Quantitative proteomic analysis to understand the mechanisms of zinc

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Ecotoxicology and Human Environmental Health

Quantitative proteomic analysis to understand the mechanisms of zinc oxide nanoparticle toxicity to Daphnia pulex (Crustacea: Daphniidae): : Comparing with Bulk Zinc Oxide and Zinc salt Li Lin, Mingzhi Xu, Huawei Mu, Wenwen Wang, Jin Sun, Jing He, Jian-Wen Qiu, and Tiangang Luan Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Environmental Science & Technology

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Quantitative proteomic analysis to understand the mechanisms

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of zinc oxide nanoparticle toxicity to Daphnia pulex (Crustacea:

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Daphniidae): Comparing with Bulk Zinc Oxide and Zinc salt

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Li Lin†, Mingzhi Xu†, Huawei Mu‡, Wenwen Wang†, Jin Sun §, Jing He†, Jian-

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Wen Qiu‖, Tiangang Luan† ,*

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†State

Key Laboratory of Biocontrol/School of Life Sciences, Sun Yat-sen University,

Guangzhou 510275, China ‡School

of Life Sciences, University of Science and Technology of China, Hefei

230071, China §Department

of Ocean Science, Hong Kong University of Science and Technology,

Hong Kong, China ‖Department

of Biology, Hong Kong Baptist University, Hong Kong, P. R. China

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* Corresponding author: Tel: +86 2084112958; E-mail address: [email protected]

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ACS Paragon Plus Environment


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Graphical abstract:

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Control

+ 114

ZnO NPs

+ 115

Bulk ZnO

+ 116

ZnSO4•7H2O

+ 117

Number of proteins

Up-regulation 200 100

Down-regulation 125

113

84

0 -100 -200 -300

178 ZnO NPs

218 235 Bulk ZnO ZnSO4·7H2O

Bulk ZnO

ZnO NPs 4

29 5

8 95

36 ZnSO4·7H2O

22 23 24 25

2


224

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ABSTRACT:

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The widespread use of zinc oxide nanoparticles (ZnO NPs) has resulted in their

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release to the environment. There has been concern about the ecotoxicity of ZnO NPs,

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but little is known about their toxic mechanisms. In the present study, we conducted

30

acute toxicity tests to show that ZnO NPs are more toxic to the freshwater crustacean

31

Daphnia pulex compared to bulk ZnO or ZnSO4·7H2O. To provide an integrated and

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quantitative insights into the toxicity of ZnO NPs, we conducted isobaric tags for

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relative and absolute quantitation (iTRAQ) proteomic analysis, which detected 262,

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331 and 360 differentially expressed proteins (DEPs) in D. pulex exposed to ZnO

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NPs, bulk ZnO and ZnSO4·7H2O, respectively. Among the DEPs, 224 were shared

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among the three treatments. These proteins were related to energy metabolism,

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oxidative stress and endoplasmic reticulum stress. The three forms of Zn all caused D.

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pulex to downregulate chitinase expression, disrupt Ca2+ homeostasis and reduce

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expression of digestive enzymes. Nevertheless, 29 proteins were expressed only in the

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ZnO NP treatment. In particular, histone (H3) and ribosomal proteins (L13) were

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obviously influenced under ZnO NP treatment. However, increased expression levels

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of h3 and l13 genes were not induced only in ZnO NP treatment, they were sensitive

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to Zn ions under the same exposure concentration. These results indicate that the three

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zinc substances have a similar mode of action and that released zinc ions are the main

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contributor to ZnO NP toxicity to D. pulex under a low concentration. Further

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investigation is needed to clarify whether a small proportion of DEPs or higher

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bioavailability cause ZnO NPs to be more toxic compared to bulk ZnO or ionic zinc.

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Keywords: Crustaceans; Ecotoxicology; Heavy metals; Proteomics; Toxicity test;

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Nanoparticles

3



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INTRODUCTION Nanomaterials having at least one dimension between 1 and 100 nm are widely

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used in many applications because of their unique physicochemical properties.

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According to the Nanotechnology Consumer Products Inventory (CPI), as many as

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1814 consumer products from 622 companies in 32 countries contain nanomaterials.1

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Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanoparticles

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and are commonly found in cosmetics, paints, textiles, sensing devices, rubber, and

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ceramics. As ZnO NPs have a broad range of applications, there has been concern

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about their release to the environment and their risk to organisms.2, 3 ZnO NPs have

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been shown to be toxic to a variety of organisms (e.g., bacteria, algae, plants, aquatic

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invertebrates, terrestrial invertebrates and aquatic vertebrates),4, 5 but the majority of

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previous studies regarding ZnO NPs have focused on their acute toxicity or the

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oxidative stress they cause.2

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The toxicity of ZnO NPs may be caused by the particles,6 by zinc ions released

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from ZnO NPs,7 or their interaction.8 ZnO NPs may produce chemical radicals or

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ROS as their surface interacts with the surrounding media.2 The particles may also

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interact with biomacromolecules such as protein and DNA, causing dysfunction.

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Although zinc is an essential trace element, the zinc ions released from ZnO NPs can

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be toxic, especially to the organisms that are susceptible to zinc. However, little is

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known about the mechanisms of ZnO NP toxicity or the relative contribution of the

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particles and the zinc ions to the overall toxicity of ZnO NPs.

