RESEARCH ARTICLE
Poly(ADP- ibose)polyme ases inhibi o s
p e en ea ly mi ochond ial agmen a ion
and hepa ocy e cell dea h induced by H
2
O
2
Sand a M. Ma ı
´n-Gue e o
1
, Jose
´A. Muñoz-Ga
´mez
2
, Ma ı
´a-Ca men Ca asco
1
,
Ja ie Salme o
´n
2
, Ma ı
´a Ma ı
´n-Es ebane
´
1
, Miguel A. Cuad os
1
, Julio Na ascue
´s
1
,
Da id Ma ı
´n-Oli a
1
*
1Depa amen o de Biologı
´a Celula , Facul ad de Ciencias, Uni e sidad de G anada, G anada, Spain,
2Ins i u o de In es igacio
´n Biome
´dica (ibsG anada), Hospi al Uni e si a io San Cecilio, G anada, Spain
*[email p o ec ed]
Abs ac
Poly(ADP- ibose)polyme ases (PARPs) a e a amily o NAD
+
consuming enzymes ha play
a c ucial ole in many cellula p ocesses, mos clea ly in main aining genome in eg i y. He e,
we p esen an ex ensi e analysis o he al e a ion o mi ochond ial mo phology and he ela-
ionship o PARPs ac i i y a e oxida i e s ess using an in i o model o human hepa ic
cells. The ollowing ou comes we e obse ed: eac i e oxygen species (ROS) induced by
oxida i e ea men quickly s imula ed PARPs ac i a ion, p omo ed changes in mi ochon-
d ial mo phology associa ed wi h ea ly mi ochond ial agmen a ion and ene gy dys unc ion
and inally igge ed apop o ic cell dea h. Pha macological ea men wi h speci ic PARP-1
( he majo NAD
+
consuming poly(ADP- ibose)polyme ases) and PARP-1/PARP-2 inhibi o s
a e he oxidan insul eco e ed no mal mi ochond ial mo phology and, hence, inc eased
he iabili y o human hepa ic cells. As he PARP-1 and PARP-1/PARP-2 inhibi o s achie ed
simila ou comes, we conclude ha mos o he PARPs e ec s we e due o PARP-1 ac i a-
ion. NAD
+
supplemen a ion had simila e ec s o hose o he PARPs inhibi o s. The e o e,
PARPs ac i a ion and he subsequen NAD
+
deple ion a e c ucial e en s in dec eased cell
su i al (and inc eased apop osis) in hepa ic cells subjec ed o oxida i e s ess. These
esul s sugges ha he al e a ions in mi ochond ial mo phology and unc ion seem o be
ela ed o NAD
+
deple ion, and show o he i s ime ha PARPs inhibi ion ab oga es mi o-
chond ial agmen a ion. In conclusion, he inhibi ion o PARPs may be a aluable he apeu-
ic app oach o ea ing li e diseases, by educing he cell dea h associa ed wi h oxida i e
s ess.
In oduc ion
The li e is a i al o gan ha plays a decisi e ole in de oxi ica ion, and he e o e hepa ic dam-
age is equen ly he cause o se e e pa hologies. One o he main ac o s p o oking hepa ocy e
degene a ion (and consequen ly li e damage) is oxida i e s ess, which is o en associa ed
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 1 / 26
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OPEN ACCESS
Ci a ion: Ma ı
´n-Gue e o SM, Muñoz-Ga
´mez JA,
Ca asco M-C, Salme o
´n J, Ma ı
´n-Es ebane
´M,
Cuad os MA, e al. (2017) Poly(ADP- ibose)
polyme ases inhibi o s p e en ea ly mi ochond ial
agmen a ion and hepa ocy e cell dea h induced
by H
2
O
2
. PLoS ONE 12(10): e0187130. h ps://doi.
o g/10.1371/jou nal.pone.0187130
Edi o : Aami Ahmad, Uni e si y o Sou h Alabama
Mi chell Cance Ins i u e, UNITED STATES
Recei ed: July 21, 2017
Accep ed: Oc obe 13, 2017
Published: Oc obe 26, 2017
Copy igh : ©2017 Ma ı
´n-Gue e o e al. This is an
open access a icle dis ibu ed unde he e ms o
he C ea i e Commons A ibu ion License, which
pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal
au ho and sou ce a e c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape and i s Suppo ing In o ma ion
iles.
Funding: This wo k was suppo ed by g an s om
Minis e io de Economia y Compe i i idad, Spain
(BFU2010-19981), CEI BioTic G anada, Spain
(BS9-2015) and Minis e io de Educacio
´n, Cul u a y
Depo e, Spain (FPU14/02219). The unde s had
no ole in s udy design, da a collec ion and
wi h he de oxi ica ion unc ion o he li e . Oxida i e s ess in a cell de elops when he e is an
imbalance be ween he amoun o eac i e oxygen species (ROS) p esen and he abili y o he
cell o elimina e i o o epai he damage esul ing om he ac ion o ROS. Oxida i e s ess
leads o mul iple ypes o cell damage, including DNA b eaks, p o ein modi ica ions, lipid pe -
oxida ion, dis up ion o calcium homeos asis, mi ochond ial ailu e, impai men o he ene gy
me abolism and NAD
+
deple ion [1]. Oxida i e s ess is appa en ly a he o igin o mos li e
diseases, such as hose caused by alcohol consump ion [2,3], hepa o oxic d ugs [4,5], en i on-
men al pollu an s [6] and o he ac o s [7,8]. Mo eo e , oxida i e s ess in pa ien s su e ing
non-alcoholic a y li e disease (NAFLD) is signi ican ly g ea e han in heal hy con ols [9].
ROS is a collec i e e m ha includes oxygen ee adicals (such as supe oxide anion,
hyd oxyl and hyd ope oxyl adical) and non adical agen s (such as hyd ogen pe oxide
[H
2
O
2
], single oxygen and pe oxyni i e) wi h oxidising capaci y [10]. ROS a e p oduced as a
consequence o oxida i e p ocesses ha ake place in a ious ypes o memb ane o ganelles,
especially in he mi ochond ia du ing ae obic me abolism [11]. As well as being c ucial o
ene gy p oduc ion, he mi ochond ia also pa icipa e in o he aspec s o cell ac i i y such as
apop osis and he biosyn hesis o ce ain molecules. Consequen ly, when ROS a e p esen in
excess, a ec ing mi ochond ial unc ion, hey may o igina e bioene ge ic and me abolic laws
ha unde lie a he e ogeneous g oup o human diseases [12,13]. In pa icula , mi ochond ial
dys unc ion and oxida i e s ess ha e been documen ed in he p og ession o a ious li e dis-
eases, including NAFLD and non-alcoholic s ea ohepa i is [14,15].
Mi ochond ia a e dynamic o ganelles ha p esen di e en mo phologies, anging om
small sphe ical o ms o long in e connec ed ubula ne wo ks, depending on he cell ype and
he physiological condi ions. In ac , hese mo phologies may be a ec ed by he al e a ion o
mi ochond ial unc ion. This beha iou is gene a ed by mi ochond ial ission, which ag-
men s he mi ochond ia, p oducing small sphe ical o ms, and also by usion p ocesses, which
gi e ise o ubula ne wo ks o in e connec ed mi ochond ia [16,17]. Inc eased ROS le els
induce mi ochond ial agmen a ion in di e en cell lines [16], an ou come ha seems o be a
hallma k o apop osis; i is widesp ead in apop o ic cell dea h and blocking mi ochond ial is-
sion delays apop osis [18].
