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T ansgene a ional inhe i ance o chemical-induced signa u e: A case s udy
wi h sim as a in
T. Neupa h
a,⁎
, A.M. Machado
a,b
, R. Mon es
c
, R. Rodil
c
, S. Ba os
a
, N. Al es
a
, R. Rui o
a
,
L. Filipe C. Cas o
a,b
, J.B. Quin ana
c
, M.M. San os
a,b,⁎
a
CIMAR/CIIMAR—In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, Uni e si y o Po o, A enida Gene al No on de Ma os, S/N, 4450-208 Ma osinhos,
Po ugal
b
FCUP –Depa men o Biology, Facul y o Sciences, Uni e si y o Po o, Po o, Po ugal
c
Depa men o Analy ical Chemis y, Nu i ion and Food Sciences, IAQBUS –Ins i u e o Resea ch on Chemical and Biological Analysis, Uni e sidade de San iago de
Compos ela, R. Cons an ino Candei a S/N, 15782 San iago de Compos ela, Spain
ARTICLE INFO
Handling Edi o : Ma i Nadal
Keywo ds:
Sim as a in
T ansgene a ional effec s
Rep oduc ion
T ansc ip omic
Epigene ics
Regula o y agencies
ABSTRACT
The hypo hesis ha exposu e o ce ain en i onmen al chemicals du ing ea ly li e s ages may dis up e-
p oduc ion ac oss mul iple non-exposed gene a ions has significan implica ions o unde s anding disease
e iology and ad e se ou comes. We demons a e he e ep oduc i e mul i and ansgene a ional effec s, a en-
i onmen ally ele an le els, o one o he mos p esc ibed human pha maceu icals, sim as a in, in a keys one
species, he amphipod Gamma us locus a. The ansgene a ional findings has majo implica ions o haza d and
isk assessmen o pha maceu icals and o he con aminan s o eme ging conce n gi en ha ansgene a ional
effec s o en i onmen al chemicals a e no add essed in cu en haza d and isk assessmen schemes. Conside ing
ha he me alona e syn hesis, one o he key me abolic pa hways a ge ed by sim as a in, is highly conse ed
among me azoans, hese esul s may also shed ligh on he po en ial ansgene a ional effec s o sim as a in on
o he animals, including humans.
1. In oduc ion
A g owing body o e idence sugges s ha he exposu e o ce ain
s esso s du ing emb yonic de elopmen may induce pe manen en-
doc ine dis u bance, which can be ansgene a ionally inhe i ed
h ough mul iple non-exposed gene a ions, e en in he absence o he
insul (Chamo o-Ga cia e al., 2017;F az e al., 2019;Klosin e al.,
2017;Ve a-Chang e al., 2018;Wang e al., 2016). This may ha e sig-
nifican implica ions o unde s anding disease e iology and ad e se
ou comes ha can be passed ac oss gene a ions (Anway e al., 2005;
Ve a-Chang e al., 2018). In he las yea s, an inc easing scien ific in-
e es on ansgene a ional inhe i ance has eme ged. Howe e , despi e
he g owing numbe o s udies claiming ansgene a ional impac s,
many p esen gaps in he ansgene a ional expe imen al design by
in es iga ing only di ec exposu e (F0, F1 and/o F2), excluding he
uly non-exposed gene a ions (F3 o abo e), o es ing chemical con-
cen a ions a abo e ecological ele ance (Je emias e al., 2020;Shaw
e al., 2017).
He e, we exposed he keys one ma ine amphipod species Gamma us
locus a (F0) (Fig. 1A) o en i onmen ally ele an concen a ions o he
hypocholes e olaemic d ug, sim as a in (SIM), and e alua ed he
ansgene a ional effec on F3. S a ins a e one o he mos p esc ibed
human pha maceu icals in wes e n Eu opean coun ies and in he
Uni ed S a es, being ubiqui ous in aqua ic en i onmen s in he icini y
h ps://doi.o g/10.1016/j.en in .2020.106020
Recei ed 20 Ap il 2020; Recei ed in e ised o m 26 June 2020; Accep ed 28 July 2020
Abb e ia ions: AACT, ace oace yl-CoA hiolase; chiA, chi inase; CYP clan 2, s e oidogenic CYP (CYP 2L1); ELOVL6, elonga ion o e y long chain a y acids p o ein
6; ELOVL7, elonga ion o e y long chain a y acids p o ein 7; FALD, a nesal dehyd ogenase; FAMET, a nesoic acid O-me hyl ans e ase; FOLD, a nosol dehy-
d ogenase; FPPP, a nesyl diphospha e py ophospha ase; FPPS, anesyl diphospha e syn hase; HEXA-B, hexosaminidase; HMGR, hyd oxyme hylglu a yl-CoA e-
duc ase; HMGS, hyd oxyme hylglu a yl-CoA syn hase; IDI1, isopen enyl diphospha e isome ase; JHAMT, ju enile ho mone acid me hyl ans e ase; JHEH, ju enile
ho mone epoxide hyd olase; LSS, lanos e ol syn hase; MDC, me alona e-5-deca boxylase; MFE, me hyl a nesoa e epoxidase; MUFA, monounsa u a ed a y acids;
MVK, me alona e kinase; PFK, 6-phospho uc okinase 1; PUFA, Polyunsa u a ed a y acids; PVK, phosphome alona e kinase; SCD, s ea oyl-CoA desa u ase; SFA,
Sa u a ed a y acids; SQLE, squalene monooxygenase; SQS, squalene syn hase; SORD, L-idi ol 2-dehyd ogenase; UGT, glucu onosyl ans e ase; xylA, xylose iso-
me ase
⁎
Co esponding au ho s a : CIMAR/CIIMAR—In e disciplina y Cen e o Ma ine and En i onmen al Resea ch, Uni e si y o Po o, A enida Gene al No on de
Ma os, S/N, 4450-208 Ma osinhos, Po ugal (T. Neupa h) and FCUP –Depa men o Biology, Facul y o Sciences, Uni e si y o Po o, Po o, Po ugal (M.M. San os).
E-mail add esses: [email p o ec ed] (T. Neupa h), [email p o ec ed] (M.M. San os).