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Branchiopod crustaceans are susceptible to environmental pollutants and are

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widely used as toxicological model organisms. Several studies have examined the

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toxicity of ZnO NPs to cladocerans. In acute toxicity tests using Daphnia magna,

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Blinova et al.9 found that the 48-h EC50 of ZnO NPs varied between 1.7 and 9.0 mg

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Zn/L, whereas Santo et al.10 reported that the 48-h EC50 of ZnO nanopowders varied

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from 1.9 to 3.1 mg/L. Adam et al. reported that D. magna can regulate its internal zinc

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concentration after exposure to ZnO nanoparticles and that toxicity was caused by the

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dissolved fraction.11 In a chronic toxicity test, Adam et al.12 found that the 4


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reproductive EC50 of ZnO NPs for D. magna was 0.112 mg Zn/L. Nevertheless, only

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a few studies have examined the molecular mechanisms of NP toxicity. Proteomic

83

tools have been used to compare the toxic mechanisms of citrate-coated silver NPs on

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D. magna compared to that of AgNO3 alone.13 Protein thiol contents and carbonyl

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levels were determined, and a two-dimensional electrophoresis (2DE) analysis was

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conducted showing that the two forms of silver had different modes of toxicity. They

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found 17 differentially expressed proteins, among which only eight were identified by

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MS/MS analysis. To our understanding, no proteomic study of the toxic mechanisms

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of ZnO NPs has been conducted.

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Daphnia pulex is a cornerstone species in freshwater food chains. Since D.

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pulex is the first crustacean whose genome has been sequenced and annotated,14 it is

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an ideal species for studies of toxic mechanisms. The present study thus determined

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the acute toxicity of ZnO NPs to D. pulex and applied isobaric tags for relative and

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absolute quantitation (iTRAQ) proteomic analysis to compare the effects of ZnO NPs,

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bulk ZnO, and ZnSO4·7H2O on its protein expression. Based on the proteomic results,

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the gene expression levels of h3, ll3, idh, scot and eip gene were analysed using q-

97

PCR to verify the molecular changes under the treatments.

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MATERIALS AND METHODS

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Test Reagents and Animal Species. Zinc oxide nanoparticles (nanopowder < 100

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nm particle size, specific surface area 15-25 m2/g, 79.1-81.5% Zn, and complex-

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ometric titration) were purchased from Sigma-Aldrich (Shanghai, China). X-Ray

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Diffraction (XRD) analysis showed that ZnO NPs were zincite structure (a = 0.325

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nm, c = 0.521 nm). ZnO NP stock suspensions were freshly prepared in reconstituted

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moderately hard water (RMHW) 15 after 30 min of sonication in a water bath

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sonicator. The RMHW (pH 7.8 ± 0.2, ionic strength 0.0051) contained NaHCO3 (96

107

mg/L), MgSO4 (60 mg/L), KCl (4 mg/L) and CaSO4·2H2O (60 mg/L).

108 109

The shape and size of ZnO NPs were characterized by using transmission electron microscopy (TEM, FEI, the Netherlands). The image demonstrates that the 5



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ZnO NPs used in this study were slender and spherical in shape (Figure S1), with a

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mean size of 61 ± 12 nm (length of longest dimension ± standard deviation),

112

determined by measuring 200 particles.

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Daphnia pulex was obtained from the School of Marine Sciences, Ningbo

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University (Ningbo, China). The culture stock originated from a lake in Minhang

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District (Shanghai, China) and has been maintained for more than three years in the

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laboratory.16 We started our lab culture with three individuals. The cladocerans were

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cultured in the RMHW with a density of 1 ind./50 mL under a 16 h light : 8 h dark

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cycle (20 ± 1 °C) in an incubator. The medium was replenished three times per week,

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and the animals were fed with Selenastrum capricornutum at concentrations of

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105~106 ind./mL.

121 122

Dissolution Measurements. A 24 h dissolution experiment was performed to

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quantify the contents of dissolved Zn released from ZnO NPs and bulk ZnO at 1

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mg/L, 10 mg/L and 100 mg/L. At 0 (immediately after mixing the chemicals with

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RMHW), 0.5, 1, 2, 4, 6, 12 and 24 h, samples were centrifuged at 10000 g for 15 min

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(Thermo Fisher Scientific, Waltham, MA). The supernatant was filtered through a

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syringe filter with a 0.22 μm pore diameter, and the Zn concentration was measured

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using inductively coupled plasma optical emission spectrometry (ICP-OES, Thermo

129

Fisher Scientific, Waltham, MA).

130 131

Acute Toxicity Test. The acute toxicity of ZnO NPs, bulk ZnO and ZnSO4·7H2O to

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D. pulex was determined according to the USEPA methods for measuring the acute

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toxicity of effluents and receiving waters to freshwater and marine organisms.15

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According to the methods, neonates were used. The effective concentration of each of

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the three chemicals was different based on preliminary experiments. For the precise

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calculation of EC50 values, exposure concentrations were 0.15, 0.3, 0.6, 1.2, 2.4, 4.8

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and 9.6 mg/L for ZnO NPs, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 mg/L for bulk ZnO, and

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0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L for ZnSO4·7H2O. Each concentration

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treatment contained 4 replicates, and each replicate comprised 10 neonates (< 24 h) 6


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and contained 50 mL of exposure solution or RMHW (control). Daphnids were

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incubated under the above mentioned culture conditions for a 48 h exposure period

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without feeding. At 24 h and 48 h, immobilized individuals in beakers were recorded.

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The median effective (immobilization) concentration (EC50) was determined

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according to Karber's method.17 The results from these toxicity tests were used to

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determine exposure concentrations for use in proteomic analysis. Because the

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neonates are usually more sensitive than the adults, the survival of the daphnids in the

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following experiments could be ensured under this concentration.

