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Exposu e o e ene, luo an hene, and hei bina y mix u e causes dis inc
ansc ip omic and apical ou comes in ainbow ou (Onco hynchus mykiss) yolk sac
ale ins
© 2022 The Au ho (s). Published by Else ie B.V.
Published e sion
E iksson, And eas N.M.; Rigaud, Cy il; K asno , Aleksei; Wincen , Emma;
Vehniäinen, Ee a-Riikka
E iksson, A. N., Rigaud, C., K asno , A., Wincen , E., & Vehniäinen, E.-R. (2022). Exposu e o
e ene, luo an hene, and hei bina y mix u e causes dis inc ansc ip omic and apical
ou comes in ainbow ou (Onco hynchus mykiss) yolk sac ale ins. Aqua ic Toxicology, 244,
A icle 106083. h ps://doi.o g/10.1016/j.aqua ox.2022.106083
2022
Aqua ic Toxicology 244 (2022) 106083
A ailable online 21 Janua y 2022
0166-445X/© 2022 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Exposu e o e ene, luo an hene, and hei bina y mix u e causes dis inc
ansc ip omic and apical ou comes in ainbow ou (Onco hynchus mykiss)
yolk sac ale ins
And eas N.M. E iksson
a
,
*
, Cy il Rigaud
a
, Aleksei K asno
b
, Emma Wincen
c
, Ee a-
Riikka Vehni¨
ainen
a
a
Depa men o Biological and En i onmen al Sciences, Uni e si y o Jy ¨
askyl¨
a, P.O. Box 35, Jy ¨
askyl¨
a FI-40014, Finland
b
Fishe ies and Aquacul u e Resea ch, No wegian Ins i u e o Food, Ås, No way
c
Ins i u e o En i onmen al Medicine, Ka olinska Ins i u e , S ockholm, Sweden
ARTICLE INFO
Keywo ds:
T ansc ip ome
Mix u e
Re ene
Fluo an hene
PAH
Rainbow ou
Onco hynchus mykiss
Ea ly li e de elopmen
ABSTRACT
Polycyclic a oma ic hyd oca bons (PAHs) a e widely sp ead en i onmen al con aminan s which a ec de el-
oping o ganisms. I is known ha imp ope ac i a ion o he a yl hyd oca bon ecep o (AhR) by some PAHs
con ibu es o oxici y, while o he PAHs can dis up cellula memb ane unc ion. The exac downs eam
mechanisms o AhR ac i a ion emain un esol ed, especially wi h ega d o ca dio oxici y. By exposing newly
ha ched ainbow ou ale ins (Onco hynchus mykiss) semi-s a ically o e ene (32 µg l
−1
; AhR agonis ), luo-
an hene (50 µg l
−1
; weak AhR agonis and CYP1a inhibi o ) and hei bina y mix u e o 1, 3, 7 and 14 days, we
aimed o unco e no el mechanisms o ca dio oxici y using a a ge ed mic oa ay app oach. A he end o he
exposu e, s anda d leng h, yolk a ea, blue sac disease (BSD) index and PAH body bu den we e measu ed, while
he hea s we e p epa ed o mic oa ay analysis. Each exposu e p oduced a unique oxici y p o ile. We obse ed
ha e ene and he mix u e, bu no luo an hene, signi ican ly educed g ow h by Day 14 compa ed o he
con ol, while exposu e o he mix u e inc eased he BSD-index signi ican ly om Day 3 onwa d. Body bu den
p o iles we e PAH-speci ic and co ela ed well wi h he exposu e-speci ic up egula ions o genes encoding o
phase I and II enzymes. Exposu e o he mix u e o e - ep esen ed pa hways ela ed o g ow h, amino acid and
xenobio ic me abolism and oxida i e s ess esponses. Ale ins exposed o he indi idual PAHs displayed o e -
ep esen ed pa hways in ol ed in ecep o signaling: e ene down egula ed genes wi h a ole in G-p o ein
signaling, while luo an hene up egula ed hose in ol ed in GABA signaling. Fu he mo e, exposu e o e ene
and luo an hene al e ed he exp ession o genes encoding o p o eins in ol ed in calcium- and po assium ion
channels, which sugges s a ec ed hea s uc u e and unc ion. This s udy p o ides deepe unde s anding o he
complexi y o PAH oxici y and he necessi y o in es iga ing PAHs as mix u es and no as indi idual
componen s.
1. In oduc ion
Polycyclic a oma ic hyd oca bons (PAHs) a e a widesp ead g oup o
en i onmen al con aminan s o ei he na u al o an h opogenic o igin.
Always occu ing as complex mix u es in na u e, PAHs a e gene a ed in
la ge quan i ies du ing combus ion o py olysis o o ganic ma e ial and
a e p esen in pe oleum p oduc s. The en i onmen al p e alence o
PAHs has inc eased o e he las cen u y due o inc eased an h opogenic
ac i i ies (Wicks ¨
om and Tolonen, 1987; Van Me e e al., 2000). The
p esence o PAHs is pa icula ly p oblema ic in anoxic s a a as any
deg ada ion o PAHs in si u equi es ae obic me abolism. Hence, PAH
con amina ion can o m legacy deposi s ha can pose long e m en i-
onmen al isks i emobilized o leached (Ha i ash and Kaushik, 2009).
Exposu e o PAHs du ing ea ly li e de elopmen o ish is well known
o esul in a b oad sui e o de ec s a mul iple le els o biological o -
ganiza ion and consequen ly, inc eased mo ali y. Hea s uc u e and
unc ion a e especially sensi i e o PAHs (Inca dona e al., 2011), which
has mul iple down-s eam consequences o he g owing ish la ae as
* Co espondence au ho .
E-mail add ess: [email p o ec ed] (A.N.M. E iksson).
Con en s lis s a ailable a ScienceDi ec
Aqua ic Toxicology
jou nal homepage: www.else ie .com/loca e/aq ox
h ps://doi.o g/10.1016/j.aqua ox.2022.106083
Recei ed 14 May 2021; Recei ed in e ised o m 11 Janua y 2022; Accep ed 18 Janua y 2022
Aqua ic Toxicology 244 (2022) 106083
2
ci cula ion o nu ien s and gas-exchange become es ic ed. Reduced
g ow h and symp oms e e ed o as he blue sac disease (BSD; yolk sac
and pe ica dial edemas, c anio acial and skele al de o mi ies, hemo -
haging and in o ) a e es ablished hallma ks o PAH oxici y (Billia d
e al., 1999; Cola ecchia e al., 2006). O he de elopmen al de ec s
associa ed wi h PAH exposu e a e geno oxici y and beha io al al e -
a ions (Rhodes e al., 2005; Geie e al., 2018).
