Mitigation in Multiple Effects of Graphene Oxide Toxicity in Zebrafish

Page 1 of 29

Environmental Science & Technology

1

Mitigation in Multiple Effects of Graphene Oxide Toxicity in

2

Zebrafish Embryogenesis Driven by Humic Acid

3 4

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

5 6

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

7

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

8

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

9 10

ABSTRACT

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

environment.

25 26

INTRODUCTION

27

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

28

considerable attention in environmental applications such as adsorbents for

29

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

ACS Paragon Plus Environment


30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

environment.

53

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

54

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

55

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

56

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

57

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

58

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

59

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


Page 2 of 29

Page 3 of 29


60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

presence of NOM.

69

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

70

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

71

development, nanomaterial−chorion interface interactions, the uptake and

72

translocation of nanomaterials, antioxidase activities, mitochondrial damage,

73

alterations of the cellular ultrastructure, modification of the protein secondary

74

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

75

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

76 77

MATERIALS AND METHODS

78

Materials

79

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

80

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

81

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

82

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

83

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

84

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

85

Other chemical reagents were of spectral or analytical grade.

86

Interactions of GO with HA

87

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

88

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

89

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



90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

CasaXPS v2.3.13 software.

109 110

Toxicological experiments

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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


Page 4 of 29

Page 5 of 29


120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

alteration of the mitochondrial membrane potential loss were measured through

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138 139

Data analyses

140

TEM and fluorescence microscopy images embyos were analyzed using Image J

141

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

142

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

143

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

144

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

145

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

146

otherwise noted.

147 148

RESULTS 5



149

Characteristics of GO and HA

150

Atomic force microscopy imaging demonstrated that the thickness of GO nanosheets

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159 160

HA alleviated GO toxicity

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

artifact of increased sorption to plastic and glass walls.

176 177

Effects of HA on interactions between GO and the chorion 6


Page 6 of 29

Page 7 of 29


178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

chorion and GO−HA.

204 205

Chorion damage

206

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

207

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



208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232 233

Uptake and translocation of nanomaterials

234

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

235

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

236

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

237

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


Page 8 of 29

Page 9 of 29


238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

and a low temperature.

251 252

Effects of HA on the mitochondrial toxicity induced by GO

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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

265

GO.

266 267

Effects of HA on the oxidative stress induced by GO 9



268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

DNA damage induced by GO in embryos.

279 280

Interactions between GO and HA

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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


Page 10 of 29

Page 11 of 29


298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

surface negative charges.

307 308

DISCUSSION

309

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

310

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

311

of nanomaterials as well as mitochondrial toxicity and oxidative stress were

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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



328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

343

that HA markedly reduces the chorion damage induced by GO.

344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

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


Page 12 of 29

Page 13 of 29


358

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

359

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

360

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

361

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

362

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

363

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

364

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

365

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

366

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

367

zebrafish embryogenesis.

368

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

369

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

370

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

371

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

372

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

373

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

374

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

375

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

376

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

REFERENCES

448

(1) Elahe, K.; Shayessteh, D.; Shabani, H.;Mohammad, A. Dispersive solid phase 15



449

microextraction with magnetic graphene oxide as the sorbent for separation and

450

preconcentration of ultra-trace amounts of gold ions. Talanta 2015, 141, 273−278.

451

(2) Li, S. L.; Li, H.; Liu, J.; Zhang, H. L.; Yang, Y. M.; Yang, Z. Y.; Wang, L. Y.;

452

Wang, B. D. Highly efficient degradation of organic dyes by palladium nanoparticles

453

decorated on 2D magnetic reduced graphene oxide nanosheets. Dalton. T. 2015, 44,

454

9193−9199.

455

(3) Chinthakindi, S.; Purohit, A.; Singh, V.; Tak, V.; Goud, D. R.; Dubey, D.K.;

456

Pardasani, D. Iron oxide functionalized graphene nano-composite for dispersive solid

457

phase extraction of chemical warfare agents from aqueous samples. J. Chromatogr. A.

458

2015, 1394, 9−17.

459

(4) Hegab, H. M.; Zou, L. D. Graphene oxide-assisted membranes: Fabrication and

460

potential applications in desalination and water purification. J. Membrane Sci. 2015,

461

484, 95−106.

462

(5) Novoselov, K. S.; Fal'ko, V. I.; Colombo, L.; Gellert, P. R.; Schwab, M., G.; Kim,

463

K. A. Roadmap for graphene. Nature 2012, 490, 192−200.

464

(6) Bianco, A. Graphene: safe or toxic? the two faces of the medal. Angew. Chem. Int.