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Acute Exposure for Proteomics. Twenty-five daphnids (5-day-old) were transferred

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into a beaker containing 250 mL of sublethal concentration (1/5th of the 24 h EC50) of

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ZnO NPs, bulk ZnO, ZnSO4·7H2O or RMHW only (control). Each treatment was

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performed in triplicate.

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The beakers were placed in the culture conditions described above. After exposure for

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24 h, the daphnids were collected into a 1.5-mL Eppendorf tube containing 8 M of

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urea and were immediately frozen with liquid nitrogen. Then, the samples were stored

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at -80°C.

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Protein Extraction, Digestion, and iTRAQ Labelling. Samples were placed on ice

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and homogenized using a plastic pestle. A mild sonication was applied using a Sonic

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Dismembrator 300 (Thermo Fisher Scientific) to break cells and release the proteins

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using the following sonication cycle: 1 min at a power setting of 28, followed by three

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bursts of 15 s at a power setting of 35, each with a 30 s pause. The samples were then

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centrifuged at 15000 g and 4°C for 15 min, and the supernatant was collected and

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purified using a methanol and chloroform method.18 For 100 μL of sample, 400 μL of

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methanol, 100 μL of chloroform and 300 μL of Milli-Q water were added

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sequentially. The mixed solution was centrifuged at 14000 g for 2 min, the top layer

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was removed, and 400 μL of methanol was added to wash the protein pellet, followed

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by a 3-min centrifugation at 14000 g. The supernatant was removed, the protein pellet

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was reconstituted with 8 M urea, and the protein concentration was determined using 7



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an RC-DC kit (Bio-Rad, Hercules, CA). Next, 100 μg of protein was reduced with 5

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mM triscarboxyethyl phosphine hydrochloride at 60°C for 60 min and alkylated with

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10 mM methylethanethio sulfonate at room temperature for 20 min. A 50 mM

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triethylammonium bicarbonate (TEAB) solution was applied to dilute sample solution

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by 8 fold. The samples were then digested using sequencing-grade trypsin (Promega,

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Madison, WI) for 16 h at 37°C with a 1: 50 trypsin to protein mass ratio. The digested

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samples were desalted with Sep-Pak C18 cartridges (Waters, Milford, MA) and dried

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in a vacuum concentrator (Eppendorf, Hamburg, Germany). Samples derived from the

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control, ZnO NP, bulk ZnO, and ZnSO4·7H2O treatments were labelled with iTRAQ

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114, 115, 116, and 117 reagents (AB Sciex, Framingham, MA), respectively. Samples

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from the four treatments were pooled together and dried in a vacuum concentrator.

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Three biological replicates were used for the following analysis.

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SCX Fractionation and LC-MS/MS Analysis. The procedure of SCX fractionation

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followed that of Mu et al.19 Dried samples were dissolved in buffer A [20%

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acetonitrile (ACN) and 10 mM KH2PO4, pH = 3.0] and fractionated using a

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PolySULFOETHYL strong cation-exchange column (5 μm particle size, 200 × 4.6

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mm, 200 Å pore size, PolyLC, Columbia, MD) on a Waters 2695 HPLC. The running

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gradient was as follows: 5 min in 100% buffer A, followed by a 28 min 0-30% linear

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gradient to buffer B (0.5 M KCl, 10 mM KH2PO4 and 20% ACN, pH = 3.0), a 5 min

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30-100% buffer B linear gradient, 5 min in 100% buffer B, and finally 7 min in 100%

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buffer A. The gradient flow rate was 1 mL/min. A total of 14 fractions were collected,

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dried in a vacuum concentrator, and desalted with Sep-Pak C18 cartridges.

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The dried fractions were dissolved in 0.1% formic acid. Each sample was

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analysed twice with an LTQ-Orbitrap Elite coupled to an Easy-nLC (Thermo Fisher,

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Bremen, Germany). Peptides were separated with a C18 capillary column (Michrom

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BioResources, CA). The following running gradient was set: 5 min in 100% solution

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A (0.1% formic acid in Milli-Q water), a 55 min 0-30% solution B (0.1% formic acid

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in ACN) linear gradient, a 10 min 30-98% solution B gradient, 10 min in 98%

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solution B, and 10 min in 100% solution A. Mass spectrometry scans were run from 8


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350 to 1600 m/z with a resolution of 60000 in the positive charge mode. The top five

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multiple-charged ions with the highest intensities and a minimum signal threshold of

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500.0 were selected for fragmentation using high-energy collision-induced

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dissociation (HCD) and collision-induced dissociation (CID). Peptides were identified

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with the CID spectra in LTQ, while iTRAQ reporters were quantified with the HCD

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spectra in C-trap. The CID and HCD scanning approaches adopted an isolation width

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of 2.0 m/z, a dynamic exclusion time of 30 s, and a mass window for precursor ion

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selection of 2.0 Th. CID fragmentation was conducted under normalized collision

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energy of 35%, activation Q of 0.25, and an activation time of 10 ms. HCD

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fragmentation was conducted under full scan with FTMS at a resolution of 15000 in

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centroid mode, a normalized collision energy of 45% and an activation time of 10 ms.

