Inhala ion o ZnO Nanopa icles: Splice Junc ion
Exp ession and Al e na i e Splicing in Mice
Pa el Rossne J ,*
,1
K is yna V bo a,* Simona S apaco a,
†
And ea Rossne o a,* An onin Amb oz,* Tana B zico a,*
,‡
Helena Libalo a,*
Eliska Ja o ko a,
§
Pa el Kulich,
†
Zbynek Vece a,
¶
Pa el Mikuska,
¶
Pa el Cou alik,
¶
Kamil K umal,
¶
Lukas Capka,
¶
Bohumil Docekal,
¶
Pa el Mo a ec,
k
Oma Se y,
kj
I an Misek,
kj
Pe Fic um,
kk
Ka el Fise ,
#
Mi osla Machala,
†
and Jan Topinka*
*Depa men o Gene ic Toxicology and Nano oxicology, Ins i u e o Expe imen al Medicine o he Czech
Academy o Sciences, P ague 14220, Czech Republic;
†
Depa men o Chemis y and Toxicology, Ve e ina y
Resea ch Ins i u e, B no 62100, Czech Republic;
‡
Depa men o Risk Resea ch and Managemen , Facul y o
Sa e y Enginee ing, VSB—Technical Uni e si y o Os a a, Os a a 700 30, Czech Republic;
§
Depa men o
T ansplan a ion Immunology, Ins i u e o Expe imen al Medicine o he Czech Academy o Sciences, P ague
14220, Czech Republic;
¶
Depa men o En i onmen al Analy ical Chemis y, Ins i u e o Analy ical Chemis y
o he Czech Academy o Sciences, B no 60200, Czech Republic;
k
Depa men o Ae osol Chemis y and
Physics, Ins i u e o Chemical P ocess Fundamen als o he Czech Academy o Sciences, P ague 16502, Czech
Republic;
kj
Depa men o Animal Emb yology, Ins i u e o Animal Physiology and Gene ics o he Czech
Academy o Sciences, B no 60200, Czech Republic;
kk
Depa men o Pa hological Mo phology and Pa asi ology,
o Ve e ina y Medicine, Uni e si y o Ve e ina y and Pha maceu ical Sciences, B no 612 42, Czech Republic;
and
#
Depa men o Pedia ic Hema ology and Oncology, 2nd Facul y o Medicine, Cha les Uni e si y P ague
and Uni e si y Hospi al Mo ol, P ague 15006, Czech Republic
1
To whom co espondence should be add essed a Depa men o Gene ic Toxicology and Nano oxicology, Ins i u e o Expe imen al Medicine, Videnska
1083, P ague 14220, Czech Republic. E-mail: [email p o ec ed].
ABSTRACT
Despi e he wide applica ion o nanoma e ials, oxici y s udies o nanopa icles (NP) a e o en limi ed o in i o cell models,
and he biological impac o NP exposu e in mammals has no been ho oughly in es iga ed. Zinc oxide (ZnO) NPs a e
commonly used in a ious consume p oduc s. To e alua e he e ec s o he inhala ion o ZnO NP in mice, we s udied
splice junc ion exp ession in he lungs as a p oxy o gene exp ession changes analysis. Female ICR mice we e ea ed wi h
6.46 10
4
and 1.93 10
6
NP/cm
3
o 3 days and 3 mon hs, espec i ely. An analysis o di e en ial exp ession and
al e na i e splicing e en s in 298 a ge s (splice junc ions) o 68 genes in ol ed in he p ocesses ele an o he biological
e ec s o ZnO NP was conduc ed using nex -gene a ion sequencing. Th ee days o exposu e esul ed in he up egula ion o
IL-6 and down egula ion o BID,GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junc ion exp ession; 3 mon hs o exposu e
inc eased he exp ession o splice junc ions in ALDH3A1,APAF1,BID,CASP3,DHCR7,GCLC,GCLM,GSR,GSS,EHHADH,FAS,
HMOX-1,IFNc,NF-kB1,NQO-1,PTGS1,PTGS2,RAD51,RIPK2,SRXN1,TRAF6, and TXNRD1. Al e na i e splicing o TRAF6 and
V
CThe Au ho (s) 2018. Published by Ox o d Uni e si y P ess on behal o he Socie y o Toxicology.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p://c ea i ecommons.o g/
licenses/by-nc/4.0/), which pe mi s non-comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Fo comme cial e-use, please con ac [email p o ec ed]
190
TOXICOLOGICAL SCIENCES, 168(1), 2019, 190–200
doi: 10.1093/ oxsci/k y288
Ad ance Access Publica ion Da e: No embe 30, 2018
Resea ch A icle
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TXNRD1 was induced a e 3 days o exposu e o 1.93 10
6
NP/cm
3
. In summa y, we obse ed changes o splice junc ion
exp ession in genes in ol ed in oxida i e s ess, apop osis, immune esponse, in lamma ion, and DNA epai , as well as he
induc ion o al e na i e splicing in genes associa ed wi h oxida i e s ess and in lamma ion. Ou da a indica e he po en ial
nega i e biological e ec s o ZnO NP inhala ion.
Key wo ds: zinc oxide nanopa icles; inhala ion; splice junc ion exp ession; al e na i e splicing.
Zinc oxide nanopa icles (ZnO NPs) a e a commonly used ma e-
ial wi h a yea ly p oduc ion o o e 30 000 ons. Because o
hei an imic obial p ope ies ZnO NP a e applied in medical
and cosme ics p oduc s, bu hey a e also ound in sunsc eens,
pigmen s, ca alys s, and in elec onic de ices. Due o he wide
applica ion o cosme ics, skin is he main ou e o exposu e o
ZnO NP o consume s. Inhala ion exposu e occu s mos ly in in-
dus ial p oduc ion by zinc oxide umes du ing ac i i ies such
as he mal cu ing, welding o gal anized s eel, o mel ing. Such
exposu e may esul in zinc e e cha ac e ized by h oa i i a-
ion, cough, and a ious espi a o y and lu-like symp oms.
Sys emic in lamma o y e ec s o ZnO NP inhala ion ha e e-
cen ly been demons a ed in human olun ee s exposed o ZnO
pa icle doses o 1.0 and 2.5 mg/m
3
, concen a ions below occu-
pa ional exposu e limi s o 5.0 mg/m
3
se in many coun ies
(Mons
ee al., 2018).
In he espi a o y sys em o expe imen al animals, he ad-
minis a ion o ZnO NP caused he induc ion o p ocesses asso-
cia ed wi h immune esponse and oxida i e s ess, al hough
pe manen pa hological changes in he o gans we e usually no
ound. In C57/Bl6 mice, sub-acu e inhala ion (2 weeks, 4 h/day,
3.5 mg/m
3
) esul ed in inc eased numbe o mac ophages in
b onchoal eola la age (BAL) luid and p oduc ion o cy okines
IL12(p40) and MIP-1a. Howe e , no signi ican his opa hological
changes in he lungs we e obse ed (Adamcako a-Dodd e al.,
2014). Inhala ion o occupa ionally ele an doses o ZnO NP (1.4
and 4.9 mg/m
3
o 2 weeks) a ec ed o al cell, neu ophil, LDH,
and o al p o ein le els in Sp ague Dawley a s. In lamma o y
pa hology in he lungs and degene a ion and nec osis o he
myoca dium we e also obse ed (Chuang e al., 2014). In F344
a s, a 4-week exposu e (6 h/day, 5 days/week) o 2 and 10 mg/
m
3
ZnO NP caused ansien inc ease in o al cell and neu ophil
coun and ele a ed le els o cy okine-induced neu ophil che-
moa ac an (CINC-1, CINC-2) and heme oxygenase 1 (HO-1) in
BAL. Howe e , no pe sis en lung in lamma ion o ib osis was
obse ed (Mo imo o e al., 2016).
