Mitigation in Multiple Effects of Graphene Oxide ... - ACS Publications

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Environmental Science & Technology

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Mitigation in Multiple Effects of Graphene Oxide Toxicity in

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Zebrafish Embryogenesis Driven by Humic Acid

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Yuming Chen, Chaoxiu Ren, Shaohu Ouyang, Xiangang Hu*, Qixing Zhou*

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Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education) /

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Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of

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Environmental Science and Engineering, Nankai University, Tianjin 300071, China

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ABSTRACT

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Graphene oxide (GO) is a widely used carbonaceous nanomaterial. To date, the

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influence of natural organic matter (NOM) on GO toxicity in aquatic vertebrates has

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not been reported. During zebrafish embryogenesis, GO induced a significant

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hatching delay and cardiac edema. The intensive interactions of GO with the chorion

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induces damage to chorion protuberances, excessive generation of •OH, and changes

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in protein secondary structure. In contrast, humic acid (HA), a ubiquitous form of

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NOM, significantly relieved the above adverse effects. HA reduced the interactions

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between GO and the chorion, and mitigated chorion damage by regulating the

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morphology, structures and surface negative charges of GO. HA also altered the

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uptake and deposition of GO and decreased the aggregation of GO in embryonic yolk

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cells and deep layer cells. Furthermore, HA mitigated the mitochondrial damage and

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oxidative stress induced by GO. This work reveals a feasible antidotal mechanism for

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GO in the presence of NOM and avoids overestimating the risks of GO in the natural

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environment.

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INTRODUCTION

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Graphene oxide (GO), a representative graphene family nanomaterial, has attracted

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considerable attention in environmental applications such as adsorbents for

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wastewater and drinking water treatment,1 catalysts for aqueous organic dyes,2 1

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function materials for solid−phase extraction,3 and membranes for desalination,4 due

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to its excellent properties and cost−effective production in substantial amounts.5-7 The

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above applications of GO make it release into the aqueous environment inevitably.

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Both in vitro and in vivo data have shown that GO might induced toxicological effects

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(including genotoxicity and cytotoxicity), and the release of GO into the ecosystem

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undoubtedly increases its environmental risks.8-11 Some researches have mentioned on

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the toxic alterations of GO−containing materials in the presence of organic

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antioxidants (e.g. ascorbic acid and polyphenols).12-14 Natural organic matter (NOM)

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is an important component of the aquatic environment, and the interactions of

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nanomaterials with NOM will be a major determinant of their fate in the

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environment.15 Compared with ascorbic acid and polyphenols, humic acid (HA), the

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main component of NOM, contains hydrophilic groups that impart a remarkable

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ability to regulate the environmental behaviors and toxicity of nanomaterials.9

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Although little is known about the effects of HA on the toxicity of carbon nanotubes,

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HA has been shown to reduce the uptake of fullerene (C60) in Daphnia and zebrafish

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as a result of the size effect and surface charge alternation of C60.16 Similarly, the

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toxicity of CeO2 nanoparticles17 to C. elegans decreases due to HA adsorption.

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However, HA adsorption increases the suspension stability and residence times of

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TiO2 nanoparticles in the water column, thus increasing the exposure of and toxicity

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to free−swimming aquatic organisms.18 Moreover, HA acts as both a source and a

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sink for reactive oxygen species (ROS) in aqueous environments.19 Therefore, HA

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likely has the potential to significantly alter the nanotoxicity of GO in an aquatic

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environment.

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Embryogenesis is a critical phase of life, and dysfunctional embryogenesis is

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associated with diseases and adverse effects such as oxidative stress, malformation,

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organ dysfunction and death.20−23 Several studies have investigated the interactions of

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carbon−based nanomaterials with embryos.24,25 For example, zebrafish embryos

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exposed to 50 mg/L of GO show significant hatching delays, and morphological

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defects are observed in the hatched larvae.24 Similarly, multiwalled carbon nanotubes

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induce significant developmental delays and abnormalities in the brains, notochords, 2


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eyes and yolk sacs of zebrafish embryos.25 In these studies, most of the attention has

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been focused on the impact of pristine carbon−based nanomaterials on embryogenesis.

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However, the alterations and mechanisms of toxicity in the presence of NOM are

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rarely reported. The chorion is an acellular envelope that surrounds the mature eggs of

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teleostean fish and provides the first barrier against nanoparticles.26 Various studies

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proposed that nanomaterials penetrate or are adsorbed by the chorion, resulting in

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negative effects on embryogenesis.24,27 Nevertheless, the interactional mechanisms of

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the chorion with graphene family nanomaterial are not clear, especially in the

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presence of NOM.

