Model Complexity as Determining Factor for In Vitro Nanosafety Studies: Effects of Silver and Titanium Dioxide Nanomaterials in Intestinal Models.

With the rising interest in the effects of orally ingested engineered nanomaterials (ENMs), much effort is undertaken to develop and advance intestinal in vitro models. The cytotoxic, proinflammatory, and DNA damaging properties of polyvinylpyrrolidone-capped silver (Ag-PVP) and titanium dioxide (TiO2 , P25) ENM in four in vitro models of increasing complexity-from proliferating Caco-2 and HT29-MTX-E12 monocultures to long-term transwell triple cultures including THP-1 macrophages to reproduce the human intestine in healthy versus inflamed-like state-are studied. Results are compared against in vivo effects of the same ENM through intestinal tissue analysis from 28-day oral exposure studies in mice. Adverse responses are only observed in monocultures and suggest toxic potential for both ENM, typically showing stronger effects for Ag-PVP than for TiO2 . By contrast, no adverse effects are observed in either the transwell cultures or the analyzed murine tissues. The data provide further support that monoculture models represent a cost and time efficient tool for early-phase hazard assessment. However, the observed similarities in morphology and ENM effects in murine intestinal tissue and the in vitro triple culture model suggest that advanced multifacetted research questions concerning oral ENM exposure are more adequately addressed by the more complex and time intensive models.

An in-depth multi-omics analysis in RLE-6TN rat alveolar epithelial cells allows for nanomaterial categorization.

BACKGROUND: Nanomaterials (NMs) can be fine-tuned in their properties resulting in a high number of variants, each requiring a thorough safety assessment. Grouping and categorization approaches that would reduce the amount of testing are in principle existing for NMs but are still mostly conceptual. One drawback is the limited mechanistic understanding of NM toxicity. Thus, we conducted a multi-omics in vitro study in RLE-6TN rat alveolar epithelial cells involving 12 NMs covering different materials and including a systematic variation of particle size, surface charge and hydrophobicity for SiO2 NMs. Cellular responses were analyzed by global proteomics, targeted metabolomics and SH2 profiling. Results were integrated using Weighted Gene Correlation Network Analysis (WGCNA). RESULTS: Cluster analyses involving all data sets separated Graphene Oxide, TiO2_NM105, SiO2_40 and Phthalocyanine Blue from the other NMs as their cellular responses showed a high degree of similarities, although apical in vivo results may differ. SiO2_7 behaved differently but still induced significant changes. In contrast, the remaining NMs were more similar to untreated controls. WGCNA revealed correlations of specific physico-chemical properties such as agglomerate size and redox potential to cellular responses. A key driver analysis could identify biomolecules being highly correlated to the observed effects, which might be representative biomarker candidates. Key drivers in our study were mainly related to oxidative stress responses and apoptosis. CONCLUSIONS: Our multi-omics approach involving proteomics, metabolomics and SH2 profiling proved useful to obtain insights into NMs Mode of Actions. Integrating results allowed for a more robust NM categorization. Moreover, key physico-chemical properties strongly correlating with NM toxicity were identified. Finally, we suggest several key drivers of toxicity that bear the potential to improve future testing and assessment approaches.

Arterial blood pressure responses to short-term exposure to fine and ultrafine particles from indoor sources - A randomized sham-controlled exposure study of healthy volunteers.

OBJECTIVES: Particulate air pollution is linked to adverse cardiovascular effects. The aim of the study was to investigate the effect of short-term exposure to indoor particles on blood pressure (BP). METHODS: We analyzed the association of particle emissions from indoor sources (candle burning, toasting bread, frying sausages) with BP changes in 54 healthy volunteers in a randomized cross-over controlled exposure study. Particle mass concentration (PMC), size-specific particle number concentration (PNC) and lung-deposited particle surface area concentration (PSC) were measured during the 2h exposure. Systolic and diastolic blood pressure were measured before, during, directly, 2, 4 and 24h after exposure. We performed multiple mixed linear regression analyses of different particle metrics and BP. RESULTS: BP significantly increased with increasing PMC, PSC and PNC resulting from toasting bread. For example, an increase per 10µg/m3 PM10 and PM2.5, systolic BP increased at all time points with largest changes 1h after exposure initiation of 1.5mmHg (95%-CI: 1.1; 1.9) and of 2.2mmHg (95%-CI: 1.3; 3.1), respectively. CONCLUSIONS: Our study suggests an association of short-term exposure to fine and ultrafine particles emitted from toasting bread with increases in BP. Particles emitted from frying sausages and candle burning did not consistently affect BP.

