Genotoxicity in the absence of inflammation after tungsten inhalation in mice.

Tungsten is used in several applications and human exposure may occur. To assess its pulmonary toxicity, we exposed male mice to nose-only inhalation of tungsten particles at 9, 23 or 132 mg/m3 (Low, Mid and High exposure) (45 min/day, 5 days/week for 2 weeks). Increased genotoxicity (assessed by comet assay) was seen in bronchoalveolar (BAL) fluid cells at Low and High exposure. We measured acellular ROS production, and cannot exclude that ROS contributed to the observed genotoxicity. We saw no effects on body weight gain, pulmonary inflammation, lactate dehydrogenase or protein in BAL fluid, pathology of liver or kidney, or on sperm counts. In conclusion, tungsten showed non-dose dependent genotoxicity in the absence of inflammation and therefore interpreted to be primary genotoxicity. Based on genotoxicity, a Lowest Observed Adverse Effect Concentration (LOAEC) could be set at 9 mg/m3. It was not possible to establish a No Adverse Effect Concentration (NOAEC).

A transcriptomic overview of lung and liver changes one day after pulmonary exposure to graphene and graphene oxide.

Hazard evaluation of graphene-based materials (GBM) is still in its early stage and it is slowed by their large diversity in the physicochemical properties. This study explores transcriptomic differences in the lung and liver after pulmonary exposure to two GBM with similar physical properties, but different surface chemistry. Female C57BL/6 mice were exposed by a single intratracheal instillation of 0, 18, 54 or 162 µg/mouse of graphene oxide (GO) or reduced graphene oxide (rGO). Pulmonary and hepatic changes in the transcriptome were profiled to identify commonly and uniquely perturbed functions and pathways by GO and rGO. These changes were then related to previously analyzed toxicity endpoints. GO exposure induced more differentially expressed genes, affected more functions, and perturbed more pathways compared to rGO, both in lung and liver tissues. The largest differences were observed for the pulmonary innate immune response and acute phase response, and for hepatic lipid homeostasis, which were strongly induced after GO exposure. These changes collective indicate a potential for atherosclerotic changes after GO, but not rGO exposure. As GO and rGO are physically similar, the higher level of hydroxyl groups on the surface of GO is likely the main reason for the observed differences. GO exposure also uniquely induced changes in the transcriptome related to fibrosis, whereas both GBM induced similar changes related to Reactive Oxygen Species production and genotoxicity. The differences in transcriptomic responses between the two GBM types can be used to understand how physicochemical properties influence biological responses and enable hazard evaluation of GBM and hazard ranking of GO and rGO, both in relation to each other and to other nanomaterials.

A response to the letter to the editor by Driscoll et al.

In response to the Letter to the Editor by Kevin Driscoll et al., we certainly agree that particle clearance halftimes are increased with increasing lung burden in rats, hamsters and mice, whereas complete inhibition of particle clearance has only been observed in rats, and only at high particle concentrations (50 mg/m3). Where we disagree with Kevin Driscoll and colleagues, is on the implications of the increased clearance halftimes observed at higher lung burden. We argue that it does not hamper the extrapolations from relatively high dose levels to lower dose levels.Furthermore, we highlight, again, the challenges of detecting particle-induced lung cancer in epidemiological studies where occupational, particle-induced lung cancer has to be detected on top of the background lung cancer incidence. Almost all available epidemiological studies on carbon black and titanium dioxide suffer from a number of limitations, including lack of control for smoking, the use of background population cancer rates as reference in the US studies, lack of information regarding particle size of the exposure, and incomplete follow-up for cause of death of the study population.

Acute Phase Response as a Biological Mechanism-of-Action of (Nano)particle-Induced Cardiovascular Disease.

