Multi-walled carbon nanotubes (MWCNTs) promoted lipid accumulation in THP-1 macrophages through modulation of endoplasmic reticulum (ER) stress.

Recent studies from us and others have shown that nanoparticle (NP) exposure could modulate endoplasmic reticulum (ER) stress. Since ER stress is closely related to lipid droplet biogenesis, in this study, we investigated if multi-walled carbon nanotubes (MWCNTs) might influence lipid accumulation in THP-1 macrophages through the modulation of ER stress. Exposure to up to 64 µg/mL pristine MWCNTs (p-MWCNTs), hydroxylated MWCNTs (h-MWCNTs), and carboxylated MWCNTs (c-MWCNTs) led to NP internalization, which was associated with a modest increase of cytotoxicity. Oxidative stress was also induced, showing as increased intracellular reactive oxygen species (ROS) and decreased glutathione. Only h-MWCNTs and c-MWCNTs significantly promoted the release of interleukin-8 (IL-8), which was significantly higher compared with that after p-MWCNT exposure. All types of MWCNTs significantly induced lipid accumulation in THP-1 macrophages and more modestly in the co-culture model consisting of 16HBE human bronchial epithelial cells and THP-1 macrophages. MWCNT-induced lipid accumulation in THP-1 macrophages was decreased modestly by antioxidant N-acetyl-l-cysteine and more effectively by ER stress inhibitor 4-phenylbutyric acid. Moreover, MWCNT exposure promoted the expression of ER stress gene DDIT3 as well as ER stress protein p-chop, whereas the transcription factor XBP-1s was decreased. In addition, the expression of scavenger receptors, namely CD36 and MSR1, was also elevated after MWCNT exposure. In conclusion, this study suggested that MWCNT exposure could promote lipid accumulation in THP-1 macrophages, which could be related to the modulation of ER stress leading to upregulation of scavenger receptors.

The toxicity of multi-walled carbon nanotubes (MWCNTs) to human endothelial cells: The influence of diameters of MWCNTs.

The biological applications of multi-walled carbon nanotubes (MWCNTs) may lead to their exposure to human blood vessels, but the influence of their physicochemical properties on toxicity to endothelial cells is incompletely known. Here, human umbilical vein endothelial cells (HUVECs) were exposed to three commercially available MWCNTs, namely XFM4, XFM22, and XFM34 (diameters XFM4 < XFM22 < XFM34), to understand the possible role of their diameter on toxicity. Based on the same mass concentration, XFM4 induced significantly higher level of cytotoxicity than the other two MWCNTs, and HUVECs internalized more XFM4. Cytokine release, monocyte adhesion, and intracellular reactive oxygen species levels were significantly induced only after XFM4 treatment. The exposure to XFM4 significantly reduced the expression of autophagic genes autophagy-related 7 (ATG7), autophagy-related 12 (ATG12), and beclin 1 (BECN1) and increased the expression of endoplasmic reticulum (ER) stress genes DNA damage inducible transcript 3 (DDIT3) and X-box binding protein 1 spliced (XBP-1s). Moreover, the modulation of autophagy-ER stress by chemicals resulted in a significant increase in the cytotoxicity of XFM4 but had minimal impact on the cytotoxicity of XFM34. These data indicate that the diameter of MWCNTs may influence their toxicity to HUVECs, probably through autophagy dysfunction and ER stress.

Palmitate enhanced the cytotoxicity of ZnO nanomaterials possibly by promoting endoplasmic reticulum stress.