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Mass Spectrometry Data Analysis. Mass spectrometry data were analysed as in Mu

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et al., 2018.20 Proteome Discoverer 1.3.0.339 (Thermo Finnigan, CA) was applied to

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convert raw data to .mgf files. Unpaired scans were filtered, and data derived from the

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CID and HCD scans were also separated with Python scripts. Reporter groups in the

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CID scans were replaced by those of HCD scans from the same parent ion. The

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intensity of ions in HCD was normalized. The new CID and HCD data files were

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submitted to Mascot version 2.3.2 (Matrix Sciences, London, UK) to search against a

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D. pulex protein database that was downloaded from NCBI and contained 32487

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protein sequences. The search parameters for CID files were set as: peptide charges of

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2+, 3+, and 4+, up to 2 missed cleavage for trypsin-digested peptides, variable

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modification: deamidated glutamine and asparagine, oxidation of methionine, fixed

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modification: methylthio of cysteine, precursor ions of ± 5 ppm, and MS/MS ion

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search with fragment ion tolerance of ± 0.6 Da. HCD parameters were similar to those

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of CID except for the fragment ion tolerance, which was set to ± 20 mmu. Data with

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ion scores lower than 27 were removed. Since short peptides might match decoy

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sequences, peptides shorter than seven amino acids were removed. The false

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discovery rate was set as 1%. Unlabelled peptides or peptides with an erratic ratio

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between any two iTRAQ reporters were removed. The median ratios between 9



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different iTRAQ reporters (i.e., 115/114, 116/114 and 117/114) were normalized to 1.

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Quantification was conducted with the proteins that were detected in all three

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biological replicates and had at least four summed matched peptides. Protein

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quantification was based on the summed intensity of matched peptides. Data from two

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technical runs of the same biological replicate were used to determine the criteria of

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significant differential expression. The cutoff values were defined as the log2-

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transformed data at which 95% of all target proteins did not deviate from each other.

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The cutoff values were 0.81 and -0.80 for 115/114, 1.01 and -0.95 for 116/114 and

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1.00 and -0.92 for 117/114, which corresponded to 1.76- and 0.57-fold for 115/114,

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2.01- and 0.52-fold for 116/114 and 2.00- and 0.53-fold for 117/114, respectively.

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These fold values were used as thresholds to define significantly up- or

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downregulated proteins between control and treatment groups.

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Bioinformatics analysis. PANTHER21 was applied to classify proteins into different

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functional groups based on their Gene Ontology (GO) annotations. Differentially

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expressed proteins (DEPs) were assigned to several level 2 Biological Process (BP)

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GO terms. DEPs without GO annotations were classified into “Others”. The

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expression levels of DEPs under each BP term were compared using log2-transformed

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data to produce a Euclidean distance similarity matrix and were clustered using the

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centroid linkage method and visualized by Java Treeview. GO enrichment was

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performed using the Gene Ontology Enrichment Analysis Software Toolkit

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(GOEAST).22 Hypothetical proteins were analysed by the Basic Local Alignment

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Search Tool (BLAST) in UniProt and searched against the UniProt Knowledgebase

253

(UniProtKB).

254 255

q-PCR analysis. Every 150 juveniles (4-6 day) of D. pulex were placed in a 500 mL

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glass beaker containing 500 mL of exposure media and exposed for 24 h to ZnO NPs,

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bulk ZnO or ZnSO4, at a concentration of 0.06 mg Zn/L, which was approximately

258

equal to 1/5th of the EC50 value of the ZnO NPs. The treatment was carried out in

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triplicate. Total RNA was extracted from 100 living animals using the E.Z.N.A. 10


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Total® RNA Kit II (OMEGA, USA) according to the manufacturer’s protocol. The

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samples were then treated with DNase. RNA integrity and purity were assessed by 1%

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agarose gel electrophoresis and a spectrophotometer (NanoDrop ND-1000, USA),

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respectively. The A260/A280 values were between 1.8 and 2.2, which indicated good

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sample quality. Total cDNA for the real-time PCRs of each sample was generated

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with a PrimeScript ™ RT reagent Kit (TaKaRa, Japan) according to the

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manufacturer's instructions. The cDNA was stored at -20℃ until further use for q-

267

PCR.

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The gapdh gene was used as a reference.23 Primer-BLAST was used to design

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primers for h3 (gi|321463426), l13 (gi|321474005), scot (gi|321478252), idh

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(gi|321478958) and eip (gi|321459564). These genes were selected from the DEPs

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according to the prior proteomic results. Primers were used for qPCR, the PCR

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products were sequenced and the results were tested using BLAST to ensure that the

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sequence homology was above 95% (Table S1).

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The final volume of each 10 μl qPCR system contained 5 μl of SYBR® Green

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Real-time PCR Master Mix, 0.4 μl of each primer (10 μm), 1 μl of diluted cDNA

276

template and 3.2 μl of PCR-grade water. The experimental protocol for qPCR

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verification consisted of a denaturation phase (10 min at 95℃), an amplification phase

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of 40 cycles (10 s at 95℃, 20 s at 58℃ and 20 s at 72℃) and a final melting phase.

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Relative mRNA expression levels of target genes were calculated using the Pfaffl

280

method.24 The data were analysed by independent sample t-tests to compare means

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between treatment and control. Statistical analysis was conducted with SPSS 22.0.

282 283 284

RESULTS

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Dissolution of ZnO NPs and bulk ZnO in exposure media. The content of

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dissolved Zn was measured for 1, 10, and 100 mg/L ZnO NPs and bulk ZnO in

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RMHW (Figure 1). In ZnO NP suspensions, the content of dissolved Zn increased

288

rapidly in 1 h and tended to become stable after 6 h for all three concentrations. At 24

289

h, the dissolved Zn was 0.47, 0.62 and 0.69 mg/L for the 1, 10 and 100 mg/L ZnO NP 11



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solutions, respectively. The amount of dissolved Zn released from the ZnO NPs was

291

in the same order of magnitude for the three concentrations. The bulk ZnO dissolved

292

in a similar pattern but had a lower dissolution rate and content of dissolved Zn. At 24

293

h, the dissolved Zn concentrations were 0.17, 0.18 and 0.23 mg/L for the 1, 10, 100

294

mg/L bulk ZnO solutions, respectively.

