The loss of hemoglobin and myoglobin does not minimize oxidative stress in Antarctic icefishes

52 Pages·2017·1.58 MB·English

©2018.PublishedbyTheCompanyofBiologistsLtd|JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
RESEARCHARTICLE
The loss of hemoglobin and myoglobin does not minimize
oxidative stress in Antarctic icefishes
KristinM.O’Brien1,*,ElizabethL.Crockett2,JacquesPhilip3,CoreyA.Oldham1,MeganHoffman1,
DonaldE.Kuhn2,RonaldBarry4andJessicaMcLaughlin1
ABSTRACT totemperature,andthelowtemperatureoftheSouthernOcean,in
conjunction with substantial vertical mixing, results in highly
Theunusualpatternofexpressionofhemoglobin(Hb)andmyoglobin
oxygenatedwater(Eastman,1993).Second,inAntarcticfishes,the
(Mb) among Antarctic notothenioid fishes provides an exceptional
tissueswithparticularlyhighenergydemands,includingoxidative
modelsystemforassessingtheimpactoftheseproteinsonoxidative
skeletal muscle and cardiac ventricles, are endowed with a large
stress. We tested the hypothesis that the lack of oxygen-binding
complementofmitochondria(Johnstonetal.,1998;Londravilleand
proteins may reduce oxidative stress. Levels and activity of pro-
Sidell,1990;O’Brien andSidell,2000), theorganelle responsible
oxidantsandsmall-moleculeandenzymaticantioxidants,andlevels
for the vast majority of ROS production in eukaryotic cells
ofoxidizedlipidsandproteinsintheliver,oxidativeskeletalmuscle
(Andreyev et al., 2005; Murphy, 2009). Third, biological
and heart ventricle were quantified in five species of notothenioid
membranes of Antarctic fishes are enriched with phospholipids
fishes differing in the expression of Hb and Mb. Levels of
withahighdegreeofunsaturation(i.e.polyunsaturatedfattyacidsor
ubiquitinated proteins and rates of protein degradation by the 20S
PUFA)(Logueetal.,2000;O’BrienandMueller,2010).Because
proteasomewerealsoquantified.Althoughlevelsofoxidizedproteins
fatty acids with high numbers of double bonds are particularly
andlipids,ubiquitinatedproteins,andantioxidantswerehigherinred-
susceptible to oxidation by ROS (Cosgrove et al., 1987), the
bloodedfishesthaninHb-lessicefishesinsometissues,thispattern
membranesofAntarcticfishesmaybeespeciallyvulnerabletolipid
did not persist across all tissues. Expression of Mb was not
peroxidation (Crockett, 2008). Finally, Antarctic notothenioids
associated with oxidative damage in the heart ventricle, whereas
possess a unique twist in the typical expression of Hb among
the activity of citrate synthase and the contents of heme were
vertebrates.ThefamilyChannichthyidae(icefishes)ischaracterized
positivelycorrelatedwithoxidativedamageinmosttissues.Despite
bytheabsenceofHb,andsixofthe16speciesoficefishesalsolack
sometissuedifferencesinlevelsofproteincarbonylsamongspecies,
expressionofMbintheheartventricle(MoylanandSidell,2000;
rates of degradation by the 20S proteasome were not markedly
Sidell et al., 1997). In addition, oxidative skeletal muscle in all
different, suggesting either alternative pathways for eliminating
species of Antarctic notothenioids is devoid of Mb (Moylan and
oxidized proteins or that redox tone varies among species.
Sidell,2000).
Together, our data indicate that the loss of Hb and Mb does not
One of our earlier studies indicated that the susceptibility of
correspondwithaclearpatternofeitherreducedoxidativedefenseor
proteins and lipids to oxidation may, in fact, be related to the
oxidativedamage.
expression of Hb and Mb in notothenioid fishes. We found
KEYWORDS:Antarcticfish,Antioxidants,Oxygen-bindingproteins, significantly lower levels of secondary products of lipid
Oxidativedamage peroxidation in the oxidative muscle of two icefishes (−Hb),
ChionodracorastrospinosusandChaenocephalusaceratus,thanin
INTRODUCTION those of the red-blooded (+Hb) species Notothenia coriiceps
Antarcticnotothenioidfishesprovideanaturalexperimentalmodel (Mueller et al., 2012). Furthermore, levels of oxidized proteins
in which questions pertaining to the risks and prevention of were lower in the heart of the −Mb icefish C. aceratus compared
oxidativestressatverylowtemperaturescanbeprobed.Alifecycle withthe+MbheartofC.rastrospinosus,andwerethehighestinthe
spent entirely at very low body temperatures may, at first glance, heartofN.coriiceps(Muelleretal.,2012).Inaddition,we(Mueller
y
suggestameanstoavoidoxidativeinjuryastheratesofproduction etal.,2012)andothers(Cassinietal.,1993;Witasetal.,1984)have g
o
ofreactiveoxygenspecies(ROS)aresignificantlyretardedatcold foundthatthecapacitiesforcentralantioxidantsinselectedtissues l
o
temperature (Abele et al., 2002). Given their habitat and their arelowerinwhite-blooded(−Hb)thaninred-blooded(+Hb)fishes, i
B
cellulararchitecture,however,theseanimalsmaybepredisposedto suggesting that the loss of Hb and Mb may permit reduced
l
a
an imbalance of ROS production and ROS degradation (i.e. antioxidantdefenses.
t
oxidative stress). First, oxygen solubility is inversely proportional The mechanism(s) responsible for elevated levels of oxidized n
e
proteinsandlipidsinred-bloodedandred-heartednotothenioidsare m
1InstituteofArcticBiology,UniversityofAlaska,Fairbanks,Alaska,99775,USA. unclear. As iron (Fe)-centered proteins, Hb and Mb may facilitate eri
2DepartmentofBiologicalSciences,OhioUniversity,Athens,Ohio,45701,USA. the production of ROS (King et al., 1964; Tappel, 1955). Any p
3CenterforAlaskaNativeHealthResearch,UniversityofAlaska,Fairbanks,Alaska, unbound (i.e. excess) Fe may promote the formation of hydroxyl x
99775,USA.4DepartmentofMathematicsandStatistics,UniversityofAlaska, radicals ((cid:129)OH) via the Fenton reaction (Halliwell and Gutteridge, E
Fairbanks,Alaska,99775,USA. f
1986),andtheseradicalsaresufficientlypotentthattheycanoxidize o
*Authorforcorrespondence([email protected]) biologicalmoleculesincludingproteinsandthelipidsthatconstitute al
n
the matrices of biological membranes (Dikalov, 2011; Lee et al.,
K.M.O.,0000-0002-3311-0690 r
2012). Autoxidation of Hb [with Fe in the ferrous (Fe2+) state] to u
o
Received8May2017;Accepted11December2017 methemoglobin (metHb) with Fein theferric (Fe3+) state leadsto J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
proteasome mayplayamoreprominent role in the degradation of
Listofabbreviations oxidizedproteins(Reinheckeletal.,2000).
CAT catalase We took a comprehensive look at relationships among pro-
CS citratesynthase oxidants, oxidative damage, antioxidant defenses, and protein
ETC electrontransportchain ubiquitination and degradation in both white- and red-blooded
GPx1 glutathioneperoxidase1
Antarctic notothenioids in order to test the hypothesis that loss of
GPx4 glutathioneperoxidase4
circulating Hb, and of Mb in the cardiac muscle of these fishes is
GSH reducedglutathione
accompanied by lower levels of oxidized proteins and lipids, a
GSSG oxidizedglutathione
GST glutathioneS-transferase reducedarsenalofantioxidantsandlowerratesofproteindegradation.
Hb hemoglobin
Mb myoglobin MATERIALSANDMETHODS
PUFA polyunsaturatedfattyacid
Animalcollection
ROS reactiveoxygenspecies
The following species were used for this study [animal masses
SOD superoxidedismutase
(means±s.e.m.) are shown in parentheses]: Chaenocephalus
TAP totalantioxidantpotential
aceratus (Lönnberg 1906) (1675±169g; n=18),
Champsocephalus gunnari Lönnberg 1905 (581±36g; n=16),
Pseudochaenichthys georgianus Norman 1937 (1528±228g;
theproductionofsuperoxide(O(cid:129)−)andhydrogenperoxide(H O ) n=8), Chionodraco rastrospinosus DeWitt & Hureau 1979 (349±
2 2 2
whenmetHbreactswithO (Winterbourn,1990).HemeintheFe3+ 30g; n=14), Gobionotothen gibberifrons (Lönnberg 1905)
2
state also reacts with H O to form two oxidants, ferryl iron (Fe4 (formerly Notothenia gibberifrons; 860±38g; n=33) and
2 2
+=O−)andaprotein-boundfreeradicalontheglobin(Reederand Notothenia coriiceps Richardson 1844 (1847±94g; n=18).