Associations between three specific a-cellular measures of the oxidative potential of particulate matter and markers of acute airway and nasal inflammation in healthy volunteers.

INTRODUCTION: We evaluated associations between three a-cellular measures of the oxidative potential (OP) of particulate matter (PM) and acute health effects. METHODS: We exposed 31 volunteers for 5 h to ambient air pollution at five locations: an underground train station, two traffic sites, a farm and an urban background site. Each volunteer visited at least three sites. We conducted health measurements before exposure, 2 h after exposure and the next morning. We measured air pollution on site and characterised the OP of PM2.5 and PM10 using three a-cellular assays; dithiotreitol (OP(DTT)), electron spin resonance (OP(ESR)) and ascorbic acid depletion (OP(AA)). RESULTS: In single-pollutant models, all measures of OP were significantly associated with increases in fractional exhaled nitric oxide and increases in interleukin-6 in nasal lavage 2 h after exposure. These OP associations remained significant after adjustment for co-pollutants when only the four outdoor sites were included, but lost significance when measurements at the underground site were included. Other health end points including lung function and vascular inflammatory and coagulation parameters in blood were not consistently associated with OP. CONCLUSIONS: We found significant associations between three a-cellular measures of OP of PM and markers of airway and nasal inflammation. However, consistency of these effects in two-pollutant models depended on how measurements at the underground site were considered. Lung function and vascular inflammatory and coagulation parameters in blood were not consistently associated with OP. Our study, therefore, provides limited support for a role of OP in predicting acute health effects of PM in healthy young adults.

Agreement of central site measurements and land use regression modeled oxidative potential of PM2.5 with personal exposure.

Oxidative potential (OP) of ambient particulate matter (PM) has been suggested as a health-relevant exposure metric. In order to use OP for exposure assessment, information is needed about how well central site OP measurements and modeled average OP at the home address reflect temporal and spatial variation of personal OP. We collected 96-hour personal, home outdoor and indoor PM2.5 samples from 15 volunteers living either at traffic, urban or regional background locations in Utrecht, the Netherlands. OP was also measured at one central reference site to account for temporal variations. OP was assessed using electron spin resonance (OP(ESR)) and dithiothreitol (OP(DTT)). Spatial variation of average OP at the home address was modeled using land use regression (LUR) models. For both OP(ESR) and OP(DTT), temporal correlations of central site measurements with home outdoor measurements were high (R>0.75), and moderate to high (R=0.49-0.70) with personal measurements. The LUR model predictions for OP correlated significantly with the home outdoor concentrations for OP(DTT) and OP(ESR) (R=0.65 and 0.62, respectively). LUR model predictions were moderately correlated with personal OP(DTT) measurements (R=0.50). Adjustment for indoor sources, such as vacuum cleaning and absence of fume-hood, improved the temporal and spatial agreement with measured personal exposure for OP(ESR). OP(DTT) was not associated with any indoor sources. Our study results support the use of central site OP for exposure assessment of epidemiological studies focusing on short-term health effects.

Proteomic analysis of protein carbonylation: a useful tool to unravel nanoparticle toxicity mechanisms.