The exac mechanisms o PAH induced oxici y emain un esol ed
o mos PAHs, al hough mul iple molecula p ocesses a e known o
con ibu e o he o ma ion o oxici y in de eloping ish. Wha is known
is ha di e en PAHs ha e di e en modes o ac ion and oxici y po-
en ial in di e en o gans and species (Timme-La agy e al., 2007; Geie
e al., 2018). Fu he mo e, he composi ion o a PAH mix u e a ec s
oxici y as he oxicological po ency o ce ain PAH mix u es has been
obse ed o induce a s onge esponse han he combined e ec o he
componen s (Sco and Hodson, 2008; B own e al., 2015). Howe e , i
mus be no ed ha ce ain componen s con ibu e mo e o oxici y han
o he s, as exempli ied by Geie e al. (2018).
The mos s udied molecula esponse in PAH exposed and de el-
oping ish is he in e ac ion be ween ce ain PAHs and he a yl hyd o-
ca bon ecep o 2 (AhR2); a ecep o which egula es no mal
de elopmen and me abolism (Billia d e al., 2002). No e ha no all
PAHs ha e a ini y o AhR2 (Ba on e al., 2004), bu hose PAHs ha
do igge i s ac i a ion as a ansc ip ion ac o . Ac i a ion induces he
exp ession o , among o he genes, cy och ome P450 (cyp1a) by associ-
a ing wi h xenobio ic esponse elemen s. The ac i a ed o m o CYP1a
unc ions as a phase I me abolic enzyme ha acili a es me abolism and
exc e ion o a la ge se o xenobio ics h ough hyd oxyla ion bu also
ac i a ion o endogenous molecules.
The PAH e ene (1-me hyl-7-isop opyl phenan h ene) is commonly
associa ed wi h e luen s om pulp- and pape mills as well as mic obial
me abolism o esin acids (Lepp¨
anen and Oika i, 1999) bu can also
unc ion as a bioma ke o o es i es (Gabos e al., 2001). Re ene is a
known AhR2 agonis wi h high a ini y (Ba on e al., 2004). Howe e ,
he exac mechanisms leading up o ca dio oxici y a e no ully unde -
s ood o e ene (Baude e al., 2005; Hodson e al., 2007), as mul iple
in e wined p ocesses a e likely o be in ol ed; exempli ied by he
Ad e se Ou come Pa hway 21 (AOP21) by Doe ing e al., 2019. F om a
oxicological pe spec i e, knockdown o ah 2, bu no cyp1a, in e ene
exposed zeb a ish emb yos (Danio e io) p e en ed he o ma ion o
ca diac de ec s (Sco e al., 2011). Howe e , as AhR2 con ols a as
numbe o genes, knockdown does no highligh any unde lying mech-
anism. One sugges ed downs eam gene is cyclooxygenase-2, bu he
e idence and quan i a i e unde s anding o his pa hway a e cu en ly
only mode a e, and he e a e also o he pa hways ha may be in ol ed
(AOP21).
Ano he PAH o in e es is luo an hene, which occu s ubiqui ously
in any na u al PAH mix u e (Page e al., 1999). Al hough a weake AhR2
agonis han e ene (Ba on e al., 2004), luo an hene can impai CYP1a
media ed me abolism by blocking he enzymes ac i e si e (Wille e al.,
1998). The ew labo a o y s udies on he impac o PAH mix u es con-
aining luo an hene on de eloping ish la ae epo inc eased oxici y
o luo an hene-con aining mix u es compa ed o he oxici y o he
indi idual componen s (Wassenbe g and Di Giulio, 2004; Geie e al.,
2018). None heless, he unde lying mechanism(s) o he s onge
oxici y o PAH mix u es con aining luo an hene, ela i e o he sum o
oxici y o he indi idual mix u e componen s, emains elusi e. Conse-
quen ly, i is impo an o unde s and how luo an hene in luences and
po en ia es he oxici y o a simple PAH mix u e, especially om he
pe spec i e o en i onmen al isk assessmen .
The e o e, by exposing newly ha ched and de eloping ainbow ou
ale ins (Onco hynchus mykiss) o sub-le hal concen a ions o e ene and
luo an hene (indi idually o as a bina y mix u e), mechanisms ela ed
o ca dio oxici y we e in es iga ed a he ansc ip omic le el which in
u n we e ela ed o e ec s on he whole o ganism. A a ge ed mic o-
a ay app oach was employed as pe ou p e ious expe ience wi h
ansc ip omic analysis (Rigaud e al., 2020; Vehni¨
ainen e al., 2016).
Rainbow ou was selec ed as es o ganism due o i s (and o he
salmonid species’) ecological, economical and scien i ic ele ance o
sub-a c ic and bo eal aqua ic ecosys ems. Ca diac issue was chosen as
he endpoin o ansc ip omic in es iga ion due o he hea being
among he mos sensi i e o gans o he exposu e wi h PAHs. Di e en ly
exp essed genes and subsequen ly o e - ep esen ed e ms and pa hways
we e hen compa ed wi h he e ec s on he whole o ganisms (g ow h
and de elopmen , body bu den and yolk consump ion, he la e unc-
ioning as a p oxy o ene gy consump ion).
2. Ma e ials and me hods
2.1. Se up, main enance, wa e analysis, animal ca e and sampling
Newly ha ched (<24 h) and heal hy ainbow ou ale ins (360◦-
days; supplied by Hanka-Taimen OY, Hankasalmi, Finland) we e
andomly selec ed and semi-s a ically exposed o dime hyl sul oxide
(DMSO, con ol), e ene, luo an hene o he bina y mix u e o he wo
PAHs (Table 1) and sampled a e 1, 3, 7 and 14 days o exposu e.
Exposu e concen a ions we e selec ed in o de o p o oke oxico-
logical esponses, while a oiding exposu e- ela ed mo ali y. Bo h Bil-
lia d e al. (1999) and Vehni¨
ainen e al. (2016) ha e epo ed ha
exposu e o 32 µg l
−1
o e ene ul ills he abo emen ioned equi emen s
while p elimina y es ing iden i ied 50 µg l
−1
o luo an hene as sui able
(unpublished da a; exposu e o 5, 50 and 500 µg l
−1
o luo an hene
esul ed in 0% mo ali y o e a 11 day pe iod; 3 eplica es pe con-
cen a ion and 10 ale ins pe eplica e).
P io o he ini ia ion o exposu e, each exposu e essel was p e-
sa u a ed o 24 h wi h he co esponding chemicals. S ock wa e ,
mean o he exposu e s udies, was deli e ed om Konne esi esea ch
s a ion (Cen al Finland) in Feb ua y and Ap il 2017; he 14 days
exposu e ook place in Feb ua y, while he 1, 3 and 7 days exposu es
we e pe o med in Ap il, using di e en ba ches o ale ins. Lake wa e
was collec ed om a dep h o 6 m and il e ed o deb is. The concen-
a ion o PAHs in he collec ed exposu e wa e was below he le el o
de ec ion. E e y exposu e was conduc ed in 1.5 L Py ex glass bowls
illed wi h 1 L o lake wa e and he co esponding exposu e compound
(s) while placed in a La in squa e sequence (Fig. s1). Exposu e was
main ained in a semi-s a ic ashion, which mean comple e enewal o
wa e and chemicals on a daily basis. Ale ins we e collec ed using a 5 ml
plas ic Pas eu pipe wi h a b oadened ip and empo a ily ans e ed o
a 50 ml plas ic cen i uga ion ube along wi h some exposu e wa e
while he exposu e medium was enewed; a p ocess ha ook less han
30 s pe eplica e.
