BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

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Nanotoxicology

ISSN: 1743-5390 (Print) 1743-5404 (Online) Journal homepage: https://www.tandfonline.com/loi/inan20

Acute phase response and inflammation following

pulmonary exposure to low doses of zinc oxide

nanoparticles in mice

Niels Hadrup, Feriel Rahmani, Nicklas R. Jacobsen, Anne T. Saber, Petra

Jackson, Stefan Bengtson, Andrew Williams, Håkan Wallin, Sabina

Halappanavar & Ulla Vogel

To cite this article:

Niels Hadrup, Feriel Rahmani, Nicklas R. Jacobsen, Anne T. Saber, Petra

Jackson, Stefan Bengtson, Andrew Williams, Håkan Wallin, Sabina Halappanavar & Ulla Vogel

(2019): Acute phase response and inflammation following pulmonary exposure to low doses of zinc

oxide nanoparticles in mice, Nanotoxicology, DOI: 10.1080/17435390.2019.1654004

To link to this article:

https://doi.org/10.1080/17435390.2019.1654004

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BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

https://doi.org/10.1080/17435390.2019.1654004

ARTICLE

Acute phase response and inflammation following pulmonary exposure to

low doses of zinc oxide nanoparticles in mice

Niels Hadrup

, Feriel Rahmani

, Nicklas R. Jacobsen

, Anne T. Saber

, Petra Jackson

, Stefan Bengtson

Andrew Williams

, Håkan Wallin

, Sabina Halappanavar

and Ulla Vogel

a,d

National Research Centre for the Working Environment, Copenhagen, Denmark;

Environmental Health Science and Research

Bureau, Health Canada, Ottawa, Canada;

Department of Biological and Chemical Work Environment, National Institute of

Occupational Health, Oslo, Norway;

DTU Health Tech, Technical University of Denmark, Lyngby, Denmark

ABSTRACT

ARTICLE HISTORY

Inhalation of nanosized zinc oxide (ZnO) induces metal fume fever and systemic acute phase

response in humans. Acute phase response activation is a cardiovascular risk factor; we investi-

gated whether pulmonary exposure of mice can be used to assess ZnO-induced acute phase

response as well as inflammation and genotoxicity. Uncoated (NM-110) and triethoxycaprylylsi-

lane-coated (NM-111) ZnO nanoparticles were intratracheally instilled once at 0.2, 0.7 or 2

mg/

mouse (11, 33 and 100

mg/kg

body weight).

Serum amyloid A3

mRNA level in lung tissue, bron-

choalveolar lavage (BAL) fluid cellularity, and levels of DNA strand breaks in BAL fluid cells, lung

and liver tissue were assessed 1, 3 and 28 days post-exposure. Global transcription patterns

were assessed in lung tissue using microarrays. The acute-phase response

serum amyloid A3

mRNA levels were increased on day 1; for uncoated ZnO nanoparticles at the highest dose and

for coated ZnO nanoparticles at medium and highest dose. Neutrophils were increased in BAL

fluid only after exposure to coated ZnO nanoparticles. Genotoxicity was observed only in single

dose groups, with no dose-response relationship. Most changes in global transcriptional

response were observed after exposure to uncoated ZnO nanoparticles and involved cell cycle

G2 to M phase DNA damage checkpoint regulation. Although, uncoated and coated ZnO nano-

particles qualitatively exerted similar effects, observed differences are likely explained by differ-

ences in solubility kinetics. The finding of

serum amyloid A3

induction at low exposure suggests

that mouse models can be used to assess the nanoparticle-mediated induction of acute phase

responses in humans.

Received 24 May 2019

Revised 5 August 2019

Accepted 5 August 2019

KEYWORDS

Gene expression; Zn; comet

assay; serum amyloid A;

body weight

Introduction

The consumer application of zinc oxide (ZnO) nano-

particles is broad and includes cosmetics, sunscreens,

biosensors, food additives, pigments, rubber manu-

facturing, electronics, agriculture, and antimicrobial

products (Burnett and Wang

2011).

Human exposure

to ZnO nanoparticles can occur in the occupational

settings during the synthesis of ZnO, manufacturing

of ZnO products and via consumer products. Thus, it

is imperative to understand the potentially harmful

effects of ZnO nanoparticles and identify exposure

levels relevant to human toxicity.

Zinc is a major constituent of welding fumes and

pulmonary toxicity is observed in welders exposed

to ZnO during welding of galvanized steel and

alloys. Inhalation of high doses of ZnO induces

metal fume fever in humans. Metal fume fever can

also be induced by other metal oxides and is char-

acterized by flu-like symptoms: fever, cough, wheez-

ing, chest tightness, fatigue, and chills (Greenberg

and Vearrier

2015).

Exposure to metal oxides at lev-

els close to the current occupational exposure limits

that do not induce metal fume fever still evoke

inflammatory and acute phase responses in

humans. Men exposed for 6 h to low doses of

Zn-containing welding fumes had increased blood

levels of interleukin (IL)-6 and the acute phase reac-

tants C-reactive protein (CRP), and serum amyloid A

CONTACT

Ulla Vogel

[email protected]

National Research Centre for the Working Environment, Lersø Parkall

105, Copenhagen Ø DK-2100, Denmark

Supplemental data for this article can be accessed

here.

2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),

which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in

any way.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

(SAA) (Baumann et al.

2016).

Increased CRP concen-

trations in blood (Hartmann et al.

2014),

elevated

BAL fluid polymorphonuclear leukocytes, tumor

necrosis factor-a (TNFa), and IL-8 levels were also

observed in welders exposed to Zn containing

welding fumes (Blanc et al.

1993;

Kuschner et al.

1995).

A positive correlation was observed between

total polymorphonuclear leukocytes count in bron-

choalveolar lavage (BAL) fluid and Zn exposure in

welders acutely exposed to welding dust (Blanc

et al.

1991).

In another study, elevated blood levels

of CRP were observed in workers exposed for 6 h to

gas brazing processes involving 1.2 or 1.5 mg Zn/m

(Brand et al.

2014).

Collectively, these observations

imply that acute phase and inflammatory reactions

can be induced in humans exposed to Zn at occu-

pationally relevant doses. In addition, welding

fumes are classified as Group 1 carcinogen by IARC

(IARC

2018).

The acute-phase response is a systemic reaction

elicited by the organism in response to tissue injury

or infection (Saber et al.

2014).

However, persistent

or recurring acute-phase responses are a risk factor

for cardiovascular diseases (Ridker et al.

2000;

Saber

et al.

2014).

In a recent study, inhalation of ZnO

nanoparticles was shown to induce the acute phase

response in humans. Human inhalation of ZnO

nanoparticles

induces

ZnO

dose-dependent

increases in body temperature and, neutrophilia at

1 and 2 mg/m

. Increased blood levels of acute-

phase response protein SAA was observed at 1 and

2 mg/m

and increased CRP already at 0.5 mg/m

(Mons

et al.

2018).

Notably, these effects were

observed at doses that are below the occupational

exposure limit for ZnO in many countries (Mons

et al.

2018;

Vogel and Cassee

2018).

In a separate

study, acute exposure of healthy human adults to

0.5 mg/m

mass concentration of ZnO (<0.1

diameter) for 2 h did not result in acute-phase

response or inflammation (Beckett et al.

2005).