The pulmona y e ec s caused by inhala ion o a ious ypes
o nanopa icles di e by he se e i y o he esponse and he du-
a ion o he e ec s. In mos cases lung in lamma ion was ob-
se ed, as eg, a e inhala ion o me al and me al oxide
nanopa icles (ZnO, TiO
2
,Al
2
O
3
,CeO
2
,Fe
2
O
3
.Fe
3
O
4
,MnFe
2
O
4
,
C OOH, Co, CuO) (Lai e al.,2018;La sen e al.,2016;P 
esum
ee al.,
2016;Wan e al.,2017) o single wall ca bon nano ubes (SWCNT)
(Me ce e al.,2008). SWNT inc eased collagen deposi ion in he
lungs bu he in lamma ion was ansien , whe eas CeO
2
inhala-
ion ga e ise o a mo e pe sis en in lamma ion. MnFe
2
O
4
and
C OOH caused mild pe ib onchiola ib osis. Co induced ex en-
si e lung in lamma ion, in e s i ial ib osis, and p oli e a ion o
in e s i ial cells. CuO p omo ed collagen accumula ion and ex-
p ession o he p og essi e ib osis ma ke a-SMA in he lungs. In
a compa ison o se e al me al oxide NP, ZnO was he only one
causing oxic e ec s in he ai ways (La sen e al.,2016). The oxic-
i y o ZnO NP is caused by he p esence o pa icles, as well as by
hei abili y o dissol e in he lungs. I has been shown ha he
pa icula e na u e o ZnO NP con ibu es o he sys emic nega i e
e ec s induced by inhala ion o hese nanopa icles. A compa i-
son o e ec s associa ed wi h ZnO NP and zinc ni a e inhala ion
e ealed acu e in lamma ion a b onchioal eola junc ions o he
lungs and cy okine sec e ion in BAL. Howe e , ac i i y o LDH in
BAL and glu ama e oxaloace a e ansaminase, glu ama e py u-
a e ansaminase, and c ea ine phosphokinase in blood we e
induced only by ZnO NP (Chen e al.,2015). The elease o Zn
2þ
ions in cy osol, and hei seques a ion by mi ochond ia ollowed
by mi ochond ial dys unc ion and apop osis, plays also an im-
po an ole in ZnO NP oxici y (Kao e al.,2012). The solubili y o
ZnO NP depends on he chemical p ope ies o he sol en : i is
lowe in RPMI medium and mode a ely ha d wa e han in
DMEM medium (Reed e al.,2012). A e in a acheal ins illa ion
in o he lungs o a s, ZnO NP emained in ac a a ound neu al
pH and apidly dissol ed unde acidic condi ions in he lyso-
somes causing lysosomal des abiliza ion and cell dea h.
In e es ingly, no dissolu ion o ZnO NP was obse ed in he a i i-
cial in e s i ial luid (Cho e al.,2011).
To in es iga e he mechanisms o NP oxici y in i o, gene
exp ession p o iling has been used. Al hough he s udies o he
pulmona y e ec s o ZnO NP exposu e in expe imen al animals
a e limi ed o in a acheal injec ion (Fukui e al., 2015) and in-
anasal ins illa ion (Sap a shi e al., 2015), gene exp ession
changes ollowing he inhala ion o o he me al-based NP (eg,
coppe , gold, i on, sil e , TiO
2
, and NiO) we e analyzed. Mos o
he s udies ocused on global gene exp ession changes using
mic oa ays, bu in some, a a ge ed analysis was pe o med
(Adamcako a-Dodd e al., 2015;Ho ie e al., 2016;Libe da e al.,
2014;Mo imo o e al., 2011). DNA damaging agen s no only al e
gene exp ession, bu may also induce al e na i e splicing which
p e e en ially a ec s he genes implica ed in DNA epai , cell-
cycle con ol and apop osis (Shk e a and Chabo , 2015).
In his s udy, we aimed o analyze splice junc ion (SJ) exp es-
sion changes in mice exposed by he inhala ion o wo concen-
a ions o ZnO NP o 3 days and 3 mon hs. The analysis o SJ
and exon exp ession is applied o de ec al e na i e splicing
e en s (Li e al., 2015). He e, we used di e en ial SJ exp ession as
a p oxy o analyze changes in he exp ession o genes in ol ed
in oxida i e s ess, immune esponse, in lamma ion, apop osis,
DNA damage and epai , and cell cycle egula ion as well as o
s udy he al e na i e splicing e en s possibly associa ed wi h
exposu e o ZnO NP.
MATERIALS AND METHODS
Animals. Adul emale ICR mice (6 weeks old, a e age weigh 24
g) ob ained om he Animal acili y o Masa yk Uni e si y
(B no, Czech Republic) we e allowed o acclima e o labo a o y
condi ions o a leas 1 week be o e he inhala ion expe i-
men s. The ICR mice we e used because as an ou b ed s ain
hey exhibi gene ic a iabili y compa able wi h ha na u ally
occu ing in human popula ions (Cui e al., 1993). Comme cial
die and wa e we e p o ided ad libi um. The expe imen s we e
app o ed by he E hical Boa d o he Ins i u e o Animal
Physiology and Gene ics (No. 081/2010; Ma ch 29, 2010).
ROSSNER ET AL. |191
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P oduc ion o ZnO nanopa icles and hei cha ac e iza ion. ZnO NPs
we e gene a ed con inuously using an e apo a ion-condensa ion-
oxida ion echnique. A ce amic c ucible con aining a small amoun
o g anula zinc was placed inside he ce amic eac o ube o a e -
ically o ien ed u nace (Ca boli e TZF 15/50/610; Ca boli e, Hope
Valley, UK). Zinc was e apo a ed a he cen e o he u nace a a
empe a u e o 465C; me al apo was ca ied ou o he u nace
wi h an ine ni ogen gas s eam a a low a eo 1.15L/min.In
he u nace ou pu zinc apo was dilu ed wi h a U-HEPA il a ed
ai s eam a a low a e o 1.80 L/min esul ing in he oxida ion o
zinc o zinc oxide and he o ma ion o ZnO NP by a chemical apo
condensa ion p ocess. The s eam wi h ZnO NP was hen spli in o
wo s eams a a a io o app oxima ely 1:30. Be o e en e ing he in-
hala ion chambe , bo h s eams we e u he dilu ed wi h a s eam
o pu i ied humidi ied ai (10 L/min) and used o whole-body inha-
la ion expe imen s.
The mo phology o ZnO NP was s udied using scanning
ansmission elec on mic oscopy (STEM). The pa icles we e
collec ed on coppe S160-4 TEM g ids (3 mm in diame e , 400
mesh g ids; Aga Scien i ic, Elec on Technology, S ans ed,
Essex, UK) by elec os a ic p ecipi a ion using a Nanome e
Ae osol Sample (model 3089; TSI, Sho e iew, Minneso a). The
size and shape o ZnO NPs we e hen analyzed using he
Magellan 400 L XHR mic oscope (FEI Company, Hillsbo o,
O egon) ope a ing in he scanning ansmission elec on mic o-
scope (STEM) mode.
