ZnO nanoparticles induced inflammatory response and genotoxicity in human blood cells: A mechanistic approach

Accepted Manuscript ZnO nanoparticles induced inflammatory response and genotoxicity in human blood cells: A mechanistic approach Violet Aileen Senapati, Ashutosh Kumar, Govind Sharan Gupta, Dr. Alok Kumar Pandey, Professor Alok Dhawan PII:

S0278-6915(15)30006-5

DOI:

10.1016/j.fct.2015.06.018

Reference:

FCT 8336

To appear in:

Food and Chemical Toxicology

Received Date: 29 April 2015 Revised Date:

26 June 2015

Accepted Date: 27 June 2015

Please cite this article as: Senapati, V.A., Kumar, A., Gupta, G.S., Pandey, A.K., Dhawan, A., ZnO nanoparticles induced inflammatory response and genotoxicity in human blood cells: A mechanistic approach, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.06.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ZnO NPs

ROS , NO GSH

M AN U

SC

RI PT

LDH release

TNF-α, IL-1β, COX-2

TE D

IκBα degradation

Translocation

EP

IκB kinase

NF-κB p50 IκBα p65 (inactive)

p50 NF-ĸB (active) p65

AC C

P IκBα

Phospho ERK, JNK, p38

Nucleus

AP-1 Proinflammatory transcripts

Inflammation

ACCEPTED MANUSCRIPT 1

ZnO nanoparticles induced inflammatory response and genotoxicity in human

2

blood cells: A mechanistic approach

3

Violet Aileen Senapatia, b, Ashutosh Kumara , Govind Sharan Guptaa,

5

Alok Kumar Pandeyb,c and Alok Dhawana, b,c

RI PT

4

6 7

9

SC

8 a

Institute of Life Sciences, School of Science and Technology, Ahmedabad

University, University Road, Ahmedabad 380009, Gujarat, India

11

b

12

Lucknow 226001, Uttar Pradesh, India

c

TE D

CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, P.O. Box 80,

EP

To whom correspondence should be addressed at: Professor Alok Dhawan Institute of Life Sciences School of Science and Technology Ahmedabad University University Road, Ahmedabad 380009, Gujarat, India Tel: +91-79-26302414; Fax: +91-79-26302419; E-mail: [email protected] and Dr. Alok Kumar Pandey Nanomaterial Toxicology Group CSIR-Indian Institute of Toxicology Research Mahatma Gandhi Marg P.O. Box 80, Lucknow226001, Uttar Pradesh, India E-mail: [email protected] ILS communication Number:- ILS-51

AC C

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

M AN U

10

1

ACCEPTED MANUSCRIPT Abstract

2

The wide application of zinc oxide nanoparticles (ZnO NPs) in cosmetics, paints,

3

biosensors, drug delivery, food packaging and as anticancerous agents has

4

increased the risk of human exposure to these NPs. Earlier in vitro and in vivo

5

studies have demonstrated a cytotoxic and genotoxic potential of ZnO NPs.

6

However, there is paucity of data regarding their immunomodulatory effects.

7

Therefore, the present study was aimed to investigate the immunotoxic potential of

8

ZnO NPs using human monocytic cell line (THP-1) as model to understand the

9

underlying molecular mechanism. A significant (p < 0.01) increase in pro-

10

inflammatory cytokines (TNF-α and IL-1β) and reactive oxygen species (ROS) was

11

observed with a concomitant concentration dependent (0.5, 1, 5, 10, 15 and 20

12

µg/mL) decrease in the glutathione (GSH) levels as compared to control. The

13

expression levels of mitogen activated protein kinase (MAPK) cascade proteins such

14

as p-ERK1/2, p-p38 and p-JNK were also significantly (p < 0.05, p < 0.01) induced.

15

Also, at the concentration tested, NPs induced DNA damage as assessed by the

16

Comet and micronucleus assays. Our data demonstrated that ZnO NPs induce

17

oxidative and nitrosative stress in human monocytes, leading to increased

18

inflammatory response via activation of redox sensitive NF-κB and MAPK signalling

19

pathways.

SC

M AN U

TE D

EP

AC C

20

RI PT

1

21

Keywords: Zinc oxide nanoparticles, cellular uptake, cytokines, oxidative stress,

22

immunotoxicity

23 24

2

ACCEPTED MANUSCRIPT 1. Introduction

2

Nanotechnology is an enabling technology due to the applications of nanoparticles

3

(NPs) in diverse commercial products like paints, sunscreens, drug delivery systems,

4

genetic engineering and other therapeutics. Metal oxide NPs have attracted

5

enormous scientific and technological interest, mainly due to their unique size

6

dependent properties that allow their use as active materials in food, cosmetic,

7

clothing, and in biomedical areas (Johnston et al., 2010; Kreuter and Gelperina,

8

2008).

9

Nanomaterials including ZnO NPs are being explored for the use in medicine,

10

biosensors, laboratory diagnostics, nanoscale devices for drug delivery and as

11

antimicrobial agents (Fortina et al., 2007; Hanley et al., 2008; Patel et al., 2014;

12

Wang et al., 2006). The total US market for nanomedicines is expected to sustain a

13

strong upward pace to $118 billion in 2019 (Nanotechnology, 2010).

14

ZnO is considered to be a “GRAS” (generally recognized as safe) substance by the

15

US Food and Drug Administration (Rasmussen et al., 2010). This holds true for

16

materials in the micron to larger size range. However, when their size is reduced to

17

nanoscale, it can result in adverse effect to human health (Rasmussen et al., 2010).

18

This has been attributed to the fact that as the size is reduced, there is a

19

corresponding increase in the surface area per unit mass (Kumar et al., 2014;

20

Oberdorster et al., 2005). ZnO NPs have been shown to elicit cytotoxic and

21

genotoxic responses in various organ specific cell lines, such as murine blood

22

macrophages, human epidermal cells and others (Hong et al., 2013; Sharma et al.,

23

2009). In an in vivo study, ZnO NPs have been shown to adversely affect the liver,

24

kidney, lung, spleen, and pancreas (Hong et al., 2013).

AC C

EP

TE D

M AN U

SC

RI PT

1

3

ACCEPTED MANUSCRIPT In humans, NPs are first recognized by the cells of the immune system (e.g.

2

monocytes) and their undesirable interactions may lead to immunostimulation or

3

immunosuppression, inflammatory or autoimmune disorders, infections and cancer

4

(Zolnik et al., 2010). Immune system can even engulf and eliminate certain NPs

5

(Dobrovolskaia et al., 2008). The prime function of the immune system is to protect

6

the host from threats caused by toxic substances and pathogens. However,

7

inadvertent recognition of NPs as a toxin, by the immune cells may result in a

8

multilevel immune response against the NPs and eventually lead to immune

9

activation. There are conflicting results in the literature regarding the inflammatory

10

potential of ZnO NPs (Roy et al., 2011; Xia et al., 2008). Studies have also

11

demonstrated that generation of ROS and oxidative stress is the key mechanism in

12

NP-induced toxicity (Park and Park, 2009; Premanathan et al., 2011); however, the

13

role of cytokines is not well understood.

