Unchanged pulmonary toxicity of ZnO nanoparticles formulated in a liquid matrix for glass coating.

The inclusion of nanoparticles can increase the quality of certain products. One application is the inclusion of Zinc oxide (ZnO) nanoparticles in a glass coating matrix to produce a UV-absorbing coating for glass sheets. Yet, the question is whether the inclusion of ZnO in the matrix induces toxicity at low exposure levels. To test this, mice were given single intratracheal instillation of 1) ZnO powder (ZnO), 2) ZnO in a glass matrix coating in its liquid phase (ZnO-Matrix), and 3) the matrix with no ZnO (Matrix). Doses of ZnO were 0.23, 0.67, and 2 µg ZnO/mouse. ZnO Matrix doses had equal amounts of ZnO, while Matrix was adjusted to have an equal volume of matrix as ZnO Matrix. Post-exposure periods were 1, 3, or 28 d. Endpoints were pulmonary inflammation as bronchoalveolar lavage (BAL) fluid cellularity, genotoxicity in lung and liver, measured by comet assay, histopathology of lung and liver, and global gene expression in lung using microarrays. Neutrophil numbers were increased to a similar extent with ZnO and ZnO-Matrix at 1 and 3 d. Only weak genotoxicity without dose-response effects was observed in the lung. Lung histology showed an earlier onset of inflammation in material-exposed groups as compared to controls. Microarray analysis showed a stronger response in terms of the number of differentially regulated genes in ZnO-Matrix exposed mice as compared to Matrix only. Activated canonical pathways included inflammatory and cardiovascular ones. In conclusion, the pulmonary toxicity of ZnO was not changed by formulation in a liquid matrix for glass coating.

Pulmonary toxicity of Fe2O3, ZnFe2O4, NiFe2O4 and NiZnFe4O8 nanomaterials: Inflammation and DNA strand breaks.

Exposure to metal oxide nanomaterials potentially occurs at the workplace. We investigated the toxicity of two Fe-oxides: Fe2O3 nanoparticles and nanorods; and three MFe2O4 spinels: NiZnFe4O8, ZnFe2O4, and NiFe2O4 nanoparticles. Mice were dosed 14, 43 or 128 µg by intratracheal instillation. Recovery periods were 1, 3, or 28 days. Inflammation - neutrophil influx into bronchoalveolar lavage (BAL) fluid - occurred for Fe2O3 rods (1 day), ZnFe2O4 (1, 3 days), NiFe2O4 (1, 3, 28 days), Fe2O3 (28 days) and NiZnFe4O8 (28 days). Conversion of mass-dose into specific surface-area-dose showed that inflammation correlated with deposited surface area and consequently, all these nanomaterials belong to the so-called low-solubility, low-toxicity class. Increased levels of DNA strand breaks were observed for both Fe2O3 particles and rods, in BAL cells three days post-exposure. To our knowledge, this is, besides magnetite (Fe3O4), the first study of the pulmonary toxicity of MFe2O4 spinel nanomaterials.

Airway exposure to TiO2 nanoparticles and quartz and effects on sperm counts and testosterone levels in male mice.

Several types of engineered nanoparticles (ENP) have been shown to adversely affect male reproduction in rodent studies, but the airway route of exposure has been little investigated. This precludes adequate risk assessment of ENP exposure in occupational settings. Titanium dioxide nanoparticles (TiO2 NP) have been shown to affect total sperm count in adult male mice after intravenous and oral administration. This study aimed to investigate whether also airway exposure would affect sperm counts in male mice. Mature C57BL/6J mice were intratracheally instilled with 63 µg of rutile nanosized TiO2, once weekly for seven weeks. Respirable α-quartz (SRM1878a) was included at a similar dose level as a positive control for pulmonary inflammation. BALF cell composition showed neutrophil granulocyte influx as indication of pulmonary inflammation in animals exposed to TiO2 NP and α-quartz, but none of the particle exposures affected weight of testes or the epididymis, sperm counts or plasma testosterone when assessed at termination of the study.

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.

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.

Epoxy composite dusts with and without carbon nanotubes cause similar pulmonary responses, but differences in liver histology in mice following pulmonary deposition.

