Evaluating the Toxicity of Silver Nanoparticles by Detecting

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Evaluating the toxicity of Ag nanoparticles by detecting phosphorylation of histone H3 in combination with flow cytometry side-scattered light Xiaoxu Zhao, and Yuko Ibuki Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00542 • Publication Date (Web): 27 Mar 2015 Downloaded from http://pubs.acs.org on March 29, 2015

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Environmental Science & Technology

Evaluating the toxicity of Ag nanoparticles by detecting phosphorylation of histone H3 in combination with flow cytometry side-scattered light Xiaoxu Zhao and Yuko Ibuki*

Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1, Yada, Suruga, Shizuoka City, Shizuoka 422-8526, Japan

*To whom correspondence should be addressed. 52-1, Yada, Suruga, Shizuoka City, Shizuoka 422-8526, Japan Phone and Fax: +81-54-264-5799 E-mail: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Post-translational modification of histones is linked to a variety of biological processes

3

and disease states. This paper focuses on phosphorylation of histone H3 at serine 10

4

(p-H3S10), induced by silver nanoparticles (AgNPs), and discusses the usefulness of

5

p-H3S10 as a marker to evaluate the toxicity of AgNPs. Cultured human cells showed

6

remarkable p-H3S10 immediately after treatment with AgNPs, but not with Ag

7

microparticles. The p-H3S10 lasts up to 24 h and strongly depends on the cellular

8

uptake of AgNPs. Removal of Ag ions suppressed p-H3S10, while adding an excess of

9

Ag ions augmented p-H3S10. We expected that p-H3S10 requires two events: cellular

10

uptake of AgNPs and continuous release of Ag ions from intracellular AgNPs. AgNPs

11

enhanced the expression of the proto-oncogene c-jun, and p-H3S10 increased in the

12

promoter sites of the gene, indicating that p-H3S10 might indicate a biological reaction

13

related to carcinogenesis. We previously showed that side-scattered light from flow

14

cytometry could be used to measure the uptake potential of nanoparticles [Suzuki et al.

15

Environ. Sci. Technol. 2007, 41, 3018-3024]. Our current findings suggest that p-H3S10

16

can be used to evaluate the toxicity of AgNPs and Ag ion release in combination with

17

detection of side-scattered light from flow cytometry.

2


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TOC art

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18

INTRODUCTION

19

Silver nanoparticles (AgNPs) with diameters less than 100 nm are currently the

20

most widely manufactured nanomaterials. They are used in nanomedical devices, and

21

consumer products such as cosmetics, clothing, household products, room sprays, and

22

even in food products.1,2 The number of products containing AgNPs has grown more

23

than 10 times between 2006 and 2011, and it has been predicted that over 1000 tons of

24

AgNPs will be produced in 2015.1 Because nano-sized particles are generally

25

considered to be more toxic than micro-sized particles owing to their small size and

26

unique physicochemical properties, such as surface area and solubility, concerns about

27

the usage of AgNPs have increased as their prevalence has grown; however, their

28

potential toxicity has yet to be fully addressed.

29

AgNPs had long been considered a relatively safe material with antibacterial

30

properties; the only known side effect of over dosage was an irreversible pigmentation

31

of skin or eyes called argyria or argyrosis.3,4 However, new evidence in recent years has

32

spurred more cautious examination of AgNPs. Colloidal AgNPs have been shown to

33

induce acute and subchronic dermal toxicity with skin inflammatory response.5

34

Inhalation of AgNPs increases inflammatory cell infiltrate in the lung and liver,6 while

35

prolonged administration leads to lung function changes.7 Several authors have reported

36

the toxicity of AgNPs in vitro.8-14 AgNPs decrease the viability of mammalian cells, and

37

their effects appear to increase as particle size decreases.8,9 The cytotoxic effect can be

38

attributed to production of reactive oxygen species (ROS) and Ag ion release.10 AgNPs

39

themselves can also directly cause adverse effects. The binding of AgNPs to RNA

40

polymerase inhibited RNA polymerase activity and overall RNA transcription, in a

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process which was distinct from the cytotoxicity pathway induced by Ag ions.11 AgNPs 4


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can induce DNA damage;12 their genotoxic activity was further confirmed by noting

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that proteins related to DNA damage, such as p53 and histone H2AX, were

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phosphorylated following treatment with AgNPs.13,14 AgNPs had a significant effect in

45

this regard; Ag ions to a lesser extent.13 Because both AgNPs and released Ag ions

46

might each pose a distinct risk to human health, it is necessary to evaluate the toxicity of

47

several types of manufactured AgNPs and released Ag ions.