14

In this study, we have addressed the relationship between ZnO NPs immunotoxicity,

15

oxidative stress and MAPK proteins. We have also assessed whether the

16

inflammatory response plays any role in genotoxicity of ZnO NPs. To achieve these

17

aims we have exposed human THP-1 monocytes to a range of concentration of ZnO

18

NPs. THP-1 cells are preferred over human primary monocytes–macrophages as

19

primary cells have the tendency to lose their characteristics under in vitro culture

20

conditions over a period of time and hence pose problems with results reproducibility

21

(Ekwall et al., 1990). Hence, in the present study it was found prudent to use THP-1

22

cells to understand the mechanism of immunotoxicity of ZnO NPs.

23

We have examined the cytokines release on ZnO NPs exposure along with the

24

generation of ROS and alteration in GSH levels. Comet and Cytokinesis- block

25

micronucleus (CBMN) assays determined the link between genotoxicity and

AC C

EP

TE D

M AN U

SC

RI PT

1

4

ACCEPTED MANUSCRIPT inflammatory response. Finally the role of MAPK proteins and transcription factor

2

nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) has been

3

elucidated.

4

We therefore designed the present study to address the immunomodulatory effects

5

of ZnO NPs and also to unravel the mechanism of inflammatory response.

RI PT

1

6

2. Materials and Methods

8

2.1. Chemicals

9

Zinc oxide nanopowder (purity > 99%), low melting point agarose, normal melting

10

agarose, ethidium bromide (EtBr), triton X-100 and poly-L-lysine (PLL) were

11

purchased

12

Ethylenediaminetetraacetic acid (EDTA) disodium salt was procured from Hi-media

13

Pvt. Ltd. (Mumbai, MH India). Foetal bovine serum (FBS), Roswell Park Memorial

14

Institute (RPMI) 1640 media, antibiotic and antimycotic solution (10,000 U/mL

15

penicillin, 10 mg/mL streptomycin, 25 µg/mL amphotericin B), phosphate buffered

16

saline (PBS; Ca2+, Mg2+ free) were purchased from Life Technologies (India) Pvt.

17

Ltd. (New Delhi, India). Cell culture plastic wares were purchased from Thermo

18

Fisher Scientific Nunc (Rochester, NY, USA).

19

2.2. Preparation and characterization of ZnO NPs

20

ZnO NPs were suspended in RPMI medium supplemented with 10% FBS at a

21

concentration of 100 µg/mL and probe sonicated (Sonics Vibra cell, Sonics &

22

Material Inc., New Town, USA) at 32 W for 4 min (1 min on and 30 s off).

23

The size of ZnO NPs was determined by transmission electron microscope (TEM;

24

TechnaiTM G2 Spirit, FEI Company, The Netherlands) by drop coating the stock

Sigma

Chemical

Co.

Ltd.

(St.

Louis,

MO,

USA).

AC C

EP

TE D

from

M AN U

SC

7

5

ACCEPTED MANUSCRIPT suspension (100 µg/mL in Milli-Q water) on formvar coated copper grids and

2

scanned at an accelerating voltage of 80 kV.

3

The size was also determined by scanning electron microscopy (SEM; Quanta FEG

4

450, FEI Company, The Netherlands) by taking ZnO NPs powder on carbon tape.

5

The average hydrodynamic size and zeta potential of ZnO NPs suspension was

6

determined by dynamic light scattering (DLS) and phase analysis light scattering

7

respectively using a Zetasizer Nano-ZS equipped with 4.0 mW, 633 nm laser (Model

8

ZEN3600, Malvern instruments Ltd., Malvern, UK).

9

2.3. Cell culture and exposure to ZnO NPs

SC

RI PT

1

The human monocytic cell line, THP-1 was obtained from National Centre for Cell

11

Sciences (NCCS), Pune, India. Cells were cultured in RPMI 1640 medium

12

supplemented with 10% FBS, 10 mL/L of antibiotic and antimycotic solution at 37 ◦C

13

under humidified conditions with 5% CO2 in cell culture incubator. Cells were plated

14

in 96, 24,12, 6 well culture plates, and 25 cm2 culture flask having a volume of 0.1,

15

0.5, 1, 2 and 5 mL respectively according to the experiments and were treated with

16

different concentrations (0.5, 1, 5, 10, 15 and 20 µg/mL) of ZnO NPs in media at 37

17

◦

18

2.4. Cellular internalization of ZnO NPs

19

2.4.1. Flow cytometry

20

The uptake of ZnO NPs in THP-1 cells was determined by flow cytometry according

21

to the method of Suzuki et al. (2007). This method is based on the principle that the

22

increase in intensity of side scattered (SSC) light with constant intensity of forward

23

scattered (FSC) light in cells reveals increased granularity of cells which is correlated

24

to cellular uptake of NPs. SSC is defined as the laser light scattered at about a 90°

25

angle to the axis of the laser beam while FSC is the laser light scattered at narrow

TE D

M AN U

10

AC C

EP

C for different time intervals.

6

ACCEPTED MANUSCRIPT angles to the axis of laser beam. Briefly 2 x 105 cells were treated with different

2

concentrations (0.5, 1, 5, 10, 15 and 20 µg/mL) of ZnO NPs for 6 h. After treatment,

3

cells were washed with 1 x PBS and the samples were evaluated by flow cytometer

4

(FACS Canto™ II, BD BioSciences, San Jose, CA, USA) equipped with a 488 nm

5

laser.

6

2.4.2. Transmission electron microscopy

7

Approximately 5 x 105 cells were treated with ZnO NPs (20 µg/mL) for 6 h; thereafter

8

the cells were centrifuged at 376 g for 5 min. The pellet formed was resuspended in

9

sodium cacodylate buffer (0.1 M, 200 µL), and fixed in 2.5% glutaraldehyde

10

overnight at 4 ◦C. The pellet was post fixed in osmium tetroxide for 1.5 h at 4 ◦C in

11

dark. Further, the pellet was dehydrated in increasing percentage of acetone,

12

ethanol and embedded in epoxy resin. The sample was then cut into thin sections by

13

diamond knife ultramicrotome and mounted on copper grids. Observations were

14

carried by TEM (TechnaiTM G2 Spirit, FEI Company, The Netherlands) at an

15

accelerating voltage of 80 kV.

16

2.5. Cytotoxicity assays

17

2.5.1. MTT assay

18

Cell viability of ZnO NP treated cells was determined by MTT assay according to the

19

method of Mossman (1983). Briefly, THP-1 cells were seeded in PLL coated plates

20

for 24 h, exposed to ZnO NPs for 3, 6 and 24 h. PLL is a polycation which enhances

21

electrostatic interaction between negatively charged ions of THP-1 cell membrane

22

and the culture surface to adhere THP-1 cells to the surface of the plate. After

23

exposure, treatment was removed and cells were incubated with MTT (3-(4,5-

24

dimethythiazoyl-2-yl)-2,5-diphenyltetrazolium bromide) dye for 3 h. Thereafter,

25

formazan

AC C

EP

TE D

M AN U

SC

RI PT

1

crystals

were

solubilised

by

dimethysulphoxide

and

measured

7

ACCEPTED MANUSCRIPT spectrophotometrically at 530 nm in a plate reader (SYNERGY-HT multiwell plate

2

reader, Bio-Tek, Winooski, VT, USA) using KC4 software. A parallel set of

3

experiment without cells were conducted to check the interference of ZnO NPs with

4

MTT dye.

5

2.5.2. LDH activity

6

Lactate dehydrogenase (LDH) release was determined by LDH assay kit (Sigma

7

Chemical Co. Ltd., St Louis, MO, USA) using manufacturer’s protocol. The

8

absorbance of resulting coloured formazan was measured at 490 and 690 nm in a

9

plate reader (SYNERGY-HT multiwell plate reader, Bio-Tek, Winooski, VT, USA)

10

using KC4 software. A parallel set of experiment without cells were conducted to

11

check the interference of ZnO NPs with dye.