The pa icle numbe concen a ion and he size dis ibu ion o
ZnO NP in he inhala ion chambe s we e measu ed con inuously
in he size ange o 7.64–229.6 nm using a Scanning Mobili y
Pa icle Size Spec ome e (SMPS Spec ome e ; model 3936L72;
DMA model 3081, CPC model 3772; TSI) a 5-min in e als.
Exposu e o ZnO NP. Adul mice we e exposed o ZnO NPs in a
whole-body inhala ion chambe desc ibed in de ail in ou p e i-
ous s udy (Vece a e al., 2011). The mice (5 animals/g oup) in
wo exposu e g oups we e exposed con inuously o 3 days, o 3
mon hs, espec i ely (24 h/day, 7 days/week). The con ol ani-
mals we e exposed o clean, il e ed ai wi hou nanopa icles
in he same inhala ion chambe . The inhala ion expe imen
s a ed a he same ime o bo h exposu e g oups and he con-
ols and was ca ied ou in pa allel wi h wo concen a ions o
ZnO NPs o ca 6.46 10
4
and 1.93 10
6
pa icles/cm
3
, espec-
i ely, co esponding o mass concen a ions o 20 mg/m
3
and
625 mg/m
3
, espec i ely. The es ima ed deposi ed dose o ZnO
o lowe NP concen a ion was 0.009 and 0.269 mg o ZnO/g o
mouse body weigh o 3 days and 3 mon hs exposu e pe iod,
espec i ely (Bide e al., 2000,Mi chell e al., 2007). Fo highe
NPs concen a ion, he es ima ed deposi ed dose o ZnO was
0.275 and 8.332 mg o ZnO/g o mouse body weigh o 3 days and
3 mon hs pe iod, espec i ely. Because no maximum human in-
hala ion esidue limi has been es ablished o zinc, he inhala-
ion doses o ou expe imen we e se up based on acu e and
in e media e o al exposu e, 300 mg Zn/kg/day (h ps://www.
a sd .cdc.go /m ls/m llis .asp; las accessed Janua y 1, 2019),
and he assump ion ha he pulmona y deposi ion ac ion o
ZnO is 100%. In his case, he inhala ion deposi ed doses o 3 mg/
kg/day o acu e, and 917 mg/kg/day o subch onic exposu e
co espond o mass concen a ion o 20 mg/m
3
and 625 mg Zn/m
3
and nanopa icle numbe concen a ion o 6.46 10
4
and 1.93 
10
6
P/cm
3
, espec i ely. The exposu e doses we e wi hin he
ange on p e ious epo s o inhala ion exposu e o ZnO NP
(Adamcako a-Dodd e al., 2014;Chuang e al., 2014;Mo imo o
e al., 2016). To p e en he adso p ion o nanopa icles on eed-
ing, a special dispense has been used. In a 10.5 cm 17 cm 2
cm poly inyl chlo ide block i e openings o a 1.5 cm diame e
we e d illed longi udinally. The openings we e illed wi h eed
and he dispense was placed, in a nea ly e ical posi ion, in a
wi e eeding compa men o he polyca bona e box in he inha-
la ion chambe . A he end o exposu e (a e 3 days, o 3
mon hs, espec i ely) he mice we e sac i iced by ce ical dislo-
ca ion. Lungs we e collec ed o biochemical, elec on mic o-
scopic, and SJ exp ession analyses.
Chemical analysis o Zn in he lungs. Fo he analysis o Zn con en ,
lung issue was collec ed om i e animals om each exposu e
g oup (a ZnO NP concen a ion o 6.46 10
4
and 1.93 10
6
NP/
cm
3
) and i e con ol animals. Samples we e insed in high pu-
i y wa e and s o ed in ials a 25C un il u he p ocessing.
The samples we e decomposed by mic owa e assis ed diges-
ion in 3 ml o concen a ed sub-boil g ade ni ic acid using
Qua z Dis illa ion Sys em (model MSBQ 2; Maasen, Eningen,
Ge many). The samples, including blanks, we e ea ed in
p ecleaned qua z ubes o closed p essu ized au ocla e sys em
(Ul aWa e, Miles one S l, I aly). The decomposi ion p og am
consis ed o ou s eps wi h he ollowing cha ac e is ics: s ep 1:
10 min wi h a empe a u e amp be ween 100 and 120C; s ep 2:
5 min wi h a empe a u e amp be ween 120 and 200C; s ep 3:
3 min wi h a empe a u e amp be ween 200 and 250C; s ep 4:
5 min a 250C. A e cooling down (10 min), diges s we e quan-
i a i ely ans e ed o ials and dilu ed wi h ul apu e wa e
o he inal mass o 10 g/ ial.
Zinc con en in diges s was de e mined by lame and elec-
o he mal a omic abso p ion spec ome y (ET AAS) using AA
3110 and AAnalys 600 (Pe kin-Elme , Inc., Shel on,
Connec icu ). Fo quan i ica ion, a me hod o s anda d addi ion
calib a ion was applied.
T ansmission elec on mic oscopy o ZnO NP in he lungs. The sam-
ples we e ixed in 3% glu a aldehyde in cacodyla e bu e , pos -
ixed in 2% OsO
4
solu ion in phospha e bu e , dehyd a ed in
50%, 70%, 90%, and 100% ace one and embedded in he Epon-
Du cupan epoxy esin mix u e (Epon 812 Se a, Ge many;
Du cupan, ACM Fluka, Swi ze land). Thin sec ions we e cu a
60 nm on an ul amic o ome Leica EM UC7 (Leica Mik osys eme
GmbH, Vienna, Aus ia) and placed on 50 mesh o m a -coa ed
nickel g ids. The sec ions we e con as ed using 2% u anyl ace-
a e and 2% lead ci a e. The samples we e obse ed a 80 kV us-
ing a Philips EM 208 ansmission elec on mic oscope (FEI
Company, Eindho en, The Ne he lands).
His opa hological examina ion o lung issue. Lung issue samples
we e ixed o e nigh in 10% bu e ed neu al o maldehyde a
4C. The samples we e hen dehyd a ed in an inc easing se ies
o e hanol, ea ed wi h xylene and embedded in pa a in. Se ial
his ological sec ions o 5 lm hickness we e p epa ed and se-
lec ed slides we e s ained in hema oxylin-eosin and G een
T ich ome using s anda d his ological echniques. Sec ions
we e examined by ligh mic oscopy in a blinded ashion.
RNA ex ac ion. Fo RNA ex ac ion lung issue samples we e lash-
ozen in liquid ni ogen and kep a 80C un il u he p ocessing.
To ex ac RNA, he AllP ep DNA/RNA/miRNA Uni e sal Ki
(Qiagen, Hilden, Ge many) was used. The issue (200–600 mg) was
homogenized unde liquid ni ogen using a mo a and pes le;
homogena es we e ans e ed o ubes con aining he lysis bu e
p o ided wi h he ki . RNA was ex ac ed om he homogena es
acco ding o he manu ac u e ’s ins uc ions and quan i ied using
a Nanod op ND-1000 Spec opho ome e (The mo Fishe Scien i ic,
192 | INHALATION OF ZnO NANOPARTICLES
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Wal ham, Massachuse s). To achie e equal RNA concen a ion,
he samples we e p ecipi a ed by sodium ace a e and e hanol, and
edissol ed in an app op ia e olume o RNase- ee wa e . The in-
eg i y o RNA was assessed wi h an Agilen 2100 Bioanalyze
(Agilen Technologies, Inc, San a Cla a, Cali o nia). An RNA
In eg i y Numbe (RIN) anged om 5.3 o 7.9. Isola ed RNA was
s o ed a 80C un il u he p ocessing.