295 296

Acute toxicity tests. The toxicity of the ZnO NPs, bulk ZnO and ZnSO4·7H2O to D.

297

pulex was determined by acute immobilization tests (Table 1). The EC50 values of all

298

three tested chemicals were below 1 mg/L. Compared to bulk ZnO and ZnSO4·7H2O,

299

the ZnO NPs had a much lower EC50. Additional details of the toxicity tests are

300

shown in Figure S2.

301 302

Proteomic analyses. In total, 1652, 1827 and 2142 proteins were identified from the

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three biological replicates. Of these proteins, 1371 were detected in all three

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biological replicates. Exposure to ZnO NPs, bulk ZnO and ZnSO4·7H2O resulted in

305

262, 331 and 360 DEPs, respectively, when compared with the control (Figure 2).

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Approximately two-thirds of DEPs were downregulated in each treatment. There was

307

a large overlap of DEPs (224 proteins) among the three treatments (Figure 3).

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Furthermore, many these shared DEPs had identical expression trends across the three

309

treatments (Figure S3). Only 29 DEPs were unique to the ZnO NP treatment.

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GO analyses were performed on the 224 shared DEPs (Figure 4). For cellular

311

components, 37.5% of them related to the cell, followed by organelles (21.3%) and

312

extracellular regions (16.3%). For molecular functions, 35.2% of DEPs were related

313

to catalytic activity, and 25.9% were related to binding. For biological processes,

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37.9% were related to metabolic processes, followed by cellular processes (16.8%)

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and localization (15.9%).

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Pathway enrichment analysis of the 224 shared DEPs resulted in 47 GO terms

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(Table S2), including 25 biological process GO terms (Table 2). The enriched

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pathways included cellular amide metabolic process, ion transport, single-organism

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transport, aminoglycan metabolic process, cellular metal ion homeostasis, organic 12


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acid metabolic process, tricarboxylic acid cycle and chitin metabolic process. Among the 29 DEPs which expressed only under the ZnO NP treatment, histone

322

(H3) and ribosomal proteins (L13) were obviously influenced under the ZnO NP

323

treatment. Isocitrate dehydrogenase (IDH), succinyl-CoA:3-ketoacid-coenzyme

324

ATransferase (SCOT) and ecdysone-induced protein (EIP) were changed also.

325 326

Gene expression. The expression levels of h3 and l13 were reduced in the ZnO NP

327

and ZnSO4 treatments (Figure 5), and the expression levels of scot, idh and eip were

328

reduced in the ZnSO4 treatment.

329 330

DISCUSSION

331

Published studies have demonstrated that cladocerans are sensitive to ZnO NPs.9, 10, 12

332

In a large proportion of the published toxicity studies on cladocerans, D. magna was

333

chosen as a model species. In our study, D. pulex showed more sensitivity to ZnO

334

NPs compared to the published results on D. magna.

335

The toxicity tests results showed that the ZnO NPs were more toxic than the

336

bulk ZnO and ZnSO4∙7H2O. However, the proteomic analyses were unable to

337

distinguish the toxicity effects among the ZnO NPs, bulk ZnO and ZnSO4∙7H2O.

338

When exposed to these three substances, a large overlap in differentially expressed

339

proteins was observed, and these common DEPs also had coincident expression

340

trends. By comparing the protein expression patterns in the three treatments, we found

341

that ZnO NPs, bulk ZnO and ZnSO4∙7H2O were acting through a similar mode of

342

action. The zinc ions may be the primary contributors to the toxic effect of ZnO NPs.

343

Similar to our conclusion, the importance of zinc ions to ZnO NP toxicity in D.

344

magna was demonstrated by Adam et al.25 in a transcriptomic study. These authors

345

reported that no significantly differentially transcribed gene fragments were found by

346

microarray when comparing daphnid exposure to ZnO NPs and the corresponding

347

metal salt. The bioavailability of ZnO NPs affected by their agglomeration and ion

348

dissolution. Agglomeration was not obvious under low concentration. Therefore, the 13



349

toxicity of ZnO nanoparticles to D. magna is mainly caused by toxic metal ions under

350

the current exposure conditions. To our knowledge, the present study is the first report

351

to use proteomic analysis to compare the toxic mechanisms of ZnO NPs to

352

crustaceans with common ZnO and Zn ions. The three treatments resulted in different

353

numbers of DEPs. This might be due to the different Zn concentrations. In this study,

354

a concentration of 1/5th of the EC50 was chosen.

355

The 224 DEPs in the present study were found to be markedly associated with

356

Ca2+ homeostasis, energy metabolism, oxidative stress, endoplasmatic reticulum (ER)

357

stress and chitin metabolism. The disruption of Ca2+ homeostasis would cause

358

interference to various physiological processes. ZnO NPs were found to interfere with

359

Ca2+ homeostasis in previous studies. Wang et al.26 found that ZnO NPs increased the

360

resting Ca2+ concentration without compromising Ca2+ homeostatic mechanisms and

361

strongly inhibited store-operated Ca2+ entry in a muscarinic receptor signalling

362

pathway. Guo et al.27 found that ZnO NPs disrupted the intracellular Ca2+ homeostasis

363

in rat retinal ganglion cells by decreasing the expression and activity of plasma

364

membrane Ca2+ ATPase. In the present study, some common DEPs in the three

365

treatments were also found to be connected with Ca2+ homeostasis (Table S3). Of

366

these, Na+/K+ ATPase was downregulated. Downregulation of Na+/K+ ATPase might

367

interfere with Ca2+ homeostasis.28 In addition, some proteins such as calmodulin and

368

annexin, which are regulated by Ca2+, were also found to be differentially expressed.