In o de o gene a e enough ca diac issue o RNA ex ac ion, 12
eplica e bowls pe ea men , (each eplica e con ained 15 ale ins)
we e main ained o exposu es las ing 1, 3 and 7 days while he expo-
su e las ing o 14 days equi ed 8 eplica es pe ea men , wi h he
same numbe o ale ins pe eplica e. This ansla ed o 180 ale ins pe
ea men las ing 1, 3 and 7 days while he 14 days exposu e equi ed
120 ale ins pe ea men . An addi ional bowl pe ea men bu wi hou
ale ins was main ained and ea ed in he same ashion as hose con-
aining de eloping ale ins o de e mine loss o PAHs due o mic obial
deg ada ion, e apo a ion o adso p ion o he glass bowl. Exposu e
wa e empe a u e, measu ed daily, was main ained a 11.7 ±0.4 ◦C
and pho ope iodici y was se a 16:8 ligh o da kness. Wa e samples
we e collec ed p io o wa e and chemical enewal on Days 1, 3, 7, 10
and 14, dilu ed 50:50 in e hanol (99.5% pu i y) and s o ed a 4 ◦C o
la e synch onous luo escence spec oscopy. Wa e quali y, wi h
ega ds o pH, conduc i i y and dissol ed oxygen con en in ae a ed
s ock wa e , was measu ed a e 1, 3, 5, 7, 10 and 14 days o exposu e.
The Finnish En i onmen Ins i u e’s da abase He a epo s ha wa e
om lake Konne esi con ains 7 o 14 µg l
−1
ammonium (as ni ogen),
0.13 o 0.19 mmol l
−1
alkalini y and has a Ca+Mg ha dness o 0.12
mmol l
−1
.
A.N.M. E iksson e al.
Aqua ic Toxicology 244 (2022) 106083
3
A sampling, exposed ale ins we e pho og aphed nex o millime e
scale pape , symp oms o BSD we e assessed and hea s excised. Hea s
om e e y ale in om 2 (ale ins exposed o 14 days; 30 hea s in o al
pe sample) o 3 (exposed o 1, 3 and 7 days; 45 hea s in o al pe
sample) exposu e eplica es o he same ea men we e pooled, snap
ozen in liquid ni ogen and s o ed a −80 ◦C o la e RNA ex ac ion.
U ilizing his app oach yielded 4 eplica es pe ea men o an-
sc ip omic analyzes, o which 3 we e p ocessed. The emaining ale ins
ca casses we e pooled based upon eplica e, snap ozen in liquid ni-
ogen and s o ed a −80 ◦C o la e p epa a ion and HPLC analysis.
2.2. Synch onous luo escence spec oscopy (SFS) and wa e quali y
This SFS p o ocol has p e iously been desc ibed by Rigaud e al.
(2020), and ou lines he analy ical p o ocol o phenan h ene, py ene
and e ene. The SFS pa ame e s, al hough sligh ly adjus ed, we e ob-
ained elsewhe e (Wa son e al., 2004; Tu co e e al., 2011) (Table 2). In
sho , exposu e wa e om 4 eplica es pe ea men (selec ed a
andom) was sampled a e 1, 3, 7, 10 and 14 days o exposu e, collec ed
in 20 ml scin illa ion bo les in a 50:50 mix u e wi h e hanol (99.5%
pu i y) and hen s o ed a 4 ◦C un il analysis. The concen a ion o
e ene and luo an hene we e measu ed using a LS55 Luminescence
Spec ome e (Pe kinElme Ins umen s, USA). E e y LS55 measu e-
men was pe o med in qua z cu e es (Qua z SUPRASIL® High P e-
cision Cell, Hellma Analy ics, Ge many) as hese PAHs ha e a low
luo escence a in es iga ed exposu e concen a ions. S anda d cu es
we e c ea ed o each PAH which we e used o calcula e concen a ion
egimes, once no malized agains hei espec i e con ol and using he
peak a ea o e ene (290–315 nm) and luo an hene (270–292 nm).
2.3. Mo phome ic analyzes
Pho os we e analyzed u ilizing ImageJ ( 1.51j8, Na ional Ins i u es
o Heal h, USA). Using he millime e pape as a known e e ence,
s anda d leng h and plana yolk a ea we e measu ed in silico wi h high
accu acy (3 decimals) and esolu ion (30 pixels pe mm). Howe e ,
empe a u e and o he abio ic ac o s a e known o a ec ish de el-
opmen and due o he a chi ec u e o he exposu e oom and he cooling
sys em, he empe a u e in he exposu e bowls a ied spa ially (1, 3 and
7 days exposu e: 99.9–101.7% ela i e o eplica e 1; 14 days exposu e:
98.5–103.6% o con ol eplica e 1; see Fig. s1). The e o e, s anda d
leng h and yolk a ea we e compensa ed o he in luence o empe a u e
by adjus ing o he numbe o deg ee-days o e e y con ol eplica e in
ela ion o con ol eplica e 1, as pe Eq. (1):
Endpoin x−adj.=Endpoin X/DD1
DDx
(1)
Whe e DD
1
is he numbe o deg ee days in con ol eplica e 1 and
DD
x
ep esen s he deg ee days in he co esponding con ol bowl
x
. The
measu ed endpoin was hen di ided by he deg ee-day quo a om he
same eplica e
x
, he eby adjus ing he measu emen s o he spa ial
empe a u e a ia ion. The unadjus ed esul s a e p esen ed in Fig. s2.
The BSD-index was es ablished pe eplica e and calcula ed acco d-
ing o es ablished con en ion (Eq. (2)) (Villalobos e al., 2000; Sco
e al., 2011). Symp oms o pe ica dial edemas (PE; sco ed 0 o 1; no
p esen o p esen ), yolk sac edema (YE; sco ed 0 o 1) and hemo hages
(HM; sco ed 0 o 1) we e assessed upon sampling. This p ocedu e ga e a
maximum sco e o 45 pe eplica e. Addi ionally, he e ec o he
mix u e on he BSD index, ela i e o he componen s ( esul s adjus ed
o baseline oxici y among DMSO exposed ale ins), was assessed as pe
combina ion index (Foucquie and Guedj, 2015).
BSD =∑PE +∑HM +∑YE
Maximum sco e (2)
Inconsis encies in he assessmen o BSD symp oms occu ed du ing
he sampling o ale ins exposed. As a consequence, only hal o he
eplica es we e included in he calcula ion o he BSD-indices o ale ins
exposed o 1, 3 and 7 days. BSD-indices among ale ins sampled a e 14
days o exposu e we e comple ely omi ed due o inconsis en sco ing.
Ale ins sampled a e 14 days o exposu e we e de elopmen ally
assessed and sco ed based upon pigmen a ion in ensi y o he do sal in
(p esen /no p esen ) and he la e al side low/high in ensi y) in acco -
dance wi h Ve nie ’s ainbow ou de elopmen al ca alog (Ve nie ,
1977), as exempli ied in Fig. s3. Fo ma ion and de elopmen o
pigmen a ion is in luenced by AhR2 and hus sensi i e o he in luence
o AhR-agonis s (Zod ow and Tanguay, 2003).