Di e en ial splice junc ion exp ession analysis by nex -gene a ion se-
quencing. T uSeq a ge ed RNA cus om-made assays we e p e-
pa ed using DesignS udio (h ps://designs udio.illumina.com/;
las accessed Janua y 1, 2019). The assays consis ed o 298 a -
ge s (SJs) in 68 selec ed genes including hose playing a ole in
oxida i e s ess, immune esponse, in lamma ion, apop osis,
DNA damage and epai , and cell cycle egula ion. Fo 16 o he
genes he a ailable assays co e ed all SJs; his allowed o he
disco e y o po en ially no el ansc ip a ian s. Fo he o he
genes, he analysis o al e na i e splicing was only possible o
known ansc ip a ian s p o ided ha he assays a ge ed SJs
ha would allow dis inguishing be ween hese a ian s (13
genes). In summa y, among he selec ed 68 genes ou cus om
made assay allowed us o analyze ansc ip a ian s in 29
(42.6%) genes. Fo a comple e lis o selec ed genes and a ge ed
SJs, see Supplemen a y Table 1.
Fo he lib a y p epa a ion, T uSeq Ta ge ed RNA Exp ession
ki (Illumina, San Diego, Cali o nia) was used. The lib a y was
p epa ed acco ding o he manu ac u e ’s ins uc ions. RNA
samples (200–600 ng, depending on he RIN) we e used o syn-
hesize cDNA, ha was u he hyb idized o he Oligo pool ( a -
ge ed SJs) and PCR ampli ied in he p esence o adap e indexes
p o ided by Illumina. A single-end sequencing eac ion was
pe o med using he MiSeq sys em (Illumina) and he MiSeq
Reagen ki 3 (150-cycle).
Analysis o sequencing da a—splice junc ion coun s. The p ima y
da a (Fas Q iles) we e used o subsequen analysis. The se-
quencing was pe o med as 150 base long single-end eads, bu
he eal leng h be ween he designed p ime s was 50 bases, so
he p ima y da a we e immed o his leng h. Quali y con ol o
he p ima y da a be o e and a e imming was done by
Fas QC. Ensembl e e ence mouse genome GRCm38 (mm10)
was downloaded om Illumina iGenomes (h ps://suppo .illu-
mina.com/sequencing/sequencing_so wa e/igenome.h ml; las
accessed Janua y 1, 2019). The app op ia e Re seq anno a ion o
mouse genes (in a g o ma ) was downloaded om UCSC Table
B owse (h ps://genome.ucsc.edu/cgi-bin/hgTables; las
accessed Janua y 1, 2019). A e e ence g ile o junc ion coun -
ing was made acco ding o he expe imen al design o co e-
spond o he con ol g oup. P ima y da a we e aligned o he
e e ence mm10 genome using Topha 2 ( e . 2.1.0). Coun s o
indi idual SJs we e ob ained by HTSeq-coun ( e . 0.6.1); da a
we e anno a ed using biomaR ( e . 2.26.1). The di e ences in
coun s be ween he con ol and exposed g oups we e analyzed
by he S uden ’s es (SPSS 20.0; IBM, A monk, New Yo k); co -
ec ion o mul iple es ing was pe o med using he S o ey
Tibshi ani FDR me hod (S o ey and Tibshi ani, 2003). Changes
in ela i e SJ exp ession, exp essed as old change, we e calcu-
la ed by he DESeq2 me hod (Lo e e al., 2014).
Analysis o sequencing da a—al e na i e splicing e en s. To analyze
al e na i e splicing e en s, he Mul i a ia e Analysis o
T ansc ip Splicing (MATS; h p:// naseq-ma s.sou ce o ge.ne ;
las accessed Janua y 1, 2019; e . 3.2.5; [Shen e al., 2014]) and
he Spliced T ansc ip s Alignmen o a Re e ence (STAR; h ps://
gi hub.com/alexdobin/STAR; las accessed Janua y 1, 2019; e .
2.5; [Dobin e al., 2013]) ools we e used. As an anno a ion ile,
mouse mm10 genome in a g o ma (gene a ed a h p://ge-
nome.ucsc.edu/cgi-bin/hgTables; las accessed Janua y 1, 2019)
was used. Files con aining sequencing da a (in a bam o ma )
we e compa ed be ween he exposed and con ol animals, o
bo h es ed concen a ions and ime in e als. The ollowing al-
e na i e splicing e en s we e analyzed: skipped exons, al e na-
i e 50and 30splice si es, mu ually exclusi e exons, and
e ained in ons. Pooled di e en ial splicing es s we e pe -
o med using MATS-STAT (h ps://gi hub.com/Xinglab/ MATS-
STAT; las accessed Janua y 1, 2019) o iden i y he e en s mod-
ula ed by ZnO NP. A g aphical p esen a ion o he esul s was
conduc ed using he Sashimi plo s (h ps://gi hub.com/Xinglab/
ma s2sashimiplo ; las accessed Janua y 1, 2019).
Real- ime quan i a i e PCR (RT-qPCR) e i ica ion o he sequencing
da a. One mic og am o RNA om each sample was used o
complemen a y DNA (cDNA) syn hesis using he T ansc ip o
High Fideli y cDNA syn hesis Ki (Roche, Basel, Swi ze land).
The o iginal p o ocol was modi ied by using 2.5 lM oligo(dT)
and 10 lM andom hexame s o p iming in a 20 ll eac ion
olume. cDNA syn hesis was un using he ollowing condi-
ions: 30 min a 55C and 5 min a 85C. RT-qPCR was pe o med
using he 7900HT Fas Real-Time PCR Sys em (Applied
Biosys ems, Ca lsbad, Cali o nia). Each RT-qPCR eac ion was
ca ied ou in a inal olume o 14 ll con aining 2.5 ll o dilu ed
cDNA, 3.8 ll o wa e and 7 ll o mas e mix. To de e mine he
le el o each a ge gene, 0.7 ll o a speci ically designed assay
(Cus om designed eal- ime PCR assay wi h Double-Dye p obe;
P ime design, Eas leigh, UK) was added o he eac ion mix u e.
Cycling condi ions we e: 2 min a 95C ollowed by 40 cycles o
ampli ica ion (10 s a 95C and 60 s a 60C). The baseline and
h eshold alues o RT-qPCR expe imen s aw da a we e
assessed wi h SDS Rela i e Quan i ica ion So wa e e sion 2.3
(Applied Biosys ems, Wal ham, Massachuse s) o de e mine C
alues. The exp ession le els o a ge genes we e no malized
o he e e ence genes (EIF4A2 and CANX). The e e ence genes
we e selec ed acco ding o he s abili y o gene exp ession du -
ing expe imen al condi ions using he geNo m Re e ence Gene
Selec ion Ki (P ime design, Eas leigh, UK). Rela i e changes in
no malized gene le els we e calcula ed using he 2
DDC
me hod
(Li ak and Schmi gen, 2001). The sequences o p ime s used in
RT-qPCR a e shown in Supplemen a y Table 2.