369

The function of mitochondria is also affected by Ca2+ homeostasis. Some key

370

enzymes of the Krebs cycle, such as pyruvate dehydrogenase, NAD+-isocitrate

371

dehydrogenase and oxoglutarate dehydrogenase, are positively regulated by Ca2+.29

372

Cells may respond to Ca2+ signals by enhancing mitochondrial ATP production to

373

balance the increased ATP demand.30 In this study, some of the 224 common DEPs

374

were found to be enzymes of energy synthesis (Table S4). These enzymes, including

375

glyceraldehyde-3-phosphate dehydrogenase, mitochondrial malate dehydrogenase and

376

ATP synthase, were all upregulated, suggesting enhanced energy synthesis. Triboulet

377

et al.31 found that carbohydrate catabolism is one of the critical determinants of

378

sensitivity to zinc when they exposed macrophages to zinc ions and ZnO NPs. 14


Page 14 of 31

Page 15 of 31

379


The formation of reactive oxygen species (ROS) and oxidative stress are

380

considered to be involved in the toxicity of ZnO NPs.8 Mwaanga et al.32 found that

381

ZnO NPs caused oxidative stress that is related to biochemical changes in D. magna.

382

The exposure caused decreased GST activity and increased GSH oxidation, TBARS

383

formation and MT induction. In our study, some proteins related to antioxidant

384

defence, such as vitellogenins fused with SOD, peroxiredoxin and glutathione S-

385

transferase, were upregulated in the three treatments (Table S5). Vitellogenins fused

386

with SOD can be found in daphnids33 and brine shrimp.34 Rainville et al.13 found that

387

vitellogenins fused with SOD were upregulated when D. magna was exposed to Ag

388

NPs, and the upregulation was believed to evolve in response to the oxidative stress

389

caused by Ag NPs. Vandegehuchte et al.35 found that Zn exposure resulted in the

390

upregulation of peroxiredoxin 6 and glutathione S-transferase in D. magna. The

391

upregulation of these proteins in the present study indicated the oxidative stress

392

response in D. pulex.

393

Oxidative stress, the inhibition of protein glycosylation, a reduction in disulfide

394

bond formation, calcium depletion and the accumulation of unfolded proteins in the

395

ER lumen may cause disruption of the ER function and induce an ER stress response

396

via the unfolded protein response (UPR).36 Chen et al.37 found that ZnO NPs activated

397

the ER stress-responsive pathway in human umbilical vein endothelial cells

398

(HUVECs), and the ER stress response might be used as an early and sensitive

399

endpoint for nanotoxicological studies. Yang et al.36 found that ER stress-induced

400

apoptosis is involved in hepato-toxicity induced by ZnO NPs in mice. In our study,

401

some DEPs were also found to be related to ER stress, indicating that ER stress was

402

involved in the toxicity (Table S6). The downregulation of glycosyl transferase

403

suggested the inhibition of protein glycosylation. The downregulation of RNA-

404

binding protein, rRNA methyltransferase, signal recognition particle receptor and

405

signal peptidase were also observed, indicating that the protein synthesis and

406

translocation across the ER was inhibited to reduce the stress of protein folding.

407

Protein disulfide isomerase (PDI) is an essential folding catalyst and chaperone of the

408

ER and is responsible for introducing disulfides into proteins and catalyses the 15



409

rearrangement of incorrect disulfides.38 The upregulation of PDI suggested an

410

enhancement of protein folding in the ER.

411

The downregulation of many digestive enzymes was observed in all three

412

treatments in this study (Table S7). The expression of digestive enzymes, such as

413

dipeptidase, serine protease, chymotrypsin, and carbohydrate digestive enzymes (e.g.,

414

beta-galactosidase, alpha-amylase and endo-beta-1,4-mannanase) were significantly

415

decreased. Since the uptake of these substances might first occur in the gut of D.

416

pulex, it might cause substantial damage to the digestive system, resulting in a

417

decrease of digestive enzymes. We also found that some chitinases were

418

downregulated in all three treatments (Table S8).

419

Notably, 29 proteins were expressed only in the ZnO NP exposure test, though

420

there was a large overlap of DEPs across the three exposure conditions. Among the

421

overlapped DEPs, histone (H3) and ribosomal proteins (L13) were obviously

422

influenced under the ZnO NP treatment. Histones are the basic structural proteins of

423

chromosomes. Histones mainly contain five kinds. DNA molecules are wrapped

424

around histone octamers formed by the polymerization of H2A, H2B, H3 and H4 to

425

form the basic structure of chromatin - nucleosomes. The binding of H1 and DNA

426

between nucleosomes controls the entry and exit of DNA in order to stabilize

427

nucleosomes.39 Ribosomal Protein (Ribosomal Protein) and ribosomal RNA (rRNA)

428

constitute ribosomes, which play an important role in the protein biosynthesis. IDH,

429

SCOT and EIP were also influenced under ZnO NPs treatment. IDH and SCOT are

430

enzymes related to the tricarboxylic acid cycle, which is a key process of body energy

431

metabolism. EIP belongs to nuclear receptor superfamily as a transcription factor and

432

plays an important role in ecdysone pathway. 40

433

Similar results have been reported by other researchers. Methylation of H3 and

434

expression of some ribosomal proteins were found to be affected by gold and silver

435

nanoparticles.41, 42 From this experimental evidence, we hypothesize that

436

nanoparticles might enter cells and affect genetic processes, such as DNA/chromatin

437

structure, transcription, and translation. This might be one of the causes of the

438

significant differences in toxicity among the three treatments. From this view, the 16


Page 16 of 31

Page 17 of 31


439

effects caused by changes in proteins that were expressed only in the ZnO NP

440

treatment group may not correlate with their respective proportion in DEPs. Omics

441

studies usually draw conclusions from the proportion of differentially expressed

442

proteins or genes. This seems to be not objective and comprehensive enough. It would

443

be valuable to investigate the contribution of the changes in this small proportion of

444

DEPs to clarify the toxicity effects of ZnO NPs.