12

2.6. Oxidative and nitrosative stress markers

13

2.6.1. ROS generation

14

ROS generation was determined using 2, 7-dichlorofluorescein diacetate (DCFDA;

15

Sigma Chemical Co. Ltd., St Louis, MO, USA). Cells (1 x 105 cells per well) were

16

treated with different concentrations (0.5, 1, 5, 10, 15 and 20 µg/mL) of ZnO NPs for

17

6 h. After exposure, cells were centrifuged and pellet was washed with 1 x PBS and

18

resuspended in 20 µM of DCFDA prepared in 1 x PBS. Cells were then incubated for

19

20 min at 37 ◦C and fluorescence intensity was detected by flow cytometer (FACS

20

Canto™ II, BD BioSciences, San Jose, CA, USA) equipped with a 488 nm laser.

21

2.6.2. Glutathione (GSH) estimation

22

Free sulphydryl content was measured as described by Ellman (1959). Briefly, cells

23

were exposed to different concentrations (0.5, 1, 5, 10, 15 and 20 µg/mL) of ZnO

24

NPs for 6 h. Thereafter, the cells were sonicated in 0.5 mL of 1 x PBS. To 0.5 mL of

25

homogenate, 0.5 mL of 5% tri-chloro acetic acid (TCA) was added and centrifuged at

AC C

EP

TE D

M AN U

SC

RI PT

1

8

ACCEPTED MANUSCRIPT 845 g for 15 min at 4 ◦C. Further, 0.5 mL of the supernatant was added to 2.5 mL of

2

0.01% 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) and the absorbance was read at

3

412 nm. The protein present in the pellet was determined by the Bradford method

4

(Bradford, 1976) and the concentration of GSH was expressed as µmol/mg protein.

5

2.6.3. Nitric oxide determination

6

Nitric oxide was estimated in cell culture medium as stable nitrite using Greiss

7

reagent as described by Green et al. (1982). 10,000 cells were treated with varying

8

concentrations of ZnO NPs for 6 h in PLL coated 96 well plates as PLL enhances the

9

adherence of THP-1 cells to the culture surface. It is based on the azo dye formation

10

from diazonium salt (formed from reaction between sulfanilic acid and nitrite)

11

coupling to naphthylethylenediamine dihydrochloride. The absorbance was read at

12

540 nm and nitric oxide concentration was determined using sodium nitrite as the

13

standard.

14

2.7. Cytokines quantitation

15

5 x 105 Cells per well in a 24 well plate were exposed to ZnO NPs for 3 h.

16

Thereafter, the supernatant was stored at -80 ◦C. The release of cytokines in the

17

supernatant was quantitated using BDTM cytometric bead array (CBA) human Th-

18

1/Th-2 Cytokine Kit II according to the manufacturer’s protocol (BD Biosciences, San

19

Jose, CA, USA). 50 µl of samples were incubated with 50 µL of mixed capture

20

antibodies along with 50 µL of Phycoerythin detection reagent to form sandwich

21

complexes. Samples were evaluated by flow cytometer (FACSCanto™ II, BD

22

BioSciences, San Jose, CA, USA) and the results were analyzed using FCAP

23

ArrayTM software.

AC C

EP

TE D

M AN U

SC

RI PT

1

24

9

ACCEPTED MANUSCRIPT 2.8. Western blot analysis

2

For western blot analysis, control and 6 h ZnO NP treated cells were pelleted and

3

lysed using CelLyticTM M Cell Lysis Reagent (Sigma Chemical Co. Ltd. St. Louis,

4

MO, USA) in the presence of Na-orthovanadate, Na-fluoride and protease inhibitor

5

cocktail. Protein isolated, was estimated by Bradford method (Bradford, 1976) and

6

bovine serum albumin (BSA) was used as standard. Thirty micrograms of total

7

protein was resolved in 10% and 12% SDS polyacrylamide gel electrophoresis at 25

8

mA/40 V. When the loading dye reaches the bottom of the gel, the power is turned

9

off. The proteins are then electrotransferred at 300 mA/100V for 3 h onto

10

polyvinylidene fluoride membranes. The blotted membrane was blocked with 5%

11

non-fat dry milk and 2% BSA in PBS containing 0.1% Tween 20 (blocking solution),

12

and incubated with specific antibodies against rabbit-monoclonal to nuclear factor

13

kappa B (NF-κB Product no. #3033; 1:1000), rabbit-monoclonal to p-ERK1/2

14

(Product no. #4376; 1:1000), rabbit-monoclonal to p-p38 (Product no. #4631:

15

1:1000), mouse-monoclonal to p-JNK (Product no. #9255; 1:1000), rabbit-polyclonal

16

to ERK1/2 (Product no. #9102; 1:1000), rabbit-polyclonal to p38 (Product no. #9212;

17

1:1000), rabbit-polyclonal to JNK (Product no. #9252; 1:1000), mouse- monoclonal

18

to IL-1β (Product no. #12242; 1:500) and rabbit-polyclonal to COX-2 (Product no.

19

#4842; 1:1000) purchased from Cell Signaling Technology (Danvers, MA, USA). The

20

blots were further incubated with horse radish peroxidase conjugated secondary

21

antibody (1:5000, anti-rabbit polyclonal IgG SAB 3700941 and anti-mouse polyclonal

22

IgG SAB 3701116; Sigma Chemical Co. Ltd., St. Louis, MO, USA) and developed in

23

gel doc (Syngene Bioimaging Private Ltd., Gurgaon, Haryana, India) by enhanced

24

chemiluminescence (Super Signal West Femto Chemiluminescent reagent, Pierce,

25

Rockford, IL, USA). All the blots were stripped and reprobed with rabbit-polyclonal to

AC C

EP

TE D

M AN U

SC

RI PT

1

10

ACCEPTED MANUSCRIPT β-actin (Product no. #4967; 1:10000,) to ensure equal loading of protein.

2

Quantification of the western blots intensity was done by Image J software (NIH,

3

USA).

4

2.9. Genotoxicity assessment

5

2.9.1. Comet assay

6

THP-1 cells (75000 cells per well) were exposed to ZnO NPs for 3 h. Cells were

7

harvested and slides were prepared according to the method described by Singh et

8

al. (1988) and modified by Bajpayee et al. (2005). Cells were lysed in chilled lysing

9

solution (2.5 M NaCl, 100 mM EDTA, 10 mM trizma base (pH 10), along with 1%

10

triton X-100 added just before use) and kept overnight at 4 ◦C. Electrophoresis was

11

performed at 24 V (~ 0.74 V/cm) and 300 mA for 30 min, neutralized with tris buffer

12

(0.4 M, (pH - 7.5)) and stained with 80 µL of 20 µg/mL EtBr. The study design was

13

as follow; Group 1 - control; Group 2 - positive control; Group 3 to Group 8– ZnO

14

NPs (0.5, 1, 5, 10, 15 and 20 µg/mL). 50 random cells (25 cells from each replicate

15

slide) were analyzed for each group as per the guidelines (Tice et al., 2000) at 400 X

16

magnification and DNA fragmentation was analyzed by using an image analysis

17

system (Komet 4.0, ANDOR technology, Belfast, UK.). 16 slides per experiment was

18

analyzed and the experiment was performed in triplicate, therefore a total of 48

19

(16X3) slides were scored.. The % tail DNA and Olive tail moment (OTM; product of

20

tail length and the fraction of total DNA in the tail) (Olive et al., 1993) was analyzed.

