Study of the Persistence of the Phytotoxicity Induced by Graphene

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Ecotoxicology and Human Environmental Health

Study of the Persistence of the Phytotoxicity Induced by Graphene Oxide Quantum Dots and of the Specific Molecular Mechanisms by Integrating Omics and Regular Analyses Weilu Kang, Xiaokang Li, Anqi Sun, Fubo Yu, and Xiangang Hu Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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Environmental Science & Technology

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Study of the Persistence of the Phytotoxicity Induced by Graphene Oxide

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Quantum Dots and of the Specific Molecular Mechanisms by Integrating Omics

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and Regular Analyses

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Weilu Kang, Xiaokang Li, Anqi Sun, Fubo Yu, Xiangang Hu*

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Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

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Education)/Tianjin Key Laboratory of Environmental Remediation and Pollution

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Control, College of Environmental Science and Engineering, Nankai University,

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Tianjin 300350, China

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Corresponding authors: Xiangang Hu Email: [email protected]

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Fax, 0086-022-23507800

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Tel, 0086-022-23507800

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ACS Paragon Plus Environment


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ABSTRACT

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Although increasing attention has been paid to the nanotoxicity of graphene oxide

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quantum dots (GOQDs) due to their broad range of applications, the persistence and

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recoverability associated with GOQDs had been widely ignored. Interestingly,

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stress-response hormesis for algal growth was observed for Chlorella vulgaris as a

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single-celled model organism. Few physiological parameters, such as algal density,

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plasmolysis and levels of reactive oxygen species, exhibited facile recovery. In

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contrast, the effects on chlorophyll a levels, permeability and starch grain

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accumulation exhibited persistent toxicity. In the exposure stage, the downregulation

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of genes related to unsaturated fatty acid biosynthesis, carotenoid biosynthesis,

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phenylpropanoid biosynthesis and binding contributed to toxic effects on

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photosynthesis. In the recovery stage, downregulation of genes related to the

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cis-Golgi network, photosystem I, photosynthetic membrane and thylakoid was linked

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to the persistence of toxic effects on photosynthesis. The upregulated galactose

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metabolism and downregulated aminoacyl-tRNA biosynthesis also indicated toxicity

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persistence in the recovery stage. The downregulation and upregulation of

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phenylalanine metabolism in the exposure and recovery stages, respectively, reflected

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the tolerance of the algae to GOQDs. The present study highlights the importance of

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studying nanotoxicity by elucidation of stress and recovery patterns with

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metabolomics and transcriptomics.

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KEYWORDS: Quantum dots; Phytotoxicity; Nanotoxicology; Chlorella vulgaris;

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Metabolomics; Transcriptomics 2


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INTRODUCTION

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Graphene oxide quantum dots (GOQDs) are a type of graphene-based nanomaterial

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with unique properties, which are associated with both graphene oxide (GO) and

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quantum dots.1, 2 GOQDs have been widely applied in various fields due to their

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excellent thermal properties, electrical conductivity and photochemical properties, for

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example, GOQDs have been used in in sensors, imaging, energy storage and catalytic

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devices.3, 4 Given the intentional or unintentional release of nanoparticles into the

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environment during their life cycle (e.g., during fabrication, use and disposal), the

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biological effects of these materials have attracted much attention.5-7 There have been

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some reports on the interactions of quantum dots with different model organisms.8-10

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Some studies have suggested that GOQDs cause distinct growth inhibition, oxidative

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stress, damage to cellular ultrastructures and defects in photosynthesis.11 In contrast, a

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few studies have concluded that GOQDs exhibit high biocompatibility and that

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organisms exhibit high tolerance to GOQD exposure,12 indicating the possibility of

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toxicity alleviation. Examination of the persistence of toxicity and recovery of

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organisms from the toxic effects of pollutants is essential for scientific assessment of

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the ecological risks of xenobiotics.13 However, the recovery and persistence patterns

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associated with GOQDs in biological systems are unclear. Compared to research on

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the biological stress response to nanoparticles, studies on defense, recovery and

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persistence mechanisms remain in the infancy stage.14 There is an emerging need to

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evaluate the patterns and mechanisms underlying the recovery from and persistence of

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adverse effects induced by GOQDs and to fill the knowledge gaps currently present in 3



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nanotoxicological studies.