48

When evaluating the toxicity of AgNPs, ease of intracellular uptake is an

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essential factor because internalization of AgNPs is the first step in their reaction with

50

cells.10,11,14 However, intracellular uptake is difficult to detect even in vitro. We have

51

recently proposed a simple method to evaluate the uptake potential of nanoparticles into

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mammalian cells using flow cytometry (FCM).15,16 The intensity of the side-scattered

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light (SS) in FCM increases when nanoparticles are taken up into cells. The advantage

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of this method is that it dispenses with the need for cumbersome treatments, and only

55

relies on the preparation of a single cell suspension. In addition, statistically valid

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information about cell populations is quickly obtained, because thousands of living cells

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are analyzed per second in FCM. This method (increase of SS) has been used to

58

measure intracellular uptake AgNPs.15 However, SS is only able to show that the

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nanoparticles were translocated to the cytoplasm, and is not able to reveal any toxic

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reactions. To screen the toxicity of several kinds of AgNPs, SS analysis should be

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complemented with methods that detect biological factors that reflect a toxic reaction.

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Post-translational modifications of histones have recently attracted research

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attention because they have been linked to a variety of biological processes and disease

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states.17,18 Carcinogenic metals, such as nickel, arsenic, and chromium, alter histone

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modifications, leading to altered gene expression and carcinogenesis.19-21 The 5



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remarkable phosphorylation of histone H3 at serine 10 (p-H3S10) has been identified

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after treatment with some kinds of metals. Arsenic-induced p-H3S10 contributed

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towards enhancing transcription of proto-oncogenes (c-jun and c-fos) and may thereby

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lead to carcinogenesis.19 Nickel ions induced p-H3S10 via activation of mitogen

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activated protein kinase pathway.20 Activation of mitogen activated protein kinase has

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also been reported to occur upon treatment with AgNPs.13,22 In addition to its activation

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by

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12-O-tetradecanoylphorbol 13-acetate and ultraviolet (UV) exposure, which are related

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to tumor promotion.23-25 Toxicological screening of nanoparticles should include testing

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for carcinogenic activity, including initiation and promotion of cancer. However,

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p-H3S10 as a potential marker for toxicity of AgNPs or Ag ions after exposure to

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AgNPs has not been investigated.

metals,

p-H3S10

is

also

induced

by

epidermal

growth

factor,

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In this study, we first found that AgNPs significantly induced p-H3S10. The

79

phosphorylation was due to the release of Ag ions from AgNPs translocated to the cell

80

interior. We discuss the possibility of using p-H3S10 as a novel candidate for evaluating

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the toxicity of AgNPs, in combination with SS analysis.

82 83

EXPERIMENTAL SECTION

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Materials. Ag particles, whose primary (listed) sizes were < 0.1 µm, 2–3.5 µm, 5–8 µm

85

and < 45 µm, were purchased from Sigma–Aldrich (St. Louis, MO). Those < 106 µm

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were purchased from Wako Pure Chemical Industries, Ltd. (Japan).

87 88

Preparation of Ag particles. Ag particles in 1.5 mL microtubes were suspended in

89

Dulbecco’s Modified Eagle Medium (DMEM; Sigma–Aldrich) with 0.5 % fetal bovine 6


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serum (FBS; Life Technologies, Grand Island, NY) at a final concentration of

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10 mg/mL. For the preparation of UVA-irradiated AgNPs, the particles suspended in

92

1 mL of water in 35 mm dishes were irradiated for 1 h (UVA tube: Hitachi Ltd. Japan;

93

350–400 nm range, 369 nm peak). For the preparation of hydrogen peroxide

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(H2O2)-treated AgNPs, the particles in 1.5 mL microtubes were treated with 1 M of

95

H2O2 for 1 h at room temperature. They were washed three times with water and

96

resuspended in DMEM to a final concentration of 10 mg/mL. The particles were

97

sonicated in a bath-type sonicator (Bioruptor; Cosmo Bio, Japan) for 1 min immediately

98

before applying them to the cells for treatment. The mean diameter of AgNPs (
5–8 µm had little or no phosphorylation (Fig. 3B). The

220

correlation between the particle size and SS or p-H3S10 (Fig. 3C) was not linear,

221

because significant p-H3S10 was detected only in the smaller Ag particles (< 0.1 µm

222

and 2–3.5 µm) for which SS values were high. These results suggested that only small

223

Ag particles, especially nano-sized particles, could incorporate into cells and induce

224

p-H3S10. Indeed, cytotoxicity of AgNPs towards various cell lines has been reported to

225

increase as particle size decreases.8,9 Liu et al.9 examined the toxicity of three types of

226

AgNPs (< 5 nm, < 20 nm and < 50 nm) using four types of cells, and concluded that

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smaller AgNPs enter cells more easily than larger ones, which might explain their

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higher cytotoxicity. In vivo, smaller AgNPs induce pulmonary inflammation more

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easily.31 Surface area characteristics of AgNPs limited the accessibility to membrane

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and changed DNA damage response.14 The size-dependent incorporation of AgNPs was