BACKGROUND: Oxidative stress, a commonly used paradigm to explain nanoparticle (NP)-induced toxicity, results from an imbalance between reactive oxygen species (ROS) generation and detoxification. As one consequence, protein carbonyl levels may become enhanced. Thus, the qualitative and quantitative description of protein carbonylation may be used to characterize how biological systems respond to oxidative stress induced by NPs. METHODS: We investigated a representative panel of 24 NPs including functionalized amorphous silica (6), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and boehmite. Surface reactivities of all NPs were studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with all NPs, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D and/or 2D immunoblotting and identified by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF). In parallel, tissue homogenates from rat lungs intratracheally instilled with silver NPs were studied. RESULTS: Eleven NPs induced elevated levels of carbonylated proteins. This was in good agreement with the surface reactivity of the NPs as obtained by ESR and the reduction in cell viability as assessed by WST-1 assay. By contrast, results obtained by DCFDA assay were deviating. Each NP induced an individual pattern of protein carbonyls on 2D immunoblots. Affected proteins comprised cytoskeletal components, proteins being involved in stress response, or cytoplasmic enzymes of central metabolic pathways such as glycolysis and gluconeogenesis. Furthermore, induction of carbonyls upon silver NP treatment was also verified in rat lung tissue homogenates. CONCLUSIONS: Analysis of protein carbonylation is a versatile and sensitive method to describe NP-induced oxidative stress and, therefore, can be used to identify NPs of concern. Furthermore, detailed information about compromised proteins may aid in classifying NPs according to their mode of action.

Mobility of coated and uncoated TiO2 nanomaterials in soil columns--Applicability of the tests methods of OECD TG 312 and 106 for nanomaterials.

Nanomaterials are commonly used in everyday life products and during their life cycle they can be released into the environment. Soils and sediments are estimated as significant sinks for those nanomaterials. To investigate and assess the behaviour of nanomaterials in soils and sediments standardized test methods are needed. In this study the applicability of two existing international standardized test guidelines for the testing of nanomaterials, OECD TG 106 "Adsorption/Desorption using a Bath Equilibrium Method" and the OECD TG 312 "Leaching in Soil Columns", were investigated. For the study one coated and two uncoated TiO2 nanomaterials were used, respectively. The results indicate that the OECD TG 106 is not applicable for nanomaterials. However, the test method according to OECD TG 312 was found to be applicable if nano-specific adaptations are applied. The mobility investigations of the OECD TG 312 indicated a material-dependent mobility of the nanomaterials, which in some cases may lead to an accumulation in the upper soil layers. Whereas no significant transport was observed for the uncoated materials for the double-coated material (coating with dimethicone and aluminiumoxide) a significant transport was detected and attributed to the coating.

Multiparametric in vitro genotoxicity assessment of different variants of amorphous silica nanomaterials in rat alveolar epithelial cells.

The hazard posed to human health by inhaled amorphous silica nanomaterials (aSiO2 NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO2 NM variants covering different sizes (7, 15, and 40 nm) and surface modifications (unmodified, phosphonate-, amino- and trimethylsilyl-modified) on rat alveolar epithelial (RLE-6TN) cells. Cytotoxicity was evaluated at 24 h after exposure to the aSiO2 NM variants by the lactate dehydrogenase (LDH) release and WST-1 reduction assays, while genotoxicity was assessed using different endpoints: DNA damage (single- and double-strand breaks [SSB and DSB]) by the comet assay for all aSiO2 NM variants; cell cycle progression and γ-H2AX levels (DSB) by flow cytometry for those variants that presented higher cytotoxic and DNA damaging potential. The variants with higher surface area demonstrated a higher cytotoxic potential (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_15_Phospho). SiO2_40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm2), a decrease in G0/G1 subpopulation was accompanied by a significant increase in S and G2/M sub-populations after exposure to all tested materials except for SiO2_40 which did not affect cell cycle progression. Based on the obtained data, the tested variants can be ranked for its genotoxic DNA damage potential as follows: SiO2_7 = SiO2_40 = SiO2_15_Unmod > SiO2_15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction.

Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects.

