Effects of Common Estuarine Pollutants on the Immune Reactions of Tunicates

11 Pages·1997·4.4 MB·English

by  D Raftos

Reference:Bio! Bull 192:62-72.(February. 1997)
Effects of Common Estuarine Pollutants on the Immune
Reactions of Tunicates
DAVID RAFTOS AND AIMEE HUTCHINSON
SchoolofBiologicalSciences, Macquarie University, North Ryde. NSW, 2109, Australia
Abstract. Tunicates are filter-feeding estuarine and son el ai, 1990; McCarthy and Shugart, 1990; Roales
marine animals that are frequently exposed to chronic and Perlmutter, 1977; Sarotand Perlmutter, 1976; Steb-
environmental pollution. This study demonstrates that bing. 1985). Some evidence suggests that the effects of
exposure to low-level (i.e., below the threshold ofacute decreased disease resistance resulting from low-level,
lethality) contamination with tributyltin, creosote, and chronic pollution may not be reflected accurately in as-
copper can have substantial effects on natural immune saysthattestacutelethality. Forinstance, heavymetalor
reactions in tunicates. Sublethal doses of toxicants ad- polychlorinated biphenyl contamination in worms can
ministered either in vitro or in vivo profoundly affected significantly inhibit lysozyme activity, wound healing,
phagocytosis, cellular cytotoxicity, and hematopoietic phagocytosis, rosette formation, and tissue transplanta-
cell proliferation. Effectswerenotalwaysinhibitory, and tion rejection at concentrations that are not acutely le-
responses often varied depending on the route oftoxi- thal (Cooper and Roch, 1992; Fitzpatrick et ai. 1992;
cantadministration.Thedatasuggestthatpollutantscan Rodrigues-Grau etai, 1989).
activatecascadesofcellularprocessesandcompensatory The aim of the current study is to demonstrate that
mechanisms, as well as directly inhibiting some ofthe subacute contamination with three common estuarine
responses tested. Some evidence indicates that toxicants pollutants the antifouling agents tributyltin (TBT),
exert their effects by altering the relative frequencies of copper,andcreosote can significantlyaffecttheimmu-
circulatory hemocytes. nological defensesoftunicates. Tunicates(Urochordata,
Ascidiacea) are aquatic filter-feeding invertebrates that
Introduction are ubiquitouscomponentsofestuarineand coastal ma-
rine systems (Berrill, 1950; Goodbody, 1974). They oc-
Marine invertebrates can be profoundly affected by cur in large populations on marinas, moorings, and
aquatic pollution. Detrimental effects have bLeDen identi- wharves that are often subjected to chronic pollution,
fied usingtests foracute lethality (e.g., 96-h 5()), toxi- particularly from antifouling treatments. The effects of
cant bioaccumulation, anatomical and biochemical ab-
pollution are likely to be compounded in tunicates by
erration, or altered biodiversity and abundance (Giam theirfilter-feeding lifestyle. Large volumesofpotentially
and Ray, 1987; Landis and Yu, 1995; Peakall, 1992). polluted water pass over the sensitive endothelial sur-
However, there is relatively little information regarding facesofthepharynx(upto 100 1ofwaterperday),greatly
the effects ofenvironmental contamination on natural increasingthepropensityoftunicatestoabsorbtoxicants
immune reactions in invertebrates, even though modu- (Goodbody, 1974).
lationoftheimmunesystem maydramaticallyalterpop- Here, we test the effects oftoxicants on a number of
ulationsbyaffectingtheirresistanceto infection (Ander- well-characterized assaysofimmunological reactivity in
tunicates. Thoseassaysquantify hemopoietic cell prolif-
eration (Raftos and Cooper, 1991; Raftos et ai. 1991),
ARbebcreeivvieadti1o7nsM:aFySW1,995fi;ltaecrceedpsteeadwa2t4erO;ctMoAbCer.1m9a9r6i.neanticoagulant; phagocytosis (Beck et ai, 1993: Kelly et ai, 1993), and
MS. marine saline; PAH. polycyclic aromatic hydrocarbon; RRBC. cellular cytotoxicity (Parrinello et ai, 1993; Peddie and
rabbitredbloodcells;TBT.tributyltin;TBS.Tris-bufferedsaline. Smith, 1993, 1994). Tunicates lack adaptive antipatho-
62
TUNICATES AND MARINE POLLUTANTS 63
genie defense mechanisms that are analogous to the Dosages
mammalian adaptive immune system (Ratios, 1994).
