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Ecotoxicology and Human Environmental Health

Molecular Toxicity of Metal Oxide Nanoparticles in Danio rerio Jing Hou, Haiqiang liu, Luyao Wang, Linshuai Duan, Shiguo Li, and Xiangke Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01464 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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Environmental Science & Technology

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Molecular Toxicity of Metal Oxide Nanoparticles in Danio rerio

2

Jing Hou a, Haiqiang Liu a, Luyao Wang a, Linshuai Duan a, Shiguo Li b*, Xiangke Wang a*

3

a

4

Beijing 102206, China

5

b

6

China

College of Environmental Science and Engineering, North China Electric Power University,

Research Center for Eco-Environmental Sciences, Chinese Academy of Science, Beijing 100085,

7 8

ABSTRACT

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Metal oxide nanoparticles can exert adverse effects on humans and aquatic organisms, however,

10

their toxic mechanisms are still unclear. We investigated the toxic effects and mechanisms of

11

copper oxide, zinc oxide, and nickel oxide nanoparticles in Danio rerio using microarray analysis

12

and the comet assay. Copper oxide nanoparticles were more lethal than the other metal oxide

13

nanoparticles. Gene ontology analysis of genes that were differentially expressed following

14

exposure to all three metal oxide nanoparticles showed that the nanoparticles mainly affected

15

nucleic acid metabolism in the nucleus via alterations in nucleic acid binding. KEGG analysis

16

classified the differentially expressed genes to the genotoxicity-related pathways “cell cycle”,

17

“Fanconi anemia”, “DNA replication”, and “homologous recombination”. The toxicity of metal

18

oxide nanoparticles may be related to impairments in DNA synthesis and repair, as well as to

19

increased production of reactive oxygen species.

20

ACS Paragon Plus Environment


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INTRODUCTION

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With the rapid development of nanotechnology, a wide range of metal oxide nanoparticles

23

(MO-NPs) have been incorporated into various commercial products such as food, sunscreen, and

24

electronic and medical devices because of their superior magnetism, conductivity, reactivity, and

25

optical properties

26

through sewage

27

tissues and cells of aquatic organisms

28

beings as the ultimate reservoir

29

biological systems, causing cytotoxicity, inflammation, and cell membrane leakage

30

thorough understanding of the toxicity of MO-NPs, from molecular mechanisms to physiology

31

and morphology, is required to mitigate their adverse effects. However, the study on the molecular

32

mechanisms of MO-NPs is not clear, which is not conducive to the scientific assessment and

33

prediction of the ecological risk of NPs.

[1, 2]

. Inevitably, MO-NPs are released into the aquatic environment, especially

[3, 4]

. The small MO-NPs (27-nm TiO2 and 24-nm CeO2) can easily enter the [5]

, and thereby accumulate up the food chain, with human

[6]

. Multiple studies have demonstrated that MO-NPs can impair [7-9]

. A

34

Many of the published studies have been devoted to assessing the environmental risk of NPs by

35

observing responses at the whole-organism level, including growth inhibition [10], reproduction [11],

36

and mortality

37

require higher exposure doses, when low doses not causing large-scale damage may in fact induce

38

effects sufficient to disrupt normal function

39

powerful experimental tool for assessing cellular alterations at the transcriptional level

40

expression analysis can provide information on low-level toxic effects at lower concentrations and

41

elucidate the underlying molecular mechanisms [15, 16]. Moreover, the complex constituents of

42

contaminants discharged into the aqueous environment render research with traditional endpoints

43

unsuited. As a sensitive high-throughput genomic technology

44

microarrays can screen multiple contaminants rapidly [18]. The responses of fish to environmental

45

toxicants have been studied in the fathead minnow Pimephales promelas

46

caeruleum

47

latipes [23].