S a is ical analysis. To compa e he Zn con en in he lungs o ex-
posed and con ol animals, compa e RT-qPCR esul s, numbe o
SJs and o calcula e co ela ions be ween da a, SPSS 20.0 (IBM)
was used. The no mali y o dis ibu ion was checked by he
Kolmogo o -Smi no es . As he da a we e dis ibu ed no mally,
he S uden ’s es was used o he compa ison o indi idual
g oups. The co ela ion be ween RT-qPCR and SJ exp ession
esul s was calcula ed using he Pea son es . The mean alues 6
s anda d de ia ions o he analyzed pa ame e s a e epo ed in
he igu es. Venn diag ams we e p epa ed in Bioin o ma ics &
E olu iona y Genomics ool (h p://bioin o ma ics.psb.ugen .be/
web ools/Venn/; las accessed Janua y 1, 2019).
RESULTS
ZnO NP Cha ac e iza ion
The size dis ibu ion o ZnO NP in he inhala ion chambe was
analyzed sepa a ely o he chambe wi h he concen a ion o
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6.46 10
4
and 1.93 10
6
ZnO NP/cm
3
, espec i ely. In he cham-
be wi h he highe concen a ion o ZnO NP, mode diame e ,
geome ic mean diame e , and a e age o al numbe concen a-
ion o nanopa icles was: 7.64 nm, 12.7 nm, and 1.93 10
6
pa -
icles/cm
3
, espec i ely; a e age mass concen a ion was 625
mg/m
3
. The size dis ibu ion o nanopa icles exp essed in num-
be concen a ion is shown in Figu e 1. In he chambe wi h he
lowe concen a ion o NPs, mode diame e , geome ic mean di-
ame e , and a e age o al numbe concen a ion o ZnO NP was
7.64 nm, 12.6 nm, and 6.46 10
4
pa icles/cm
3
, espec i ely; a -
e age mass concen a ion was 20.2 mg/m
3
. The mic og aphs
om STEM analysis showed ha he nanopa icles a e o med
by agglome a es in he size ange o abou 6–21 nm (Figu e 2).
Zinc Concen a ion, ZnO NP De ec ion, and His opa hological
Changes in he Lungs
The concen a ion o zinc in he lungs di e ed signi ican ly be-
ween he con ols, and animals exposed o 3 days o 1.93 10
6
ZnO NP/cm
3
(Zn con en [mg/g issue]: 13.1 60.10 s 20.6 60.38, p<
.05). Howe e , no such di e ence was obse ed o he 3-mon h ex-
posu e pe iod (Figu e 3). The chemical analysis o he Zn con en in
he lungs o mice exposed o 6.46 10
4
ZnO NP/cm
3
did no show a
signi ican di e ence be ween he exposed and con ol animals.
Scanning ansmission elec on mic oscopy showed he p esence
o ZnO NP in pneumocy es a e 3 mon hs o exposu e o 1.93 10
6
ZnO NP/cm
3
(Figu e 4); howe e , o a lowe concen a ion and/o
sho e inhala ion pe iod no ZnO NP we e de ec ed.
His opa hological e alua ion e ealed a goble cell hype plasia a
he e minal b onchioli o he lungs o animals exposed o 1.93 
10
6
ZnO NP/cm
3
o 3 mon hs (Figu e 5).
Di e en ial Splice Junc ion Exp ession Analysis
To analyze he e ec o ZnO NP exposu e on he exp ession o
he s udied genes, we assessed he di e ences in he abundan-
ces ( ead coun s) o indi idual SJs wi hin he selec ed genes.
Th ee days o exposu e esul ed in signi ican changes in he
exp ession o 8 SJs in 7 genes (Supplemen a y Table 3,Figs. 6A
and 6B), whe eas 3 mon hs o exposu e signi ican ly a ec ed 54
SJs in 22 genes (Supplemen a y Table 4,Figs. 6A and 6B). Fo he
sho e ea men pe iod, he exp ession was mos ly down egu-
la ed; a e he longe inhala ion o ZnO NP all signi ican
changes we e associa ed wi h he up egula ion o SJ exp ession.
In e es ingly, he signi ican esul s ob ained o 3 mon hs ex-
posu e we e mos ly hose induced by 6.46 10
4
ZnO NP/cm
3
,a
lowe es ed concen a ion; his was no con i med o 3 days o
exposu e. The e was e y li le o e lap o de egula ed SJs and
espec i e genes be ween bo h exposu e pe iods and es ed
doses (Figs. 6A and 6B). Signi ican e ec s o bo h concen a-
ions o ZnO NP we e ound only o he exp ession o SJs in BID
(3 days exposu e), FAS,NQO1, and TXNRD1 (3 mon hs exposu e).
O e all, he a ec ed genes included hose pa icipa ing in apo-
p o ic (APAF1,BID,CASP3,FAS), oxida i e s ess (ALDH1A3,
DHCR7,EHHADH,GCLC,GCLM,GSR,GSS,HMOX1,NQO1,PTGS1,
PTGS2,SRXN1,TXNRD1) and immunological esponse (IFN-c,IL-
6,NF-jB1,NF-jB2,RIPK2,TRAF6), ion anspo (SLC11A2), and
DNA epai (RAD51). A comple e lis o de egula ed SJs wi h a
leas one signi ican esul o he gi en gene is p o ided in
Supplemen a y Tables 5A and 5B.
To alida e ou app oach o using SJ exp ession as a p oxy
o gene exp ession changes analysis, he di e en ial SJ exp es-
sion da a ob ained o GCLC,GSR,HMOX,NQO1,NF-jB2,PTGS2,
and TXNRD1 a e exposu e o bo h concen a ions o ZnO NP in
bo h ime in e als we e co ela ed wi h mRNA exp ession
assessed using RT-qPCR (Figu e 7). The esul s showed a e y
good ag eemen be ween he me hods: hey signi ican ly co e-
la ed (R¼0.961, p<.001) and o 96.4% o he gene/ZnO NP con-
cen a ion/exposu e ime combina ions he da a we e
compa able o bo h app oaches. The di e ence was obse ed
only o he NF-jB2 exp ession a e 3 mon hs o exposu e o
he highe concen a ion o ZnO NP (a nonsigni ican
Figu e 1. Size dis ibu ion o ZnO NP exp essed in numbe concen a ion o nanopa icles in he inhala ion chambe , wi h a concen a ion o 1.93 10
6
ZnO NP/cm
3
(mode diame e 7.64 nm, geome ic mean diame e 12.7 nm).
Figu e 2. The scanning ansmission elec on mic og aph showing he agglom-
e a es o p ima y ZnO NP.
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up egula ion de ec ed by RT-qPCR in con as wi h a down egu-
la ion obse ed by he nex -gene a ion sequencing [NGS] ap-
p oach; da a no shown).