445

The expression levels of five related genes were selected for investigation among

446

the above mentioned 29 proteins. However, the results of the gene expression level

447

detection were not consistent with the results of the proteomics research. These 5

448

genes were not particularly induced in the ZnO NP treatment. Instead, they were

449

sensitive to Zn ions under the same exposure concentration. The gene expression

450

levels of h3 and l13 were reduced under ZnO NP and ZnSO4 exposure (Figure 5).

451

However, no difference was found between ZnO NP and Zn ion exposure. Expression

452

levels of scot, idh (energy metabolism-related genes) and eip (moulting-related gene)

453

mRNA showed no difference between the ZnO NP treatment and the control, but they

454

were inhibited under the ZnSO4 exposure (Figure 5). Poynton et al.43 also found that

455

Zn caused a decrease in chitinase activity in D. magna. Unlike ZnO NPs, non-

456

dissociation TiO2 NPs can promote moulting of D. magna.44 From the gene

457

expression results, it seems that Zn ions, rather than nanoparticles, are the main reason

458

for the toxic effects on moulting and genetic energy metabolism at relatively lower

459

treatment concentrations.

460

Combining the results of the acute toxicity tests and proteomic analyses, we

461

hypothesize that ZnO NPs have a higher bioavailability to D. pulex than bulk ZnO

462

and ZnSO4∙7H2O, although the three substances have a similar mode of action. The

463

relatively higher bioavailability endows the ZnO NPs with a higher toxicity, even at

464

lower concentrations, compared to bulk ZnO and ZnSO4∙7H2O. The marked

465

bioavailability of ZnO NPs was also mentioned in previously published studies.

466

Poynton et al.45 found that ZnO NPs were more toxic to the epibenthic crustacean

467

Hyalella azteca compared with zinc sulfate. However, gene expression analysis is

468

unable to distinguish between ZnO NP exposure and zinc sulfate exposure. The 17



469

authors hypothesized that ZnO NPs provide an enhanced exposure route for Zn2+

470

uptake in H. azteca. By synthesizing 65Zn tracer, Li et al. demonstrated a distinctive

471

uptake mode of ZnO NPs compared to zinc ions.46 However, after assessing the

472

results of the present study and the previous reports, we believe that there is a need for

473

more evidence to confirm this.

474

In conclusion, proteomic research provided an integrated and quantitative view of

475

toxic mechanism at the protein level. The present study firstly revealed that the

476

pathway of ZnO NPs were the same with common ZnO and Zn ions to daphnids

477

under a low concentration by using proteomic analysis.

478 479

Implications for Toxicity Mechanisms of ZnO NPs. In the present study, we

480

investigated the toxicity of ZnO NPs, bulk ZnO and ZnSO4∙7H2O to the freshwater

481

crustacean D. pulex by proteomic analysis. The results revealed that ZnO NPs, bulk

482

ZnO and ZnSO4∙7H2O were acting through a similar mode of action and that the zinc

483

ions may primarily contribute to the toxic effect of the ZnO NPs. There were 29

484

proteins that were expressed only upon ZnO NP exposure. Though these made up a

485

small proportion of total DEPs, the contribution from the changes of these proteins are

486

valuable for further investigation of the toxic effects of ZnO NPs. Our results suggest

487

that ZnO NPs might have a higher bioavailability to D. pulex than does the bulk ZnO

488

and ionic Zn. Increased attention should be given to the difference in bioavailability

489

of ZnO NPs and Zn from other sources when investigating ZnO NP toxicity. This

490

concept could also apply to other metal-based nanoparticles with dissolving capacity.

491 492

Supporting Information

493

Materials including dose-response curves, expression patterns of the common DEPs,

494

GO enrichment analysis results, and details of the common DEPs associated with

495

different physiological processes are freely available via the Internet at

496

http://pubs.acs.org.

497 18


Page 18 of 31

Page 19 of 31


498

ACKNOWLEDGMENTS

499

This research was supported by the National Key R & D Program of China

500

(2018YFD0900803, 2018YFD0900604), National Science Foundation of China

501

(Project No. 21777197, 21625703), and the Science Foundation of Guangdong

502

Province (Project No. 2016A030313337).

503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537

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640 641

22


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642


Figure Captions

643 644

Figure 1 The concentration of dissolved Zn released from ZnO NPs and bulk ZnO at

645

a 1, 10, or 100 mg/L during 24-h of incubation in RMHW. Data are shown

646

as the mean ± SD (n=3).

647 648 649 650 651 652

Figure 2 Number of differentially expressed proteins in D. pulex exposed to ZnO NPs, bulk ZnO and ZnSO4∙7H2O. Figure 3 Venn diagram showing the overlap of differentially expressed proteins of D. pulex exposed to ZnO NPs, bulk ZnO and ZnSO4∙7H2O. Figure 4 Gene ontology (GO) assignment of the shared differentially expressed proteins in D. pulex exposed to ZnO NPs, bulk ZnO and ZnSO4∙7H2O.

653

The meanings in the figure of molecular function were as follows, AC:

654

antioxidant activity (GO:0016209), NABTFA: nucleic acid binding transcription

655

factor activity (GO:0001071), PBTFA: protein binding transcription factor

656

activity (GO:0000988), RA: receptor activity (GO:0004872), TRA: translation

657

regulator activity (GO:0045182).

