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Jun 1, 2017 - Graphene oxide nanosheets induce DNA. 2 damage and activate the base excision repair. 3. BER signaling pathway both in vitro and in. 4 vivo.
Accepted Manuscript Graphene oxide nanosheets induce DNA damage and activate the base excision repair (BER) signaling pathway both in vitro and in vivo Chun-Jiao Lu, Xue-Feng Jiang, Muhammad Junaid, Yan-Bo Ma, Pan-Pan Jia, HuaBin Wang, De-Sheng Pei PII:

S0045-6535(17)30951-7

DOI:

10.1016/j.chemosphere.2017.06.049

Reference:

CHEM 19443

To appear in:

ECSN

Received Date: 13 March 2017 Revised Date:

1 June 2017

Accepted Date: 13 June 2017

Please cite this article as: Lu, C.-J., Jiang, X.-F., Junaid, M., Ma, Y.-B., Jia, P.-P., Wang, H.B., Pei, D.-S., Graphene oxide nanosheets induce DNA damage and activate the base excision repair (BER) signaling pathway both in vitro and in vivo, Chemosphere (2017), doi: 10.1016/ j.chemosphere.2017.06.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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Original Research Article

Graphene oxide nanosheets induce DNA

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damage and activate the base excision repair BER

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signaling pathway both in vitro and in vivo

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Chun-Jiao Lu1¶, Xue-Feng Jiang1¶, Muhammad Junaid1, 2¶,

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Yan-Bo Ma1, Pan-Pan Jia1, 2, Hua-Bin Wang1*, De-Sheng Pei1*

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Sciences, Chongqing 400714, China.

Chongqing Institute of Green and Intelligent Technology, Chinese Academy of

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Emails: [email protected] (H.B.W) and [email protected] (D.S.P)

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University of Chinese Academy of Sciences, Beijing 100049, China

These authors contributed equally to this work. Corresponding authors.

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Abstract Graphene oxide (GO) has widespread concerns in the fields of biological

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sciences and medical applications. Currently, studies have reported that excessive GO

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exposure can cause cellular DNA damage through reactive oxygen species (ROS)

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generation. However, DNA damage mediated response of the base excision repair

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(BER) pathway due to GO exposure is not elucidated yet. Therefore, we exposed

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HEK293T cells and zebrafish embryos to different concentrations of GO for 24 h, and

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transcriptional profiles of BER pathway genes, DNA damage, and cell viability were

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analyzed both in vitro and in vivo. Moreover, the deformation of HEK293T cells

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before and after GO exposure was also investigated using atomic force microscopy

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(AFM) to identify the physical changes occurred in the cells’ structure. CCK-8 and

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Comet assay revealed the significant decrease in cell viability and increase in DNA

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damage in HEK293T cells at higher GO doses (25 and 50 µg/mL). Among the

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investigated genetic markers in HEK293T cells, BER pathway genes (APEX1, OGG1,

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CREB1, UNG) were significantly up-regulated upon exposure to higher GO dose (50

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µg/mL), however, low exposure concentration (5, 25 µg/mL) failed to induce

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significant genetic induction except for CREB1 at 25 µg/mL. Additionally, the

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viscosity of HEK293T cells decreased upon GO exposure. In zebrafish, the results of

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up-regulated gene expressions (apex1, ogg1, polb, creb1) were consistent with those

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in the HEK293T cells. Taken all together, the exposure to elevated GO concentration

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could cause DNA damage to HEK293T cells and zebrafish embryos; BER pathway

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could be proposed as the possible inner response mechanism.

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Keywords: Atomic force microscopy; Base excision repair; Graphene oxide;

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HEK293T cells; Zebrafish embryos

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1. Introduction Nanomaterials have abundant applications in medical, biological, and

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pharmaceutical fields (Ahlin et al., 2002; Kukowska-Latallo et al., 2005; Chong et al.,

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2006). Graphene is a two-dimensional plane carbon nanomaterial, and graphene oxide

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(GO) is a single carbon layer graphene derivative (Cushing et al., 2014). GO has both

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remarkable physical properties and stable chemical properties (Zhang et al., 2005;

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Novoselov et al., 2007). Due to unique physicochemical properties, GO, similar to

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other nanomaterials has a broad spectrum of applications in biology and medicine

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such as disease diagnosis, anticancer drugs delivery, molecular probes, and biological

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sensors (Lee et al., 2005; Mohanty and Berry, 2008; Yang et al., 2008; Choi et al.,

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2010; Wang et al., 2010b; Zhang et al., 2010; Akhavan et al., 2012; Ni et al., 2013).