En i onmen In e na ional 144 (2020) 106020
A ailable online 26 Augus 2020
0160-4120/ © 2020 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
T
o u ban a eas, wi h concen a ions abo e 100 ng/L being de ec ed in
su ace wa e s and up o 2650 and 11,700 ng/L in was e wa e ea -
men plan effluen s and influen s, espec i ely (Pe ei a e al., 2015;
San os e al., 2016;Te e e al., 2020). This he apeu ic class dis up s
choles e ol syn hesis by inhibi ing he enzyme 3-hyd oxy-3-me hyl-
glu a yl-CoA educ ase (HMGR), esponsible o he a e limi ing s ep in
he me alona e syn hesis, a pa hway highly conse ed among me-
azoans, including humans (Fig. 1B). Gi en ha SIM was p e iously
ound o impac G. locus a ep oduc ion in he ange o concen a ions
epo ed in su ace wa e s (Neupa h e al., 2014), and i is a known
modula o o epigene ic ma ke s in mammalian cells (Ka lic e al.,
2015), we in es iga ed he mul igene a ional and ansgene a ional (F0
o F3) effec s o en i onmen ally ele an concen a ions o SIM, using
as model species he amphipod G. locus a (Fig. 1C).
2. Ma e ials and me hods
2.1. Tes species
The amphipod Gamma us locus a was he o ganism selec ed o in-
es iga e he ansgene a ional effec s o Sim as a in (SIM) conside ing
i s ad an ages in compa ison wi h o he animal models, i.e., i s sho
li e-cycle (35 days a 20 °C), educed logis ical suppo o es ing and
b eeding and e hical cons ain s. They a e excellen models o oxici y
es ing and eco oxicological isk assessmen due o hei high sensi i i y
o an h opogenic s esso s and wide geog aphic dis ibu ion, bu also
because hey display a key ole in he aqua ic ecosys em (Cos a and
Cos a, 2000;Neupa h e al., 2002). A deep knowledge on he biology
and ecology o he species has been ob ained o e he pas wo decades
(Cos a and Cos a, 1999, 2000;Neupa h e al., 2002, 2005, 2014).
2.2. Expe imen al design
All expe imen s conduc ed in his s udy we e ca ied ou a Bio é io
de O ganismos Aquá icos (BOGA, CIIMAR) aqua ic animal acili ies and
ha e been app o ed by he CIIMAR e hical commi ee and by CIIMAR
Managing Animal Wel a e Body (ORBEA) acco ding o he Eu opean
Union Di ec i e 2010/63/EU on he p o ec ion o animals used o
scien ific pu poses.
To in es iga e he mul igene a ional and he ansgene a ional (F0
o F3) effec s o en i onmen ally ele an concen a ions o SIM (CAS
n°. 79902-63-9; Sigma Ald ich), G. locus a was con inuously main ained
unde SIM exposu e du ing ou successi e gene a ions (F0E, F1E, F2E
and F3E) –exposed g oup (Fig. 1C). Addi ionally, in o de o e alua e
he pu a i e SIM ansgene a ional effec s, in pa allel o he con inuous
SIM exposu e, a sub-se o offsp ing p oduced in F0E we e aised unde
SIM- ee wa e (con ol wa e ) o he subsequen h ee gene a ions
e.g., F1T, F2T, F3T – ansgene a ional g oup (Fig. 1C).
To conduc he expe imen , 7L aqua ia in a semi-s a ic sys em we e
se -up in con olled labo a o y condi ions (20 °C, 16 h ligh /8 h da k).
Each aqua ium was filled wi h 5L o na u al fil e ed seawa e (33-35‰)
dosed wi h SIM –exposed g oup –o ee o SIM – ansgene a ional
g oup. A 1 cm-deep laye o na u al clean sedimen and small s ones
we e supplied o p o ide shel e and ae a ion was p o ided wi h plas ic
ips. Each gene a ion (F0 o F3) was ini ia ed wi h exac ly fi y offsp ing
ha we e alloca ed o each ea men aqua ia, and las ed o 55 o
Fig. 1. Ra ionale o he s udy. (A) Gamma us locus a in amplexus. (B) Simplified scheme o he Me alona e pa hway. S a ins inhibi he enzyme 3-hyd oxy-3-me hyl-
glu a yl-CoA educ ase (HMGR), esponsible o he a e limi ing s ep in he me alona e pa hway. The dime hylallyl py ophospha e syn hesis om ace yl-CoA is
conse ed o all me azoan (g ay box). The a nesyl py ophospha ase syn hesis (g een box) is es ablished in e eb a es and a h opods. Downs eam a nesyl
py ophospha ase, he e a e g oup-specific a ia ions: choles e ol biosyn hesis in e eb a es (blue box) and he sesquis e penoid biosyn hesis in a h opods wi h he
p oduc ion o me hyl a nesoa e (yellow box). Beyond choles e ol and me hyl a nesoa e, o he isop enoids a e syn hesized in he me alona e pa hway and a nesyl
and ge anyl moie ies also pa icipa e in p o ein p enyla ion (g een Box). (C) Expe imen al Design: Mul igene a ional (o ange) and ansgene a ional (blue) s udies o
in es iga e he effec s o Sim as a in (SIM) on he amphipod G. locus a du ing 4 consecu i e gene a ion (F0 o F3). Mul igene a ional s udy (F0E-F3E), ansge-
ne a ional s udy (F1T-F3T). (Fo in e p e a ion o he e e ences o colou in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
2
65 days. Th oughou he expe imen amphipods we e ed ad libi um
wi h Ul a sp. collec ed om a coas al a ea in he Aguda beach, Po ugal
−41°02′55.2″N 8°39′16.6″W–a si e de oid o di ec con amina ion
sou ces. The aqua ia we e inspec ed daily o eeding equi emen s and
ae a ion and he pH, conduc i i y, dissol ed oxygen, ammonia le el,
salini y and empe a u e we e s ic ly moni o ed and con olled a each
wa e change.