2
Wilson, 2005). However, most cells, particularly red blood cells, Animals were collected off the southwestern shore of Low Island
have an abundance of low molecular weight antioxidants and (63°25′S, 62°10′W) and in Dallmann Bay (64°10′S, 62°35′W)
antioxidant enzymes that detoxify ROS and maintain iron in the using a benthic otter trawl and fish pots between April and June,
ferrous state (Cohen and Hochstein, 1963; Johnson et al., 2005; 2011 and 2013. Animals collected in 2013 were used for
Lopez-Torres et al., 1993; Low et al., 2008; Perez-Campo et al., quantifying levels of ubiquitinated proteinsand the activityof the
1993;vanZwietenetal.,2014).Thecentralantioxidantssuperoxide 20S proteasome. All other measurements were completed using
dismutase (SOD) and catalase (CAT) are able to detoxify two animalscollectedin2011.Fishwereheldinrecirculatingseawater
common ROS, O (cid:129)− and H O , respectively (Finkel and tanks on board the US ARSV Laurence M. Gould and then
2 2 2
Holbrook, 2000). H O may also be detoxified by several transferred to the aquarium at the US Antarctic Research Station,
2 2
antioxidant enzymes that require reduced glutathione (GSH), Palmer Station, where they were held in tanks with recirculating
including the glutathione peroxidases (GPx), with glutathione seawaterat0.1±0.5°C.Animalswerekilledwithasharpblowtothe
reductase (GR) having the important role of recycling the GSH head followed by cervical transection. Samples of the heart
(Brigelius-Flohé, 1999). Another family of enzymes, the ventricle,pectoraladductormuscleandliverwereharvested,flash
glutathione S-transferases (GST), also depends on GSH for the frozen in liquid nitrogen and stored at −80°C. Icefishes and G.
catalytic conjugation of a variety of potentially deleterious gibberifronswereharvestedwithin2weeksofcapture.Notothenia
electrophilic compounds and xenobiotics prior to excretion coriicepswerefedadietoffishmuscleapproximatelyevery3days
(Hayes and Pulford, 1995). Additionally, any extracellular Hb and were harvested within approximately 3weeks of capture. All
shouldbeboundbyhaptoglobin(Alayashetal.,2013),andfree protocols were approved by the University of Alaska Fairbanks
heme by hemopexin (Smith and McCulloh, 2015), minimizing InstitutionalCareandUseCommittee(1374774-2).
Fe-mediated ROS formation. An alternative explanation for the
highlevelsofoxidizedmacromoleculesinred-bloodedfishesis Hematocrit,hemoglobinandhemequantification
that they are caused by high rates of aerobic metabolism, Hematocrit (Hct) measurements were obtained by drawing blood
supportedby theexpressionofoxygen-bindingproteins, which intoheparinizedcapillarytubesandcentrifugingfor5mininanHct
can enhance ROS formation, rather than by Fe-mediated ROS centrifuge.Eachindividualwasmeasuredintriplicate.
formationviaHbandMb. Whole bloodwasmixedwith3.2%sodiumcitrate(9:1 forred-
y
Lowerlevelsofoxidizedproteinsinicefishescomparedwithred- blooded species and 4:1 for icefishes) to prevent clotting. Hb g
bloodedspeciesmayreduceratesofproteindegradation.Oxidized concentration was determined by mixing 20μl of citrated blood lo
o
proteinsaremarkedfordegradationbytheATP-dependentcovalent with5mlDrabkin’sreagent(Sigma-Aldrich,St.Louis,MO,USA), i
B
conjugation of ubiquitin (Dudek et al., 2005; Shang et al., 1997). incubating the mixture at room temperature for 30min and
l
a
Ubiquitinated proteins are degraded by the 26S proteasome in measuring absorbance at 540nm. Samples and a standard curve
t
anotherATP-dependentprocess(Davies,2001;Shringarpureetal., with concentrations between 0 and 180mgml−1 of Hb were n
e
2003). Oxidized proteins may also be degraded by the 20S measuredintriplicate. m
proteasomeinaubiquitin- andATP-independentprocess(Davies, Heme concentration in blood diluted with sodium citrate (as ri
e
2001; Reinheckel et al., 2000). Although both the 26S and 20S described above) was determined using the Quantichrome Heme
p
proteasome degrade oxidatively modified proteins (Shang and Assay Kit according to the manufacturer’s instructions (BioAssay x
E
Taylor,2011), the20Sproteasome doesnotrequireproteinstobe Systems, Hayward, CA, USA). Samples were measured in
f
ubiquitinated to degrade them and can, therefore, degrade duplicate. o
oxidativelydamagedproteinsincellswithcompromisedubiquitin al
n
conjugation systems (Davies, 2001; Shringarpure et al., 2003). Myoglobinquantification r
Additionally,the26Sproteasome ismoresusceptible tooxidative Heart ventricles were homogenized (10% w/v) in 20mmoll−1 u
o
damage than the 20S proteasome, suggesting that the 20S Hepes buffer (pH 7.8 at 4°C) using a Tenbroeck ground-glass J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
homogenizer(Wheaton,Millville,NJ,USA)onice.Homogenates USA).ProteinswereseparatedusingTSKgelGuardSW andQC–
XL
were centrifuged at 10,000g for 10min at 4°C, the supernatant PAKGFC200columns(TOSOHBioscience,KingofPrussia,PA,
collected and protein concentration determined using the USA)andarunningbufferof6moll−1guanidiniumhydrochloride,
bicinchoninic acid assay (Sigma-Aldrich) with bovine serum pH2.5.SpectrawereanalyzedwithWatersEmpowerProAnalysis
albumin(BSA)asastandard(Smithetal.,1985).Thesupernatant software(WatersCorporation).Fromtheaconitasestandard,100μg
wasmixedwithloadingbuffer(150mmoll−1Tris–HCl,4%SDS, wasresuspendedtoafinalconcentrationof300ngproteinμl−1in
12% glycerol, 100mmoll−1 dithiothreitol, 0.01% Bromophenol 50mmoll−1KH PO (pH7.8);300μlofthestandardwasmixed
2 4
Blue, pH 6.8) and heated at 98°C for 10min. Proteins were with 70μl of 10% streptomycin sulfate and incubated at room
separatedon12%Tris-Tricinegelsasdescribedpreviously(Moylan temperaturefor15mintoprecipitateDNA.Thesampleswerethen
and Sidell, 2000). Each gel included a standard curve of known centrifuged at 10,000g for 10min at 4°C, the supernatant was
amounts of purified Mb from N. coriiceps. Proteins from each collected and 1200μl of ice-cold 100% acetone was added. The
individual were run in triplicate on two separate gels. Gels were sampleswerethenvortexedandincubatedfor30minat−20°Cto
stainedin0.10%(w/v)CoomassieBrilliantBlueR-250stain(50% precipitateprotein.Sampleswerecentrifugedat16,000gfor15min
methanol, 10% acetic acid), destained in 5% methanol and 7% at4°Candrinsedwithice-cold80%acetone.Thepelletwasrinsed
aceticacidandscannedwithanImageScanner(GEHealthcareBio- inacetoneandcentrifugedat16,000gfor15minat4°C.Acetone
Sciences, Pittsburgh, PA, USA). Images were quantified using wasdiscardedandthepelletwasresuspendedin100μlofrunning
ImageQuant(GEHealthcareBio-Sciences)andtheconcentrationof buffer.Theaconitasestandardwasdividedintofour25μlaliquots.