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GO is widely used in biological sciences and medical applications and becomes a

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potential emerging pollutant in the environment (Deng et al., 2017; Hazeem et al.,

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2017). Simultaneously, a great deal of interest in the scientific community has been

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generated on understanding the potential adverse effects of GO. Previous studies

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indicated that GO has cytotoxicity at high concentration by causing apoptosis and

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granulomas formation in lungs (Wang et al., 2010a; Chang et al., 2011; Hu et al.,

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2011; Zhang et al., 2011), but no obvious cytotoxicity for SH-SY5Y cell after 96 h

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exposure at low concentration (< 80 µg/mL) (Lv et al., 2012). It has also been

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reported that GO can induce an immune response, oxidative stress and immune

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toxicity in adult zebrafish (Chen et al., 2016). Furthermore, GO exposure affects the

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activity of lactate dehydrogenase, induces cell immune toxicity, and generates

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reactive oxygen species, which ultimately lead to the oxidative stress and DNA

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damage (Ma et al., 2012; Zhi et al., 2013).

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The base excision repair (BER) pathway is a major naturally existing mechanism

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for DNA repair in organisms, which is responsible for repairing the damaged bases of

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DNA (Kawamura, 2010; Menezo et al., 2010; Pei et al., 2011). The adverse effects of

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nanomaterials on BER signaling pathway could cause serious consequences in

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exposed organisms and cells. For example, Kovvuru et al. indicated that the oral

ACCEPTED MANUSCRIPT ingestion of silver nanoparticles (AgNPs) induced permanent genome alterations and

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caused BER pathway defects in mice (Kovvuru et al., 2015). Asharani et al. reported

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that AgNPs exposure caused a downregulation of BER pathway key genes in human

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lung IMR 90 cell lines (Asharani et al., 2012). Moreover, It has also been confirmed

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that nanoparticles can cause oxidative stress-mediated mitochondrial DNA damage in

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C. elegans (Ahn et al., 2014). However, so far the GO’s studies of adverse effects on

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BER pathway are elusive, which impedes the applications of GO. Therefore,

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elucidation of GO exposure associated activation BER pathway is essential to get

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complete insights of GO toxicity, which is crucial for the rational use of GO

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nanomaterials.

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This study evaluated the toxicity of GO nanosheets to the BER pathway of human

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embryonic kidney 293 transformed cells (HEK 293T cells) in vitro and zebrafish

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embryos in vivo. HEK 293T cells and zebrafish embryos are often used as model

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systems in molecular biology and environmental toxicology research (Cui et al., 2005;

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Lindberg et al., 2009; Kim et al., 2013; Jia et al., 2016). The cell counting assay

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(CCK-8), Comet assay, qRT-PCR and atomic force microscopy (AFM) techniques

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were employed to investigate cell viability, DNA damages and genetic induction in

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HEK 293T cells. Further, developmental parameters and response of the BER

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pathway genes were also quantified in zebrafish embryos after exposure to GO

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nanosheets.

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2. Materials and Methods

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2.1 Ethics statement

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All animal experiments were performed according to the “Guide for the Care and

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Use of Laboratory Animals” (Eighth Edition, 2011. ILARCLS, National Research

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Council, Washington, D.C.). The animal protocol was approved by the Institutional

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Animal Care and Use Committee of Chongqing Institute of Green and Intelligent

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Technology, Chinese Academy of Sciences (Approval ID: ZKCQY00186).

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2.2 Preparation and characterization of GO

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GO (Sigma-Aldrich, USA) powder was suspended in ultrapure water (Millipore,

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Billerica, MA), then sonicated in an ultrasonic machine (50 W/L, 40 kHz) for 50 min

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to prepare for 1 g/L stock solution, which was further diluted to desired final

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concentrations in the experiments.

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2.3 AFM characterization of GO

AFM experiments were carried out at room temperature using Dimension Edge

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Instrument (Bruker Nano Surfaces, Santa Barbara, CA). Tapping mode AFM with

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optimized operational parameter was used to characterize GO in air using doped

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silicon cantilever (RTESP, Camarillo, CA) with a nominal spring constant of 40 N/m

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and a nominal probe curvature radius of 10 nm.

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2.4 Cells culture, GO exposure and cell viability measurement HEK293T cells (ATCC CRL-11268) were cultured in RPMI Medium 1640

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(Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Life Technologies,

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Australia) and 1% Penicillin-Streptomycin Solution (Beyotime, Jiangsu, China) at

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37 °C in a humidified incubator with an atmosphere consisting of 5% CO2 in air.