In animal experimental models, pulmonary

exposure to ZnO nanoparticles has been shown to

induce strong dose-dependent toxic responses

including pulmonary cytotoxicity and mortality in

mice (Jacobsen et al.

2015).

The toxicity has been

attributed to ZnO dissolution and the release of

zinc ions and was observed at dose levels that are

well-tolerated for insoluble nanoparticles (Cho et al.

2011;

Kao et al.

2012;

Jacobsen et al.

2015).

ZnO

nanoparticles undergo dissolution in water, cell cul-

ture medium and in biological fluids (Reed et al.

2012;

Kermanizadeh et al.

2013;

Da Silva et al.

2019),

the rate of which are influenced by pH, the

primary particle size and different surface coatings

(Cho et al.

2011;

Rathnayake et al.

2014).

Thus, there is evidence to support a pulmonary

acute phase and inflammogenic potential of ZnO

and potentially inflammogenic responses to ZnO

nanoparticle exposure in humans. Given the diver-

sity of nanoparticles, it is not clear if all variants

(sizes, different surface coatings or surface proper-

ties) of ZnO nanoparticles are equally toxic to

humans and human studies are not an option.

Rodent inhalation studies have shown pulmonary

inflammation following ZnO nanoparticle exposure

(Ho et al.

2011;

Adamcakova-Dodd et al.

2014a;

Chuang et al.

2014;

Chen et al.

2015;

Larsen et al.

2016).

However, animal studies investigating the

effects of ZnO on acute phase response are absent

and only a few studies included genotoxic effects

(Ho et al.

2011;

Chuang et al.

2014;

Larsen

et al.

2016).

Thus, the main objective of the present study

was to explore the possibilities of using mice to

investigate the ZnO-induced pulmonary and sys-

temic toxicity including the acute phase, pro-inflam-

matory and genotoxic effects of ZnO nanoparticles

at low doses relevant to human occupational scen-

arios. Pristine uncoated (uncoated ZnO) and a trie-

thoxycaprylylsilane coated ZnO (coated ZnO)

nanoparticles were used in the study. The rationale

for also including a coated ZnO nanoparticle was

that coatings such as the investigated, triethoxycap-

rylylsilane, are used in the cosmetics industry for

improving the ability of nanoparticles to mix with

other ingredients (KOBO_Products_Inc.

2017).

Mice

were exposed via single instillation to 0.2, 0.7 and

mg/mouse

of the coated and uncoated ZnO. The

relatively low doses were selected based on a previ-

ous study demonstrating that higher doses induce

acute toxicity in mice (Jacobsen et al.

2015).

BAL

fluid, lung and liver tissues were collected 24 h, 3

and 28 days post-instillation. Local lung inflamma-

tion by BAL fluid cellularity, pulmonary and sys-

temic inflammation by acute phase reactant

measurement and genotoxicity by comet assay

were measured. Global gene expression changes

were profiled in the lungs to identify mechanisms

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

Material and methods

Description of nanomaterials

ZnO NM-110 (CAS number 1314-13-2) and NM-111

selected by the OECD Working Party for

Nanomaterials and were generously given by the

EU Joint Research Centre, ISPRA, Italy. The uncoated

NM-110 has a primary diameter pf 100 nm and was

designated:

‘uncoated

ZnO’ in the current work.

The NM-111 has a primary diameter of 130 nm par-

ticle and consists of ZnO NM-110 as the core par-

ticle treated with a triethoxycaprylylsilane surface

coating, to form a poly-octyl-siloxy-coating and was

designated

‘coated

ZnO’ in the current work.

Dissolution studies of these materials, from the lit-

erature, have shown that the uncoated ZnO dis-

solves somewhat faster in water as compared to

the coated ZnO (Table

1).

Approximately 50% of

the uncoated ZnO particle mass is dissolved after

24 h, as published by Kermanizadeh et al. (2013).

Dispersion procedures

The nanoparticles were pre-wetted by ethanol 0.5%

(v/w) and suspended in 0.2

filtered,

c-irradiated

Nanopure Diamond UV water (Pyrogens:

<0,001

EU/ml, total organic carbon:

<3.0

ppb), at a mass

concentration of 3.24 mg/mL. The stock suspension

was further diluted 81-fold to obtain the high dose

exposure concentration 0.04 mg/mL (2

mg/mouse

a volume of 50

ml).

This high-dose suspension was

placed in an ice-bath and continuously sonicated

for 16 min using a 13 nm disruptor horn equipped

Branson Sonifier (Prod. no. disruptor horn: 101-147-

037, Prod. No. Sonifier: S-450D, Branson Ultrasonics

Corp., Danbury, CT, USA) equipped with a 13 nm

disruptor horn (Prod. no.: 101-147-037, Branson

Ultrasonics Corp., Danbury, CT, USA). Lower dose

dilutions were obtained by subsequent three- and

nine-fold dilution, respectively

–

of the high-dose

suspension and further sonicated for an additional

2 min before exposure. Vehicle solution was pre-

pared as described for the high-dose dilution

without nanoparticles. To ensure nanoparticle

homogeneity, all suspensions were administered to

the mice within one hour of preparation.

Z average

size at

mg/mouse

(nm) Dispersity

0.15

0.13

468

727

70 to

>100

(Kermanizadeh

et al.

2013)

58–93

(Kermanizadeh

et al.

2013)

130

Zincite (Kermanizadeh

et al.

2013)

Triethoxycaprylylsilane

ZnO / NM-111 / 1314-

13-2 (particle), 2943-

75-1 (coating)

XRD size (nm)

Table 1.

Physical chemical characteristics of the ZnO and carbon black nanomaterials.

Vendor

reported

size (nm)

100

Zincite (Kermanizadeh

et al.

2013)

Phase

Surface coating

No coating reported

Particle name / NM

number / CAS number

Carbon black Printex 90

(included as a bench-

mark positive control

nanoparticle)

ZnO / NM-110 / 1314-

13-2

No coating

Not reported

14 from (Saber

et al.

2005;

Jacobsen

et al.

2008)

254

0.55

295–338 from (Saber

et al.

2005, 2012;

Jacobsen et al.

2008)

BET surface

area (m

/g)

14 (Kermanizadeh

et al.

2013)

18 (Kermanizadeh

et al.

2013)

60 in H

O; 47 in C3A cell

culture medium

(Kermanizadeh

et al.

2013)

18 in H

O; 39 in C3A cell

culture medium

(Kermanizadeh

et al.

2013)

n/a

Solubility (%) at 24 h

at 37

C, at 1

mg/mL

underlying the observed toxicological effects in

lung tissue.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

Material characterization

The hydrodynamic size distributions of the instilla-

tion suspensions were determined by Dynamic

Light Scattering (DLS) using a Malvern Zetasizer

Nano ZS (Malvern Instruments, UK) at 25

Analysis was done using the Dispersion Technology

Software

v5.0

(Malvern

Instruments,

UK).

Hydrodynamic size was calculated using a disper-

sion refractive index of 1.33, a materials refractive

index of 2.1, a viscosity 0.89 cP and a material

absorption value of 2.0.

Animal exposures

Nanoparticles were administered by a single intra-

tracheal instillation as described previously (Jackson

et al.

2011).

Doses were 0.2, 0.7 and 2

mg/mouse

(or: 11, 33 and 100

mg/kg

bw). These low doses

were based on previously conducted pilot studies.