The ranges ofdoses used for the three toxicants were
Hence, innate (or natural) immunological reactions,
such as phagocytosis and cellular cytotoxicity, are prob- sneilfeiccatnetd dtioffienrceonrcpeosrfatreomconnocnetnrteraatteidoncsontthraotlsyiienldaetdlesaisgt-
ablythe major protective responsesofthese animals.
oneoftheassaystested. Copperwasthe onlycompound
that proved to be acutely toxic at high doses. All tuni-
Materialsand Methods cates (n = 8) died within 8 days ofexposure to >5 Mg/ml
Tunicates copper. Mortality wasassessed by the sensitivity oftuni-
catestotouch (failureto retract siphons)andbyan anal-
Specimens ofStyela plicata were collected from two ysisofhemocyte (blood cell) viability. None ofthe other
sites on Sydney Harbor. Australia (Balmoral Beach and dosestested, including <5 Mg/ml copper, caused mortal-
Birkenhead Point). After collection, tunicates were ity within 20 days(n = 4 perdose).
maintained in 40-1 aquaria filled with seawater. Aquaria
wereheld at 14C underconstant aeration. Thesecondi- Hemocyteharvestingandmanipulation
tions can maintain S. plicata for up to 2 months with
limited mortality. In vivo exposures were conducted in Hemocytes were harvested from incisions in the buc-
partitioned aquariacontaining40 1 ofseawaterpercom- (cablloosdi)phwoansscoolfleSc.tedpliincateaq.ualThveoluemxuedseodfihcee-mcoolldymmpah-
partment.
rine anticoagulant buffer (MAC; 0.1 A/glucose. 15 mA/
trisodium citrate, 13mA/ citric acid, 10mA/ EDTA.
Toxicants
0.45 A/NaCl, pH 7.0; Peddie and Smith, 1994)or FSW.
Tributyltin oxide and copper sulfate were purchased Debris and cell aggregates were removed from the hem-
from ICN Chemicals (Sydney, NSW, Australia)and cre- olymph by sedimentation for5 min(1 Xg). Asrequired,
osote was obtained from BBC Pty Ltd (Chatswood, hemocytes were washed by centrifugation (400 X g,
NSW,Australia). Tributyltinandcopperwerediluteddi- 5 min, 4C) through either MAC or FSW.
rectlyin filteredseawater(FSW;0.45-^m filtration).Cre-
osote was prepared asa saturated stock solution in FSW Cellviabilityandmorphology
by vigorously agitating 1% v/v creosote in toxicant-free, Hemocyte viabilities and the relative frequencies of
filteredseawaterovernightandthen filteringthesolution distinct hemocyte subpopulations were determined us-
(0.45-Mm) to remove insoluble material. Concentrations ing a FACScan flow cytometer with an argon-ion laser
ofcreosotearecited aspercentages(v/v)offiltered, satu- tuned to 488 nm (Becton Dickenson, Mountain View,
rated creosote solutions. Chemical analysis (Australian CA). Hemocytesforflowcytometrywereobtainedeither
NSW,
Analytical Laboratories, Asquith, Australia) re- by bleedingtunicatesthat had been exposed totoxicants
vealed that 5% v/v of a saturated solution of creosote in aquaria or by harvestingcellsthat had migrated from
contained 1 mg/1 total polycyclic aromatic hydrocar- cultured pharyngeal explants during in vitro exposures
bons(PAH)(napthalene, 280 /ig/1: anthracene, 250^g/1; ("emigrant hemocytes"; seeProliferativeactivityofio.\i-
phenanthrene. 150/wg/l; acenapthene, 93 /ig/1; remain- cant-treated tunicate cells section). In viability studies,
liinmgitP=AH1sMg</08w0 Meg/'d)-etNecotePdAinHsthe(pnroacrtmiaclalsequaawnattietrautsieodn hmeimdoecy(t0e.s1%(1v/Xv)10i6m/mmeld)iwaetreelystapirnieodrwtiothaneatlhyisidsi.umDberaod-
inaquaria. cells were detected by their increased red (800 nm) flu-
orescence reflecting the intercalation of ethidium bro-
Treatmentprotocols mide into cellular DNA. The relative frequencies ofdis-
Two forms oftoxicant treatment were applied. Tuni- tinct hemocyte subpopulationsweredetermined byana-
lyzingforwardangle versus90 light-scatterplots.
