Appl Biochem Biotechnol DOI 10.1007/s12010-014-1351-y
Cytotoxicity of Zinc Oxide Nanoparticles on Antioxidant Enzyme Activities and mRNA Expression in the Cocultured C2C12 and 3T3-L1 Cells Muthuraman Pandurangan & Muthuviveganandavel Veerappan & Doo Hwan Kim
Received: 8 June 2014 / Accepted: 28 October 2014 # Springer Science+Business Media New York 2014
Abstract The present study was aimed to investigate the dose-dependent effect of zinc oxide (ZnO) nanoparticles on antioxidant enzyme activities and messenger RNA (mRNA) expression in the cocultured C2C12 and 3T3-L1 cells. Coculturing experiments are 3D and more reliable compared to mono-culture (2D) experiment. Even though, there are several studies on ZnO nanoparticle-mediated cytotoxicity, but there are no studies on the effect of ZnO nanoparticle on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles on the C2C12 and 3T3-L1 cell viability. At higher concentration of ZnO nanoparticles, C2C12 and 3T3-L1 cells almost die. ZnO nanoparticles increased reactive oxygen species (ROS) and lipid peroxidation and reduced glutathione (GSH) levels in a dose-dependent manner in the C2C12 and 3T3-L1 cells. In addition, ZnO nanoparticles increased antioxidant enzyme activities and their mRNA expression in the C2C12 and 3T3L1 cells. In conclusion, the present study showed that ZnO nanoparticles increased oxidative stress, antioxidant enzyme activities, and their mRNA expression in the cocultured C2C12 and 3T3-L1 cells. Keywords C2C12 . 3T3-L1 . Antioxidant . ZnO nanoparticles . Cytotoxicity . Enzymes . mRNA
Introduction Nanostructural zinc oxide (ZnO) attracts several researchers because of its potent biomedical applications in the modern world. ZnO nanoparticles were synthesized by several methods including physical and chemical methods, and their properties are determined by the size, chemical composition, and surface chemistry [1]. ZnO is used in cosmetic lotions as UV blockers [2], increases the antibacterial activity [3], and used in the cotton fabric, rubber, and food packaging industry [4]. Cytotoxicity of nanoparticles and their interaction with biological M. Pandurangan : D. H. Kim (*) Department of Bioresources and Food Science, Konkuk University, Seoul, South Korea e-mail: [email protected] M. Veerappan Department of Zoology, Tagore Arts College, Pondicherry, India
Appl Biochem Biotechnol
systems is still unclear [5]. ZnO nanoparticles showed its distribution in the mice tissue such as the liver, spleen, kidneys, and adipose tissue [6]. Zinc level increased in the liver, adipose tissue, and pancreas because ZnO nanoparticles were systemically absorbed [7]. DNA damage and cell death are reported to occur due to oxidative stress induced by ZnO nanoparticles [8]. However, there are no studies on the effect of ZnO nanoparticles on the cocultured C2C12 and 3T3-L1 cells. The present study was aimed to investigate the dose-dependent effect of ZnO nanoparticles on reactive oxygen species (ROS), lipid peroxidation, reduced glutathione, antioxidant enzyme activities, and messenger RNA (mRNA) expression in the C2C12 and 3T3-L1 cells.
Materials and Methods ZnO nanoparticles were synthesized and characterized by SEM and TEM analysis. All chemicals and laboratory wares were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA), respectively. Cell Culture C2C12 and 3T3-L1 cells were incubated at a density of 7000 cells/cm2 and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and 1 % antibiotics at 37 °C in 5 % CO2. Confluent 3T3-L1 cells were induced to differentiate with a standard differentiation medium consisting of DMEM medium supplemented with 10 % FBS, 250 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μg/ml insulin, and 1 % antibiotics. Cultures were re-fed every 2 days to allow 90 % cells to reach fully differentiation before coculturing. C2C12 cells were grown to 90 % confluence and changed into differentiation medium and fed with fresh differentiation medium everyday. Cell Coculture C2C12 and 3T3-L1 cells were cocultured by using Transwell inserts with a 0.4-μm porous membrane to separate the cells. Each cell type was grown independently on the Transwell plates. After cell differentiation, inserts containing adipocytes were transferred to myotube plates and inserts containing myotubes were transferred to adipocyte plates [9]. Treatment of Cells C2C12 and 3T3-L1 cells were treated with various concentrations of ZnO nanoparticles ranging from 10 to 30 μg/ml. ZnO nanoparticle suspension was sonicated for 10 min prior to start of each experiment and freshly diluted in the medium before treatment. C2C12 and 3T3-L1 cells were incubated with medium containing ZnO nanoparticles for 48 h at 37 °C in 5 % CO2 prior to harvesting. MTT Assay Cytotoxicity of ZnO nanoparticle was determined by MTT assay [10]. C2C12 and 3T3-L1 cells were seeded at seeding density of 2.5×104 cells/ml into 96-well microplates and allowed to adhere for 24 h and treated with various concentrations of ZnO nanoparticles ranging from 10 to 30 μg/ml. Cells were observed under light microscope to determine cellular morphology
Appl Biochem Biotechnol