48

[12]

. These traditional toxicological endpoints are time-consuming and generally

[20]

, Cyprinus carpio

[18]

[13]

. In this context, microarray technology is a

, Gasterosteus aculeatus

[14]

. Gene

[17]

, expression profiling with

[19]

, Etheostoma

[21]

, Salmo trutta [22], and Oryzias

Understanding the toxic effects and mechanisms of MO-NPs requires the use of representative [24]

49

research species. Fish are a preferred model in the study of NP toxicity to aquatic organisms

50

Danio rerio has long been used as a model organism of vertebrate molecular developmental


.

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biology [25]. As a model organism recommended by the Organization for Economic Co-operation

52

and Development (OECD)

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[27, 28]

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genome is 70% homologous to the human genome, permitting some extrapolations to humans [31].

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D. rerio adults are highly fecund, and embryos are transparent and easy to handle [32], allowing the

56

study of embryonic toxicity and malformation [13].

[26]

, D. rerio is gaining recognition in the fields of aquatic toxicology

drug discovery, and disease research

[29]

because of its characteristics [30]. The D. rerio

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The aim of this study was to investigate the transcriptional toxicity of MO-NPs on D. rerio and

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to explore its toxicity mechanisms of various MO-NPs in the same valence state. We used

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microarrays to identify differentially expressed genes (DEGs) common to copper oxide (CuO),

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zinc oxide (ZnO), and nickel oxide (NiO) NP exposure. Next, we used bioinformatics approaches

61

to suggest the molecular mechanism of toxicity. These findings contribute to our understanding of

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the toxicity of MO-NPs to aquatic organisms, and are of significance in evaluating the ecological

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risk of MO-NPs.

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MATERIALS AND METHODS

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D. rerio maintenance. All experimental D. rerio were kindly provided by the Chinese Research

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Academy of Environmental Sciences. D. rerio were 5 months old and had an average length of 2.8

67

cm and an average weight of 12.5 g. Fish were healthy and free of any signs of disease. During the

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acclimation period (not less than 14 days), D. rerio were housed in 60-L tanks (40 cm height × 30

69

cm width × 50 cm length) at a maximum density of seven D. rerio per liter. The tanks were filled

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with water previously treated by aeration and kept under filtration at a temperature of 24 ± 2°C

71

and pH of 7.0-8.0. Fish were fed commercially available dry flakes twice a day. Dead fish were

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removed in a timely manner and the cumulative mortality did not exceed 2%.

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Preparation of NPs. CuO NPs (< 50 nm), ZnO NPs, (< 50 nm), and NiO (< 50 nm) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were prepared by

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dispersing nanopowders in distilled water and sonicating at 600 W and 40 kHz for 30 min to

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decompose aggregates prior to dilution to exposure concentrations. The hydrodynamic size

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distribution and zeta potential of freshly prepared and 96-h aged NP solutions were determined

78

using a Malvern Zetasizer Nano-ZS instrument (Malvern, UK).

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Experimental design. After two weeks of acclimation, D. rerio were exposed to eight

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concentrations of each NP solution (ZnO: 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, and 128.0 mg/L; CuO:



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0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L; NiO: 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0,

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and 256.0 mg/L) with a day/night cycle of 16/8 h. Three biological replicates were used in each

83

treatment and each replicate consisted of 10 individuals. D. rerio were exposed for 96 h without

84

feeding. Survival rates were recorded each day and the median lethal concentration (LC50) was

85

calculated using the probit method. All subsequent exposures for the microarray, real-time

86

quantitative PCR (RT-qPCR), and comet assays were performed using one-tenth of the LC50 for

87

each NP solution (0.2 mg/L CuO, 4.8 mg/L ZnO, and 17.5 mg/L NiO). These concentrations have

88

been shown to fall below the level causing specific gene expression responses

89

were sacrificed for each treatment, and the gills were pooled as a sample. Three biological

90

replicates were used for each treatment.