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For GO exposure, cells were seeded in 6-well plates (Approximately 2.4 × 105

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cells/well, 3 mL/well) and cultured at 37 °C for 12 h. Then, cells were exposed to the

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medium containing different concentrations of GO (0, 5, 25, 50 µg/mL). Each

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treatment was repeated 3 times, and all cells were incubated for 24 h at 37 °C. CCK-8 (Beyotime, China) assay was used to explore the viability of HEK293T

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cells after exposure to GO. The cells were seeded in 96-well plates (Approximately

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1×104 cells/well, 100 uL/well) and cultured at 37 °C for 12 h. The supernatant was

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removed and cells were washed with PBS (HyClone, Beijing, China) twice.

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Afterward, the blank medium (control group) and the medium containing different

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concentrations of GO (test group) were added. Each treatment was repeated 6 times,

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and all cells were incubated for 24 h at 37 °C in a humidified incubator with an

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atmosphere of 5% CO2. Then the CCK-8 solution was added and the absorbance was

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measured after 4 h using a microplate reader at the wavelength of 450 nm.

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2.5 Comet assay In this study, DNA damage was measured using an alkaline comet assay

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(Trevigen, USA) according to the manufacturer’s instructions. Briefly, 500 uL of

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LMAgarose (Low melting point agarose, Trevigen, USA) was poured into a 1.5 mL

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EP tube, and heated in boiling water for 5 min; Then, the temperature was reduced to

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37 °C, and melting gel was mixed with 50 uL of cell suspension (Approximately

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1 × 105 to 2 × 105 cells /mL). Each well of the CometSlide (Trevigen, USA) was filled

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with 50 µL of the cell/agarose suspension. The slides were placed at 4 °C in the dark

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for 10 min and then immersed into 4 °C Lysis Solution (Trevigen, USA) for 60 min.

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After removed excess buffer, slides were immersed in freshly prepared Alkaline

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Unwinding Solution (200 mmol/L NaOH, 1mmol/L EDTA, pH > 13), and incubated

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at room temperature in the dark for 60 min.

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Subsequently, alkaline electrophoresis solution was prepared for electrophoresis

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in the CometAssay ES tank (Trevigen, USA) at 21 V for 30 min. The slides were first

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immersed twice in dH2O for 5 min and then fixed in 70% ethanol for another 5 min.

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Then slides were dried and stained for 20 min at 4 °C with SYBR Gold (Trevigen,

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USA) (diluted 1: 30 000 in 10 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA). Finally,

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excess staining solution was removed, and slides were dried at room temperature in

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the dark for microscopy.

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2.6 GO mechanical effects on cells using AFM

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Previous studies revealed alterations in the viscosity and elasticity of the cells

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due to the cellular deformations caused by internal disturbance and such alterations

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were measured through AFM (O. Klymenko et al., 2009; Fernandes et al., 2012;

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Wang et al., 2017). Therefore, the cellular viscosity is a good physical parameter that

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can be used to evaluate the biological changes induce in the cytoskeleton of cells upon

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GO treatment. The factor of viscosity was obtained by analyzing the force curves

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measured in the cells using the protocol previously described (O. Klymenko et al.,

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2009; Fernandes et al., 2012; Wang et al., 2017). Briefly, force curves were acquired

ACCEPTED MANUSCRIPT using the Dimension Edge instrument with a contact mode in PBS buffer. Silicon

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nitride cantilevers were purchased from Bruker Corporation (DNP-10, Camarillo, CA)

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with a nominal spring constant of 0.06 N/m, and their respective spring constants

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were calibrated on the surface of the bottom of a petri dish in PBS buffer through the

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thermal tune function using AFM software. The AFM combined with an optical

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microscope allowed the precise lateral positioning of the AFM tip over the central

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region of the cell. After calibration, force curves were recorded at a speed of 1 µm/s.

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At least 10 force curves were collected on each cell, and more than 30 cells from three

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independent experiments were measured. The raw force curves were converted to

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force-distance curves by correcting cantilever bending and removing it from the piezo

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displacement with a self-written procedure based on Igor Pro (Version 6.04,

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Wavemetrics Inc., USA). The deformation of the cells was evaluated based on the

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obtained force-distance curves and quantified using the factor of viscosity (P), which

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was calculated according to the equation: P= viscous deformation/(viscous

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deformation + elastic deformation). A typical force-distance curve is shown in the Fig.

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1, in which the viscous deformation and elastic deformation are marked with olive

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and yellow colors, respectively. All calculations were performed using a self-written

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procedure based on Matlab (Version R2010a, Mathworks).