The immense diversity and constant development of nanomaterials (NMs) increase the need for a facilitated risk assessment, which requires knowledge of the modes of action (MoAs) of NMs. This necessitates a comprehensive data basis, which can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is essential to follow the 3R concept and to cope with the high number of NMs. In the present study, we aimed to compare NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data analysis strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For the in vitro investigations, rat alveolar epithelial cells (RLE-6TN) and rat alveolar macrophages (NR8383) were treated with different doses of NMs, and the results were compared with the effects on rat lungs after short-term inhalations and instillations. Since reactive oxygen species (ROS) production has been described as a critical biological effect of NMs, we focused on different levels of oxidative stress. Thus, we found opposite changes in proteins and metabolites related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, demonstrating that the MoAs of NMs depend on the model system used. Interestingly, in vivo, pathways related to inflammation were more affected than oxidative stress responses. Hence, the assignment of the observed effects to levels of oxidative stress was also different in vitro and in vivo. However, the overall classification of "active" and "passive" NMs was consistent in vitro and in vivo, suggesting that both cell lines tested are suitable for the assessment of NM toxicity. In summary, the results presented here highlight the need to carefully review model systems to decipher the extent to which they can replace in vivo assays.

Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM).

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

Evaluation of the NLRP3 Inflammasome Activating Effects of a Large Panel of TiO2 Nanomaterials in Macrophages.

TiO2 nanomaterials are among the most commonly produced and used engineered nanomaterials (NMs) in the world. There is controversy regarding their ability to induce inflammation-mediated lung injuries following inhalation exposure. Activation of the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome and subsequent release of the cytokine interleukin (IL)-1ß in pulmonary macrophages has been postulated as an essential pathway for the inflammatory and associated tissue-remodeling effects of toxic particles. Our study aim was to determine and rank the IL-1ß activating properties of TiO2 NMs by comparing a large panel of different samples against each other as well as against fine TiO2, synthetic amorphous silica and crystalline silica (DQ12 quartz). Effects were evaluated in primary bone marrow derived macrophages (BMDMs) from NALP3-deficient and proficient mice as well as in the rat alveolar macrophage cell line NR8383. Our results show that specific TiO2 NMs can activate the inflammasome in macrophages albeit with a markedly lower potency than amorphous SiO2 and quartz. The heterogeneity in IL-1ß release observed in our study among 19 different TiO2 NMs underscores the relevance of case-by-case evaluation of nanomaterials of similar chemical composition. Our findings also further promote the NR8383 cell line as a promising in vitro tool for the assessment of the inflammatory and inflammasome activating properties of NMs.

A multi-omics approach reveals mechanisms of nanomaterial toxicity and structure-activity relationships in alveolar macrophages.

In respect to the high number of released nanomaterials and their highly variable properties, novel grouping approaches are required based on the effects of nanomaterials. Proper grouping calls for a combination of an experimental setup with a higher number of structurally similar nanomaterials and for employing integrated omics approaches to identify the mode of action. Here, we analyzed the effects of seven well-characterized NMs comprising different chemical compositions, sizes and chemical surface modifications on the rat alveolar macrophage cell line NR8383. The NMs were investigated at three doses ranging from 2.5 to 10 µg/cm2 after 24 h incubation using an integrated multi-omics approach involving untargeted proteomics, targeted metabolomics, and src homology 2 (SH2) profiling. By using Weighted Gene Correlation Network Analysis (WGCNA) for the integrative data, we identified correlations of molecular pathways with physico-chemical properties and toxicological endpoints. The three investigated SiO2 variants induced strong alterations in all three omics approaches and were, therefore, be classified as "active." Two organic phthalocyanines showed minor responses and Mn2O3 induced a different molecular response pattern than the other NMs. WGCNA revealed that agglomerate size and surface area as well as LDH release are among the most important parameters correlating with nanotoxicology. Moreover, we identified key drivers that can serve as representative biomarker candidates, supporting the value of multi-omics approaches to establish integrated approaches to testing and assessment (IATAs).

Metabolomics profiling to investigate nanomaterial toxicity in vitro and in vivo.

Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure-activity relationship and into the underlying MoA.

Multi-walled carbon nanotubes induce stronger migration of inflammatory cells in vitro than asbestos or granular particles but a similar pattern of inflammatory mediators.