Two weeks be o e he beginning o he assay, wen y couples o
sexually ma u e males and emales wi h abou 12 week old, om he
main cul u ing sys em, we e sepa a ed and used o p oduce he F0
gene a ion. Each emale a his age p oduce in a e age 48 offsp ing
(Neupa h e al., 2002). These sub se o adul s we e main ained a he
same empe a u e and salini y o he bioassay, wi h unlimi ed ood
(Ul a sp.). The bioassay s a ed wi h fi y, one-week old, offsp ing pe
aqua ium, andomly isola ed om he cul u ing sub-se and we e
con inually exposed up o adul hood o h ee en i onmen al con-
cen a ions o SIM (32, 64 and 320 ng/L) plus con ol (fil e ed na u al
seawa e ) and sol en con ol (0.0005% ace one), ou eplica e
aqua ia o each condi ion (F0E). The concen a ions o SIM used in he
p esen s udy we e based upon ou p e ious esea ch (Neupa h e al.,
2014) whe e SIM was ound o impac G. locus a ep oduc ion in he
ange o concen a ions epo ed in su ace wa e s (ng/L ange). The
SIM solu ions o he diffe en ea men g oups, 32, 64 and 320 ng/L,
we e p epa ed by se ial dilu ions o a 0.64 mg/mL s ock solu ion p e-
pa ed in ace one (0.0005%). These solu ions we e s o ed in he da k
a −20 °C. A each wa e enewal o he expe imen al aqua ia, ha
occu ed e e y h ee days, SIM solu ions we e applied di ec ly in he
middle o each aqua ium and he final SIM concen a ions we e e-
es ablished by p ope ly s i ed. This app oach was p e iously alida ed
(Neupa h e al., 2014), hus a oiding he s ess o daily wa e change.
Gi en ha in p e ious expe imen s SIM was ound o be s able in wa e
unde a simila expe imen al design (Neupa h e al., 2014,Ba os
e al., 2018), he ac ual concen a ion o SIM in each ea men /gen-
e a ion was quan ified using LC-MS/MS a e one o he wa e change
(0 h, SIM addic ion) and be o e he nex wa e change (72 h pos SIM
addic ion) (see Neupa h e al., 2020, Sec ion 2.2).
Gi en ha no significan effec s in g ow h and ep oduc ion we e
obse ed a he lowes SIM exposu e concen a ion (32 ng/L) (Fig. 2),
he ea men s a 64 and 320 ng/L we e selec ed o con inue he mul i
and ansgene a ional s udies (F1, F2 and F3 exposed g oup –64E and
320E; and ansgene a ional g oup –64T and 320T). A ma u i y and
a e h ee consecu i e ep oduc ions, males and emales we e sampled
(see nex sec ion) and he offsp ing o each ea men we e di ided in o
wo b anches: one was main ained unde con inuous SIM exposu e and
aised o mo e h ee consecu i e gene a ions (F1E, F2E and F3E) and
he o he was main ained in SIM- ee wa e (na u al fil e ed seawa e
and SIM ee sedimen s, simila o he con ol g oup) o h ee con-
secu i e gene a ions (F1T, F2T and F3T).
2.3. Sample collec ion, ecological endpoin s
In each gene a ion, a e he second/ hi d ep oduc ion, he o e -
lying wa e o each ea men eplica e (exposed o ansgene a ional
g oups) was sie ed h ough 1000 and 250 µm sc eens, o collec he
adul s and hei offsp ing, espec i ely, ollowing he p o ocol o
Neupa h e al. (2014). Sedimen s we e washed fi e imes o assu e ha
all o ganisms we e collec ed. Fi y offsp ing om each ea men e-
plica e we e andomly selec ed o s a he subsequen gene a ion. The
adul s’(males and emales) su i al, body leng h, as well as emale
ecundi y, we e quan ified in each o he ou eplica e aqua ia pe
condi ion. Su i al and body leng h we e de e mined sepa a ely o
each sex and exp essed as pe cen age ela i e o con ol g oups. Simila
o ou p e ious s udy (Neupa h e al., 2014), su i al did no diffe
among SIM ea men s and con ol g oups. A e being anes he ized in
ice wa e , he amphipodśbody leng h was measu ed o he nea es
0.1 mm using a s e eomic oscope (Leica EZ4) by he dis ance be ween
he an e io end o he os um and he pos e io end o he las me-
asoma ic segmen –me asoma ic leng h (Neupa h e al., 2014). A e
being measu ed, adul s we e indi idually p ese ed in RNA la e o
liquid ni ogen and s o ed a −80 °C un il u he use in RNA-seq
analyses and me hyl a nesoa e quan ifica ion, espec i ely. The e-
cundi y (a e age numbe o offsp ing p oduced by emale) was quan-
ified a e each ep oduc ion. All offsp ing p esen in each aqua ium
we e collec ed and p ese ed in 70% e hanol wi h Bengal Rose o la e
quan ifica ion o ep oduc i e success.
2.4. Quan ifica ion o me hyl a nesoa e le els in Gamma us locus a
The concen a ion o me hyl a nesoa e (MF) in G. locus a was de-
e mined o each ea men and sex o exposed and ansgene a ional
g oups in he ou gene a ions by ma ix-solid phase dispe sion (MSPD)
and gas ch oma og aphy-chemical ioniza ion mass spec ome y (GC-
CI-MS). This analy ical me hodology was de eloped as an al e na i e o
hemolymph ex ac ion and is desc ibed in de ail in Mon es e al.
(2017). The MF le els (ng/g) we e de e mined in ou male and ou
emale samples, each composed by h ee pooled indi iduals belonging
o diffe en eplica es o each ea men o he exposed and ansge-
ne a ional g oups om he ou gene a ions. The sample weigh was
eco ded, and he esul s we e exp essed as we weigh concen a ion.
Deu e a ed me hyl hep adecanoa e (Sigma-Ald ich) was used as in-
e nal s anda d (IS). Each sample (a e age weigh 0.302 g and 0.147 g
o males and emales, espec i ely) was dispe sed in a mo a wi h
0.5 g o PSA (p ima y-seconda y amine) and loaded in o a MSPD ca -
idge p e iously filled wi h 1.5 g o Flo isil (co-so ben ) and 0.5 g o
sodium sul a e anhyd ous (desiccan ). Then, MF and IS we e elu ed
om he MSPD ca idges using 1.5 mL o e hyl ace a e. The final ex-
ac s we e concen a ed o 20 µL and injec ed in he GC-CI-MS sys em
(Agilen ) using isobu ane as eagen gas. Bo h MF and he IS we e
measu ed in he single ion moni o ing mode using he p o ona ed
molecula ion o quan ifica ion (Q) and wo addi ional ions as quali-
fie s (q), pe compound. These ions (m/z) we e 251 (Q) and 219, 191
(q) o MF and 317.5 (Q) and 267.5, 282.5 (q) o IS. The concen a ion
o he ex ac s was calcula ed by in e nal s anda d calib a ion in a
concen a ion ange be ween 5 and 250 ng/mL and hen exp essed as
we weigh concen a ion (ng/g) o each sample. The MQLs we e
3.1 ng/g o males 2 ng/g o emales. The eco e ies we e e alua ed a
wo diffe en spiked le els (2 and 10 ng o emales and 1 and 5 ng o
males, o e he na i e concen a ion) and u ned ou 96 and 103% o
emales and 81 and 85% o males wi h a maximum ela i e s anda d
de ia ion (RSD) o 16%.