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2.7 Zebrafish husbandry and exposure to GO nanosheets Adult

three-month-old

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Hydrobiology, Chinese Academy of Sciences, Wuhan, China) were kept in the

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automatic water cycle system on a 14 h light: 10 h dark period and fed with brine

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shrimp (Artemia nauplii) three times daily. Normal adult zebrafish were selected to

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spawn in tanks overnight with 2 males and 1 female. After spawning, 150 embryos at

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the blastula stage were identified and transferred to 10 cm Petri dishes and exposed to

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medium containing different concentrations of GO (0, 5, 25, 50 µg/mL). Each

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treatment was repeated 3 times independently in the illumination light incubator at 28

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± 5 °C. The survival and hatching rates at 72 hpf (hours post fertilization) were

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calculated using the following equation;

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Survival/hatching rate =

  /  ! "   !

× 100 (1)

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2.8 Gene expression profiling in HEK293T cells and zebrafish embryos After exposure to GO for 24 h, the total RNAs of HEK293T cells were extracted

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from 6-well plates (approximately 2.4 × 105 cells/well, 3 mL/well) by using RNAiso

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Plus reagent (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions.

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The synthesis of cDNA was performed by using the Primer Script RT reagent Kit

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(Takara Bio, Shiga, Japan). qRT-PCR was performed using the SYBR Green RCR Kit

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(Toyobo, Tokyo, Japan) on the ABI 7300 System (PerkinElmer Applied Biosystems,

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Foster City, CA, USA). The primers of the selected genes and the sequences are listed

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in the Table 1. The housekeeping gene β-actin was chosen as the internal control as

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recommended previously (Feng et al., 2009). All the samples were run in triplicates.

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The expression levels of genes were normalized to β-actin by using the 2-∆∆CT

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method.

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The zebrafish embryos were cultured in 10 cm Petri dishes and exposed to GO

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for 24 h. Each treatment was repeated 3 times. Total RNAs were extracted from 30

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homogenized zebrafish embryos. cDNAs were synthesized for qRT-PCR following

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the same protocol as used for HEK293T cells.

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2.9 Statistical analysis

The normality of the data and the homogeneity of variances were analyzed by

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Kolmogorov-Smirnov test and Leven’s test. The difference between the variables was

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calculated by one-way analysis of variance (ANOVA) followed by Dunnet test using

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SPSS17.0 software (SPSS, Chicago, IL, USA). A value of p0.05), compared with

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the controls at different exposure concentrations (Fig. 6A).

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Interestingly, we found significant positive correlation among DNA damage

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parameters and BER pathway gene expressions (Table 4). For example, percentage

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tail DNA revealed significant positive correlated with OGG1 (r = 0.92), CREB1 (r =

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0.99) and UNG (r = 0.97). Similarly, other DNA damage parameters (tail length, tail

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moment and olive tail movement) also expressed significant positive correlations (p
0.05). Moreover, using

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DNA damage as predictor of gene expressions, revealed significantly higher linear

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regression coefficients (r2) in the following order: OGG1 (0.965) > UNG (0.962) >

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CREB1 (0.883) > APEX1 (0.578) > POLB (0.549). These statistical interpretations

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implied that the gene expression of BER pathway was an attribution of the DNA

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damage in HEK293T cells, caused by GO exposure.

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In zebrafish embryos, the transcriptional level of the apex1 gene was

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significantly up-regulated by 1.57 folds and 1.31 folds after exposure to 25 and 50

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µg/mL of GO, respectively (Fig. 6B). When exposed to 50 µg/mL GO, the expression

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levels of ogg1, polb, and creb1 were also significantly augmented by 1.36 folds, 1.47

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folds and 1.77 folds, respectively (Fig. 6B). However, for other GO concentrations,

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no obvious difference was observed for the expressions of these genes.

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4. Discussion

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Previous studies revealed that GO nanosheets have potential to induce in vivo

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and in vitro mutagenesis (Liu et al., 2013). Park et al. reported the high concentration

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of GO (50-400 µg/mL) and its derivatives could induce severe toxicity in A549 cells

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(Park et al., 2015). Nguyen et al. found that higher doses of GO had mild cytotoxic

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effects on Caco-2 cells, but no toxicity was observed in the selected bacteria after

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exposure to GO (10-500 µg/mL) for 24 h (Nguyen et al., 2015). Chatterjee et al.

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found graphene-family nanomaterials (GFNs) caused DNA damage to human

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bronchial epithelial cells(Chatterjee et al., 2014). In accordance with these findings,

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our results showed that GO could induce cytotoxic effects to HEK293T cells, and

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exposure to higher concentrations of GO (25-50 µg/mL) significantly reduced the cell

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viability (Fig 3A, 3B) and induced DNA damage to HEK293T cells (Fig 4), which

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consequently altered the gene expression levels and the factor of viscosity in cells at a

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dose-dependent manner (Fig. 5, 6).

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In the current study, the cell viability was decreased from 87.7% to 75% (p