Mbwascalculatedfromthestandardcurve. Twoaliquotspersamplewereincubatedwith35μlof10mmoll−1
2,4-dinitrophenylhydrazine (DNPH) in running buffer and two in
Citratesynthaseactivity runningbufferalone.Absorptionwasmeasuredat280nmfortotal
Themaximalactivityofcitratesynthase(EC2.3.3.1)wasmeasured protein and 366nm for the carbonyl hydrozone. Carbonyl
at5°C±0.5°CusingaLambda25spectrophotometer(PerkinElmer, concentration was determined using the equations described in
Waltham,MA,USA)andamodificationoftheprotocoldescribed Levineetal.(2000).
bySrereetal.(1963).Frozentissueswerefinelychoppedonanice- Tissueswerehomogenizedin50mmoll−1KH PO pH7.8and
2 4
coldstageandthenhomogenizedin19volumesofice-coldbuffer centrifuged at 4°C for 10min at 9000g. Bradford assays, as
(75mmoll−1 Tris-HCl, 1mmoll−1 EDTA, 2mmoll−1 MgCl , describedabove,wereusedtodeterminetheproteinconcentration
2
pH 8.2 at 5°C) using Tenbroeck ground-glass homogenizers. The ofthesupernatant;50μgofproteinwasbroughttoafinalvolumeof
final reaction mixture contained 0.25mmoll−1 5,5′-dithiobis-2- 100μlwithMQH Oanddriedinavacuumcentrifuge(CentriVap
2
nitrobenzoic acid (DTNB), 0.40mmoll−1 acetyl coenzyme A Concentrator,LabconcoCorporation,KansasCity,MO,USA)for
(CoA), 0.5mmoll−1 oxaloacetate,75mmoll−1Tris-HCl, pH8.2, 2hatroomtemperature.Samplesand100μgofaconitasestandard
with0.025%tissue.Backgroundactivitywasmeasuredfor5minin were resuspended to a final concentration of 300ngμl−1 in a
theabsenceoftheinitiatingsubstrateoxaloacetate.Theprogressof derivitization solution consisting of 20mmoll−1 DNPH, 0.5%
thereactionwasmonitoredbyfollowingthereductionofDTNBat trifluoroaceticacid(TFA)and 92.5%dimethylsulfoxide(DMSO).
412nmfor5minfollowingtheadditionofoxaloacetate.Activityis Sampleswerevortexed atroomtemperaturefor45–60min before
expressedasµmolproductmin−1g−1wetmass. being spotted onto a PVDF membrane. A standard curve of
aconitase containing 0–300ngμl−1 protein was loaded onto each
Proteincarbonylation membraneintriplicate.Thesamplesandaconitaseweredilutedin
Proteincarbonylationwasquantifiedusinganimmunochemicaldot derivitizationsolutionfollowedbyphosphate-bufferedsaline(PBS)
blot method as described by Wehr and Levine (2012). A to a minimum volume of 200μl. The final concentration of each
carbonylated aconitase standard was prepared by resuspending sample and standard was determined using a Bradford assay as
aconitase(Sigma-Aldrich)atafinalconcentrationof5mgml−1in described above but with BSA resuspended in derivitization
buffer (10mmoll−1 MgCl , 50mmoll−1 Hepes, 100mmoll−1 solutionasthestandard. Allsampleswereprepared andloaded in
2
KCl, pH 7.2) and dialyzed against 25mmoll−1 ascorbate and triplicate.
0.1mmoll−1 FeCl for 24h at room temperature using a Pur-A- PVDF membranes (GE Healthcare Bio-Sciences) were wetted
3
Lyzer Maxi 12000 Dialysis Kit (Sigma-Aldrich) as described by using 100% methanol and soaked for at least 10min in transfer
RivettandLevine(1990).Ondaytwo,thebufferwasreplacedwith buffer (25mmoll−1 Tris, 192mmoll−1 glycine, 0.1% SDS, 20%
y
onecontaining1mmoll−1EDTAtostopthecarbonylationreaction MeOH)beforeloadingsamplesusingavacuum-drawnslotblotter g
andthen placedat4°C withstirring.Ondaythree,thebufferwas (Bio-DotSFCell,Bio-Rad,Hercules,CA,USA).Atotalof200μl lo
o
replaced with the original dialysis buffer to remove EDTA. All oftransferbufferwaspipettedintoeachslotanddrawnacrossthe i
B
bufferswerechangedthreetimesdaily.Proteinconcentrationofthe membraneusingavacuum,followedbysamples,andthenanother
l
aconitase carbonyl standard was determined using the Bradford 200μloftransferbuffer.Blotsweredriedfor15min,washedwith a
t
Assay with BSA asthe standard (Bradford, 1976). Absorbance at glacialaceticacid,twicefor2minwith100%aceticacid,andonce n
e
595nm was measured using a Spectramax Plus 384 microplate for2minin5mlaceticacidwithasufficientamountofMQH O m
2
reader (Molecular Devices, Sunnyvale, CA, USA). The aconitase added to cover the membrane (approximately 60–70ml total ri
standard was then divided into 50μg aliquots, brought to a final volume),andthenoncefor5mininMQH O,driedandthenstored e
2 p
volume of 100μl with MQ H O, dried in a vacuum centrifuge at−80°Cforupto2daysbeforedeveloping. x
2 E
(CentriVapConcentrator,LabconcoCorporation,KansasCity,MO, Membraneswereblockedwith5%non-fatdrymilkin0.1%PBS-
f
USA)atroomtemperaturefor2handstoredat−80°Cuntiluse. Tweenfor1handthenwashedwith0.1%PBS-Tween(twicequick, o
Levels of protein carbonylation in the aconitase standard were oncefor15min,andtwicefor5min).Membraneswereincubated al
n
determinedusinghighperformanceliquidchromatography(HPLC) with the primary antibody goat anti-DNPH (Bethyl Laboratories,
r
withaWaters1525BinaryHPLCequippedwithanin-linedegasser Montgomery,TX,USA),diluted1:5000in5%non-fatdrymilkin u
o
and photodiode arraydetector(Waters Corporation, Milford,MA, 0.1% PBS-Tween for 2h, and then washed as described above. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
Membranes were then incubated with the secondary antibody of CAT (EC 1.11.1.6) activity was measured at 5°C using a
donkeyanti-goatHRP(SC2020;SantaCruzBiotechnology,Dallas, Beckman DU-640 spectrophotometer (Brea, CA, USA) according
TX,USA),diluted1:10,000in5%non-fatdrymilkin0.1%PBS- tothemethodofBeersandSizer(1952).
Tween for 1h and washed as described above. Membranes were Glutathione reductase (EC 1.8.1.7) activities in tissue
developedusingAmershamECLPrimeWesternBlottingDetection supernatants were measured at 5°C using a slightly modified
Reagent(GEHealthcareBio-Sciences),viewedusingAlphaImager versionofaprotocoldescribedbySigma-Aldrich(GRSATechnical
3300 Imaging System (Protein Simple, San Jose, CA, USA) and Bulletin).Tissueswerehomogenizedinassaybuffer(125mmoll−1
analyzedwithImageQuantTL(GEHealthcareBio-Sciences). potassium phosphate, 1.25mmoll−1 EDTA, pH 7.5) using a
BulletBlenderStorm24fromNextAdvance(Averill,NY,USA)ata
Lipidperoxidation 2:1 (w/w) bead to tissue ratio. The resulting homogenates were
Tissue homogenates were used to measure malondialdehyde, a centrifuged at 21,000g for 15min. Supernatants were collected
thiobarbituric acid-reactive substance (TBARS). Tissues were and, if not used immediately, stored at –70°C. The assay was
processed and analyzed according to the method of Mihara and performed in assay buffer at a final volume of 200µl containing
Uchiyama(1978)andasdescribedpreviouslybyourgroup(Mueller 0.15mmoll−1 DTNB, 1.5mmoll−1 GSSG and 0.215mmoll−1
et al., 2012). For the purpose of normalizing malondialdehyde to NADPH.Tenmicrolitersoftissuesupernatantswereusedforeach
lipid content, total lipids were determined gravimetrically. Lipids sample. Reactions were initiated by the addition of NADPH and
were extracted from tissues using methanol:chloroform:water as absorbance values were recorded every 2min overa 20min time
describedbyBlighandDyer(1959).Afterlipidswereextracted,a period.SamplesrunintheabsenceofGSSGservedascontrols.
fivedecimalplacebalance(MettlerH54AR,Columbus,OH,USA) Enzymaticactivitiesofbothglutathioneperoxidase(EC1.11.1.9)
wasusedtoquantifythefinallipidmassofeachsample. and GST (EC 2.5.1.18) were quantified as previously described
(Grim et al., 2013), with the following exceptions: (1) all assays
Glutathione were conducted at 5°C and in 96-well microplates and (2) the
Tissuecontentsofglutathioneweremeasuredusinganassaybasedon concentration of 1-chloro-2,4-dinitrobenzene (CDNB) used in the
the2-vinylpyridine(2VP)methoddescribedbyGriffith(1980).Total GSTassaywas0.83mmoll−1.
glutathione (GST =GSH+GSSG) was measured, along with Levels of glutathione peroxidase 4 (GPx4) (EC 1.11.1.12)
tot
oxidized glutathione (GSSG) following the elimination of GSH were quantified using western blot analysis. Tissue samples were
using2VP.Tissueswerehomogenizedinfivevolumesofice-cold5% homogenizedat10%(w/v)inanice-coldradioimmunoprecipitation
5′-sulfosalicylic acid in 100mmoll−1 potassium phosphate and assay(RIPA)buffer(150mmoll−1NaCl,1%TritonX-100,0.5%
1mmoll−1 EDTA (pH 7.0) using a BioHomogenizer tissue Na deoxycholate, 0.1% SDS, 50mmoll−1 Tris, pH 8.0) using a
homogenizer (BioSpec Products, Bartlesville, OK, USA). Tenbroeck ground-glass homogenizer. Homogenates were
Followingcentrifugationat10,000gfor5minat4°C,supernatants centrifuged at 21,000g for 15min at 4°C. The resulting
werecollectedandusedimmediatelyorstoredat−70°C.Supernatants supernatants were used immediatelyor stored at −80°C for future
were added to a buffered 2VP solution (0.3moll−1 2VP final use. Protein concentrations of samples were determined using the
concentration)orbufferaloneandincubatedatroomtemperaturefor bicinchoninic acid method (Smith et al., 1985). For each sample,
10–15min.Anassaysolutioncontaining100mmoll−1K-phosphate, 40µg protein was diluted with RIPA buffer to avolume of 10µl.