Complete immobility and breathing difficulty was

observed at 162

of the coated ZnO NP per

mouse (8.2 mg/kg bw). Mortality was observed at

1.4 mg/kg bw as reported by (Jacobsen et al.

2015).

In addition, body weight decrease was observed at

doses above 6

mg/mouse

of other ZnO nanoparticles

(1) ZincoxTM 10, IBUtec advanced materials AG,

Weimar, Germany, and (2) Alfa Aesar (ID 43141, A

Johnson Matthey Company, Karlsruhe, Germany;

and (3) the current coated ZnO (NM-111) (Jacobsen

et al.

2015).

Carbon black Printex 90 (162

mg/mouse)

was tested head-to-head with the ZnO nanopar-

ticles in the current experiment; the carbon black

data were included as reference in other studies

that were performed in parallel with the current

study (Kyjovska et al.

2015a, 2015b;

Wallin

et al.

2017).

In brief, mice were anaesthetized by inhalation of

4% isoflurane. Next, 50

of particle suspension or

vehicle control followed by 100

of air was

instilled using a SGE glass syringe (250F-LT-GT,

MicroLab, Aarhus, Denmark). Breathing was moni-

tored post-instillation to ensure that airways were

not blocked by instillation fluid. The animals were

weighed on the day of exposure as well as on day

2 and on day 27 post-exposure. Mice were killed on

day 1, day 3 and day 28 post-exposure by subcuta-

neous injection of Hypnorm (fentanyl citrate

0.315 mg/mL and Fluanisone 10 mg/mL, Janssen

Pharma,

Beerse,

Belgium)

and

Dormicum

(Midazolam

5 mg/mL

from

Roche,

Basel,

Switzerland), which both were mixed with an equal

volume of sterile water.

BAL fluid collection and cellularity

BAL fluid was recovered by flushing the lungs twice

using 1 mL saline/25 g bw per each flush. BAL fluid

was kept on ice for a maximum of 2 h, at which the

samples were centrifuged at 400

g at 4

C for

10 min to recover cells. The cells were re-suspended

in 100

HAM-F12 medium (Prod no. 21765037,

Animal housing

All animal procedures complied with the EC

Directive 86/609/EEC and Danish law regulating

experiments with animals (The Danish Ministry of

Justice, Animal Experiments Inspectorate permission

2010/561-1779), and were approved by the local

animal ethical committee. Female C57BL/6J BomTac

mice, 7 weeks of age, were purchased from Taconic

Europe (Ejby, Denmark). For logistic reasons, only

female mice were used in the study. Compared to

male mice, female mice are less aggressive towards

each other, and can, therefore, be housed in groups

of 6–8 mice. At arrival, the mice were randomly dis-

tributed to cages containing either nanomaterial

administered animals or vehicle control animals.

The number of animals (N) was 8 per cage for

nanomaterial dosed animals and 6 per cage for

controls. Each experimental group consisted of a

minimum of 8 animals; 8 mice were included in

uncoated ZnO, coated ZnO and Printex 90 carbon

black groups; and 12 mice used for the vehicle con-

trol group. The mice had

ad libitum

access to tap

water and food (Altromin no. 1324, Christian

Petersen, Denmark). Housing was in polypropylene

cages with Enviro-Dri bedding (Brogaarden,

Gentofte, Denmark). MS wood blocks (Brogaarden,

Gentofte, Denmark) and hides (Mouse House,

Scanbur, Karlslunde, Denmark) served as enrich-

ment. The room temperature was kept at 20 ± 2

and the humidity at 50 ± 20%. The animals were

housed under a 12 h light: 12 h dark cycle (on from

6 a.m. to 6 p.m.) and allowed to acclimatize for

one week.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

Invitrogen, Carlsbad, CA, USA) containing 10% fetal

bovine serum (Prod no. 10106169, Invitrogen,

Carlsbad, CA, USA). For differential counting of

immune cells in BAL fluid, 40

of the fresh resus-

pension was collected on microscope slides by cen-

trifugation at 60

g, 4 min by use of a Cytofuge 2

(StatSpin, Bie and Berntsen, Rødovre, Denmark).

Cells were fixed by the addition of 96% ethanol

€

and incubated with May-Grunwald-Giemsa stain.

The total number of cells in the resuspension was

measured

with

NucleoCounter

NC-100

(Chemometec, Allerød, Denmark) Live/Dead Assay.

The differential cell count was carried out on a total

of 200 cells per sample. For the analysis by comet

assay, 160

of 90% HAMF12, 10% FBS, 1%

Dimethyl sulfoxide was added to 40

of the BAL

fluid cell suspension. Lung and liver samples were

snap-frozen in liquid nitrogen. Comet assay samples

were stored at

À80

C until analyses.

Measurement of SAA-3 protein levels in plasma

The levels of SAA-3 protein were determined in

blood plasma from mice exposed to high and

medium doses of uncoated or coated ZnO nanoma-

terials, as well as for carbon black exposed mice.

This was carried out by ELISA in accordance with

the manufacturer’s instructions (Mouse Serum

Amyloid A-3, Cat.#EZMSAA3-12K, Millipore) and has

been described in detail by (Poulsen, Saber,

Mortensen, et al.

2015;

Poulsen et al.

2017).

The

level of detection (LOD) was 0.08

mg/mL.

For sam-

ples that were below LOD a value of

LOD was

used (0.04

mg/mL).

RNA isolation and quantitative PCR measurement

of the Saa3 mRNA level

Purification of mRNA and the subsequent quantita-

tive PCR was conducted as previously described

(Saber et al.

2006).

In brief, RNA was recovered

from the left lung by use of the NucleoSpin 96 RNA

€

kit (Macherey-Nagel, Duren, Germany). The tissue

was lysed in 2 mL RLT buffer, by homogenizing for

60 s using a Tissuelyser (Qiagen, Denmark) con-

taining a 5 mm stainless steel bead. The samples

were then run through a QIAshredder (Qiagen,

USA). The remaining purification steps were con-

ducted according to the description of the

NucleoSpin 96 RNA kit. Next, cDNA was prepared

using the reverse transcription reagents from

TaqMan (Applied Biosystems, USA) as described by

the manufacturer. The quantitative PCR was per-

formed on an ABI PRISM

7500 sequence detector

(PE Biosystems, Foster City, CA, USA), using

Universal Mastermix (Applied Biosystems, Naerum,

Denmark). The sequence of

Saa3

forward primer

was: 5 GCC TGG GCT GCT AAA GTC AT 3, that of

the

Saa3

reverse primer: 5 TGC TCC ATG TCC CGT

GAA C 3, and that of the

Saa3

probe: 5 FAM-TCT

GAA CAG CCT CTC TGG CAT CGC T-TAMRA 3. Data

were normalized to 18S rRNA (prod. no.

Mm03024053_m1 from Applied Biosystems) and

multiplied with 10

to provide values that were

more readable (0 to 2500).

Levels of DNA strand breaks

Levels of DNA strand breaks were determined in

BAL fluid cells, lung and liver tissue. The percentage

of DNA in the tail was determined by comet assay

using the IMSTAR Pathfinder

system as previously

described (Jackson et al.

2013).