Inhaled nanoparticles constitute a potential health hazard due to their size-dependent lung deposition and large surface to mass ratio. Exposure to high levels contributes to the risk of developing respiratory and cardiovascular diseases, as well as of lung cancer. Particle-induced acute phase response may be an important mechanism of action of particle-induced cardiovascular disease. Here, the authors review new important scientific evidence showing causal relationships between inhalation of particle and nanomaterials, induction of acute phase response, and risk of cardiovascular disease. Particle-induced acute phase response provides a means for risk assessment of particle-induced cardiovascular disease and underscores cardiovascular disease as an occupational disease.

Commentary: the chronic inhalation study in rats for assessing lung cancer risk may be better than its reputation.

Recently, Borm and Driscoll published a commentary discussing grouping of Poorly Soluble particles of Low Toxicity (PSLTs) and the use of rats as an animal model for human hazard assessment of PSLTs (Particle and Fibre Toxicology (2019) 16(1):11). The commentary was based on the scientific opinion of several international experts on these topics. The general conclusion from the authors was a cautious approach towards using chronic inhalation studies in rats for human hazard assessment of PSLTs. This was based on evidence of inhibition of particle clearance leading to overload in the rats after high dose exposure, and a suggested over reactivity of rat lung cancer responses compared to human risk.As a response to the commentary, we here discuss evidence from the scientific literature showing that a) diesel exhaust particles, carbon black nanoparticles and TiO2 nanoparticles have similar carcinogenic potential in rats, and induce lung cancer at air concentrations below the air concentrations that inhibit particle clearance in rats, and b) chronic inhalation studies of diesel exhaust particles are less sensitive than epidemiological studies, leading to higher risk estimates for lung cancer. Thus, evidence suggests that the chronic inhalation study in rats can be used for assessing lung cancer risk insoluble nanomaterials.

Physicochemical predictors of Multi-Walled Carbon Nanotube-induced pulmonary histopathology and toxicity one year after pulmonary deposition of 11 different Multi-Walled Carbon Nanotubes in mice.

Multi-walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity-inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well-characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 µg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m3 ). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT-related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical-dependent difference in MWCNT-induced long-term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure.

Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been linked to an increased risk of developing cardiovascular disease in addition to the well-documented physicochemical-dependent adverse lung effects. A proposed mechanism is through a strong and sustained pulmonary secretion of acute phase proteins to the blood. We identified physicochemical determinants of MWCNT-induced systemic acute phase response by analyzing effects of pulmonary exposure to 14 commercial, well-characterized MWCNTs in female C57BL/6J mice pulmonary exposed to 0, 6, 18 or 54 µg MWCNT/mouse. Plasma levels of acute phase response proteins serum amyloid A1/2 (SAA1/2) and SAA3 were determined on day 1, 28 or 92. Expression levels of hepatic Saa1 and pulmonary Saa3 mRNA levels were assessed to determine the origin of the acute phase response proteins. Pulmonary Saa3 mRNA expression levels were greater and lasted longer than hepatic Saa1 mRNA expression. Plasma SAA1/2 and SAA3 protein levels were related to time and physicochemical properties using adjusted, multiple regression analyses. SAA3 and SAA1/2 plasma protein levels were increased after exposure to almost all of the MWCNTs on day 1, whereas limited changes were observed on day 28 and 92. SAA1/2 and SAA3 protein levels did not correlate and only SAA3 protein levels correlated with neutrophil influx. The multiple regression analyses revealed a protective effect of MWCNT length on SAA1/2 protein level on day 1, such that a longer length resulted in lowered SAA1/2 plasma levels. Increased SAA3 protein levels were positively related to dose and content of Mn, Mg and Co on day 1, whereas oxidation and diameter of the MWCNTs were protective on day 28 and 92, respectively. The results of this study reveal very differently controlled pulmonary and hepatic acute phase responses after MWCNT exposure. As the responses were influenced by the physicochemical properties of the MWCNTs, this study provides the first step towards designing MWCNT that induce less SAA.

Surface modification does not influence the genotoxic and inflammatory effects of TiO2 nanoparticles after pulmonary exposure by instillation in mice.