Poly(ADP- ibose)polyme ases (PARPs) a e a amily o nuclea p o eins ha consume
NAD
+
o modi y a ge p o eins. In humans, PARPs amily membe s a e encoded by 17 genes
and he main membe o his amily is he enzyme poly(ADP- ibose)polyme ase-1 (PARP-1),
which is esponsible o 85–90% o PARPs ac i i y (and NAD
+
consump ion), ollowed by
PARP-2, which accoun s o he emaining 10–15% o ac i i y [19]. Only hese wo membe s
o he PARPs amily ha e been implica ed in DNA damage sensing and epai ing [20]. Thus,
ROS exposu e, inducing s and b eaks in DNA, inc eases he ac i a ion o PARP-1 and PARP-
2 (he ea e e e ed o, join ly, as PARPs). Ac i a ed PARPs clea e NAD
+
subs a es o o m
poly-ADP- ibose (PAR) polyme s ha a e added o hemsel es and o o he p o ein accep o s.
Many PAR-modi ied p o eins a e ela ed o DNA epai [21]. O e ac i a ion o PARPs
deple es he NAD
+
om he cell and consequen ly induces a d ama ic educ ion in ATP le els
[22]. Thus, ex ensi e PARPs ac i a ion could esul in a global ene gy ailu e, causing nec osis
[23], which is implica ed in many in lamma o y diseases [24,25].
In he p esen s udy, an in i o model o human hepa ic cells exposed o sho imes o
high le els o a p o-oxidan agen was used o de e mine whe he PARPs inhibi ion exe s a
p o ec i e ole agains mi ochond ial dys unc ion and cell dea h. Ou esul s show ha PARPs
inhibi o s inc ease he iabili y o hepa ocy es and enable he eco e y o no mal mi ochon-
d ial mo phology a e he oxidan insul . Thus, PARPs inhibi ion could be a no el a ge o
supp ess bo h he cell dea h associa ed wi h oxida i e s ess and he pa hological ou come o
ela ed hepa ic diseases.
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 2 / 26
analysis, decision o publish, o p epa a ion o he
manusc ip .
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
Ma e ials and me hods
Cell cul u e and ea men s
WRL68 human li e cells (hepa ic cells) we e pu chased by he Scien i ic Ins umen a ion Se -
ices (CIC) o he Uni e si y o G anada om he Eu opean Collec ion o Cell Cul u es (ca #
89121403). Hepa ic cells we e main ained as an adhe en cul u ed monolaye in DMEM
(Sigma, S . Louis, MO) added wi h 10% oe al bo ine se um (Sigma), L-glu amine solu ion (2
mM; Sigma) con aining s ep omycin (100 μg/ml) and penicillin (100 U/ml), and incuba ed a
37˚C in 5% CO
2
.
In o de o induce ROS p oduc ion and oxida i e insul , hepa ic cell cul u es we e ea ed
wi h a single dose o H
2
O
2
( om a 30% s ock solu ion, Sigma) dilu ed o concen a ions ang-
ing om 0.25 o 5 mM o 0–30 min. The medium was hen eplaced wi h esh g ow h
medium, and he cells we e u he incuba ed o 2, 4 o 24 h depending on he expe imen
(Fig 1).
PJ34 (Enzo Li e Sciences, San Diego, CA) and AG14361 (Selleck Chemicals, Hous on, TX)
we e used as PARPs inhibi o s. PJ34 is a wa e -soluble cell-pe meable phenan h idinone de i -
a i e which selec i ely inhibi s he ca aly ic ac i i y o PARP-1 and PARP-2 (EC
50
= 20 nM)
[26]; while AG14361, a icyclic benzimidazole, is an ex emely po en inhibi o speci ic o
PARP-1 (K
i
<5 nM) [27]. The cells we e p e-incuba ed ( his is e med he Inhibi o p e- ea -
men s ep in Fig 1) wi h he PJ34 o AG14361 a a concen a ion o 1 μM o 16 h be o e he
oxida i e ea men ; he same PARPs inhibi o and concen a ion we e also p esen in he
medium o H
2
O
2
incuba ion and in he esh medium added pos - ea men ( e med he Pos -
incuba ion ime s ep in Fig 1).
To a oid NAD
+
deple ion, exogenous NAD
+
(0.25 mM, Sigma) was added o he cell cul-
u e du ing he H
2
O
2
ea men and o he esh medium du ing pos - ea men . Some cell cul-
u es we e ea ed wi h 4 mM o sodium py u a e (Sigma), a ROS sca enge , du ing he H
2
O
2
ea men and pos - ea men .
Fig 1. Schema ic summa y o expe imen al p ocedu es. Oxida i e ea men was applied as a single dose o H
2
O
2
o 30 min. PARPs ac i i y was
analysed du ing oxida i e ea men and 15 minu es a e wa ds. ROS p oduc ion was analysed du ing H
2
O
2
ea men and a e 30 min o pos -incuba ion.
Cell iabili y and cha ac e isa ion o cell dea h we e de e mined a e 24 h o pos -incuba ion. Mi ochond ial mo phology was s udied a 2 h o 4 h a e H
2
O
2
exposu e. Cellula ATP con en was s udied a 2 h a e he end o he H
2
O
2
ea men . PARPs inhibi o s we e added o cul u e media 16 h be o e oxida i e
ea men , du ing he ea men and du ing pos -incuba ion. Media we e supplemen ed wi h NAD
+
du ing H
2
O
2
ea men and du ing pos -incuba ion.
h ps://doi.o g/10.1371/jou nal.pone.0187130.g001
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 3 / 26
Measu emen s o in acellula ROS
The hepa ic cells we e seeded in 6-well cul u e pla es a a densi y o 2.0 x 10
5
cells/well. Be o e
he H
2
O
2
ea men , he cells we e incuba ed wi h 20 μM o dichlo odihyd o luo escein diace-
a e (H
2
DCFDA) o 30 min. This p obe is cell-pe meable and is hyd olysed in acellula ly o
he DCFH ca boxyla e anion, which is e ained in he cell. Oxida ion o DCFH by in acellula
ROS esul s in he o ma ion o he luo escen p oduc dichlo o luo escein (DCF) [10]. A e
H
2
DCFDA incuba ion, he medium was emo ed and he cells we e ea ed wi h H
2
O
2
o
0–30 min (Fig 1). The cells we e hen de ached wi h ypsin-EDTA solu ion (Sigma), cen i-
uged a 300 g and washed wi h ice-cold PBS. Finally, he luo escen s aining was de ec ed by
low cy ome y analysis in a Bec on Dickinson FACSA ia III cy ome e using FACSDi a so -
wa e (BD Biosciences, E embodegem, Belgium). The numbe o luo escen posi i e-cells was
exp essed as a pe cen age o he o al numbe o cells. I is impo an o no e ha DCFH does
no di ec ly eac wi h H
2
O
2
o o m he luo escen p oduc DCF [10].