465

Edit. 2013, 52, 4986−97.

466

(7) Hu, X. H.; Zhou, Q. X. Health and ecosystem risks of graphene. Chem. Rev.

467

2013,113, 3815−35.

468

(8) Akhavan, O.; Ghaderi, E.; Akhavan, A. Size-dependent genotoxicity of graphene

469

nanoplatelets in human stem cells. Biomaterials 2012, 33, 8017−8025.

470

(9) Hu, X. H.; Mu, L.; Kang, J.; Lu, K. C.; Zhou, R. R.; Zhou, Q. X. Humic acid acts

471

as a natural antidote of graphene by regulating nanomaterial translocation and

472

metabolic fluxes in vivo. Environ. Sci. Technol. 2014, 48, 6919−6927.

473

(10) Akhavan, O.; Ghaderi, E.; Emamy, H.; Akhavan, F. Genotoxicity of graphene

474

nanoribbons in human mesenchymal stem cells. Carbon 2013, 54, 419−431.

475

(11) Yang, K.; Li, Y. J.; Tan, X. F.; Peng, R. ; Liu, Z. Behavior and toxicity of

476

graphene and its functionalized derivatives in biological systems. Small 2013, 9,

477

1492−1503

478

(12) Akhavan, O.; Ghaderi, E. Flash photo stimulation of human neural stem cells on 16


Page 16 of 29

Page 17 of 29


479

graphene/TiO2 heterojunction for differentiation into neurons. Nanoscale 2013, 5,

480

10316−10326.

481

(13) Lee, B. M.; Seo, Y. S.; Hur, J. Investigation of adsorptive fractionation of humic

482

acid on graphene oxide using fluorescence EEM-PARAFAC. Water Res. 2015, 7 3,

483

242−251.

484

(14) Akhavan, O.; Ghaderi, E. Differentiation of human neural stem cells into neural

485

networks on graphene nanogrids. J. Mater. Chem. B. 2013, 1, 6291−6301.

486

(15) Chowdhury, I.; Duch, M. C.; Mansukhani, N. D.; Hersam, M. C.; Bouchard, D.

487

Interactions of graphene oxide nanomaterials with natural organic matter and metal

488

oxide Surfaces. Environ. Sci. Technol. 2014, 48, 9382−9390.

489

(16) Chen, Q. Q.; Yin, D. Q.; Li, J.; Hu, X. L. The effects of humic acid on the uptake

490

and depuration of fullerene aqueous suspensions in two aquatic organisms. Environ.

491

Toxicol. Chem. 2014, 33, 1090−1097.

492

(17) Collin, B.; Oostveen, E.; Tsyusko, O. V.; Unrine, J. M. Influence of natural

493

organic matter and surface charge on the toxicity and bioaccumulation of

494

functionalized ceria nanoparticles in Caenorhabditis elegans. Environ. Sci. Technol.

495

2014, 48, 1280−1289.

496

(18) Yang, S. P.; Bar-Ilan, O.; Peterson, R. E.; Heideman, W.; Hamers, R. J.;

497

Pedersen, J.A. Influence of humic acid on titanium dioxide nanoparticle toxicity to

498

developing zebrafish. Environ. Sci. Technol. 2013, 47, 4718−4725.

499

(19) Latch, D. E.; McNeill, K. Microheterogeneity of singlet oxygen distributions in

500

irradiated humic acid solutions. Science 2006, 311, 1743−1747.

501

(20) Chen, P. H.; Hsiao, K. M.; Chou, C. C. Molecular characterization of toxicity

502

mechanism of single-walled carbon nanotubes. Biomaterials 2013, 34, 5661−5669.

503

(21) Ribas Ferreira, J. L.; Lonne, M. N.; Franca, T. A.; Maximilla, N. R.; Lugokenski,

504

T. H.; Costa, P. G.; Fillmann, G.; Antunes Soares, F. A.; de la Torre, F. R.; Monserrat,

505

J. M., Co-exposure of the organic nanomaterial fullerene C-60 with benzo a pyrene in

506

Danio rerio (zebrafish) hepatocytes: Evidence of toxicological interactions. Aquat.

507

Toxicol. 2014, 147, 76-83.

508

(22) Ilan, O. B.; Chuang, C. C.; Schwahn, D. J.; Yang, S.; Joshi, S.; Pedersen, J. A.; 17



509

Hamers, R.J.; Peterson, R. E.; Heideman, W. TiO2 Nanoparticle exposure and

510

illumination during zebrafish development: mortality at parts per billion

511

concentrations. Environ. Sci. Technol. 2013, 47, 4726–4733.