2.5. S a is ical analysis
Fo each gene a ion (F0 o F3), he ecological da a (males and
emaleśleng h and ecundi y) and he me hyl a nesoa e le els om he
exposed and ansgene a ional g oups we e fi s checked o no mali y
(Kolmogo o -Smi no es ) and homogenei y o a iances (Le ene's
es ). I he ANOVA assump ions we e ulfilled, each endpoin da a om
he exposed and ansgene a ional g oups we e analyzed oge he by
one-way analysis o a iance (ANOVA). When significan diffe ences
we e ound by ANOVA, Fishe ’s leas significan diffe ence (LSD) es
was used o disc imina e s a is ically significan diffe ences be ween
exposed o ansgene a ional ea men s and he con ol. Fo males and
emales leng h, as he no mali y o he da a we e no ulfilled, e en
a e da a ans o ma ion, a K uskal-Wallis ANOVA by anks was used.
Significan diffe ences we e es ablished a p < 0.05 o e e y analysis
and all s a is ics we e compu ed wi h S a is ica 12.5 (S a -so , USA).
Simila o ou p e ious s udy (Neupa h e al., 2014), he sol en used,
ace one a 0.0005%, ende ed no significan effec (P > 0.05) in
compa ison o he wa e con ol a any o he gene a ions/sex. The e-
o e, ollowing G een and Wheele (2013) p oposal, he da a om hese
g oups we e pooled o inc ease s a is ical powe and e e ed o as
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
3
con ol g oup.
2.6. RNA ex ac ion, lib a y p epa a ion and Illumina sequencing o G.
locus a samples
RNA-seq was pe o med independen ly in h ee indi idual G. locus a
emales, pe g oup, o e he ou gene a ions: sol en con ol g oups
(F0.C, F1.C, F2.C and F3.C), F0 320 exposed g oup (F0.320E) and F1 o
F3 320 ansgene a ional g oup (F1.320T; F2.320T and F3.320T)
(De ailed in o ma ion displayed in Neupa h e al., 2020, Annex 1).
To al RNA om h ee emales pe g oup was ex ac ed using he Illus a
RNAspin Min RNA Isola ion Ki (GE Heal hca e), acco ding o he
manu ac u e ’s p o ocol. RNA sample in eg i y and concen a ion we e
ini ially checked by aga ose gel elec opho esis and hen by an Agilen
Bioanalyze 2100 (Agilen Technologies, USA), wi h RIN alues a ying
om 6.3 o 7.7. RNA-seq lib a ies and sequencing, Illumina HiSeq2500
pai ed-end (2x150), we e ob ained comme cially a No ogene (Hong
Kong).
2.7. In silico clean-up o aw da ase s and de no o ansc ip ome assembly
The 563 M o PE sequenced eads we e analysed using se e al gold
s anda d me hods. Ini ially, o ha e a quali y o e iew o he da ase s,
he Fas QC ( .0.11.8) so wa e was applied (h p://www.
bioin o ma ics.bab aham.ac.uk/p ojec s/ as qc/). A e ha , he
Rco ec o ( .1.0.3) (Song and Flo ea, 2015) was applied o e o
co ec ion, wi h he de aul s, and Cen i uge ( .1.0.3-be a) (Kim e al.,
2016) o axonomically fil e each ead da ase . To pe o m he e-
e ence ansc ip ome assembly o G. locus a he de no o assembly
me hod T ini y ( .2.8.4) (G abhe e al., 2011) was applied (De ailed
in o ma ion displayed in Neupa h e al., 2020, sec ion 2.3).
2.8. Decon amina ion, open eading ame (ORF) p edic ion, and
assessmen o ansc ip ome assembly
The ansc ip ome assembly decon amina ion and assessmen s we e
done using h ee dis inc app oaches, blas sea ches agains NCBI
Fig. 2. Mul igene a ional and ansgene a ional impai men o sim as a in ac oss mul iple gene a ions (F0 o F3). (A) G ow h, exp essed as pe cen age ela i e o
con ol g oup, n = 52–58. (B) Fecundi y, exp essed as pe cen age ela i e o con ol g oup, n = 4 eplica e anks pe ea men . (C) me hyl a nesoa e (MF) le els
(ng/g) exp essed as pe cen age ela i e o con ol, n = 4 pools o males and emales. Da a a e p esen ed as he means ± s anda d e o and he as e isks ep esen
significan diffe ence in he exposed g oup (E) o ansgene a ional g oup (T) compa ed wi h he con ol (CTR): *p < 0.05 and **p ≤0.01.
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
4
da abases, calcula ion o basic s a is ics wi h T ini y and T ans a e
sc ip s ( .1.0.3) (Smi h-Unna e al., 2016) and compa isons wi h da-
abases o conse ed o hologs genes, h ough he Benchma king Uni-
e sal Single –Copy O hologs so wa e (BUSCO .3.0.2) (Simão e al.,
2015). The open eading ames we e p edic ed using he T anDecode
so wa e ( .5.3.0) (Haas e al., 2013). This p ocess gene a ed h ee
e sions o he ansc ip ome assembly; he 1s e sion –Raw an-
sc ip ome assembly –Decon amina ed; 2nd e sion –P o ein coding
assembly –All ansc ip s wi h ORF; 3 d e sion –P o ein coding as-
sembly –Unigenes wi h ORF (De ailed in o ma ion displayed in
Neupa h e al., 2020, sec ion 2.4).
