Distribution, metabolism, excretion, and toxicity of implanted silver: a review.

Some implantable medical devices contain silver. We aimed to assess at what amount implanted silver becomes toxic. Silver was elevated in bodily fluids and tissues surrounding silver-containing implants. Silver released from implants also distributes to blood and other tissues; there is evidence to suggest silver can pass the blood-brain-barrier. Silver can be deposited as nano-sized particles in various tissues. Such particles, in addition to silver, often contain other elements too, e.g., selenium and sulfur. Silver released from implants seems to stay in the body for long periods (years). Reported excretion pathways following implantation are urinary and fecal ones. Reported toxicological effects were virtually all local reactions surrounding the implants. Argyria is a blue-gray discoloration of the skin due to deposited silver granules. Localized argyria has been described after the implantation of acupuncture needles and silver-coated prostheses, although the presence of silver was tested only for and shown in the former. Other toxicological effects include local tissue reactivity and examples of neurotoxic and vascular effects. We did not include genotoxicity studies in the present publication as we recently evaluated silver to be genotoxic. Carcinogenicity studies were absent. We conclude that local toxicity of implanted silver can be foreseen in some situations.

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.

Pulmonary toxicity of silver vapours, nanoparticles and fine dusts: A review.

Silver is used in a wide range of products, and during their production and use, humans may be exposed through inhalation. Therefore, it is critical to know the concentration levels at which adverse effects may occur. In rodents, inhalation of silver nanoparticles has resulted in increased silver in the lungs, lymph nodes, liver, kidney, spleen, ovaries, and testes. Reported excretion pathways of pulmonary silver are urinary and faecal excretion. Acute effects in humans of the inhalation of silver include lung failure that involved increased heart rate and decreased arterial blood oxygen pressure. Argyria-a blue-grey discoloration of skin due to deposited silver-was observed after pulmonary exposure in 3 individuals; however, the presence of silver in the discolorations was not tested. Argyria after inhalation seems to be less likely than after oral or dermal exposure. Repeated inhalation findings in rodents have shown effects on lung function, pulmonary inflammation, bile duct hyperplasia, and genotoxicity. In our evaluation, the range of NOAEC values was 0.11-0.75 mg/m3. Silver in the ionic form is likely more toxic than in the nanoparticle form but that difference could reflect their different biokinetics. However, silver nanoparticles and ions have a similar pattern of toxicity, probably reflecting that the effect of silver nanoparticles is primarily mediated by released ions. Concerning genotoxicity studies, we evaluated silver to be positive based on studies in mammalian cells in vitro and in vivo when considering various exposure routes. Carcinogenicity data are absent; therefore, no conclusion can be provided on this endpoint.

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.

Influence of dispersion medium on nanomaterial-induced pulmonary inflammation and DNA strand breaks: investigation of carbon black, carbon nanotubes and three titanium dioxide nanoparticles.

Intratracheal instillation serves as a model for inhalation exposure. However, for this, materials are dispersed in appropriate media that may influence toxicity. We tested whether different intratracheal instillation dispersion media influence the pulmonary toxicity of different nanomaterials. Rodents were intratracheally instilled with 162 µg/mouse/1620 µg/rat carbon black (CB), 67 µg/mouse titanium dioxide nanoparticles (TiO2) or 54 µg/mouse carbon nanotubes (CNT). The dispersion media were as follows: water (CB, TiO2); 2% serum in water (CB, CNT, TiO2); 0.05% serum albumin in water (CB, CNT, TiO2); 10% bronchoalveolar lavage fluid in 0.9% NaCl (CB), 10% bronchoalveolar lavage (BAL) fluid in water (CB) or 0.1% Tween-80 in water (CB). Inflammation was measured as pulmonary influx of neutrophils into bronchoalveolar fluid, and DNA damage as DNA strand breaks in BAL cells by comet assay. Inflammation was observed for all nanomaterials (except 38-nm TiO2) in all dispersion media. For CB, inflammation was dispersion medium dependent. Increased levels of DNA strand breaks for CB were observed only in water, 2% serum and 10% BAL fluid in 0.9% NaCl. No dispersion medium-dependent effects on genotoxicity were observed for TiO2, whereas CNT in 2% serum induced higher DNA strand break levels than in 0.05% serum albumin. In conclusion, the dispersion medium was a determinant of CB-induced inflammation and genotoxicity. Water seemed to be the best dispersion medium to mimic CB inhalation, exhibiting DNA strand breaks with only limited inflammation. The influence of dispersion media on nanomaterial toxicity should be considered in the planning of intratracheal investigations.

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.

A Multilaboratory Toxicological Assessment of a Panel of 10 Engineered Nanomaterials to Human Health--ENPRA Project--The Highlights, Limitations, and Current and Future Challenges.

ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of "nanotoxicology research" based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

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.

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.

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 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.

Tissue distribution and elimination after oral and intravenous administration of different titanium dioxide nanoparticles in rats.