512

(23) Lee, K. J.; Nallathamby, P. D.; Browning, L. M.; Osgood, C. J.; Xu, X. H. N. In

513

vivo imaging of transport and biocompatibility of single silver nanoparticles in early

514

development of zebrafish embryos. ACS Nano 2007, 1, 133–143.

515

(24) Chen, L. Q, Hu, P. P.; Zhang, L.; Huang, S. Z.; Luo, L. F.; Huang, C. Z. Toxicity

516

of graphene oxide and multi-walled carbon nanotubes against human cells and

517

zebrafish. Sci. China Chem. 2012, 55, 2209–2216.

518

(25) Cheng J, Cheng SH. Influence of carbon nanotube length on toxicity to zebrafish

519

embryos. Int. J. Nanomed. 2012, 7, 3731–3739.

520

(26) Cheng, J. P.; Flahaut, E.; Cheng, S. H. Effect of carbon nanotubes on developing

521

zebrafish (Danio rerio) embryos. Environ. Toxicol. Chem. 2007, 26, 708−716.

522

(27) Gollavelli, G.; Ling, Y. C. Multi-functional graphene as an in vitro and in vivo

523

imaging probe. Biomaterials 2012, 33, 2532–2545.

524

(28) Galluzzi, L.; Chiarantini, L.; Pantucci, E.; Curci, R.; Merikhi, J.; Hummel, H.;

525

Bachmann, P. K.; Manuali, E.; Pezzotti, G.; Magnani, M. Development of a

526

multilevel approach for the evaluation of nanomaterials’ toxicity. Nanomedicine 2012,

527

7, 393−409.

528

(29) Chen, Y. M.; Hu, X. G.; Sun, J.; Zhou, Q. X. Specific nanotoxicity of graphene

529

oxide during zebrafish embryogenesis. Nanotoxicology 2015,

530

doi:10.3109/17435390.2015.1005032.

531

(30) Steinberg, C. E. W.; Kamara, S.; Prokhotskaya, V. Y.; Manusadzianas, L.;

532

Karasyova, T. A.; Timofeyev, M. A.; Jie, Z.; Paul, A.; Meinelt, T.; Farjalla, V. F.;

533

Matsuo, A. Y. O.; Burnison, B. K.; Menzel, R. Dissolved humic substances ecological

534

driving forces from the individual to the ecosystem level? Freshwater Biol. 2006, 51,

535

1189−1210.

536

(31) Choi, V. W. Y.; Cheng, S. H.; Yu, K. N. Radioadaptive response induced by

537

alpha-particle-induced stress communicated in vivo between zebrafish embryos.

538

Environ Sci. Technol. 2010, 44, 8829-8834. 18


Page 18 of 29

Page 19 of 29


539

(32) Usenko, C. Y.; Harper, S. L.; Tanguay, R. L. Fullerene C-60 exposure elicits an

540

oxidative stress response in embryonic zebrafish. Toxicol. Appl. Pharm. 2008, 229,

541

44−55.

542

(33) Fenoglio, I.; Aldieri, E.; Gazzano, E.; Cesano, F.; Colonna, M.; Scarano, D.;

543

Mazzucco, G.; Attanasio, A.; Yakoub, Y.; Lison, D.; Fubini, B. Thickness of

544

multiwalled carbon nanotubes affects their lung toxicity. Chem. Res. Toxicol. 2012,

545

25, 74-82.

546

(34) Yang, H. Y.; Yang, S. N.; Kong, J. L.; Dong, A. C.; Yu, S. N. Obtaining

547

information about protein secondary structures in aqueous solution using Fourier

548

transform IR spectroscopy. Nat. Protoc. 2015, 10, 82−96.

549

(35) Deng, J.; Yu, L.Q.; Liu, C.S.; Yu, K.; Shi, X.J.; Zhou, B.S.

550

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish

551

embryos. Aquat. Toxicol. 2009, 93, 29–36.

552

(36) Stensberg, M. C.; Madangopal, R.; Yale, G.; Wei, Q. S.; Ochoa-Acuña, H,; Wei,

553

A.; Mclamore, E. S.; Rickus1, J.; Porterfield1, D. M.; Sepúlveda, M. S. Silver

554

nanoparticle-specific mitotoxicity in Daphnia magna. Nanotoxicology 2014, 8,

555

833–842.

556

(37) Biswal, H. S.; Loquais, Y; Tardivel, B.; Gloaguen, E.; Mons, M. Isolated

557

monohydrates of a model peptide chain: effect of a first water molecule on the

558

secondary structure of a capped phenylalanine. J. Am. Chem. Soc. 2011, 133,

559

3931−3942.