In brief, BAL cells

were thawed at 37 C. Frozen lung and liver pieces

were homogenized in Merchant’s medium (140 mM

NaCl, 1.5 mM KH

, 2.7 mM KCl, 8.1 mM Na

HPO

10 mM Na

-EDTA, pH 7.4) through a steel mesh

within a syringe. Obtained cells were next sus-

pended in 0.6% agarose at 37

C, followed by

embedding on Trevigen CometSlides

. The slides

were cooled and incubated at 4

C overnight in lysis

buffer. Next, the slides were rinsed in electrophor-

esis buffer (pH

>13)

followed by 40 min of alkaline

treatment. The electrophoresis was run for 25 min

at an applied voltage of 1.15 V/cm and a current of

300 mA. After pH neutralization, the slides were

fixed in 96% ethanol and placed on a 45

C heating

plate for 15 min. The cells were next stained with

SYBR

Green fluorescent stain. After the addition of

a UV-filter and coverslips, levels of DNA strand

breaks were analyzed using the IMSTAR

Pathfinder

system. The results are calculated as

an average percentage tail DNA value for all cells

scored in each Trevigen CometSlide well. Negative

and positive controls included on all slides were

non-exposed A549 cells, and A549 cells exposed to

respectively. The data were normalized

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

to the negative controls in order to adjust for day-

to-day variation.

Statistical analysis

Total protein, neutrophil cells, DNA strand breaks,

and Saa3 mRNA levels

The Graph Pad Prism 7.02 software package (Graph

Pad Software Inc., La Jolla, CA, USA) was used for

statistical calculations. Data were tested for normal-

ity with the Shapiro-Wilk test. The

t-test

and the

ANOVA test are robust against deviations in normal-

ity and were used for inter-group comparisons,

except when the

p-value

of the Shapiro Wilks test

was very low (p

0.001); or when the standard

deviations of the groups were determined to be

very different. Differences in standard deviations

were assessed using the F test (for two-sample

comparisons) or Brown-Forsythe test for three or

more treatment groups (p

0.001). The latter tests

were applied because the

t-test

and the ANOVA are

somewhat sensitive to differences in data variability.

In case of such deviations in normality or in inter-

group standard deviations, a non-parametric test in

the form of the Mann–Whitney test (two groups) or

the Kruskall–Wallis test (more than two groups) was

calculated. In order to assess inter-group differences

in one-way ANOVA or the Kruskall–Wallis test, mul-

tiple comparisons post-tests were applied. These

were Holm–Sidak’s multiple comparisons test

(ANOVA) or Dunn’s multiple comparisons test

(Kruskall–Wallis test). The data were tested so that

each particle type was tested independently against

vehicle control.

Microarray data

The microarray data were statistically analyzed as

described previously (Husain et al.

2013,

Labib et al.

2013).

In brief, a reference randomized block design

was used to analyze gene expression microarray

data. Data were normalized using LOcally WEighted

Scatterplot Smoothing (LOWESS) regression model-

ing method and statistical significance of the differ-

entially expressed genes was determined using

MicroArray ANalysis Of VAriance (MAANOVA)

(Rahman et al.

2017)

in R statistical software (R Core

Team

2012).

The Fs statistic (Rahman et al.

2017)

was used to test the treatment effects compared to

the control vehicle, and

p-values

were estimated by

the permutation method using residual shuffling. In

order to minimize any false positives, the false

Total RNA extraction and purification for

microarray analysis

Random sections of the left lungs were used to iso-

late total RNA (n

5 per experimental group) using

TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and

purified using RNeasy Plus Mini kits (Qiagen,

Mississauga, ON, Canada) according to the manufac-

turer’s instruction. NanoDrop 2000 spectrophotom-

eter (Thermo Fisher Scientific Inc., Wilmington, DE,

USA) was used to quantify the total RNA concentra-

tion and RNA quality and integrity was assessed

using an Agilent 2100 Bioanalyzer (Agilent

Technologies, Mississauga, ON, Canada) according

to the manufacturer’s instruction. All samples had

RNA integrity numbers above 7.0.

Microarray hybridization

Double-stranded cDNA was synthesized using the

total RNA (250 ng) from individual mice (n

5 per

experimental or control group) and Universal

Mouse Reference total RNA (UMRR) (Agilent

Technologies, Mississauga, ON, Canada). Cyanine-

labelled cRNAs were synthesized from the cDNA

using

Quick

Amp Labelling

Kit

(Agilent

Technologies, Mississauga, ON, Canada). cRNAs from

control and ZnO nanoparticle-treated samples were

labeled with Cyanine 5-CTP, and reference cRNAs

were labeled with Cyanine 3-CTP using a T7 RNA

polymerase

in vitro

transcription kit (Agilent

Technologies, Mississauga, ON, Canada) and purified

using RNeasy Mini kits (Qiagen, Mississauga, ON,

Canada). An equimolar amount of reference cRNA

was mixed with each experimental cRNA sample

and was hybridized to Agilent mouse 8

60 k oligo-

nucleotide microarrays (Agilent Technologies Inc.,

Mississauga, ON, Canada) for 17 h in a hybridization

chamber at 65

C with a rotation speed of 10 rpm.

Following hybridization, arrays were scanned on an

Agilent G2505B scanner according to manufac-

turer’s protocols. Gene expression data from the

scanned images were extracted using Agilent

Feature Extraction software version 9.5.3.1.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

discovery rate (FDR) multiple testing correction

(Rahman et al.

2017)

was applied. The fold changes

of gene expression were calculated considering the

least-square means. Genes with FDR

p-values

of less

than or equal to 0.05 (p 0.05) were considered

significantly differentially expressed and were used

in all downstream analyses. Since the gene list was

small, the fold-change based filtering was not

conducted.

Functional and pathway analyses of differentially

regulated mRNAs

These analyses were done as previously described

in (Rahman et al.

2017).

In brief, Ingenuity Pathway

Analysis (IPA, Ingenuity Systems, Redwood City, CA,

USA) was used to identify the pathways associated

with differentially expressed genes. Pathway signifi-

cance was defined using Fisher’s exact

p-value

0.05.

a dispersity of 0.55. These data, as well as other

physical chemical characteristics obtained from the

literature, are presented in

Table 1.

Body weight

A decrease in body weight gain was observed in

mice exposed to the highest dose (2

mg/mouse)

coated ZnO nanoparticles at 2 days after exposure

(the mice were weighted one day before euthaniza-

tion). The effect was reversed at 27 days post-expos-

ure. Body weight was unaffected in mice exposed

to uncoated ZnO nanoparticles, whereas the posi-

tive control carbon black resulted in a lower weight

gain as compared to control (Figure

1).

BAL fluid cellularity and protein content

Total protein in BAL fluid was increased in mice

exposed to a high dose of uncoated or coated

ZnO nanoparticles at day 1 and 3 but not at day

28. Total protein was increased for the positive

control carbon black at all three time-points, as

previously reported (Kyjovska et al.

2015b)

(Figure

2).

Total cell counts in BAL fluid, as well as cell dis-

tribution by cell type, are summarized in

Supplementary Materials, Table S1.

Increased neu-

trophil numbers were observed only in the lungs

Results

Physical chemical characterization

The uncoated ZnO had a Z average of 468 and a

dispersity of 0.15. The coated ZnO had a Z average

of 727 and a dispersity of 0.13. The positive control,

carbon black Printex 90 had a Z average of 128 and

Body weight gain

Body weight gain (g)

-2

* ****

Figure 1.