OBJECTIVE: The aim of this study was to obtain kinetic data that can be used in human risk assessment of titanium dioxide nanomaterials. METHODS: Tissue distribution and blood kinetics of various titanium dioxide nanoparticles (NM-100, NM-101, NM-102, NM-103, and NM-104), which differ with respect to primary particle size, crystalline form and hydrophobicity, were investigated in rats up to 90 days post-exposure after oral and intravenous administration of a single or five repeated doses. RESULTS: For the oral study, liver, spleen and mesenteric lymph nodes were selected as target tissues for titanium (Ti) analysis. Ti-levels in liver and spleen were above the detection limit only in some rats. Titanium could be detected at low levels in mesenteric lymph nodes. These results indicate that some minor absorption occurs in the gastrointestinal tract, but to a very limited extent.Both after single and repeated intravenous (IV) exposure, titanium rapidly distributed from the systemic circulation to all tissues evaluated (i.e. liver, spleen, kidney, lung, heart, brain, thymus, reproductive organs). Liver was identified as the main target tissue, followed by spleen and lung. Total recovery (expressed as % of nominal dose) for all four tested nanomaterials measured 24 h after single or repeated exposure ranged from 64-95% or 59-108% for male or female animals, respectively. During the 90 days post-exposure period, some decrease in Ti-levels was observed (mainly for NM-100 and NM-102) with a maximum relative decrease of 26%. This was also confirmed by the results of the kinetic analysis which revealed that for each of the investigated tissues the half-lifes were considerable (range 28-650 days, depending on the TiO(2)-particle and tissue investigated). Minor differences in kinetic profile were observed between the various particles, though these could not be clearly related to differences in primary particle size or hydrophobicity. Some indications were observed for an effect of crystalline form (anatase vs. rutile) on total Ti recovery. CONCLUSION: Overall, the results of the present oral and IV study indicates very low oral bioavailability and slow tissue elimination. Limited uptake in combination with slow elimination might result in the long run in potential tissue accumulation.

Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation.

We investigated gene expression, protein synthesis, and particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO2). Female C57BL/6 mice were exposed to rutile nano-TiO2 via single intratracheal instillations of 18, 54, and 162µg/mouse. Mice were sampled 1, 3, and 28days post-exposure. The deposition of nano-TiO2 in the lungs was assessed using nanoscale hyperspectral microscopy. Biological responses in the pulmonary system were analyzed using DNA microarrays, pathway-specific real-time RT-PCR (qPCR), gene-specific qPCR arrays, and tissue protein ELISA. Hyperspectral mapping showed dose-dependent retention of nano-TiO2 in the lungs up to 28days post-instillation. DNA microarray analysis revealed approximately 3000 genes that were altered across all treatment groups (±1.3 fold; p<0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein level. Although no influx of neutrophils was detected at the low dose, changes in the expression of several genes and proteins associated with inflammation were observed. Resolving inflammation at the medium dose, and lack of neutrophil influx in the lung fluid at the low dose, were associated with down-regulation of genes involved in ion homeostasis and muscle regulation. Our gene expression results imply that retention of nano-TiO2 in the absence of inflammation over time may potentially perturb calcium and ion homeostasis, and affect smooth muscle activities.

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.

Toxicity dose descriptors from animal inhalation studies of 13 nanomaterials and their bulk and ionic counterparts and variation with primary particle characteristics.

This study collects toxicity data from animal inhalation studies of some nanomaterials and their bulk and ionic counterparts. To allow potential grouping and interpretations, we retrieved the primary physicochemical and exposure data to the extent possible for each of the materials. Reviewed materials are compounds (mainly elements, oxides and salts) of carbon (carbon black, carbon nanotubes, and graphene), silver, cerium, cobalt, copper, iron, nickel, silicium (amorphous silica and quartz), titanium (titanium dioxide), and zinc (chemical symbols: Ag, C, Ce, Co, Cu, Fe, Ni, Si, Ti, TiO2, and Zn). Collected endpoints are: a) pulmonary inflammation, measured as neutrophils in bronchoalveolar lavage (BAL) fluid at 0-24 hours after last exposure; and b) genotoxicity/carcinogenicity. We present the dose descriptors no-observed-adverse-effect concentrations (NOAECs) and lowest-observed-adverse-effect concentrations (LOAECs) for 88 nanomaterial investigations in data-library and graph formats. We also calculate 'the value where 25% of exposed animals develop tumors' (T25) for carcinogenicity studies. We describe how the data may be used for hazard assessment of the materials using carbon black as an example. The collected data also enable hazard comparison between different materials. An important observation for poorly soluble particles is that the NOAEC for neutrophil numbers in general lies around 1 to 2 mg/m3. We further discuss why some materials' dose descriptors deviate from this level, likely reflecting the effects of the ionic form and effects of the fiber-shape. Finally, we discuss that long-term studies, in general, provide the lowest dose descriptors, and dose descriptors are positively correlated with particle size for near-spherical materials.

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).

Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver.

BACKGROUND: Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic in vitro but less is known about their genotoxicity in various organs in vivo. METHODS: We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by Saa3 mRNA real-time quantitative PCR. RESULTS: Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on post-exposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of Saa3 mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver. CONCLUSIONS: Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the initial exposure. Our results demonstrate that CBNPs may cause genotoxicity both in the primary exposed tissue, lung and BAL cells, and in a secondary tissue, the liver.