Immuno luo escence and PARPs ac i i y
Hepa ic cells seeded on co e slips in 6-well cul u e pla es we e ea ed o 0–30 min wi h H
2
O
2
and ixed wi hin 15 min wi h ice-cold me hanol-ace one 1:1 o 10 min o de e mine he ime-
cou se o maximum ac i i y o PARPs (Fig 1). The co e slips we e incuba ed o e nigh a 4˚C
wi h he p ima y mouse monoclonal an i-PAR an ibody. Two an ibodies we e indis inc ly
used o hese immuno luo escence s udies: ca # 4335-MC-100 (T e igen, Gai he sbu g, MD)
and ca # ALX-804-220-R100 (Enzo Li e Sciences, San Diego, CA) [28], bo h a a dilu ion o
1:400. Then, he co e slips we e incuba ed wi h he seconda y an ibody (Alexa luo 488-con-
juga ed goa an i-mouse IgG; Molecula P obes, Eugene, OR; ca # A-11001; dilu ion 1:1000)
a oom empe a u e o 2 h and cell nuclei we e coun e s ained wi h he dye Hoechs 33342
(Sigma) o 2 min. Finally, he co e slips we e moun ed on slides using Fluo omoun G
moun ing medium (Sou he n Bio ech, Bi mingham, AL), and analysed using an Axiopho
mic oscope (Zeiss, Obe kochen, Ge many). To de e mine he le el o PARPs ac i a ion, he
numbe o PAR-posi i e nuclei de ec ed a 400X magni ica ion was coun ed in a o al o 200
nuclei o each condi ion, om h ee sepa a e expe imen s.
De e mina ion o cell iabili y and cell dea h
Cell iabili y was de e mined using he MTT (3-[4,5-dime hyl hiazol-2-yl]-2,5-diphenyl e azo-
lium b omide, Sigma) me hod [29]. The cells we e seeded in 96-well cul u e pla es a an ini ial
densi y o 8.0 x 10
3
cells/well. A e 30 min o H
2
O
2
ea men , he cells we e incuba ed o 24 h
(pos -incuba ion ime) in esh medium o eco e y, and 5 mg/ml MTT was hen added o 3 h
o measu e cell iabili y (Fig 1). A e his, he cul u e medium was emo ed and 100 μl/well o
DMSO (Sigma) we e added o dissol e he o mazan c ys als. The abso bance o each well a
595 nm was measu ed in a mic opla e spec opho ome e eade (Mul iskan Ascen , The mo
Scien i ic, Rock o d, lL). Cell iabili y in each well was exp essed as a pe cen age o he abso -
bance o each expe imen al condi ion in ela ion o he con ol wells (un ea ed cells), assum-
ing ha un ea ed cells ep esen ed 100% iabili y.
Apop o ic cell dea h was measu ed using an Annexin-V-FITC ki wi h p opidium iodide
solu ion (InmunoS ep, Salamanca, Spain; ca # ANXVF-200T) o de ec phospha idylse ine
exposu e [30]. Hepa ic cells, seeded in 6-well cul u e pla es, we e ea ed o 30 min wi h
H
2
O
2
, and a e 24 h o pos -incuba ion ime (Fig 1) we e de ached, labelled ollowing he
manu ac u e ’s p o ocol and analysed wi h a Bec on Dickinson FACSA ia III cy ome e using
FACSDi a so wa e (BD Biosciences). Cells ea ed wi h 6 μM o s au ospo ine (Sigma) o 24
h se ed as posi i e con ols o apop osis [31].
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 4 / 26
Nec o ic cell dea h was e alua ed by examining he elease o lac a e dehyd ogenase (LDH)
[32]. In his me hod, he cells we e seeded in 96-well cul u e pla es a an ini ial densi y o 12.0
x 10
3
cells/well and cul u ed o e nigh a 37˚C be o e H
2
O
2
ea men (Fig 1); a e 24 h o
pos -incuba ion, LDH elease was assessed by measu ing LDH ac i i y in cul u e medium
using a LDH Cy o oxici y Assay Ki (The mo Scien i ic; ca # 88953) acco ding o he manu-
ac u e ’s ins uc ions. The abso bance (Abs) a 490 nm o cell-cul u e medium om each
expe imen al condi ion (Abs exp) was measu ed in he mic opla e spec opho ome e eade
(Mul iskan Ascen , The mo Scien i ic); o de e mine he Abs a ia ion due o he expe imen al
condi ions, he backg ound Abs alues we e sub ac ed in each Abs exp. Some cul u es we e
ea ed wi h 1% T i on (Sigma) o 10 min as a posi i e con ol o nec osis: abso bance co e-
sponding o he spon aneous LDH elease (Abs spon ) was de e mined in un ea ed cells,
while ha o he T i on- ea ed cells co esponded o he maximum LDH elease (Abs max).
The pe cen ages (%) o LDH elease we e de e mined as LDH elease (%) = (Abs exp—Abs
spon ) / (Abs max–Abs spon ).
Mi ochond ial mo phology quan i ica ion and ul as uc u al s udy
Changes in mi ochond ial mo phology and ul as uc u e we e s udied in hepa ic cells seeded
on co e slips in 6-well cul u e pla es subjec ed o 30 min o hyd ogen pe oxide ea men , and
pos -incuba ed in esh medium o 2 o 4 h (Fig 1).
Two s a egies we e employed in his s udy: i s , he luo escen mi ochond ial p obe Mi o-
acke Red CMXROS (Cell Signaling Technology, Dan e s, MA) was added a a concen a-
ion o 125 nM o he cells du ing he las 45 min o pos -incuba ion. Then, he cells we e ixed
wi h ice-cold me hanol-ace one 1:1 o 10 min, moun ed on slides using Fluo omoun G
(Sou he nBio ech), and analysed wi h a con ocal Leica TCS-SP mic oscope (Leica, We zla ,
Ge many). The mi ochond ia we e analysed and classi ied using Mic oP so wa e, a use ul
ool ha has been used p e iously o s udy mi ochond ial agmen a ion [33]. Two majo
ypes o mi ochond ia we e conside ed, in e ms o he mo phology obse ed: a) Type I, small
mi ochond ia wi h globula mo phology; b) Type II, mi ochond ia wi h ubula mo phologies
(including b anched, wis ed o s aigh ubules). App oxima ely 7,000 mi ochond ia we e
analysed in 10 high-powe ields pe condi ion in each sepa a e expe imen a 600x magni ica-
ion. The elonga ion index and he mi ochond iala ea we e also measu ed wi h Mic oP so -
wa e. The elonga ion index was calcula ed as he leng h o he mi ochond ia di ided by hei
wid h. A la ge elonga ion index co esponded o ubula mi ochond ia mo phology.
In he second s a egy, he cells we e p ocessed o ansmission elec on mic oscopy
(TEM) by ixing hem in 2% glu a aldehyde in 0.05 M cacodyla e bu e (pH 7.4) supple-
men ed wi h 2 mM Cl
2
Mg and 0.03 g/L suc ose o 2 h, pos ixed in 1% osmium e oxide o
1 h, dehyd a ed in g aded se ies o e hanol, and embedded in epoxy esin. Ul a hin sec ions
(50–70 nm) we e moun ed on coppe g ids and examined unde a Zeiss Lib a 120 EDX elec-
on mic oscope (Zeiss, Obe kochen, Ge many).