91

Microarray Analysis. Agilent zebrafish (V3) gene expression microarray (4x44K) were used in

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our study. Total RNA was isolated from the gills of D. rerio exposed to the NPs and the controls

93

using Trizol reagent (Invitrogen, Carlsbad, CA, USA). To remove contaminated DNA, the

94

isolated RNA sample was digested with DNase I at 37°C for 15 min. To avoid dye-based bias, the

95

RNA sample was then labeled with cyanine 5 and cyanine 3 using the Low Input QuickAmp

96

Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) with a dye swap design. RNeasy

97

Plant Mini Kit (Qiagen, Hilden, Germany) was used to purify the labeled cRNA, which was then

98

fragmented and hybridized on a D. rerio microarray. Fifteen micrograms of the fragmented cRNA

99

were hybridized to each microarray at 45°C for 15 h in an Affymetrix GeneChip Hybridization

100

Oven 640 (Affymetrix, Santa Clara, CA, USA). The microarray was washed after hybridization.

101

Streptavidin phycoerythrinonan was used to stain the microarray on an Affymetrix Fluidics

102

Station 450 (Affymetrix), and a GeneChip Scanner 3000 7G (Affymetrix) was used to scan the

103

microarray. Three replicate experiments were conducted using cRNA prepared independently

104

from individual D. rerio. Microarray data were further analyzed with Microarray Suite version 5.0

105

at the default settings, and global scaling was used as the normalization method. Genes with a

106

false discovery rate-adjusted fold-change ≥ 2 and p-value < 0.05 were considered differentially

107

expressed.

108

Bioinformatics Analysis. Gene ontology (GO) enrichment of the obtained DEGs was analyzed

109

using R and corrected for gene-length bias. The DEGs were then checked against the Kyoto

110

Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) to identify


[33]

. Ten D. rerio

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significantly enriched KEGG pathways (p-value < 0.01). Principal component analysis (PCA) was

112

used to plot numeric data from experimental observations.

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RT-qPCR. We performed RT-qPCR to validate the reliability of the microarray data. Total RNA

114

was isolated from D. rerio samples and purified according to the methods described above.

115

First-strand cDNA was synthesized using a Superscript III First-Strand Synthesis kit (Invitrogen).

116

RT-qPCR was performed using a reaction system of 2 × SYBR Premix Ex TaqII (Takara, Shiga,

117

Japan) with 0.2 µM of each primer, 100 ng cDNA, and PCR-grade water on a 7500 Fast

118

Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The reaction conditions

119

were as follows: One cycle of 55°C for 3 min and 95°C for 3 min for initial denaturation, and 40

120

cycles of 95°C for 15 s and 60°C for 30 s. Each sample was analyzed three times. Dr-ACTIN was

121

used as a housekeeping reference gene. Primers used in RT-qPCR were all designed using the

122

Primer3 online program (http://primer3.ut.ee/). The 2-∆∆CT method was used to calculate the

123

fold-changes of the selected genes in D. rerio exposed to the MO-NPs. Fold-change data

124

are presented as the mean ± standard error from three biological replicates.

125

Comet assay. The assay was performed following the method of Collins

126

modification. Briefly, after treatment according to the protocol above, 1% trypsin was added to

127

cells, and the supernatant was removed after centrifugation for 4 min at 500 × g. Cells were

128

washed and re-suspended in 85 µL phosphate-buffered saline. More than 2.5 × 104 cells were

129

obtained in 200 µL of 0.5% low-melting agarose gel. Before solidification, classic slides were

130

treated and covered with a thin layer of normal-melting-point agarose. After preheating to 37°C in

131

a water bath, a total of 85 µL of sample were dripped onto slides, coverslipped, and refrigerated at

132

4°C for 5 min to solidify. The gels were then incubated in a freshly prepared lysis solution (pH

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10.0; 2.5 M NaCl, 10% dimethyl sulfoxide, 1% Na lauroylsarcosinate, 100 mM Na2 EDTA, 10

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mM Tris-HCl, and 1% Triton X-100) at 4°C overnight. After lysis, running buffer (300 mM

135

NaOH, 1 mM EDTA, pH > 13) was added onto the slides. The slides were incubated for 20 min

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and then subjected to horizontal electrophoresis at 10°C and 200 mA for 25 min. Finally, the

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slides were neutralized in a solution of 0.4 mol/L Tris-HCl (pH 7.5), washed, and dried at 37°C.

