SCREENING FOR OXIDATIVE STRESS ELICITED BY ENGINEERED NANOMATERIALS: EVALUATION OF ACELLULAR DCFH ASSAY

The DCFH assay is commonly used for measuring free radicals generated by engineered nanomaterials (ENM), a well-established mechanism of ENM toxicity. Concerns exist over susceptibility of the DCFH assay to: assay conditions, adsorption of DCFH onto ENM, fluorescence quenching and light scattering. These effects vary in magnitude depending on ENM physiochemical properties and concentration. A rigorous evaluation of this method is still lacking. The objective was to evaluate performance of the DCFH assay for measuring ENM-induced free radicals. A series of diverse and well-characterized ENM were tested in the acellular DCFH assay. We investigated the effect of sonication conditions, dispersion media, ENM concentration, and the use of horseradish peroxidase (HRP) on the DCFH results. The acellular DCFH assay suffers from high background signals resulting from dye auto-oxidation and lacks sensitivity and robustness. DCFH oxidation is further enhanced by HRP. The number of positive ENM in the assay and their relative ranking changed as a function of experimental conditions. An inverse dose relationship was observed for several Carbon-based ENM. Overall, these findings indicate the importance of having standardized assays for evaluating ENM toxicity and highlights limitations of the DCFH assay for measuring ENM-induced free radicals

Characterization of PARIS LaBr3_3(Ce)-NaI(Tl) phoswich detectors upto EγE_\gamma ∼\sim 22 MeV

In order to understand the performance of the PARIS (Photon Array for the studies with Radioactive Ion and Stable beams) detector, detailed characterization of two individual phoswich (LaBr$_3$(Ce)-NaI(Tl)) elements has been carried out. The detector response is investigated over a wide range of $E_{\gamma}$ = 0.6 to 22.6 MeV using radioactive sources and employing $^{11}B(p,\gamma)$ reaction at $E_p$ = 163 keV and $E_p$ = 7.2 MeV. The linearity of energy response of the LaBr$_3$(Ce) detector is tested upto 22.6 MeV using three different voltage dividers. The data acquisition system using CAEN digitizers is set up and optimized to get the best energy and time resolution. The energy resolution of $\sim$ 2.1% at $E_\gamma$ = 22.6~MeV is measured for the configuration giving best linearity upto high energy. Time resolution of the phoswich detector is measured with a $^{60}$Co source after implementing CFD algorithm for the digitized pulses and is found to be excellent (FWHM $\sim$ 315~ps). In order to study the effect of count rate on detectors, the centroid position and width of the $E_{\gamma}$ = 835~keV peak were measured upto 220 kHz count rate. The measured efficiency data with radioactive sources are in good agreement with GEANT4 based simulations. The total energy spectrum after the add-back of energy signals in phoswich components is also presented.Comment: Accepted in JINS

Heterogeneous solar photo-fenton degradation of reactive Black 5 using foundry sand and fly ash: value addition to waste

This work evaluates the scope of alternative sources of iron i.e. Foundry Sand (FS), Fly Ash (FA) and mixture of FS and FA in heterogeneous photo-Fenton process for the degradation and decolourization of the dye Reactive Black5 (RB5). SEM-EDS characterization of FS and FA confirmed the presence of iron and aluminium which are required for photo-Fenton. In case of FS as an iron source, results confirmed the degradation and decolourization efficiency of 90% in 70 min and 45 min respectively when the reaction parameters were H2O2 = 2.2 mm, FS dose = 0.5 g, ph 3. With FA 90% degradation was achieved within 30 min and 93% decolourization rate within 15 min. When a mixture of FS (0.1 gm) and FA (0.05 gm) is used as source of iron 99% degradation and decolourization of the dye was achieved under the reaction conditions where H2O2 = 2.2 mm, FA:FS  = 1:2, ph 3

A living cell quartz crystal microbalance biosensor for continuous monitoring of cytotoxic responses of macrophages to single-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs).</p> <p>Results</p> <p>We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 μg/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 μg/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Δf reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 μg/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays.</p> <p>Conclusion</p> <p>These data suggest that the new QCMB detects and provides unique information about peak, sub-lethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.</p

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

Advanced computational modeling for in vitro nanomaterial dosimetry

Background: Accurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition. Methods: Here we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time. Results: Dose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material. Conclusions: The advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures. Electronic supplementary material The online version of this article (doi:10.1186/s12989-015-0109-1) contains supplementary material, which is available to authorized users

Titanium dioxide mediated photocatalytic degradation of malathion in aqueous phase

46-51The photocatalytic degradation of technical grade pesticide ‘malathion’ has been studied in aqueous suspensions of TiO2. The main aim of using this process is to degrade the toxic complex structure of the pesticide to harmless products by treating the sample in the UV-reactor. The COD measurements are carried out in order to study the percentage mineralization and degradation is studied under different parameters like varying pH, catalyst concentration, and oxidant addition (H2O2). The degradation of the pesticide is found to be maximum at optimum catalyst loading of 3.0 g/L, pH of 6.0 and oxidant concentration of 2.5 mL/200 mL. Adsorption is negligible as compared to photocatalytic degradation of the pesticide