2.4. High-Pe o mance liquid ch oma og aphy analysis
The body bu den o e ene and luo an hene was assessed using a
High-Pe o mance Liquid Ch oma og aphy (HPLC) app oach. Fo a
de ailed desc ip ion on ma e ial p epa a ion, analysis and eco e y
assessmen , see supplemen a y ma e ial s1.1. In sho , pooled ale in
ca casses (10–13 pe eplica e) we e homogenized in 70% ace oni ile
(ACN; Fishe Scien i ic). The homogena e was cen i uged o 15 min a
14,000 pm in 4 ◦C (Cen i uge 5415 R, Eppendo , Ge many) and he
supe na an collec ed. The pelle was esuspended in 70% CAN,
cen i uged and he supe na an collec ed and pooled. The esuspension
Table 1
DMSO, e ene and luo an hene s ock solu ion concen a ions (µM; dissol ed in DMSO), olume o s ock solu ion added pe exposu e bowl (µL), nominal exposu e
concen a ion (nM), chemical pu i y (%), supplie and CAS-numbe . No e: 136.6 nM o e ene equals 32 µg L
−1
while 247.2 nM o luo an hene equals 50 µg L
−1
. The
nominal concen a ion o DMSO co esponds o 0.002% which is below he h eshold o 0.01% sol en ecommended by he OECD (2013) and is hus unlikely o
con ibu e o, o inc ease, he oxici y o PAHs (Ch is ou e al., 2020; Kais e al., 2013; Maes e al., 2012).
Exposu e S ock solu ion concen a ion
(µM)
Volume added
(µL)
Nominal PAH +DMSO exposu e concen a ions
(nM)
Pu i y
(%)
Supplie CAS
Numbe
DMSO Pu e DMSO 20 256 ≥99.9 Sigma Ald ich 67-68-5
Re ene +DMSO 13,655 10 +10 136.6 +128 98 MP
Biomedical
483-65-3
Fluo an hene +
DMSO
24,720 10 +10 247.2 +128 ≥98 Sigma Ald ich 206-44-0
Re ene +
Fluo an hene
As abo e 10 +10 136.6 +247.2 As abo e As abo e As abo e
Table 2
LS55 luminescence synch onous luo escence spec oscopy pa ame e s used o he iden i ica ion o e ene and luo an hene.
PAH Wa eleng hs measu ed (nm) Del a wa eleng h (Δλ; nm) Peak (nm) Exci a ion sli (nm) Emission sli (nm) Scan speed (nm/min)
Re ene 250–350 50 290–315 5 5 300
Fluo an hene 200–500 155 270–292 2.5 5 240
A.N.M. E iksson e al.
Aqua ic Toxicology 244 (2022) 106083
4
p ocesses o he pelle we e epea ed wice.
An aliquo o he collec ed supe na an (100 µl; he emaining su-
pe na an was s o ed a −20 ◦C) was ans e ed o a 250 µL glass inse
(Agilen Technologies, Ge man) placed in an ambe glass ial (Agilen
Technologies, Poland). Ten µl o he aliquo we e analysis using a Shi-
madzu U-HPLC Nexe a sys em connec ed o a RF-20A xs P ominence
luo escence de ec o (Shimadzu, Japan) wi h a 150 mm long ACE C18-
AR column wi h a pa icle size o 5 µm (Ad anced Ch oma og aphy
Technologies LTD, Sco land, UK). The analy ical p o ocol de eloped
allowed o simul aneous measu emen o bo h PAHs in one un, he eby
enabling de ec ion o possible PAH c oss-con amina ion. Re ene was
measu ed a exci a ion 259 nm and emission 370 nm wi h a e en ion
ime o 16.71 ±0.09 min while luo an hene was measu ed a exci a ion
288 nm and emission 525 nm a e 13.5 ±0.21 min. Ch oma og am a ea
unde he cu e (AUC) was manually adjus ed and backg ound- (by
sub ac ing he a e age AUC om DMSO exposed ale ins) and eco e y
compensa ed. The concen a ions we e hen calcula ed acco ding o he
s anda d cu es and he a e age PAH body bu den (amoun ) calcula ed
pe ish.
2.5. T ansc ip omic analysis
RNA was ex ac ed om he pooled ish hea s wi h TRI Reagen
(Molecula Resea ch Cen e , USA). RNA was quan i ied using a Nano-
D op 1000 .3.8.1 and NanoD op 2000 (The mo Fishe Scien i ic, USA)
and RNA in eg i y numbe (RIN) sco ed using Bioanalyze RNA 6000
Nano assay ki (Agilen Technologies) acco ding o manu ac u e ’s in-
s uc ions; lowes measu ed RIN was 9.8. Ex ac ed RNA was hen
di ided in o wo aliquo s (one o mic oa ay and one o qPCR ali-
da ion analysis) and s o ed a −80 ◦C.
In b ie , qPCR analysis was p ima ily mean as mic oa ay alida-
ion, while me hodology was i s desc ibed by Rigaud e al. (2020). In
o al, 8 di e en genes ela ed o xenobio ic, ene gy and i on me a-
bolism, as well as oxida i e s ess, we e analyzed, using ndu a8 and l17
as e e ences due o hei ansc ip s abili y. Fo de ails, see supple-
men a y ma e ial s1.2.
Mic oa ay (Agilen 4 ×44 K, Salgeno Design ID 082,522) p epa-
a ion and analysis we e pe o med a NOFIMA (Ås, No way) acco ding
o a p e iously de eloped p o ocol (K asno e al., 2011). Fi s , 220 ng
o RNA was labeled wi h one-colo Cy3 dye (Agilen Low Inpu Quick
Amp Labeling Ki ; p oduc numbe 5190–2305), ampli ied, and pu i ied
using Qiagen RNeasy Mini Ki (Qiagen, Ge many). Based upon Nano-
D op measu emen he cRNA concen a ion (a e age 266.3 ±48.9 ng
µl
−1
) and speci ic Cy3 ac i i y was calcula ed (a e age 12.9 ±2.5
ρ
mol
pe µg cRNA). Samples we e hyb idized o e nigh a 65 ◦C using Agilen
Gene Exp ession Hyb idiza ion Ki (p oduc numbe 5188–5242), and
he hyb idized mic oa ays we e washed using he Agilen Gene
Exp ession Wash Bu e Ki (p oduc numbe 5188–5327). The hyb id-
ized mic oa ays we e hen analyzed using he Agilen Su eScan
Mic oa ay Scanne and he gene exp ession eadings we e p ocessed,
analyzed and hei in ensi y le els es ablished using No ima’s bioin-
o ma ics package (K asno e al., 2011). A e age mic oa ay in ensi y
le els we e no malized and log
2
- ans o med compa ed o con ols. The
bioin o ma ics package iden i ied di e en ially exp essed genes ela i e
o con ol using S uden ’s - es (p ≤0.05). Mic oa ay aw da a ou pu
is a ailable a A ayExp ess (E-MTAB-8980).
