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 (TiO(2) NPs) following a single intratracheal instillation. NRCWE-001 was unmodified rutile TiO(2) 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 TiO(2) 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 TiO(2) to positively charged did not consistently influence pulmonary toxicity of the studied TiO(2) NPs

Particle-induced pulmonary acute phase response may be the causal link between particle inhalation and cardiovascular disease

Inhalation of ambient and workplace particulate air pollution is associated with increased risk of cardiovascular disease. One proposed mechanism for this association is that pulmonary inflammation induces a hepatic acute phase response, which increases risk of cardiovascular disease. Induction of the acute phase response is intimately linked to risk of cardiovascular disease as shown in both epidemiological and animal studies. Indeed, blood levels of acute phase proteins, such as C-reactive protein and serum amyloid A, are independent predictors of risk of cardiovascular disease in prospective epidemiological studies. In this review, we present and review emerging evidence that inhalation of particles (e.g., air diesel exhaust particles and nanoparticles) induces a pulmonary acute phase response, and propose that this induction constitutes the causal link between particle inhalation and risk of cardiovascular disease. Increased levels of acute phase mRNA and proteins in lung tissues, bronchoalveolar lavage fluid and plasma clearly indicate pulmonary acute phase response following pulmonary deposition of different kinds of particles including diesel exhaust particles, nanoparticles, and carbon nanotubes. The pulmonary acute phase response is dose-dependent and long lasting. Conversely, the hepatic acute phase response is reduced relative to lung or entirely absent. We also provide evidence that pulmonary inflammation, as measured by neutrophil influx, is a predictor of the acute phase response and that the total surface area of deposited particles correlates with the pulmonary acute phase response. We discuss the implications of these findings in relation to occupational exposure to nanoparticles. How to cite this article: WIREs Nanomed Nanobiotechnol 2014, 6:517–531. doi: 10.1002/wnan.127

Airway exposure to multi-walled carbon nanotubes disrupts the female reproductive cycle without affecting pregnancy outcomes in mice

Abstract Background The use of multiwalled carbon nanotubes (MWCNT) is increasing due to a growing use in a variety of products across several industries. Thus, occupational exposure is also of increasing concern, particularly since airway exposure to MWCNTs can induce sustained pulmonary acute phase response and inflammation in experimental animals, which may affect female reproduction. This proof-of-principle study therefore aimed to investigate if lung exposure by intratracheal instillation of the MWCNT NM-400 would affect the estrous cycle and reproductive function in female mice. Results Estrous cycle regularity was investigated by comparing vaginal smears before and after exposure to 67 μg of NM-400, whereas reproductive function was analyzed by measuring time to delivery of litters after instillation of 2, 18 or 67 μg of NM-400. Compared to normal estrous cycling determined prior to exposure, exposure to MWCNT significantly prolonged the estrous cycle during which exposure took place, but significantly shortened the estrous cycle immediately after the exposed cycle. No consistent effects were seen on time to delivery of litter or other gestational or litter parameters, such as litter size, sex ratio, implantations and implantation loss. Conclusion Lung exposure to MWCNT interfered with estrous cycling. Effects caused by MWCNTs depended on the time of exposure: the estrous stage was particularly sensitive to exposure, as animals exposed during this stage showed a higher incidence of irregular cycling after exposure. Our data indicates that MWCNT exposure may interfere with events leading to ovulation

DNA Damage Following Pulmonary Exposure by Instillation to Low Doses of Carbon Black (Printex 90) Nanoparticles in Mice

We previously observed genotoxic effects of carbon black nanoparticles at low doses relative to the Danish Occupational Exposure Limit (3.5 mg/m(3)). Furthermore, DNA damage occurred in broncho-alveolar lavage (BAL) cells in the absence of inflammation, indicating that inflammation is not required for the genotoxic effects of carbon black. In this study, we investigated inflammatory and acute phase response in addition to genotoxic effects occurring following exposure to nanoparticulate carbon black (NPCB) at even lower doses. C57BL/6JBomTac mice were examined 1, 3, and 28 days after a single instillation of 0.67, 2, 6, and 162 µg Printex 90 NPCB and vehicle. Cellular composition and protein concentration was evaluated in BAL fluid as markers of inflammatory response and cell damage. DNA strand breaks in BAL cells, lung, and liver tissue were assessed using the alkaline comet assay. The pulmonary acute phase response was analyzed by Saa3 mRNA real-time quantitative PCR. Instillation of the low doses of NPCB induced a slight neutrophil influx one day after exposure. Pulmonary exposure to small doses of NPCB caused an increase in DNA strand breaks in BAL cells and lung tissue measured using the comet assay. We interpret the increased DNA strand breaks occurring following these low exposure doses of NPCB as DNA damage caused by primary genotoxicity in the absence of substantial inflammation, cell damage, and acute phase response. Environ. Mol. Mutagen. 56:41–49, 2015. © 2014 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Societ