21

2.9.2. Cytokinesis- block micronucleus (CBMN) assay

22

CBMN assay was carried according to the method of Fenech (2000). Cells were

23

treated with ZnO NPs for 3 h, centrifuged, washed with 1 x PBS and suspended in

24

complete medium containing cytochalasin-B at a final concentration of 3 µg/mL, in an

25

incubator for 23 h. The cells were harvested and slides for each concentration were

AC C

EP

TE D

M AN U

SC

RI PT

1

11

ACCEPTED MANUSCRIPT prepared by centrifuging cells using the cytospin (Thermo Shandon, Hampshire,

2

UK). Two thousand binucleate cells from each concentration (1000 binucleate cells

3

from each slide, 500 cells per dot) were scored. Total of 16 slides in each experiment

4

was analyzed and experiment was performed thrice. The slides were fixed in chilled

5

methanol for 5 min, air dried and stored until staining. The slides were stained with

6

20 µg/mL acridine orange and observed for the presence of micronuclei (MN) in

7

2000 binucleated cells (BNCs) using fluorescent microscope (DMLB, Leica, Wetzlar,

8

Germany) at 400 X magnification. The presence of micronucleus (MN) in 2000 BNCs

9

was counted and the result for the MN frequency was expressed as MN/1000 BNCs.

10

The nuclear division index (NDI) was also calculated from 500 cells per

11

concentration according to the formula:

12

NDI = (No. of mononucleate cells + 2 x No. of binucleate cells + 3 x No. of trinucleate

13

cells + 4 x No. of quadrinuclear cells) / Total No. of cells

14

2.10. Statistical analysis

15

Results were expressed as mean ± standard error of mean (S.E.M.) of three

16

experiments and data were analyzed using one way analysis of variance (ANOVA)

17

followed by a Dunnett’s post hoc multiple comparison test from GraphPad Prism-3.0

18

(GraphPad Prism software, San Diego, California, USA). In all cases, p < 0.05 and p

19

< 0.01 was considered significant.

SC

M AN U

TE D

EP

AC C

20

RI PT

1

21

3. Results

22

3.1. Particle characterization

23

ZnO NPs was in the form of white solid nanopowder. The specific surface area was

24

15 - 25 m2/g (as described in the product specification sheet obtained from Sigma

25

Chemical Co. Ltd., St. Louis, MO, USA). The mean hydrodynamic size of ZnO NPs 12

ACCEPTED MANUSCRIPT 1

as determined by DLS is 264.4 nm in RPMI medium and 297 nm in Milli-Q water.

2

The zeta potential in RPMI medium is -13.4 mV and in Milli-Q water is -25.3 mV. The

3

average size of ZnO NPs measured by TEM and SEM was 30 nm (Table 1).

4

Nanopa

TEM

SEM

DLS

DLS

Zeta

Zeta

rticle

size

size

(Hydrodynamic

(Hydrodynamic

potential

potential

diameter in

diameter in

in Milli-Q

in media

media)

water

ZnO

~30 nm

~30 nm

297 nm

264.4 nm

-25.3 mV

-13.4 mV

TE D

6

M AN U

Milli-Q water)

7

RI PT

Table 1. Characterization of ZnO NPs by TEM, SEM and DLS.

SC

5

3.2. Cellular uptake of ZnO NPs

9

A significant (p < 0.01) concentration dependent increase in SSC intensity was

10

observed (Figure 1 A) in ZnO NP treated cells indicating their internalization. At

11

exposure concentration 15 and 20 µg/mL the mean SSC intensity (arbitrary unit) was

12

1662 and 2182.6 respectively, as compared to the control (1242). Internalization of

13

the NPs in cells was also observed in TEM, where ZnO NPs were found to be

14

localised in the endosomes (Figure 1 C-E) as compared to control (Figure 1 B). A

15

distinct endosome formation as well as disintegration of the mitochondrial membrane

16

was observed in ZnO NP treated cells (Figure 2 A-B).

AC C

EP

8

13

1

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

2

Figure 1. Internalization of ZnO NPs in THP-1 cells. (A) Analysis of internalization of

3

ZnO NPs by flow cytometric parameter viz. SSC intensity (B) Control cells, (C) ZnO

4

NPs treated cells and (D-E) are higher magnification of area marked with black

5

boxes in (C); white arrows indicate accumulation of NPs inside endosomes. 14

RI PT

ACCEPTED MANUSCRIPT

1

Figure 2. TEM photomicrographs showing ultrastructural changes in ZnO NP treated

3

cells. The white arrows indicate (A) endosome formation and (B) mitochondrial

4

membrane disintegration.

5

3.3. Cytotoxicity assessment

6

MTT and LDH assays demonstrated a significant (p < 0.05, p < 0.01) concentration

7

and time dependent increase in the cytotoxicity in THP-1 cells after ZnO NPs

8

exposure.

9

The mitochondrial succinate dehydrogenase activity was reduced to 83.8%, 59.7%

10

and 47.4% respectively after 3, 6 and 24 h of exposure to 20 µg/mL ZnO NPs in

11

THP-1 cells, as compared to control (Figure 3 A).

M AN U

TE D

EP

13

A significant (p < 0.05, p < 0.01) increase (181.2% and 195.1%) in LDH release was observed in cells treated with 15 and 20 µg/mL ZnO NPs after 24 h (Figure 3 B).

AC C

12

SC

2

15

ACCEPTED MANUSCRIPT

120

3h

6h

24h

100 80

**

60

**

RI PT

% MTT reduction

A

**

40

**

20 0 0.5

1

5

10

15

20

SC

Control

**

ZnO NPs (µg/ml) 3h

B

6h

24h

M AN U

250

**

*

150

** **

*

100 50 0 Control

TE D

% LDH release

200

0.5

1

5

10

15

20

EP

ZnO NPs (µg/ml)

1

Figure 3. Concentration and time dependent cytotoxicity of ZnO NPs in THP-1 cells,

3

(A) % MTT reduction, (B) % LDH release. Results represent mean ± S.E.M. of three

4

independent experiments. *p < 0.05, **p < 0.01, when compared to control.

5

3.4. Evaluation of oxidative stress

6

ZnO NPs (15 and 20 µg/mL) treated cells showed a significant (p < 0.05)

7

concentration dependent increase in the intracellular ROS as observed by the

8

increase in the intensity of fluorescence of DCFDA dye (Figure 4 A). A significant (p

9

< 0.05) concentration dependent reduction in GSH levels (65.6% and 61.2%) was

AC C

2

16

ACCEPTED MANUSCRIPT observed in cells treated with ZnO NPs (15 and 20 µg/mL) for 6 h (Figure 4 B). Also,

2

a concentration dependent significant (p < 0.05) increase in nitrite levels was

3

observed in cells treated with ZnO NPs (15 and 20 µg/mL) when compared to control

4

(Figure 4 C).