1mmoll−1 EDTA, 0.1mmoll−1 DTNB and 0.164unitsml−1 Laemmlibuffer(5µl)containing5%β-mercaptoethanolwasadded
glutathionereductase(400unitsml−1)(pH7.0)wasadded(150µl) and the sample mixture was heated at 96°C for 10min. Ten
tothewellsofa96-wellmicroplate.Tenmicrolitersofdilutedsample microlitersofeachsample,ormolecularweightstandard(Precision
(typically20-fold)orstandardwasaddedtoeachwellinduplicateand Plus Protein), was added to one of each lanes in a 10-well Mini-
the reaction was initiated by adding 50µl of an NADPH solution ProteanTGXprecastgelandproteinswereseparatedat200V.The
(0.05mmoll−1finalconcentration).Absorbancereadingsat412nm runningsolutionwasastandardSDS-PAGEbuffer(192mmoll−1
were begun immediately thereafter and taken every 20s for 3min glycine, 0.1% w/v SDS, 25mmoll−1 Tris, pH 8.3). Following
usingaSpectraMaxM2emicroplatereader(MolecularDevices)and electrophoresis, gels were rinsed several times and transferred to
theaccompanyingSoftMaxProsoftwarekineticprogram. PVDF membranes using a transfer buffer (SDS-PAGE buffer
described above containing 20% methanol). Proteins were
Enzymaticantioxidants transferred using a mini Trans-Blot electrophoretic transfer cell
y
SOD(EC1.15.1.1)activitywasmeasuredusingakitfromSigma (Bio-RadLaboratories)at1.3A/1minigelor2.5A/2minigelsanda g
o
Life Sciences (catalog no. 19160) by following the extent of maximumof25Vfor7min.PVDFmembraneswererinsedseveral l
o
inhibition of the reduction of xanthine by xanthine oxidase, timesandthenincubatedfor1hatroomtemperature(orovernightat i
B
coupledtothereductionofthechromogen2-(4-iodophenyl)-3-(4- 4°C) in blocking buffer (0.5mmoll−1 NaCl, 1% casein,
l
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) at 20mmoll−1 Tris, pH 7.4). Blocked membranes were rinsed a
t
440nm. Oneunit of activity wasdefinedastheamountof SOD briefly and then incubated for 1h at room temperature in an n
e
necessary to inhibit the reduction of xanthine by 50%. Tissues antibodysolution(20-folddilutionoftheblockingbufferdescribed m
werehomogenized(10%,w/v)inice-cold50mmoll−1potassium above)containinga1:1000dilutionofprimaryantibody(ab40993; ri
e
phosphatebuffer(pH7.2)asdescribedabove,followedbyprobe Abcam,Cambridge,MA,USA).Thissolutionwasremovedandthe
p
sonication (three pulses of 4s duration, 2mm probe, output at membranesrinsedbriefly,afterwhichthemembraneswerewashed x
E
20% of maximum). Aliquots of each sample were removed and four times for 5min each in blocking buffer. Washed membranes
f
centrifuged at2000g for 10min at4°C. Supernatants werethen were next incubated for 1h at room temperature in an antibody o
dilutedasnecessaryinhomogenizationbufferforuseintheassay. solution containing a 1:3000 dilution of secondary antibody al
n
All samples were measured at 5°C in triplicate using a (ab6721; Abcam). The secondary antibody solution was removed
r
SpectraMax M2e microplate spectrophotometer (Molecular and the membranes rinsed and washed as described above. u
o
Devices). Antibody binding was visualized using a horseradish peroxidase J
4
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
(HRP) detection kit (Pierce ECL2 Western Blotting Substrate, (Amersham ECL Prime Western Blotting Detection Reagent, GE
ThermoFisher Scientific, Waltham, MA, USA). Two milliliters of Healthcare Bio-Sciences) according to the manufacturer’s
finalHRPsubstratesolutionwereaddedtocovereachmembraneand specifications. Chemiluminescencewas detected for 15min using
themembranewasimmediatelyplacedinaBio-RadImageDocphoto an AlphaImager3300ImagingSystem (Protein Simple, San Jose,
imager. The signal was read every 20s for 3min. GPx4-specific CA, USA) and quantified using ImageQuant TL software (GE
bands were analyzed using GelAnalyzer 2010 after removing the Healthcare Bio-Sciences). Levels of ubiquitinated proteins were
non-specific background with ImageJ. Individual samples from all normalizedtolevelsintheheartventriclesofN.coriiceps(n=6)and
specieswererunonasinglegelforcomparisonpurposesandeach were blotted in triplicate on membranes for measurements in the
sample was run in duplicate. Proportions of sample GPx4 were heartventricleandinduplicateonmembranesformeasurementsin
quantifiedasapercentoftotalGPx4fromeachgel. thepectoraladductorandliver.
Totalantioxidantpower 20Sproteasomeactivity
Total antioxidant power was measured using a colorimetric Activityofthe20Sproteasomewasmeasuredbasedonthemethod
microplate assay kit from Oxford Biomedical Research (TA02; developedbyShibataniandWard(1995)andadaptedforuseinfish
Oxford, MI, USA) according to the manufacturer’s instructions. by Doblyet al. (2004). Frozen tissues were finely chopped on an
Values for total antioxidant power are expressed as Trolox ice-cold stage and then homogenized in five volumes of ice-cold
equivalents(µmolg−1wetmassoftissue). lysis buffer (50mmoll−1 Tris-HCl, 0.1mmoll−1 EDTA,
1.0mmoll−1 β-mercaptoethanol, pH 8.0) using a Tissuemizer
Levelsofubiquitinatedproteins homogenizer (Tekmar) and Tenbroeck ground-glass tissue
Levels of ubiquitinated protein were measured based on the homogenizers. The homogenate was centrifuged at 20,000g for
methods used by Hofmann and Somero (1995) and modified by 1hat4°Candthesupernatantwasretained.Proteincontentofthe
Todgham et al. (2007). Frozen tissues were finely chopped on an supernatantwasdeterminedusingaBradfordproteinassay(1976)
ice-cold stage and then homogenized in five volumes of ice-cold withBSAusedforthestandardcurve.
homogenization buffer [4% SDS (w/v), 1mmoll−1 EDTA, Proteasomeactivitywasmeasuredusingtheproteasome-specific
50mmoll−1 Tris-HCl, pH 6.8] supplemented with protease fluorogenic substrate LLVY-7-amino-4-methlycoumarin (LLVY-
inhibitors (cOmplete Protease Inhibitor Cocktail Tablets, Roche, AMC; Enzo). The substrate was dissolved in DMSO
Indianapolis,IN,USA)usingaTissuemizerhomogenizer(Tekmar, (5.71mmoll−1), then aliquoted and stored at −80°C until use.