The influence of surface charge of nanomaterials on toxicological effects is not yet fully understood. We investigated the inflammatory response, the acute phase response and the genotoxic effect of two different titanium dioxide nanoparticles (TiO2 NPs) following a single intratracheal instillation. NRCWE-001 was unmodified rutile TiO2 with endogenous negative surface charge, whereas NRCWE-002 was surface modified to be positively charged. C57BL/6J BomTac mice received 18, 54 and 162 µg/mouse and were humanely killed 1, 3 and 28 days post-exposure. Vehicle controls were tested alongside for comparison. The cellular composition and protein concentration were determined in bronchoalveolar lavage (BAL) fluid as markers for an inflammatory response. Pulmonary and systemic genotoxicity was analysed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. The pulmonary and hepatic acute phase response was analysed by Saa3 mRNA levels in lung tissue or Saa1 mRNA levels in liver tissue by real-time quantitative polymerase chain reaction. Instillation of NRCWE-001 and -002 both induced a dose-dependent neutrophil influx into the lung lining fluid and Saa3 mRNA levels in lung tissue at all assessed time points. There was no statistically significant difference between NRCWE-001 and NRCWE-002. Exposure to both TiO2 NPs induced increased levels of DNA strand breaks in lung tissue at all doses 1 and 28 days post-exposure and NRCWE-002 at the low and middle dose 3 days post-exposure. The DNA strand break levels were statistically significantly different for NRCWE-001 and -002 for liver and for BAL cells, but no consistent pattern was observed. In conclusion, functionalisation of reactive negatively charged rutile TiO2 to positively charged did not consistently influence pulmonary toxicity of the studied TiO2 NPs.

Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity.

Lung deposition of multi-walled carbon nanotubes (MWCNT) induces pulmonary toxicity. Commercial MWCNT vary greatly in physicochemical properties and consequently in biological effects. To identify determinants of MWCNT-induced toxicity, we analyzed the effects of pulmonary exposure to 10 commercial MWCNT (supplied in three groups of different dimensions, with one pristine and two/three surface modified in each group). We characterized morphology, chemical composition, surface area and functionalization levels. MWCNT were deposited in lungs of female C57BL/6J mice by intratracheal instillation of 0, 6, 18 or 54 µg/mouse. Pulmonary inflammation (neutrophil influx in bronchoalveolar lavage (BAL)) and genotoxicity were determined on day 1, 28 or 92. Histopathology of the lungs was performed on day 28 and 92. All MWCNT induced similar histological changes. Lymphocytic aggregates were detected for all MWCNT on day 28 and 92. Using adjusted, multiple regression analyses, inflammation and genotoxicity were related to dose, time and physicochemical properties. The specific surface area (BET) was identified as a positive predictor of pulmonary inflammation on all post-exposure days. In addition, length significantly predicted pulmonary inflammation, whereas surface oxidation (-OH and -COOH) was predictor of lowered inflammation on day 28. BET surface area, and therefore diameter, significantly predicted genotoxicity in BAL fluid cells and lung tissue such that lower BET surface area or correspondingly larger diameter was associated with increased genotoxicity. This study provides information on possible toxicity-driving physicochemical properties of MWCNT. The results may contribute to safe-by-design manufacturing of MWCNT, thereby minimizing adverse effects.

MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs.