BACKGROUND: The toxicity of dusts from mechanical abrasion of multi-walled carbon nanotube (CNT) epoxy nanocomposites is unknown. We compared the toxic effects of dusts generated by sanding of epoxy composites with and without CNT. The used CNT type was included for comparison. METHODS: Mice received a single intratracheal instillation of 18, 54 and 162 µg of CNT or 54, 162 and 486 µg of the sanding dust from epoxy composite with and without CNT. DNA damage in lung and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Furthermore, the mRNA expression of interleukin 6 and heme oxygenase 1 was measured in the lungs and serum amyloid A1 in the liver. Printex 90 carbon black was included as a reference particle. RESULTS: Pulmonary exposure to CNT and all dusts obtained by sanding epoxy composite boards resulted in recruitment of inflammatory cells into lung lumen: On day 1 after instillation these cells were primarily neutrophils but on day 3, eosinophils contributed significantly to the cell population. There were still increased numbers of neutrophils 28 days after intratracheal instillation of the highest dose of the epoxy dusts. Both CNT and epoxy dusts induced DNA damage in lung tissue up to 3 days after intratracheal instillation but not in liver tissue. There was no additive effect of adding CNT to epoxy resins for any of the pulmonary endpoints. In livers of mice instilled with CNT and epoxy dust with CNTs inflammatory and necrotic histological changes were observed, however, not in mice instilled with epoxy dust without CNT. CONCLUSIONS: Pulmonary deposition of epoxy dusts with and without CNT induced inflammation and DNA damage in lung tissue. There was no additive effect of adding CNT to epoxies for any of the pulmonary endpoints. However, hepatic inflammatory and necrotic histopathological changes were seen in mice instilled with sanding dust from CNT-containing epoxy but not in mice instilled with reference epoxy.

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.

Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation.

Inhalation of carbon black nanoparticles (CBNPs) causes pulmonary inflammation; however, time course data to evaluate the detailed evolution of lung inflammatory responses are lacking. Here we establish a time-series of lung inflammatory response to CBNPs. Female C57BL/6 mice were intratracheally instilled with 162 µg CBNPs alongside vehicle controls. Lung tissues were examined 3h, and 1, 2, 3, 4, 5, 14, and 42 days (d) post-exposure. Global gene expression and pulmonary inflammation were assessed. DNA damage was evaluated in bronchoalveolar lavage (BAL) cells and lung tissue using the comet assay. Increased neutrophil influx was observed at all time-points. DNA strand breaks were increased in BAL cells 3h post-exposure, and in lung tissues 2-5d post-exposure. Approximately 2600 genes were differentially expressed (± 1.5 fold; p ≤ 0.05) across all time-points in the lungs of exposed mice. Altered transcript levels were associated with immune-inflammatory response and acute phase response pathways, consistent with the BAL profiles and expression changes found in common respiratory infectious diseases. Genes involved in DNA repair, apoptosis, cell cycle regulation, and muscle contraction were also differentially expressed. Gene expression changes associated with inflammatory response followed a biphasic pattern, with initial changes at 3h post-exposure declining to base-levels by 3d, increasing again at 14 d, and then persisting to 42 d post-exposure. Thus, this single CBNP exposure that was equivalent to nine 8-h working days at the current Danish occupational exposure limit induced biphasic inflammatory response in gene expression that lasted until 42 d post-exposure, raising concern over the chronic effects of CBNP exposure.

DNA strand breaks, acute phase response and inflammation following pulmonary exposure by instillation to the diesel exhaust particle NIST1650b in mice.

