Computational protocol: Exposure to Palladium Nanoparticles Affects Serum Levels of Cytokines in Female Wistar Rats

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Protocol publication

[…] As a first step, 300 μL of a freshly prepared 0.029 molar sodium borohydride solution, obtained by dissolution of 11 mg of sodium borohydride (p.a., Merck, Darmstadt, Germany) in 10 mL of ultrapure water, were diluted in 100 mL of ultrapure water. Then, 500 μL of a Pd stock standard solution (1000 mg/L, Pd(NO3)2 in 0.5 mol/L HNO3, Merck, Darmstadt, Germany) were added and the mixture was shaken thoroughly. The immediate color change from transparent to dark grey indicated the formation of Pd-NPs. The mixture was kept in the dark at room temperature for 12 hours to allow complete reaction.The Pd-NPs hydrosol obtained was characterized by continuum source—graphite furnace atomic absorption spectrometry (CS-GFAAS; contrAA 600, Analytik Jena, Jena, Germany) and transmission electron microscopy (TEM; Zeiss EM 10, Carl Zeiss Microscopy GmbH, Jena, Germany) operating at 80 kV. The Pd concentration of the stock hydrosol was determined in a 100-fold dilution of the stock hydrosol in ultra pure water by means of CS-GFAAS using the spectral line at 244.791 nm. Calibration was performed in a concentration ranging from 20 to 80 μg Pd/L by applying adequate dilutions of a Pd stock standard solution (1000 mg/L, Pd(NO3)2 in 0.5 mol/L HNO3, traceable to Standard Reference Materials from the National Institute of Standards and Technology, Merck, Darmstadt, Germany) in 0.5 mol/L HNO3. This resulted in a linear calibration function with a correlation coefficient of 0.986. The stock hydrosol Pd concentration was found to be 4.71 ± 0.05 mg/L. The measurement of 500 individual particles depicted by TEM images using ImageJ software (National Institutes of Health, Bethesda, MD) revealed the size distribution of the particles to be 10 ± 6 nm (). The hydrosol served as a stock solution for all experiments and is stable for at least 2 weeks when stored in refrigerators at 4°C. Before use, the stored Pd-NP hydrosol was homogenized by shaking vigorously. Finally, aliquots of the stock solution were diluted in ultrapure water to obtain the final concentrations used in the experiments. [...] Statistical analysis was carried out by IBM SPSS statistics software (IBM Statistical Package for Social Sciences for Windows, Version 22.0. Armonk, New York, USA). Levels of cytokines IL-1α, IL-2, IL-4, IL-6, IL-10, IL-12, GM-CSF, INF-γ and TNF-α were measured after the four levels of exposure on day 14. The normal distribution of observed values was checked using the non-parametric Kolmogorov–Smirnov Z test and variance homogeneity was tested using the Levene test. One-way analysis of variance (ANOVA) was then performed to test the significance of differences in parameter means in the exposed and control rat groups. The Dunnett post hoc multiple comparison test was used to test the significance (p value Dunnett t test <0.05) of differences in values for each parameter at different exposure levels against the control group. Box-plot or linear graphs were obtained for all analyzed parameters at different exposure levels. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, cryo-EM, Microscopic phenotype analysis
Organisms Rattus norvegicus
Chemicals Palladium