An integrated approach for the in vitro dosimetry of engineered nanomaterials

Background: There is a great need for screening tools capable of rapidly assessing nanomaterial toxicity. One impediment to the development of reliable in vitro screening methods is the need for accurate measures of cellular dose. We present here a methodology that enables accurate determination of delivered to cell dose metrics. This methodology includes (1) standardization of engineered nanomaterial (ENM) suspension preparation; (2) measurement of ENM characteristics controlling delivery to cells in culture; and (3) calculation of delivered dose as a function of exposure time using the ISDD model. The approach is validated against experimentally measured doses, and simplified analytical expressions for the delivered dose (Relevant In Vitro Dose (RID)f function) are derived for 20 ENMs. These functions can be used by nanotoxicologists to accurately calculate the total mass (RIDM), surface area (RIDSA), or particle number (RIDN) delivered to cells as a function of exposure time. Results: The proposed methodology was used to derive the effective density, agglomerate diameter and RID functions for 17 industrially-relevant metal and metal oxide ENMs, two carbonaceous nanoparticles, and non-agglomerating gold nanospheres, for two well plate configurations (96 and 384 well plates). For agglomerating ENMs, the measured effective density was on average 60% below the material density. We report great variability in delivered dose metrics, with some materials depositing within 24 hours while others require over 100 hours for delivery to cells. A neutron-activated tracer particle system was employed to validate the proposed in vitro dosimetry methodology for a number of ENMs (measured delivered to cell dose within 9% of estimated). Conclusions: Our findings confirm and extend experimental and computational evidence that agglomerate characteristics affect the dose delivered to cells. Therefore measurement of these characteristics is critical for effective use of in vitro systems for nanotoxicology. The mixed experimental/computational approach to cellular dosimetry proposed and validated here can be used by nanotoxicologists to accurately calculate the delivered to cell dose metrics for various ENMs and in vitro conditions as a function of exposure time. The RID functions and characterization data for widely used ENMs presented here can together be used by experimentalists to design and interpret toxicity studies

The effect of short-term changes in air pollution on respiratory and cardiovascular morbidity in Nicosia, Cyprus.

Presented at the 6th International Conference on Urban Air Quality, Limassol, March, 2007. Short-paper was submitted for peer-review and appears in proceedings of the conference.This study investigates the effect of daily changes in levels of PM10 on the daily volume of respiratory and cardiovascular admissions in Nicosia, Cyprus during 1995-2004. After controlling for long- (year and month) and short-term (day of the week) patterns as well as the effect of weather in Generalized Additive Poisson models, some positive associations were observed with all-cause and cause-specific admissions. Risk of hospitalization increased stepwise across quartiles of days with increasing levels of PM10 by 1.3% (-0.3, 2.8), 4.9% (3.3, 6.6), 5.6% (3.9, 7.3) as compared to days with the lowest concentrations. For every 10μg/m3 increase in daily average PM10 concentration, there was a 1.2% (-0.1%, 2.4%) increase in cardiovascular admissions. With respects to respiratory admissions, an effect was observed only in the warm season with a 1.8% (-0.22, 3.85) increase in admissions per 10μg/m3 increase in PM10. The effect on respiratory admissions seemed to be much stronger in women and, surprisingly, restricted to people of adult age

High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO[subscript 2], ZnO, Fe[subscript 2]O[subscript 3], Ag, and CeO[subscript 2]) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO > Ag > Fe[subscript 2]O[subscript 3] > CeO[subscript 2] > SiO[subscript 2] in TK6 cells at 4 h and Ag > Fe[subscript 2]O[subscript 3] > ZnO > CeO[subscript 2] > SiO[subscript 2] in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies.National Science Foundation (U.S.) (Grant 1235806)National Institutes of Health (U.S.) (Grant P30ES000002

An Online Nanoinformatics Platform Empowering Computational Modeling of Nanomaterials by Nanostructure Annotations and Machine Learning Toolkits.