Cellula ATP assay
WRL68 cells we e seeded in a 25 cm
2
lask a a densi y o 1.0 x 10
6
cells/ lask and allowed o
g ow o 24 h. The cells we e hen ea ed wi h H
2
O
2
o 30 min and pos -incuba ed o 2 h in
esh medium (Fig 1). A e his, he cells we e eco e ed, cen i uged a 300 g o 10 min and
washed wi h PBS. In acellula ATP was ex ac ed using he boiling wa e p ocedu e desc ibed
by Yang and cowo ke s [34]: 400 μl o boiling wa e was added o each cellula pelle , which
was subsequen ly o exed and incuba ed o 5 min; he esul ing suspension was cen i uged
a 12000 g o 5 min. ATP con en in he supe na an was de e mined using he Adenosine 5’-
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 5 / 26

iphospha e (ATP) Bioluminescen Assay Ki (Sigma, ca # FLAA-1KT) acco ding o he man-
u ac u e ’s ins uc ions. Luminescence was measu ed om 96-well pla es using a mic opla e
eade (TRIAD se ies mul imode de ec o , Dynex Technologies, Chan illy, VA) and he quan-
i y o ATP was calcula ed using a s anda d cu e. P o eins we e measu ed using he B ad o d
me hod (Bio ad). ATP con en was exp essed as nmol pe mg o o al p o ein.
S a is ical analysis
Da a a e exp essed as mean ±SEM om a leas h ee independen expe imen s. S uden ’s -
es was used o de e mine signi ican di e ences. S a is ical analyses we e pe o med using
IBM-SPSS S a is ics e sion 19.0 so wa e (IBM Co p., A monk, NY). A alue o P<0.05 was
conside ed s a is ically signi ican .
Resul s
A single sho exposu e o an oxidizing agen educe hepa ic cell iabili y
The esponse o hepa ic cells o expe imen al oxida i e s ess was assessed by de e mining he
iabili y o WRL68 cells a 24 h a e ea men o 30 min wi h a single dose o H
2
O
2
anging
om 0.25 o 5 mM. The MTT assay showed ha cell iabili y dec eased in a dose-dependen
manne a e oxida i e ea men . Pa icula ly, concen a ions o less han 2.5 mM o H
2
O
2
educed cell iabili y by 30–40%, compa ed o he con ol (Fig 2A; 29.4 ±2.7%, 32.8 ±2.8%
and 37.7 ±1.1% educ ion o 0.25, 0.5 and 1 mM, espec i ely). Howe e , ea men s a a con-
cen a ion exceeding 2.5 mM o H
2
O
2
p o oked a dec ease in cell su i al o o e 60% (Fig
2A; 63.2 ±2.5% and 90.3 ±0.2% educ ion o 3.5 and 5 mM, espec i ely; 43.4 ±4.4% educ-
ion o 2.5 mM). To e i y he gene a ion o ROS in hepa ic cells du ing H
2
O
2
ea men , we
analysed he con e sion o H
2
DCFDA o DCF (see Me hods). Time-cou se analysis o his oxi-
da i e ea men showed ha he la ges amoun s o cells showing in acellula ROS we e
de ec ed as ea ly as 15 min a e incuba ion wi h 3.5 mM o H
2
O
2
and ha hese p opo ions
o labelled cells we e main ained un il 30 min o H
2
O
2
incuba ion, when he oxida i e ea -
men concluded (Fig 2B). The numbe o cells wi h in acellula ROS d ama ically dec eased
jus a ew minu es a e he oxida i e ea men . I is o no e ha he signi ican inc ease in
cells wi h in acellula ROS was seen only a concen a ions o H
2
O
2
equal o o highe han
2.5 mM (Fig 2C; 84.6 ±7.6% and 96.8 ±4.7% inc ease o 2.5 and 3.5 mM, espec i ely). The e-
o e, he inc eased p oduc ion o ROS obse ed a e ea men wi h H
2
O
2
(concen a ion 2.5
mM) co ela es wi h a s ong dec ease in cell iabili y wi hin 24 h.
ROS induced by oxida i e ea men ac i a es PARPs
Oxida i e s ess can ac i a e PARPs due o i s abili y o cause DNA b eaks [35]. The e o e, we
s udied he le els o PARPs ac i a ion, measu ed by PAR polyme o ma ion using immuno-
luo escence analysis, when ROS we e p esen (Fig 3). We con i med ha mos PAR polyme
o ma ion had aken place a 15 min om he beginning o H
2
O
2
(Fig 3A), coinciding wi h he
p e iously desc ibed ea ly in acellula ROS p oduc ion; hence, PARPs ac i a ion is an ea ly
e en in hese oxida i e ea men . Pa icula ly, PARPs ac i a ion inc eased as H
2
O
2
concen-
a ion augmen ed (Fig 3B and 3C; 20.7 ±5.5%, 22.0 ±4.6%, 42.3 ±3.5%, 45.3 ±4.7%, 50.6 ±
4.0% and 72.6 ±5.7% inc ease in PAR-posi i e cells o 0.25, 0.5, 1, 2.5, 3.5 and 5 mM H
2
O
2
ea men compa ed o un ea ed con ol cells, espec i ely). So, ea men wi h H
2
O
2
3.5 mM
inc eased ea ly ROS p oduc ion (96.8 ±4.7% inc ease; Fig 2C), PARPs ac i a ion (50.6 ±4.0%
inc ease in PAR-posi i e cells; Fig 3) and s ongly educed cell iabili y a 24 h a e he ea -
men (63.2 ±2.5% educ ion; Fig 2A).
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
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Fig 2. Dec eased cell iabili y and ROS p oduc ion induced by oxida i e ea men . (A) Twen y- ou hou s a e exposu e o di e se
concen a ions o H
2
O
2
o 30 min, he iabili y o WRL68 cells was analysed wi h he MTT assay. Cell iabili y was exp essed as he pe cen age
o iable cells wi h espec o hose de e mined in he con ols (H
2
O
2
non- ea ed cells), which was conside ed o be 100%. Ba g aph shows he
mean ±SEM o h ee independen expe imen s. Signi ican di e ences wi h espec o he con ols (H
2
O
2
non- ea ed cells): *P<0.05,
**P<0.01 and ***P<0.001. (B) ROS p oduc ion by WRL68 cells was e alua ed by incuba ing he cells wi h H
2
DCFDA o 30 min, be o e
exposing hem o H
2
O
2
. The le el o DCF luo escence was e alua ed by low cy ome y a 0, 5, 15 and 30 min du ing exposu e and a 15 and 30
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
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PARPs inhibi o s inc ease cell iabili y and dec ease he a e o cell
dea h a e oxida i e ea men
We show abo e ha cell iabili y s ongly dec eases wi h ROS induc ion (Fig 2) and PARPs
ac i a ion (Fig 3) a e 3.5 mM o ea men wi h H
2
O
2
. The e o e, we will use his concen a-
ion o H
2
O
2
o s udy he ole o PARPs ac i a ion on he hepa ocy es su e ing an oxida i e
insul .
In i s place we de e mined whe he PARPs inhibi o s would ab oga e he dec ease in cell
iabili y (Fig 4A). To do so, we used AG14361 (a po en and selec i e inhibi o o PARP-1
ac i i y), which is known o inhibi o e 90% o PARP-1 ac i i y in human cells [36], and PJ34
(an inhibi o o PARP-1 and PARP-2 ac i i y) a 1 μM concen a ion; highe concen a ions
we e ound o be cy o oxic in his cell line (S1 Fig). Bo h PARPs inhibi o s p oduced a signi i-
can inc ease in he a e o cell su i al a e H
2
O
2
ea men , wi h 27.3 ±6.1% and 32.7 ±5.5%
inc ease in cell iabili y o PJ34 and AG14361 espec i ely (Fig 4A). Immuno luo escence
echniques showed ha PAR polyme o ma ion was s ongly diminished in hepa ic cells
ea ed wi h H
2
O
2
and PARPs inhibi o s (S2 Fig).