Al e na i e Splicing E en s Analysis
Al e na i e splicing (AS) e en s we e analyzed based on he
coun s o SJs in he indi idual genes ollowing inhala ion expo-
su e o ZnO and in he con ols. O e all, we ound changes in
AS e en s associa ed wi h 3 days o exposu e o ZnO NP, whe e-
as 3 mon hs o exposu e had no e ec s on he p ocess. Exon
skipping, bu no o he AS e en , was a ec ed a e exposu e o
1.93 10
6
ZnO NP/cm
3
; we obse ed no e ec o he lowe ZnO
NP concen a ion. We de ec ed an inc eased equency o AS o
TRAF6 and TXNRD1, genes pa icipa ing in he in lamma o y
and oxida i e s ess esponse, espec i ely, ollowing exposu e
o ZnO NP. Fo TRAF6, wo ansc ip a ian s (a longe
[NM_009424.3; 6188 bp] and a sho e [NM_001303273.1; 5985 bp]
one) ha e been desc ibed. The exposu e o ZnO NP inc eased
he ac i i y o AS and hus he equency o he sho e
NM_001303273.1 a ian . The a e age inclusion le el o he
skipped exon was 37.3% o he exposed and 59.7% o he
con ol animals (p<.001) (Table 1). Fou ansc ip a ian s o
TXNRD1 ha e been epo ed: NM_001042523.1, NM_001042513.1,
NM_015762.2, and NM_001042514.1. In ou s udy, we iden i ied
wo o hese a ian s: a longe a ian (NM_001042513.1; 3417
bp) and a sho e a ian (NM_015762.2; 3310 bp). We ound sig-
ni ican di e ences be ween he equencies o hese a ian s:
he exposu e o 1.93 10
6
ZnO NP/cm
3
was associa ed wi h an
inc eased equency o he sho e a ian when compa ed wi h
he con ols indica ing he induc ion o AS. The a e age inclu-
sion le el o he skipped exon in he exposed animals was
9.9%, whe eas in he con ols i eached 31.1% (p<.001)
(Table 1).
DISCUSSION
As ZnO NPs a e widely used in nume ous consume p oduc s
and indus ial applica ions, i is impo an o ob ain su icien
in o ma ion abou he biological impac o ZnO NP exposu e o
iden i y po en ial heal h isks. In gene al, exposu e o nanopa -
icles depends on he me hod o hei p oduc ion and hei eal-
li e applica ions. Thus, he same ype o NP can en e he body
by di e en ou es and he espi a o y ac is he main and yp-
ical ou e o en y o human o ganism o many NPs (Cho e al.,
2012;Kendall e al., 2011;Li e al., 2013). The e o e, he lungs o -
en ep esen an o gan wi h maximum exposu e o nanopa -
icles and inc eased suscep ibili y o hei oxic e ec s.
Al hough zinc is an essen ial mine al ha plays an impo -
an ole in cellula me abolism, and is p esen in ac i e si es o
enzymes and ac s as an an ioxidan (Coope , 2008), i has nu-
me ous oxic e ec s a highe concen a ions. In he case o ai -
way exposu e o expe imen al animals o ZnO NP, hese e ec s
a e mos ly associa ed wi h in lamma o y esponses and nec o-
sis caused by he abili y o ZnO NP o gene a e eac i e oxygen
species (Cho e al., 2010,2011;Chuang e al., 2014;Luy s e al.,
2014;Vandeb iel and De Jong, 2012;Xu e al., 2014).
In his s udy, we aimed o e alua e he e ec s o ZnO NP in-
hala ion in mice. Pa icula ly, we ocused on SJ exp ession
changes in he lungs as a p ima y a ge o gan a e he inhala-
ion o wo concen a ions o ZnO NP (6.46 10
4
and 1.93 10
6
ZnO NP/cm
3
) o 3 days and 3 mon hs o mimic acu e and sub-
ch onic exposu e. As al e na i e splicing is a mechanism ha
Figu e 4. The ansmission elec on mic og aph o ZnO NP (a ows) in pneumo-
cy es a e 3 mon hs exposu e o 1.93 10
6
ZnO NP/cm
3
. Abb e ia ions: E, e y h-
ocy e; N, nucleus; Pn, pneumocy e.
Figu e 5. A goble cell hype plasia a he e minal b onchioli o he lungs (indi-
ca ed by he a ow) o animals a e 3 mon hs exposu e o 1.93 10
6
ZnO NP/
cm
3
. This inding, no obse ed in he con ol g oup, may be ela ed o i i a ion
as a consequence o ZnO NP inhala ion. Abb e ia ions: TB, e minal b onchioli;
V, essel.
Figu e 3. Mean Zn con en (6SD) in he lungs o animals exposed o 1.93 10
6
ZnO NP/cm
3
o 3 days and 3 mon hs, espec i ely.
ROSSNER ET AL. |195
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con ibu es o ansc ip omic and p o eomic di e si y in highe
euka yo es a e eg, DNA damage, we u he concen a ed on
he induc ion o AS in he lungs o exposed animals.
Conside ing he physicochemical p ope ies o ZnO NP, hei
oxici y may be associa ed wi h bo h he p esence o pa icles
and he dissolu ion o ZnO ollowed by he elease o Zn
2þ
in
he o ganism. Cu en ly, i is belie ed ha he nega i e biologi-
cal e ec s o ZnO NP a e mos ly associa ed wi h Zn
2þ
elease
(Reed e al., 2012). A s udy by Gilbe e al. (2012) sugges s ha in
he human b onchial epi helial cells (BEAS-2B) ZnO NP a e i s
accumula ed in acellula ly and hen comple ely dissol ed gen-
e a ing Zn
2þ
ha is complexed by molecula ligands. The da a
Figu e 6. Venn diag ams epo ing numbe s o unique and common de egula ed splice junc ions (A) and espec i e genes (B) in he lungs a e inhala ion exposu e o
ZnO NP. Th ee days/low—exposu e o 6.46 10
4
NP/cm
3
o 3 days; 3 days/high—exposu e o 1.93 10
6
NP/cm
3
o 3 days; 3 mon hs/low—exposu e o 6.46 10
4
NP/
cm
3
o 3 mon hs; 3 days/high—exposu e o 1.93 10
6
NP/cm
3
o 3 mon hs.
Figu e 7. mRNA exp ession le els assessed by RT-qPCR o selec ed genes. The exp ession signi ican ly co ela ed wi h di e en ial SJ exp ession da a ob ained o
hese genes (R¼0.961, p<.001) indica ing ha SJ exp ession could be used as a alid app oach o s udy di e en ial gene exp ession. Th ee days/low—exposu e o
6.46 10
4
NP/cm
3
o 3 days; 3 days/high—exposu e o 1.93 10
6
NP/cm
3
o 3 days; 3 mon hs/low—exposu e o 6.46 10
4
NP/cm
3
o 3 mon hs; 3 days/high—exposu e
o 1.93 10
6
NP/cm
3
o 3 mon hs.
Table 1. Al e na i e Splicing E en s in Lung Tissues o Mice Following 3 Days Exposu e o 1.93 10
6
ZnO NP/cm
3
Gene Re Seq ID
(Longe /Sho e
T ansc ip
Va ian )
Exposed (N¼5) Con ols (N¼5) Inclusion le el
di e ence (%)
Numbe o
junc ions
included
Numbe o
junc ions
skipped
Mean
inclusion
le el (%)
Numbe o
junc ions
included
Numbe o
junc ions
skipped
Mean
inclusion
le el (%)
TRAF6 NM_009424.3/NM_001303273.1 62 638
a
54 633
b
37.3
e
136 694 42 620 59.6 22.3
TXNRD-1 NM_001042513.1/NM_015762.2 20 613
c
89 660
d
9.9
e
33 620 42 631 31.1 21.2
“Numbe o junc ions included” epo s he mean numbe o eads (6SD)/animal mapped o he splice junc ions included in he longe ansc ip a ian ; “numbe o
junc ions skipped” epo s he mean numbe o eads (6SD)/animal mapped o he skipped splice junc ions; “mean inclusion le el” is he no malized mean pe cen -
age o splice junc ions included in he longe ansc ip a ian ; “inclusion le el di e ence” epo s he di e ence be ween he mean inclusion le el in exposed and
con ol animals, a nega i e alue indica es a highe p opo ion o ansc ip a ian s wi h skipped exons and ac i i y o al e na i e splicing in he exposed animals;
de ails o he calcula ion a e p o ided in Shen e al. (2014).
a
p¼.145,
b
p¼.508,
c
p¼.252,
d
p¼.161 o a compa ison o numbe o junc ions included o numbe o junc ions skipped be ween he exposed and he con ol animals.