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Most environmental studies test only a few metabolites or genes as biological

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endpoints to examine toxicological mechanisms.10, 15 However, alterations in only a

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few metabolites or genes may miss important biological information. Metabolomics

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and transcriptomics, as untargeted measurements, are sensitive techniques that can

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provide detailed insights into the mechanisms underlying biological alterations.16, 17

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Integrated omics techniques, such as the use of both metabolomics and

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transcriptomics, can lead to comprehensive and definitive evaluation of nanotoxicity

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better than the use of a single omics technique.18 Moreover, omics analysis should not

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be seen as merely the generation of lists of genes, proteins, or metabolites by using

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omics platforms.19, 20 Herein, the association between omics and conventional

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techniques will be studied to explore the toxicological mechanisms underlying the

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occurrence and termination of nanotoxicity.

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Chlorella vulgaris (C. vulgaris), a freshwater algal species, serves as a model

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organism for toxicity assessment.21 Herein, the interactions of nanomaterials with

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algal cells, and the associated adverse effects (e.g., morphological damage, oxidative

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stress and growth viability), will be investigated under GOQD exposure and after

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recovery. Then, by integrating omics (metabolomics and transcriptomics) analyses of

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algal responses with conventional methods (e.g., analysis of algal growth, oxidative

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stress, ultrastructure, photosynthesis and nanoparticle uptake), the specific

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toxicological mechanisms will be explored under GOQD exposure and after recovery.

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The present study provides insights into the persistence of, and recoverability of 4


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organisms from, the nanotoxicity associated with GOQDs and provides new routes for

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scientific evaluation of nanotoxicity.

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Materials and Methods

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Characterization of GOQDs

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GOQDs (product number XF042) were obtained from Nanjing XFNANO Materials

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Tech Co., Ltd., China. Details regarding nanomaterial characterization were described

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in our recent study.22 To study the morphology of the nanomaterial, field-emission

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transmission electron microscopy (TEM) and atomic force microscopy (AFM) were

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conducted with a JEM-2010 FEF (JEOL, Japan) and a Nanoscope IV (VEECO,

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USA), respectively. Raman spectra were obtained on a micro-Raman spectroscopy

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system (RTS-HiR-AM, TEO, China) using an excitation wavelength of 532 nm at

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ambient temperature. The surface functional groups of the GOQDs were analyzed via

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Fourier-transform infrared (FTIR) spectroscopy (Bruker Tensor 27, Germany) at a

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resolution of 4 cm−1 from 400 to 4000 cm−1. The hydrodynamic diameters and zeta

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potentials were determined by dynamic light scattering measurement on a ZetaSizer

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Nano instrument equipped with a 30-mW 635-nm laser (BI-200SM, Brookhaven,

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USA).23

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Exposure and Recovery Experiments with C. vulgaris

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C. vulgaris (product number FACH13-8) and culture medium (product number

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BG-11) were obtained from the Freshwater Algae Culture Collection at the Institute

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of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. Due to the short 5



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history of GOQD use, the current environmental concentrations should be very low,

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except for locations of GOQD fabrication and application. For other engineered

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nanoparticles (ENPs) with long-term applications, such as SiO2 nanoparticles, Ag

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nanoparticles and TiO2 nanoparticles, environmental concentrations have reached ppb

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or ppm levels.24 Herein, GOQDs were tested at 0.1-10 mg/L to investigate the

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recoverability of the organism under phytotoxic conditions and to compare the

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underlying molecular mechanisms to the reported literature.25,26 GOQDs were

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prepared in BG-11 culture medium at a pH of 7.2. The initial density of the algal cells

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was approximately 3 × 105 cells/mL. The algal suspensions were shaken once per 8 h

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and placed in an artificial light growth chamber (light cycle, 14 h irradiation and 10 h

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dark) with 80% humidity at 26 °C. Toxicity experiments were carried out using a 96-h

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period of exposure, in accordance with the conditions commonly used for studies of

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algal growth inhibition (algal growth would be inhibited at exposure periods greater

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than 96-h due to the high density of algal cells).27,28 A 96-h period of exposure was