Body weight gain in mice exposed to ZnO nanoparticles. Uncoated or triethoxycaprylylsilane-coated ZnO nanoparticles

were administered by intratracheal instillation at 0.2, 0.7 or 2

mg/mouse.

Low, medium and high designates low-dose, medium-

dose and high-dose, respectively. Two or twenty-seven days later the body weight was measured and body weight gain com-

pared to weight at exposure was calculated. Data are mean and bars represent SD.

designates a

p-value

<0.05

vs.

vehicle of

one way ANOVA with Holm–Sidak’s multiple comparisons test. In the case of carbon black

ÃÃÃÃ

and

designates

p-values

<0.0001

and

<0.05

respectively

vs.

vehicle of the

t-test.

nc co

oa ate Veh

Z cl

Zn nO e

m ow

oa ZnO m

at ted hi

Z gh

Zn nO

m ow

Zn ium

bo hig

nc co

at Ve k

te ed hic

Zn nO e

m ow

oa ZnO m

te ed hig

Zn nO

m ow

Zn ium

bo hig

2 days

27 days

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

Total protein in BAL fluid

2000

Total protein ( g/mL)

****

1500

1000

****

500

***

Figure 2.

Total protein in BAL fluid at 1, 3 and 28 days of ZnO nanoparticle exposure. Uncoated or triethoxycaprylylsilane-coated

ZnO nanoparticles were administered by intratracheal instillation at 0.2, 0.7 or 2

mg/mouse.

Low, medium and high designates

low-dose, medium-dose and high-dose, respectively. One, three or twenty-eight days after exposure, BAL fluid was prepared and

total protein determined. Data are mean and bars represent SD.

ÃÃÃÃ

ÃÃÃ

ÃÃ

and

designates

p-values

<0.0001, <0.001,

<0.01

and

<0.05

respectively

vs.

vehicle of one way ANOVA with Holm–Sidak’s multiple comparisons test. In the case of carbon

black

ÃÃÃ

, and

ÃÃ

designates

p-values

<0.001,

and

<0.01

respectively

vs.

vehicle of the

t-test.

Neutrophils in BAL fluid

(total number of cells)

200000

Figure 3.

Neutrophil numbers in BAL fluid at 1, 3 and 28 days of ZnO nanoparticle exposure. Uncoated (uncoated ZnO) or trie-

thoxycaprylylsilane-coated ZnO nanoparticles (coated ZnO) were administered by intratracheal instillation at 0.2, 0.7 or 2

mg/

mouse. Low, medium and high designates low-dose, medium-dose and high-dose, respectively. Carbon black at 162

mg/mouse

served as positive control. One, three or twenty-eight days post-exposure, BAL fluid was prepared and the number of neutrophils

established by differential counting. Data are mean and bars represent SD.

ÃÃÃ

ÃÃ

and

designates

p-values

<0.001, <0.01

and

<0.05

respectively of one way ANOVA with Holm–Sidak’s multiple comparisons test in case of data approaching normality

and not having a highly different variation (details given in the methods section), otherwise by Kruskall–Wallis test with Dunn’s

multiple comparisons test. In the case of carbon black

ÃÃÃÃ

ÃÃÃ

ÃÃ

and

designates

p-values

<0.0001, <0.001, <0.01

and

<0.05

respectively

vs.

vehicle of the Mann Whitney test.

of mice exposed to a high dose of coated ZnO

nanoparticles on day 1 and 3 post-exposure.

Notably, at 28 d post-exposure, increased neutro-

phil count was observed in coated ZnO nanopar-

ticles low and medium dose groups only. In

comparison, no significant increases in the BAL

Un U

co nco

at at

U ed ed Veh

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

U U

nc nc

n ig

oa oa

b h

te te V lac

U d d eh k

nc Z Z i

oa nO nO cle

Co C ted me low

at oat Zn diu

ed ed O m

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

U U

nc nc

n igh

oa oa

t t

Un ed ed Veh ck

Zn Zn ic

a O O le

C C ted me low

oa oa Z d

te te nO ium

d d

Co Zn Zn hig

at O O h

ed m lo

e w

C Zn diu

ar O m

bo h

n ig

bl h

nc Unc

oa oa

t t

U ed ed Veh

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

Un U

n igh

co nco

at at

U ed ed Veh ck

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

Co Zn Zn hig

at O O h

ed m lo

e w

C Zn diu

ar O m

bo h

U U

nc nc

n igh

oa oa

te te V lac

U d d eh k

nc Z Z i

oa nO nO cle

Co C ted me low

at oat Zn diu

ed ed O m

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

n igh

1 day

3 days

28 days

****

150000

100000

50000

****

***

****

* **

****

1 day

3 days

28 days

neutrophil populations were observed in mice

exposed to uncoated ZnO nanoparticles. Mice

instilled with positive control carbon black (162

mg/

mouse) showed increased neutrophil influx at all

three post-exposure time points, as previously

reported (Kyjovska et al.

2015b)

(Figure

3).

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

Figure 4.

Saa3

mRNA levels in lung 1, 3 and 28 days of ZnO nanoparticle exposure. Uncoated (uncoated ZnO) or triethoxycapry-

lylsilane-coated ZnO nanoparticles (coated ZnO) were administered by intratracheal instillation at 0.2, 0.7 or 2

mg/mouse.

Low,

medium and high designates low-dose, medium-dose and high-dose, respectively. Carbon black at 162

mg/mouse

served as posi-

tive control. One, three or twenty-eight days post-exposure lung tissue was recovered and

Saa3

mRNA levels measured by quanti-

tative real time PCR. Data are mean and bars represent SD.

ÃÃÃÃ

ÃÃÃ

ÃÃ

and

designates

p-values

<0.0001, <0.001, <0.01

and

<0.05

respectively

vs.

vehicle of one way ANOVA with Holm–Sidak’s multiple comparisons test.

Saa3 mRNA levels in lung tissue and SAA-3

protein levels in plasma

Acute increases in the

Saa3

mRNA level were

observed on day 1 following exposure to the high-

est dose of uncoated ZnO, and medium and high

dose of coated ZnO (Figure

4).

There were no

effects on the protein level of SAA-3 in plasma of

mice exposed to uncoated ZnO, coated ZnO or in

mice exposed to carbon black (data not shown).

Levels of DNA strand breaks

Levels of DNA strand breaks measured as percent-

age DNA in the tail was assessed in BAL fluid, lung

and liver tissues. In BAL fluid, increased levels of

DNA strand breaks were observed only for coated

ZnO at low-dose at 28 days post-exposure

(Figure

5).

In lung tissue, increased levels of DNA

strand breaks was observed for both ZnO nanopar-

ticles at day 28, but only in the medium-dose

groups (Figure

6).

No increases in DNA strand break

levels were observed in liver tissue (Figure

S1).

The

positive control, carbon black, induced an elevated

DNA strand break levels in the lung at 1 and 28

days post-exposure, as previously reported

(Kyjovska et al.

2015b, 2015a)

(Figure

6).