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To evaluate nanotoxicity in a natural aquatic system, one option is to develop a

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multilevel approach for measuring the toxicity of nanomaterials.28 Herein, embryonic

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development, nanomaterial−chorion interface interactions, the uptake and

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translocation of nanomaterials, antioxidase activities, mitochondrial damage,

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alterations of the cellular ultrastructure, modification of the protein secondary

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structure and interactions of nanomaterials with HA were investigated. This work

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proposes feasible antidotal paths for teleostean embryos in the presence of HA.

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MATERIALS AND METHODS

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Materials

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GO (purity of 99%) was obtained from the Nanjing XFNANO Materials Tech Co.,

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Ltd., China, and synthesized by the classical Hummers’ method. HA (biological grade,

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extracted from lignite) was purchased from the Shanghai Hui Cheng Biological

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Technology Co., Ltd., China. 5,5−dimethyl−1−pyrroline−N−oxide (DMPO),

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5,5',6,6'−tetrachloro−1,1',3,3'−tetraethyl-imidacarbocyanine iodide (JC−1) and 2',

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7'−dichlorofluofescein-diacetate (DCFH−DA) were purchased from Sigma−Aldrich.

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Other chemical reagents were of spectral or analytical grade.

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Interactions of GO with HA

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Given that HA is easy to dissolve in alkaline environment, HA suspension (500 mg/L)

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was initially adjusted to pH 11.0 using 1 M NaOH and then magnetically stirred at

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1,200 rpm for 2 h, after which the solution was filtered through a 0.45 µm polytetra 3



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fluoroethylene membrane. A further HA solution (100 mL, 10 mg/L, adjusted to pH =

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7.4 with 1 M HCl) was then prepared and gently mixed with 0.01 g GO for 24 h

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(light/dark =14 h/10 h). Subsequently, the suspension was centrifuged (3,500 g, 30

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min) and filtered (0.2 µm polytetrafluoro−ethylene membrane) to collect the

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hybridized GO−HA after gentle washing with water. Various techniques were used to

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characterize HA, GO and GO−HA. Atomic force microscopy (AFM) images were

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obtained in tapping mode using an Agilent 5420 AFM instrument (Agilent, CA).

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Scanning electron microscopy (SEM) was conducted using a Hitachi SU8010

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microscope (Japan). Transmission electron microscopy (TEM) images were recorded

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using a Hitachi HT7700 high−resolution transmission electron microscope (Japan).

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Raman spectrometry (RS) with a 514 nm laser (Thermo Scientific, DXR) was

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performed to analyze the structures of GO and GO−HA. Fourier transform infrared

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spectroscopy (FT−IR) spectra were recorded on a Bruker Tensor 27 infrared

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spectrometer with a resolution of 2 cm−1 step from 4,000 to 400 cm−1. The ζ−potential

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and hydrodynamic diameter were obtained using dynamic light scattering with a 30

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mW 657 nm laser (ZetaPALS, Brookhaven, USA). X−ray photoelectron spectroscopy

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(XPS) measurements were performed using an Axis Ultra XPS system (Kratos) with a

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monochromatic Al KαX-ray source (1486.6eV). The spectra were analyzed using

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CasaXPS v2.3.13 software.

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Toxicological experiments

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For all of the experiments conducted in this study, zebrafish embryos (AB strains)

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were maintained as described in a previous report.29 Embryos were collected

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immediately after fertilization and sorted to remove feces and infertile eggs. Then, the

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collected embryos were exposed to HA, GO or GO−HA in 96−well plates from 2.5 h

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post−fertilization (hpf) until 72 hpf. To achieve comparable doses of carbon

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nanomaterials to those used in previous developmental toxicity studies,30 the

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suspensions ([GO] = 0 − 100 mg/L; [HA] = 0 − 100 mg/L) were prepared in E3

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medium (5 mM NaCl, 0.33 mM CaCl2, 0.17 mM KCl, 0.33 mM MgSO4, pH 7.4).