Biopersistent pro-inflammatory fibers are suspected human carcinogens. Cytotoxicity and transcription of pro- and anti-inflammatory mediators of different fibers were investigated in functional relationship to chemotaxis in vitro as a model for fiber-induced inflammation of the lung. We challenged NR8383 rat macrophages with multi-walled carbon nanotubes (MWCNT) and various asbestos fibers. The resulting cell supernatants were than studied using the Particle-induced Cell Migration Assay (PICMA) and cytotoxicity was determined using the LDH test. Expression of inflammatory mediators was analyzed with qPCR and verified by ELISA. Chrysotile A and the rigid, needle-shaped NM-401 caused the strongest cytotoxic effects and the largest number of migrated cells. In contrast, the MWCNT NM-400, NM-402, and NM403 were apparently non-cytotoxic but induced pronounced cell migration showing a very steep dose response. However, the strength of cell migration and cytotoxicity of the asbestos fibers were correlated. The expression profile of inflammatory mediators was comparable, although cytotoxicity of the MWCNT NM-401 and NM-403 differed strongly. Induction of the corresponding proteins was confirmed for CCL2, CCL3, CXCL1, CXCL3, IL1RA (IL1RN), CSF1, GDF15 and TNFa. Chrysotile A and NM-401 induced much stronger chemotaxis than the non-fibrous particles reported in our previous study. Cytotoxic and chemotactic effects correspond to the induction of inflammatory mediators.

Effects of short-term exposure to fine and ultrafine particles from indoor sources on arterial stiffness - A randomized sham-controlled exposure study.

OBJECTIVES: Particulate air pollution is linked to adverse cardiovascular effects, including arterial stiffness. The aim of the study was to investigate the effect of short-term exposure to indoor fine and ultrafine particles on augmentation index (AIx), augmentation pressure (AP), and pulse wave velocity (PWV), early signs of vascular damage. METHODS: We analyzed the association of particle emissions from typical indoor sources (candle burning - CB, toasting bread - TB, and frying sausages - FS) with changes in pulse wave analysis indices in 55 healthy adults in a randomized cross-over controlled exposure study. Particle mass concentration (PMC), size-specific particle number concentration (PNC) and lung-deposited particle surface area concentration (PSC) were measured during the 2 h exposure. AIx and AP were measured before, directly, 2, 4 and 24 h after exposure. PWV was measured directly and 24 h after exposure. We performed multiple mixed linear regression analyses of different particle metrics and AIx, AP and PWV. RESULTS: The highest mean PMC was observed during FS reaching a maximum of 210 µg/m3 PM10. The maximal PNC for UFP <100 nm was reached during CB with 2.3 million particles/cm3. PSC was similar across all three exposures (about 3000 µm2/cm³). Strongest associations between different particles metrics and arterial stiffness indices could be observed for UFP from CB and FS and for PMC from TB. The highest mean increase could be observed for the UFP fraction <10 nm, measured during CB, and AIx with an increase of 9.5%-points (95%-CI: 3.1; 15.9). PSC seemed to follow the pattern of PNC. PM10 and PM2.5 from TB led to clear changes in AIx with biggest increases for PM10 of 5.8%-points (95%-CI: 3.2; 8.4) 2 h after exposure and for PM2.5 of 8.1%-points (95%-CI: 2.5; 13.7) directly after exposure. CONCLUSIONS: Our study indicates effects of indoor exposure to fine and ultrafine particles on systemic arterial stiffness indices that depend on the indoor source as well as on particle metric. Differences in size-specific physical characteristics of source-specific particles might account for these differential effects. We did not observe clear and stable associations of indoor particle exposure and PWV.

The nanoGRAVUR framework to group (nano)materials for their occupational, consumer, environmental risks based on a harmonized set of material properties, applied to 34 case studies.