Saa3

mRNA level normalised to 18S rRNA x10

U Un

nc c

oa oa

te te Ve

U d Z d Z hic

nc n n l

oa O O e

te m lo

C Co d Z ed w

oa a n iu

te ted O m

Z h

C Zn nO igh

oa O

te m low

d ed

Zn iu

U Un

O m

nc c

oa oa

te ted Ve gh

U d Z Z hic

nc n n l

oa O O e

te m lo

C Co d Z ed w

oa a n iu

te ted O m

C Zn ZnO igh

oa O

te m low

d ed

Zn iu

U Un

O m

nc c

oa oa

te ted Ve gh

U d Z Z hic

nc n n l

oa O O e

te m lo

C Co d Z ed w

oa a n iu

te ted O m

Z h

C Zn nO igh

oa O

te m low

d ed

Zn iu

O m

Saa3

mRNA level

2500

2000

1500

1000

500

***

****

1 day

3 days

28 days

Gene expression analysis

In addition to the targeted gene expression ana-

lysis, global gene expression profiling was con-

ducted using microarrays to identify the genes and

pathways perturbed by ZnO exposure. In general,

the response at the gene expression level was

larger in lungs of mice exposed to uncoated ZnO as

compared to the coated ZnO treated lungs on day

1, some of which was still observed at day 28.

However, the magnitude of the response was small

with most genes showing fold changes around or

less than 1.5. In uncoated ZnO-treated groups, a

dose-dependent increase in the number of differen-

tially expressed genes was observed (Table

2);

total of 49, 86 and 128 genes (day 1) and, 46, 74

and 85 genes (day 28) were upregulated and, a

total of 55, 54, 54 genes (day 1) and 70, 64 and 75

genes (day 28) were downregulated at the low,

medium and high doses, respectively. In compari-

son, coated ZnO treated groups did not show dose-

dependency; on day 1, there were 30, 100 and 37

genes upregulated and 40, 47, 25 genes downregu-

lated and, on day 28, there were 117, 188, and 107

genes upregulated and 70, 82 and 76 genes down-

regulated. In the coated ZnO-treated groups, the

response was large at 28 days post-exposure.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

Levels of DNA strand breaks in BAL fluid cells

Tail %DNA

Figure 5.

Levels of DNA strand breaks in BAL fluid cells at 1, 3 and 28 days of ZnO nanoparticle exposure. Uncoated (uncoated

ZnO) or triethoxycaprylylsilane-coated ZnO nanoparticles (coated ZnO) were administered by intratracheal instillation at 0.2, 0.7 or

mg/mouse.

Low, medium and high designates low-dose, medium-dose and high-dose, respectively. Carbon black at 162

mg/

mouse was included as reference material. One, three or twenty-eight days later BAL fluid cells were prepared and levels of DNA

strand breaks measured as percent DNA in the tail by comet assay. Data are mean and bars represent SD.

designates

p-values

<0.05

vs.

vehicle of one way ANOVA with Holm–Sidak’s multiple comparisons test. In the case of carbon black data were

tested with

t-test.

Tail %DNA

Figure 6.

Levels of DNA strand breaks in lung tissue at 1, 3 and 28 days of ZnO exposure. Uncoated (uncoated ZnO) or triethoxy-

caprylylsilane-coated ZnO nanoparticles (coated ZnO) were administered by intratracheal instillation at 0.2, 0.7 or 2

mg/mouse.

Low, medium and high designates low-dose, medium-dose and high-dose, respectively. Carbon black at 162

mg/mouse

served as

positive control. One, three or twenty-eight days later, lung tissue was recovered and levels of DNA strand breaks measured as

percent DNA in the tail by comet assay. Data are mean and bars represent SD.

ÃÃÃÃ

ÃÃÃ

ÃÃ

and

designates

p-values

<0.0001, <0.001, <0.01

and

<0.05

respectively

vs.

vehicle of one way ANOVA with Holm–Sidak’s multiple comparisons test. In

the case of carbon black data were tested with

t-test.

A further analysis of the differentially expressed

genes and the associated canonical pathways

revealed that the

cell cycle G2M DNA damage check-

point regulation pathway

and functions related to

cell cycle progression, segregation of chromosomes

and alignment of chromosomes were the most

Un U

co nco

at at

U ed ed Veh

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

Co Zn Zn hig

at O O h

ed m lo

e w

C Zn diu

ar O m

bo h

U U

nc nc

n igh

oa oa

te te V lac

U d d eh k

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

U U

nc nc

n ig

oa oa

bl h

t t

Un ed ed Veh ck

Zn Zn ic

a O O le

Co C ted me low

at oat Zn diu

ed ed O m

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

n igh

Un U

co nco

at at

U ed ed Veh

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

Co Zn Zn hig

at O O h

ed m lo

e w

C Zn diu

ar O m

bo h

U U

nc nc

n igh

oa oa

te te V lac

U d d eh k

nc Z Z i

oa nO nO cle

C C ted me low

oa oa Z d

te te nO ium

d d

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

U U

nc nc

n ig

oa oa

bl h

t t

Un ed ed Veh ck

Zn Zn ic

a O O le

Co C ted me low

at oat Zn diu

ed ed O m

C Zn Zn hig

oa O O h

te m lo

d e w

C Zn diu

ar O m

bo h

n igh

1 day

3 days

28 days

Levels of DNA strand breaks in lung

****

***

*******

1 day

3 days

28 days

significantly affected in mice exposed to medium

and high doses of uncoated ZnO nanoparticles on

day 1 (Figure

7).

The other pathways significantly

altered following exposure to coated or uncoated

ZnO nanoparticles included

Circadian rhythm signal-

ing; Protein ubiquitination pathway; Unfolded protein

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

response;

and

AMPK signaling.

Although acute phase

signaling was one of the significantly affected path-

ways, the number of differentially expressed genes

associated with this pathway was very low.

Saa3

was the only acute phase gene that showed dose-

dependent increases in mRNA level, which was

observed only in mice exposed to uncoated ZnO

nanoparticles.

phase response, inflammogenic and genotoxic

effects of the ZnO nanoparticles. Whole genome

microarrays were used to identify the underlying

mechanisms of toxicity in lungs.

Effects on the acute-phase response

We have previously shown pulmonary acute phase

response in response to nanomaterial exposure. In

our previous studies of global transcriptional

responses to inhaled or instilled nanomaterials, we

found dose-dependent pulmonary acute phase

responses both in terms of the number of differen-

tially regulated acute phase genes and fold

increases of

Saa3

mRNA levels (Halappanavar et al.

2011, 2015, 2019;

Bourdon, Halappanavar, et al.

2012;

Husain et al.

2013;

Saber et al.

2014;

Poulsen,

Saber, Williams, et al.

2015).

Saa3

is among the

most differentially regulated genes 1-day post

exposure to various nanomaterials (Halappanavar

et al.

2019).

Pulmonary

Saa3

mRNA levels correlate

closely with neutrophil influx (Saber et al.

2013,

2014;

Poulsen et al.

2017)

and SAA3 levels in

plasma (Poulsen et al.

2017).

In the current study,

we, therefore, used

Saa3

mRNA levels as a sensitive

biomarker of the pulmonary acute-phase response.

The

Saa3

mRNA expression was increased in

uncoated ZnO nanoparticle-treated groups at the

highest dose (2

mg/mouse;

100

mg/kg

bw) and for

the coated ZnO nanoparticles at medium and high-

doses (0.7 and 2

mg/mouse).

