Elsevier

Toxicology Letters

Volume 230, Issue 1, 1 October 2014, Pages 69-79
Toxicology Letters

Sheet-type titania, but not P25, induced paraptosis accompanying apoptosis in murine alveolar macrophage cells

https://doi.org/10.1016/j.toxlet.2014.07.027Get rights and content

Highlights

  • We compared the toxic effects of sheet-type titania (TNS) and P25 in MH-S cells.

  • TNS, but not P25, formed large vacuoles, and dilated the ER and mitochondria.

  • P25 decreased the level of an ER membrane protein and increased the ROS generation.

  • TNS increased the secretion of NO and pro-inflammatory cytokines, but not of ROS.

  • TNS, but not P25, induced paraptosis accompanied by apoptosis in MH-S cells.

Abstract

In this study, we identified the toxic effects of sheet-type titania (TNS), which are being developed as a material for UV-blocking glass, comparing with P25, a benchmark control for titania, in MH-S cells, a mouse alveolar macrophage cell line. After 24 h exposure, the TNS-exposed cells formed large vacuoles while the P25-exposed ones did not. The decreased levels of cell viability were similar between the P25 and TNS groups, but ATP production was clearly lower in cells exposed to the TNS. P25 decreased the expression of calnexin protein, an endoplasmic reticulum (ER) membrane marker, and increased the number of cells generating ROS in a dose dependent manner. Meanwhile, TNS dilated the ER and mitochondria and increased the secretion of NO and pro-inflammatory cytokines, but not of ROS. Subsequently, we studied the molecular response following TNS-induced vacuolization. TNS started to form vacuoles in the cytosol since 20 min after exposure, and the expression of the mitochondria function-related genes were down-regulated the most in the cells exposed for 1 h. After 24 h exposure, the number of apoptotic cells and the relative levels of BAX to Bcl-2 increased. The expression of SOD1 protein, but not of SOD2, also dose-dependently increased with an increase in caspase-8 activity. Additionally, the MAPK pathway was significantly activated, even though the expression of p-EGFR did not change significantly. Furthermore, the number of apoptotic cells increased rapidly with time and with the inhibition of vacuole formation. Taken together, we suggest that P25 and TNS may target different organelles. In addition, TNS, but not P25, induced paraptosis accompanied by apoptosis in MH-S cells, and the formation of the cytoplasmic vacuoles allowed delay apoptosis following TNS exposure.

Introduction

Manufactured nanomaterials (MNs) are man-made materials produced on a nanoscale, and their market size is expected to reach a $1 trillion by 2015 because of the surprising growth in the nanotechnology industry (Nel et al., 2006). Thus, their prevalence in the environment and the potential risk to human health is a growing concern (Oberdörster et al., 2005, Nohynek and Dufour, 2012, Elsaesser and Howard, 2012, Koivisto et al., 2012). Titanium dioxide nanoparticles (hereafter, titania) are widely used in various products, including paper, plastics, cosmetics, photocatalysts, and paints due to their excellent brightness, their ability to filter ultraviolet (UV) light, superior biocompatibility, and safety (Yin et al., 2013, Earle, 1942, Chen et al., 2013, Skocaj et al., 2011). In contrast, many researchers have reported the hazardous effects of titania in vivo and in vitro (Trouiller et al., 2009, Gui et al., 2013, Zhao et al., 2013, Gao et al., 2012). Hence, the safety of titania is still controversial. In addition, the toxicity of nanoparticles is expected to be dependent on their physicochemical properties. Thus, the toxicity of modified nanoparticles should be investigated independently with raw materials (Hamzeh and Sunahara, 2013, Bolis et al., 2012, Nagy et al., 2012, Nel et al., 2009).

Cellular uptake of nanoparticles and the subsequent biological responses depend on the interactions between unique properties, such as the size, shape, surface chemistry, and crystal structure of the nanoparticles and the specific properties of the biological system used in the experiments. Therefore, we must simultaneously consider both properties for a more accurate understanding of nanoparticle toxicity (Verma and Stellacci, 2010, Akatsuka et al., 2009). In addition, many researchers reported that nanoparticles induce autophagy along with apoptosis (Park et al., 2014a, Zhao et al., 2013, Hussain and Garantziotis, 2013, Hussain et al., 2012, Afeseh Ngwaq et al., 2011). Both types of cell death represent programmed cell death with paraptosis based on morphological features and proteins involved in these pathways (Sperandio et al., 2000, Wang et al., 2012b, Yoon et al., 2010). In our previous study, non-coated iron oxide nanoparticles (FeNPs, rod-type) induced autophagic cell death in RAW264.7 cells, a murine macrophage cell line, whereas they induced paraptosis-like cell death in MH-S cells, a mouse alveolar macrophage cell line (Park et al., 2014a, Park et al., 2014b). Phospholipid-coated FeNPs (sphere-type) did not increase the protein levels of autophagic cell death in MH-S cells despite observed autophagic morphological changes. Herein, we investigated both inherent gene profiles of MH-S cells and the physicochemical properties of sheet-type titania (TNS). Then, we compared the toxicity of TNS and P25, a benchmark control for titania, in MH-S cells, and further explored the molecular response following TNS exposure.

Section snippets

Preparation of TNS

Titania (average primary particle size, 50 nm) and cesium carbonate were obtained from SukgyungAT (Ansan, Korea) and Sigma–Aldrich (St. Louis, MO, USA), respectively. Starting materials were calcined at high temperature to prepare powder of CsxTi2  x/4x/4O4, and 1 M HCl solution was added to produce protonic oxide HxTi2  x/4x/4O4·yH2O (□, vacancy; x = 0.7, Gao et al., 2008). Then, 2.51 mM tetrabutylammonium hydroxide was added to produce a colloidal suspension of single Ti0.91O2 nanosheet. The

Characterization of TNS

The X-ray diffraction pattern reveals that TNS has a lepidocrocite-type structure with lattice spacing of 0.89 nm, which corresponds to the (1 0 1) reflection peak of anatase TiO2 (data not shown). TNS also had a two-dimensional architecture (Fig. 1A) with a thickness of ∼2 nm, an average lateral size of 390.7 ± 292.8 nm, and an average area of 0.141 ± 0.053 μm2 (data not shown). In addition, TNS (stock concentration: 2331 mg/L) was well dispersed in the vehicle control and was thereby applied to the

Discussion

The respiratory system is a major exposure route to MNs, and alveolar macrophages are one of the primary responders to MNs entering the body. In addition, certain biological events, such as the penetration of nanoparticles into cell membranes and the phagocytosis of antigens, can be induced through a charge-matching mechanism between the cells and the target. Thus, membrane receptors play a key role in mediating the uptake of nanoparticles (Verma and Stellacci, 2010, Akatsuka et al., 2009,

Conflict of interest

The authors declare no conflict of interest.

Transparency document

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Acknowledgements

We would like to thank Bengt Fadeel from the Karolinska Institute for helpful discussions. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF-2011-35B-E00011). This work was also partly supported by the National Platform Technology Programs of the Korean Ministry of Knowledge Economy (grant 10034751).

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