The project nanoGRAVUR (BMBF, 2015-2018) developed a framework for grouping of nanomaterials. Different groups may result for each of the three distinct perspectives of occupational, consumer and environmental safety. The properties, methods and descriptors are harmonised between the three perspectives and are based on: Tier 1 intrinsic physico-chemical properties (what they are) or GHS classification of the non-nano-form (human tox, ecotox, physical hazards); Tier 2 extrinsic physico-chemical properties, release from nano-enabled products, in vitro assays with cells (where they go; what they do); Tier 3 case-specific tests, potentially in vivo studies to substantiate the similarity within groups or application-specific exposure testing. Amongst all properties, dissolution and transformation are least modulated by different nanoforms within one substance, whereas dustiness, dispersion stability, abiotic and especially in vitro surface reactivity vary more often between different nanoforms. The methods developed or selected by nanoGRAVUR fill several gaps highlighted in the ProSafe reviews, and are useful to implement (i) the concept of nanoforms of the European Chemicals Agency (ECHA) and (ii) the concept of discrete forms of the United States Environmental Protection Agency (EPA). One cannot assess the significance of a dissimilarity, if the dynamic range of that property is unknown. Benchmark materials span dynamic ranges that enable us to establish bands, often with order-of-magnitude ranges. In 34 case studies we observed high biological similarity within each substance when we compared different (nano)forms of SiO2, BaSO4, kaolin, CeO2, ZnO, organic pigments, especially when we compared forms that are all untreated on the surface. In contrast, different Fe2O3 or TiO2 (nano)forms differ more significantly. The same nanoforms were also integrated in nano-enabled products (NEPs) for automotive coatings, clinker-reduced cements, cosmetic sunscreen, and lightweight polymers.

Ultrafine and Fine Particle Number and Surface Area Concentrations and Daily Cause-Specific Mortality in the Ruhr Area, Germany, 2009-2014.

BACKGROUND: Although epidemiologic studies have shown associations between particle mass and daily mortality, evidence on other particle metrics is weak. OBJECTIVES: We investigated associations of size-specific particle number concentration (PNC) and lung-deposited particle surface area concentration (PSC) with cause-specific daily mortality in contrast to PM10. METHODS: We used time-series data (March 2009-December 2014) on daily natural, cardiovascular, and respiratory mortality (NM, CVM, RM) of three adjacent cities in the Ruhr Area, Germany. Size-specific PNC (electric mobility diameter of 13.3-750 nm), PSC, and PM10 were measured at an urban background monitoring site. In single- and multipollutant Poisson regression models, we estimated percentage change (95% confidence interval) [% (95% CI)] in mortality per interquartile range (IQR) in exposure at single-day (0-7) and aggregated lags (0-1, 2-3, 4-7), accounting for time trend, temperature, humidity, day of week, holidays, period of seasonal population decrease, and influenza. RESULTS: PNC100-750 and PSC were highly correlated and had similar immediate (lag0-1) and delayed (lag4-7) associations with NM and CVM, for example, 1.12% (95% CI: 0.09, 2.33) and 1.56% (95% CI: 0.22, 2.92) higher NM with IQR increases in PNC100-750 at lag0-1 and lag4-7, respectfully, which were slightly stronger then associations with IQR increases in PM10. Positive associations between PNC and NM were strongest for accumulation mode particles (PNC 100-500 nm), and for larger UFPs (PNC 50-100 nm). Associations between NM and PNC<100 changed little after adjustment for O3 or PM10, but were more sensitive to adjustment for NO2. CONCLUSION: Size-specific PNC (50-500 nm) and lung-deposited PSC were associated with natural and cardiovascular mortality in the Ruhr Area. Although associations were similar to those estimated for an IQR increase in PM10, particle number size distributions can be linked to emission sources, and thus may be more informative for potential public health interventions. Moreover, PSC could be used as an alternative metric that integrates particle size distribution as well as deposition efficiency. https://doi.org/10.1289/EHP2054.

Silver nanoparticles induce hormesis in A549 human epithelial cells.

Despite the gaps in our knowledge on the toxicity of silver nanoparticles (AgNPs), the application of these materials is fast expanding, from medicine, to food as well as the use in consumer products. It has been reported that prolonged exposure might make cells more resistant to AgNPs. This prompted us to investigate if AgNPs may give rise to a hormetic response. Two types of AgNPs were used, i.e. colloidal AgNPs and an AgNP powder. For both types of nanosilver it was found that a low dose pretreatment of A549 human epithelial cells with AgNPs induced protection against a toxic dose of AgNPs and acrolein. This protection was more pronounced after pretreatment with the colloidal AgNPs. Interestingly, the mechanism of the hormetic response appeared to differ from that of acrolein. Adaptation to acrolein is related to Nrf2 translocation, increased mRNA expression of γGCS, HO-1 and increased GSH levels and the increased GSH levels can explain the hormetic effect. The adaptive response to AgNPs was not related to an increase in mRNA expression of γGCS and GSH levels. Yet, HO-1 mRNA expression and Nrf2 immunoreactivity were enhanced, indicating that these processes might be involved. So, AgNPs induce adaptation, but in contrast to acrolein GSH plays no role.