560

(38) Radu, I.; Bamann, C.; Nack, M.; Nagel, G.; Bamberg, E.; Heberle, J.

561

Conformational changes of channelrhodopsin-2. J. Am. Chem. Soc. 2009, 131,

562

7313−7319.

563

(39) Lorenz-Fonfria, V. A.; Schultz, B. J.; Resler, T.; Schlesinger, R.; Bamann, C.;

564

Bamberg, E .; Heberle, J. Pre-gating conformational changes in the ChETA variant of

565

channelrhodopsin-2 monitored by nanosecond IR spectroscopy. J. Am. Chem. Soc.

566

2015, 137, 1850-1861.

567

(40) Palaniappan P. L. R. M.; Vijayasundaram, V. The effect of arsenic exposure and

568

the efficacy of DMSA on the proteins and lipids of the gill tissues of Labeo rohita. 19



569

Food Chem. Toxicol. 2009, 47, 1752–1759.

570

(41) Yang, S.; Li, L. Y.; Pei, Z. G.; Li, C. M.; Shan, X. Q.; Wen, B.; Zhang, S. Z.;

571

Zheng, L. R.; Zhang, J.; Xie, Y. N.; Huang, R. X. Effects of humic acid on copper

572

adsorption onto few-layer reduced graphene oxide and few-layer graphene oxide.

573

Carbon 2014, 75, 227−235.

574

(42) Gao, J.; Powers, K.; Wang, Y.; Zhou, H.; Roberts, S. M.; Moudgil, B. M.;

575

Koopman, B.; Barber, D. S. Influence of Suwannee River humic acid on particle

576

properties and toxicity of silver nanoparticles. Chemosphere 2012, 89, 96−101.

577

(43) Mold, D. E.; Dinitz, A. E.; Sambandan, D. R. Regulation of zebrafish zona

578

pellucida gene activity in developing oocytes. Biol. Reprod. 2009, 81, 101−110.

579

(44) Bonin, J.; Costentin, C.; Robert, M.; Saveant, J. M.; Tard, C. Hydrogen-bond

580

relays in concerted proton-electron transfers. Accounts Chem. Res. 2012, 45,

581

372−381.

582

(45) Ong, K. J.; Zhao, X. Thistle, M .E.; MacCormack, T. J.; Clark, R. J.; Ma, G.;

583

Martinez-Rubi, Y.; Simard, B.; Loo, J. S. C.; Veinot, J. G. C.; Goss, G. G.

584

Mechanistic insights into the effect of nanoparticles on zebrafish hatch.

585

Nanotoxicology 2014, 8, 295−304.

586

(46) Huang, J.; Zong, C.; Shen, H.; Liu, M.; Chen, B. A.; Ren, B.; Zhang, Z. J.

587

Mechanism of cellular uptake of graphene oxide studied by surface-enhanced raman

588

spectroscopy. Small 2012, 8, 2577−2584.

589

(47) Akhavan, O.; Ghaderi, E.; Esfandiar, A. Wrapping bacteria by graphene

590

nanosheets for isolation from environment, reactivation by sonication, and

591

inactivation by near-infrared irradiation. J. Phys. Chem. B. 2011, 115, 6279–6288.

592

(48) Hashemi, E.; Akhavan, O.; Shamsara, M.; Rahighi, R.; Esfandiar, A.; Tayefeh, A.

593

R. Cyto and genotoxicities of graphene oxide and reduced graphene oxide sheets on

594

spermatozoa. RSC Adv. 2014, 4, 27213–27223.

595

(49) Palaniappan, P. L. R. M.; Krishnakumar, N.; Vadivelu, M. FT-IR study of the

596

effect of lead and the influence of chelating agents, DMSA and D-Penicillamine, on

597

the biochemical contents of brain tissues of Catla catla fingerlings. Aquat. Sci. 2008,

598

70, 314−322. 20


Page 20 of 29

Page 21 of 29


599

(50) Chu, H. L.; Lin, T.Y.; Lin, S.Y. Effect of cyanide concentrations on the

600

secondary structures of protein in the crude homogenates of the fish gill tissue. Aquat.

601

Toxicol. 2001, 55, 171–176.

602

(51) Toyran, N.; Zorlu, F.; Severcan, F. Effect of stereotactic radiosurgery on lipids

603

and proteins of normal and hypofused rat brain homogenates: A Fourier transform

604

infrared spectroscopy study. Int. J. Radia. Biol. 2006, 81: 911–918.