We specula ed ha PARPs inhibi o s migh inc ease cell su i al a es due o he di ec
neu alisa ion o ROS o , al e na i ely, by p e en ing he deple ion o NAD
+
and hence he
educ ion in ATP, as a esul o PARPs ac i a ion. To decide his ques ion, we i s examined
whe he ROS we e o med in he p esence o PARPs inhibi o s. We ound ha ROS con inued
o be p oduced a no mal le els despi e he p esence o he inhibi o s o PARPs ac i i y (Fig
4B); he e o e bo h PJ34 and AG14361 ha e no sca enge capaci y pe se and, hence, hei
e ec in eco e ing cell iabili y a e oxida i e damage is de i ed exclusi ely om he inhibi-
ion o PARPs ac i i y. As a second app oach, we conside ed whe he NAD
+
supplemen a ion
p e en ed a dec ease in cell iabili y a e he oxida i e damage. To de e mine his poin ,
hepa ic cells we e exposed o 3.5 mM H
2
O
2
o 30 min, oge he wi h NAD
+
incuba ion (0.25
mM), ollowed by 24 h o NAD
+
pos -incuba ion. NAD
+
supplemen a ion was ound o signi -
ican ly inc ease he cell su i al a e o hepa ic cells exposed o H
2
O
2
ea men (27.2 ±4.8%
inc ease; Fig 4C), o a deg ee simila o ha p esen ed by he PARPs inhibi o s.
The dec ease in cell iabili y o hepa ic cells exposed o H
2
O
2
ea men was mainly co e-
la ed wi h a signi ican inc ease in Annexin V-posi i e cells (a ma ke o apop osis; 60.5 ±
10.9% inc ease; Fig 5A and 5B). This apop o ic iden i y was co obo a ed by using s au ospo -
ine, a well-known apop osis induce [31], which ma kedly inc eased he numbe o Annexin
V-posi i e cells (29.6 ±4.7% inc ease; Fig 5B). In con as , he e was no such inc ease in LDH
elease, conside ed o indica e nec osis, a e H
2
O
2
ea men (6.4 ±2.5% inc ease; Fig 5C).
This sugges s ha a cell dea h mechanism simila o apop osis (iden i y by he phospha idylse -
ine exposu e de ec ed wi h Annexin V and he low LDH elease) may ha e occu ed in hepa ic
cells when oxida i e s ess was induced. In e es ingly, he PJ34 inhibi o signi ican ly sup-
p essed he inc ease in Annexin V-posi i e cells induced by H
2
O
2
(52.9 ±8.8% educ ion; Fig
5A and 5B) bu did no a ec he LDH elease a e he oxida i e insul (Fig 5C).
Taken oge he , hese esul s show ha a c i ical ole is played by PARPs ac i a ion and
NAD
+
deple ion in dec easing he su i al a es o hepa ic cells subjec ed o oxida i e s ess.
min a e 30 min exposu e o H
2
O
2
( ime poin o 45 min and 60 min, espec i ely, in he igu e). The linea g aph shows he pe cen age o
luo escen cells (DCF posi i e cells) a each ime poin . Mean ±SEM o h ee independen expe imen s. Signi ican di e ences be ween each
ea men and hei espec i e con ols (H
2
O
2
non- ea ed cells): *P<0.05, **P<0.01, and ***P<0.001. (C) Ba g aph showing he p opo ion
o DCF- luo escen cells (indica ing ROS p oduc ion) a 15 minu es o exposu e o di e en doses o H
2
O
2
ea men . Ba s ep esen he
mean ±SEM o h ee independen expe imen s. Signi ican di e ences wi h espec o H
2
O
2
non- ea ed cells: ***P<0.001.
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PARPs inhibi o s p e en he ea ly al e a ions in mi ochond ial o m
induced by ROS
Oxida i e s ess induced by ROS o e p oduc ion has been ela ed wi h mi ochond ial damage
and cell dea h [37,38]. In ou s udy, he apid onse o ROS gene a ion ollowing exogenous
Fig 3. PARPs ac i a ion in esponse o oxida i e ea men . (A) PARPs ac i i y was measu ed by de ec ing PAR p oduc ion by means o
immuno luo escence. Cells we e ea ed wi h H
2
O
2
and PAR p oduc ion was de ec ed a 0, 15 and 30 min o exposu e, and 15 minu es a e 30 min o
exposu e o H
2
O
2
( ime poin o 45 min in he g aph). The linea g aph shows he pe cen age o PAR posi i e nuclei (a leas 200 cells we e coun ed o
each poin and condi ion) a di e en ime poin s; he highes le el o PARPs ac i a ion was a 15 min o H
2
O
2
ea men . Mean ±SEM o h ee
independen expe imen s. Signi ican di e ences be ween each H
2
O
2
ea men and hei espec i e con ols: **P<0.01 and ***P<0.001. (B) Ba
g aph e ealing he e ec o di e en concen a ions o H
2
O
2
on he pe cen age o PAR posi i e nuclei de ec ed a 15 min o H
2
O
2
exposu e; a leas 200
cells pe condi ion we e coun ed. Ba s ep esen he mean ±SEM o h ee independen expe imen s. Signi ican di e ences wi h espec o H
2
O
2
non-
ea ed cells: *P<0.05, **P<0.01, and ***P<0.001. (C) Rep esen a i e immuno luo escence images showing he inc ease in PAR polyme (g een)
when WRL68 cells we e ea ed wi h di e en doses o H
2
O
2
o 15 min. Nuclei we e s ained wi h Hoechs (blue). Scale ba : 50 μm.
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PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
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In summa y, he abo e esul s clea ly sugges ha PARPs inhibi o s p e en he ea ly
changes in mi ochond ial and ul as uc u al mo phologies associa ed wi h oxida i e s ess
induced by a single sho exposu e o H
2
O
2
.
Fig 9. Analysis o mi ochond ial ul as uc u e. T ansmission elec on mic oscope pho omic og aphs
co esponding o hin sec ions o WRL68 cells a 4000x (A) and 8000x magni ica ion (B). Cells we e p e-
incuba ed, ea ed wi h H
2
O
2
o 30 min and hen pos -incuba ed o 2 h (in he p esence o absence o PJ34
inhibi o ) be o e being analysed by elec on mic oscopy. The H
2
O
2
ea ed cells, in he absence o PJ34
inhibi o (lowe le pho omic og aphs), show mi ochond ia wi h small g anula mo phology and sca ce
ans e sal c is ae, while he p esence o he inhibi o in oxida i e ea men (lowe igh pho omic og aphs)
es o es he ubula -like mo phology and ans e sal c is ae dis ibu ion. Scale ba : 1 μm in (A) and 0.5 μm in
(B).