% ROS generation

A

*

200

* 150

100

50

0 Control

0.5

1

5

10

15

0.6

B 0.5

*

0.4 0.3 0.2 0.1 0.0

20

Control

0.5

1

5

10

15

*

20

ZnO NPs (µg/ml)

0.14

*

C

M AN U

Concentration of nitrite (µM)

ZnO NPs (µg/ml)

*

0.12 0.10 0.08 0.06 0.04 0.02 0.00 Control

0.5

1

5

10

15

20

ZnO NPs (µg/ml)

TE D

5

RI PT

250

SC

Glutathione (µmol/mg protein)

1

Figure 4. Effect of ZnO NPs on, (A) Reactive oxygen species (ROS), (B) Glutathione

7

(GSH) and (C) Nitrite levels. Data represents the mean ± S.E.M. of three

8

independent experiments. *p < 0.05 when compared with control.

9

3.5. Cytokine quantitation

AC C

EP

6

10

A statistically significant (p < 0.01) increase in the level of tumour necrosis factor-

11

alpha (TNF-α; Figure 5) was observed in cells treated with ZnO NPs at

12

concentrations ranging from 5-20 µg/mL. Release of IL-6 and INF-γ was also

13

obtained but the results were not significant compared to control (data not shown).

14

17

250

** 200

**

**

**

*

150

50

0 Control

0.5

1

5

10

ZnO NPs (µg/ml) 1

15

RI PT

100

20

SC

Concentration of TNF-α(pg/ml)

ACCEPTED MANUSCRIPT

Figure 5. Effect of ZnO NPs on TNF-α release in THP-1 cells. Data represents the

3

mean ± S.E.M. of three independent experiments. **p < 0.01 when compared with

4

control.

5

3.6. Western blot analysis

6

A significant (p < 0.05, p < 0.01) increase (1.9-2.6 fold) in the levels of inflammatory

7

marker proteins (IL-1β, COX-2) and transcription factor (NF-κB) in THP-1 cells was

8

observed after treatment with ZnO NPs (20 µg/mL; Figure 6 A and C).

9

Also, a significant (p < 0.05, p < 0.01) increase (1.67-2.25 fold) in phosphorylation of

10

proteins viz extracellular signal-regulating kinase (ERK1/2), Jun-N-terminal kinase

11

(JNK) and p38 was observed in cells treated with 20 µg/mL ZnO NPs. However, no

12

significant change was observed in the expression levels of total ERK1/2, JNK and

13

p38 which also confirmed equal protein loading. The expression profile of β actin

14

was used as an internal control for protein loading in control and ZnO NPs treated

15

samples (Figure 6 B and D).

AC C

EP

TE D

M AN U

2

18

1

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 6. Western blot analysis of proteins from THP-1 cells treated with ZnO NPs

3

(1, 10 and 20 µg/mL). (A) IL-1β, COX-2 and NF-κB, (B) MAPK signalling proteins

4

(phospho- ERK1/2, phospho-JNK and phospho-p38) and the corresponding (C and

5

D) bar graphs showing their densitometric analysis. β-actin was used as internal

6

control. Results represent mean ± S.E.M. of three independent experiments. *p <

7

0.05, **p < 0.01, when compared to control.

AC C

2

8 9 19

ACCEPTED MANUSCRIPT 3.7. Genotoxic potential of ZnO NPs

2

Comet assay data showed a significant (p < 0.05) increase in DNA damage as

3

evident from the Olive tail moment (OTM) and tail DNA (%) values. The cells treated

4

with ZnO NPs (20 µg/ml) exhibited an OTM of 3.11 in comparison to control (0.84;

5

Table 2).

6

Table 2. DNA damage in THP-1 cells after 3 h exposure to ZnO NPs as evident by

7

Comet parameters. Groups

OTMa (arbitrary unit)

Control

0.84 ± 0.08

1 mM EMSb

19.00 ± 0.53**

ZnO NPs (0.5 µg/mL)

1.83 ± 0.46

15.86 ± 2.27*

2.13 ± 0.43

18.63 ± 2.08**

2.46 ± 0.46

20.67 ± 1.56**

2.58 ± 0.49

21.54 ± 1.97**

2.69 ± 0.48

21.87 ± 2.04**

3.11 ± 0.85*

23.59 ± 3.59**

ZnO NPs (10 µg/mL) ZnO NPs (15 µg/mL)

SC

Tail DNA (%) 7.92 ± 0.60

64.75 ± 0.90**

EP

ZnO NPs (20 µg/mL)

TE D

ZnO NPs (5 µg/mL)

M AN U

ZnO NPs (1 µg/mL)

8

RI PT

1

Values are expressed as mean ± S.E.M. of three experiments; aOTM- Olive tail

10

moment; bEMS- ethyl methanesulphonate- positive control; *p < 0.05, **p < 0.01

11

when compared with control.

12

Increase in number of micronucleated cells was also observed with increasing

13

concentration of ZnO NPs (10, 15 and 20 µg/mL) when compared to control. At the

14

highest concentration of ZnO NPs (20 µg/ml) a significant (p < 0.01) increase in the

15

micronucleus frequency (20.67 MN/1000 BNCs) was observed as compared to the

16

control (7.33 MN/1000 BNCs; Table 3).

AC C

9

20

ACCEPTED MANUSCRIPT 1 2

Table 3. Effect of ZnO NPs on micronucleus formation in THP-1 cells. Groups

No. of MN/1000 BNCs

NDIa

% BNCs/ 500 viable cells

7.33 ± 0.33

1.99 ± 0.002

100 ± 0.00

1 mM EMSb

24.67 ± 0.33**

1.82 ± 0.001**

82.90 ± 0.36**

ZnO NPs (0.5 µg/mL)

7.67 ± 0.67

1.98 ± 0.003

99.12 ± 0.18

ZnO NPs (1 µg/mL)

8.67 ± 1.45

1.98 ± 0.002

98.38 ± 0.12**

ZnO NPs (5 µg/mL)

11.67 ± 1.45

1.96 ± 0.003**

95.89 ± 0.24**

ZnO NPs (10 µg/mL)

15.00 ± 1.53**

1.96 ± 0.005**

93.60 ± 0.24**

ZnO NPs (15 µg/mL)

17.67 ± 1.45**

1.95 ± 0.005**

93.27 ± 0.35**

ZnO NPs (20 µg/mL)

20.67 ± 0.67**

1.91 ± 0.005**

89.16 ± 0.36**

TE D

M AN U

SC

RI PT

Control

3

Values are expressed as mean ± S.E.M. of three experiments; aNDI- Nuclear division

5

index; bEMS- ethyl methane sulphonate- positive control; *p < 0.05, **p < 0.01 when

6

compared with control.

AC C

7

EP

4

21

ACCEPTED MANUSCRIPT 4. Discussion

2

Monocytes play a significant role in the host defence against any foreign body

3

including microorganisms. Therefore, in the present study it was thought prudent to

4

use monocytes as a model to investigate the immunotoxicity of ZnO NPs. The innate

5

immune mechanism detects the nonself antigens, resulting in imbalance of pro-

6

inflammatory and cell signalling molecules. Therefore, the evaluation of pro-

7

inflammatory response in monocytes treated with ZnO NPs is important in

8

nanotoxicological assessments.

9

Exposure of ZnO NPs elicits cytotoxic, genotoxic and pro-inflammatory responses

10

(Hackenberg et al., 2011; Sharma et al., 2009). Prach et al. (2013) demonstrated

11

that ZnO NPs exposure to THP-1 cells did not induce the release of pro-

12

inflammatory cytokines. This was due to the fact that the cytokine got bound to ZnO

13

NPs after 24 h of incubation and was masked from being detected in the ELISA. To

14

overcome the shortcomings, the present study was designed to evaluate the

15

immunomodulatory effect of ZnO NPs in THP-1 cells after 3 h and the mechanism of

16

inflammatory response was also determined.