2.6. O e - ep esen a ion analyzes
O e - ep esen a ion analyzes (ORAs) we e pe o med using R
( .3.5.1., R Co e Team, USA); ex ended by he packages Bioconduc o
3.7 and clus e P o ile 3.8.1 (Boyle e al., 2004; Yu e al., 2012). Gene
anno a ion in clus e P o ile was es ablished using he Anno a ionHub
da abase. Howe e , in acco dance wi h Rigaud e al. (2020), as no gene
anno a ion da abase we e a ailable o ainbow ou in Anno a ionHub
a he ime o da a analysis, we used zeb a ish Re Seq gene ID and
symbols and hen a ibu ed hese o each ea u e o he mic oa ay. This
was achie ed using he NCBI BLAST so wa e 2.7.1 (Na ional Cen e o
Bio echnology In o ma ion, Be hesda, MD, USA). F om he BLAST
ou pu , we gene a ed an ORAs backg ound da abase con aining 19,025
unique zeb a ish gene IDs (E- alues ≥10
−3
we e emo ed).
O e - ep esen a ion- and pa hway analysis we e pe o med and include
Gene On ologies e ms (GO), and KEGGs pa hways. P- alue cu -o was
se o 0.05 and adjus ed o mul iple compa isons using Benjamini and
Hochbe g me hod (Benjamini and Hochbe g, 1995). Once ORAs we e
pe o med, co esponding genes in ela ion o en ichmen pa hways
we e so ed based upon he exposu e du a ion as o iden i y he speci ic
genes in ol ed in e m- and pa hways o e - ep esen a ions (Supple-
men a y ile 1).
The mic oa ay p o iles we e alida ed by co ela ion analysis
(Spea man’s) wi h he co esponding qPCR esul s as well as he
mic oa ay esul s o e ene, ob ained om Rigaud e al. (2020)
(Fig. s4).
2.7. S a is ics
S a is ical analyzes we e pe o med in R-s udio e sion 3.5.1. (R
Co e Team, USA). Gaussian dis ibu ion was es ed using Shapi o-Wilk
es and a iance compa ison was es ed using one-way ANOVA wi h
Tukey’s pos -hoc es (Tukey) i he da a was no mally dis ibu ed,
o he wise, da a we e subjec ed o K uskal–Wallis (KW) analysis o
a iance wi h Dunne ’s pos -hoc es (Dunn). Mo ali y and pigmen a-
ion we e assessed using Fishe ’s exac es . Compa ing he body bu den
be ween ale ins exposed o e ene and luo an hene alone and pa o
he mix u e was pe o med using - es i he da a was no mally
dis ibu ed and Mann-Whi ney es (MW) i no . Signi icance- and cu -
o alue was se a p ≤0.05. All nume ical da a a e p esen ed as
mean ±s anda d de ia ion.
3. Resul s
3.1. Exposu e pa ame e s
Measu ed concen a ions o PAHs in exposu e wa e a ied wi h ime
and ea men (Table 3). The concen a ions o he wo PAHs in expo-
su e wa e we e highe when co-exposed compa ed o he indi idual
exposu es. A e 7 and 14 days o exposu e, signi ican ly mo e e ene
was measu ed in he samples om mix u e exposu e wa e han hose
om e ene only exposu es. The same pa e n was obse ed ega ding
luo an hene bu only a e 1 day o exposu e. Wa e quali y was wi hin
accep able limi s: conduc i i y 26.83 ±4.35 mS m
−1
; oxygen sa u a ion
103.89 ±5.32%; and pH 7.16 ±0.10. The high oxygen sa u a ion was
due o cons an ae a ion o s ock wa e .
Table 3
A e age measu ed (nM ±s anda d de ia ion) and a e age pe cen ile o nominal
concen a ion (wi hin pa en heses) o e ene (Re ) and luo an hene (Flu) in
exposu e wa e measu ed by synch onous luo escence spec oscopy a e 1, 3,
7, 10 and 14 days o exposu e. Fo compa ison, he de eloping ale ins we e
exposed o 247.21 nM o luo an hene and 136.55 nM o e ene (nominally).
S a is ical di e ences be ween measu ed concen a ions a e deno ed wi h ¤, *
and # o luo an hene Day 1, e ene Day 7 and 14, espec i ely ( - es ).
Concen a ion in nM, mean ±SD (% o nominal)
Day 1 Day 3 Day 7 Day 10 Day 14
Re
alone
35 ±11
(26)
35 ±11
(26)
30 ±10
(22)*
26 ±20
(19)
19 ±14
(14)
#
Re mix 32 ±7 (23) 39 ±8
(29)
53 ±28
(39)*
35 ±12
(26)
47 ±14
(35)
#
Flu
alone
37 ±17
(15)
¤
62 ±32
(25)
88 ±33
(36)
99 ±75
(40)
113 ±36
(46)
Flu mix 74 ±38
(30)
¤
76 ±22
(31)
103 ±44
(42)
108 ±71
(44)
113 ±42
(46)
A.N.M. E iksson e al.

Aqua ic Toxicology 244 (2022) 106083
5
3.2. Mo ali y and mo phome ics
Mo ali y ne e exceeded 6% in any ea men o eplica e. A sig-
ni ican di e ence was obse ed be ween ale ins exposed o e ene and
luo an hene indi idually and sampled on Day 7 (Table S2). Exposu e o
he bina y mix u e esul ed in a signi ican ly inc eased BSD-index on
Day 3 and onwa d compa ed o con ol and he indi idual compounds
on day 3 ( e ene) and 7 ( luo an hene), espec i ely (Table 4). The BSD-
indices among mix u e exposed ale ins we e, on a e age, g ea e han
he combined addi i e e ec exe ed by he componen s ollowing
exposu e o 3 and 7 days, when adjus ed o baseline oxici y among
DMSO exposed ale ins.
Fu he mo e, ale ins exposed o e ene ( o 14 days) and mix u e
(Days 3 and 14) we e signi ican ly sho e compa ed o con ol ale ins
(Fig. 1a). Fluo an hene exposed ale ins we e signi ican ly sho e a e 3
days o exposu e compa ed o con ol. Re ene-exposed ale ins had
signi ican ly smalle plana yolk a ea han hose exposed o mix u e on
Day 14 (Fig. 1b). Fu he mo e, no di e ences wi h ega ds o signi i-
cances be ween he di e en ea men s we e obse ed be ween em-
pe a u e adjus ed and un-adjus ed s anda d leng hs (Fig. s2a) and plana
yolk a eas (Fig. s2b). Ale ins exposed o 14 days o luo an hene and
mix u e had educed pigmen a ion in ensi y on hei la e al side, and all
PAH exposu es caused hypopigmen a ion o he do sal in (Table 5;
Fig. s3).