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correlated to the extent of p-H3S10, meaning that p-H3S10 is induced by either

232

processes that occur during uptake of AgNPs, or reactions that occur once AgNPs are

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inside the cell. 12


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Cells can take up particles by endocytosis, phagocytosis, and related

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methods.32,33 Greulich et al.34 reported that cells took up AgNPs by macropinocytosis

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and endocytosis dependent on clathrin. To further investigate how uptake of AgNPs

237

affects p-H3S10, we performed a series of experiments using the uptake inhibitor 2-DG

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and NaN3, which have been shown to inhibit cellular uptake of CuO nanoparticles.32

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Reduction of ATP level by both inhibitors suppresses endocytosis, an ATP-dependent

240

process. 35 Treatment with NaN3 and 2-DG suppressed the increase in SS intensity that

241

had occurred upon treatment with AgNPs (Fig. 4A). When uptake was inhibited,

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p-H3S10 was clearly suppressed (Fig. 4B). The lines showing the correlation between

243

the dosage of AgNPs (X-axis) and the values of SS or p-H3S10 (Y-axis), showed an

244

upward slope in the absence of inhibitors but a flat slope, parallel to the X-axis, in the

245

presence of the inhibitors (Fig. 4C). The slight downward slope in the presence of

246

inhibitors in HaCaT cells is due to a high background value of p-H3S10 in

247

AgNPs-untreated cells. These results suggested that incorporation of AgNPs into cells is

248

necessary for AgNPs to induce p-H3S10.

249 250

Release of Ag ions from AgNPs and p-H3S10. We previously detected Ag

251

ions released from AgNPs (5 µmol/L from 10 mmol/L of AgNPs at 24 h) using

252

inductively coupled plasma atomic-emission spectrometry.36 Ag ions react with

253

biological molecules, which sometimes reveals their toxicity.37-39 Park et al.40 suggested

254

that AgNPs might act as a “Trojan horse”, that is, after becoming incorporated into cells

255

the AgNPs release Ag ions that in turn damage cell machinery. In contrast, some reports

256

suggest that AgNPs induce toxicity independently of Ag ions.11,41 We conducted

257

experiments to clarify the mechanism by which AgNPs induce p-H3S10, to determine 13



258

whether the p-H3S10 arises from uptake of AgNPs to the cytoplasm or Ag ion release

259

from intracellular AgNPs.

260

First, to clarify the role of Ag ions released from AgNPs in the p-H3S10, both

261

cell lines were treated with AgNO3 (50 µM) for 10 h. The p-H3S10 was temporarily

262

induced after treatment with AgNO3, but rapidly ceased (Fig. 5A). In addition, the

263

p-H3S10 was inhibited by NAC, whose thiol group has high affinity for Ag ions (Fig.

264

5B). NAC does not affect the uptake of AgNPs by cells (Fig. 5C). Figure 5D shows the

265

dosage of AgNPs plotted against the values of SS in the absence and presence of NAC.

266

The correlation between the dosage of AgNPs (X-axis) and the values of SS (Y-axis)

267

was similar in the presence and absence of NAC, as evident in the similarity between

268

the slopes of the lines, indicating that NAC does not influence the extent to which

269

AgNPs were incorporated into the cells. However, NAC decreased the slope of the line

270

demonstrating the correlation between dosage of AgNPs (X-axis) and p-H3S10 (Y-axis),

271

and that between the value of SS (X-axis) and p-H3S10 (Y-axis). This indicated that Ag

272

ions are required for the p-H3S10 to occur.

273

We thus hypothesized that two events might be required for AgNPs to induce

274

p-H3S10; 1) AgNPs are incorporated into cells, and 2) Ag ions are continuously

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released from AgNPs within the cells. To test this hypothesis, AgNPs were pretreated

276

with UVA or H2O2 to enhance the release of Ag ions from their surface. AgNPs oxidized

277

by UVA or H2O2 release 2 to 6 times more Ag ions than untreated AgNPs.36 In vivo, the

278

reaction of AgNPs with intracellular H2O2 was presumed to be among the factors

279

causing the release of Ag ions.42 The UVA-irradiated AgNPs and H2O2-treated AgNPs

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generated remarkable levels of p-H3S10 compared with untreated AgNPs (Fig. 6A).

281

However, both untreated and oxidized AgNPs were incorporated into cells to a similar 14


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extent (Fig. 6B).

283

The GSH assay was used to examine the amounts of Ag ions released from

284

oxidized AgNPs within cells (Fig. 6C). Ag ions interact with GSH in a 1:1 stoichiometry.

285

If a large amount of Ag ions are released inside cells, the level of GSH in the cell

286

decreases because of the reaction with Ag ions. The amounts of GSH decreased in

287

AgNPs exposed to UVA and H2O2. These results suggest that p-H3S10 is caused by Ag

288

ions, and the lasting release of Ag ions from AgNPs inside cells might generate

289

p-H3S10 for a long time after initial uptake of AgNPs.