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AG14361 inhibi o coun e ac s he dec ease in ATP induced by ROS
Mi ochond ial agmen a ion and he appa en educ ion in mi ochond ial c is ae obse ed in
H
2
O
2
ea ed cells ha e been closely ela ed o de ec s in ene gy p oduc ion and, hence, o
mi ochond ial dys unc ion. In o de o de e mine he impai men o mi ochond ial unc ion,
we nex analysed cellula ATP con en , using an ATP bioluminescence assay pe o med on
in ac cells ea ed o 30 min wi h H
2
O
2
ei he in he absence o p esence o PARPs inhibi o s
(see Me hods). A signi ican dec ease in ATP con en was obse ed a 2 h a e he H
2
O
2
ea -
men (a 4.7 ±1.4 nmol/mg educ ion compa ed wi h non- ea ed cells; Fig 10A), which was
signi ican ly neu alised wi h he AG14361 inhibi o (2.6 ±0.2 nmol/mg inc ease in ATP con-
en compa ed wi h H
2
O
2
ea men ; Fig 10A). Conco dan ly, NAD
+
supplemen a ion in oxi-
da i e ea men also caused an inc ease in ATP con en simila o hose obse ed o he
AG14361 inhibi o (2.4 ±0.6 nmol/mg inc ease compa ed wi h H
2
O
2
ea men ; Fig 10B), sug-
ges ing ha NAD
+
deple ion caused by PARPs ac i a ion con ibu es o mi ochond ial ene -
ge ic dys unc ion.
Discussion
The p esen s udy in es iga es he e ec o he PARPs inhibi o s PJ34 (which inhibi s PARP-1
and PARP-2) and AG14361 (which speci ically inhibi s PARP-1) on cell su i al and dea h
a e oxida i e insul . The oxida i e insul o WRL68 cells, used as an in i o hepa ic model
[43], consis ed in a sho ea men wi h H
2
O
2
. As PARP-1 and PARP-2, whose o e ac i a ion
p oduces he deple ion o cellula NAD
+
, a e in ol ed in he sensing and epai o DNA, hei
e ec on cell dea h and su i al was analysed by inhibi ing hei ac i i y. The su i al o
WRL68 cells ollowing H
2
O
2
ea men inc eased when he PARPs we e inhibi ed. Mo eo e ,
since bo h inhibi o s eco e ed simila amoun s o cell iabili y, we concluded ha his e ec
Fig 10. PARPs inhibi ion o NAD
+
supplemen a ion inc eases he ATP cell con en a e H
2
O
2
ea men . (A) The ba g aph
shows he ATP con en (in nmol pe mg o o al p o ein) ob ained in soluble ex ac s om WRL68 cells in each expe imen al condi ion.
Hepa ic cells we e p e-incuba ed, ea ed wi h H
2
O
2
o 30 min and pos -incuba ed o 2 h (always in he p esence o AG14361 inhibi o )
be o e being p ocessed o ob ain he cell ex ac s. (B) NAD
+
was supplemen ed du ing oxida i e ea men (30 min) and du ing he pos -
incuba ion ime (2h). Then, cell ex ac s we e ob ained and p ocessed o measu e ATP con en (in nmol pe mg o o al p o ein). Ba s
ep esen he mean ±SEM o ou (A) and h ee (B) independen expe imen s. Signi ican di e ences: *P<0.05 and **P<0.01 e sus
con ol (non- ea ed cells);
#
P<0.05 and
##
P<0.01 e sus 3.5 mM H
2
O
2
.
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PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
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is essen ially due o he inhibi ion o PARP-1 ac i i y. In addi ion, we demons a e ha he
inc eased cell su i al could be due o p e en ing he NAD
+
deple ion p oduced a e he
PARPs ac i a ion induced by oxida i e s ess. Simila esul s ha e been ob ained in chick
emb yo li e and o he ypes o cells cha ac e ised by PARP-1 o e ac i a ion and supple-
men ed wi h NAD
+
[44–46]. As he deple ion o NAD
+
has been ela ed o a loss o abili y o
egene a e ATP wi hin he cells, i s es o a ion o he p e en ion o i s deple ion by inhibi ing
PARPs enzymes could a oid ene gy ailu e in hepa ic cells.
We used millimola concen a ions o H
2
O
2
o induce oxida i e s ess in ou in i o model,
which a e highe han physiological and mos pa hological concen a ions. We made his choice
a e inding ha WRL68 hepa ic cells we e esis an o cell dea h induced by mic omola H
2
O
2
concen a ions. This esis ance may be ela ed o he elabo a e de oxi ica ion and an ioxidan
sys ems o hepa ic cells, likely de eloped o main ain cellula homeos asis in he li e [47]. In
con as , o he cell ypes a e mo e sensi i e o H
2
O
2
ea men , and concen a ions abo e
50 μM ha e been desc ibed as cy o oxic o a wide ange o animal cells in cul u e[48]. Hence,
he H
2
O
2
dosage mus be adap ed o each cell ype. In summa y, he concen a ion o H
2
O
2
selec ed (3.5 mM concen a ion o 30 min) esul ed in a s ong educ ion in cell iabili y (Fig
2A), ROS accumula ion in all cells (Fig 2C), an inc ease in PARPs ac i a ion (Fig 3) and mi o-
chond ial al e a ions (Fig 6). These mi ochond ial al e a ions we e simila o hose documen ed
in a ious li e diseases[14,15,49,50]. The p esen s udy p esen s a possible expe imen al
model o he s udy o hese changes and he ela ionships among ROS p oduc ion, PARPs ac i-
a ion and mi ochond ial al e a ion. Fu he esea ch is wa an ed on he po en ial ole o
PARPs in he supp ession o oxida i e s ess-associa ed cell dea h in hepa ic diseases ha
de elop wi h ea ly mi ochond ial changes.
The inhibi ion o PARPs is o clinical in e es . These inhibi o s ha e been desc ibed as a
powe ul he apy o pa ien s wi h he edi a y b eas o o a ian cance con aining mu a ions in
he BRCA-1 and BRCA-2 genes ha a e essen ial o epai ing DNA lesions by he homolo-
gous ecombina ion pa hway [51,52]. The inhibi ion o PARPs in hese gene ic condi ions
enhances he o ma ion o non- epai ed double s and b eaks, p oducing cy o oxic lesions
ha induce cell dea h in he cance cells [53]. Addi ionally, PARPs inhibi o s ha e been ela ed
o po en ial he apeu ic e ec s in non-oncological diseases [54]; in an i-in lamma o y he apy
[22,24,55] hey educe he NAD
+
deple ion ha igge s nec o ic cell dea h and an agonise
he ansc ip ion o p o-in lamma o y genes p omo ed by PARPs [24,25]. Ou indings, om
using WRL68 cells (as an in i o model o hepa ic cells), co obo a e he he apeu ic possibili-
ies o PARPs inhibi o s in diso de s linked o oxida i e s ess as a means o educing NAD
+
and ATP deple ion. The e o e, ou esul s ag ee wi h ha o o he au ho s, ha he pha maco-
logical inhibi ion o PARP-1 is a p omising he apy o hepa ic cells su e ing oxida i e s ess,
as well as o in lamma o y diso de s and bioene ge ic dys unc ion [56–58].