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Embryos were analyzed daily, and the survival rate, heartbeat and malformations were 4


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recorded at the end points of toxicity through light microscopy (Olympus ZL 61,

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Olympus, Japan).31 The activities of superoxide dismutase (SOD) and glutathione

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(GSH) and the contents of malondialdehyde (MDA) were analyzed as described in a

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previous report.31,32 Hydroxyl radicals (•OH) were collected using DMPO at room

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temperature (296 K) and quantified using an electron paramagnetic resonance (EPR)

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spectrometer (Magnettech MiniScope 400, Germany).33 The oxygen concentrations in

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the chorion space were measured using a Unisense oxygen microsensor.29 The

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contents of biochemical constituents of proteins and DNA were recorded based on

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FT−IR spectra (Bruker Tensor 27, Germany).34 The generation of ROS and the

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alteration of the mitochondrial membrane potential loss were measured through

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DCFH−DA 35 and JC−1 fluorescence staining,36 respectively, using a fluorescence

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microscope (Olympus X71; Olympus, Japan). TEM (Hitachi HT7700, Japan) was

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employed to investigate the cellular ultrastructure and chorionic variations, and the

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suspected GO-containing regions in the TEM images were confirmed using a RS

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(Thermo Scientific, DXR) with a 780 nm laser was used to analyze GO. The details

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are presented in the Supporting Information. Sorption of GO on plastic and glass

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wells, uptake of GO in vivo, protein carbonyls and 8-hydroxy-2-deoxy-guanosine

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(8−OHdG) measurements were provided in Supporting Information.

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Data analyses

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TEM and fluorescence microscopy images embyos were analyzed using Image J

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(Collins 2007). The FT−IR spectra were analyzed with Origin 8.5 and Peak Fit_ v4.12

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software. IBM SPSS 22.0 statistical software was used for statistical analyses. Each

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experiment was performed in triplicate or more, unless otherwise noted. One−way

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analysis of variance with Dunnett’s test was employed to analyze the significance

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level at p < 0.05. Data are presented as the means ± standard deviation (SD) unless

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otherwise noted.

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RESULTS 5



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Characteristics of GO and HA

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Atomic force microscopy imaging demonstrated that the thickness of GO nanosheets

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were approximately 0.8−1.2 nm, with sharp zigzag edges being observed (Figure S1).

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SEM and TEM images, the hydrodynamic diameter, Zeta potential, surface chemistry

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and RS spectra are presented in the section “Interactions between GO and HA”. HA

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contains 77.85% C1s, 20.49% O1s and 1.66% N1s, as shown in Figure S2. The TEM

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and AFM images of Figure S3 show that the diameters of HA range from

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approximately 2 to 12 nm (centered on approximately 7 nm). Table S1 and Figure

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S17 demonstrate the functional groups and chemical bonds HA involve C=C, C-O,

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C-N, C-C, O-C=O, C-H and O-H.

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HA alleviated GO toxicity

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The effects of GO with and without HA on the development of zebrafish embryos

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were studied, as shown in Figures S4-S6. In the control groups, the hatching rate,

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pericardial edema and heart beat were 78.0 ± 4.7%, 4.8 ± 1.5% and 160.0 ± 9.0%,

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respectively. Compared with the control, HA only (0.01 – 100 mg/L) did not induce

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significant adverse effects on embryonic development. However, 100 mg/L GO

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(GO100) did induce significant toxicity on the embryos compared with the control (p

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< 0.05), and the hatching rate, pericardial edema and heart beat shifted to 37.0 ± 5.0%,

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34.8 ± 11.7% and 197.0 ± 14.0 beat/min from 78.0 ± 4.7%, 4.8 ± 1.5% and 160.0 ±

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9.0 beat/min, respectively. In contrast, in the presence of 10 mg/L HA, the hatching

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rate, pericardial edema and heart beat were recovered to 70.0 ± 8.0%, 7.6 ± 2.3% and

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168.0 ± 11.0%, respectively, which indicate that the adverse effects of GO100 on the

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development of zebrafish embryos were markedly mitigated (p < 0.05). Furthermore,

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Figure S7 shows that neither GO or GO-HA has a significant sorption on the plastic

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and glass wells (p > 0.05), verifying that GO toxicity is mitigated by HA and is not an

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artifact of increased sorption to plastic and glass walls.

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Effects of HA on interactions between GO and the chorion 6


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The chorion, as the first barrier against external risk, directly contacts nanoparticles.17

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Therefore, the interactions between GO and the chorion in the absence and presence

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of HA were detected using TEM and FT−IR. GO adhered to and enveloped the

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chorion, forming a layer with a thickness ranging from approximately 50 to 600 nm in

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the absence of HA and from approximately 800 to 1,200 nm in the presence of HA

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(Figure 1a and b). The ratios of the length of junction lines between chorion and GO

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to the overall length of chorion were measured to analyze the interactional interface

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between the chorion and nanomaterials in TEM images. The ratio of interactional

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interface was 86.7% in the absence of HA, whereas it significantly decreased to

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26.4% in the presence of HA (p < 0.05) (Figure 1e). In Figure 1c, it can be seen that

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the width of the electron−dense structure between the chorions and GO was

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approximately 20 nm, as indicated by the red arrows. However, the electron−dense

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structure was not obvious between the chorions and GO−HA (Figure 1d).