290 291

p-H3S10 and expression of proto-oncogene. Some studies report a relationship

292

between p-H3S10 and the induction of proto-oncogenes, such as c-fos and c-jun.

293

Several

294

12-O-tetradecanoylphorbol 13-acetate also generate p-H3S10.23,24,26 We also have

295

reported that the carcinogenic agent formaldehyde is able to induce p-H3S10 and

296

enhance expression of the proto-oncogenes.45 Treatment with AgNPs significantly

297

induced the c-jun gene in a dose- and time-dependent manner (Fig. 7A). To examine

298

whether AgNPs stimulated p-H3S10 at c-jun loci, ChIP assays were performed using an

299

antibody that recognized p-H3S10. Genomic DNA present in the immunoprecipitates

300

was extracted and analyzed by real-time PCR using primers specific to the gene, as

301

shown in Fig. 7B. AgNPs enhanced the p-H3S10 on the c-jun promoter region (-80 and

302

+150). In other regions, no significant differences were observed upon treatment with

303

AgNPs. These results indicated that the induction of p-H3S10 by AgNPs would involve

304

the induction of some genes relating to carcinogenesis. However, the induction of c-fos

305

gene, which has been reported to accompany the induction of c-jun, was not observed

tumor-promoters

such

as

epidermal

15


growth

factor

43, 44

and


306

after treatment with AgNPs (Supporting Information, Figure 2).

307 308

The application of p-H3S10 for toxicological screening of nanoparticles in

309

combination with SS. In this study, we found that AgNPs induced significant levels of

310

p-H3S10, due to the release of Ag ions from AgNPs incorporated into cells. Since our

311

report that outlined the use of SS in FCM to evaluate the incorporation of

312

nanoparticles,15,16 many researchers have used this method to study the uptake of

313

several kinds of nanoparticles.46-48 However, this method was limited to evaluating only

314

the incorporation of nanoparticles and does not reflect any toxicological reaction. In

315

addition, SS does not evaluate the toxicity of ion release from particles. Because

316

p-H3S10 requires Ag ion release following incorporation of AgNPs into cells,

317

simultaneous detection of SS and p-H3S10 would produce more meaningful analytical

318

results, compared with detection via SS alone. One advantage of SS analysis is its

319

ability to probe living cells. Expression of fluorescence-tagged proteins which have

320

high affinity with p-H3S10 would enable the simultaneous measurement of SS and

321

p-H3S10 in living cells using FCM. This is the subject of our subsequent ongoing

322

research. Moreover, in this study, we found that p-H3S10 is related to the expression of

323

proto-oncogenes, which suggests that p-H3S10 may be a good marker for detecting

324

biological factors relating to carcinogenesis. As toxic responses might occur without the

325

pathway of p-H3S10 in other nanoparticles, the particle specificity for p-H3S10 is

326

needed to examine.

327 328

ACKNOWLEDGEMENT

329

This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour 16


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and Welfare, Japan, and by a Grant-in-Aid from JSPS KAKENHI (24510084).

331 332 333

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FIGURE LEGENDS

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Fig.1 p-H3S10 after treatment with AgNPs

496

(A) p-H3S10 after treatment with AgNPs. HaCaT and A549 cells were treated with

497

AgNPs (1 mg/mL) for ~24 h. H3 (CBB staining) was used as a standard for the

498

equal loading of proteins for SDS-PAGE.

499

(B) Images of p-H3S10 after treatment with AgNPs. HaCaT cells treated with AgNPs

500

(1 mg/mL) for 1 h were stained with the antibody for p-H3S10 and DAPI. Cells

501

indicated by arrows were magnified.

502 503

Fig. 2 Incorporation of AgNPs into cells, and subsequent p-H3S10

504

HaCaT and A549 cells were treated with several doses (1–1000 µg/mL) of AgNPs for 1

505

h.

506

(A) The intercellular uptake of AgNPs. FS and SS were analyzed using FCM.

507

(B) p-H3S10 after treatment with AgNPs. H3 (CBB staining) was used as a standard

508

for the equal loading of proteins for SDS-PAGE.

509

(C) Correlation between intercellular uptake of AgNPs (average values of SS) and

510

p-H3S10. p-H3S10 was determined using western blotting, where the intensity of

511

each band was extracted using Image J version 1.38. The extent of p-H3S10 in cells

512

treated with AgNPs, versus untreated control cells, was calculated. Correlations

513

were calculated using the least-squares method.

514 515

Fig. 3 Size-dependent incorporation of Ag particles, and its influence on p-H3S10

516

HaCaT and A549 cells were treated with Ag particles (1 mg/mL) of different sizes (

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