The mean inclusion le els o he skipped exons s a is ically di e ed (
e
p<.001) be ween he s udied g oups o bo h genes.
196 | INHALATION OF ZnO NANOPARTICLES
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ob ained in Wis a a s indica e ha ollowing dissolu ion in
he lungs Zn
2þ
ansloca e o he blood (Wang e al., 2010). In ou
s udy, we obse ed an inc eased con en o Zn in he lungs o
he animals exposed o 1.93 10
6
NP/cm
3
o 3 days when com-
pa ed wi h he con ols; a e 3 mon hs o exposu e, he e was
no di e ence be ween he g oups. A he same ime, STEM
de ec ed he p esence o ZnO NP in he lungs a e 3 mon hs,
bu no a e 3 days o exposu e. This obse a ion sugges s ha
a longe inhala ion pe iod may ha e esul ed in he sa u a ion
o mechanisms esponsible o ZnO NP dissolu ion in lyso-
somes, causing he shi o balance be ween he pa icula e and
dissol ed o m o ZnO NP owa d he pa icula e o m. This ac
may ha e con ibu ed o he di e ences in he SJ exp ession
and AS e en s induced a e acu e and subch onic exposu e o
ZnO NP.
Gene exp ession changes in he lungs ollowing ZnO expo-
su e ha e been in es iga ed in a s and mice in se e al s udies.
The inhala ion o ZnO umes induced mRNA le els o me allo-
hionein, a p o ein esponsible o binding hea y me als, hus
p o iding p o ec ion agains hei oxici y (Cosma e al., 1992).
The inc ease in exp ession le els was obse ed a ZnO concen-
a ions o 1–5 mg/m
3
and e u ned o he con ol le els 24 h a e
exposu e. The in a acheal ins illa ion o ZnO NP inc eased
mRNA le els o heme oxygenase-1 (HMOX1), in e leukin-6 (IL-6),
cy okine-induced neu ophil chemoa ac an (CINC)-1 and -3,
and me allo hionein-1 24 h a e exposu e. The ele a ed le els
we e no de ec ed in he samples collec ed 1 week a e ins illa-
ion (Fukui e al., 2015). In ano he s udy, in anasal ins illa ion
o ZnO NP esul ed in he inc eased exp ession o eo axin mRNA
24 h a e exposu e; he exp ession o monocy e chemo a ac-
an p o ein (MCP)-1 and umo nec osis ac o alpha (TNF-a)
mRNA was no a ec ed (Sap a shi e al., 2015). Al hough he da a
a e limi ed and ou es o ZnO deli e y di e , he esul s indica e
ha in he pulmona y sys em ZnO induces he exp ession o
genes associa ed wi h immune esponse and oxida i e s ess.
In ou s udy, we in es iga ed he changes o exp ession o a
la ge se o genes pa icipa ing in oxida i e s ess, immune e-
sponse, in lamma ion, apop osis, DNA damage and epai , and
cell cycle egula ion. We used a ge ed RNA sequencing ha
allowed us no only o assess changes in he exp ession o se-
lec ed SJs bu also o iden i y he induc ion o AS ollowing expo-
su e o ZnO NP. The analysis o SJ exp ession e ealed he
ollowing no able esul s: (1) The numbe o de egula ed SJs was
lowe a e 3 days exposu e han a e he longe inhala ion pe-
iod. (2) The exp ession o mos o he SJs was down egula ed a e
3dayso exposu e oZnONP,whe eas he3mon hso exposu e
caused an up egula ion o SJ exp ession. (3) Mos o he signi ican
esul s ob ained a e he longe ZnO NP ea men we e obse ed
o helowe concen a ion(6.4610
4
NP/cm
3
); he concen a ion
o 1.93 10
6
NP/cm
3
had mos ly no signi ican e ec .
The acu e exposu e caused a signi ican dec ease in SJs ex-
p ession le els in genes associa ed wi h apop osis (BID, BH3
in e ac ing-domain dea h agonis ), oxida i e s ess esponse
(GSR, glu a hione educ ase; PTGS2, p os aglandin syn hase 2;
TXNRD1, hio edoxin educ ase 1), me al anspo a ion
(SLC11A2, solu e ca ie amily 11 membe 2), and in lamma-
ion/immuni y (NF-jB2). In e leukin 6, an impo an cy okine
sec e ed by T cells and mac ophages o modula e immune e-
sponse, was he only p o ein whose SJ exp ession was induced
a e 3 days o ZnO NP inhala ion. In con as o his da a, he
subch onic 3 mon hs o ZnO NP inhala ion induced he SJ ex-
p ession in genes encoding p o eins in ol ed in he execu ion
o he apop o ic esponse (APAF1, a p o ein esponsible o he
ini ia ion o apop osis; CASP3, a membe o caspase cascade
ha ac i a es caspase 6 and 7; FAS, a ecep o playing a cen al
ole in he ini ia ion o apop osis) and oxida i e s ess including
HMOX1 (an enzyme esponsible o con e sion o heme o bili-
e din, ha also possesses an i-in lamma o y p ope ies),
ALDH3A1 (a p o ein ha plays a ole in esponse o lipid pe oxi-
da ion), NQO1 (a quinone educ ase pa icipa ing in de oxi ica-
ion eac ions), EHHADH (a p o ein in ol ed in he pe oxisomal
oxida ion o a y acids), enzymes associa ed wi h glu a hione
me abolism (GSS, GSR, GCLM, GCLC), SRXN1 (an enzyme ha
educes cys eine-sul inic acid o med as a esul o exposu e o
oxidan s), PTGS1 (a key p o ein in p os aglandin biosyn hesis
ha is exp essed as a esul o in lamma ion), and DHCR7 (a
p o ein pa icipa ing in choles e ol biosyn hesis ha educes
oxida i e s ess by dehyd ocholes e ol educ ion). O he
de egula ed SJs included hose in genes pa icipa ing in im-
mune esponse and in lamma ion (NF-jB1 and TRAF6, a signal
ansduce in he NF-jB pa hway; RIPK2 and IFN-c ha play a
ole in inna e and adap i e immune esponse) and DNA epai
(Rad51 ha plays a ole in homologous ecombina ion). Thus,
ou esul s sugges ha he e is a s a k di e ence in he e-
sponse o he o ganism o acu e and subch onic exposu e o
ZnO NP. Whe eas sho - e m inhala ion seemed o ail o ac i-
a e p o ec i e mechanisms in he lungs, long- e m exposu e
was associa ed wi h he induc ion o such p ocesses. Indeed,
some epo s indica e de elopmen o clinical ole ance a e e-
pea ed inhala ion exposu e o ZnO umes and ZnO NP
(Adamcako a-Dodd e al., 2014). Thus, ou obse a ions may
ep esen a molecula mani es a ion o such p ocesses.