We recently synthesized ZnO nanomaterials (denoted as ZnO nanorods [NRs] and Mini-NRs) and suggested that their cytotoxicity could be related with the activation of endoplasmic reticulum (ER) stress apoptosis. However, in a complex biological microenvironment, the ER stress-apoptosis pathway could also be modulated by biological molecules, such as free fatty acids, leading to unpredicted biological effects. In this study, we investigated the combined toxicity of ZnO NRs/Mini-NRs and palmitate (PA) to THP-1 macrophages. PA influenced the zeta potential and solubility of ZnO NRs and ZnO Mini-NRs in water, which indicated a change of colloidal stability. Exposure to ZnO NRs and Mini-NRs dose-dependent decreased cellular viability and release of soluble monocyte chemotactic protein 1 (sMCP-1), and these effects were significantly promoted with the presence of PA. However, ZnO NR- and Mini-NR-induced intracellular Zn ions or reactive oxygen species were not significantly affected by PA. ZnO NRs and ZnO Mini-NRs significantly promoted the expression of ER stress genes HSPA5, DDIT3, XBP-1s and apoptotic gene CASP3, whereas PA also modestly promoted the expression of HSPA5, DDIT3 and CASP3. Interestingly, the ER stress inducer thapsigargin showed a similar effect as PA to promote the cytotoxicity of ZnO NRs and ZnO Mini-NRs. It is suggested that PA might promote the cytotoxicity of ZnO NRs and ZnO Mini-NRs possibly by promoting ER stress.

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

Understanding the possible role of physicochemical properties in determining the toxicity of ZnO nanomaterials (NMs) is crucial for the safe use of ZnO-based materials. In this study, we synthesized four types of ZnO NMs, and characterized them as ZnO nanorods (NRs; length 400-500 nm, diameter 150-200 nm), ZnO Mini-NRs (length 50-100 nm, diameter 15-20 nm), amorphous ZnO microspheres (a-ZnO MS) and crystalline ZnO MS (c-ZnO MS; the a/c-ZnO MS are nanoflowers with an extensive growth of sheet-like structures). ZnO NMs and ZnO Mini-NRs were significantly more cytotoxic than a/c-ZnO MS, and this trend was similar in both HepG2 cells and human umbilical vein endothelial cells. Intracellular reactive oxygen species was only modestly induced by c-ZnO MS, whereas intracellular Zn ions were dose-dependently increased in HepG2 cells by the exposure of all types of ZnO NMs. The expression of endoplasmic reticulum stress marker DDIT3 was induced following an order of ZnO NRs > a-ZnO MS > c-ZnO MS > ZnO Mini-NRs, and the apoptosis gene CASP12 was induced following an order of a-ZnO MS > ZnO NRs > c-ZnO MS > ZnO Mini-NRs. Combined, these results suggested that ZnO NM-induced cytotoxicity and expression of endoplasmic reticulum stress-apoptosis genes could be influenced by the size and shape of ZnO NMs.

A comparative study of toxicity of TiO2, ZnO, and Ag nanoparticles to human aortic smooth-muscle cells.

PURPOSE: To evaluate the adverse vascular effects of nanoparticles (NPs) in vitro, extensive studies have investigated the toxicity of NPs on endothelial cells, but the knowledge of potential toxicity on human smooth-muscle cells (SMCs) is currently limited. METHODS: This study compared the toxicity of TiO2, ZnO, and Ag NPs to human aortic SMCs. RESULTS: Only ZnO NPs significantly induced cytotoxicity, accompanied by increased intracellular reactive oxygen species, Zn ions, and endoplasmic reticulum stress biomarkers (DDIT3 expression and p-Chop proteins). All the NPs significantly promoted the release of soluble VCAM1 and soluble sICAM1, but not IL6, which suggested that metal-based NPs might promote inflammatory responses. Furthermore, KLF4 expression (a transcription factor for SMC-phenotype switch) was significantly induced by TiO2 NPs and modestly by ZnO NPs, but the expression of CD68 remained unaltered. CONCLUSION: Our data indicated that ZnO NPs were more cytotoxic to human aortic SMCs than TiO2 and Ag NPs at the same mass concentrations, which might have been associated with intracellular reactive oxygen species, Zn ions, and endoplasmic reticulum stress.

Lipid accumulation in multi-walled carbon nanotube-exposed HepG2 cells: Possible role of lipophagy pathway.