We investigated the inflammatory response, acute phase response and genotoxic effect of diesel exhaust particles (DEPs, NIST1650b) following a single intratracheal instillation. C57BL/6J BomTac mice received 18, 54 or 162 µg/mouse and were killed 1, 3 and 28 days post-exposure. Vehicle controls and the benchmark particle carbon black (CB, Printex 90; 162 µg/mouse) 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 acute phase response was analysed by Saa3 mRNA levels by real-time quantitative polymerase chain reaction. Instillation of DEP induced a strong neutrophil influx 1 and 3 days, but not 28 days post-exposure. Saa3 mRNA levels were increased at all time point for the highest dose and 28 days post-exposure for the middle dose. DEP increased levels of DNA strand breaks in lung tissue for all doses 1 day post-exposure and after 28 days for mid- and high-dose groups. Pulmonary exposure to DEP induced transient inflammation but long-lasting pulmonary acute phase response as well as genotoxicity in lung tissue 28 days post-exposure. The observed long-term pulmonary genotoxicity by DEP was less than the previously observed genotoxicity for CB using identical experimental set-up.

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.

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.

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.

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.

Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay.

The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R (2) = 0.93 for %DNA in tail (%TDNA) and R (2) = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57-77% and the day-to-day variation was only 2-12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor.

Effects of lung exposure to carbon nanotubes on female fertility and pregnancy. A study in mice.

We studied the effects of preconceptional exposure to multiwalled carbon nanotubes (MWCNTs): mature, female C57BL/6J mice were intratracheally instilled with 67µg NM-400 MWCNT, and the following day co-housed with mature males, in breeding pairs. Time to delivery of the first litter, litter parameters, maternal inflammation and histopathology of lung and liver were recorded. In male offspring, locomotor activity, startle response, and daily sperm production (DSP) were assessed. In the dams, lung and liver bore evidence of MWCNT exposure when assessed 6 weeks and 4 months after exposure. A short delay in the delivery of the first litter was observed in exposed females. Litter parameters, behavior and DSP were similar in control and exposed groups. In conclusion, instillation of a single dose of MWCNT induced long lasting pathological changes in dam lung and liver. Theoretically, lung inflammation due to particle exposure could interfere with female reproductive parameters. Whether the observed lag in delivery of a first litter was in fact caused by exposure to MWCNT should be addressed in a study designed specifically to elucidate effects on the early processes involved in establishment of pregnancy. Exposure was not associated with changes in the assessed gestational or offspring parameters.

Daily sperm production: application in studies of prenatal exposure to nanoparticles in mice.

We investigated the influence of maternal airway exposure to nanoparticulate titanium dioxide (TiO2, UV-Titan) and carbon black (CB, Printex90), on male reproductive function in the two following generations. Time-mated C57BL/6J mice were exposed by inhalation to UV-Titan, or by intratracheal instillation with Printex90. Body and testicle weight, sperm content per g testicular parenchyma and daily sperm production (DSP) were assessed. The protocol for assessment of DSP was optimized for application in mice (C57BL/6J) and the influence of different parameters was studied. Maternal particulate exposure did not affect DSP statistically significantly in the F1 generation, although TiO2 tended to reduce sperm counts. Overall, time-to-first F2 litter increased with decreasing sperm production. There was no effect on sperm production in the F2 generation originating after TiO2 exposure. F2 offspring, whose fathers were prenatally exposed to Printex90, showed lowered sperm production. Furthermore, we report statistically significant differences in sperm production between mouse strains.

New embryo lethals in Arabidopsis thaliana: basic genetic and morphological study.

Six different mutations with defects in immature seed development have been identified during screening of a T-DNA collection of Arabidopsis thaliana. The mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development, and therefore they belong to the embryo-lethal class of mutants. The genetic and morphological studies of T-DNA mutations affecting embryo development are presented. The youngest embryos with a defect were observed at the globular stage in the VIII-64 mutation. Externally located cells, precursor of the protoderm, were characterised by abnormal cell division. VIII-41 mutation with a defect at the late globular stage was arrested at the globular-heart stage transition. VIII-111 mutation showed defect at heart stage of embryogenesis with atypical development of cotyledon primordia. The defect was associated with abnormal pattern of cell division constituting the precursor of the shoot apical meristem. In VIII-82 mutation defect in torpedo stage with asymmetric cotyledons was observed. Cotyledon stage of embryos and chlorophyll defect were observed in VIII-75 mutant. Abnormal suspensor consisting of two columns of cells was observed in 280-4-4 mutation. Newly identified embryo-lethals can serve as starting material for more detailed genetic and molecular studies.