The esul o PARP-1 o e ac i a ion is cell dea h. Deple ion o NAD
+
and/o ATP a e
PARPs ac i a ion may o igina e nec o ic cell dea h [23]. In addi ion, PAR is in ol ed in he
elease o AIF om mi ochond ia o he nucleus ha induces apop osis [59]. In ou s udy, 30
min o exposu e o 3.5 mM H
2
O
2
( e e ed o in he ex as sho -exposu e ime) caused dea h
in WRL68 li e cells ha was cha ac e ised as apop osis, as he cells exp essed biochemical
ma ke s o apop osis, such as phospha idylse ine exposu e on he ou e lea le o he plasma
memb ane; howe e , ansloca ion o he AIF o he nucleus was no de ec ed (da a no
shown). Acco ding o a s udy in which he same hepa ic cell line was ea ed o 3 h wi h 0.3
mM H
2
O
2
, he PJ34 inhibi o inc eases Ak ac i a ion, which in u n inhibi s he p oapo o ic
molecule BAD [60]. I should be no ed ha bo h he la e s udy and ou own eco ded an
inc ease in he iabili y (e alua ed by he MTT assay) o cells ea ed wi h a PARPs inhibi o ,
al hough we did no obse e a ela ion be ween he ac i a ion o Ak and cell su i al (da a no
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
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shown). Finally, a hi d s udy [56], which used mouse p ima y hepa ocy es ea ed wi h 3 mM
H
2
O
2
o 16 h (long exposu e ime), desc ibed an inc ease in nec osis ha was educed by
PARPs inhibi o s. Hence, i seems ha he mechanisms implica ed in H
2
O
2
induced cell dea h
in hepa ic cells depend on he concen a ion and du a ion o he oxida i e ea men .
Oxida i e s ess induced by exogenous H
2
O
2
ea men leads o mul iple cell damage, p o-
ducing an inc ease in ROS and in i s eac ion wi h cellula componen s such as p o ein, lipids
and nucleic acids. Hence, oxida i e s ess and ROS can ha e a ious biological consequences,
which include non-su i al o su i al acco ding o he ex en o damage and he cellula con-
ex [61]. The esul an DNA oxida i e damage can lead o mild ac i a ion o o e ac i a ion o
PARPs. In he i s ins ance, PARPs ac i a ion induced by mode a e oxida i e damage does
no cause deple ion o NAD
+
, and he cell dea h p oduced would no depend on PARPs ac i a-
ion. Howe e , excessi e oxida i e s ess can elici ex ensi e DNA damage associa ed wi h a
s ong NAD
+
deple ion gene a ed by PARPs o e ac i a ion, and he ensuing cell dea h would
be PARPs-dependen [22,62,63]. The p esen esul s a e consis en wi h his p oposi ion,
gi en ha lowe doses o H
2
O
2
(0.25 mM) we e associa ed wi h low ROS gene a ion (Fig 2C),
mode a e PARPs ac i a ion (Fig 3B) and no mi ochond ia al e a ion (da a no shown), p o-
ducing a lesse dec ease in cell iabili y (Fig 2A), whe eas highe H
2
O
2
concen a ions (3.5
mM; Fig 2A) ma kedly educed he p opo ion o iable cells, gene a ing ele a ed ROS p o-
duc ion (Fig 2C), PARPs o e ac i a ion (Fig 3B) and mi ochond ial dys unc ion (Fig 6). These
da a sugges ha he dec eased cell iabili y a low H
2
O
2
concen a ions is independen o
PARPs ac i a ion, while he inc eased cell dea h a highe H
2
O
2
concen a ions esul s om
PARPs o e ac i a ion. The e o e, PARPs a e no in ol ed in all o he cell dea h phenomena,
as indica ed by he ac ha PARPs inhibi o s did no escue all cell dea h, and PARPs-inde-
penden cell dea h mechanisms would also play a ole a e oxida i e damage.
Ne e heless, PARPs inhibi o s con e ed p o ec ion agains cell dea h o a subs an ial pe -
cen age o cells, and esea ch is wa an ed on i s use ulness in he apies in which hepa ic cells
a e exposed o high ROS concen a ions.
A majo poin o ou indings is he ela ionship obse ed be ween al e a ions in mi ochon-
d ial mo phology and he ene ge ic dys unc ion de ec ed a e oxida i e ea men . In i o
obse a ions sugges ha mi ochond ial dys unc ion is in ima ely linked o he bioene ge ics
s a us in me abolic diseases o hepa ic cells [15]. Mi ochond ial dys unc ion is ela ed o
changes in he mo phology o mi ochond ia, which anges om long in e connec ed ubules
o small globula o ms, depending on he physiological condi ion [17], wi h he small globula
o ms being ela ed o he p ocess o mi ochond ial agmen a ion. Ou ex ensi e analysis o
he mi ochond ial al e a ions induced by oxida i e s ess and ROS in hepa ic cells shows ha
mi ochond ial agmen a ion and loss o c is ae a e an ea ly and p og essi e e en a e oxida-
i e s ess, and ha hese al e a ions a e coun e ac ed by PARPs inhibi ion.
Mi ochond ial ission lowe s espi a o y ac i i y, ATP cell con en , mi ochond ial dys unc-
ion and cell dea h [16]. P e ious in i o s udies e ealed ha mi ochond ial agmen a ion is
enhanced by oxida i e s ess and ROS [64–68]. In e es ingly, we show, oo, ha he mi ochon-
d ial agmen a ion and ene ge ic dys unc ion (loss o ATP con en ) induced by ROS p oduc-
ion a e H
2
O
2
ea men a e inhibi ed by PARPs inhibi o s in an in i o model o hepa ic
cells. In sho , ROS o ma ion, PARPs ac i a ion, mi ochond ial agmen a ion and cell dea h
appea o be causally ela ed. A ecen wo k using e inal cells epo ed a simila co ela ion
[69].
PARPs ac i a ion appa en ly impai s mi ochond ial unc ion [70–74], bu he mechanism
unde lying his e ec has no been elucida ed and se e al possibili ies ha e been p oposed. In
he i s place, as he NAD
+
coenzyme is essen ial o glycolysis, he ica boxylic acid cycle
and mi ochond ial espi a ion, i s deple ion a e PARPs ac i a ion would esul in an
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 19 / 26
ene ge ic ca as ophe wi hin he cell, and, hence, in mi ochond ial dys unc ion. Secondly,
some au ho s ha e p oposed ha , in addi ion o nuclea ac i i y, he e exis s a mi ochond ial
PARPs ac i i y ha could p omo e mi ochond ial dys unc ion by he poly-ADP- ibosyla ion
o mi ochond ial p o eins [73,74]. Thi d, we also hypo hesise ha mi ochond ial impai men
Fig 11. Scheme showing he p oposed mechanism by which PARPs inhibi o s p e en mi ochond ial agmen a ion and dec ease cell dea h
induced by ROS in hepa ic cells. Reac i e oxygen species (ROS) induced by oxida i e ea men (H
2
O
2
) s imula ed PARPs ac i a ion, gene a ing NAD
+
deple ion and causing an al e a ion in he NAD
+
/NADH pool. This al e a ion p oduces changes in mi ochond ial mo phology, esul ing in mi ochond ial
agmen a ion and cell dea h as a consequence. The inhibi ion o PARPs ac i i y by PARPs inhibi o s a oid NAD
+
deple ion and he e o e p e en s
mi ochond ial agmen a ion and cell dea h.