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also recommended by the Organization for Economic Cooperation and Development

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guidelines (OECD201, 2011).29

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The algal cell density at 96 h was determined using flow cytometry (CyFLOW

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Space, Partec, Germany). After 96 h of exposure to GOQDs, the algal recovery

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experiment was conducted. Before being transferred to fresh BG-11 culture medium

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without additional GOQDs, the treated algal cells were centrifuged at 3000×g for 10

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min and washed with BG-11. In the recovery experiments, the C. vulgaris cell culture

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was diluted to the initial density of the exposure experiment (3 × 105 cells/mL) using 6


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fresh medium, and then, the algal cells were cultured in fresh medium for 96 h. The

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culture conditions in the recovery experiment were the same as those in the exposure

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experiment.

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Reactive Oxygen Species and Chlorophyll Assay

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The generation of intracellular reactive oxygen species (ROS) was detected by using

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2’,7’-dichlorodihydro fluorescein diacetate (DCFH-DA) as a fluorescent probe.

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Nonfluorescent DCFH-DA entered the algal cells and, upon reacting with intracellular

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ROS, produced dichlorofluorescein (DCF), which had detectable fluorescence. Algal

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cells were collected at 96 h, centrifuged (9000×g, 5 min) and washed with PBS. Then,

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the algal cells were incubated with DCFH-DA (10 mM) in the dark at 25 °C for 30

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min and rinsed again with PBS. The fluorescence intensity of DCF was measured

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using a fluorescence spectrophotometer (LS55, PerkinElmer, USA) with an excitation

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wavelength of 485 nm and an emission wavelength of 530 nm. The chlorophyll a

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content was measured using a UV-via spectrophotometer (TU-1900, Purkinje General

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Instrument, China), as described previously in the literature.30 Five milliliters of the

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algal cell suspension was centrifuged at 9000×g for 15 min, treated with 5 mL of 95%

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methanol, and incubated in the dark. After being centrifuged again, the supernatants

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were subjected to absorbance measurements at 652 nm and 665 nm. The following

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formula was used for calculations:

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Chlorophyll a (μg/mL) = (16.52A665−8.096A652)/5

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Cell Permeability and Ultrastructure

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Algal cell suspensions (1 mL) were centrifuged at 9000×g for 5 min, washed with 7



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fresh BG-11 medium and then stained with fluorescein diacetate (FDA, 10 μg/mL) in

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the dark at 25 °C for 30 min. After staining, the cells were centrifuged (9000×g, 2

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min) and then washed using PBS. The fluorescence intensity of the cell samples was

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determined by a fluorescence microscope (LS55, PerkinElmer, USA) with an

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excitation wavelength of 485 nm and an emission wavelength of 520 nm. For TEM

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imaging, 5 mL of algal cells was centrifuged at 9000×g for 5 min and fixed with 2.5%

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glutaraldehyde for 2 h. Subsequently, the fixed cell suspensions were washed using

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fresh BG-11 medium and postfixed in 1% osmium tetroxide for 2 h. A graded ethanol

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series (30%, 50%, 70%, 80%, 90%, 95% and 100%) was used to dehydrate the

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samples, and then, the cell samples were embedded in epoxy resin. Finally, ultrathin

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sections (thickness, 90 nm) were obtained using a diamond knife (EMFC7, Leica,

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Germany) and stained with uranyl acetate and lead citrate for 15 min. The alterations

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in cellular ultrastructure and cellular uptake of GOQDs were then observed using

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TEM (HT7700, Hitachi, Japan) and Raman spectroscopy (RTS-HiR-AM, TEO,

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China), respectively.

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GOQD Uptake and Excretion

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To investigate the uptake of GOQDs into algal cells, the treated algal cells were

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further visualized using confocal laser scanning microscopy (CLSM; LSM880 with

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Airyscan, ZEISS, Germany). After 4 and 24 h of exposure, the cells in both the

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control and exposure groups (GOQDs at 10 mg/L) were collected, rinsed and

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suspended in BG-11 medium. Meanwhile, algal cells were transferred to fresh BG-11

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culture medium after 24 h of exposure and then cultured for an additional 4 h. Direct 8


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visualization was performed at an excitation wavelength of 405 nm and emission

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wavelength of 462 nm. To analyze the effect of intracellular substances on GOQDs

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stability, 50 mL of algal suspension without nanomaterial exposure was centrifuged at

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9000×g for 10 min, and the cytoplasm was extracted by sonication in an ice-water

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bath at 150 W for 30 min with PBS. The GOQDs (10 mg/L) were placed in this

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extracted cytoplasm for 4 h, and then, the fluorescence of the quantum dots was

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measured using CLSM. Untreated pristine GOQDs were used as a control.