Also in the microarray

Discussion

In this study, we investigated the acute-phase

response, inflammation, and genotoxicity after pul-

monary exposure to relatively low doses of ZnO

nanoparticles. Mice were exposed via intratracheal

instillation to 0.2, 0.7 or 2

mg/mouse,

corresponding

to 0.01, 0.33, and 0.1 mg/kg bw, of coated or

uncoated ZnO nanoparticles. The tissue mRNA and

plasma protein levels of SAA-3, BAL fluid cellularity

and DNA strand breaks in BAL fluid, lung and liver

tissues were measured to investigate the acute

Table 2.

Numbers of genes regulated with a

p-value

of less

than 0.05.

1 day

Dose

Uncoated ZnO

Upregulated

Downregulated

Total

Coated ZnO

Upregulated

Downregulated

Total

Low

104

Medium

140

100

147

High

128

182

Low

116

117

187

28 days

Medium

138

188

270

High

160

107

183

Figure 7.

Canonical pathways affected by 1 or 28 days of ZnO nanoparticle exposure. Uncoated (uncoated ZnO) or triethoxycapry-

lylsilane-coated ZnO nanoparticles (coated ZnO) were administered by intratracheal instillation at 0.2, 0.7 or 2

mg/mouse

(desig-

nated: low, medium, and high). The deeper the coloring is, the higher the effect is on the specific canonical pathway. No effects

were observed at the medium dose for the coated ZnO, thus this group is not included in the figure.

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

analysis, acute phase signaling was an affected

pathway (data not shown), showing dose-depend-

ent increases in

Saa3

mRNA levels in mice exposed

to uncoated ZnO nanoparticles (data not shown).

The increased

Saa3

mRNA expression was not

reflected in an increased protein plasma level of

SAA3. This may reflect that the contribution to the

systemic circulation is too low to be detected.

Notably, we have previously found a close positive

correlation between pulmonary

Saa3

mRNA levels

and plasma SAA3 in mice exposed to MWCNTs

(Poulsen et al.

2017).

In humans, increased SAA and

CRP levels are observed in the circulation as early

as 4 h following inhalation of 0.5, 1 or 2 mg ZnO

nanoparticles/m

. With an estimated inhalation vol-

ume of 20 m

/day during light activity and bw of

70 kg, this amounts to a cumulative exposure of

mg/kg

bw/day (at 0.5 mg/m

for 4 h) or to 10

mg/

kg bw taking the alveolar deposition rate of

$50%

reported in (Monse et al.

2018)

into account.

Likewise, the 1 and 2 mg/m

mass concentration

doses correspond to 20 and 40

mg/kg

bw, respect-

ively. The medium- and high-doses used in the pre-

sent study are equal to 33 and 100

mg/kg

bw,

respectively. Thus, the medium dose corresponds to

the highest doses given by (Monse et al.

2018)

mass. It has been proposed, however, that a more

correct conversion of doses from studies in animals

to studies in humans is to normalize by body sur-

face area (Reagan-Shaw, Nihal, and Ahmed

2008).

we scale by body surface area according to Reagan-

Shaw, Nihal, and Ahmed (2008), the high dose in

the present study corresponds to 8

mg/kg

bw in a

Human Equivalent Dose and thus, corresponds to

the lowest mass concentration studied by Mons

et al. (2018). Based on these calculations and the

results obtained, humans may activate acute phase

response more readily following exposure to ZnO

compared to the C57BL/6 mice used in the current

study. In these mice, the lowest-observed-adverse-

effect level (LOAEL) for induction of SAA3 in lung

tissue was 100

mg/kg

bw for the uncoated ZnO and

mg/kg

bw for the coated ZnO, whereas the

LOAEL for SAA in blood is calculated as 20

mg/kg

bw for the human volunteers (Mons

et al.

2018).

Several studies have demonstrated induction of

localized acute phase response in lungs following

inhalation exposure to nanomaterials and combus-

tion particles (Halappanavar et al.

2011;

Bourdon,

Saber, et al.

2012;

Saber et al.

2013, 2014;

Poulsen

et al.

2017).

The activation of the systemic acute

phase response in the liver in these studies was

more limited and dependent on the type of nano-

material and the magnitude of the lung acute

phase and pro-inflammatory response. In humans,

the liver is believed to be the major organ associ-

ated with acute phase reactions (Sack

2018),

although SAA is also expressed in a range of nor-

mal human tissues including lung (Urieli-Shoval

et al.

1998;

Calero et al.

2014).

Albeit these differen-

ces, the results from the present study suggests

that mouse models can serve as the first pass

screen in the investigation of particle-induced

acute-phase response. The acute phase response,

and especially serum amyloid A is causally impli-

cated in atherosclerosis and cardiovascular disease

(Saber et al.

2013, 2014).

Mice have 3 inducible SAA

isoforms,

Saa1, Saa2

and

Saa3

and simultaneous

inactivation of all 3 isogenes lowers plaque forma-

tion in APOE knockout mice, whereas overexpres-

sion of

Saa1

Saa3

increases plaque formation

(Dong et al.

2011;

Thompson et al.

2018).

The ZnO-dependent systemic acute phase

response observed in human volunteers (Mons

et al.

2018)

constitutes a causal link between inhal-

ation of ZnO particles and cardiovascular diseases.

In addition to ZnO, also other metals and metal

oxides induce metal fume fever and acute phase

response (Greenberg and Vearrier

2015)

and thus,

occupational exposure to these metals may cause

cardiovascular disease. Welding has been associated

with increased risk of cardiovascular disease (Ibfelt,

Bonde, and Hansen

2010);

and acute phase

response could potentially be used as a biomarker

of cardiovascular risk in risk assessment and

regulation.

Effects on pulmonary inflammation

The two ZnO nanoparticles investigated showed dif-

ferential potential to induce pulmonary inflamma-

tion as increased neutrophil influx was only

observed in the coated ZnO group. In agreement

with this, inhalation exposure of male C56Bl/6 mice

to 3.5 mg/m

4 h/day, 5 days/week for two weeks to

uncoated ZnO nanoparticles (15 nm in diameter)

only increased neutrophil influx from 0.2% to 1.7%

of the BAL cells, immediately after exposure

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

NANOTOXICOLOGY

(Adamcakova-Dodd et al.

2014).

In the present

study, such a small increase is expected to be

below the detection limit because the procedure of

instillation causes a low neutrophil influx 1 day

post-exposure (Jackson et al.

2011).

However, a

similar, but non-statistically significant increase in

neutrophil influx from 0.5% to 3.9% was seen for

the uncoated ZnO nanoparticle on day 3 post-

exposure. The observed differences in inflammatory

response could be due to the differential solubility

of the two ZnO types and the high level of protein

in BAL from the ZnO-exposed mice could be a

€

result of dissolution-mediated cytotoxicity (Muller

et al.

2010;

Eixenberger et al.

2017).

ZnO nanopar-

ticles of different sizes have been reported to

induce neutrophil influx in BAL fluid in rat and

mouse models following inhalation exposure to

mass concentrations of 1 to 12 mg/m

of ZnO nano-

particles (Conner et al.

1988;

Ho et al.

2011;

Adamcakova-Dodd et al.

2014;

Chuang et al.

2014;

Chen et al.

2015;

Larsen et al.

2016).

Following

intratracheal instillation, increased neutrophil num-

bers in BAL fluid have been reported with ZnO par-

ticles of different sizes. Here, the LOAELs were in

the range of 0.3 to 1 mg/kg bw in rat and mouse

(Warheit, Sayes, and Reed

2009;

Cho et al.

2010,

2011, 2012;

Jacobsen et al.

