CAs have limitations (i.e., toxicity, negative contrast, label instability, and/or inappropriate size). Therefore, a naturally derived hemin-L-lysine (HL) complex is used as a potential CA to label collagen-based templates for magnetic resonance imaging (MRI). Labeling does not change the basic characteristics of the collagen templates. When hybrid templates composed of collagen type I reinforced with degradable polymers are subcutaneously implanted in mice, longitudinal visualization by MRI is possible with good contrast and in correlation with template remodeling. In contrast, unlabeled collagen templates are hardly detectable and the fate of these templates cannot be monitored by MRI. Interestingly, tissue remodeling and vascularization are enhanced within HL-labeled templates. Thus, HL labeling is presented as a promising universal imaging marker to label tissue-engineered implants for MRI, which additionally seems to accelerate tissue regeneration.

An updated review of the genotoxicity of respirable crystalline silica.

Human exposure to (certain forms of) crystalline silica (CS) potentially results in adverse effects on human health. Since 1997 IARC has classified CS as a Group 1 carcinogen [1], which was confirmed in a later review in 2012 [2]. The genotoxic potential and mode of genotoxic action of CS was not conclusive in either of the IARC reviews, although a proposal for mode of actions was made in an extensive review of the genotoxicity of CS by Borm, Tran and Donaldson in 2011 [3]. The present study identified 141 new papers from search strings related to genotoxicity of respirable CS (RCS) since 2011 and, of these, 17 relevant publications with genotoxicity data were included in this detailed review.Studies on in vitro genotoxic endpoints primarily included micronucleus (MN) frequency and % fragmented DNA as measured in the comet assay, and were mostly negative, apart from two studies using primary or cultured macrophages. In vivo studies confirmed the role of persistent inflammation due to quartz surface toxicity leading to anti-oxidant responses in mice and rats, but DNA damage was only seen in rats. The role of surface characteristics was strengthened by in vitro and in vivo studies using aluminium or hydrophobic treatment to quench the silanol groups on the CS surface.In conclusion, the different modes of action of RCS-induced genotoxicity have been evaluated in a series of independent, adequate studies since 2011. Earlier conclusions on the role of inflammation driven by quartz surface in genotoxic and carcinogenic effects after inhalation are confirmed and findings support a practical threshold. Whereas classic in vitro genotoxicity studies confirm an earlier no-observed effect level (NOEL) in cell cultures of 60-70 µg/cm2, transformation frequency in SHE cells suggests a lower threshold around 5 µg/cm2. Both levels are only achieved in vivo at doses (2-4 mg) beyond in vivo doses (> 200 µg) that cause persistent inflammation and tissue remodelling in the rat lung.

Magnetic Particle Imaging: A Resovist Based Marking Technology for Guide Wires and Catheters for Vascular Interventions.

Magnetic particle imaging (MPI) is able to provide high temporal and good spatial resolution, high signal to noise ratio and sensitivity. Furthermore, it is a truly quantitative method as its signal strength is proportional to the concentration of its tracer, superparamagnetic iron oxide nanoparticles (SPIOs), over a wide range practically relevant concentrations. Thus, MPI is proposed as a promising future method for guidance of vascular interventions. To implement this, devices such as guide wires and catheters have to be discernible in MPI, which can be achieved by coating already commercially available devices with SPIOs. In this proof of principle study the feasibility of that approach is demonstrated. First, a Ferucarbotran-based SPIO-varnish was developed by embedding Ferucarbotran into an organic based solvent. Subsequently, the biocompatible varnish was applied to a commercially available guidewire and diagnostic catheter for vascular interventional purposes. In an interventional setting using a vessel phantom, the coating proved to be mechanically and chemically stable and thin enough to ensure normal handling as with uncoated devices. The devices were visualized in 3D on a preclinical MPI demonstrator using a system function based image reconstruction process. The system function was acquired with a probe of the dried varnish prior to the measurements. The devices were visualized with a very high temporal resolution and a simple catheter/guide wire maneuver was demonstrated.

Comparative performance of a panel of commercially available antimicrobial nanocoatings in Europe.