3.3. Body bu den
Each PAH ea men p oduced exposu e speci ic body bu den p o-
iles as exposu e o he mix u e al e ed he accumula ion pa e n o he
PAHs compa ed o ale ins exposed o he indi idual componen s. In he
case o e ene, he body bu den was signi ican ly inc eased by 353%
a e 1 day o exposu e, which eached 364% by Day 14, compa ed o
ale ins exposed o e ene alone (Fig. 2a). The body bu den o luo-
an hene by con as was educed signi ican ly by 60% a e 1 day o
exposu e and by Day 14, he educ ion was 94% compa ed o ale ins
exposed o luo an hene alone (Fig. 2b).
Low eco e y a es o he wo PAHs highligh ha he absolu e
quan i ica ion should be conside ed ca e ully. Ye , he ch oma og ams
yielded clea exposu e speci ic peaks, which we e 122 and 602 imes
g ea e o e ene and luo an hene, espec i ely, han backg ound
(con ol), al eady a e 1 day o exposu e.
3.4. T ansc ip omic esponses
Al oge he , 1896 di e en ly exp essed genes (DEGs) we e iden i ied
in ainbow ou hea issue, independen o exposu e du a ion and
ac oss all PAH ea men s. I espec i e o exposu e du a ion, exposu e o
luo an hene, e ene and he mix u e esul s in 344, 937 and 615 DEGs,
espec i ely. Few DEGs we e sha ed among he di e en exposu es and
hose ha we e sha ed we e p ima ily be ween e ene and mix u e
exposed ale ins (Fig. 3a–d). The ca diac mic oa ay esul s we e ali-
da ed by qPCR analysis as well as by compa ing he sha ed DEGs e-
po ed o e ene by Rigaud e al. (2020) wi h ou s. In hei s udy,
ainbow ou ale ins we e exposed o e ene unde nea ly iden ical
condi ions as hose epo ed in his p esen s udy; he only di e ence
being he addi ional 10 µl o DMSO added o he exposu e eplica e as
pe his s udy. Co ela ion analysis (Spea man) o he DEGs iden i ied by
mic oa ay analysis p o ided a eliable app oach o alida ion (R
2
=
0.79; p <0.0001; Fig. s4) compa ed o qPCR analysis, which su e ed
om poo e ma ching (R
2
=0.39, −0.05, 0.65 and 0.37 on Day 1, 3, 7
and 14, espec i ely; Table s3). Poo e co ela ion be ween he mic o-
a ay and qPCR could s em om mic oa ay p obe sensi i i y and
speci ici y in ela ion o he qPCR p ime s bu also possible deg ada ion
o he ozen ma e ial used o qPCR alida ion.
O he 1896 DEGs iden i ied by he mic oa ay app oach, only 6
DEGs we e sha ed among he ea men s; cyp1a being he only gene
consis en ly up egula ed by e e y ea men . The emaining 5 DEGs
sha ed by all ea men s we e nebule e (nebl, down egula ed, day 3),
slow myosin hea y chain b (smyhc2, down egula ed, Day 3), choles e yl
es e ans e p o ein (ce p, up egula ed, Day 7), zinc inge p o ein
(Danio e io gene id: 103,910,593; down egula ed by e ene and up e-
gula ed by luo an hene and mix u e, Day 14) and C1q and TNF-like
domains (cbln11, down egula ed, Day 14).
O e - ep esen a ion analysis e ealed ha exposu e o luo an hene
al e ed he ewes numbe o pa hways (Figs. 4, 5 and s5). Re ene o e -
ep esen ed a as numbe o GOs and KEGGs, al hough se e al we e o
low gene coun and wi h o e lapping unc ions. An in e es ing obse -
a ion was ha e ene and luo an hene o e - ep esen ed di e en
signaling pa hways in a unique ashion and in opposi e di ec ions;
e ene o e - ep esen ed G-p o ein signaling (down egula ion) whe eas
luo an hene a ec ed GABA-signaling (up egula ion). Ano he no e-
wo hy e ec o exposu e o he indi idual componen s was ha bo h
exposu es esul ed in al e ed exp ession o genes encoding o compo-
nen s ela ed o ca diac po assium- (kcnq) and calcium ion channels
(cacna).
Exposu e o he bina y mix u e did no o e - ep esen any unc ions
ela ed o ca diac ion channels o signaling. Howe e , he mix u e o e -
ep esen ed a wide epe oi e o e ms and pa hways ela ed o me a-
bolism o xenobio ics, amino acid me abolism and biosyn hesis, oxido-
educ ase ac i i y as well as g ow h and de elopmen (de ails a e
p esen ed in Supplemen a y ile 1). The mos p onounced e ec o
exposu e o he mix u e was he s onge and b oade up egula ion o
genes ela ed o phase I and II me abolism compa ed o he indi idual
PAHs: exposu e o e ene up egula ed cyp1a, cy osolic sul o ans e ase
(sul 1), UDP-glucu onosyl ans e ase (ug p1a1) and ca bonyl educ ase
(cb 1l) while luo an hene only up egula ed cyp1a and glu a hione S-
ans e ase P (gs p). Consequen ly, he mix u e, bu no he componen s,
up egula ed p ocesses aimed a coun e ac ing oxida i e s ess which
Table 4
A e age blue sac disease index (BSD; ±SD) pe ea men (con ol =DMSO, e ene =Re , Fluo an hene =Flu and mix u e =Mix) sampled a e 1, 3 and 7 days o
exposu e. Occu ence o BSD symp oms (pe ica dial and yolk sac edemas and hemo hages) we e sco ed du ing sampling (Villalobos e al., 2000; Sco e al., 2011).
Assessmen o BSD among ale ins sampled on Day 14 a e omi ed due o inconsis en sco ing du ing sampling. Signi ican di e ences be ween ea men s a e deno ed
wi h lowe case (Day 3) and uppe case le e (Day 7) (p <0.05; KW +Dunn). Baseline adjus ed BSD indices (a e age BSD index among con ol ale ins sub ac ed om
he a e age index among PAH exposed ale ins) we e u ilized o assess he e ec o he mix u e ela i e o he componen s (combina ion index <1; deno ed +
(Foucquie and Guedj, 2015)). No e, due o inconsis encies be ween he sample s, only hal o he eplica es a e included in he calcula ion o BSD-index.
Exposu e du a ion BSD indices Baseline adjus men
DMSO Flu Re Mix Flu Re Mix Numbe o ale ins
Day 1 0.03 ±0.03 0.08 ±0.10 0.07 ±0.09 0.10 ±0.06 0.05 0.04 0.07 90
Day 3 0.20 ±0.08
a
0.34 ±0.17
ab
0.25 ±0.08
a
0.49 ±0.09
b
0.14 0.05 0.29
+
90*
Day 7 0.23 ±0.11
A
0.25 ±0.06
A
0.29 ±0.16
AB
0.43 ±0.10
B
0.02 0.06 0.20
+
80–90**
*) Flu: n =89.
**) Re : n =80; DMSO and Mix: n =87; Flu: n =90.
+) Mix u e induced a BSD index g ea e han he addi i e e ec o he componen s; combina ion index <1.
A.N.M. E iksson e al.