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DNA damage in individual cells was identified by an epifluorescence microscope (Olympus BX50,

139

Tokyo, Japan) under 400× magnification. The tail moment was used to indicate the degree of


[34]

with slight


140

DNA damage. Each sample was randomly measured for 30 tail lengths, and the data were

141

averaged.

142

RESULTS

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Lethality. D. rerio were exposed to eight concentrations of CuO, ZnO, and NiO NPs for 96 h. The

144

survival rate following exposure to low concentrations was comparable to that of the control

145

(Figure 1). Survival decreased sharply in a concentration-dependent manner. The 96-h LC50 values

146

for CuO, ZnO, and NiO NPs were 2.25, 48.2, and 175 mg/L, respectively, demonstrating that CuO

147

NPs were the most toxic.

148

NP characteristics. Table 1 shows that the 96-h hydrodynamic diameter of the NPs was greater

149

than the diameter at baseline. The absolute value of the 96-h zeta potential also increased from

150

baseline measurements. These results indicate that the MO-NPs aggregate and tend to stabilize

151

after 96 h of exposure. In addition, the 96-h zeta potential measurements suggest that the CuO NPs

152

were more stable than the other NPs and could resist aggregation, resulting in smaller particles.

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Differential expression. We used volcano plots to illustrate the distribution of DEGs by statistical

154

significance and fold-change between treated and control D. rerio. A total of 10,671, 8,680, and

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8,382 DEGs were identified for CuO, ZnO, and NiO NPs, respectively (fold-change > 2.0 and

156

p-value < 0.5) (Figure 2A–C, File S1). The number of down-regulated genes was greater than the

157

number of up-regulated genes for all NP treatments. We also identified 4,792 DEGs that

158

overlapped in all three treatments (Figure 2D), accounting for 33.5% of the total DEGs detected

159

(File S2). Moreover, we identified 2,976 (20.8%), 1,434 (10.0%), and 1,237 (8.7%) DEGs that

160

were specific for only CuO, ZnO, and NiO NP exposures, respectively.

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GO enrichment analysis. To assess the putative mechanisms of MO-NP toxicity, the 4,792 genes

162

differentially expressed in all three treatments were subjected to GO enrichment analysis, which is

163

used to predict the functions of DEGs. The results of the GO enrichment analysis were classified

164

into three categories: 3,793 GO terms for biological processes, 615 terms for cellular components,

165

and 1,370 terms for molecular function (File S3). The top terms in each category, sorted by

166

p-value, demonstrated that nucleic acid metabolism (GO: 0090304) (p = 1.4 × 10−14) was the most

167

enriched biological process term, the nucleus (GO: 0005634) (p = 5.8 × 10−14) was the most

168

enriched cellular component term, and nucleic acid binding (GO: 0003676) (p = 8.3 × 10−15) was

169

the most enriched molecular function term (Figure 3).


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KEGG Pathway Analysis of DEGs. Further insight into the biological function of the 4792

171

co-expressed DEGs was gained by examining the distribution of DEGs across KEGG pathway

172

analysis. A total of 147 KEGG pathways were obtained (File S4). Taking p-value < 0.01 as a

173

threshold, four significantly enriched pathways cell cycle pathway (Figure S1, p=5.3×10-5),

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fanconi anemia pathway (Figure S2, p=0.1×10-2), DNA replication pathway (Figure S3, p=0.1×

175

10-2) and homologous recombination pathway (Figure S4, p=0.2×10-2) were identified.