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Furthermore, the oxygen concentration in the chorion space was analyzed using a

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microsensor (Figure 1f). In the control sample, the oxygen concentration near the

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chorion was 269 µM and gradually decreased to 266 µM in the chorion space when

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the sensor was inserted to a depth of 250 µm, near the embryo. Similar to the control

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results, the oxygen concentrations in the GO−HA groups decreased slightly, from 270

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to 264 µM, with increasing depth. In the embryos exposed to 100 mg/L GO, the

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oxygen concentration decreased significantly, with concentrations of 233 and 227 µM

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being recorded at depths of 100 and 250 µm, respectively. These data demonstrate

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that the tight envelopment of the chorion by GO produced a hypoxic environment in

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the chorion. Conversely, in the presence of HA, the chorion was oxygenated and did

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not exhibit remarkable hypoxic phenomena. The above results suggest that the

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interactions between the chorion and GO are more intensive than those between the

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chorion and GO−HA.

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Chorion damage

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The •OH near the chorion was trapped by DMPO and determined via EPR, as shown

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in Figure 2a. There was an intensive •OH signal in the samples treated with 7



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nanomaterials, whereas the signal intensity decreased significantly in the presence of

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HA, suggesting that HA reduced the generation of •OH. In Figure 2b, it can be seen

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that the protuberances on the chorion surface were irregular and misshapen in the

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GO100−treated samples, and the relative number decreased to 42.0% of the number

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observed in untreated samples. In contrast, the protuberances were inerratic and intact

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in the presence of HA, and the number observed in untreated samples increased to

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84.2% (Figure 2 b). These data suggest that the interactions between GO and the

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chorion trigger the generation of •OH and result in chorion damage, but these effects

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are mitigated by HA. FT−IR spectroscopy is a powerful tool for determining

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biochemical constituents, protein patterns and secondary structures.37−39 Table S2 lists

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the tentative assignments of the various FT−IR bands according to previous

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reports.34,40 As shown in Figure 2d, the percentages of α−helixes, β−sheets, random

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coils, and turns & bends in the chorions were 15.7 ± 2.3%, 52.6 ± 4.4%, 16.1 ± 1.5%

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and 15.6 ± 1.3%, respectively, in the control; and those protein secondary structures

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were not interfered significantly by 10 mg/L HA (p > 0.05). Upon GO exposure, the

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percentages of both β−sheets and turns & bends increased to 67.3 ± 5.2% and 24.2 ±

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1.7%, respectively; while those of α−helixes decreased to 8.5 ± 1.1% and random

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coils disappeared completely. However, the alterations of the protein secondary

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structure recovered and did not significant difference compared with the control in the

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presence of HA (p > 0.05), as shown in Figure 2d and S8. The ratios of the bands at

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1,543 cm−1 (amideⅡ) and 1,681 cm−1 (amideⅠ) (I1543/I1681) are linked to the

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composition of the overall protein pattern.38 The I1543/I1681 ratios were 0.97, 0.60 and

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1.05 in the control, GO and GO−HA groups, respectively, as shown in Figure S9.

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These results confirm that HA reduced the chorion damage induced by GO.

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Uptake and translocation of nanomaterials

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The uptake and translocation of GO and GO−HA were detected using TEM with RS

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(the specific D and G bands of GO in embryos were observed), as shown in Figure 3.

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Both GO1 (1 mg/L GO) and GO1−HA (1 mg/L GO with 0.1 mg/L HA) penetrated

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the chorion. However, more GO−HA than GO entered the chorion through the 8


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chorion pores, which was confirmed furthermore by the results of the real−time

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monitoring of FITC−labeled GO using a laser scanning confocal microscopy, as

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shown in Figure S10. Compared with the control shown in Figure 3d, there were

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obvious dark deposits in the yolk cells and deep layer cells of the embryos in the

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GO−treated samples, as indicated by the red arrows in Figure 3e, especially for the

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inserted image in Figure 3e. In the GO−HA treated samples, the size of these dark

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deposits became smaller, and they were mainly located in the epithelial enveloping

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layer cells of the embryos, as can be observed in Figure 3f. As shown in Figure 3g,

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the remarkable G and D bands observed via RS confirmed that the dark deposits were

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GO. In addition, the overall protein pattern of the chorion varied significantly when

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embryos were incubated with GO or at 4.0 °C (Figure S9, S11 and S12), suggesting

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that the protein pattern of the chorion could be altered by nanomaterial stimulation

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and a low temperature.