Nanoparticle (NP) exposure might promote hepatic steatosis, but relatively few studies investigated the influence of multi-walled carbon nanotubes (MWCNTs) on lipid accumulation in hepatocytes in vitro. This study investigated lipid accumulation and the possible role of lipophagy (autophagic degradation of lipid droplets) in MWCNT-exposed HepG2 cells. Pristine (XFM19) and carboxylated MWCNTs (XFM21) were internalized, accompanying cytotoxicity, lysosomal destabilization, and intracellular reactive oxygen species (ROS) production. Compared with XFM21, XFM19 promoted lipid accumulation in HepG2 cells more effectively, which was further enhanced by pre-incubation with autophagy inhibitor NH4Cl. In addition, MWCNTs increased the expression of lipophagy genes PLIN2 and BECN1 but decreased that of ATG7. The expression of endoplasmic reticulum (ER) stress regulators, namely DDIT3, HSPA5, and XBP-1s, was also altered in MWCNT exposed HepG2 cells. Combined, these results suggested that MWCNT exposure might promote lipid accumulation in hepatocytes probably through the modulation of lipophagy pathway.

Cytotoxicity, cytokine release and ER stress-autophagy gene expression in endothelial cells and alveolar-endothelial co-culture exposed to pristine and carboxylated multi-walled carbon nanotubes.

Recently we found that direct exposure of human umbilical vein endothelial cells (HUVECs) to multi-walled carbon nanotubes (MWCNTs) might induce toxicological responses through the modulation of ER stress gene expression, but whether this signal could be transferred from other cells to endothelial cells (ECs) is unknown. This study investigated the toxicity of pristine and carboxylated MWCNTs to HUVECs and alveolar-endothelial co-culture, the later of which could mimic the possible signaling communications between ECs and MWCNT exposed alveolar cells. The results showed that direct contact with high levels of MWCNTs induced cytotoxicity and modulated expression of genes associated with ER stress (HSPA5, DDIT3 and XBP-1s) and autophagy (BECN1 and ATG12) both in A549-THP-1 macrophages cultured in the upper chambers as well as HUVECs. However, most of these responses were minimal or negligible in HUVECs cultured in the lower chambers. Moreover, significantly increased cytokine release (interleukin-6 and soluble vascular cell adhesion molecule-1) was only observed in MWCNT exposed HUVECs (p < 0.01) but not HUVECs cultured in the lower chambers (p > 0.05). The minimal or even absent response was likely due to relatively low translocation of MWCNTs from upper chambers to lower chambers, whereas A549-macrophages cultured in the upper chambers internalized large amount MWCNTs. The results indicated that ER stress-autophagy signaling might not be able to transfer from alveolar cells to endothelial cells unless sufficient MWCNTs are translocated.

Influence of bovine serum albumin pre-incubation on toxicity and ER stress-apoptosis gene expression in THP-1 macrophages exposed to ZnO nanoparticles.

When entering a biological environment, proteins could be adsorbed onto nanoparticles (NPs), which can potentially influence the toxicity of NPs. This study used bovine serum albumin (BSA) as the model for serum protein and investigated its interactions with three different types of ZnO NPs, coded as XFI06 (pristine NPs of 20 nm), NM110 (pristine NPs of 100 nm) and NM111 (hydrophobic NPs of 130 nm). Atomic force microscope indicated the adsorption of BSA to ZnO NPs, leading to the increase of NP diameters. Pre-incubation with BSA did not significantly affect hydrodynamic size but decreased Zeta potential of NM110 and NM111. The fluorescence and synchronous fluorescence of BSA were quenched after pre-incubation with ZnO NPs, and the quenching effects were more obvious for XFI06 and NM110. Exposure to all types of ZnO NPs significantly induced cytotoxicity and lysosomal destabilization, which was slightly alleviated when NPs were pre-incubated with BSA. However, ZnO NPs with or without pre-incubation of BSA resulted in comparable intracellular Zn ions, glutathione and reactive oxygen species in THP-1 macrophages. Exposure to ZnO NPs promoted the expression of endoplasmic reticulum (ER) stress markers (DDIT3 and XBP-1s) and apoptosis genes (CASP9 and CASP12). Pre-incubation with BSA had minimal impact on ER stress gene expression but decreased apoptosis gene expression. Combined, these results suggested that pre-incubation with BSA could modestly alleviate the cytotoxicity and reduce ER stress related apoptosis gene expression in THP-1 macrophages after ZnO NP exposure.

