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

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

Engineered Nanomaterials: Linking Physiochemical Properties with Biology

Serious concerns over potential toxicity of new engineered nanomaterials (ENMs) require the development of reliable and high throughput screening assays that impart critical information regarding potential toxicity, guide in-depth toxicity evaluations, justify exposure controls, and aid in the development of sustainable nanomanufacturing. Reactive oxygen species (ROS) generation or biological oxidative damage (BOD) is an important mechanism of ENM toxicity. Several acellular assays have been used to screen for ENMs toxicity based on ROS generation. The dichlorofluorescin (DCFH) assay has gained popularity to determine the degree of ROS generation due to its low cost and automation. However, a rigorous evaluation of this method is lacking. Our study; (i) evaluates the performance of the acellular DCFH assay; (ii) compares the performance of the DCFH and the newly developed Ferric Reducing Ability of Serum (FRAS) assay; (iii) explores the variability in physicochemical characterizations (PCs) of ENMs and their relationship to ROS generation and FRAS-measured BOD; (iv) validates BOD as a metric to determine the effects of individual PCs and their interplays, and further exams the possible utility of ENM-induced BOD for hazard identification of these materials. This study demonstrates that FRAS-measured BOD is a highly informative biologically relevant metric for ENM hazard screening, reflecting the combined effects of multiple PCs. Further, FRAS shows significant potential for hazard identification, as a direct exposure metric, and as a useful tool in responsible nanomanufacturing efforts, providing an opportunity for engineering redesign with the goal of producing less toxic materials while maintaining targeted functional properties

Evaluation of the DCFH assay for ROS measurement

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

Photocatalytic degradation of 3,4-dichlorophenol using TiO₂ in a shallow pond slurry reactor

75-81In the present study, the TiO2 mediated photocatalytic degradation of 3,4-dichlorophenol, as a model compound, has been investigated using a low cost non-concentrating shallow pond slurry reactor at laboratory scale under a variety of conditions. The degradation was studied by monitoring the change in substrate concentration employing UV-spectroscopic analysis, decrease in COD values and increase in chloride formation as a function of irradiation time. The effect of pH, catalyst loading, substrate concentration, UV intensity, aperture to volume ratio of the reactor and presence of electron acceptors such as hydrogen peroxide besides molecular oxygen, on degradation, was studied. The degradation rates were strongly influenced by some of these parameters. The optimum parameters for maximum degradation were determined. The degradation of 3,4-dichlorophenol can be emulated in sunlight using a similar large-scale shallow pond reactor for the solar detoxification in open atmosphere