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

<p>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.</p

Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide

We investigated toxicity of 2-3 layered >1 μm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 μg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis

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 extensively produced and used in composite materials and electronic applications, thus increasing risk of worker and consumer exposure. MWCNTs are an inhomogeneous group of nanomaterials that come in various lengths, shapes and with different metal contaminations, which makes hazard evaluation difficult. However, several studies suggest that length plays an important role in the toxicity induced by MWCNTs. How the length influences toxicity at the molecular level is yet to be characterized. Female C57BL/6 mice were exposed by single intratracheal instillation to 18, 54 or 162 µg/mouse of a short MWCNT (NRCWE-026, 847±102 nm in length) or long MWCNT (NM-401, 4048±366 nm in length). The two MWCNTs were extensively characterized. Lung tissues were harvested 24 h, 3 d and 28 d after exposure. We employed DNA microarrays, bronchoalveolar lavage fluid analysis, comet assay and dichlorodihydrofluorescein assay in order to profile the pulmonary responses. Bioinformatics tools were then applied to compare and contrast the expression profiles and to build a length dependent property-response matrix for gene-by-gene comparison. The toxicogenomic analysis of the global mRNA changes after exposure to the short, entangled NRCWE-026 or the longer, stiffer NM-401 showed high degree of similarities. The toxicity of both MWCNTs was driven by strong inflammatory and acute phase responses, which peaked at day 3 and was observed both in bronchoalveolar lavage cell influx and in gene expression profiles. The inflammatory response was sustained at post-exposure day 28. Also, at the sub-chronic level, we identified a sub-set of 14 fibrosis related genes that were uniquely differentially regulated after exposure to NM-401. Acellular ROS production occurred almost exclusively with NRCWE-026, however the longer NM-401 induced in vivo DNA strand breaks and differential regulation of genes involved in free radical scavenging more readily than NRCWE-026. Our results indicate that the global mRNA response after exposure to MWCNTs is length independent at the acute time points, but that fibrosis may be length dependent sub-chronic end point.JRC.H.6-Digital Earth and Reference Dat

Transcriptomic Analysis Reveals Novel Mechanistic Insight into Murine Biological Responses to Multi-Walled Carbon Nanotubes in Lungs and Cultured Lung Epithelial Cells

There is great interest in substituting animal work with in vitro experimentation in human health risk assessment; however, there are only few comparisons of in vitro and in vivo biological responses to engineered nanomaterials. We used high-content genomics tools to compare in vivo pulmonary responses of multiwalled carbon nanotubes (MWCNT) to those in vitro in cultured lung epithelial cells (FE1) at the global transcriptomic level. Primary size, surface area and other properties of MWCNT- XNRI -7 (Mitsui7) were characterized using DLS, SEM and TEM. Mice were exposed via a single intratracheal instillation to 18, 54, or 162 μg of Mitsui7/mouse. FE1 cells were incubated with 12.5, 25 and 100 μg/ml of Mitsui7. Tissue and cell samples were collected at 24 hours post-exposure. DNA microarrays were employed to establish mechanistic differences and similarities between the two models. Microarray results were confirmed using gene-specific RT-qPCR. Bronchoalveolar lavage (BAL) fluid was assessed for indications of inflammation in vivo. A strong dose-dependent activation of acute phase and inflammation response was observed in mouse lungs reflective mainly of an inflammatory response as observed in BAL. In vitro, a wide variety of core cellular functions were affected including transcription, cell cycle, and cellular growth and proliferation. Oxidative stress, fibrosis and inflammation processes were altered in both models. Although there were similarities observed between the two models at the pathway-level, the specific genes altered under these pathways were different, suggesting that the underlying mechanisms of responses are different in cells in culture and the lung tissue. Our results suggest that careful consideration should be given in selecting relevant endpoints when substituting animal with in vitro testing