Internalization, cytotoxicity, oxidative stress and inflammation of multi-walled carbon nanotubes in human endothelial cells: influence of pre-incubation with bovine serum albumin.

When entering circulation, multi-walled carbon nanotubes (MWCNTs) will inevitably adsorb proteins, which can consequently influence their toxicity to cells lining human blood vessels. In this study, we investigated the influence of pre-incubation with bovine serum albumin (BSA) on internalization, cytotoxicity, oxidative stress and inflammation induced by pristine/carboxylated MWCNTs to human umbilical vein endothelial cells (HUVECs). Atomic force microscopy (AFM) indicated the adsorption of proteins onto the surface of MWCNTs, which consequently increased the diameter. Pre-incubation with BSA did not obviously influence the hydrodynamic sizes, but decreased the zeta potential of MWCNTs. Transmission electron microscopy (TEM) indicated the internalization of both types of MWCNTs into HUVECs, whereas pre-incubation with BSA appeared to enhance the internalization. MWCNT exposure induced cytotoxicity and oxidative stress, as well as a modest inflammatory response shown as an increased THP-1 adhesion to HUVECs, but not release of interleukin 6 (IL-6) or tumor necrosis factor (TNFα). Exposure to MWCNTs pre-incubated with BSA induced less cytotoxicity to HUVECs, associated with increased intracellular glutathione (GSH). However, MWCNTs induced IL-6 and TNFα release, as well as THP-1 adhesion to HUVECs, were enhanced after pre-incubation with BSA. In summary, these data indicated that pre-incubation with BSA could enhance the internalization of MWCNTs to HUVECs, which consequently reduces the cytotoxicity and oxidative stress, but enhances the inflammatory response of MWCNTs. The reduced cytotoxicity and oxidative stress, and enhanced inflammatory responses are likely due to a combined effect of BSA and MWCNTs, which suggests that when assessing the biological effects of MWCNTs in circulation, it is necessary to consider the interactions between MWCNTs and serum proteins.

The adverse vascular effects of multi-walled carbon nanotubes (MWCNTs) to human vein endothelial cells (HUVECs) in vitro: role of length of MWCNTs.

BACKGROUND: Increasing evidences indicate that exposure to multi-walled carbon nanotubes (MWCNTs) could induce adverse vascular effects, but the role of length of MWCNTs in determining the toxic effects is less studied. This study investigated the adverse effects of two well-characterized MWCNTs to human umbilical vein endothelial cells (HUVECs). METHODS: The internalization and localization of MWCNTs in HUVECs were examined by using transmission electron microscopy (TEM). The cytotoxicity of MWCNTs to HUVECs was assessed by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays. Oxidative stress was indicated by the measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS). ELISA was used to determine the release of inflammatory cytokines. THP-1 monocyte adhesion to HUVECs was also measured. To indicate the activation of endoplasmic reticulum (ER) stress, the expression of ddit3 and xbp-1s was measured by RT-PCR, and BiP protein level was measured by Western blot. RESULTS: Transmission electron microscopy observation indicates the internalization of MWCNTs into HUVECs, with a localization in nuclei and mitochondria. The longer MWCNTs induced a higher level of cytotoxicity to HUVECs compared with the shorter ones. Neither of MWCNTs significantly promoted intracellular ROS, but the longer MWCNTs caused a higher depletion of GSH. Exposure to both types of MWCNTs significantly promoted THP-1 adhesion to HUVECs, accompanying with a significant increase of release of interleukin-6 (IL-6) but not tumor necrosis factor α (TNFα), soluble ICAM-1 (sICAM-1) or soluble VCAM-1 (sVCAM-1). Moreover, THP-1 adhesion and release of IL-6 and sVCAM-1 induced by the longer MWCNTs were significantly higher compared with the responses induced by the shorter ones. The biomarker of ER stress, ddit3 expression, but not xbp-1s expression or BiP protein level, was significantly induced by the exposure of longer MWCNTs. CONCLUSIONS: Combined, these results indicated length dependent toxic effects of MWCNTs to HUVECs in vitro, which might be associated with oxidative stress and activation of ER stress.