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PCA. We used PCA to compare the similarities and differences of treatment effects, and determine

177

which treatments were most responsible for the variations observed. Figure 4 illustrates a

178

three-dimensional PCA plot of the 12 replicates. The control group was significantly different

179

from the treatment groups, and the results from the three NP exposures are located in a relatively

180

concentrated region, suggesting that the toxic effects of CuO, ZnO, and NiO NPs on D. rerio were

181

similar at the molecular level. The contributions of the first three principal components were

182

35.21%, 25.62%, and 9.83%. The higher loads of samples from CuO and ZnO NP exposures

183

treatment on the first component reflect the contribution of CuO and ZnO NPs to D. rerio toxicity.

184

Microarray validation. Fifteen DEGs were selected at random from each treatment for a total of

185

45 DEGs for RT-qPCR analysis to verify the reliability of microarray results. Table S1 gives

186

detailed information on these genes and their fold-changes. Trends in all tested DEGs were

187

consistent with their trends in microarray analysis (Figure 5). The results from the two methods

188

correlated well, with correlation coefficients of 0.958, 0.935, and 0.929 for CuO, ZnO, and NiO

189

NP exposures, respectively, confirming the accuracy and reliability of the microarray data.

190

Comet Assay. All D. rerio treated with MO-NPs showed a tailing phenomenon, while the control

191

group did not (Figure 6). The DNA tail moment caused by CuO NPs was 1.92 times greater than

192

that of ZnO NPs and 2.09 times greater than that of NiO NPs. These results indicate that MO-NPs

193

can damage the DNA structure of D. rerio cells, and are consistent with the results obtained from

194

GO and KEGG analysis.

195

DISCUSSION

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The present study found that the order of toxicity to D. rerio was CuO NPs > ZnO NPs > NiO NPs.

197

Particle size is one of the factors that affect its toxicity. The negative charge on the surface of NPs

198

increases as the zeta potential increases, leading to greater electrostatic repulsive force between

199

NPs and to reduce aggregation. The 96-h zeta potential of CuO NPs was higher than that of ZnO



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and NiO NPs, indicating that CuO NPs were more stable and could resist aggregation, resulting in

201

smaller particles. NiO NPs had the lowest zeta potential, indicating a tendency to coagulate or

202

aggregate, resulting in larger particles. Aggregation can reduce the toxicity of NPs

203

lowest toxicity was indeed observed for NiO NPs.

[35, 36]

, and the

204

The expression of multiple genes involved in nucleic acid metabolism was dysregulated, which

205

may lead to impairments in the synthesis and/or degradation of nucleic acids. The nucleotide is the

206

basic nucleic acid unit, and nucleotide synthesis generally includes the chemical reaction of

207

pentose sugar, phosphate, and nitrogenous base in the nucleus. In contrast, degradation of nucleic

208

acid is a catabolic nuclear reaction, whereby partial nucleobases and nucleotides can be recycled

209

to create new nucleotides, suggesting a complex relationship between degradation and synthesis.

210

The expression of multiple genes involved in nucleic acid binding was also dysregulated by

211

exposure to MO-NPs. Studies have demonstrated that DNA or RNA binding to proteins affects

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protein structure and function, leading to dysregulation of transcription, translation, DNA

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replication, DNA repair and chromosomal recombination, and RNA processing and translocation

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[37-39]

215

rerio.

. Therefore, the GO enrichment analysis provided insights into the effects of MO-NPs on D.

216

KEGG pathway analysis indicated that cell cycle, Fanconi anemia, DNA replication, and

217

homologous recombination may be involved in the toxicity induced by MO-NPs. These four

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pathways are closely linked. The cell cycle in D. rerio incorporates several successive events,

219

including the S phase (DNA replication), M phase (mitosis), and G1 and G2 phases.

220

Cyclin-dependent kinases (CDKs) are important regulators of the cell cycle and typically contain

221

two subunits, the activating cyclin subunit and the catalytic CDK subunit. CDKs can control the

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cell cycle phases by regulating the activities of their key substrates. Targets downstream of CDKs

223

include multiple proteins such as origin recognition complex (ORC), minichromosome

224

maintenance (MCM), cell division control (CDC), transcription factor E2F, and its regulator Rb.