Size matters--The phototoxicity of TiO2 nanomaterials.

Under solar radiation several titanium dioxide nanoparticles (nano-TiO2) are known to be phototoxic for daphnids. We investigated the influence of primary particle size (10, 25, and 220 nm) and ionic strength (IS) of the test medium on the acute phototoxicity of anatase TiO2 particles to Daphnia magna. The intermediate sized particles (25 nm) showed the highest phototoxicity followed by the 10 nm and 220 nm sized particles (median effective concentrations (EC50): 0.53, 1.28, 3.88 mg/L). Photoactivity was specified by differentiating free OH radicals (therephthalic acid method) and on the other hand surface adsorbed, as well as free OH, electron holes, and O2(-) (electron paramagnetic resonance spectroscopy, EPR). We show that the formation of free OH radicals increased with a decrease in primary particle size (terephthalic acid method), whereas the total measured ROS content was highest at an intermediate particle size of 25 nm, which consequently revealed the highest photoxicity. The photoactivities of the 10 and 220 nm particles as measured by EPR were comparable. We suggest that phototoxicity depends additionally on the particle-daphnia interaction area, which explains the higher photoxicity of the 10 nm particles compared to the 220 nm particles. Thus, phototoxicity is a function of the generation of different ROS and the particle-daphnia interaction area, both depending on particle size. Phototoxicity of the 10 nm and 25 nm sized nanoparticles decreased as IS of the test medium increased (EC50: 2.9 and 1.1 mg/L). In conformity with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory we suggest that the precipitation of nano-TiO2 was more pronounced in high than in low IS medium, causing a lower phototoxicity. In summary, primary particle size and IS of the medium were identified as factors influencing phototoxicity of anatase nano-TiO2 to D. magna.

Oxidative potential of particulate matter at a German motorway.

Ambient particulate matter (PM10) was sampled alongside a motorway in North-Rhine Westphalia, Germany, during a one-year period. In sum, 120 PM10 samples on quartz fibre filters, 60 samples at each side of the motorway, were taken during clear cross-wind direction situations, i.e. upwind (local background situation) and downwind (traffic influenced). To quantify the traffic-related oxidative potential (OP), or more precisely the hydroxyl radical (OH˙) generation potency, these samples were analysed to study their hydrogen peroxide dependent oxidant generation by Electron Paramagnetic Resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. In addition the PM10 mass, the chemical composition and the NOx concentrations were determined. For PM10 mass and traffic tracers like Sb, Ba, elemental and organic carbon as well as for NOx, an additional contribution to the background concentration caused by the traffic was observed (factor: 1.3-6.0). The downwind measurements showed in 72% of cases higher OH˙ generation potencies with an average factor of 1.4. Significant correlations to OH˙ were detected for Fe (r > 0.58) and Cu (r > 0.57) for the upwind and overall (upwind + downwind, r > 0.44) dataset. At the downwind side these correlations were absent and are assumed to be covered by the interferences with additional soot particles leading to a quenching of OH˙. Accordingly, no significant overall correlation of the OH˙ generation potency with the traffic intensity was detected. The suggested quenching effect was confirmed via standard diesel soot (SRM 2975) measurements using the EPR approach. In summary, the traffic related PM causes an intrinsic OH˙ generation via Fenton-like reaction but obviously also leads to interferences and scavenging by traffic related carbonaceous compounds. In consequence, for future studies that would link the intrinsic OP and adverse health effects we suggest to analyse the relationship to EC/OC and to use in parallel also a further OP detection method.