Modern nanotechnology has generated numerous datasets from in vitro and in vivo studies on nanomaterials, with some available on nanoinformatics portals. However, these existing databases lack the digital data and tools suitable for machine learning studies. Here, we report a nanoinformatics platform that accurately annotates nanostructures into machine-readable data files and provides modeling toolkits. This platform, accessible to the public at https://vinas-toolbox.com/, has annotated nanostructures of 14 material types. The associated nanodescriptor data and assay test results are appropriate for modeling purposes. The modeling toolkits enable data standardization, data visualization, and machine learning model development to predict properties and bioactivities of new nanomaterials. Moreover, a library of virtual nanostructures with their predicted properties and bioactivities is available, directing the synthesis of new nanomaterials. This platform provides a data-driven computational modeling platform for the nanoscience community, significantly aiding in the development of safe and effective nanomaterials

Estimating the effective density of engineered nanomaterials for in vitro dosimetry

The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by bench-top centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro

A 10-year time-series analysis of respiratory and cardiovascular morbidity in Nicosia, Cyprus: the effect of short-term changes in air pollution and dust storms

<p>Abstract</p> <p>Background </p> <p>To date, a substantial body of research has shown adverse health effects of short-term changes in levels of air pollution. Such associations have not been investigated in smaller size cities in the Eastern Mediterranean. A particular feature in the region is dust blown from the Sahara a few times a year resulting in extreme PM₁₀concentrations. It is not entirely clear whether such natural phenomena pose the same risks.</p> <p>Methods </p> <p>The effect of changes in daily levels of particulate matter (PM₁₀) and ozone (O₃) on hospitalization for all, cardiovascular and respiratory causes in the two hospitals in Nicosia during 1 January 1995 and 30 December 2004 was investigated using generalized additive Poisson models after controlling for long- and short-term patterns as well as for the effect of weather. Meteorological records were reviewed to identify dust-storm days and analyses were repeated to quantify their effect on cardio-respiratory morbidity.</p> <p>Results </p> <p>For every 10 μg/m³increase in daily average PM₁₀concentrations, there was a 0.9% (95%CI: 0.6%, 1.2%) increase in all-cause and 1.2% (95%CI: -0.0%, 2.4%) increase in cardiovascular admissions. With respect to respiratory causes, an effect was observed only in the warm months. No lagged effects with levels of PM₁₀were observed. In contrast, positive associations with levels of ozone were only observed the two days prior to admission. These appeared stronger for cardiovascular causes and independent of the effect of PM. All-cause and cardiovascular admissions were 4.8% (95%CI: 0.7%, 9.0%) and 10.4% (95%CI: -4.7%, 27.9%) higher on dust storm days respectively. In both cases the magnitude of effect was comparable to that seen on the quartile of non-storm days with the highest levels of PM₁₀.</p> <p>Conclusion </p> <p>We observed an increased risk of hospitalization at elevated levels of particulate matter and ozone generally consistent with the magnitude seen across several European cities. We also observed an increased risk of hospitalization on dust storm days, particularly for cardiovascular causes. While inference from these associations is limited due to the small number of dust storm days in the study period, it would appear imperative to issue health warnings for these natural events, particularly directed towards vulnerable population groups.</p

Bioavailability, distribution and clearance of tracheally-instilled and gavaged uncoated or silica-coated zinc oxide nanoparticles

Background: Nanoparticle pharmacokinetics and biological effects are influenced by several factors. We assessed the effects of amorphous SiO2 coating on the pharmacokinetics of zinc oxide nanoparticles (ZnO NPs) following intratracheal (IT) instillation and gavage in rats. Methods: Uncoated and SiO2-coated ZnO NPs were neutron-activated and IT-instilled at 1 mg/kg or gavaged at 5 mg/kg. Rats were followed over 28 days post-IT, and over 7 days post-gavage. Tissue samples were analyzed for 65Zn radioactivity. Pulmonary responses to instilled NPs were also evaluated at 24 hours. Results: SiO2-coated ZnO elicited significantly higher inflammatory responses than uncoated NPs. Pulmonary clearance of both 65ZnO NPs was biphasic with a rapid initial t1/2 (0.2 - 0.3 hours), and a slower terminal t1/2 of 1.2 days (SiO2-coated ZnO) and 1.7 days (ZnO). Both NPs were almost completely cleared by day 7 (>98%). With IT-instilled 65ZnO NPs, significantly more 65Zn was found in skeletal muscle, liver, skin, kidneys, cecum and blood on day 2 in uncoated than SiO2-coated NPs. By 28 days, extrapulmonary levels of 65Zn from both NPs significantly decreased. However, 65Zn levels in skeletal muscle, skin and blood remained higher from uncoated NPs. Interestingly, 65Zn levels in bone marrow and thoracic lymph nodes were higher from coated 65ZnO NPs. More 65Zn was excreted in the urine from rats instilled with SiO2-coated 65ZnO NPs. After 7 days post-gavage, only 7.4% (uncoated) and 6.7% (coated) of 65Zn dose were measured in all tissues combined. As with instilled NPs, after gavage significantly more 65Zn was measured in skeletal muscle from uncoated NPs and less in thoracic lymph nodes. More 65Zn was excreted in the urine and feces with coated than uncoated 65ZnO NPs. However, over 95% of the total dose of both NPs was eliminated in the feces by day 7. Conclusions: Although SiO2-coated ZnO NPs were more inflammogenic, the overall lung clearance rate was not affected. However, SiO2 coating altered the tissue distribution of 65Zn in some extrapulmonary tissues. For both IT instillation and gavage administration, SiO2 coating enhanced transport of 65Zn to thoracic lymph nodes and decreased transport to the skeletal muscle