after 48-h incubation. Cells were labeled with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution for 4 h, and the resulting formazan was solubilized in DMSO. The absorption was measured at 570 nm by plate reader. Determination of Cellular ROS Cellular level of ROS was determined based on the measurement of 2,7-dichlorofluorescein diacetate [11]. After treatment, cells were washed twice with HBSS and then incubated in 1 ml of 2,7-dichlorofluorescein diacetate at 37 °C for 30 min. C2C12 and 3T3-L1 cells were lysed in alkaline solution and centrifuged for 10 min. Supernatant was transferred to a 96-well plate, and fluorescence was measured at 485-nm excitations and 520-nm emissions with a microplate reader. Values were expressed as a percentage of intensity. Determination of MDA and GSH Malondialdehyde (MDA) was determined by the method of Umrani and Paknikar [7] in the C2C12 and 3T3-L1 cells. Reduced glutathione (GSH) was determined by the method of Owen and Allan Butterfield [11]. Antioxidant Enzymes Glutathione peroxidase, glutathione reductase, superoxide dismutase (SOD), and catalase were assayed as by Weydert and Cullen [12]. Lactate dehydrogenase (LDH) enzyme activity was measured by the method of Dawei et al. [13]. qPCR Total RNA was isolated from the control and treated C2C12 and 3T3-L1 cells with Trizol reagent according to the manufacturer’s protocol. The first-strand complementary DNA (cDNA) was synthesized from 1 μg of the total RNA using the M-MLV reverse transcriptase with the anchored oligo d(T)12–18 primer. qPCR was performed using a cDNA equivalent of 10 ng of total RNA from each sample with primers specific for glutathione reductase (GenBank NM_053906.1), glutathione peroxidase (GenBank NM_030826.3), SOD (GenBank NM_017051.2), catalase (GenBank NM_012520.1), and a housekeeping gene GAPDH. The reaction was carried out in 10 μl using SYBR Green Master Mix (Invitrogen) according to the manufacturer’s instructions. Relative ratios were calculated based on the 2−△△CT method [14]. PCR was monitored using the MiniOpticon Real-Time PCR System (Bio-Rad). Statistical Analysis All values are expressed as means±SEM. Student’s t test was performed to determine the differences between control and treatments. p
Cytotoxicity of Zinc Oxide Nanoparticles on Antioxidant Enzyme Activities and mRNA Expression in the Cocultured C2C12 and 3T3-L1 Cells Muthuraman Pandurangan & Muthuviveganandavel Veerappan & Doo Hwan Kim
Received: 8 June 2014 / Accepted: 28 October 2014 # Springer Science+Business Media New York 2014
Abstract The present study was aimed to investigate the dose-dependent effect of zinc oxide (ZnO) nanoparticles on antioxidant enzyme activities and messenger RNA (mRNA) expression in the cocultured C2C12 and 3T3-L1 cells. Coculturing experiments are 3D and more reliable compared to mono-culture (2D) experiment. Even though, there are several studies on ZnO nanoparticle-mediated cytotoxicity, but there are no studies on the effect of ZnO nanoparticle on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles on the C2C12 and 3T3-L1 cell viability. At higher concentration of ZnO nanoparticles, C2C12 and 3T3-L1 cells almost die. ZnO nanoparticles increased reactive oxygen species (ROS) and lipid peroxidation and reduced glutathione (GSH) levels in a dose-dependent manner in the C2C12 and 3T3-L1 cells. In addition, ZnO nanoparticles increased antioxidant enzyme activities and their mRNA expression in the C2C12 and 3T3L1 cells. In conclusion, the present study showed that ZnO nanoparticles increased oxidative stress, antioxidant enzyme activities, and their mRNA expression in the cocultured C2C12 and 3T3-L1 cells. Keywords C2C12 . 3T3-L1 . Antioxidant . ZnO nanoparticles . Cytotoxicity . Enzymes . mRNA
Introduction Nanostructural zinc oxide (ZnO) attracts several researchers because of its potent biomedical applications in the modern world. ZnO nanoparticles were synthesized by several methods including physical and chemical methods, and their properties are determined by the size, chemical composition, and surface chemistry [1]. ZnO is used in cosmetic lotions as UV blockers [2], increases the antibacterial activity [3], and used in the cotton fabric, rubber, and food packaging industry [4]. Cytotoxicity of nanoparticles and their interaction with biological M. Pandurangan : D. H. Kim (*) Department of Bioresources and Food Science, Konkuk University, Seoul, South Korea e-mail: [email protected] M. Veerappan Department of Zoology, Tagore Arts College, Pondicherry, India
Appl Biochem Biotechnol
systems is still unclear [5]. ZnO nanoparticles showed its distribution in the mice tissue such as the liver, spleen, kidneys, and adipose tissue [6]. Zinc level increased in the liver, adipose tissue, and pancreas because ZnO nanoparticles were systemically absorbed [7]. DNA damage and cell death are reported to occur due to oxidative stress induced by ZnO nanoparticles [8]. However, there are no studies on the effect of ZnO nanoparticles on the cocultured C2C12 and 3T3-L1 cells. The present study was aimed to investigate the dose-dependent effect of ZnO nanoparticles on reactive oxygen species (ROS), lipid peroxidation, reduced glutathione, antioxidant enzyme activities, and messenger RNA (mRNA) expression in the C2C12 and 3T3-L1 cells.
