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Pinsino A, Russo R, Bonaventura R, Brunelli A, Marcomini A, Matranga V.
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Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue.
Figure 1. TiO2 nanoparticles, adult sea urchins and immune cells under the microscope.(A) TiO2NP agglomerates/aggregates of different sizes observed 60 minutes after suspension in ASW. (B) Paracentrotus lividus sea urchins after 24-hour exposure to TiO2NP suspension (1 μg/ml concentration). (C) Living immune cells in a Fast-Read chamber harvested as a total cell population by bleeding sea urchins exposed for 24 hour through a cut in the peristomal membrane. (D) Graphic representation of the total cell concentration (cell/ml, upper graph) and the number of phagocytes (in percent, lower graph). Cell types are indicated by captions of different colors and corresponding pointing arrows: red amoebocyte (red arrows), white/colourless amoebocyte (white arrow), phagocyte (black arrows), and vibratile cell (blue arrow). Images were captured by Zeiss Axioskop 2 Plus microscope (Zeiss, Arese, Italy).
Figure 2. Neutral Red for live cell imaging of the phagocytic activity of exposed immune cells.(A) The pH-indicator dye NR is concentrated in lysosomes forming typical small red vesicles within all three different cell types of freely circulating immune cells present in Paracentrotus lividus (phagocytes, amoebocytes and vibratile cells), most evident in the vibratile cells. Cell types are indicated by captions of different colors and corresponding pointing arrows as described in Fig. 1. (B) Petaloid and filopodial phagocytes show areas of high lysosomal and phagocytic activity in which NR became concentrated. (C) Schematic model and demonstrative pictures representing an immune cell undergoing phagosomal maturation. Arrows indicate lysosomes (orange arrows), phagosomal/lysosomal fusion (yellow arrows), phagolysosome (red arrows). Bar 5 μm.
Figure 3. Dihexyloxacarbocyanine iodide to monitor lysosomal internal membrane stability of TiO2 exposed immune cells.(A) The green fluorescent membrane dye DiOC6 is detected within the cytoplasm, as a dense signal around the nuclei of both the non-activate filopodial and petaloid phagocytes. (B) A few cells showed a growing network of vesicles (green arrows), validating a phagocytic activity in progress. (C) Representative image of the HSP70 levels of 6 control specimens, 6 specimens exposed to TiO2NP and 1 to LPS, evaluated by immunoblotting. Contrary to LPS exposure, TiO2NP exposure do not stimulate the activation of the HSP70-dependent stress response in the sea urchin immune cells.
Figure 4. TiO2 nanoparticles stimulate a member of the Toll-like receptor family.(A) Levels of expression of Pl-TLR gene analysed by comparative qPCR with total RNA isolated from control (injected only with ASW) and exposed immune cells. Levels are expressed in arbitrary units as fold increase compared to controls assumed as 1, using the endogenous gene Pl-Z12-1 for normalization. Each bar represents the mean of three independent experiments ±SE. (B) Representative images of the TLR4-like protein levels of 3 control specimens and 3 specimens exposed to TiO2NPs evaluated by immunoblotting. Histograms represent the means of the three independent experiments (18 specimens obtained from 3 independent experiments) ±SE after normalization with actin levels.
Figure 5. TiO2 nanoparticles affect phosphorylation levels of the p38 MAPK.(A) Immunoblotting analysis with anti-P-p38 MAPK on total cell lysates (upper panel) and subcellular fractions (lower panel) of sea urchin immune cells exposed to TiO2NPs, shows a significant reduction (50%) in the in the levels of the phosphorylated form of p38 MAPK. (B) Immunoblotting analysis with anti-p38 MAPK on total cell lysates of sea urchin immune cells (upper panel) and comparative qPCR analysis of Pl-p38 MAPK gene expression (lower panel). (C) Immunoblotting analysis with anti-P-ERK (upper panel) and representative image of 3 control specimens, and 3 specimens exposed to TiO2NPs (lower panel). Histograms represent the means of the three independent experiments ±SE after normalization with tubulin or actin levels for the proteins and Pl-Z12-1 gene for the gene expression. Cy: cytosol; M/O: membrane/organelles; N: nuclei; Ct: cytoskeleton.
Figure 6. Signal transduction downstream to p38 MAPK in response to TiO2 nanoparticles.(A) Comparative qPCR analysis show levels of expression of Pl-NF-kB gene 2-fold lower than those measured in controls (injected only with ASW) (upper panel), while the transcript of Pl-Jun showed a weak increase in its levels (lower panel). Levels are expressed in arbitrary units as fold increase compared to controls assumed as 1, using the endogenous gene Pl-Z12-1 for normalization. Results were compared to those obtained in response to LPS (2 μg/ml in ASW, 24 h): in this case, the levels of expression of Pl-NF-kB gene were found comparable to controls while the transcript of Pl-Jun was found strongly over-expressed (2.56 ± 0.71-fold). Each bar represents the mean of three independent experiments ±SE. (B) Immunoblotting analysis with anti-IL6 subcellular fractions of sea urchin immune cells. Cy: cytosol; M/O: membrane/organelles; N: nuclei; Ct: cytoskeleton.
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