658

The meanings in the figure of biological process were as follows, BA: biological

659

adhesion (GO:0022610), BG: biological regulation (GO:0065007), CCO: cellular

660

component organization or biogenesis (GO:0071840), DP: developmental process

661

(GO:0032502), ISP: immune system process (GO:0002376), R: reproduction

662

(GO:0000003), RS: response to stimulus (GO:0050896).

663

Figure 5 Relative expression level of h3, l13, scot, idh and eip mRNA of D. pulex

664

exposed to ZnO NPs, bulk ZnO and ZnSO4∙7H2O at 0.06 mg Zn/L

665

(approximately equal to 1/5th of the ZnO NP EC50). Data are shown as the

666

mean ± SD (n = 3). * indicates P < 0.05, ** indicates P < 0.01

667 668 23



669 670 671 672 673

674 675

Figure 1

24


Page 24 of 31

Page 25 of 31


676

Number of proteins

Up-regulation 200 100

125

113

84

0 -100 -200 -300

178 ZnO NPs

218 Bulk ZnO

677 678

Figure 2

679

680 681

Down-regulation

Figure 3 25


235 ZnSO4·7H2O


682

Cellular component extracellular matrix (GO:0031012), 6.3%

membrane (GO:0016020), 6.3% macromolecular complex (GO:0032991), 12.5%

cell part (GO:0044464), 37.5%

extracellular region (GO:0005576), 16.3%

683 684

organelle (GO:0043226), 21.3%

Molecular function AC, 1.0%

NABTFA, 1.6% TRA, 2.6%

PBTFA, 0.5%

ERA, 4.1%

catalytic activity (GO:0003824), 35.2%

transporter activity (GO:0005215), 9.3% structural molecule activity (GO:0005198), 9.8%

685 686

RA, 9.8%

binding (GO:0005488), 25.9%

Biological process BA, 2.6%

ISP, 2.6%

DP, 2.2%

multicellular organismal process (GO:0032501), 3.9%

CCO, 3.9%

R, 0.4% metabolic process (GO:0008152), 37.9%

RS, 6.0%

BG, 7.8%

687 688

Page 26 of 31

cellular process (GO:0009987), 16.8%

localization (GO:0051179), 15.9%

Figure 4 26


Page 27 of 31


689 690 691

692 693 694

Figure 5

27



695 696 697

Table 1. Acute toxicity of ZnO NPs, bulk ZnO and ZnSO4∙7H2O to D. pulex (calculated from 4 replicates)

Treatment ZnO NPs Bulk ZnO ZnSO4·7H2O

24 h toxicity test (mg Zn/L) EC50 95% CI 0.32 0.27-0.39 0.76 0.63-0.92 0.98 0.80-1.18

48 h toxicity test (mg Zn/L) 　 EC50 95% CI 0.19 0.16-0.23 0.46 0.37-0.56 　 0.50 0.42-0.60 　

698

28


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Page 29 of 31


699

Table 2. Significantly enriched biological process Gene Ontology (GO) terms for the common differentially expressed proteins of D.

700

pulex exposed to ZnO NPs, bulk ZnO and ZnSO4∙ 7H2O GO ID

Term

Level

Number of overlapped DEPs

Number of proteins P value in database

GO:0006099

tricarboxylic acid cycle

6

4

27

4.72E-03

GO:0006879

cellular iron ion homeostasis

6

3

19

2.50E-02

GO:0015988

energy coupled proton transmembrane transport, against electrochemical gradient

5

5

18

5.95E-05

GO:0015991

ATP hydrolysis coupled proton transport

5

5

18

5.95E-05

GO:0006030

chitin metabolic process

5

9

184

1.28E-03

GO:0006101

citrate metabolic process

5

4

28

5.20E-03

GO:1902600

hydrogen ion transmembrane transport

5

5

55

5.20E-03

GO:0072350

tricarboxylic acid metabolic process

5

4

29

5.80E-03

GO:0019752

carboxylic acid metabolic process

5

12

461

1.27E-02

GO:0043436

oxoacid metabolic process

5

12

468

1.42E-02

GO:0006082

organic acid metabolic process

5

12

489

1.97E-02

GO:0046916

cellular transition metal ion homeostasis

5

3

20

2.73E-02

29



701

Page 30 of 31

Table 2 (Continued) GO ID

Term

Level

Number of overlapped DEPs

GO:0043648

dicarboxylic acid metabolic process

5

3

20

2.73E-02

GO:0006875

cellular metal ion homeostasis

5

3

25

4.89E-02

GO:0090662

ATP hydrolysis coupled transmembrane transport

4

5

18

5.95E-05

GO:0006022

aminoglycan metabolic process

4

9

196

1.84E-03

GO:0015992

proton transport

4

6

85

4.23E-03

GO:0006812

cation transport

3

11

194

6.24E-05

GO:0044765

single-organism transport

3

20

907

1.44E-03

GO:0015672

monovalent inorganic cation transport

3

8

144

1.45E-03

GO:0006818

hydrogen transport

3

6

85

4.23E-03

GO:0006811

ion transport

3

13

472

4.72E-03

GO:0006826

iron ion transport

3

3

17

1.97E-02

GO:0000041

transition metal ion transport

3

3

22

3.50E-02

GO:0043603

cellular amide metabolic process

3

8

261

4.11E-02

702 30


Number of proteins in database

P value

Page 31 of 31


Abstract graphic 254x190mm (96 x 96 DPI)


Quantitative proteomic analysis to understand the mechanisms of zinc

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