17

Size of the NPs is a crucial parameter that determines their potential to induce

18

cytokine production; hence, the particle needs to be characterized thoroughly in

19

suspension and powder form (Zolnik et al., 2010). In the present study, the size of

20

ZnO NPs as measured by DLS in complete RPMI media and Milli-Q water was 264.4

21

and 299 nm respectively. The decrease in size of NPs in media has been attributed

22

to the presence of serum which coats the particles and thus decreases

23

agglomeration (Dhawan and Sharma, 2010; Kumar et al., 2011b).

24

Since these NPs fall within a small size distribution range, they can easily enter into

25

the cells and localise in the organelles. The present study, through TEM

AC C

EP

TE D

M AN U

SC

RI PT

1

22

ACCEPTED MANUSCRIPT measurements clearly shows that ZnO NPs enter the cells through endocytosis or

2

phagocytosis as evidenced by the formation of endosomes (Figure 2 A). The

3

internalization was further confirmed by the flow cytometry data where a

4

concentration-dependent increase in the intensity of SSC was observed. It has been

5

shown that SSC intensity is an indicator for the uptake of NPs (Kumar and Dhawan,

6

2013; Kumar et al., 2011a; Shukla et al., 2013; Suzuki et al., 2007). Distribution of

7

these NPs inside cells would therefore enable their interactions with biological

8

macromolecules, including lipids, proteins and nucleic acids thereby eliciting toxic

9

responses.

SC

RI PT

1

In the present study, THP-1 cells were exposed to ZnO NPs at different

11

concentrations (0.5, 1, 5, 10, 15 and 20 µg/mL) and time points (3, 6 and 24 h). The

12

endocytosed particles showed significant cytotoxicity at concentration 10, 15 and 20

13

µg/mL after 6 and 24 h of exposure as observed in MTT and LDH release assay.

14

However, the cells were more than 80% viable after 3 h exposure of ZnO NPs.

15

Hence, this time point was used for the immunomodulatory and genotoxicity studies.

16

The entry of ZnO NPs into the cells induced mitochondrial membrane damage as

17

observed in TEM (Figure 2 B). This led to ROS generation, oxidative stress and

18

inflammatory response. This is in accordance with similar studies reported earlier

19

(Sharma et al., 2009; 2011; Xia et al., 2008) which show that such responses lead to

20

protein, membrane and DNA damage. Our data showed a significant increase in

21

ROS generation and depletion in GSH levels with a concomitant increase in nitrite

22

production revealing that ZnO NPs induce oxidative and nitrosative stress. The

23

semiconductor property of ZnO NPs is also considered as one of the probable

24

reason for ROS generation. Crystalline ZnO NPs have a band gap of ~ 3.3 eV (Lany

25

et al., 2007). Due to transfer of electrons from valence band to conduction band,

AC C

EP

TE D

M AN U

10

23

ACCEPTED MANUSCRIPT electron holes are created in the valence band. Electrons and holes sometimes

2

reach the surface of NPs where, electrons react with oxygen and holes react with

3

hydroxyl ions or water forming superoxide and hydroxyl radicals. As the size of ZnO

4

NPs decreases, crystal defects are created, forming a large number of electron-hole

5

pairs. The holes act as oxidants and can split water molecules into H+ and OH- while

6

the conduction band electrons act as reducers and reacts with dissolved oxygen

7

molecules to generate superoxide radical anions (Rasmussen et al., 2010). NO and

8

ROS are known to cooperatively act in the in vivo vasodilation of endothelium walls

9

thus leading to the release of pro-inflammatory mediators (Kvietys and Granger,

SC

RI PT

1

2012).

11

Our study also showed that ZnO NPs stimulated the release of pro-inflammatory

12

cytokines, IL-1β and TNF-α. These results are consistent with the data reported

13

earlier on nasal mucosa cells (Hackenberg et al., 2011). Increase in expression of

14

inflammatory marker, COX-2 was obtained on ZnO NPs treatment in cells. The

15

oxidative and nitrosative stress response upregulates the expression of the

16

inflammatory mediators such as IL-1β, TNF-α and COX-2 via the activation of the

17

transcription factor NF-кB (Lu and Wahl, 2005). NF-κB gets activated by the

18

phosphorylation of inhibitory protein (IκBα) by IκB Kinase and concomitant

19

translocation of NF-κB (p50, p65) from cytoplasm to nucleus. A significant

20

concentration dependent increase in the expression of NF-κB was obtained, which is

21

a pivotal mediator involved in multiple cellular responses and regulates the release

22

of pro-inflammatory mediators (Bonizzi and Karin, 2004). Therefore, the activation of

23

NF-κB explains the enhanced expression of inflammatory cytokines.

24

The present study showed that ZnO NPs cause DNA damage as evident from the

25

data of the Comet and CBMN assays. This could be attributed to the uptake of NPs

AC C

EP

TE D

M AN U

10

24

ACCEPTED MANUSCRIPT as evident by TEM in conjunction with an increase in ROS and nitrite levels along

2

with decrease in GSH (AshaRani et al., 2009; Kumar and Dhawan, 2013; Sharma et

3

al., 2012; Sharma et al., 2011). The release of inflammatory cytokines could also be

4

a probable reason for DNA damage caused by ZnO NPs (Totsuka et al., 2014).

5

Additionally, the role of signalling pathways involved in inflammatory response was

6

explored. In the present study, it was observed that the JNK, ERK1/2 and p38 that

7

constitute the MAPK cascade play a key role in the inflammatory response of ZnO

8

NPs as evident from the increased expression levels of phosphorylated JNK,

9

phosphorylated ERK1/2 and phosphorylated p38. The increase in oxidative and

10

nitrosative stress induced the activation of MAPK proteins (Kumar et al., 2015;

11

Mccubrey et al., 2006). MAPKs phosphorylate specific serine, threonine and tyrosine

12

residues of these proteins thereby modulating their function. Studies have shown

13

that JNK, a major subgroup of MAPK, is activated primarily by inflammatory

14

cytokines and environmental stress and phosphorylated on threonine-183 and

15

tyrosine-185 residues by the dual specific kinases MKK4 and MKK7 (Davis, 2000).

16

p38 is phosphorylated by MKK3 on threonine-180 and tyrosine-182 and MAPK/ERK

17

kinases (MEK1 and MEK2) phosphorylate ERK1/2 within a conserved threonine-

18

glutamine-tyrosine motif (Raingeaud et al., 1995; Roux and Blenis, 2004). Activation

19

of both ERK1/2 and p38 kinase is necessary in cytokine gene expression (Carter et

20

al., 1999). Therefore, corroborating with the earlier studies our results demonstrate

21

that ZnO NPs induced inflammatory cytokines mediated the activation of MAPK

22

proteins. The phosphorylation of JNK, ERK1/2 and p38 could further activate the

23

transcription factor, activator protein 1 (AP-1), consequently leading to enhanced

24

pro-inflammatory transcripts causing inflammation.

AC C

EP

TE D

M AN U

SC

RI PT

1

25

ACCEPTED MANUSCRIPT In conclusion, our study determined the immunomodulatory effect of ZnO NPs and

2

led to the determination of the possible pathway of ZnO NPs induced inflammatory

3

response as depicted in figure 7. Therefore the indiscriminate use of NPs in

4

cosmetics, paints and other consumer goods should be avoided and guidelines

5

should be implemented for NPs usage.