Cincinnati, OH, USA). Homogenization was completed using Althoughmaximalactivityofthe20Sproteasomehasbeenshown
Tenbroeckground-glasstissuehomogenizers.Tissuehomogenates torequireSDS(ShibataniandWard,1995),wefound,aftertesting
wereboiledfor5mintodenatureproteins.Homogenateswerethen foractivity in SDSconcentrations ranging from 0 to 0.025%, that
centrifuged at 12,000g for 15min at room temperature and the the maximal activity of the 20S proteasome was obtained in Tris
supernatant was retained. Protein content of the supernatant was buffer lacking SDS and, thus, we omitted it from the reaction
determined usingaBradford proteinassay(1976) with BSA used mixture. Activity was measured by incubating 50µg protein from
forthestandardcurve.Supernatantswerestoredat−80°C. the supernatant with 40µmol l−1 LLVY-AMC in 22.5µl
Samples were diluted with Tris-buffered saline solution (TBS) 100mmoll−1 Tris-HCl (pH 8.0) for 60min at 5°C. The reaction
(20mmoll−1 Tris-HCl, 140mmoll−1 NaCl, pH 7.6) to a wasdeterminedtobelinearfor90min.Thereactionwasstoppedby
concentration of 0.5µg µl−1 for ventricle and pectoral adductor adding225µlof0.1moll−1sodiumborate(pH9.1)and65µl1%
samples, and to 0.25µg µl−1 for liver samples. One µl of each SDS. Fluorescence of AMC was determined at excitation or
samplewaspipettedontoa12×10cmsheetof0.2µmnitrocellulose emissionwavelengthsof380and460nm,respectively,onaGemini
membrane(GEHealthcareBio-Sciences)intriplicate.Theprotein EMMicroplateReader(MolecularDevices).Parallelsampleswere
washeat-fixedtothemembraneat65°Cfor20min.Themembrane prepared by adding the proteasome inhibitor MG-132
wasthenblockedwith5%non-fatmilkpowderdissolvedinTween- (133µmol l−1; Enzo) prior to incubation. Activity was calculated
20 Tris-buffered saline solution (TTBS) (20mmoll−1 Tris-HCl, bydeterminingtheconcentrationofAMCinthesamplesusingthe
140mmoll−1NaCl,0.01%Tween-20,pH7.6,roomtemperature) standard curve minus the activity in the presence of MG-132.
for 1h. After blocking, the membranes were rinsed twice briefly Activity is expressed as pmol AMC h−1 50µg−1 protein. The
with TTBS and then three times for 5min each with TTBS. The standard curve was prepared using eight AMC concentrations
y
membranes were incubated at 4°C with the ubiquitin conjugate between3.3and44µmoll−1.Thesamplesandstandardcurvewere g
o
primary antibody (mono- and poly-ubiquitinylated conjugates measuredintriplicate. l
o
monoclonal antibody produced in mice; Enzo Life Sciences, i
B
BML-PW8810, Farmdale, NY, USA) diluted 1:5000 in 5% non- Statisticalanalysis
l
a
fatmilkpowderdissolvedin TTBS.Incubation timeswere12.5h Differences in the mean values of antioxidants, pro-oxidants,
t
forventricle, 15hfor pectoraladductorand2hfor liversamples. ubiquitinatedproteinsand20Sproteasomeactivityamongspecies n
e
ThemembraneswerethenrinsedbrieflytwicewithTTBSandthen andwithineachtissueweredeterminedusingaone-wayANOVA m
rinsedthreetimesfor5mineachwithTTBS.Themembraneswere followedbyaTukey’sposthoctest.Equalvariancewasconfirmed ri
incubatedatroomtemperaturewiththesecondaryantibody(rabbit andnormalitytestedusingaShapiro–Wilktest.AKruskal–Wallis e
p
anti-Mouse IgG peroxidase antibody A9044; Sigma-Aldrich) testfollowedbyaDunn’stestonrankeddatawasusedtodetermine x
E
diluted 1:10,000 in 5% non-fat dry milk powder in TTBS. differences among species for data that failed to meet the
f
Membranes with ventricle samples were incubated for 1.5h assumptions of the ANOVA. Outliers were identified by o
whereas those with pectoral adductor samples and liver samples calculating quantiles and samples with values outside the third al
n
wereincubatedfor2and2.25h,respectively.Themembraneswere quantilewereremovedfromtheanalysisofproteasomeactivityand
r
thenrinsedbrieflytwicewithTTBSandthenwashedthreetimesfor levelsofubiquitinatedproteins.Simplelinearregressionswereused u
o
5mineachinTTBSanddevelopedusingachemiluminescencekit to assess correlations between pro-oxidants and oxidative damage J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
Table1.Parametersmeasuredinthebloodofnotothenioids
Chaenocephalus Champsocephalus Chionodracorastrospinosus Nototheniacoriiceps Gobionotothengibberifrons
aceratus(−Hb/−Mb) gunnari(−Hb/−Mb) (−Hb/+Mb) (+Hb/+Mb) (+Hb/+Mb)
Heme(mmoll−1) 0.04±0.01a(6) 0.25±0.01a,b(6) 0.09±0.05a(6) 5.53±0.55b(9) 5.55±0.25b(16)
Hb(mgml−1) 119.81±11.03*(8) 95.54±2.81(16)
Hct(%) 32.91±1.78(8) 37.02±1.16(16)
Hb,hemoglobin;Hct,hematocrit;Mb,myoglobin.Valuesaremeans±s.e.m.DifferentsuperscriptlettersindicatesignificantdifferencesasdeterminedbyaDunn’s
posthoctest(P<0.05).TheasteriskindicatesasignificantdifferencebasedonaStudent’st-test(P<0.05).Thenumberofindividuals(n)isindicatedin
parentheses.
(levelsofTBARSandcarbonyls)ineachtissue.Significancewas icefishspeciesC.gunnari(Table1).Mblevelswere3.4-foldhigher
set at P<0.05. ANOVA tests and regression analyses were in the heart ventricles of red-blooded N. coriiceps than in red-
conducted using Sigma Plot 11.0 (Systat Software,San Jose, CA, blooded G. gibberifrons and white-blooded C. rastrospinosus
USA)andJMP7(SASInstitute,Cary,NC,USA);allotheranalyses (Table2).AlthoughHctlevelsweresimilarinthetwored-blooded
wereconductedusingR(www.r-project.org). species, Hb levels were higher in N. coriiceps than in G.
Tocharacterizetheclusteringofantioxidantsamongspeciesfor gibberifrons(Table1).
each tissue, we conducted a principal component analysis (PCA) MaximalactivityofCS,ameasureoffluxthroughthecitricacid
afterimputingmissingvalues(JosseandHusson,2013)followedby cycle, was lowest in the heart of white-blooded C. aceratus
ahierarchicalclusteringonprincipalcomponents(HCPC)usingthe (Table2).However,lowactivityofCSintheC.aceratusventricle
PCA and HCPC functions from the FactoMineR R package, wasnotassociatedwiththelackofbothoxygen-binding proteins,
respectively. The first five components of the PCA were used as Hb and Mb, because CS activity in the hearts of the icefish
input for the HCPC, and the number of clusters retained was C. gunnari, a species which lacks Mb, was similar to that in the
determinedbylookingathowspeciesclusteredonthePCAplots. hearts of red-blooded notothenioidsthat express Mb (Table 2). In
Usingonlythefirstfivecomponentsallowsustoseparatethenoise thepectoraladductormuscle,CSactivitywashighestinthetwored-
fromthestructureofthedatabeforeperformingtheHCPC.Aχ2test bloodedfishesandintheicefishC.gunnari(Table3).CSactivityin
wasperformedbetweenspeciesandgroups(clusters)ofindividuals. theliverwashighestinC.gunnari (Table4).
Finally, foreach group,thegroup andoverall meanvalue of each
antioxidantwerecomparedbycalculatingastatisticcorresponding Oxidativedamage
to their standardized difference, which follows a standard normal The ANOVA indicated that differences in levels of both oxidized
distribution under the null hypothesis that the means are equal. proteins (carbonyls) and lipids (TBARS normalized to total lipid)
Individualsthatweremissingmorethanfourofthenineantioxidant didnotalwayscorrespondwiththepresenceorabsenceofHband
measurements were excluded, and individuals missing values for Mb(Tables2–4).Intheheartventricle,TBARSwerelowerinred-
antioxidants (17 for liver, 14 for adductor muscle and heart bloodedfishescomparedwithicefishes,whereasproteincarbonyls
ventricle) were imputed using the regularized iterative PCA werelowestintheicefishC.aceratus(Table2).Levelsofprotein
algorithm (Josse and Husson, 2013), resulting in a sample size of carbonyls and TBARS were higher in the tissues of red-blooded
38foreachtissue. fishes compared with icefishes only in oxidative skeletal muscle
tissue(Table3).Intheliver,TBARSwerehighestinC.gunnariand
RESULTS G.gibberifrons,andproteincarbonylswerehighestinC.gunnari
Pro-oxidants andC.aceratus(Table4).