Aqua ic Toxicology 244 (2022) 106083
6
included bo h an ioxida i e- and hea shock p ocesses. Fu he mo e,
exposu e o he mix u e also o e -p esen ed se e al e ms ela ed o
g ow h and de elopmen which shi ed om emb yonic mo phogenesis
(GO:0048598) by Day 1 o o ma ion- and de elopmen o ex acellula
s uc u es (GO:0005615) and g ow h and de elopmen (GO:0040007
and GO:0048589) by Day 3.
4. Discussion
Exposu e o e ene and luo an hene, alone o as a bina y mix u e,
p oduced exposu e speci ic oxici y p o iles; bo h a he whole o ganism
le el and a he ansc ip omic le el. As expec ed pe p e ious esea ch
(Billia d e al., 2008; Van Tiem and Di Giulio, 2011), he bina y mix u e
p oduced a s onge oxici y esponse and a modula ed body bu den
p o ile compa ed o exposu e o he indi idual componen s (while
e ene being s onge han luo an hene). Subsequen
o e - ep esen a ion analysis o he ca diac ansc ip ome p o ided in-
sigh s on mul iple po en ial molecula esponses ha can explain he
body bu den p o iles and con ibu e o he unde s anding on how PAH
induced ca dio oxici y as well as how exposu e impac s g ow h and
de elopmen . The changes in gene exp ession a e no , howe e , neces-
sa ily speci ic o he ca diac issue, and may he e o e sugges o he
ypes o oxici y as well.
4.1. E ec o exposu e on genes in ol ed in hea unc ion and
de elopmen
Exposu e o he PAHs alone, bu no he mix u e, p oduced se e al
DEGs ha a e known o be in ol ed in main aining hea unc ion and
de elopmen . Th oughou he exposu e du a ion, luo an hene down-
egula ed kcnq5b (Day 3; a componen o he slow po assium ion channel
which s abilizes he memb ane po en ial (Jen sch, 2000)) and up egu-
la ed cacna1 (Day 7; linked o calcium ion channel unc ion (G an ,
2009)). Exposu e o e ene esul ed in down egula ion o 6 ypes o kcnq
genes and 4 ypes o cacna genes by Day 14. These al e a ions sugges a
delayed dis up ion o he ac ion po en ial and hea unc ion compa ed
o luo an hene. I is known ha PAHs in e ac wi h ca diac ion channels
which esul s in al e a ions o he ac ion po en ial in ca diomyocy es
(Inca dona e al., 2011). Ven icula ca diomyocy es ( om ju enile
ainbow ou ) exposed o e ene, in i o, esul ed in sho e and al e ed
ac ion po en ial du a ion while exposu e o phenan h ene had less
impac (Vehni¨
ainen e al., 2019). I is possible ha al e ed exp ession
pa e n o cacna- and kcnq genes a e compensa o y mechanisms as o
main ain no mal hea unc ions. This assump ion is suppo ed by p e-
ious esea ch whe e mice we e adminis a ed wi h he d ug i ab adine
Fig. 1. Boxplo o ainbow ou ale in s anda d leng h (g ow h) (a) and plana yolk a ea (b) a e 1, 3, 7 and 14 days o exposu e o DMSO (Con ), e ene (Re ),
luo an hene (Flu) and he bina y mix u e o he wo PAHs (Mix). Signi ican di e ences (p <0.05; depending on no mali y, ei he KW +Dunn o ANOVA +Tukey)
a e deno ed using: (1) di e en lowe case La in le e s o s anda d leng h a Day 3; (2) uppe case La in le e s o s anda d leng h a Day 14; (3) G eek le e s o yolk
a ea a Day 7; and (4) numbe s o yolk a ea a Day 14. A o al o 18 ale ins (3 ale ins sampled om un-e enly numbe ed eplica es) we e analyzed pe ea men and
sampled a e 1, 3 and 7 o exposu e while 24 ale ins (6 ale ins sampled om un-e enly numbe ed eplica es) exposed o 14 days we e measu ed pe ea men .
Table 5
De elopmen al obse a ions (% ±SD) measu ed as pigmen a ion in ensi y o
he la e al side (low - high) and he do sal in (yes - no) a sampling a e 14 days
o exposu e o con ol (DMSO), luo an hene (Flu), e ene (Re ) and he bina y
mix u e o he wo PAHs (Mix). Signi ican di e ences a e deno ed using lowe
(la e al side) and uppe case le e s (do sal in), as pe Fishe ’s exac es . N pe
ea men =24; 8 eplica es and 3 ale ins pe eplica e.
Tissue ype Ou come DMSO Flu Re Mix
Pigmen a ion in ensi y
o he la e al side
High (%) 83.3 ±
38.1
a
66.6 ±
48.2
b
70.8 ±
46.4
ab
62.5 ±
45.5
b
Pigmen a ion o he
do sal in
Yes (%) 100
A
87.5 ±
33.8
B
41.7 ±
50.4
C
29.2 ±
46.4
C
A.N.M. E iksson e al.
Aqua ic Toxicology 244 (2022) 106083
7
( educes he hea a e by a ec ing he ca diac pacemake cells (Bois
e al., 1996)), which esul ed in al e ed exp ession o a numbe o genes
encoding o ion channels in he sinoa ial node o mouse hea and o a
lesse ex en , he en icle (Leoni e al., 2006). In con as o he indi-
idual PAHs, exposu e o he mix u e, independen o exposu e du a-
ion, did no o e - ep esen o al e he exp ession o any gene ela ed o
ca diac ion channels. Howe e , i canno be uled ou ha exposu e o
he mix u e a ec ed hea unc ion h ough o he mechanisms wi hou
gi ing ise o a ansc ip omic esponse(s).
Simila ca diac ansc ip omic al e a ions ha e p e iously been e-
po ed in ish exposed o e ene o luo an hene. Jayasunda a e al.
(2014) exposed newly ha ched zeb a ish o a combina ion o benzo[a]
py ene and luo an hene (albei a much highe concen a ions han
used in his s udy) and iden i ied a numbe o exposu e-speci ic changes
in he ca diac o e - ep esen a ion p o iles and DEGs ela ed o calcium
ion homeos asis, emb yonic de elopmen and ca dio ascula sys em
de elopmen and unc ion. Bo h Vehni¨
ainen e al. (2016) and Rigaud
e al. (2020) exposed newly ha ched ainbow ou ale ins o e ene
(using he same se up as p esen ed in his s udy) and assessed he ca diac
ansc ip ome ( he esul s epo ed by he la e co ela ed well wi h
hose p esen ed he e). Among he nume ous o e - ep esen a ions
ela ed o ca diac unc ion and de elopmen , he o me epo ed
o e - ep esen a ion o signaling ansduc ion, g ow h, and ca dio as-
cula de elopmen , while he la e epo ed o e - ep esen a ion o
calcium, sodium, and po assium ion channel unc ion, homeos asis and
muscle con ac ion. The e o e, hei esul s, when conside ed along
hose p esen ed he e, p o ide ample suppo o he no ion ha PAHs
a ec hea unc ion h ough mul iple pa hways and ha he e is a
ansc ip omic componen in ol ed in he de elopmen o PAH induced
ca dio oxici y. Howe e , i is unknown i hese DEGs a e compensa o y
wi h ega ds o exposu e o a di ec e ec o exposu e, no i hey a e
speci ic o he hea only o di e en ly exp essed in a simila ashion in
o he issues.
