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ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells.

Wang C, Hu X, Gao Y, Ji Y - Biomed Res Int (2015)

Bottom Line: Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method.Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure.The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, 17 Chunhui Road, Laishan District, Yantai 264003, China ; University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT
Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

No MeSH data available.


Related in: MedlinePlus

ZnO nanoparticles-induced apoptosis in LTEP-a-2 cells after 4 h of exposure. (a) Morphologic examination of LTEP-a-2 cells by AO/EB fluorescence staining ((A) 0 μg/mL, control, (B) 0.05 μg/mL, (C) 0.1 μg/mL, and (D) 0.2 μg/mL) and (b) caspase-3 activity. ∗ versus control, P < 0.05; ∗∗ versus control, P < 0.01 by Student's t-test.
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fig5: ZnO nanoparticles-induced apoptosis in LTEP-a-2 cells after 4 h of exposure. (a) Morphologic examination of LTEP-a-2 cells by AO/EB fluorescence staining ((A) 0 μg/mL, control, (B) 0.05 μg/mL, (C) 0.1 μg/mL, and (D) 0.2 μg/mL) and (b) caspase-3 activity. ∗ versus control, P < 0.05; ∗∗ versus control, P < 0.01 by Student's t-test.

Mentions: AO/EB staining is considered an ideal method for distinguishing apoptotic cells from necrotic ones [29, 30]. Here we use AO/EB staining to verify whether ZnO NPs inhibit proliferation of LTEP-a-2 cells by inducing apoptosis or killing cells directly (necrosis). During morphologic examinations, normal viable LTEP-a-2 control cells were stained green (0 μg/mL ZnO NPs), and the apoptotic cells exposed to ZnO NPs (0.05, 0.1, and 0.2 μg/mL) appeared as bright green arcs in an early stage and with condensed, yellow/orange nuclei in the late stage (Figure 5(a)). Additionally, the results of caspase-3 activity assay showed that exposure of ZnO NPs induced significant increases in caspase-3 activity compared to the control treatment (P < 0.05 for 0.01–0.05 μg/mL ZnO NPs, P < 0.01 for 0.1–0.5 μg/L ZnO NPs; Figure 5(b)). As a key mediator, caspase-3 plays a pivotal role in caspase-dependent apoptosis [31]. Together, these results confirm that exposure of ZnO NPs induced substantial apoptosis in LTEP-a-2 cells even at low concentrations (e.g., 0.01 μg/mL), thus inhibiting cell proliferation.


ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells.

Wang C, Hu X, Gao Y, Ji Y - Biomed Res Int (2015)

ZnO nanoparticles-induced apoptosis in LTEP-a-2 cells after 4 h of exposure. (a) Morphologic examination of LTEP-a-2 cells by AO/EB fluorescence staining ((A) 0 μg/mL, control, (B) 0.05 μg/mL, (C) 0.1 μg/mL, and (D) 0.2 μg/mL) and (b) caspase-3 activity. ∗ versus control, P < 0.05; ∗∗ versus control, P < 0.01 by Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: ZnO nanoparticles-induced apoptosis in LTEP-a-2 cells after 4 h of exposure. (a) Morphologic examination of LTEP-a-2 cells by AO/EB fluorescence staining ((A) 0 μg/mL, control, (B) 0.05 μg/mL, (C) 0.1 μg/mL, and (D) 0.2 μg/mL) and (b) caspase-3 activity. ∗ versus control, P < 0.05; ∗∗ versus control, P < 0.01 by Student's t-test.
Mentions: AO/EB staining is considered an ideal method for distinguishing apoptotic cells from necrotic ones [29, 30]. Here we use AO/EB staining to verify whether ZnO NPs inhibit proliferation of LTEP-a-2 cells by inducing apoptosis or killing cells directly (necrosis). During morphologic examinations, normal viable LTEP-a-2 control cells were stained green (0 μg/mL ZnO NPs), and the apoptotic cells exposed to ZnO NPs (0.05, 0.1, and 0.2 μg/mL) appeared as bright green arcs in an early stage and with condensed, yellow/orange nuclei in the late stage (Figure 5(a)). Additionally, the results of caspase-3 activity assay showed that exposure of ZnO NPs induced significant increases in caspase-3 activity compared to the control treatment (P < 0.05 for 0.01–0.05 μg/mL ZnO NPs, P < 0.01 for 0.1–0.5 μg/L ZnO NPs; Figure 5(b)). As a key mediator, caspase-3 plays a pivotal role in caspase-dependent apoptosis [31]. Together, these results confirm that exposure of ZnO NPs induced substantial apoptosis in LTEP-a-2 cells even at low concentrations (e.g., 0.01 μg/mL), thus inhibiting cell proliferation.

Bottom Line: Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method.Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure.The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, 17 Chunhui Road, Laishan District, Yantai 264003, China ; University of Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT
Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

No MeSH data available.


Related in: MedlinePlus