Toxicity of ZnO nanoparticles (NPs) to A549 cells and A549 epithelium in vitro: Interactions with dipalmitoyl phosphatidylcholine (DPPC).

Once inhaled, nanoparticles (NPs) will first interact with lung surfactant system, which may influence the colloidal aspects of NPs and consequently the toxic potential of NPs to pulmonary cells. In this study, we investigated the effects of dipalmitoyl phosphatidylcholine (DPPC), the major component in lung surfactant, on stability and toxicity of ZnO NPs. The presence of DPPC increased the UV-vis spectra, hydrodynamic size, Zeta potential and dissolution rate of ZnO NPs, which indicates that DPPC might interact with NPs and affect the colloidal stability of NPs. Exposure to ZnO NPs induced cytotoxicity associated with increased intracellular Zn ions but not superoxide in A549 cells. In A549 epithelium model, exposure to ZnO NPs induced cytotoxicity and decreased the release of interleukin 6 (IL-6) without a significant effect on epithelial permeability rate. Co-exposure of A549 cells or A549 epithelium model to DPPC and ZnO NPs induced a higher release of lactate dehydrogenase (LDH) and interleukin-6 (IL-6) compared with the exposure of ZnO NPs alone. We concluded that the presence of DPPC could influence the colloidal stability of ZnO NPs and increase the damage of NPs to membrane probably due to the increased positive surface charge.

A review of endoplasmic reticulum (ER) stress and nanoparticle (NP) exposure.

Understanding the mechanism of nanoparticle (NP) induced toxicity is important for nanotoxicological and nanomedicinal studies. Endoplasmic reticulum (ER) is a crucial organelle involved in proper protein folding. High levels of misfolded proteins in the ER could lead to a condition termed as ER stress, which may ultimately influence the fate of cells and development of human diseases. In this review, we summarized studies about effects of NP exposure on ER stress. A variety of NPs, especially metal-based NPs, could induce morphological changes of ER and activate ER stress pathway both in vivo and in vitro. In addition, modulation of ER stress by chemicals has been shown to alter the toxicity of NPs. These studies in combination suggested that ER stress could be the mechanism responsible for NP induced toxicity. Meanwhile, nanomedicinal studies also used ER stress inducing NPs or NPs loaded with ER stress inducer to selectively induce ER stress mediated apoptosis in cancer cells for cancer therapy. In contrast, alleviation of ER stress by NPs has also been shown as a strategy to cure metabolic diseases. In conclusion, exposure to NPs may modulate ER stress, which could be a target for future nanotoxicological and nanomedicinal studies.

Foam cell formation by particulate matter (PM) exposure: a review.

Increasing evidence suggests that exposure of particulate matter (PM) from traffic vehicles, e.g., diesel exhaust particles (DEP), was associated with adverse vascular effects, e.g., acceleration of atherosclerotic plaque progression. By analogy, engineered nanoparticles (NPs) could also induce similar effects. The formation of lipid laden foam cells, derived predominately from macrophages and vascular smooth muscle cells (VSMC), is closely associated with the development of atherosclerosis and adverse vascular effects. We reviewed current studies about particle exposure-induced lipid laden foam cell formation. In vivo studies using animal models have shown that exposure of air pollution by PM promoted lipid accumulation in alveolar macrophages or foam cells in plaques, which was likely associated with pulmonary inflammation or systemic oxidative stress, but not blood lipid profile. In support of these findings, in vitro studies showed that direct exposure of cultured macrophages to DEP or NP exposure, with or without further exposure to external lipids, promoted intracellular lipid accumulation. The mechanisms remained unknown. Although a number studies found increased reactive oxygen species (ROS) or an adaptive response to oxidative stress, the exact role of oxidative stress in mediating particle-induced foam cell formation requires future research. There is currently lack of reports concerning VSMC as a source for foam cells induced by particle exposure. In the future, it is necessary to explore the role of foam cell formation in particle exposure-induced atherosclerosis development. In addition, the formation of VSMC derived foam cells by particle exposure may also need extensive studies.