cate cells were exposed to toxicants in vitro to assess
effects on isolated tissues, and live tunicates were ex-
Proliferativeactivityoftoxicant-treatedtunicatecells
posed to toxicants in aquariato identifyeffectsthat were
derived from interactions between organ or physiologi- To quantify hemopoietic cell proliferation, tunicate
cal systems. Where possible, theeffectsoftoxicantswere tissue cultures were established by excising small por-
tested over a range of doses and at a number of time tions (2x2 mm) ofthe pharynx for explant culture in
points forexposure periods ofup to 9 days. For brevity, tunicate tissue culture medium (T-RPMI; Raftos and
onlyrepresentativedatareflectingtrendsin bothdose re- Cooper, 1990). Each 100 mlofT-RPMIcontained 10 ml
sponse and kinetic analysisare presented here. RPMI-1640 tissue culture medium (with sodium bicar-
64 D. RAFTOS AND A. HUTCHINSON
bonate, without L-glutamine; Sigma Chemicals, St. tunicatesandcultured overnight (15C)inT-RPMI con-
Louis, MO), 1 ml 20% w/v NaCl, 1 ml antibiotic stock taining various concentrations oftoxicants before being
solution (4 mg/ml strepMtomycinsulfate, 103 lU/ml peni- tested forcytotoxicactivity.
cillin sulfate), 100/il 1 L-glutamine, and 88 ml FSW. Cytotoxic activities of hemocytes were tested in two
Cultures were maintained at 15C without CO2 supple- assays that used either K.562 human chronic myeloge-
mentation. Undertheseconditionsexplantculturesnor- nous leukemia cells or rabbit red blood cells (RRBC) as
mally maintain cell viability and function for up to targets.Thecapacityofhemocytestokill K-562cellswas
70 days(RaftosandCooper, 1990). assessed by a modification ofthe method ofPeddie and
Forin vitroexposuretrials, explantswerecultured for Smith (1993). Hemocyte suspensions were adjusted to
upto8 daysin 96-well flat-bottomedtissuecultureplates 4 X 107 cells/ml in MAC; 50-100 n\ ofthese hemocyte
containing 200 jul/well T-RPMI and various concentra- suspensions were then mixed in round-bottomed 5-ml
tionsoftoxicants. Explantsweremovedtofresh medium flow cytometry tubes with equal volumes ofK-562 cells
every2-4 days. Afterappropriateexposureperiodsofup (4 X 10h cells/ml) suspended in marine saline (MS;
to8 days,explantswereincubated(overnight, 15C)with 12mA/ CamClM2-6H:O, 11mA/ KC1, 26mA/ MgCl2-
18.5MBq/ml 3H-thymidine (Amersham, NSW, Aus- 6H2O, 45 Tris, 38 mA/ HC1, 400 mA/ NaCl, pH
tralia, 740GBq/mmol). Non-incorporated 3H-thymi- 7.4). The cell mixtures were incubated at 15C for
dinewasremoved afterincubation byextensivewashing 90 min, stained with ethidium bromideand then imme-
in FSW. Explantswerethendigested(37C, overnight)in diately tested, using a FACScan flow cytometer, for the
2.0% w/v trypsin (Sigma Chemicals) to facilitate liquid uptake ofred fluorescence (800 nm). Specific cytotoxic
scintillation counting in Ecolite scintillation cocktail activitieswerecalculatedasthepercentageofdead K562
(ICN, Seven Hills, NSW, Australia)(Raftoset a/., 1991). cells in a particularsample minusthepercentage ofdead
Explants from tunicates exposed to toxicants in cells in controls that contained K562 cells but no hemo-
aquariawereexcised, incubatedimmediatelyinT-RPMI cytes. K562cellswereobtainedfromtheAmericanType
containing 18.5 MBq/ml 3H-thymidine (overnight, 15C), Tissue Culture Collection (Rockville, MD) and were
and then washed anddigested asdescribed above. grown in RPMI-1640 tissue culture medium. Immedi-
ately prior to their use in cytotoxicity assays, K562 cells
MS
Phagocvticactivityoftoxicant-exposedhemocytes were conditioned to the high tonicity of by incuba-
tion (30 min, 20C) in an intermediate saline solution
btreeeTanoteedtxetsputonsitechadeteptsohawtgeoorxceiycthaiancrtvsaecsittnievdaiqtiuynatrooifaF,tSunhWiecmaaontcedystthteehsiartfdrheoanmd- (61H220m,A/45CmaAC/lTr2i.s6,H23O8,mA1/1HmCAl/, 3K0C01,mA2/6NmaCMl,MpgHC7l.24;-
Peddieand Smith, 1993).