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Effects of HA on the mitochondrial toxicity induced by GO

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The mitochondrial toxicity induced by GO and GO−HA was investigated via TEM, as

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shown in Figure S13a. In embryonic cells, the mitochondria were dispersed regularly

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and showed intact structures in the control and GO100−HA groups. However, the

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mitochondria became swollen and loose, and the integrity of the membrane and

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cristae was damaged under GO100 exposure. These results demonstrate that HA can

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reduce the mitochondrial toxicity of GO. Furthermore, JC−1 staining confirmed this

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hypothesis, as shown in Figure S13 b and c. The images from the control and

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GO100−HA groups were dominated by red fluorescence (healthy mitochondria), and

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the corresponding ratios of the intensity at 590 nm to that at 520 nm (I590/I520) were

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6.7 and 6.3, respectively. In contrast, the embryos exposed to GO100 exhibited strong

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green fluorescence (damaged mitochondria), and the I590/I520 ratio decreased to 1.8.

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The above results suggest that HA mitigates the mitochondrial toxicity induced by

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GO.

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Effects of HA on the oxidative stress induced by GO 9



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Compared with the control, 10 mg/L HA did not significantly alter SOD, GSH, MDA

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or ROS levels over 72 hpf, as shown in Figure S14. GO100 significantly increased

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GSH, MDA and ROS levels and inhibited SOD activity. In contrast, GO−HA

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triggered low oxidative stress, consistent with the FT−IR results. As shown in Figure

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S15, the peak of −CH2 at 2,853 cm-1 in GO−treated embryos was decreased compared

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with the control and GO−HA groups, suggesting that long−chain structures were

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cracked due to GO exposure. The peaks at both 1,681 and 1,025 cm-1 were weakened,

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implying the existence of protein and DNA damage, respectively, in GO−treated

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samples, which supported by a significant increase of carbonyl protein and 8−OHdG,

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respectively (p < 0.05) (Figure S16). Conversely, HA reduced the lipid, protein and

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DNA damage induced by GO in embryos.

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Interactions between GO and HA

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Compared with the GO spectrum, the C=C groups of GO−HA exhibited a blue shift of

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approximately 40 cm−1 (from1,608 to 1,645 cm-1), and the hydroxyl peak at 1,396

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cm-1 became weak, as shown in Figure S17. The FT−IR spectra suggested that HA

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may localize to the sp2 plane of GO through π−π interactions, in agreement with the

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proposals of Hu9 and Yang41 for graphene and few−layer reduced graphene oxide,

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respectively. To further analyze the interactions between GO and HA, TEM, SEM and

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AFM were performed. As illustrated in Figures 4a, 4b, S1 and S18, GO sheets were

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smooth and transparent, whereas GO−HA presented black spots and particles on the

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nanosheets in the TEM and SEM images, respectively. Moreover, the edges of the GO

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sheets become smooth and the thickness shifted to 2.0 − 8.9 nm. These data

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confirming the immobilization of HA on the surface of GO sheets. In the RS spectra

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shown in Figure 4c, the G and D bands reflect the sp2 (1,358 cm-1) and sp3 (1695 cm-1)

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carbon systems of GO, respectively.29 The ratios of the D to G band intensity were

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0.88 ± 0.03 and 0.76 ± 0.02 for GO and GO−HA, respectively, suggesting that the

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adsorption of HA significantly reduced the disordered structure of GO (p < 0.05). The

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ζ−potentials (dispersity) of GO and GO−HA were evaluated by performing dynamic

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light scattering, as shown in Figure 4d. The ζ−potentials of GO and GO−HA 10


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decreased with the pH, demonstrating that the surfaces of both materials showed

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negative charges at pH 2.0 − 12.0 in E3 solution. The results regarding the ζ−potential

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also indicated that GO−HA exhibited more negative charges and was more dispersive

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than GO. The size distribution results are presented in Figure 4e. Initially, the size

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distributions of GO and GO−HA were 192.8 − 531.25 nm (mean size, 323.2 ± 23.7

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nm) and 220.2 − 481.4 nm (mean size, 356.1 ± 29.1 nm), respectively. After 24 h, the

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mean sizes of GO and GO−HA had increased to 825.8 and 394.4 nm, respectively.

