The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO₂ nanoparticles to human endothelial cells

Abstract

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO NPs were used to compare the effects. Exposure to 32 μg/mL ZnO NPs (p < 0.05), but not TiO NPs (p > 0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO NP exposure (p > 0.05). The presence of 250 nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p < 0.01), but did not significantly affect intracellular ROS or release of TNFα (p > 0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p > 0.05) except neutral red uptake assay (p < 0.01). We concluded ER stress is probably not associated with ZnO NP exposure induced oxidative stress and inflammatory responses in HUVECs.

The use of human umbilical vein endothelial cells (HUVECs) as an in vitro model to assess the toxicity of nanoparticles to endothelium: a review

“…For example, Montiel-Davalos et al (2012) reported that TiO 2 , an insoluble NP, could inhibit HUVEC proliferation and promote apoptosis and necrosis. However, we and others did not observe cytotoxic effects in HUVECs after TiO 2 NP exposure (Suzuki et al, 2014;Gu et al, 2017). The exact reason for the inconsistent reports remains unclear, but it has been suggested the relevant factors associated with the toxicological responses of NPs should be considered (Azhdarzadeh et al, 2015).…”

“…Similarly, Chen et al () showed that exposure to ZnO NPs induced the expression of a panel of ER stress markers before inducing apoptosis. However, in our recent study, stressing HUVECs with ER stress inducer did not significantly affect the toxicity of ZnO NPs (Gu et al, ). Rather, activation of macrophages with a ER stress inducer promoted the toxicity of ZnO NPs to macrophages, which could be further transferred to HUVECs in macrophage–endothelial co‐cultures (Chen et al, ).…”

“…Some types of NPs, particular metal‐based NPs that are partially soluble to release metal ions, have been convincingly shown to be cytotoxic to HUVECs (Supplementary Information Table 2). For example, ZnO NPs, which could dissolute to release high levels of Zn ions, have been shown to induce cytotoxicity of HUVECs as reduced mitochondrial viability and membrane integrity, inhibited proliferation as well as increased lysosomal destabilization and apoptosis (Tsou et al, ; Chen et al, ; Suzuki et al, ; Danielsen et al, ; Gong et al, ; Gu et al, ). Similar effects were also observed in HUVECs after exposure to Ag NPs, which can lead to intracellular Ag accumulation (Shi et al, ; Ucciferri et al, ; Danielsen et al, ; Huo et al, ; Guo et al, ), as well as Cd‐based QD, which can release the highly cytotoxic Cd ions (Yan et al, , ; Soenen et al, ;).…”

“…The different data obtained by different groups could be due to the different properties of NPs used. Nevertheless, all the three studies showed that the soluble ZnO NPs, but not insoluble TiO 2 NPs, induced the cytotoxicity of HUVECs associated with increased intracellular Zn ions, which indicated a role of solubility of NPs (Suzuki et al, ; Gong et al, ; Gu et al, ). Huhn et al () showed higher cellular interactions, cytotoxicity and oxidative stress in HUVECs after exposure to the positively charged Au NPs compared with the negatively charged ones, which suggested a role of surface charge.…”

The toxicity of ZnO nanomaterials to HepG2 cells: the influence of size and shape of particles

“…Exposure to ZnO nanoparticles (NPs; definition similar to NMs) has been shown to activate ER stress‐apoptosis pathways both in vivo and in vitro, which could be the molecular mechanisms underlying the toxicity of ZnO NPs (Chen et al, ; Yang et al, ). Moreover, modulation of ER stress by chemicals could alter ZnO NP‐induced cytotoxicity and/or inflammatory responses, which further suggested a role of ER stress in determining the toxic effects of ZnO NPs (Chen et al, ; Gu et al, ). However, relatively few studies assessed the possible role of physicochemical properties in ZnO NP‐induced ER stress and toxicity.…”