spiinetnivesistiroaondesjxupshotaserudvreetssot,e3tdoXxfir1c0aon6mtcsenlwolensr/eemxlapdowdisetedhdotuottuhnweiamcsaohtcieynstge.(3sFuosXr- wbeyrTethheewaambsiehltiethdyoodofnhocefemPotachryrrtioneuesglhltooTlreyitssea-lbR.uRf(f1Be9rC9e3d)w.assaHlqeiunmaeontc(iyTftBieSes;d
106 cells/ml in T-RPMI). Aliquots (200 n\) ofhemocyte 10 mA/Tris, 150 mA/NaCl, pH 7.4)andresuspendedin
csouvsepresnlsiiposns(2w2erXe c2u2ltmurme)d (f1o5rC)eitohnerau2thocl(aavqeudargliausms T2BXS1s0u6pcpelllesm/emnl.teOdnew-ithhun1d0rmedMCmiacrCo!li2t(erTsBSpe-rCawe)ltlooyfitehled
ehxepmooscuyrteess) woreroevewransihgehdt w(iinthvi4tr0o0e^x\poFsSurWes)b.efoArdehebreeinntg sequusaplenvsoiolnusmeweorfeRthReBnCin(c4ubXat1e0d7(c6e0llms/imnl,i3n7TCB)S-wCitah)ainn
oCvheermliaciadlsw;it5hX5010n6\yeyeaasstt/m(lB)atkheart'shaydeabsetentyppreepIaI,reSdigamnad c96u-bwaetlilonrotuhnedp-lbaotetstowmeerdetciesnstureifcuulgteudreanpdlat1e0s.0^A1ftoefrtihne-
opsonized with S. plicata plasma according to the resulting supernatant was transferred to flat-bottomed
mmeotvheoddboyfeBxetceknseitvael.w(a1s9h93i)n.gNwointihngFeSstWedafyteearstaw3e0r-emrien- plates so that the absorbance (405 nm) of hemoglobin
released from lysed cells could be quantified on a mi-
incubation (15C), and phagocytic activity was quanti-
croplate spectrophotometer. Specific cytotoxic activities
fied microscopically (Beck et al.. 1993; Kelly et til., were calculated as percentages relative to maximum re-
1993). lease (4 X 10" RRBC/well in H2O) and spontaneous re-
lease (4 X 10" RRBC/well in TBS-Ca. no hemocytes)
Cytotoxicactivityojtreatedcells
values.
Hemocytes from tunicates that had been exposed to
toxicants in aquaria were harvested in MAC and tested Statisticalanalysis
immediately in cytotoxicity assays. For /;; vitro Nontreatedcontrolswere included in all experiments.
exposures, hemocytes were harvested from nontreated A minimum offour tunicates were tested for each dose
TUNICATES AND MARINE POLLUTANTS 65
TableI copper (ug/ml)
PercentageviahililienofhemocylesafterNJaysn/invitrooraquarium 0.01 0.1 1 10 100
c.v/'r>\ureinavarietyojtoxicants
viablecells SEM(n>4)
Treatment Aquarium
Control(notreatment)
66 D. RAFTOS AND A. HUTCH1NSON
TUNICATES AND MARINE POLLUTANTS 67
TableIII
Phhaagg(ocyticactivities, relativetonontreaiedcontrols, "Ininicute
he<:mmocvle\thaihadbeenexposedtovarioustoxicantsforeither24hin
vitroorSdaysinaquaria
%ofnontreatedcontrol1 SEM(n>4)
Treatment
68 D. RAFTOS AND A. HUTCHINSON
TableIV
Effectiijrtreatmentwithvarioustoxicantseitherovernightfin vitro
exposure)orforSclays(aquariumexposures)oncytoloxicactivityof
tunicatehemocylestowardk-562orrabbitredbloodcells(RRBC)
%ofnontreatedcontrol1 SEM(n>4)
TUNICATES AND MARINE POLLUTANTS 69
B
70 D. RAFTOS AND A. HUTCHINSON
have been due to the rapid clearance ofdead hemocytes
//; vivo. Those dead cells may not have appeared in the
circulating hemolymph, and so may not have been de-
tectedin viabilityassays. Second,tunicatescould possess
mechanismsto detoxify, sequester, or prevent the pene-
tration ofcopper and creosote in vivo. This possibility is
not, however, supported by the observation that some
immunological reactionsweresimilarlyaffectedbyiden-
tical doses of toxicants applied in vitro and in aquaria
(e.g.. copper's inhibitory effect on cell proliferation).