Multi-walled carbon nanotubes (MWCNTs) are an inhomogeneous group of nanomaterials that vary in lengths, shapes and types of metal contamination, which makes hazard evaluation difficult. Here we present a toxicogenomic analysis of female C57BL/6 mouse lungs following a single intratracheal instillation of 0, 18, 54 or 162 µg/mouse of a small, curled (CNT(Small), 0.8 ± 0.1 µm in length) or large, thick MWCNT (CNT(Large), 4 ± 0.4 µm in length). The two MWCNTs were extensively characterized by SEM and TEM imaging, thermogravimetric analysis, and Brunauer-Emmett-Teller surface area analysis. Lung tissues were harvested 24h, 3 days and 28 days post-exposure. DNA microarrays were used to analyze gene expression, in parallel with analysis of bronchoalveolar lavage fluid, lung histology, DNA damage (comet assay) and the presence of reactive oxygen species (dichlorodihydrofluorescein assay), to profile and characterize related pulmonary endpoints. Overall changes in global transcription following exposure to CNT(Small) or CNT(Large) were similar. Both MWCNTs elicited strong acute phase and inflammatory responses that peaked at day 3, persisted up to 28 days, and were characterized by increased cellular influx in bronchoalveolar lavage fluid, interstitial pneumonia and gene expression changes. However, CNT(Large) elicited an earlier onset of inflammation and DNA damage, and induced more fibrosis and a unique fibrotic gene expression signature at day 28, compared to CNT(Small). The results indicate that the extent of change at the molecular level during early response phases following an acute exposure is greater in mice exposed to CNT(Large), which may eventually lead to the different responses observed at day 28.

Changes in cholesterol homeostasis and acute phase response link pulmonary exposure to multi-walled carbon nanotubes to risk of cardiovascular disease.

Adverse lung effects following pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) are well documented in rodents. However, systemic effects are less understood. Epidemiological studies have shown increased cardiovascular disease risk after pulmonary exposure to airborne particles, which has led to concerns that inhalation exposure to MWCNTs might pose similar risks. We analyzed parameters related to cardiovascular disease, including plasma acute phase response (APR) proteins and plasma lipids, in female C57BL/6 mice exposed to a single intratracheal instillation of 0, 18, 54 or 162µg/mouse of small, entangled (CNTSmall, 0.8±0.1µm long) or large, thick MWCNTs (CNTLarge, 4±0.4µm long). Liver tissues and plasma were harvested 1, 3 and 28days post-exposure. In addition, global hepatic gene expression, hepatic cholesterol content and liver histology were used to assess hepatic effects. The two MWCNTs induced similar systemic responses despite their different physicochemical properties. APR proteins SAA3 and haptoglobin, plasma total cholesterol and low-density/very low-density lipoprotein were significantly increased following exposure to either MWCNTs. Plasma SAA3 levels correlated strongly with pulmonary Saa3 levels. Analysis of global gene expression revealed perturbation of the same biological processes and pathways in liver, including the HMG-CoA reductase pathway. Both MWCNTs induced similar histological hepatic changes, with a tendency towards greater response following CNTLarge exposure. Overall, we show that pulmonary exposure to two different MWCNTs induces similar systemic and hepatic responses, including changes in plasma APR, lipid composition, hepatic gene expression and liver morphology. The results link pulmonary exposure to MWCNTs with risk of cardiovascular disease.

Time-dependent subcellular distribution and effects of carbon nanotubes in lungs of mice.

BACKGROUND AND METHODS: Pulmonary deposited carbon nanotubes (CNTs) are cleared very slowly from the lung, but there is limited information on how CNTs interact with the lung tissue over time. To address this, three different multiwalled CNTs were intratracheally instilled into female C57BL/6 mice: one short (850 nm) and tangled, and two longer (4 µm and 5.7 µm) and thicker. We assessed the cellular interaction with these CNTs using transmission electron microscopy (TEM) 1, 3 and 28 days after instillation. RESULTS: TEM analysis revealed that the three CNTs followed the same overall progression pattern over time. Initially, CNTs were taken up either by a diffusion mechanism or via endocytosis. Then CNTs were agglomerated in vesicles in macrophages. Lastly, at 28 days post-exposure, evidence suggesting CNT escape from vesicle enclosures were found. The longer and thicker CNTs more often perturbed and escaped vesicular enclosures in macrophages compared to the smaller CNTs. Bronchoalveolar lavage (BAL) showed that the CNT exposure induced both an eosinophil influx and also eosinophilic crystalline pneumonia. CONCLUSION: Two very different types of multiwalled CNTs had very similar pattern of cellular interactions in lung tissue, with the longer and thicker CNTs resulting in more severe effects in terms of eosinophil influx and incidence of eosinophilic crystalline pneumonia (ECP).