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Comet assay

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Comet assay was conducted to detect algal DNA damage in the exposure and

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recovery stages31 using a comet assay kit (Catalog number, KGA240-50, Keygen

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Biotech, China) according to the manufacturer's instructions. The detailed procedure

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is described in the Supporting Information.

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Metabolic Analysis

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Detailed information regarding the metabolic analysis was provided in our recent

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work.32 After 96 h of exposure and recovery, the algal suspension (10 mL) was

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collected by centrifugation (9000×g, 5 min) and rapidly frozen in liquid nitrogen. A

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precooled methanol/chloroform/water (4.5 mL, v/v/v=2.5/1/1) mixture was added to

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the cell suspensions, and then, the cells were completely lysed in an ice-water bath at

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150 W for 10 min. Subsequently, the metabolites were extracted using sonication at

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200 W for 30 min in an ice-water bath, and the suspension was centrifuged at 9000×g

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for 20 min. The supernatant was collected, and the pellet was extracted using 4.5 mL

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of the mixed solution (methanol/chloroform/water, v/v/v=2.5/1/1) again. The 9



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supernatant was mixed with the aforementioned supernatant. Deionized water (1.5

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mL) was added to the total supernatant, and then, centrifugation was performed as

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described above. The lower phase was filtered through a 5-cm silica gel column and

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dried by a nitrogen stream. For the upper phase, organic solvents and water were

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removed by a nitrogen stream and lyophilization, respectively. The treated upper and

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lower phases were pooled, followed by derivatization with methoxamine

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hydrochloride (20 mg/mL, 50 μL) and N-methyl-N-(trimethylsilyl)-trifluoroacetamide

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(80 μL). The metabolites were detected using gas chromatography coupled with mass

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spectrometry (GC-MS, Agilent 6890N/5973, USA). The metabolites were identified

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using full-scan mode with a detection range of m/z 50-650, based on the National

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Institute of Standards and Technology (NIST08) mass spectrum library in

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ChemStation software.

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Transcriptomic Analysis

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Transcriptomic analysis was performed at Novogene Bioinformatics Technology Co.,

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Ltd, Tianjin, China. After 96 h of exposure, total RNA was extracted from the algal

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cells using TRIzol reagent (Life Technologies, Carlsbad, Canada). The RNA

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preparation procedures were performed as previously described.33 RNA integrity was

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determined using an RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system

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(Agilent Technologies, USA). Following the manufacturer’s instructions, 3 μg of each

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RNA sample was used as a template with random hexamer primers for cDNA

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synthesis. The sequencing library was constructed by polymerase chain

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reaction-based amplification, quantified with the Agilent Bioanalyzer 2100 system, 10


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and sequenced with the Illumina HiSeq platform (Agilent Technologies, USA).

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Clustering of the index-coded samples was performed on a cBot cluster generation

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system with a TruSeq PE Cluster Kit v3-cBot-HS. After cluster generation, the

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prepared libraries were sequenced on an Illumina HiSeq platform, and 125-bp/150-bp

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paired-end reads were generated. Analysis of differentially expressed genes (DEGs)

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was performed using the DEGseq R package (1.20.0). Validation of the

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transcriptomic analysis was performed with quantitative real-time PCR (qRT-PCR),

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and the details are provided in the Supporting Information.

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Data Analysis

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The toxicological experiments were conducted in at least triplicate in each group, and

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the results are presented as the means with standard deviations. Data analysis was

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performed using OriginPro 9.0 software. One-way analysis of variance (ANOVA)

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was employed using SPSS 18.0 to determine statistical significance. The differences

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between the two groups were assessed by p-values, and p

Study of the Persistence of the Phytotoxicity Induced by Graphene

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