2.9. T ansc ip ome anno a ion
To pe o m he ansc ip ome anno a ion, he second e sion o he
ansc ip ome assembly and T ino a e ool ( .3.1.1) we e used (B yan
e al., 2017). T ino a e uses se e al me hods o unc ionally anno a e
ansc ip ome assemblies. He e, a bunch o ools we e used o anno a e
he ansc ip ome o G. locus a: blas sea ches, iden ifica ion o p o ein
domains (PFAM Da abase) (39), classifica ion o o hologous g oups o
genes (eggNOG) (Powell e al., 2012) and a ibu ion o Gene On ology
(GO) e ms (Ashbu ne e al., 2000) (De ailed in o ma ion displayed in
Neupa h e al., 2020, sec ion 2.5).
2.10. Diffe en ial gene exp ession analyses
Fo he diffe en ial gene exp ession (DEGs) analyses he second
e sion o ansc ip ome assembly and he clean eads we e used. The
second e sion o he ansc ip ome was used o maximize mapping o
he aw eads agains he p o ein coding ansc ip ome. The ead
mapping and ansc ip quan ifica ion we e done wi h he abundan-
ce_es ima es_ o_ma ix.pl sc ip o T ini y he pipeline, unde he de-
aul s (Haas e al., 2013). He e, he Bow ie2 ( .2.3.5) (Langmead and
Salzbe g, 2012) so wa e was applied o map he eads and he RSEM
( .1.3.0) (Li and Dewey, 2011) ool o es ima e he ansc ip abun-
dance. The diffe en ial gene exp ession analyses we e ca ied ou in
Degus ( .4.1.1) (Powell e al., 2015) pla o m (h p://degus .e c.
monash.edu/) being he edgeR ( .3.26.8) package (Robinson e al.,
2010) o R ( .3.6.1) used o calcula e diffe en ial gene exp ession. To
de e mine DEG, he exposed o ansgene a ional samples o each
gene a ion we e compa ed agains he espec i e con ol (F0.C s
F0.320E; F1.C s F1.320T; F2.C s F2.320T; F3.C s F3.320T), and all
genes wi h False Disco e y Ra e –co ec ed (FDR) p- alue < 0.05 and
log2| old change| ≥2 we e conside ed diffe en ially exp essed (De-
ailed in o ma ion displayed in Neupa h e al., 2020, sec ion 2.6).
2.11. KEGG pa hways analyses
The main me abolic pa hways affec ed by he ansgene a ional
effec s o SIM ac oss all gene a ions we e iden ified using he KAAS
webse e (Mo iya e al., 2007). The DGEs genes we e blas ed on o
KEGG pa hways da abase using he single-di ec ional bes hi (SBH)
me hod, he p o eins co esponding o he diffe en ial exp essed genes
(collec ed om he hi d e sion o he ansc ip ome), and 710,890
e e ence sequences o 40 manually selec ed species (De ailed in-
o ma ion displayed in Neupa h e al., 2020, sec ion 2.7).
3. Resul s and discussion
3.1. Mul igene a ional and ansgene a ional impai men o sim as a in
ac oss gene a ions: effec s on G. locus ag ow h and ep oduc ion and
me hyl a nesoa e (MF) le els
We fi s exposed G. locus a ju eniles (one-week old) om ou la-
bo a o y cul u e o en i onmen ally ele an concen a ions o SIM (32,
64 and 320 ng/L) (Neupa h e al., 2020, Table A) up o adul hood (F0)
and e alua ed ecologically ele an endpoin s ( emale ecundi y and
g ow h). A he F0 gene a ion, we ound significan effec s a 320 ng/L,
i.e., dec ease o ep oduc i e ou pu and g ow h, and educ ion o e-
male g ow h a 64 ng/L (Fig. 2A and B). We hen e alua ed hese
endpoin s in he h ee subsequen gene a ions (F1 o F3), bo h in ani-
mals con inuously exposed o SIM (exposed g oup, 64E and 320E) and
in he offsp ing o animals exposed in F0 he ea e main ained in
con ol wa e ( ansgene a ional g oup, 64T and 320T). We ound a
significan impac in ecundi y, in se e al ea men s in compa ison
wi h con ol, up o F3. A significan dec ease in ep oduc ion was ob-
se ed o 64E, 320E and 320T (p < 0.05) in he F1 and o 320E
(p < 0.01) in he F2. In he F3, he ansgene a ional and exposed
g oups (64T and 320T and 320 E) also p esen ed a significan dec ease
in he ep oduc i e ou pu i.e., 40% dec eased ecundi y o 64T, 50%
o 320T and 48% o 320E (Fig. 2B). G ow h was also impac ed in he
exposed g oup (320E) and in he ansgene a ional g oup (320T), bu in
he la e only in F2 and F3 (Fig. 2A). Al hough 64T displayed a sig-
nifican impac in he F3 ecundi y, animals om his ea men did no
p esen , in any o he gene a ions o sexes, g ow h impai men . This
suppo s he hypo hesis ha he ecundi y dec ease obse ed o 64T in
F3 is independen om emale leng h.
The final s eps o he me alona e pa hway display g oup-specific
a ia ions, i.e., choles e ol syn hesis in e eb a es and syn hesis o he
isop enoid me hyl a nesoa e (MF) in a h opods (Fig. 1B; San os e al.,
2016). Gi en ha in c us aceans MF has a cen al ole in mol ing and
ep oduc ion (Lau e and Bigge s, 2001), we nex de eloped a highly
sensi i e analy ical me hod (ma ix solid-phase dispe sion wi h gas
ch oma og aphy coupled o mass spec ome y) o de ec MF in a -
h opod issues, including G. locus a (Mon es e al., 2017). Bo h males
and emales om all gene a ions (F0 o F3) we e sc eened o MF issue
le els, wi h his isop enoid being de ec ed in all samples. Le els o MF
in males we e significan ly inc eased a 32E and 320E in F0 and 64T in
F1 (Fig. 2C). Fo F2 and F3, bo h in he exposed and he ansgene a-
ional g oups, male MF le els did no diffe significan ly om con ol
(P > 0.05) (Fig. 2C). In con as , a significan dec ease o MF le els
was eco ded in emales o mos o he ea men s and gene a ions
(Fig. 2C). Wi h excep ion o he emales om 320E in F0 and F3 ha
did no diffe significan ly om con ol, emale MF le els we e sig-
nifican ly educed in all F0, F1 and F3 g oups wi h a dec ease in
compa ison o con ol anging be ween 27 and 45%. In F2, he excep-
ion was he emales om 64T and 320T. In ac , in F2, simila o e-
males’ ecundi y, le els o emale MF in he ansgene a ional g oups
(64T and 320T) did no diffe significan ly om con ol. In F3, emales
om bo h ansgene a ional g oups (64T and 320T) p esen ed a sig-
nifican educ ion on MF le els in compa ison wi h con ol, wi h he
dec ease anging om 25% (64T, p < 0.05) o 35% (320T, p < 0.01).