225

Cyclins usually form a complex with CDKs prior to involvement in the cell cycle process.

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Therefore, orderly cell division requires regular inactivation and activation of CDKs at specific

227

points in the cell cycle process. We found that MO-NPs inhibited the expression of CDK1, CDK2,

228

and CDK7 and their downstream target genes ORC, CDC6, CDC4, CDC5, CDC7, and MCM, but

229

had no significant effect on the expression of CDK4, CDK6, E2F, or Rb. While MO-NPs are


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selective for the CDK gene family, they can inhibit key regulatory enzymes in the cell cycle,

231

affecting DNA synthesis. These enzymes are involved in all phases of the cell cycle, suggesting

232

that the effect of MO-NPs on D. rerio would not be isolated to one phase. In particular, the

233

CycA-CDK2-Cdc6-ORC process has a more direct inhibitory effect on DNA synthesis.

234

DNA replication is mainly accomplished during the S phase, and is termed “semi-conservative

235

replication” because it contains both newly synthesized and original DNA. Both strands of the

236

original DNA serve as templates for the opposite strands. DNA polymerases are enzyme families

237

involved in DNA replication and are the rate-limiting factor in DNA replication. These enzymes

238

synthesize DNA molecules from deoxyribonucleotides to create uniform DNA strands. Three

239

DNA polymerase complexes have been identified in eukaryotes. The genes encoding DNA

240

polymerases were down-regulated following exposure to MO-NPs, indicating that enzyme

241

production decreased, reducing the efficiency of DNA replication and leading to a blocked S

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phase.

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Numerous studies have reported that NPs penetrating the nucleus through a nuclear pore may

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interact directly with chromosomal DNA. During interphase, NPs can bind or interact with DNA

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molecules, interrupting DNA replication and transcription

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NPs can bind to single-stranded DNA and then merge into a double-strand structure [43] In addition,

247

NPs can disrupt mitosis or chromosomes by mechanical or chemical binding, leading to some

248

vertebrate diseases. NPs can also induce primary genotoxicity by indirect contact with DNA, via

249

interaction with nuclear proteins involved in DNA replication, transcription, and repair processes,

250

or by interfering with cell cycle checkpoint functions and generating mitochondrial reactive

251

oxygen species. Oxidative stress has been proposed as a mechanism of MO-NP toxicity in various

252

organisms

253

epithelial cells

254

with acidic conditions (pH 4.5) that can dissolve MOs and release them as metal ions

255

possible that CuO NPs may easily cross the cell membrane, and once inside the cell, may release

256

Cu2+ and induce the production of reactive oxygen species or directly affect intracellular proteins.

257

Cu2+ may also decrease cell viability by binding to DNA directly, resulting in DNA damage and

258

cell death [47].

259

[40-42]

. During DNA replication, carbon

[44]

. Studies have shown that MO-NPs can cross the cell membrane of respiratory [45]

. MO-NPs entering the cell will be transported to the lysosome, an organelle [46]

. It is

Several studies have suggested that the toxicity of MO-NPs is associated with oxidative stress,



[48-50]

260

leading to DNA damage

and constant activation of DNA repair processes. DNA damage

261

from environmental stressors typically includes double-strand breaks (DSBs). Homologous

262

recombination is critical for the accurate repair of DSBs, and is a process of nucleotide sequence

263

exchange between two identical or similar DNA molecules. Cells usually repair DSBs rapidly by

264

homologous recombination. Recombinases are key rate-limiting enzymes in the homologous

265

recombination process, and the RecA recombinase protein family is thought to have been inherited

266

from a common ancestral recombinase. RecA is a 38-KDa recombinase necessary for repairing

267

DNA damage and maintaining DNA integrity, and a functional and structural homolog of RecA

