Cerium Oxide Nanoparticle Administration to Skeletal Muscle Cells under Different Gravity and Radiation Conditions

Abstract

For their remarkable biomimetic properties implying strong modulation of the intracellular and extracellular redox state, cerium oxide nanoparticles (also termed "nanoceria") were hypothesized to exert a protective role against oxidative stress associated with the harsh environmental conditions of spaceflight, characterized by microgravity and highly energetic radiations. Nanoparticles were supplied to proliferating C2C12 mouse skeletal muscle cells under different gravity and radiation levels. Biological responses were thus investigated at a transcriptional level by RNA next-generation sequencing. Lists of differentially expressed genes (DEGs) were generated and intersected by taking into consideration relevant comparisons, which led to the observation of prevailing effects of the space environment over those induced by nanoceria. In space, upregulation of transcription was slightly preponderant over downregulation, implying involvement of intracellular compartments, with the majority of DEGs consistently over- or under-expressed whenever present. Cosmic radiations regulated a higher number of DEGs than microgravity and seemed to promote increased cellular catabolism. By taking into consideration space physical stressors alone, microgravity and cosmic radiations appeared to have opposite effects at transcriptional levels despite partial sharing of molecular pathways. Interestingly, gene ontology denoted some enrichment in terms related to vision, when only effects of radiations were assessed. The transcriptional regulation of mitochondrial uncoupling protein 2 in space-relevant samples suggests perturbation of the intracellular redox homeostasis, and leaves open opportunities for antioxidant treatment for oxidative stress reduction in harsh environments.

Keywords: cerium oxide nanoparticles; gene ontology; microgravity; radiations; skeletal muscle cells; transcriptome; uncoupling protein 2 (Ucp2).

Figure 1

In-flight experiment. (A) Experiment timeline with the indication of relevant time points before and after launch. (B) Diagram of the flow circuit of experiment units, where “CCC” stands for the cell culture chamber, “M” for the cell culture medium, “S” for saline, and “F” for the fixative solution. M1: ±NC for cells that were either treated (+ NC) or not (−NC) with nanoceria, M2: −NC, and M3: −NC.

Figure 2

Characterization of cerium oxide nanoparticles (NC) and of their dispersions. (A) Electron microscopy image of nanoparticles before and after coating with fetal bovine serum (FBS, respectively indicated as −FBS and +FBS). (B) X-ray diffraction pattern of uncoated NC. “I” stands for intensity. (C) Wide X-ray photoelectron spectroscopy (XPS) spectrum of uncoated NC. “CPS” stands for counts per second. (D) Narrow XPS spectrum of the Ce 3d peaks of uncoated NC, with peak deconvolution. (E) Thermogravimetric analysis of uncoated and FBS-coated NC. (F) Dynamic light scattering analysis of NC dispersions in complete cell culture medium after exposure to normal gravity and to simulated microgravity. “HD” stands for hydrodynamic diameter.

Figure 3

Nanoceria and cell cultures in-flight. (A) Thermal profile of the experiment, where “S” stands for space and “E” for Earth samples. Duplicates are due to the number of data loggers. (B) Representative images, obtained by phase-contrast optical microscopy upon recovery from experiment units (EUs), of cultures exposed to different gravity (g) and radiation (rad) levels, either treated (+NC) or not (−NC) with nanoceria.

Figure 4

RNA next-generation sequencing heatmaps for the following experimental classes: A (−NC, μg in space), B (+ NC, μg in space), C (−NC, 1 g in space), D (+NC, 1 g in space), E (−NC, 1 g on ground), and F (+NC, 1 g on ground). Heatmaps for the nine selected comparisons among experimental classes (mRNAs only, control class reported as the first term of each comparison). For every comparison, a map features (left side of each graph) hierarchical clustering of expression variations for the top 24 genes. Single replicas (EU number is indicated) for the two experimental classes (each in pink or sky blue) are also hierarchically clustered according to the overall expression profile of the 24 genes considered. Expression values for such genes are reported as colored bars. The hotter the color, the higher the fold change (FC; most over-expressed genes within each map are indicated in red, and most under-expressed genes are shown in blue, see the color scale on the left of the panel).

Figure 5

Scatter plots. Volcano plots for the nine selected comparisons among experimental classes (mRNAs only, control class reported as the first term of each comparison). Experimental classes: A (−NC, μg in space), B (+NC, μg in space), C (−NC, 1 g in space), D (+NC, 1 g in space), E (−NC, 1 g on ground), and F (+NC, 1 g on ground). Each volcano plot shows significance (y-axis, as log₁₀ of the adjusted p-value, p_adj) and overexpression change (x-axis, as log₂ FC, where “FC” stands for fold change) for all genes (each represented by a dot) in a given comparison. Significantly up- or downregulated genes are reported in red (clustering on the right/top corner of the plot) or blue (clustering on the left/top corner of the plot), respectively. The remaining genes are indicated in black.

Figure 6

RNA next-generation sequencing Venn diagrams, featuring one to four among the nine selected comparisons among experimental classes (mRNAs only, control class reported as the first term of each comparison). The number of differentially expressed genes (DEGs) residing in each subset is reported. Compatibly with room availability, the composition in terms of up- (↑) or downregulation (↓) is specified after an opening brace: up-only or down-only genes at the intersection between two or more parent sets are deemed coherent. The noncoherent fraction of DEGs at a given intersection, when present and shown, is boxed in white; in that case, to understand in which parent sets a group of DEGs is up- or downregulated, one should assign arrow signs to the relevant parent sets starting from top-left and proceeding in a clockwise order. Intersections for “Effects of nanoceria in different environments” and “Effects of space in different environments” have been simplified, the former not including comparison (C vs D) and the latter not considering comparisons (C vs A and D vs B). Red shadowed boxes, each associated with a Greek letter, highlight particular subsets/parent sets, also depicted by REVIGO interactive graphs.

Figure 7

RNA next-generation sequencing gene ontology (GO). REVIGO interactive graphs incorporate enriched (p, q < 0.05) GO terms for general biological processes (process), specific molecular functions (function), or cell/tissue compartment, niche and thereof (component). Each term is associated with a red circle, which becomes smaller for more specific terms and more saturated for more supported ones. Terms were initially positioned to reflect semantic analogies but were adjusted as needed for graphical reasons. Gray lines connect similar GO terms, with line width reflecting similarity levels.