These findings sugges ha he ansgene a ional inhe i ance o he
ep oduc ion impai men in he 64T and 320T g oups could be asso-
cia ed wi h a dec ease o he MF le els in emales, a key egula o o
ep oduc ion in c us aceans.
In ou p e ious s udy o add ess he ch onic li e-cycle effec o SIM
in G. locus a ep oduc ion (Neupa h e al., 2014), we obse ed a simila
impac o ha epo ed he e in F0 emales exposed o SIM a 320 ng/L.
In o de o u he unde s and he effec s in ep oduc ion, Neupa h
e al., 2014 pe o med a de ailed his ological analysis o male and e-
male gonads. Whe eas emale gonad li e-cycle exposed o 320 ng/L
displayed delayed ma u a ion e ealing p edominance o p ima y oo-
cy es, he majo i y o con ol emales we e ully ma u e. In con as ,
spe ma ogenesis was no affec ed a SIM concen a ions anging om
320 ng/L o 8000 ng/L. Hence, aken oge he , ou p e ious findings
(Neupa h e al., 2014) and he esul s epo ed he e o MF le els and
ecundi y suppo he hypo hesis ha SIM effec s on ep oduc ion seem
o be associa ed wi h emales a he han males.
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
5
3.2. T ansc ip omic analysis o emales ac oss gene a ions
To gain addi ional insigh s in o he biological unc ions and cano-
nical pa hways ansgene a ionally dis up ed by SIM a comp ehensi e
ansc ip ome assembly was p oduced and he ansc ip omic p ofiles
o emales om he con ol g oup (F0 o F3), he 320E g oup (F0) and
he 320T g oup (F1 o F3) we e analyzed (Fig. 3,Fig. 4 and Neupa h
e al., 2020, Annex 1 and Annex 2).
In o de o mine he la ge se o mul igene a ional da a, we ocused
on he in e connec ions and he ne wo k o genes sha ing common
biological p ocesses and unc ions a he han on single genes. We ad-
d essed in pa icula he common gene ne wo k pa hways affec ed in
emales om F0 (320E) and F3 (320T), ha can con ibu e o he un-
de s anding o he ansgene a ional impai ed ecundi y and g ow h on
F3 (Fig. 3 and Fig. 4).
The RNA sequencing (RNA-seq) analysis (Neupa h e al., 2020,
Annexes 1 o 8, 12 o 14 and 16) iden ified a o al o 3785 diffe en ially
exp essed unigenes in emales ac oss all gene a ions (F0 o F3), wi h a
False Disco e y Ra e –co ec ed (FDR) p- alue < 0.05 and log2| old
change| ≥2(Neupa h e al., 2020, Annexes 4 o 7). O hese, 1482 and
2017 genes we e al e ed in F0 and F3, espec i ely (Fig. 3C and
Neupa h e al., 2020, Annex 3 and Annex 11). Hie a chical clus e ing
hea map (Fig. 3A and Neupa h e al., Annex 9) e ealed a dis inc
pa e n o gene ansc ip ion be ween con ol and 320E o 320T, in
bo h F0 and F3, which is consis en wi h he mul idimensional scaling
analysis (Fig. 3B and Neupa h e al., 2020, Annex 10) ha g ouped
con ol and exposed emales in diffe en axes. The Venn diag am co -
obo a es his pa e n iden i ying o e 800 DEGs common o bo h F0
and F3 (Fig. 3C and Neupa h e al., 2020, Annex 11).
We u he in es iga ed he mos p ominen o e ep esen ed cano-
nical pa hways using KEGG Mappe – econs uc pa hway. Twen y ou
pa hways con ained DEGs in F3 gene a ion (320T e sus con ol):
me abolism o e penoids and polyke ides (me alona e pa hway/ses-
qui e p enoid biosyn hesis and deg ada ion, ecdys e oid biosyn hesis),
ca bohyd a e me abolism, lipid me abolism, ene gy me abolism, amino
acid me abolism, glycan biosyn hesis and me abolism, me abolism o
co ac o s and i amins, xenobio ics biodeg ada ion and me abolism,
ansc ip ion, olding so ing and deg ada ion, signal ansduc ion,
signaling molecules and in e ac ion, anspo and ca abolism, cell
g ow h and dead, cell mo ili y, immune sys em, endoc ine sys em, di-
ges i e sys em, ci cula o y sys em, ne ous sys em, senso y sys em,
de elopmen and egene a ion, aging and en i onmen al adap a ion
(Fig. 4 and Neupa h e al., 2020, Annex 7 and Annex 15). Impo an ly,
all F3 DEGs ound in hese pa hways, be ween 320T and con ol, we e
also diffe en ially exp essed in F0 emales exposed o SIM (320E)
(Neupa h e al., 2020, Annexes 4, 8 and 15). We nex e alua ed
whe he impai ed MF le els and ep oduc ion caused by exposu e o
SIM in F0 we e associa ed wi h shi s in he ansc ip ional p ofiles o
me alona e biosyn hesis modules, as well as he downs eam sesqui-
e penoid biosyn hesis and deg ada ion and/o he ecdys e oid bio-
syn hesis in F3 (Fig. 4A and Neupa h e al., 2020, Annex 16). Th ee
genes we e diffe en ially exp essed in F3 320T in compa ison wi h
con ol emales: me hyl a nesoa e epoxidase (MFE), ju enile ho mone
epoxide hyd olase (JHEH) and CYP-2L1 (Fig. 4A). In e es ingly JHEH
ha ca alyzes an impo an s ep o sesqui e penoid deg ada ion is
modula ed in F0 and F3 (Fig. 4A). In insec s, JHEH is in ol ed in he
egula ion o ju enile ho mone (JH) i e and me amo phosis/mol (Sin
e al., 2015). Al hough JH has no been iden ified in non-insec a -
h opods, se e al gene o hologues in ol ed in JH p oduc ion and de-
g ada ion, as JHEH, p e iously hough o be insec -specific, we e
iden ified in wa e flea, sh imp and cen ipede (Gui a d e al., 2011;Lee
e al., 2011;Sin e al., 2015). In c us aceans, simila ly o insec s, he
deg ada ion o MF appea s o occu h ough es e hyd olysis by specific
ca boxyles e ases (Lee e al., 2011). The e o e, as c us aceans lack JH,
he po en ial subs a e o he hyd olysis eac ion o JHEH could be he
MF (Lee e al., 2011). The CYP-2L1, a s e oidogenic enzyme om he
CYP clan 2, was also ound o be diffe en ially exp essed in bo h F0 and
F3 in he ecdys e oid biosyn hesis pa hway. Al hough his enzyme has
no been unc ionally cha ac e ized in c us aceans, he unc ion o
CYP18A1, belonging also o he CYP clan 2, has been cha ac e ized in
D osophila (Gui a d e al., 2011), displaying a cen al ole in he
main enance o ecdys e oids wi hin physiological le els, egula ing
Fig. 3. T ansc ip omic analysis. (A) Hie a chical clus e ing hea map depic ing he pa e ns o gene ansc ip ion be ween F0 con ol (F0.C.R), 320E (F0.320E.R), F3
con ol (F3.C.R) and 320T (F3.320T.R), n = 3 pe ea men ; p- alue < 0.05 and old change ≥2. (B) mul idimensional scaling analysis be ween F0 and F3 (C)
Venn diag am o he o e lapping significan ly (p < 0.05) modula ed common genes o F0 and F3.