h ps://doi.o g/10.1371/jou nal.pone.0187130.g011
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 20 / 26
a e PARPs ac i a ion may be ela ed o al e a ions in he NAD
+
/NADH pool, as NAD
+
enhances he biogenesis o mi ochond ial espi a o y chain complexes [75] in addi ion o play-
ing a ole in mi ochond ial ene gy p oduc ion [76]. NAD
+
is educed o NADH, and NADH,
in u n, enhances he impo and biogenesis o espi a o y chain complexes; he e o e, he
educ ion o NADH a e NAD
+
deple ion would a ec he mi ochond ia. Finally, PARPs ac i-
a ion-induced NAD
+
deple ion may in e e e wi h NAD
+
-dependen deace ylase si uin-3
(SIRT3), which is a membe o he si uin enzyme amily loca ed in he mi ochond ia. SIRT3
is highly exp essed in he li e and is c ucial o he main enance o mi ochond ial unc ions
[77]. SIRT3 unc ion in he mi ochond ia is essen ial o egula ion o he ac i i ies o espi a-
o y complexes, he an ioxidan enzyme MnSOD and o he enzymes in ol ed in egene a ing
educed co ac o s in o de o main ain a p ope glu a hione edox s a us[77]. SIRT3 he e o e
has a c ucial ole in ene gy p oduc ion and ROS de oxi ica ion. SIRT3 p obably compe es wi h
PARPs o NAD
+
, and PARPs o e ac i a ion has been ound o co ela e wi h si uin ac i i y
down egula ion[78].Con e sely, PARP-1 inhibi ion es o es he ac i i y o SIRT3 and a ious
mi ochond ial an ioxidan enzymes[79]. The e o e, he mechanism by which PARPs inhibi-
ion could p e en mi ochond ial dys unc ion and agmen a ion migh be ela ed o he pa -
icipa ion o SIRT3 in main aining mi ochond ial unc ions. Fu he s udies a e necessa y o
es ablish he pa icula ole played by PARPs ac i a ion in he agmen a ion o mi ochond ia
and in ene ge ic dys unc ion.
In conclusion, his s udy p esen s some no el and in e es ing da a ega ding he change in
mi ochond ial mo phology a e PARPs inhibi ion and i s ela ionship wi h he dec ease o
hepa ic cell dea h induced by ROS, which a e summa ized in Fig 11. We epo ha mi ochon-
d ial agmen a ion is an ea ly e en in hepa ic cells su e ing oxida i e s ess causing cell
dea h. PARPs inhibi o s educe his cell dea h, by p o ec ing agains mi ochond ial agmen a-
ion and NAD
+
deple ion, and es o e mi ochond ial unc ion.
Suppo ing in o ma ion
S1 Fig. S udy o cy o oxic e ec s o PARPs inhibi o s in WRL68 cells. E ec s o PARPs
inhibi o s on he cell iabili y o WRL68 cells assessed by he MTT me hod. Cell iabili y was
de e mined in WRL68 cells incuba ed wi h PJ34 o AG14361. To do so, hepa ic cells we e
exposed o 40 h (16 h o p e- ea men and 24 h o pos -incuba ion ime) o di e en concen-
a ions (0.1, 0.5, 1, 10 mM) o PARPs inhibi o s as indica ed. Cell iabili y was exp essed as
pe cen ages o he con ol, which was conside ed o be 100%. Mean ±SEM o h ee indepen-
den expe imen s. Signi ican di e ences wi h espec o he con ol (non- ea ed cells):
P<0.05.
(TIF)
S2 Fig. PARPs inhibi o s block cellula PAR polyme o ma ion. Immuno luo escence
de ec ion o PAR polyme (g een) in WRL68 cells ea ed wi h H
2
O
2
o 15 min in he absence
o p esence o he PJ34 inhibi o . Immuno luo escence images show ha PAR polyme o ma-
ion is blocked when PARPs inhibi o s we e used in oxida i e ea men . Nuclei we e coun e -
s ained wi h Hoechs (blue). Rep esen a i e images om h ee independen expe imen s.
Scale ba : 50 μm.
(TIF)
S3 Fig. Mo phology o mi ochond ia 4 h a e H
2
O
2
ea men . (A) Pe cen age o ype I
(small globula ; le g aph) and ype II ( ubula including linea , wis ed, b anched and looped
o ms; igh g aph) o mi ochond ial mo phology. Cells we e ea ed wi h H
2
O
2
o 30 min
and hen pos -incuba ed o 4 h p io o quan i ying mi ochond ial mo phology wi h Mic oP
PARPs inhibi o s educe mi ochond ial agmen a ion and cell dea h induced by ROS
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0187130 Oc obe 26, 2017 21 / 26

so wa e. (B) Mi ochond ial elonga ion index ( ela ion be ween majo and mino axis leng hs).
(C) Mi ochond ial a ea. Ba s ep esen he mean ±SEM o ou independen expe imen s; a
leas 7,000 mi ochond ia we e analysed in each condi ion and in each expe imen . Signi ican
di e ences wi h espec o non- ea ed cells: P<0.001.
(TIF)
S4 Fig. PARP inhibi ion es o es mi ochond ial mo phology 4 h a e H
2
O
2
ea men . (A)
Pe cen age o ype I (small globula ; le g aph) and ype II ( ubula including linea , wis ed,
b anched and looped o ms; igh g aph) o mi ochond ial mo phology p io o quan i ying
mi ochond ial mo phology wi h Mic oP so wa e. WRL68 cells p e-incuba ed 16 h wi h
AG14361 we e ea ed wi h H
2
O
2
o 30 min and hen pos -incuba ed o 4 h. (B) Elonga ion
index o mi ochond ia. (C) A ea o mi ochond ia. A leas 7000 mi ochond ia we e analysed
wi h Mic oP so wa e in each condi ion and in each expe imen . Ba s ep esen he mean ±
SEM o h ee independen expe imen s. Signi ican di e ences: P<0.01 wi h espec o he
con ol (non- ea ed cells);
#
P<0.05 wi h espec o 3.5 mM H
2
O
2
.
(TIF)
Acknowledgmen s
We hank Concepcio
´n He na
´ndez and Da id Po cel (CIC, Uni e sidad de G anada) o hei
aluable assis ance in elec onic mic oscope echniques, Rosa io Sepu
´l eda (Uni e sidad de
G anada) o e ising he manusc ip and p o iding c i ical commen s, and Glenn Ha ding
and Richa d Da ies o p oo eading he English-language manusc ip .
Au ho Con ibu ions
Concep ualiza ion: Da id Ma ı
´n-Oli a.
Fo mal analysis: Sand a M. Ma ı
´n-Gue e o, Da id Ma ı
´n-Oli a.
Funding acquisi ion: Jose
´A. Muñoz-Ga
´mez, Ma ı
´a-Ca men Ca asco, Julio Na ascue
´s.
In es iga ion: Sand a M. Ma ı
´n-Gue e o, Jose
´A. Muñoz-Ga
´mez, Ma ı
´a-Ca men Ca asco,
Ma ı
´a Ma ı
´n-Es ebane
´, Da id Ma ı
´n-Oli a.
P ojec adminis a ion: Miguel A. Cuad os, Da id Ma ı
´n-Oli a.
Resou ces: Jose
´A. Muñoz-Ga
´mez, Ja ie Salme o
´n.
Visualiza ion: Sand a M. Ma ı
´n-Gue e o, Miguel A. Cuad os, Da id Ma ı
´n-Oli a.
W i ing – o iginal d a : Sand a M. Ma ı
´n-Gue e o, Da id Ma ı
´n-Oli a.
W i ing – e iew & edi ing: Miguel A. Cuad os, Julio Na ascue
´s, Da id Ma ı
´n-Oli a.
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