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The above results confirm that HA enhanced the dispersity of GO by increasing its

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surface negative charges.

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DISCUSSION

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To explore the effects of HA on GO nanotoxicity, the interactions of GO/GO−HA

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with the chorion interface, alterations of chorionic proteins and uptake and deposition

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of nanomaterials as well as mitochondrial toxicity and oxidative stress were

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investigated. The tested concentrations (0.01 − 100 mg/L) of HA are environmentally

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relevant.30,42 The presented work indicated that HA greatly reduced the potential risk

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of GO. The zebrafish chorion, which is composed of glycosylated proteins,43 protects

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embryos and serves as the first barrier against nanoparticles.26 In the present study,

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GO was observed to adhere tightly to the chorion surface, with an interactional

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interface of up to 86.7% being observed, which blocked the chorion pores and

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resulted in a hypoxic microenvironment in the chorionic space. Hypoxia induces

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ischemia, hatching delays and malformations during zebrafish embryogenesis.44−46

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ROS is not the only factor for nanotoxicity, and the envelopment of GO on the surface

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of the organisms also were found in bacteria47 and spermatozoa.48 Hence, trapping

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embryo within aggregating GO sheets for isolation from environment is suggested a

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possible mechanism of GO toxicity to organisms.29 However, in the presence of HA,

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the interactional interface of GO with the chorion became looser and was markedly

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decreased, and the oxygen concentration in the chorionic space recovered to a normal

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level. Accordingly, the hatching rate increased, and the incidence of pericardial edema

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decreased in the embryos incubated with GO−HA. Furthermore, the protein 11



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secondary structure of the chorion was modified significantly in the GO−treated

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embryos. The increased β–sheet structure and decreased α−helical structure observed

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in these embryos were similar to the adverse effects of lead,49 cyanide50 and

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radiation51 exposure. Earlier work suggested that all of the constituent amino acid side

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chains in proteins are susceptible to free radicals, and their secondary structures may

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be modified due to attack by free radicals.52,53 In the present study, the generated •OH

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might have attacked the side−chain amino acids of proteins, resulting in the

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modification of protein secondary structures. Moreover, the g−value of GO was

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2.0031, similar to that of free electrons (2.0023), suggesting that GO has the potential

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to trigger ROS formation due to carbon defects such as dangling bonds, turns and

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kinks on the edges or inner plane.54 In contrast, in the presence of HA, the generation

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of •OH and the interactional interface of GO−HA with the chorions were markedly

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decreased. Accordingly, the chorionic protein secondary structure recovered in the

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embryos exposed to GO−HA. In addition, the chorion protuberances were destroyed

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by GO and recovered in the presence of HA. Taken together, these results indicate

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that HA markedly reduces the chorion damage induced by GO.

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The natural pore size in the chorion ranges from approximately 0.5 to 1 µm, which

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is sufficiently large to allow many nanoparticles to enter the chorionic sac.23,55 Given

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the sharp edges in TEM images and excellent mechanical properties of GO, GO easily

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entered the chorion through the pores and by penetrating the biological membrane,

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which has been widely proposed.7 Following HA immobilization, the thickness of GO

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became greater, and the zigzag edge disappeared with an enhanced folding; and

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membrane penetration was mitigated, resulting in the uptake of GO−HA mainly

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through the chorion pores. In addition, the chorionic damage induced by GO, such as

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the modification of protein secondary structures and the collapse of protuberances,

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facilitated the uptake of nanomaterials in the chorionic sac. In contrast, less GO

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entered the chorionic sac in the presence of HA, as shown by TEM, RS and confocal

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microscopy. These data imply that chorions may have the potential to control the

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uptake of GO and GO−HA. To check the possibility that GO-HA sorption to plastic

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wells, the sorption of GO-HA to plastic wells are investigated. Figure S7 supports that 12


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there on obvious sorption of GO-HA to plastic wells, demonstrating that sorption of

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GO-HA to plastic wells has not accounted for decreased embryonic uptake of GO, and

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the chorion controls the uptake of GO. A recent report 56 found that beyond serving as

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a physical barrier, the chorion is a chemically reactive membrane that controls the in

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situ speciation of silver, supporting our proposal. Moreover, the chorion might

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respond to external stimuli via variations of the overall protein pattern, which might

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involve a biochemical process, although the underlying mechanisms are not clear.