Materials and Methods ZnO nanoparticles were synthesized and characterized by SEM and TEM analysis. All chemicals and laboratory wares were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA), respectively. Cell Culture C2C12 and 3T3-L1 cells were incubated at a density of 7000 cells/cm2 and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and 1 % antibiotics at 37 °C in 5 % CO2. Confluent 3T3-L1 cells were induced to differentiate with a standard differentiation medium consisting of DMEM medium supplemented with 10 % FBS, 250 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 5 μg/ml insulin, and 1 % antibiotics. Cultures were re-fed every 2 days to allow 90 % cells to reach fully differentiation before coculturing. C2C12 cells were grown to 90 % confluence and changed into differentiation medium and fed with fresh differentiation medium everyday. Cell Coculture C2C12 and 3T3-L1 cells were cocultured by using Transwell inserts with a 0.4-μm porous membrane to separate the cells. Each cell type was grown independently on the Transwell plates. After cell differentiation, inserts containing adipocytes were transferred to myotube plates and inserts containing myotubes were transferred to adipocyte plates [9]. Treatment of Cells C2C12 and 3T3-L1 cells were treated with various concentrations of ZnO nanoparticles ranging from 10 to 30 μg/ml. ZnO nanoparticle suspension was sonicated for 10 min prior to start of each experiment and freshly diluted in the medium before treatment. C2C12 and 3T3-L1 cells were incubated with medium containing ZnO nanoparticles for 48 h at 37 °C in 5 % CO2 prior to harvesting. MTT Assay Cytotoxicity of ZnO nanoparticle was determined by MTT assay [10]. C2C12 and 3T3-L1 cells were seeded at seeding density of 2.5×104 cells/ml into 96-well microplates and allowed to adhere for 24 h and treated with various concentrations of ZnO nanoparticles ranging from 10 to 30 μg/ml. Cells were observed under light microscope to determine cellular morphology
Appl Biochem Biotechnol
after 48-h incubation. Cells were labeled with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) solution for 4 h, and the resulting formazan was solubilized in DMSO. The absorption was measured at 570 nm by plate reader. Determination of Cellular ROS Cellular level of ROS was determined based on the measurement of 2,7-dichlorofluorescein diacetate [11]. After treatment, cells were washed twice with HBSS and then incubated in 1 ml of 2,7-dichlorofluorescein diacetate at 37 °C for 30 min. C2C12 and 3T3-L1 cells were lysed in alkaline solution and centrifuged for 10 min. Supernatant was transferred to a 96-well plate, and fluorescence was measured at 485-nm excitations and 520-nm emissions with a microplate reader. Values were expressed as a percentage of intensity. Determination of MDA and GSH Malondialdehyde (MDA) was determined by the method of Umrani and Paknikar [7] in the C2C12 and 3T3-L1 cells. Reduced glutathione (GSH) was determined by the method of Owen and Allan Butterfield [11]. Antioxidant Enzymes Glutathione peroxidase, glutathione reductase, superoxide dismutase (SOD), and catalase were assayed as by Weydert and Cullen [12]. Lactate dehydrogenase (LDH) enzyme activity was measured by the method of Dawei et al. [13]. qPCR Total RNA was isolated from the control and treated C2C12 and 3T3-L1 cells with Trizol reagent according to the manufacturer’s protocol. The first-strand complementary DNA (cDNA) was synthesized from 1 μg of the total RNA using the M-MLV reverse transcriptase with the anchored oligo d(T)12–18 primer. qPCR was performed using a cDNA equivalent of 10 ng of total RNA from each sample with primers specific for glutathione reductase (GenBank NM_053906.1), glutathione peroxidase (GenBank NM_030826.3), SOD (GenBank NM_017051.2), catalase (GenBank NM_012520.1), and a housekeeping gene GAPDH. The reaction was carried out in 10 μl using SYBR Green Master Mix (Invitrogen) according to the manufacturer’s instructions. Relative ratios were calculated based on the 2−△△CT method [14]. PCR was monitored using the MiniOpticon Real-Time PCR System (Bio-Rad). Statistical Analysis All values are expressed as means±SEM. Student’s t test was performed to determine the differences between control and treatments. p