268

has been identified in multiple species. Investigations into the evolutionary relationships between

269

these recombinases in bacteria, eukaryotes, and archaea have indicated that they are monophyletic

270

and may have originated from a common ancestor. Several recA-like genes have been identified in

271

D. rerio, and all are essential for cell division, differentiation, and proliferation. In the present

272

study, the RecA-like genes RAD51B, RAD52, RAD54, and XRCC2 were significantly

273

down-regulated following exposure to MO-NPs. The protein encoded by RAD51B is a member of

274

the RAD51 family, evolutionarily conserved proteins responsible for homologous recombination

275

in DNA damage repair. RAD51B can interact with RAD51C to form a stable heterodimer, which

276

can interact with other members of this protein family to effect DNA single-strand invasion.

277

Moreover, RAD54 can closely interact with RAD51s to form a complex that participates in

278

homologous recombination and DNA damage repair. Alterations in the expression of these genes

279

are known to delay the G1 phase and cell apoptosis, suggesting a role for these proteins in DNA

280

damage-sensing. RAD52 is a key regulator in DSB repair and homologous recombination via

281

binding to the ends of single-stranded DNA, a process crucial to annealing complementary DNA

282

strands. RAD52 can also interact with the DNA recombination protein RAD51, suggesting an

283

underlying role of this complex in RAD51-related DNA recombination and repair.

284

Down-regulating the expression of these genes can reduce the levels of the response proteins,

285

affect the stability of the DNA strand and the single-strand invasion process during repair, and

286

prevent efficient repair of damaged DNA. The comet assay results showed that the DNA tail

287

moment increased significantly following exposure to MO-NPs, indicating that DNA damage

288

occurred. DNA damage from environmental stressors can often be repaired effectively by

289

homologous recombination. In this study, the MO-NPs not only influenced the cell cycle and


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DNA replication processes, but also suppressed homologous recombination, amplifying the effects

291

on DNA replication and the cell cycle because the DSBs could not be effectively repaired by

292

homologous recombination.

293

Our analysis indicated effects on the Fanconi anemia pathway, suggesting that in addition to

294

DSB damage, MO-NP exposure may also lead to DNA cross-link damage in D. rerio. DNA

295

cross-link damage can block the formation of the DNA replication fork and affect the replication

296

process. The DNA cross-link repair process is relatively complex, and the Fanconi anemia

297

pathway is one effective repair method. Fanconi anemia is a rare genetic disease that can cause an

298

impaired response to DNA damage, and each of its subtypes corresponds to a gene mutation. At

299

least 12 protein families encoded by Fanconi anemia genes have been described. In the present

300

study, the proteins FANCB, FANCC, FANCF, FANCG, and FANCI, as well as their interacting

301

centromere protein MHF, core complex-associated protein FAAP24, and downstream target

302

replication protein RPA, were down-regulated following exposure to MO-NPs. This indicates that

303

the Fanconi anemia pathway was significantly inhibited by the exposure, hindering the repair of

304

DNA cross-link damage. In particular, the decrease in RPA expression can establish a relationship

305

between the Fanconi anemia pathway and homologous recombination or the repair of DSBs.

306

DSBs in Fanconi anemia have been shown to occur in response to cross-linking agents

307

indicating that the Fanconi anemia pathway is involved in repairing DSBs by homologous

308

recombination. The Fanconi anemia, DNA replication, cell cycle, and homologous recombination

309

pathways may therefore be closely related to the normal physiological processes in D. rerio, and

310

impairment of these pathways may explain the toxic effects of MO-NPs at the molecular level.

[36]

,

311

In this study, MO-NPs mainly affected nucleic acid metabolism in the nucleus via alterations in

312

nucleic acid binding. The DEGs were mainly classified into the genotoxicity-related pathways

313

“cell cycle”, “Fanconi anemia”, “DNA replication”, and “homologous recombination”. These

314

results will help us to understand the nature of aquatic toxicity of NPs with different properties

315

and have important scientific significance. In order to reduce the toxicity, the interaction and

316

integration of NPs should be paid more attention in the future research.