EP

TE D

M AN U

SC

RI PT

1

6

Figure 7. A schematic showing possible mechanism of ZnO NPs induced

8

inflammatory response in human monocytic cells. The dotted lines represent the

9

pathway of other inflammatory mediators activated during inflammation. Activator

10

AC C

7

protein 1 (AP-1).

11

26

ACCEPTED MANUSCRIPT 1

Acknowledgements

2

This work was supported by the Council of Scientific and Industrial Research, New

3

Delhi, India through network projects NWP35 and NanoSHE (BSC0112), and the EU

4

Framework

5

(NanoValid- Development of reference methods for hazard identification, risk

6

assessment and LCA of engineered nanomaterials) is acknowledged. The funding

7

received from the Gujarat Institute of Chemical Technology, India under CENTRA

8

(Centre for Nanotechnology and Research Application) project in grant agreement

9

number (ILS/GICT/2013/003) is also gratefully acknowledged. Violet A. Senapati

10

thanks Council of Scientific and Industrial Research (CSIR), New Delhi for the award

11

of Junior and Senior Research Fellowship.

(FP7/2007-2013)

under

Grant

Agreement

263147

M AN U

SC

RI PT

Programme

AC C

EP

TE D

12

27

ACCEPTED MANUSCRIPT 1

References

2

AshaRani, P. V., Low, K. M. G., Hande, M. P., Valiyaveettil, S., 2009. Cytotoxicity

3

and genotoxicity of silver nanoparticles in human cells. ACS Nano. 3, 279-

4

290. Bajpayee, M., Pandey, A. K., Parmar, D., Mathur, N., Seth, P. K., Dhawan, A., 2005.

6

Comet assay responses in human lymphocytes are not influenced by the

7

menstrual cycle: a study in healthy Indian females. Mutat. Res. 565, 163-172.

9

Bonizzi, G., Karin, M., 2004. The two NF-kappa B activation pathways and their role

SC

8

RI PT

5

in innate and adaptive immunity. Trends in Immunology. 25, 280-288. Bradford, M. M., 1976. A rapid and sensitive method for the quantitation of

11

microgram quantities of protein utilizing the principle of protein-dye binding.

12

Anal. Biochem. 72, 248-254.

M AN U

10

Carter, A. B., M., M. M., Hunninghake, G. W., 1999. Both Erk and p38 Kinases are

14

necessary for cytokine gene transcription. Am J. Respir. Cell Mol. Biol. 20,

15

751–758.

18 19

239-252.

EP

17

Davis, R. J., 2000. Signal transduction by the JNK group of MAP kinases. Cell. 103,

Dhawan, A., Sharma, V., 2010. Toxicity assessment of nanomaterials: methods and

AC C

16

TE D

13

challenges. Anal. Bioanal. Chem. 398, 589-605.

20

Dobrovolskaia, M. A., Aggarwal, P., Hall, J. B., McNeil, S. E., 2008. Preclinical

21

studies to understand nanoparticle interaction with the immune system and its

22

potential effects on nanoparticle biodistribution. Mol. Pharm. 5, 487-495.

23

Ekwall, B., Silano, V., Paganuzzi-Stammati, A., Zucco, F., 1990. Chapter 7- Toxicity

24

Tests with Mammalian Cell Cultures. Short-term Toxicity Tests for Non-

28

ACCEPTED MANUSCRIPT 1

genotoxic Effects. Edited by P. Bourdeau et al John Wiley & Sons, Inc; New

2

York. 75-97. Ellman, G. L., 1959. Tissue sufhydryl groups. Arch. Biochem. Biophys. 82, 70-77.

4

Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res. 455, 81-95.

5

Fortina, P., Kricka, L. J., Graves, D. J., Park, J., Hyslop, T., Tam, F., Halas, N.,

6

Surrey, S., Waldman, S. A., 2007. Applications of nanoparticles to diagnostics

7

and therapeutics in colorectal cancer. TRENDS in Biotechnology. 25, 145-

8

152.

SC

RI PT

3

Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S.,

10

Tannenbaum, S. R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in

11

biological samples. Anal. Biochem. 126, 131-138.

M AN U

9

Hackenberg, S., Scherzed, A., Technau, A., Kessler, M., Froelich, K., Ginzkey, C.,

13

Koehler, C., Burghartz, M., Hagen, R., Kleinsasser, N., 2011. Cytotoxic,

14

genotoxic and pro-inflammatory effects of zinc oxide nanoparticles in human

15

nasal mucosa cells in vitro. Toxicol. In Vitro. 25, 657-663.

TE D

12

Hanley, C., Layne, J., Punnoose, A., Reddy, K. M., Coombs, I., Coombs, A., Feris,

17

K., Wingett, D., 2008. Preferential killing of cancer cells and activated human

18

T cells using ZnO nanoparticles. Nanotechnology. 19, 295103.

AC C

EP

16

19

Hong, T. K., Tripathy, N., Son, H. J., Ha, K. T., Jeong, H. S., Hahn, Y. B., 2013. A

20

comprehensive in vitro and in vivo study of ZnO nanoparticles toxicity. J.

21

Mater. Chem. B. 1, 2985-2992.

22

Johnston, H. J., Hutchison, G., Christensen, F. M., Peters, S., Hankin, S., Stone, V.,

23

2010. A review of the in vivo and in vitro toxicity of silver and gold particulates:

24

particle attributes and biological mechanisms responsible for the observed

25

toxicity. Crit. Rev. Toxicol. 40, 328-346.

29

ACCEPTED MANUSCRIPT 1 2 3 4

Kreuter, J., Gelperina, S., 2008. Use of nanoparticles for cerebral cancer. Tumori. 94, 271-277. Kumar, A., Dhawan, A., 2013. Genotoxic and carcinogenic potential of engineered nanoparticles: an update. Arch. Toxicol. 87, 1883-1900. Kumar, A., Khan, S., Dhawan, A., 2014. Comprehensive molecular analysis of the

6

responses induced by titanium dioxide nanoparticles in human keratinocyte

7

cells. J. Transl. Toxicol. 1, 28-39.

RI PT

5

Kumar, A., Najafzadeh, M., Jacob, B. K., Dhawan, A., Anderson, D., 2015. Zinc

9

oxide nanoparticles affect the expression of p53, Ras p21 and JNKs: an ex

10

vivo/in vitro exposure study in respiratory disease patients. Mutagenesis. 30,

11

237-245.

M AN U

SC

8

Kumar, A., Pandey, A. K., Singh, S. S., Shanker, R., Dhawan, A., 2011a. A flow

13

cytometric method to assess nanoparticle uptake in bacteria. Cytometry Part

14

A. 79A, 707-712.

TE D

12

Kumar, A., Pandey, A. K., Singh, S. S., Shanker, R., Dhawan, A., 2011b. Engineered

16

ZnO and TiO2 nanoparticles induce oxidative stress and DNA damage

17

leading to reduced viability of Escherichia coli. Free Radic. Biol. Med. 51,

18

1872-1881.

20

AC C

19

EP

15

Kvietys, P. R., Granger, D. N., 2012. Role of reactive oxygen and nitrogen species in the vascular responses to inflammation. Free Radic. Biol. Med. 52, 556-592.