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
6
ecdys e oids inac i a ion, hus affec ing mol ing and me amo phosis
(Gui a d e al., 2011). Addi ionally, he ecdysone inducible gene (E75),
a p ima y a ge o ecdysone ecep o wi h a c i ical ole in he mol ing
p ocess o a h opods (P iya e al., 2010), by ac i a ing he deg ada ion
o old cu icle and he o ma ion o a new cu icle, was ound o be down-
egula ed in he F3 (Neupa h e al., 2020, Annex 7). In amphipods,
emales’ ep oduc i e cycles a e co ela ed wi h he mol cycle and
ep oduc ion is p eceded by a mol (Hyne, 2011). The mol cycle is
egula ed by se e al ho mones (including MF), unde di ec con ol o
he s e oid ho mone, 20-hyd oxyecdysone, he pu a i e subs a e o
CYP-2L1. Thus, dis up ion o CYP-2L1 ansc ip ion could also affec
his p ocess.
The ansc ip ion o genes coding o o he enzymes, i.e., phos-
pho uc okinase (PFK), chi inase (CHIT1) and elonga ion o e y long
Fig. 4. Schema ic ep esen a ion o selec ed me abolic pa hways diffe en ially exp essed in emales om F0 (320E e sus con ol) and F3 (320T e sus con ol). (A)
Ca bohyd a e me abolism (Glycolysis/gluconeogenesis, Pen ose phospha e pa hway, Pen ose and gluco ona e in e con e sions, F uc ose and mannose me abolism,
Galac ose me abolism and Amino suga and nucleo ide suga me abolism), Me abolism o e penoids and polyke ides (Me alona e pa hway/Sesqui e penoid bio-
syn hesis and deg ada ion, and Ecdys e oid biosyn hesis). (B) Lipid me abolism (Biosyn hesis o unsa u a ed a y acids). The genes modula ed by SIM acco ding o
KEGG anno a ion a e ep esen ed in ec angles.
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
7
chain a y acids p o ein 7 (ELOVL 7), wi h a key unc ion in ca bohy-
d a e and lipid me abolism, we e ound o be dis up ed in he F3 be-
ween 320T and con ol (Fig. 4A and B and Neupa h e al., 2020,
Annex 7). PFK, ELOVL 7 and se e al o he s genes we e also impac ed in
he F0 (320E e sus con ol) (Fig. 4A and B). The findings o changes on
ca bohyd a e and lipid me abolism in eg a e well wi h ou p e ious
his ological obse a ion (Neupa h e al., 2014): emales’li e-cycle ex-
posed o SIM concen a ions anging om 320 o 8000 ng/L yielded
educed glycogen and lipid s o age. In e es ingly, a ecen s udy wi h
sho - e m exposu e o mussel (My ilus edulis) o a o as a in a
1200 ng/L, led o deple ion o lipids and ca bohyd a es (Fal ushynska
e al., 2019). The e o e, ep oduc i e impai men and/o g ow h may
also be linked o me abolic dys unc ion.
Addi ional pa hways modula ed by SIM ela e o he cu icle me a-
bolism. The ansc ip ion o wen y genes we e down- egula ed in F3
(Neupa h e al., 2020, Annex 12). In e es ingly, mos o hese genes
al e ed in F3 we e also down- egula ed in F0 (Neupa h e al., 2020,
Annex 12). This diffe en ial gene exp ession p ofile sugges s ha SIM
ansgene a ional exposu e modula es exoskele on main enance and
mol ing. Since he cu icle deg ada ion p ocess is closely linked o he
mol cycle and synch onized wi h amphipod’s g ow h and ep oduc ion
(T app e al., 2014), he diffe en ial exp ession o genes coding o
cu icle p o eins could also be linked o he obse ed effec s on g ow h
and ep oduc ion o he F3 exposed and ansgene a ional g oups
(Fig. 2).