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318

ASSOCIATED CONTENT

319

Supporting Information Available: File S1 lists differentially expressed genes for CuO NPs, ZnO

320

NPs, and NiO NPs. File S2 lists 4792 differentially expressed genes co-expressed in CuO NPs,

321

ZnO NPs, and NiO NPs treatments. File S3 lists of enrichment GO terms for CuO NPs, ZnO NPs,

322

and NiO NPs. File S4 lists of enriched KEGG pathways for CuO NPs, ZnO NPs, and NiO NPs.

323

The Supporting Information is available free of charge on the ACS Publications website at DOI:

324

10.1021/acs.est.XXXXX.

325 326

AUTHOR INFORMATION

327

* Corresponding authors: [email protected] (Shiguo Li); [email protected] (Xiangke Wang).

328

Tel(Fax):+86-10-61772890

329 330

Conflict of Interest: The authors declare no competing financial interest.

331 332

Acknowledgments. Financial support from NSFC (21607043), the Youth Innovation Promotion

333

Association, Chinese Academy of Sciences (2018054), the Open Project of Key Laboratory of

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Environmental Biotechnology, CAS (kf2016009), the Fundamental Research Funds for the

335

Central Universities (2016ZZD06, 2018ZD11) are acknowledged.

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Table 1. The mean hydrodynamic diameter and zeta potential of copper oxide (CuO), zinc oxide

339

(ZnO), and nickel oxide (NiO) nanoparticles (NPs) at baseline (0 h) and 96 h. Hydrodynamic diameter (nm)

ζ-potential (mV)

0h

96 h

0h

96 h

CuO NPs

45±11

348±33

-18.2±3.3

-36.6±5.6

ZnO NPs

55±9

686±59

-19.6±3.8

-29.6±4.8

NiO NPs

60±12

1225±96

-13.6±2.6

-23.9±4.9

340 341



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Figure 1. Survival curves of Danio rerio exposed to copper oxide (CuO), zinc oxide (ZnO), and

344

nickel oxide (NiO) nanoparticles (NPs) for 96 h.

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Figure 2. Volcano plots illustrating the distribution of differentially expressed genes (DEGs) in

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Danio rerio treated with (A) copper oxide (CuO) nanoparticles (NPs), (B) zinc oxide (ZnO) NPs,

349

and (C) nickel oxide (NiO) NPs relative to the control. The x axis indicates the fold-change of

350

each gene on a log2 scale (fold change > 2). The y axis indicates the statistical significance of each

351

gene on a log10 scale (p < 0.05). Each dot represents one gene. Green dots

352

represent down-regulated DEGs. Red dots represent up-regulated DEGs. Black dots represent

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genes that were not differentially expressed. (D) Venn diagram illustrating the overlap of DEGs

354

between the three treatments.

355



356 357

Figure 3. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs). The

358

top 30 GO terms were sorted by p-value. The x-axis represents p-value on a log10 scale, and the

359

y-axis is the GO functional classification. The GO enrichment analysis is divided into three

360

categories: Biological processes, cellular components, and molecular functions.


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Figure 4. Three-dimensional principal component analysis score plot of Danio rerio exposure to

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copper oxide (CuO), zinc oxide (ZnO), and nickel oxide (NiO) nanoparticles (NPs). Each color

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represents an exposure and each point represents a replicate sample.

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366 367

Figure 5. Fifteen genes that were differentially expressed following exposure to (A) copper oxide,

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(B) zinc oxide, and (C) nickel oxide nanoparticles were selected at random for RT-qPCR analysis

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to verify the accuracy of microarray data. Each dot represents a gene.

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Figure 6. Effects of copper oxide (CuO), zinc oxide (ZnO), and nickel oxide (NiO) nanoparticles

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(NPs) on DNA damage in Danio rerio cells, detected by the comet assay.

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