A s iking di e ence in gene exp ession and o e - ep esen a ion was
ound in ale ins exposed o luo an hene o e ene alone. Bo h exposu es
o e - ep esen ed se e al e ms and pa hways ela ed o cellula - and
ansmemb ane signaling (high gene coun and signi icance), including
signaling ecep o ac i i y (GO:0038023) and neu oac i e ligand e-
cep o in e ac ions (d e04080). The di ec ion o exp ession o he
in ol ed DEGs cons i u ing hese e ms and pa hways we e bo h expo-
su e and signaling pa hway speci ic, as exposu e o luo an hene (Day 7)
esul ed in he up egula ion o in ol ed DEGs in ol ed in GABA e-
cep o complex signaling (GO:1902710), while exposu e o e ene
esul ed in down egula ion o G p o ein-coupled ecep o signaling
(GO:0007186; Day 14). These exposu e speci ic impac s highligh po-
en ial exposu e speci ic signaling dis up ion. O e - ep esen a ion o
GABA ecep o complex and up egula ion o he in ol ed DEGs,
ollowing exposu e o luo an hene, sugges s an inc ease in GABA e-
cep o ac i a ion and consequen ly less pa asympa he ic in luence on
blood p essu e and hea unc ion h ough he agus ne e (Lei e e al.,
2009; Ben zen and G unne , 2011). By con as , p olonged sa u a ion
and ac i a ion o G-p o ein ecep o s can lead o down egula ion o
genes encoding o he ecep o s (Tsao and on Zas ow, 2000). Why
Fig. 2. Boxplo o e ene (a; Re ; pMol ish
−1
) and luo-
an hene (b; Flu; pMol ish
−1
) body bu den in whole body
ainbow ou ale in ca casses (backg ound compensa ed)
exposed o 1, 3, 7 and 14 days o he PAHs indi idually ( illed
boxes) o he bina y mix u e o he wo (Mix, un illed boxes).
Signi ican di e ences (p <0.05) in he body bu den o e ene
and luo an hene among mix u e exposed ale ins a e deno ed
wi h lowe case le e s, while uppe case le e s deno e signi i-
can di e ences in body bu den among ale ins exposed o he
indi idual PAHs (Fluo an hene alone and in mix u e: KW +
Dunn; Re ene alone and in Mix: ANOVA +Tukey). Signi ican
di e ences in PAH body bu den be ween single and mix u e
exposed ale ins a e deno ed wi h a * unde nea h mix u e box
plo s (Re : - es ; Flu: MW). No e, ale ins we e exposed o
136.56 nM o e ene and 247.21 nM o luo an hene.
Fig. 3. Venn-diag ams ep esen ing he numbe o di e en ly exp essed ca -
diac genes a ec ed by exposu e o e ene (Re ; ed), luo an hene (Flu; blue)
and he mix u e o he wo PAHs (Mix; g een) ollowing 1 (a), 3 (b), 7 (c) and 14
(d) days o exposu e. The Venn-diag am was c ea ed using he online pla o m
p o ided by Bioin o ma ics & E olu iona y Genomics.
A.N.M. E iksson e al.
Aqua ic Toxicology 244 (2022) 106083
8
exposu e o e ene and luo an hene o e - ep esen ed signaling pa h-
ways so speci ically is cu en ly unknown bu equi es u he in es i-
ga ion. Howe e , i may be ha he speci ici y is linked o how he PAHs
in e ac wi h ca diomyocy e ion-channels and subsequen ly al e he
epola iza ion o ca diac ac ion po en ial, hus making hese
o e - ep esen a ions compensa o y a he han a di ec consequence o
exposu e. Ye , i is unknown i his phenomenon is hea -speci ic o i i
could ake place in o he issues as well. Addi ionally, he ac ha hese
wo o e - ep esen a ions only occu ed among ale ins exposed o 7 and
14 days suppo s he iew ha ce ain endpoin s equi e ex ended
exposu e du a ion (o ha e o ake place a a speci ic de elopmen al
s age) be o e he compensa o y e en s become mani es ed in he
de eloping o ganism (P ice and Mage , 2020).
4.2. Body bu den and xenobio ic me abolism
Unique and exposu e speci ic body bu den p o iles we e ob ained
al eady a e 1 day o exposu e and h oughou he exposu e du a ion,
sugges ing exposu e speci ic phase I and II me abolic p o iles. The body
bu den o e ene was signi ican ly educed by Day 14, compa ed o Day
1, ollowing exposu e o e ene alone o as a mix u e (albei signi ican ly
mo e abundan in mix u e exposed ale ins). By con as , exposu e o
luo an hene alone esul ed in a signi ican ly inc eased body bu den by
Day 7 and onwa d, which can be a ibu ed o pa ial inhibi ion o he
ca aly ic unc ion o CYP1a by luo an hene, alongside ac i a ion o a
na ow sui e o phase II me abolic p ocesses. When co-adminis e ed
wi h e ene, he body bu den o luo an hene inc eased non-
signi ican ly om Day 1 o Day 3 be o e dec easing signi ican ly wi h
ime. Simila changes in he body bu den p o iles o PAHs ha e p e i-
ously been obse ed in ainbow ou exposed o a bina y mix u e o
alpha-naph ho la one (ANF; a CYP1a inhibi o ) and e ene (Hodson
e al., 2007). Thei speci ic bina y mix u e esul ed in inc eased body
bu den o e ene wi h inc eased dose o ANF. Changes in he body
bu den p o iles o speci ic PAHs do no seem o be species speci ic as pe
p e ious obse a ions in zeb a ish exposed o a complex mix u e o
PAHs and a bina y mix u e o he AhR2 agonis be a-naph ho la one
and ANF (Timme-La agy e al., 2007; Geie e al., 2018). Hence, he
body bu den o PAHs is bo h PAH and mix u e speci ic while he un-
de lying molecula p ocesses esul ing in he subsequen body bu den
p o iles a e ela ed o accumula ion in ela ion o me abolism. Howe e ,
due o low eco e y a es o he PAHs, he quan i ica ion o body bu den
should be conside ed ca e ully. None heless, he low eco e y a es
appea jus i iable in ligh o he simila eco e y a es o e ene (14.3%)
and luo an hene (14.6%) as well as he a io be ween he subsequen
Fig. 4. O e - ep esen a ion analysis o gene on ology (GO) g oups ollowing exposu e o e ene (Re ), luo an hene (Flu) and he bina y mix u e (Mix) las ing o 1,
3, 7 and 14 days. Da a was analyzed, p- alue adjus ed and plo ed using clus e P o ile . The size o each do equals he numbe o genes in ol ed in each GO- e m.
No e: due o igu e size limi a ions, Fig. 3 con ains 54 ou a o al o 135 o e - ep esen ed GO- e m (whole da a is p esen ed in Fig. s5).
A.N.M. E iksson e al.