BACKGROUND: Bacterial resistance against the classic antibiotics is posing an increasing challenge for the prevention and treatment of infections in health care environments. The introduction of antimicrobial nanocoatings with active ingredients provides alternative measures for active killing of microorganisms, through a preventive hygiene approach. PURPOSE: The purpose of this study was to investigate the antimicrobial activity of a panel of antimicrobial coatings available on the European market. METHODS: A comparative, biased selection of commercially available antimicrobial coatings was tested for antimicrobial efficiency. Suppliers were contacted to deliver their coatings on glass and/or stainless steel substrates. In total, 23 coatings from eleven suppliers were received, which were investigated for their effect on the growth of Escherichia coli, using the International Organization for Standardization (ISO) 22196 protocol. RESULTS: The majority of nanomaterial-containing coatings (n=13) contained nanosilver (n=12), while only one had photocatalytic TiO2 as the active particle. The differences in antimicrobial activity among all of the coatings, expressed as log reduction values, varied between 1.3 and 6.6, while the variation within the nanomaterial-based group was between 2.0 and 6.2. Although nanosilver coatings were on average very effective in reducing the number of viable bacteria after challenge, the strongest log reduction (6.6) was seen with a coating that has immobilized, covalently bound quaternary ammonium salt in its matrix. Besides these two compounds, coatings containing TiO2, poly(dimethylsiloxane), triclosan, or zinc pyrithione evoked 100% killing of E. coli. CONCLUSION: Our findings indicate that nanosilver dominates the nanoparticle-based coatings and performs adequately. However, considering the unknowns in relation to ecotoxicological emission and effects, it needs further consideration before widespread application into different environments.

Silica-induced NLRP3 inflammasome activation in vitro and in rat lungs.

RATIONALE: Mineral particles in the lung cause inflammation and silicosis. In myeloid and bronchial epithelial cells the inflammasome plays a role in responses to crystalline silica. Thioredoxin (TRX) and its inhibitory protein TRX-interacting protein link oxidative stress with inflammasome activation. We investigated inflammasome activation by crystalline silica polymorphs and modulation by TRX in vitro, as well as its localization and the importance of silica surface reactivity in rats. METHODS: We exposed bronchial epithelial cells and differentiated macrophages to silica polymorphs quartz and cristobalite and measured caspase-1 activity as well as the release of IL-1ß, bFGF and HMGB1; including after TRX overexpression or treatment with recombinant TRX. Rats were intratracheally instilled with vehicle control, Dörentruper quartz (DQ12) or DQ12 coated with polyvinylpyridine N-oxide. At days 3, 7, 28, 90, 180 and 360 five animals per treatment group were sacrificed. Hallmarks of silicosis were assessed with Haematoxylin-eosin and Sirius Red stainings. Caspase-1 activity in the bronchoalveolar lavage and caspase-1 and IL-1ß localization in lung tissue were determined using Western blot and immunohistochemistry (IHC). RESULTS: Silica polymorphs triggered secretion of IL-1ß, bFGF and HMGB1 in a surface reactivity dependent manner. Inflammasome readouts linked with caspase-1 enzymatic activity were attenuated by TRX overexpression or treatment. At day 3 and 7 increased caspase-1 activity was detected in BALF of the DQ12 group and increased levels of caspase-1 and IL-1ß were observed with IHC in the DQ12 group compared to controls. DQ12 exposure revealed silicotic nodules at 180 and 360 days. Particle surface modification markedly attenuated the grade of inflammation and lymphocyte influx and attenuated the level of inflammasome activation, indicating that the development of silicosis and inflammasome activation is determined by crystalline silica surface reactivity. CONCLUSION: Our novel data indicate the pivotal role of surface reactivity of crystalline silica to activate the inflammasome in cultures of both epithelial cells and macrophages. Inhibitory capacity of the antioxidant TRX to inflammasome activation was evidenced. DQ12 quartz exposure induced acute and chronic functional activation of the inflammasome in the heterogeneous cell populations of the lung in associated with its crystalline surface reactivity.

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements.

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.