605

(52) Nukuna, B. N.; Sun, G.; Anderson, V. E. Hydroxyl radical oxidation of

606

cytochrome c by aerobic radiolysis. Free Radical Bio. Med. 2004, 37, 1203−1213.

607

(53) Moskovitz, J.; Yim, M. B.; Chock, P. B. Free radicals and disease. Arch.

608

Biochem. Biophys. 2002, 397, 354−359.

609

(54) Mu, L.; Gao, Y.; Hu, X. G. L-Cysteine: A biocompatible, breathable and

610

beneficial coating for graphene oxide. Biomaterials 2015, 52, 301−311.

611

(55). Rawson, D. M.; Zhang, T.; Kalicharan, D.; Jongebloed, W. L. Field emission

612

scanning electron microscopy and transmission electron microscopy studies of the

613

chorion, plasma membrane and syncytial layers of the gastrula-Stage embryo of the

614

zebrafish brachydanio rerio: A consideration of the structural and functional

615

relationships with respect to cryoprotectant penetration. Aquacult. Res. 2000, 31,

616

325–336.

617

(56) Auffan, M.; Matson, C. W.; Rose, J.; Arnold, M.; Proux, O.; Fayard, B.; Liu, W.;

618

Chaurand, P.; Wiesner, M. R.; Bottero, J. Y.; Di Giulio, R. T. Salinity-dependent

619

silver nanoparticle uptake and transformation by Atlantic killifish (Fundulus

620

heteroclitus) embryos. Nanotoxicology 2014, 8 167−176.

621

(57) Sagerstrom, C. G.; Gammill, L. S.; Veale, R.; Sive, H. Specification of the

622

enveloping layer and lack of autoneuralization in zebrafish embryonic explants. Dev.

623

Dynam. 2005, 232, 85−97.

624

(58) Kimmel, C. B.; Warga, R. M.; Schilling, T. F. Origin and organization of the

625

zebrafish fate map. Development 1990, 108, 581−94.

626

(59) Babin, P. J.; Thisse, C.; Durliat, M.; Andre, M.; Akimenko, M. A.; Thisse, B.

627

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and

628

are highly expressed during embryonic development. P. Natl. Acad. Sci. U. S. A. 1997, 21



629

94, 8622−8627.

630

(60) Yan, L.; Zheng, Y. B.; Zhao, F.; Li, S. J.; Gao, X. F.; Xu, B. Q.; Weiss, P. S.;

631

Zhao, Y. L. Chemistry and physics of a single atomic layer: strategies and challenges

632

for functionalization of graphene and graphene-based materials. Chem. Soc. Rev. 2012,

633

41, 97−114.

634

(61) Akhavan, O.; Ghaderi, E. Toxicity of graphene and graphene oxide nanowalls

635

against bacteria. ACS nano 2010, 4, 5731−5736.

636

(62) Liu, S. B.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R. R.; Kong,

637

J.; Chen, Y. Antibacterial activity of graphite, graphite oxide, graphene oxide, and

638

reduced graphene oxide: membrane and oxidative stress. ACS nano 2011, 5,

639

6971−6980.

640

(63) Shim, G.; Kim, J. Y.; Han, J.; Chung, S. W.; Lee, S.; Byun, Y .; Oh, Y. K.

641

Reduced graphene oxide nanosheets coated with an anti-angiogenic anticancer

642

low-molecular-weight heparin derivative for delivery of anticancer drugs. J. Control.

643

Release 2014, 189, 80−89.

644

(64) Bexiga, M. G.; Varela, J. A.; Wang, F. J.; Fenaroli, F.; Salvati, A.; Lynch, I.;

645

Simpson, J. C.; Dawson, K. A. Cationic nanoparticles induce caspase 3-, 7- and

646

9-mediated cytotoxicity in a human astrocytoma cell line. Nanotoxicology 2011, 5,

647

557−567.

648

(65) Li, X. Y.; He, Q. J.; Shi, J. L. Global gene expression analysis of cellular death

649

mechanisms induced by mesoporous silica nanoparticle-based drug delivery system.

650

ACS nano 2014, 8, 1309−1320.

651 652 653 654 655 656 657 658 22


Page 22 of 29

Page 23 of 29


659

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


Page 24 of 29

Page 25 of 29


Figures

719 720

721 722

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

723

25



724 725

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

726

26


Page 26 of 29

Page 27 of 29


727 728

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

729

27



730 731

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

732

28


Page 28 of 29

Page 29 of 29



Mitigation in Multiple Effects of Graphene Oxide Toxicity in Zebrafish

Recommend Documents