Levels of heme were significantly higher in the blood of red- Although neither the presence nor the absence of Hb and Mb
blooded fishes than in the blood of most icefishes, except for the correspondedwithlevelsofoxidativedamage,regressionanalyses
Table2.Levelsofpro-oxidants,antioxidantsandoxidizedmacromoleculesintheheartventriclesofnotothenioids
C.aceratus C.gunnari C.rastrospinosus N.coriiceps G.gibberifrons
(−Hb/−Mb) (−Hb/−Mb) (−Hb/+Mb) (+Hb/+Mb) (+Hb/+Mb) gy
o
Pro-oxidants
l
CS(Ug−1wetmass) 9.32±0.17a(5) 12.93±0.92b(5) 9.72±0.63a,c(4) 12.58±0.75b,c(5) 10.75±0.62a,b,c(5) o
i
Mb(mgg−1) 0.95±0.20a(6) 3.57±0.30b(6) 0.96±0.12a(5) B
Antioxidants al
SOD(Ug−1) 1836.63±105.77a(8) 1692.25±86.56a(8) 1811.83±150.92a(6) 2278.75±99.37b(8) 2708.53±80.14c(8) t
n
CAT(µmolmin−1) 142.69±15.20a(8) 260.86±9.40a,b(8) 269.23±16.10a,b(6) 461.66±42.15b(9) 243.27±43.20a,b(7) e
GST(Ug−1) 1.13±0.10a(8) 0.85±0.07a(8) 0.67±0.10b(6) 1.10±0.12a,b(9) 1.13±0.10a(8) m
TAP(µmolg−1) 6.65±0.19a,c(8) 6.42±0.35a(8) 5.99±0.12a(6) 8.26±0.27b(9) 7.59±0.29b,c(8) i
r
GPx1(Ug−1) 0.84±0.07a,b(8) 1.06±0.10a(8) 0.55±0.12b(6) 0.96±0.04a,b(8) 0.83±0.10a,b(7) e
p
GPx4(%oftotal) 19.04±3.83(6) 16.74±1.77(6) 18.46±5.71(6) 21.65±2.54(6) 18.86±1.73(6) x
GR(mUg−1) 280.89±15.74a(8) 177.93±13.65b(8) 235.75±14.73a,b(6) 297.95±22.07a(8) 461.06±17.57c(8) E
2GSSG:GSH 0.097±0.008a(8) 0.148±0.009b(8) 0.119±0.007a,b(6) 0.112±0.016a(8) 0.109±0.006a,b(8) f
o
Oxidativedamage
TBARS(nmolg−1lipid) 132.67±20.40a(8) 167.05±10.69a(8) 142.80±16.71a(6) 81.82±7.40b(8) 76.93±4.40b(9) al
Carbonyls(mmolmol−1) 17.44±5.15a(7) 85.56±17.02b(8) 43.40±10.54a,b(6) 33.50±3.67a,b(7) 73.38±12.77b(7) n
r
u
Valuesaremeans±s.e.m.Differentsuperscriptlettersindicatesignificantdifferencesasdeterminedbyaone-wayANOVAorKruskal–Wallistestfollowedbya o
Tukey’sorDunn’sposthoctest(P<0.05).Thenumberofindividuals(n)isindicatedinparentheses. J
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RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
Table3.Levelsofpro-oxidants,antioxidantsandoxidizedmacromoleculesinthepectoraladductormuscleofnotothenioids
C.aceratus C.gunnari C.rastrospinosus N.coriiceps G.gibberifrons
(−Hb/−Mb) (−Hb/−Mb) (−Hb/+Mb) (+Hb/+Mb) (+Hb/+Mb)
Pro-oxidants
CS(Ug−1wetmass) 13.40±1.69a(6) 23.13±1.18b(4) 15.44±1.40a(4) 26.13±1.67b(5) 22.30±1.85b(5)
Antioxidants
SOD(Ug−1) 1513.13±170.99a(8) 2500.36±127.71b(8) 1810.50±203.29c(6) 2031.88±107.19a,b,c(8) 2271.35±108.32b,c(8)
CAT(µmolmin−1) 131.31±13.34a(8) 243.04±8.42b(8) 324.54±22.27c(6) 409.24±19.90d(8) 218.46±17.67b(7)
GST(Ug−1) 0.97±0.13(8) 1.34±0.17(8) 1.11±0.19(6) 1.57±0.12(8) 1.54±0.27(8)
TAP(µmolg−1) 5.37±0.39(8) 4.61±0.24(8) 5.60±0.48(6) 4.59±0.60(8) 5.66±0.49(8)
GPx1(Ug−1) 0.55±0.07a,b(8) 0.85±0.06c(8) 0.29±0.06a(6) 0.64±0.10b,c(8) 0.66±0.06b,c(8)
GPx4(%oftotal) 8.89±0.49a(6) 20.64±2.32b(6) 15.48±0.99b(6) 27.70±1.58c(6) 28.09±1.71c(6)
GR(mUg−1) 127.29±10.52a(8) 134.67±10.48a,b,c(8) 111.16±6.58b(6) 286.48±17.15d(8) 177.37±7.19c(8)
2GSSG:GSH 0.131±0.007a(8) 0.172±0.008a,b(8) 0.181±0.020b(6) 0.138±0.008a,b(8) 0.143±0.010a,b(8)
Oxidativedamage
TBARS(nmolg−1lipid) 50.79±4.40a(8) 75.52±6.33a,b(8) 74.38±12.73a,b(6) 240.19±42.19b,c(8) 279.22±15.95c(8)
Carbonyls(mmolmol−1) 4.77±0.88a(7) 5.70±1.21a,c(6) 7.15±1.70a,b,c(5) 41.98±9.99b(7) 25.38±3.53b,c(6)
Valuesaremeans±s.e.m.Differentsuperscriptlettersindicatesignificantdifferencesasdeterminedbyaone-wayANOVAorKruskal–Wallistestfollowedbya
Tukey’sorDunn’sposthoctest(P<0.05).Thenumberofindividuals(n)isindicatedinparentheses.
indicated that maximal activity of CS was significantly and alignedmostcloselywiththeresultsfromthehierarchicalclustering.
positively correlated with levels of protein carbonyls and TBARS Therewasasmalldifference(4%)betweenthevariationexplained
in both oxidative skeletal muscle (Fig. S2A,B) and the liver by components 2 and 3 in thistissue (Fig. 1B). Only in the heart
(Fig.S3A,B)butnotintheheartventricle(Fig.S1A,B).Hemewas ventriclewerelevelsofantioxidantssimilarinthethreespeciesof
alsopositivelycorrelatedwithTBARSandcarbonylsinoxidative icefishes and lower than in the two red-blooded species (Fig. 1A,
skeletalmuscle(Fig.S2C,D). TableS1).Inthepectoralmuscleandliver,antioxidantlevelsinthe
twoicefishesC.aceratusandC.rastrospinosusweresimilartoeach
Antioxidants other (Fig. 1B,C) and lower than in the two red-blooded species
TheANOVAindicatedthatantioxidants(capacitiesorlevels)were (Tables S2, S3). However, in the liverand pectoral muscle of the
higher in red-blooded fishes than in icefishes (Tables 2–4). icefish C.gunnari,antioxidantlevelsweredifferentfromthosein
However, in the pectoral adductor muscle, activities of SOD and theothericefishesaswellasthoseinthered-bloodedspecies,with
GPx1 were higher in C. gunnari than in other icefishes, and some antioxidants higher than the overall mean and some lower
equivalenttothoseinthetwored-bloodedspecies(Table3).Also, (Fig. 1B,C, Tables S2, S3). Notably, SOD activities in the C.
intheliver,activitiesofSODandCATwerehigherinC.gunnari gunnaripectoralmuscleandliverwerehigherthantheoverallmean
thaninothericefishesandsimilartothoseinG.gibberifrons(SOD) (TablesS2,S3),whichcorrespondswiththehighactivityofCSin
(Table4). thesetissues(Tables3,4).