21

Lany, S., Osorio-Guillen, J., Zunger, A., 2007. Origins of the doping asymmetry in

22

oxides: Hole doping in NiO versus electron doping in ZnO. Phys. Rev. B. 75,

23

2031-2034.

24

Lu, Y., Wahl, L. M., 2005. Oxidative stress augments the production of

25

matrixmetalloproteinase-1, cyclooxygenase-2, and prostaglandin E2 through

30

ACCEPTED MANUSCRIPT 1

enhancement of NF-Kappa B activity in lipopolysaccharide-activated human

2

primary monocytes. J. Immunol. 175, 5423-5429. Mccubrey, J. A., Lahair, M. M., Franklin, R. A., 2006. Reactive Oxygen Species-

4

Induced Activation of the MAP Kinase Signaling Pathways. Antioxidants &

5

redox signaling. 8, 1775-1789.

RI PT

3

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:

7

application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65,

8

55-63.

9

SC

6

Nanotechnology. (2010) Nanotechnology in health care: US industry study with forecasts

for

2014

11

http://www.freedoniagroup.com/brochure/26xx/2622smwe.pdf. In.

M AN U

10

&

2019.

Oberdorster, G., Oberdorster, E., Oberdorster, J., 2005. Nanotoxicology: an

13

emerging discipline evolving from studies of ultrafine particles. Environ. Health

14

Perspect. 113, 823-839.

TE D

12

Olive, P. L., Frazer, G., Banath, J. P., 1993. Radiation-induced apoptosis measured

16

in Tk6-human B-lymphoblast cells using the comet assay. Radiat. Res. 136,

17

130-136.

19

Park, E. J., Park, K., 2009. Oxidative stress and pro-inflammatory responses induced

AC C

18

EP

15

by silica nanoparticles in vivo and in vitro. Toxicol. Lett. 184, 18-25.

20

Patel, P., Kansara, K., Shah, D., Vallabani, N. V., Shukla, R. K., Singh, S., Dhawan,

21

A., Kumar, A., 2014. Cytotoxicity assessment of ZnO nanoparticles on human

22

epidermal cells. Mol. Cytogenet. 7(Suppl 1), 81.

23

Prach, M., Stone, V., Proudfoot, L., 2013. Zinc oxide nanoparticles and monocytes:

24

impact of size, charge and solubility on activation status. Toxicol. Appl.

25

Pharmacol. 266, 19-26.

31

ACCEPTED MANUSCRIPT 1

Premanathan, M., Karthikeyan, K., Jeyasubramanian, K., Manivannan, G., 2011.

2

Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and

3

cancer cells by apoptosis through lipid peroxidation. Nanomed-Nanotechnol.

4

Biol. Med. 7, 184-192. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., Davis,

6

R. J., 1995. Pro-inflammatory cytokines and environmental stress cause p38

7

mitogen-activated protein kinase activation by dual phosphorylation on

8

tyrosine and threonine. J. Biol. Chem. 270, 7420-7426.

SC

RI PT

5

Rasmussen, J. W., Martinez, E., Louka, P., Wingett, D. G., 2010. Zinc oxide

10

nanoparticles for selective destruction of tumor cells and potential for drug

11

delivery applications. Expert Opin. Drug Deliv. 7, 1063-1077.

M AN U

9

Roux, P. P., Blenis, J., 2004. ERK and p38 MAPK-Activated Protein Kinases: a

13

Family of Protein Kinases with Diverse Biological Functions. Microbiol. Mol.

14

Biol. Rev. 68, 320-344.

TE D

12

Roy, R., Tripathi, A., Das, M., Dwivedi, P. D., 2011. Cytotoxicity and uptake of zinc

16

oxide nanoparticles leading to enhanced inflammatory cytokines levels in

17

murine macrophages: comparison with bulk zinc oxide. J. Biomed.

18

Nanotechnol. 7, 110-111.

20

AC C

19

EP

15

Sharma, V., Kumar, A., Dhawan, A., 2012. Nanomaterials: exposure, effects and toxicity assessment. Proc. Natl. Acad. Sci. India Sect. B Biol. Sci. 82, 3-11.

21

Sharma, V., Shukla, R. K., Saxena, N., Parmar, D., Das, M., Dhawan, A., 2009. DNA

22

damaging potential of zinc oxide nanoparticles in human epidermal cells.

23

Toxicol. Lett. 185, 211-218.

32

ACCEPTED MANUSCRIPT 1

Sharma, V., Singh, S. K., Anderson, D., Tobin, D. J., Dhawan, A., 2011. Zinc oxide

2

nanoparticle induced genotoxicity in primary human epidermal keratinocytes.

3

J. Nanosci. Nanotechnol. 11, 3782–3788. Shukla, R. K., Kumar, A., Gurbani, D., Pandey, A. K., Singh, S., Dhawan, A., 2013.

5

TiO2 nanoparticles induce oxidative DNA damage and apoptosis in human

6

liver cells. Nanotoxicology. 7, 48-60.

RI PT

4

Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L., 1988. A simple technique

8

for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res.

9

175, 184-191.

SC

7

Suzuki, H., Toyooka, T., Ibuki, Y., 2007. Simple and easy method to evaluate uptake

11

potential of nanoparticles in mammalian cells using a flow cytometric light

12

scatter analysis. Environ. Sci. Technol. 41, 3018–3024.

M AN U

10

Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H.,

14

Miyamae, Y., Rojas, E., Ryu, J. C., Sasaki, Y. F., 2000. Single cell gel

15

electrophoresis/Comet assay: guidelines for in vitro and in vivo genetic

16

toxicology testing. Environ. Mol. Mutagen. . 35, 206-221.

TE D

13

Totsuka, Y., Ishino, K., Kato, T., Goto, S., Tada, Y., Nakae, D., Watanabe, M.,

18

Wakabayashi, K., 2014. Magnetite nanoparticles induce genotoxicity in the

20 21

AC C

19

EP

17

lungs of mice via inflammatory response. Nanomaterials. 4, 175-188.

Wang, L., O'Donoghue, M. B., Tan, W., 2006. Nanoparticles for multiplex diagnostics and imaging. Nanomed. 1, 413-426.

22

Xia, T., Kovochich, M., Liong, M., Madler, L., Gilbert, B., Shi, H., Yeh, J. I., Zink, J. I.,

23

Nel, A. E., 2008. Comparison of the mechanism of toxicity of zinc oxide and

24

cerium oxide nanoparticles based on dissolution and oxidative stress

25

properties. ACS Nano. 2, 2121-2134.

33

ACCEPTED MANUSCRIPT 1

Zolnik, B. S., Gonzalez-Fernandez, A., Sadrieh, N., Dobrovolskaia, M. A., 2010.

2

Minireview: Nanoparticles and the Immune System. Endocrinology. 151, 458-

3

465.

4

AC C

EP

TE D

M AN U

SC

RI PT

5

34

ACCEPTED MANUSCRIPT Highlights

RI PT

SC M AN U

• •

TE D

•

EP

•

Zinc oxide (ZnO) nanoparticles (NPs) are widely used in cosmetics, food packaging, medicine etc but their unique properties cause deleterious health effects This study was performed to evaluate the immunomodulatory and genotoxic effect of ZnO NPs in human monocytic cell line (THP-1) ZnO NPs exposure to THP-1 cells leads to the release of pro-inflammatory cytokines with increase in oxidative and nitrosative stress ZnO NPs induced activation of NF-κB and MAPK signalling pathway There is a need to implement guidelines for NPs usage in consumer goods

AC C

•