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However, these data indicated that HA can regulate the morphology and uptake of GO,

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enhance the protective effect of the chorion and reduce the toxicity of GO during

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zebrafish embryogenesis.

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After the nanomaterials entered the embryos, GO and GO−HA displayed different

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deposition patterns, as shown in Figure 3. The aggregation of GO was obvious in the

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yolk cells and deep layer cells of the embryos. In contrast, the GO−HA deposits were

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smaller and fewer and were mainly located in epithelial enveloping layer cells, which

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play a protective role and inhibit the exclusive generation of periderm.57,58 Compared

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with epithelial enveloping layer cells, both yolk cells and deep layer cells play

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important roles in embryogenesis. The former supply nutrition and mediate cell

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proliferation and differentiation in early stages, and the latter are the primary regions

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of embryogenesis.58,59 These data indicated that the presence of HA caused GO to

377

translocate to relatively unimportant tissues, consistent with the HA−mediated

378

enhancement of graphene storage in wheat vacuoles.9 The translocation mechanism

379

may involve the shape and hydrophilia of nanomaterials, as GO exhibits better

380

lipophilic characteristics and sharp zigzag edges, thus potentially allowing it to

381

penetrate the cytomembrane and aggregate in the cytoplasm more readily than

382

GO−HA.

383

Both the hypoxia from the envelopment of GO and physical damage with the

384

zigzag edges may result in mitochondrial impairment.29,60-62 In fact, 100 mg/L GO did

385

induce a significant mitochondrial impairment, whereas HA significantly relieved the

386

above adverse effects. In addition, maintaining a stable dispersion of graphene based

387

materials is recommended to minimize their toxicity.63 ζ−potential showed that 13



388

GO−HA exhibited more negative charges and was more stable than GO under the

389

physiological relevant pH range of 6 − 10. Therefore, GO−HA might also reduce the

390

toxicity of GO during zebrafish embryogenesis by increasing negative charges on the

391

surface and decreasing its contact with embryo, mitochondria and other cell structures

392

via charge repulsion.

393

Mitochondrial damage is linked to the generation of free radicals and the

394

enhancement of oxidative stress, which has been proposed as the primary mechanism

395

of nanotoxicity.64,65 In the GO exposure group, the generation of ROS in embryos was

396

increased significantly compared with the control and GO−HA groups (p < 0.05),

397

which may disturb the dynamic equilibrium of free radicals maintained by enzymatic

398

and non-enzymatic defensive systems. Consistently, GO markedly induced increased

399

SOD levels and decreased GSH levels compared with the control and GO−HA groups

400

(p < 0.05). Furthermore, the FT−IR results demonstrated that both DNA and proteins

401

were damaged in GO−treated embryos, which consistent with the data of protein

402

carbonylation and 8−HOdG adducts. Recent works showed that GO trigger the

403

8−OHdG modification in DNA54 in Danio rerio which consistent to our results.

404

However, in the presence of HA, the above negative effects induced by GO were

405

relieved, and the activities of the tested antioxidant enzymes recovered. These data

406

indicates that HA decrease the oxidative stress induced by GO via recovery of

407

mitochondrial impairment.

408 409

ENVIRONMENTAL IMPLICATIONS

410

This work found that HA altered the surface morphology of GO and increased its

411

stability under the physiology and environment relevant pH range of 6 – 10. HA

412

recovered the oxygen contents in the intra–chorionic microenvironment of embryos

413

via mitigating the interactions of GO with the chorions. Furthermore, HA influenced

414

the uptake and translocation of GO, indicating a detoxification for teleostean

415

embryogenesis. HA reduced the damage of mitochondria and morphology, and the

416

oxidative stress of embryos induced by GO. The above results proposed that HA

417

altered the fate and biological responses of GO. Given the wide distribution of HA in 14


Page 14 of 29

Page 15 of 29


418

natural water, HA could be employed as a natural antidote of GO. This work also

419

implies that the previous studies of nanomaterial ecological risks without HA

420

exposure should be reconsidered.

421 422

ASSOCIATED CONTENT

423

Supporting Information Available

424

Additional figures and table regarding the characteristics of GO and HA, uptake in

425

vivo, development toxicity, the contents of biochemical constituents, mitochondria

426

toxicity and oxidative stress. This information is available free of charge via the

427

Internet at http://pubs.acs.org/.

428 429 430

AUTHOR INFORMATION

431

Corresponding author

432

* E-mail: [email protected] (Q.Z.); [email protected] (X.H.). Phone:

433

+86-022-23507800; fax: +86-022-66229562.