Whe eas i is di icul o explain his da a, we should s ill bea
in mind ha he SJ exp ession was assessed and he p o ein le -
els o he espec i e ma ke s may ha e been di e en . Such
analyses, howe e , we e ou o he scope o ou s udy. O e all,
mos o he de egula ed SJs we e de ec ed in he genes associ-
a ed wi h oxida i e s ess esponse and/o in lamma ion. Bo h
p ocesses, cha ac e is ic o NP exposu e, a e known o play an
impo an ole in induc ion o neu odegene a i e and ca dio-
ascula diseases, as well as in cance (Madl e al., 2014). We
may hus assume, ha acu e inhala ion o ZnO NP may inc ease
he isk o hese diseases, whe eas a long- e m, ch onic expo-
su e may induce p o ec i e mechanisms ha dec ease he isk
o ZnO NP exposu e.
DNA damage is one o he ac o s a ec ing al e na i e splic-
ing, pa icula ly in he genes in ol ed in DNA epai , cell cycle
con ol and apop osis (Shk e a and Chabo , 2015). Va ious DNA
damaging agen s ha e been shown o induce AS ( e iewed in
[Shk e a and Chabo , 2015]), bu he e ec o nanopa icles ex-
posu e has no ye been s udied. A ecen s udy e ealed ha
ROS p oduc ion induced by pa aqua a ec ed he AS o DNA e-
pai genes including APAF-1,H- as,ERCC1,SKP2, and BIN1
(Vi a elli e al., 2013). The gene a ion o ROS is a mechanism
also implica ed in he nega i e e ec s o NP exposu e. To in es-
iga e AS, me hods based on RT-PCR o mic oa ay echniques
ha e been used. Howe e , hese app oaches only allow o he
de ec ion o known, epo ed ansc ip a ian s. A ecen de-
elopmen o NGS b ough he oppo uni y o disco e no el
ansc ip a ian s p o ided ha he whole-genome ansc ip-
ome was analyzed. This app oach has ecen ly been used o
de ec AS e en s in HepG2 cells ollowing exposu e o benzo[a]-
py ene, a human ca cinogen ( an Del e al., 2012). In his s udy,
we used he MiSeq sys em and assays p o ided in Illumina
DesignS udio o de ec SJs in he selec ed genes.
We ound AS e en s (speci ically exon skipping) o be in-
duced a e 3 days o exposu e o 1.93 10
6
NP/cm
3
. Longe
ea men and/o lowe concen a ion o ZnO NP had no impac
ROSSNER ET AL. |197
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on he p ocess. Fo bo h a ec ed genes (TRAF6 and TXNRD1),
he equency o he sho e splicing a ian inc eased ollowing
he ea men . TRAF6 encodes umo nec osis ac o ecep o -
associa ed ac o 6, a p o ein ha se es as a downs eam ac o
o mul iple ecep o amilies wi h immuno egula o y unc ions
(Walsh e al., 2015), as well as in NF-jB ac i a ion ollowing gen-
o oxic s ess, esul ing in double s and DNA damage (Hadian
and K appmann, 2011). The NCBI da abase epo s wo an-
sc ip a ian s o TRAF6, bo h de ec ed in ou s udy, di e ing in
one exon bu encoding he same p o ein (NM_009424.3, 6188 bp;
NM_001303273.1, 5985 bp; h ps://www.ncbi.nlm.nih.go /gene/
22034; las accessed Janua y 1, 2019). Howe e , no in o ma ion
on he possible unc ional impac o he p esence/absence o
he exon is a ailable. TXNRD1 encodes hio edoxin educ ase 1,
an enzyme ha uses elec ons om NADPH o educe oxidized
hio edoxin and o he p o ein and nonp o ein subs a es, and
hus helps o p o ec he o ganism agains oxida i e s ess
(A n
e 2009;Cebula e al., 2015). Fou ansc ip a ian s o
TXNRD1 a e epo ed a he NCBI da abase (h ps://www.ncbi.
nlm.nih.go /gene/50493; las accessed Janua y 1, 2019): a ian
1 encoding he longe cy osolic iso o m 1 (NM_001042523.1); and
a ian 2 (NM_001042513), a ian 3 (NM_015762.2), and a ian
4 (NM_001042514.1), all encoding he same cy osolic iso o m 2.
Al hough no in o ma ion on he unc ional di e ences o bo h
iso o ms in mice is a ailable, in human cells s ably o e exp ess-
ing indi idual iso o ms, he al e a ion o genes associa ed wi h
di e en ia ion has been obse ed (Nal a e e al., 2015). In ou
s udy, we we e able o dis inguish be ween wo ansc ip a -
ian s di e ing in one exon (NM_001042513 and NM_015762.2).
Simila ly o TRAF6, he exposu e o ZnO NP esul ed in highe
equency o he sho e a ian (NM_015762.2). Thus, al hough
we showed ha inhala ion o ZnO NP a ec s AS in he exposed
mice, he unc ional impac o such changes, i any, could no
be de e mined based on ou da a.
LIMITATIONS OF THE STUDY
Al hough we used NGS o de ec di e en ial SJ exp ession, due
o echnical easons ( he capaci y o he MiSeq sys em) we had
o limi he numbe o genes o which his analysis has been
conduc ed. Due o budge es ic ions, p o ein exp ession analy-
ses in he lungs ha e no been conduc ed; hese would po en-
ially shed mo e ligh on he mechanisms o esponse o ZnO
NP inhala ion. Finally, ou esul s migh be a ec ed by he ac
ha due o logis ics easons ( he size o he inhala ion chambe
and he need o sepa a e cages in case o bo h gende s) only e-
male mice we e exposed. As in gene al, gende di e ences in
gene exp ession o mice ha e been obse ed (Sh edo a e al.,
2015), a s udy on bo h gende s would p o ide mo e comple e in-
o ma ion on he biological e ec s on ZnO NP inhala ion.
CONCLUSIONS
In ou s udy, we aimed o e alua e he biological consequences
o acu e and subch onic exposu e o mice o ZnO NP. To he
bes o ou knowledge, his is he i s s udy analyzing he
e ec s o inhala ion exposu e o ZnO NP using NGS echnology,
allowing no only he analysis o di e en ial SJ exp ession, bu
also he de ec ion o al e na i e splicing. Ou da a showed SJ ex-
p ession changes in he lungs, conce ning pa icula ly he p o-
cesses associa ed wi h oxida i e s ess, immune esponse, and
in lamma ion a e subch onic exposu e o ZnO NP. We u he
de ec ed he induc ion o al e na i e splicing ollowing acu e
exposu e. In summa y, exposu e o ZnO NP by inhala ion
caused po en ially nega i e biological esponse in he expe i-
men al animals.
SUPPLEMENTARY DATA
Supplemen a y da a a e a ailable a Toxicological Sciences
online.
ACKNOWLEDGMENTS
The au ho s acknowledge he assis ance p o ided by he
Resea ch In as uc u e NanoEn iCZ, suppo ed by he
Minis y o Educa ion, You h, and Spo s o he Czech
Republic unde P ojec No. LM2015073. The au ho s epo
no con lic o in e es .
FUNDING
This s udy was suppo ed by Czech Science Founda ion
(P503/12/G147, 18-02079S), he Minis y o You h, Educa ion,
and Spo s o he Czech Republic (LO1508, CZ.02.1.01/0.0/0.0/
16_013/00 01821), and Ope a ional P og am P ague—
Compe i i eness (CZ.2.16/3.1.00/21528).
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198 | INHALATION OF ZnO NANOPARTICLES
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