Third, differences between in vitro and aquarium trials
mighthavebeenduetotheexistenceofcompensatoryor
interactive mechanisms that cannot operate in isolated
/// vitrosystems.
The latterexplanation issupported by differencesthat
wereevidentbetweentheeffectsofin vitroandaquarium
exposureson immunological parameters such as phago-
cytosis, cell proliferation, and cytotoxicity. Forinstance,
in ritm creosote treatment inhibited phagocytic activity
andcell proliferation, whereastunicatestreatedwithcre-
osote in aquaria had an enhanced capacity for phagocy-
tosis and a transient increase in proliferative activity.
Suchcontrastingresultsindicatethatsomeeffectsin vivo
may result from interactive mechanisms rather than
fromdirecttoxicitytowardtheresponsebeingexamined.
Creosote poisoning, for instance, may have stimulated
regulatoryactivitythat specificallyenhanced phagocyto-
sis and proliferation. Mechanisms that are capable of
such cellular regulation are well characterized in tuni-
cates. Regulatory molecules in the hemolymph can en-
hance phagocytosis and cell proliferation in a manner
analogous to the activities ofvertebrate cytokines (Beck
etai. 1993;Raftos, 1994; Raftostf ai. 1991).
The data also suggest that tunicates have mechanisms
TableV
Summaryoftheeffectsofdifferenttoxicantsonaninetyolresponses
thaiweretestedeitherinvitro(vit)orhyaquariumexposure
Toxicant/treatment
TUNICATES AND MARINE POLLUTANTS 71
inowitz, 1993; Wright, 1981). The regulatory activity cyclicaromatic hydrocarbon contaminated environments. Immii-
that may have activated cell proliferation might also nopharmacol. Immunotoxicol, 13:311-327.
haveaffected phagocyticactivity. Tunicatecytokine-like Fit/patrick, L.C., R. Sassani, B.J. Venables, and A.J. Goven. 1992.
moleculeshavepleiotropiceffectsthat includethesimul- CwoomrpmasraEtiisveeniatoxfioceintdyaoafnpdolLyucmhhlroirciiniastelderbriespthnesn.ylEsnvtiorotnh.ePeoalrltuht.-
taneous activation of phagocytosis and proliferation 77:65-79.
(Beck ela/.. 1993; Raftos, 1994; Raftosetat.. 1991). This George, S.G. 1990. Biochemical and cytological assessments of
role ofaltered hemocyte frequencies on immune func- metaltoxicity in marineanimals. Pp. 123-142 in HeavyAletalxin
tionsremainsspeculative, but iscurrently beingtested. llic Environment. R. W. Furness and P. S. Rainbow, eds. CRC
In conclusion, it is clearthat environmental contami- Press. BocaRaton,FL.
nantshave profoundeffectson immunological reactions Giamma,lsG..CS.R,CanPdresLs..EB.oRcaay.Rat19o8n7,.FL.Pollution Studies in MarineAni-
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National Research Council, U.S.A. 1972. PaniculatePolrcyclic Or-
Thisstudywasfunded in partbyagrant from theAus- ganic Mailer. U.S. National Academy of Sciences. Washington.
DC.
tralian Research Council and an Internal Research
Grant from the University ofTechnology, Sydney. We ParrCiynteolltoo,xiNc.,acVt.ivAirtiyzozfa,doMn.aCainmtmeastriantalai,shaenmdocDy.tMes.:Pparrorpienretllioe.so1f99t3h.e
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