We used PCA followed by hierarchical clustering analysis to
identify differences in antioxidant levels among species. The χ2 Levelsofubiquitinatedproteins
analysis indicated that there was a significant difference in Levelsofubiquitinatedproteinswerehigherintheheartventricles
antioxidant levels among the species in all tissues (P<0.001; ofthered-bloodedfishesN.coriicepsandG.gibberifronsthan in
TablesS1–S3).Components1and2ofthePCAanalysisexplained thoseoficefishes,andlevelswerehigherintheheartofN.coriiceps
mostofthevariationamongthespeciesinantioxidantlevels(37– than in thatof G.gibberifrons(Fig. 2A). Therewasnodifference
40% and 16–17%, respectively) (Fig. 1). The PCA plot for the between levels of ubiquitinated proteins in the heart ventricles of
pectoral muscle is shown for components 1 and 3 because this icefishesthat express Mb compared with those that do not. In the
Table4.Levelsofpro-oxidantsantioxidantsandoxidizedmacromoleculesintheliversofnotothenioids
y
C.aceratus C.gunnari C.rastrospinosus N.coriiceps G.gibberifrons g
(−Hb/−Mb) (−Hb/−Mb) (−Hb/+Mb) (+Hb/+Mb) (+Hb/+Mb) o
l
o
Pro-oxidants
i
CS(Ug−1wetmass) 0.29±0.01a(5) 0.79±0.11b(5) 0.40±0.05a(5) 0.42±0.03a(5) 0.38±0.03a(5) B
Antioxidants al
SOD(Ug−1) 4492.63±376.14a(8) 9542.63±338.19b(8) 6090.67±246.47c(6) 6847.75±357.20c(8) 8713.80±168.19b(8) t
n
CAT(µmolmin−1) 1563.06±195.11a(7) 3880.22±488.35b(6) 1806.98±308.02a(6) 5460.75±342.61c(9) 5526.70±327.11c(7) e
GST(Ug−1) 13.20±2.45a(8) 15.92±1.88a,b(8) 13.41±2.03a,b(6) 20.68±1.67a,b,c(8) 29.97±1.54c(8) m
TAP(µmolg−1) 4.87±0.27a(8) 4.82±0.69a,b(8) 4.06±1.40a,b(6) 7.84±0.54b(8) 6.89±0.67a,b(8) ri
GPx1(Ug−1) 0.48±0.11(8) 0.46±0.06(8) 0.60±0.10(6) 0.70±0.06(8) 0.66±0.05(8) e
p
GPx4(%oftotal) 19.78±1.60(6) 21.75±1.94(6) 18.30±2.19(6) 19.67±1.50(6) 20.49±2.32(6) x
GR(mUg−1) 241.16±31.06a(8) 116.66±18.77b(8) 245.45±26.55a(6) 276.97±31.10a(8) 397.83±31.53a(8) E
2GSSG:GSH 0.192±0.020a(8) 0.171±0.017a(8) 0.211±0.024a,b(6) 0.285±0.030b(8) 0.203±0.028a,b(8) f
o
Oxidativedamage
TBARS(nmolg−1lipid) 117.66±13.80a(8) 245.38±18.78b(8) 94.93±28.91a(6) 114.13±27.09a(8) 149.98±9.86a,b(8) al
Carbonyls(mmolmol−1) 19.78±4.20a,b(6) 30.14±7.81a(8) 4.43±1.19c(6) 5.61±1.24b,c(6) 8.04±3.46b,c(6) n
r
u
Valuesaremeans±s.e.m.Differentsuperscriptlettersindicatesignificantdifferencesasdeterminedbyaone-wayANOVAorKruskal–Wallistestfollowedbya o
Tukey’sorDunn’sposthoctest(P<0.05).Thenumberofindividuals(n)isindicatedinparentheses. J
7
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
A B
2
2
8%) 1 Groups %) 1
14. ACE 13
mponent 2 ( –01 GGCRUOIABNSR mponent 3 0
Co Co –1
–2
–2
2
–2 0 2 –4 –2 0 2
Component 1 (38.6%) Component 1 (37%)
C
3
%) 2
6
1
2 ( 1
ent 0
n
o
p
m
o–1
C
–2
–2 0 2 4
Component 1 (39.7%)
Fig.1.Antioxidantsintheheartventricle,pectoraladductorandliverofnotothenioids.Distributionofantioxidantlevelsofindividualfishbyspecies
(ACE:Chaenocephalusaceratus,GUN:Champsocephalusgunnari,RAS:Chionodracorastrospinosus,COR:Nototheniacoriiceps,GIB:Gobionotothen
gibberifrons),overprincipalcomponents1and2for(A)heartventricleand(C)liver,and1and3for(B)pectoraladductormuscle.Theclusteringofantioxidants
isvisualizedby0.95confidenceellipsesaroundthebarycenterforeachspecies.Individualsthatweremissingmorethanfourofthenineantioxidant
measurementswereexcluded,andindividualmissingvaluesforantioxidants(17forliver,14foradductormuscleandheartventricle)wereimputedusingthe
regularizediterativePCAalgorithm(JosseandHusson,2013),resultinginasamplesizeof38foreachtissue.
pectoral adductor muscle, levels of ubiquitinated proteins were inC.gunnariandwas1.7–2.9-foldhigherthanintheotherspecies
highestinN.coriiceps(1.6–2.4-foldhigherthaninotherspecies). (Fig. 3C). There was no significant difference in 20S proteasome
Levels of ubiquitinated proteins were mostly similar in the other activity in the liver between red-blooded notothenioids and the
y
species, with the exception that levels were lower in C. icefishesC.aceratus,P.georgianusandC.rastrospinosus(Fig.3C). g
o
rastrospinosus than in C. aceratus (Fig. 2B). Similar to the heart l
o
ventricle,levelsofubiquitinatedproteinswerehigherintheliversof DISCUSSION i
B
thetwored-bloodedspeciesthaninthoseoftheicefishesexceptC. We had originally predicted that the loss of the iron-centered
l
a
aceratus(Fig.2C). oxygen-binding proteins, Hb and Mb, would result in lower
t
levels of oxidized biological macromolecules, capacities of n
e
20Sproteasomeactivity antioxidants and rates of protein degradation by the 20S m
In the heart ventricle, the chymotrypsin-like activity of the 20S proteasome. Our data demonstrate that levels of oxidized ri
e
proteasomedidnotshowacleartrendassociatedwiththeexpression proteins and lipids and capacities for antioxidant activity are
p
of oxygen-binding proteins. The only significant differences in indeedlowerinsometissuesofHb-lessspecies,yetthispatternis x
E
proteasomeactivitywerebetweenthetwored-bloodedspeciesand not observed in all tissues, nor does the activity of the 20S
f
theicefishC.aceratus,withtheactivitybeingonaverage1.6-fold proteasome show a trend with the expression of Hb and Mb. o
higher in the hearts of the red-blooded fishes (Fig. 3A). In the Neither Hb nor Mb is associated with oxidative damage across al
n
pectoralmuscle,20Sproteasomeactivitywasmostlysimilarinall tissues. However, the activity of CS is positively correlated with
r
species, although activity was higher in C. aceratus than in N. levels of oxidized proteins and lipids in both the pectoral u
o
coriiceps(Fig.3B).Intheliver,20Sproteasomeactivitywashighest adductor muscle and the liver (Figs S2–S3). J
8
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
1.2 A –Hb/–Mb
d 700 A
–Hb/+Mb
1.0 –Hb/–Mb 600 b +Hb/+Mb
–Hb/+Mb b,c
0.8 +Hb/+Mb 500 b,c
400 a,b,c a,b,c
0.6
c
a
300
0.4
b 200
0.2 a a,b a,b
100
n)
0 ei
s 0.25 B c prot 0
ein –1 500 B
ated prot 0.20 –+HHbba –1h 50 µg 400 a a,b a,b a,b a,b –+HHbb
uitin 0.15 a a,b a,b MC
q A 300 b
ubi b ol
of 0.10 pm
e levels 0.05 activity ( 200
ativ me 100
el o
R 0 s
a
0.35 C ote 0
0.30 –Hb a 0S pr 800 C b
+Hb a 2 –Hb
0.25 +Hb
600
0.20
a,b a
0.15 400
b,c
a
0.10 b,c a a a
c
0.05 200
0
Fig.2.LeveC.l sacoerfatuubsiqC. ugiutinnnaarPti. egdeoprgrioCa. tnreuaissntrsosipnintoissGu.s sugiebbseroifrfonnosNt.o ctohriiecneiposidsthat 0 C. aceratus C. gunnaPr.i georgCi. arnaustsrospinosGu. sgibberifronsN. coriiceps
y
varyintheexpressionofhemoglobin(Hb)andmyoglobin(Mb).Relative g
levelsofubiquitinatedproteinsinthe(A)heartventricle,(B)pectoraladductor Fig.3.Activityofthe20Sproteasomeintissuesofsixspeciesof o
muscleand(C)liverofsixspeciesofnotothenioids.Levelsofubiquitinated notothenioidsthatvaryintheexpressionofHbandMb.(A)Heartventricle, ol
proteinswerenormalizedtolevelsintheheartventricleofN.coriiceps.Values (B)pectoraladductormuscleand(C)liver.Valuesaremeans±s.e.m.(n=6–8). Bi
aremeans±s.e.m.(n=6).Significantdifferencesamongspeciesandwithin Significantdifferencesamongspeciesandwithineachtissueareindicatedby l
eachtissueareindicatedbydifferentletters(P<0.05). differentletters(P<0.05).AMC,7-amino-4-methylcoumarin. ta
n
e
Heartventricle:evidencethatMbdoesnotamplifyoxidative Driedzic, 1983). The loss of Mb expression has occurred four m
damage times during the radiation of the Channichthyidae family, ri
e
Mb stores intracellular oxygen and may facilitate its transport to suggesting weakened selective pressure maintaining the Mb gene
p
mitochondria (Wittenberg, 1970). The importance of cardiac Mb (Moylan and Sidell, 2000). The oxygen-rich environment of the x
E
under normoxic conditions in teleost fishes is equivocal, as Southern Ocean is likely to have relaxed selective pressure to
f
evidenced by in vitro studies (Canty and Driedzic, 1987) and the maintainMbexpression,makingthelossofMbaneutralmutation, o
widespread loss of Mb among teleost species (Grove and Sidell, and yet in vitro studies have shown that Mb enhances cardiac al
n
2002; Macqueen et al., 2014). Under hypoxic conditions, Mb is performance in notothenioids at high afterload pressures (Acierno
r
clearly advantageous and has been shown to be essential for et al., 1997). Alternatively, if Mb contributes to ROS formation, u
o
maintaining cardiac performance (Bailey and Driedzic, 1992; perhapsthroughthereactionofH O withMetMb,formingaferryl J
2 2
9
RESEARCHARTICLE JournalofExperimentalBiology(2018)221,jeb162503.doi:10.1242/jeb.162503
speciesandtyrosineperoxylradical(Newmanetal.,1991),lossof proteins werealso observed in othertissues (e.g. the liverand the
Mb may be beneficial, thereby reducing selective pressure to heart)fromanimalslackingHb,thesedataindicatethatproductsof
maintain the protein. Heart ventricles of N. coriiceps have a Mb oxidative stress can also arise from other processes (Tables 1–4).