2015).

However, the

doses used in these studies were higher than the

highest dose used in the current study (100

mg/

kg bw).

Genotoxicity

IARC has classified welding fumes as a Group 1 car-

cinogen (IARC

2018).

As welding fumes contain

zinc, the endpoints of genotoxicity and carcinogen-

icity are of interest in the hazard identification of

ZnO nanoparticles. In the current investigation, we

found no dose-response relationship for genotoxic-

ity by comet assay across time points: increased lev-

els of DNA strand break was only observed at

single dose levels in BAL fluid cells and in lung tis-

sue following exposure to both ZnO types.

Nonetheless, genes and pathways associated with

cell cycle progression and cell cycle checkpoint

were identified in the microarray analysis (Figure

and dysregulation of these processes potentially

induce genotoxicity. This could, for example, involve

cytotoxicity. We observed ZnO instillation to

increase the protein content in BAL fluid indicating

increased cellular membrane permeability and cyto-

toxicity. The damaged cells could be arrested in G2/

M phase of the cell cycle and eventually undergo

senescence by apoptosis if not repaired.

There is evidence from others to suggest that

ZnO nanoparticles are genotoxic; however, the

results are not consistent. Increased oxidative dam-

age (8-oxo-2’-dG) was observed following 3.7 and

45 mg/m

of ZnO nanoparticles of 35 and 250 nm,

respectively, in rats following 6 h inhalation (Ho

et al.

2011).

Similarly, increased 8-oxo-2’-dG levels

were observed in rats intratracheally exposed to a

high dose of 33 mg/kg bw of 50 nm ZnO nanopar-

ticles (Chuang et al.

2014).

In another study, one

hour of inhalation exposure to 58 or 53 mg/m

ZnO nanoparticles of 13 and 36 nm did not exert

increased genotoxicity as measured by the comet

assay in mice (Larsen et al.

2016).

Collectively, these

results suggest that ZnO nanoparticles have the

potential to induce genotoxicity; however, studies

incorporating the diverse physical-chemical proper-

ties and, a range of dose and post-exposure time

points are warranted.

Differences in toxicity of the coated and uncoated

ZnO nanoparticles

The uncoated and the coated ZnO qualitatively

induced similar responses. Both coated and

uncoated ZnO nanoparticles induced the pulmonary

acute phase response, increased the levels of total

protein in BAL fluid, and genotoxicity at single

doses and time points. However, only the coated

ZnO affected body weight gain (day 2) and pul-

monary inflammation in terms of neutrophil influx.

In contrast, in the microarray analysis, the uncoated

ZnO nanoparticle induced more differentially

expressed genes day 1 post-exposure as compared

to the coated ZnO nanoparticle, although the two

nanoparticles perturbed the same pathways. On

day 28 post-exposure, the highest number of differ-

entially expressed genes was observed in the

coated ZnO group. Again, it can be speculated that

the differential transcriptional response to the two

different ZnO nanoparticles could be caused by the

differences in dissolution rates. The coated ZnO

may dissolve slower than the uncoated ZnO nano-

particle, which is reflected by the increased

BEU, Alm.del - 2018-19 (2. samling) - Bilag 73: Orientering om resultater om zinkoxid, fra beskæftigelsesministeren

N. HADRUP ET AL.

differential gene expression on day 28. ZnO nano-

particles have been shown to quickly dissolve in

biological tissues (Xia et al.

2008;

Adam et al.

2014;

Eixenberger et al.

2017).

The dissolution behavior of

the uncoated ZnO (NM-110) and the coated ZnO

(NM-111) in water and cell culture medium have

been previously studied; at the 1

mg/mL

concentra-

tion, 60% of the uncoated ZnO dissolves in water at

24 h

vs.

18% for the coated ZnO nanoparticle. In

the cell culture medium, at the same concentration

of 1

mg/mL,

the dissolution rates of the uncoated

ZnO were reduced to 47%, whereas, 39% of the

coated ZnO was dissolved at 24 h, which is higher

than the dissolution observed in water for this ZnO

type (Kermanizadeh et al.

2013).

In another study,

the time-resolved dissolution kinetics in lysosomal

fluid showed a transient delay (10 h) of the coated

ZnO (NM-111) dissolution in comparison to the

€

uncoated ZnO (NM-110) (Koltermann-Jully et al.

2018).

Thus, the observed differences in pulmonary

responses to uncoated and coated ZnO exposure

are consistent with the uncoated being more sol-

uble than the coated. It has previously been shown

that at Zn doses of 40, 100 and 400

mg/rat

of either

ZnO nanoparticles or Zn ions induce similar pul-

monary toxicity in terms of neutrophil influx and

protein in BAL, suggesting that zinc ions are at least

partially responsible for the effects of pulmonary

ZnO nanoparticle exposure (Jeong et al.

2016).

did not assess the ZnO content in the lungs of the

exposed mice due to the low dose levels used

mg/mouse

at the highest dose), which in combin-

ation with the endogenous Zn levels limits the

chance of detection of treatment-related Zn.

Nevertheless, despite this potential difference in dis-

solution kinetics, pulmonary exposure to both ZnO

nanoparticles still induced differential gene expres-

sion 28 days after exposure and the coated ZnO

nanoparticles induced increased neutrophil influx at

this time point. This suggests that even the rela-

tively low doses used in this study (0.1 mg/kg bw)

induced long-term effects in mice.

terms of increased total protein in BAL fluid. At the

doses investigated, only weak non-dose-dependent

genotoxic effects were observed. Only the coated

ZnO nanoparticles induced pulmonary inflammation

as measured by BAL fluid neutrophil influx and

decreased body weight gain at day 2. The two ZnO

nanoparticles perturbed similar pathways in the

microarray analysis. Thus, the uncoated and coated

ZnO nanoparticles overall induced similar responses

and observed differences can most likely be attrib-

uted to differences in solubility kinetics. The pul-

monary

Saa3

response in mice was induced at ZnO

dose levels that were comparable to the ZnO doses

that induce systemic acute phase response in

humans after inhalation exposure. This suggests

that the murine pulmonary acute phase response

may be used as a model to predict human acute

phase response following exposure to metal oxides,

including ZnO. Future studies looking into mecha-

nisms of metal nanomaterial-induced acute phase

response are warranted.

Acknowledgments

Michael Guldbrandsen, Lisbeth Meyer Petersen, Anne-Karin

Asp, Yasmin Akhtar, Elzbieta Christiansen, Zdenka O

Kyjovska, and Lourdes M. Pedersen are thanked for excellent

technical assistance. The authors would like to acknowledge

the contributions of Dr. Luna Rahman and Alex Lim-Sersan

in the microarray data analysis and Dongmei Wu for her

assistance in planning the microarray experiments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by Danish Centre for Nanosafety

[grant number 20110092173-3 from the Danish Working

Environment Research Foundation]; Danish Centre for

Nanosafety 2; the European Union’s Horizon 2020 research

and innovation programme [grant numbers 686098

(SmartNanoTox), 646221 (NanoReg

)]; and Chemicals

Management

Plan

and

Genomics

Research

and

Development Initiative of Health Canada.

Conclusion

Pulmonary exposure to relatively low doses of

uncoated and triethoxycaprylylsilane-coated ZnO

nanoparticles induced dose-dependent pulmonary

acute phase response and pulmonary cytotoxicity in

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