Evaluation of cytotoxic, genotoxic and inflammatory responses of nanoparticles from photocopiers in three human cell lines

Background: Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. Methods: Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 μg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 μg nanoparticles/mL for 6-hr exposure for confirmation purposes. Results: Multiple cytokines, GM-CSF, IL-1β, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles. Conclusions: Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted

Silica coating influences the corona and biokinetics of cerium oxide nanoparticles

Background The physicochemical properties of nanoparticles (NPs) influence their biological outcomes. Methods We assessed the effects of an amorphous silica coating on the pharmacokinetics and pulmonary effects of CeO2 NPs following intratracheal (IT) instillation, gavage and intravenous injection in rats. Uncoated and silica-coated CeO2 NPs were generated by flame spray pyrolysis and later neutron-activated. These radioactive NPs were IT-instilled, gavaged, or intravenously (IV) injected in rats. Animals were analyzed over 28 days post-IT, 7 days post-gavage and 2 days post-injection. Results Our data indicate that silica coating caused more but transient lung inflammation compared to uncoated CeO2. The transient inflammation of silica-coated CeO2 was accompanied by its enhanced clearance. Then, from 7 to 28 days, clearance was similar although significantly more 141Ce from silica-coated (35 %) was cleared than from uncoated (19 %) 141CeO2 in 28 days. The protein coronas of the two NPs were significantly different when they were incubated with alveolar lining fluid. Despite more rapid clearance from the lungs, the extrapulmonary 141Ce from silica-coated 141CeO2 was still minimal (\u3c1 %) although lower than from uncoated 141CeO2 NPs. Post-gavage, nearly 100 % of both NPs were excreted in the feces consistent with very low gut absorption. Both IV-injected 141CeO2 NP types were primarily retained in the liver and spleen. The silica coating significantly altered the plasma protein corona composition and enhanced retention of 141Ce in other organs except the liver. Conclusion We conclude that silica coating of nanoceria alters the biodistribution of cerium likely due to modifications in protein corona formation after IT and IV administration

Silica coating influences the corona and biokinetics of cerium oxide nanoparticles

Background The physicochemical properties of nanoparticles (NPs) influence their biological outcomes. Methods We assessed the effects of an amorphous silica coating on the pharmacokinetics and pulmonary effects of CeO2 NPs following intratracheal (IT) instillation, gavage and intravenous injection in rats. Uncoated and silica-coated CeO2 NPs were generated by flame spray pyrolysis and later neutron-activated. These radioactive NPs were IT-instilled, gavaged, or intravenously (IV) injected in rats. Animals were analyzed over 28 days post-IT, 7 days post-gavage and 2 days post-injection. Results Our data indicate that silica coating caused more but transient lung inflammation compared to uncoated CeO2. The transient inflammation of silica-coated CeO2 was accompanied by its enhanced clearance. Then, from 7 to 28 days, clearance was similar although significantly more 141Ce from silica-coated (35 %) was cleared than from uncoated (19 %) 141CeO2 in 28 days. The protein coronas of the two NPs were significantly different when they were incubated with alveolar lining fluid. Despite more rapid clearance from the lungs, the extrapulmonary 141Ce from silica-coated 141CeO2 was still minimal (\u3c1 %) although lower than from uncoated 141CeO2 NPs. Post-gavage, nearly 100 % of both NPs were excreted in the feces consistent with very low gut absorption. Both IV-injected 141CeO2 NP types were primarily retained in the liver and spleen. The silica coating significantly altered the plasma protein corona composition and enhanced retention of 141Ce in other organs except the liver. Conclusion We conclude that silica coating of nanoceria alters the biodistribution of cerium likely due to modifications in protein corona formation after IT and IV administration