content 3.4-fold greater than those of G. gibberifrons and Two factors that could potentially contribute to higher levels of
C. rastrospinosus; however, levels of oxidized and ubiquitinated oxidized proteins in skeletal muscle compared with both the liver
proteins are similar among red- and white-blooded species, and andtheheart,andtothedifferencesamongspecies,areratesof
levelsofTBARSarelowerinthetwored-bloodedspeciescompared ROS formation and protein turnover. As described above,
withicefishes,arguingagainstaredoxadvantageconferredbythe although rates of ROS formation may correlate with maximal
lossofMb.Asadditionalsupportforthis,thePCAandhierarchical CS activity, ROS generation is also affected by the degree of
clusteringanalysesshowthatthepresenceofMbintheheartofthe coupling between the activity of the ETC and ATP synthesis,
icefish C. rastrospinosus is not associated with higher levels of which cannot be inferred from CS activity. Mitochondria from
antioxidantscomparedwithicefishesthatlacktheprotein(Fig.1A, the liver, in general, have a lower respiratory control ratio than
TableS1). those from muscle (e.g. Jørgensen et al., 2012), and
mitochondria from skeletal muscle have a lower RCR than
OxidativedamageinskeletalmusclereflectsactivityofCS, thosefromcardiacmuscle(Parketal.,2014).Thus,highlevels
Hbandhemelevels of oxidized macromolecules in the pectoral adductor may be
MaximalactivitiesofCS,aregulatoryenzymeoftheKrebscycle,is driven,inpart,bygreaterratesofmitochondrialROSproduction
oftenusedasamarkerforoxidativecapacitypergramoftissueand comparedwiththoseintheliver.
mitochondrial volume density (e.g. Lucassen et al., 2003; Rates ofproteinturnovermayalso impactsteadystatelevels of
Orczewskaetal.,2010)and,asthemajorityofROSareproduced oxidized proteins. Rates of protein synthesis in Antarctic
bymitochondria(BoverisandChance,1973),CSactivitycanalso notothenioids are approximately fivefold lower in the pectoral
be used to infer capacity for ROS production. Levels of oxidized adductorthanintheheartventricleandapproximately20-foldlower
proteinsandlipidsarethreefoldtoeightfoldhigherinthepectoral compared with rates in the liver, consistent with the higher
adductor muscle of red-blooded notothenioids compared with metaboliccostofproteinsynthesisinthelivercomparedwiththat
icefishes, and correspond with the higher activities of CS in red- in cardiac myocytes (Lewis et al., 2015; J. M. Lewis and
blooded species compared with most icefishes (Table 3). The K.M.O., unpublished data). Despite higher levels of ubiquitinated
approximatelytwofoldhighermaximalactivityofCSinoxidative proteins in the pectoral muscle of red-blooded species compared
skeletalmuscleofred-bloodedfishescomparedwithmosticefishes withicefishes(Fig.2B),ratesofdegradationbythe20Sproteasome
makesittempting toconclude thatelevatedcapacityforoxidative arelowestinN.coriicepsandsimilarinallotherspecies(Fig.3B).
metabolism is afactorcontributing to the high levels of oxidative If the rates of oxidative damage are similar in tissues, lower rates
damageinthisparticulartissue. of protein turnover in the pectoral muscle could result in higher
Wesuggest,however,thatthisisprobablynotthecasebecause levelsofoxidizedproteins.
theicefishC.gunnaridisplaysCSactivitysimilartothatofthered-
bloodedspeciesbutmaintainslowlevelsofoxidizedproteins.We AbsenceofHbisnotaccompaniedbyreductioninoxidative
positthatamoreprobableexplanationisthatROSproducedbythe stressinnotothenioidlivers
electrontransportchain(ETC)areamplifiedinreactionsmediated Theparticularlyhighlevelofoxidativedamage intheliverof the
byiron. This is supported bythe observation thatheme levels are icefish C. gunnari provides compelling evidence that the absence
associatedwithoxidativedamageinthetissuesofsomespecies(e.g. of oxygen-binding proteins does not confer an advantage for
pectoral adductor muscle for G. gibberifrons and N. coriiceps) white-blooded notothenioids by reducing oxidative stress in
(Table3;Fig.S2).Themechanismbywhichhemepotentiatesthe hepatic tissue (Table 4). In fact, CS activity appears to be the
formation of intracellular ROS is unclear, especially given that best predictor of levels of oxidative damage in the livers of all
transcript levels of haptoglobin, ferritin, serotransferrin and species, with the liverof C. gunnari possessing the highest levels
hepcidin,whichareinvolvedinFetransportandsequestration,are or activities of CS, protein carbonyls, TBARS and the 20S
higherintheAntarcticfishDissostichusmawsonithanintemperate proteasome among hepatic tissues of all the species studied
species(Chenetal.,2008).Also,inourpreviouswork,wefound (Table 4, Fig. 3C).
that tissue levels of the proteins ferritin and ceruloplasmin were Several antioxidants (SOD, CAT and GST) are highest in the
y
generallyhigherinred-bloodedspeciesthaninicefishes,although liver, compared with other tissues, among all notothenioid tissues g
o
transferrin levels were no different among species (Kuhn et al. studiedandyettheliverdisplaysthelowestmaximalactivityofCS l
o
2016). (Table4).Thesedataindicatethatinnotothenioidfishes,asinother i
B
Despitesignificantlyhigherlevelsofsomeantioxidants(GRand organisms,ROScanbegeneratedbybothmitochondrialandnon-
l
a
GPx4) in the pectoral adductor muscle of N. coriiceps and mitochondrial sources. The liver contains a large complement of
t
G. gibberifrons compared with most icefishes, it appears that peroxisomes,anorganellewithvariousoxidaseenzymes,which n
e
antioxidant levels are insufficient to prevent oxidative damage, produceasignificantportionofthetotalcellularH O (Boveris m
2 2
giventhatlevelsofbothproteincarbonylsandperoxidizedlipidsare et al., 1972; Schrader and Fahimi, 2006). Red-blooded G. ri
e
up to eightfold higher in red-blooded species compared with gibberifrons has exceptionally high capacities for peroxisomal
p
icefishes(Table3).Thehighlevelsofcarbonylatedproteinsinthe β-oxidation(CrockettandSidell,1993),andthefirststepinthis x
E
pectoral adductor muscle of N. coriiceps are also associated pathway is catalyzed by acyl CoA oxidase, which, like other
f
with higher levels of ubiquitinated proteins compared with other peroxisomal oxidases, generates H O (Lazarowand De Duve, o
2 2
species(Fig.2B). 1976). In addition, high levels of cytochrome P450s, enzymes al
n
Itisclearfromourresultsthatthehighlevelsofoxidizedproteins that detoxify xenobiotics and generate ROS as a by-product
r
in the pectoral adductor of red-blooded notothenioids cannot be (Hrycay and Bandiera, 2012), may warrant high levels of u
o
attributed to either heme or Hb alone. As high levels of oxidized antioxidants. J
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