SIM exposu e also induced he ansc ip ion modula ion o se e al
p ocesses in ol ed in epigene ic egula ion, no ably, his one pos -
ansla ional modifica ions such as me hyla ion, ace yla ion, c o ony-
la ion and sumonyla ion, me hyla ion index modifica ion and nucleo-
some assembly (Table 1 and Neupa h e al., Annex 13). In e es ingly,
he p o ein FAM98A (FAM98A) and YEATS domain-con aining p o ein
2-like (YEATS2) we e down- egula ed in 320T emales om F0, F1 and
F3 gene a ions (Table 1 and Neupa h e al., 2020, Annex 13). Since
hese pa hways a e implica ed in epigene ic modifica ions, hei mod-
ula ion may be associa ed wi h he obse ed ansgene a ional
dis up ion o he ep oduc ion and g ow h in F3 (A is izaba e al.,
2019). P e ious s udies wi h mammalian cells demons a ed ha SIM
modula es DNA me hyla ion, his one modifica ions and miRNAs (Ka lic
e al., 2015), hus suppo ing he ansc ip omic esponses obse ed
he e. Gi en he complex ne wo k o pa hways in ol ed in s a in-in-
duced epigene ic modifica ions (Ka lic e al., 2015), u u e expe imen s
a e needed o un a el he molecula mechanisms unde lying SIM
modula ion o epigene ic ma ke s and how hese ela e wi h he ob-
se ed finge p in be ween F0 and F3.
3.3. Implica ions o haza d and isk assessmen
O e all, his s udy p o ides he fi s e idence ha one o he mos
p esc ibed pha maceu icals in wes e n Eu opean coun ies, a en-
i onmen al ele an concen a ions, induces ansgene a ional effec s
in he ep oduc ion o a keys one ma ine amphipod species. This is e y
ele an , since he effec s o he SIM exposu e expe ienced by G. locus a
du ing i s li e ime in he pa en al gene a ion (F0E) we e uly inhe i ed
o non-exposed gene a ions (F3T) and we e no a simply esul o di ec
exposu e. These esul s ha e b oad implica ions o ou unde s anding
o he haza d o ansgene a ional impai men and how en i onmen al
pollu an s may con ibu e o such dis up ion. The cu en chemical isk
assessmen pa adigm includes s eps o haza d iden ifica ion, dose- e-
sponse assessmen , exposu e assessmen , and isk cha ac e iza ion
(Shaw e al., 2017). A ailable es guidelines al eady add ess mul i-
gene a ional effec s, bu , he po en ial ansgene a ional effec s o en-
i onmen al pollu an s a e s ill no in eg a ed in haza d and isk as-
sessmen amewo ks. Thus, he esul s epo ed he e highligh he
impo ance o in eg a ing chemical-induced ansgene a ional effec s in
oxici y es ing s a egies and isk assessmen .
CRediT au ho ship con ibu ion s a emen
T. Neupa h: Concep ualiza ion, Me hodology, Valida ion, Fo mal
analysis, In es iga ion. :Resou ces, Da a cu a ion, W i ing - o iginal
Fig. 4. (con inued)
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
8
d a , W i ing - e iew & edi ing, Supe ision, P ojec adminis a ion.
A.M. Machado: Me hodology, Valida ion, Fo mal analysis,
In es iga ion. :Da a cu a ion, W i ing - e iew & edi ing. R. Mon es:
Me hodology, Fo mal analysis, In es iga ion, Da a cu a ion. R. Rodil:
Fo mal analysis, In es iga ion, Da a cu a ion. S. Ba os: Fo mal ana-
lysis, In es iga ion, Da a cu a ion. N. Al es: Fo mal analysis,
In es iga ion, Da a cu a ion. R. Rui o: Fo mal analysis, In es iga ion,
Da a cu a ion, W i ing - e iew & edi ing. L. Filipe C. Cas o:
Valida ion, Fo mal analysis, In es iga ion, Resou ces, Da a cu a ion,
W i ing - e iew & edi ing. J.B. Quin ana: Fo mal analysis,
In es iga ion, Da a cu a ion, W i ing - e iew & edi ing, Funding ac-
quisi ion. M.M. San os: Concep ualiza ion, Me hodology, Valida ion,
Fo mal analysis, In es iga ion, Resou ces, Da a cu a ion, W i ing -
o iginal d a , W i ing - e iew & edi ing, Supe ision, P ojec admin-
is a ion, Funding acquisi ion.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing financial
in e es s o pe sonal ela ionships ha could ha e appea ed o influ-
ence he wo k epo ed in his pape .
Acknowledgmen s
This esea ch was unded by COMPETE 2020, Po ugal 2020, he
Eu opean Union h ough he ERDF and he Po uguese Founda ion o
Science and Technology –FCT (T ansobesogen p ojec –T ans-phyle ic
obesogenic esponses: om epigene ic modules o ansgene a ional
en i onmen al impac s, e e ence: PTDC/CTA-AMB/31544/2017 –
NORTE-01-0145-FEDER-031544). This esea ch was also suppo ed by
he Na ional Funds h ough FCT unde he p ojec s (UIDB/04423/2020;
UIDP/04423/2020), by he Spanish Agencia Es a al de In es igación
(CTM2017-84763-C3-2-R), and by he Galician Council o Cul u e,
Educa ion and Uni e si ies (ED431C2017/36), co ounded by ERDF. A
PhD g an awa ded o Susana Ba os (PD/BD/143090/2018) was
unded by he FCT.
Da a a ailabili y
All e sions o he ansc ip ome we e submi ed o he online
Figsha e eposi o y and can be consul ed in he ollowing link: h ps://
figsha e.com/s/1110e0d14 cc6a275acb (a ailable i eques ed). All he
clean ead da ase s we e submi ed o Sequence Read A chi e (SRA)
da abase o NCBI, and can be consul ed unde he Bio p ojec numbe
PRJNA600472.
Re e ences
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Table 1
O e iew o he genes in ol ed in epigene ic egula ion al e ed in F0 o F3.
F0/F3 indica es genes common o bo h F0 and F3; Red indica es up egula ion, blue indica es down egula ion; (-) indica es no changes in gene exp ession; DDB –
pu a i e me hyl ans e ase DDB; FAM98A –p o ein FAM98A; YEATS2 –YEATS domain-con aining p o ein 2-like; PRMT1 –p o ein a ginine N-me hyl ans e ase 1-
like; NAA40 –N-alpha-ace yl ans e ase 40-like iso o m X1; AHCY –adenosylhomocys einase; INO80C –INO80 complex subuni C; HN1N2 –his one-binding p o ein
N1/N2-like iso o m X1; SAE1 –SUMO-ac i a ing enzyme subuni 1-like; SAM –S-adenosyl me hionine; SAH –S-adenosyl homocys eine; HCY –homocys eine; Ado –
adenosine.
T. Neupa h, e al. En i onmen In e na ional 144 (2020) 106020
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