434 435

Notes

436

The authors declare no competing financial interest.

437 438

ACKNOWLEDGMENTS

439

This work was financially supported by the Ministry of Education of China as an

440

innovative team project (grant No. IRT 13024), the National Natural Science

441

Foundation of China (grant Nos. 31170473, U1133006, 21307061 and 21407085), the

442

Tianjin Natural Science Foundation (grant No. 14JCQNJC08900), the Specialized

443

Research Fund for the Doctoral Program of Higher Education of China (grant No.

444

2013003112016) and the Postdoctoral Science Foundation of China (grant No.

445

2014M550138).

446 447

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Figure Legends

660 661

Figure 1. Interactions between the chorion and GO/GO−HA. (a−d), Transmission

662

electron microscopy images. (c) and (d) are enlargements of (a) and (b), respectively

663

(n = 3). Green arrows indicate the interactional interface of the chorion with

664

GO/GO−HA. Red arrows indicate the electron density between the chorion and the

665

nanomaterials. In, inside space of chorion; Po, pore of chorion; Out, outside

666

environment of chorion. (e) The ratios of the length of junction lines between chorion

667

and GO to the overall length of chorion were measured to analyze the interactional

668

interface between the chorion and nanomaterials in TEM images. Six TEM images

669

were analyzed from three embryos in each treated group. *, significant at the p < 0.05

670

level. (f) The concentration of oxygen in the chorion. *, significant at the p < 0.05

671

level with n = 6. GO, 100 mg/L graphene oxide; HA, 10 mg/L humic acid.

672 673

Figure 2. Effects of HA on the chorion damage induced by GO. (a) Generation of

674

hydroxyl radicals determined from electron paramagnetic resonance spectra. S I, the

675

signal intensity of the electron paramagnetic resonance spectra. Each treatment group

676

contained 40 embryos. (b) TEM images of GO/GO−HA interacting with the chorion.

677

(c) Semi−quantitative analysis of chorion protuberances. The relative number is the

678

ratio of the number of protuberances in the nanomaterial−treated groups to that in the

679

control groups. Green arrows indicate chorion protuberances. Six TEM images were

680

analyzed from three embryos in each treated group. *, significant at the p < 0.05 level.

681

(d) Variations in chorion protein secondary structure. There were 25 embryos in each

682

treatment groups. *, significant at the p < 0.05 level, n = 3. Cho, chorion; GO, 100

683

mg/L graphene oxide. HA, 10 mg/L humic acid.

684 685

Figure 3. Effects of HA on the uptake and translocation of GO in zebrafish embryos.

686

(a) Transmission electron microscopy images of chorions exposed to GO (n = 3). (b)

687

Transmission electron microscopy images of chorions exposed to GO−HA (n = 3). (c)

688

Ratio of the area of dark deposits to total chorion area. Six TEM images were 23



689

anlyazed from three embryos in each treatment group. *, significant at the p < 0.05

690

level. (d), (e) and (f) Transmission electron microscopy images of translocation of GO

691

in zebrafish embryos (n = 3). (g) Raman spectra of embryos (n = 3). Green lines

692

indicate the regions of the pores on the chorions. Red arrows indicate nanomaterials.

693

In, the inside space of chorions; Por, the pores of chorions; Out, the outside

694

environment of chorions; GO, graphene oxide at 1 mg/L; HA, humic acid at 0.1 mg/L.

695

Scale bars, 1 µm.

696 697

Figure 4. Interactions between GO and HA. (a) Transmission electron microscopy

698

image of GO−HA. (b) Scanning electron microscopy images of GO and GO−HA. (c)

699

Atomic force microscopy image of GO−HA. (d) Raman spectra of GO and GO−HA.

700

*, significant at the p < 0.05 level (n = 3). (e) Variation of the ζ-potential versus pH (n

701

= 3). The blue scatter indicates the threshold for good colloidal dispersion at −30 mV.

702

(f) Variation of the size distributions of GO and GO−HA at 0 h and 24 h (n = 3).

703

Green arrows indicate HA. GO, graphene oxide at 100 mg/L; HA, humic acid at 10

704

mg/L.

705 706 707 708 709 710 711 712 713 714 715 716 717 718 24


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Figures

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721 722

Figure 1. ---------------------

723

25



724 725

Figure 2. ---------------------

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Figure 3. ---------------------

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27



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Figure 4. ---------------------

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Mitigation in Multiple Effects of Graphene Oxide ... - ACS Publications

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