Evaluation of a Reporter Gene System Biomarker for

303

ECOLOGY AND EVOLUTION Reference: Bid. Bull. 197: 303-306. (October

1999)

Evaluation of a Reporter Gene System Biomarker for Detecting Contamination in Tropical Marine Sediments Lisa M. Kerr (Biology Department, University of Massachusetts, 100 Morrissey Boston, Massachusetts 02125), Phillip S. Lobel’, and J. Mark Ingoglia2

Blvd.,

AhR complexbindsto the xenobioticresponsiveelementin the promoterregionof theCYPlAl gene,resultingin its transcription. Compoundsthat are not AhR ligands,suchasmetalsand pesticides,do not causeinductionin the system(1,7). Briefly, the RGS methodologyinvolves exposinghuman1OlL cells to 10 ~1 of solventextractsfor 16 hours(1, 5). Sedimentextractswere preparedaccordingto EPA method3540.The cellsare stablytransfected with a plasmidcontainingfirefly luciferaselinked to the humanCYPlAl promotersequence. Thispromotersequence contains 1800bp of flanking regulatoryDNA with three xenobiotic responsiveelements(1). Exposureto Ah receptorligandsinduces luciferaseactivity which is quantifiedwith a luminometer(7, 8). Previousstudieshave detexminedthat RGSdetectionlimits in sedimentare8 pg/gfor dioxin, 63 pg/gfor furan,6.2 to 7500rig/g for specificPAHs, and250rig/gfor a mixtureof PAHs(7). Solvent blankswere usedas negativecontrolswhile extractsfrom sedimentsspikedwith 2 rig/g TCDD were usedaspositive controls (n = 6). From the level of inductiongeneratedby eachsample, equivalentdosesof TCDD or benzo(a)pyrene(BaP) that would resultin the observedlevel of inductionwerecalculated.While the level of inductionis the samein eachsample,the amountof pure compound(eitherTCDD or BaP)requiredto producethe observed inductionlevel differs betweenthe compounds.Equivalentdoses for total PAHs anddioxins/furanscalculatedfrom RGSinduction are designatedas“RGS BaPEQ” and “RGS TEQ” respectively. For the GC/MS analyses,sediments were extractedusingEPA Method 3540.PAHs anddioxins/furansweremeasured usingEPA Methods 8270 and 8290. Detectionlimits were from 0.0638to 0.777pg/g for the 17 individual dioxin or furan congeners measured.Total PAHs measuredincluded 14 individual compounds with detectionlimits ranging from 19.1 to 40.7 rig/g. Toxicity equivalents(TEQs) basedon the 17 dioxin and furan congeners measured were calculatedusinghuman/mammalian toxic equivalency factors(TEFs; 9). TEQs calculatedfrom the chemicalanalysis of dioxins andfuransaredesignatedas “ChemTEQ.” Total PAHs measuredby chemicalmethodsare referredto as “Chem PAHS.” Linear regressionanalysiswas usedto determineif significant relationshipsexisted betweenthe RGS TEQ and Chem TEQ. Similarly, the relationshipbetweenChemPAH and RGS BaPEQwasdetermined.Data were log transformedto equalize variances. Therewasa significantstatisticalrelationship betweenChemTEQ andRGSTEQs(3 = 0.774;P < 0.001)(Fig. 1).However,41.9%of the pointsfall outsidethe 95% confklenceintervals.Additionally, betweenChemPAHs ’ BostonUniversityMarineProgram,MarineBiologicalLaboratory, therewasa significantstatisticalrelationship andRGSBaPEQ(r? = 0.564,P < 0.001).In thiscase,35.5%of the WoodsHole,Massachusetts 02543. pointsfall outsideof the 95%conlidenceintervals.While therewas *UnitedStatesAir Force,Environmental Restoration, HickamAFB, significantcovariancebetweenChemand RGS vahtes,the actual Hawaii96853. A majorchallengein conductingfield assessments of potential ecologicalimpactsis optimizingthe numberof samplesand the costs.This is especiallyimportantin light of the growingconcern over the presenceof persistentorganic contaminants,such as PCBs,dioxins, furans,and PAHs in sediments.A reportergene system(RGS)assaythat measures inductionof the CYPlAl gene andtranscriptionof P450enzymesystemsis often usedto assess potentialtoxicity of thesecompounds in environmentalsamples (1, 2,3). RGShasgainedacceptance asaninexpensive,rapid method for screeningenvironmentalsamples for contaminants (4, 5). The RGS approachhas beenvalidated in the laboratory with pure compounds,known chemicalmixtures,and from field-collected sediments by comparingRGSsystemresponse andchemicalconcentrations(1, 2, 3, 6). Our study differed from other validation studiesin two respects:we usedfield-collectedsedimentsover a wide range of contaminantconcentrationsand evaluatedRGS responseto sedimentsamplescontaininga mixture of dioxins, furans,and PAHs. With few exceptions,the previousvalidation studiesusingfield-collectedsamples usedfairly smallsamplesizes and generallyevaluatedone chemicalgroup (3, 6). Few studies have comparedlargenumbersof samplescontainingboth PAHs and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) over the range reportedhere.The purposeof this study wasto determineif there is a high correlationbetweenRGSresponse andchemistryresults from the samesamples.If this provesto be the case,the assay could be used to screenlarge areasat a relatively low cost. Samplesexhibiting high responses could be targetedfor further characterizationusingmoreprecise,but costly, GC/MS methods. Matched sedimentsamples(n = 31) were collectedoff the northwesternshore of JohnstonIsland, adjacent to potential sourcesof PAHs and2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) (Site 1), andat sitesfarther removedfrom the potentialcontaminantsources(Sites3,4). To ensureblind analysisof the samples, P450 RGS assayswere conductedby MEC Analytical Systems (Carlsbad,California) and ColumbiaAnalytical Services(Vista, California)while chemicalanalyseswerecompletedby the Toxic ContaminantResearchLaboratory at Wright State University (Dayton, Ohio). The RGS assayusedin this study wasdevelopedby Anderson et al. (1) anddetailedmethodshavebeendescribed(1, 7). Induction in the assayis dependentuponthe aryl hydrocarbonreceptor (AhR) activation pathway. AhR ligands,includingplanarPCBs, PAHs, andTCDD, bind to AhR, activatingit andresultingin its translocation to thenucleusof the cell. In thenucleus,the activated

REPORTS

304

FROM

THE

ME%L GENERAL

A. 10000

CHEM

PAHs

(nglg)

B.

CHEM

TEQ (pg/g)

Figure 1. (A) Comparison of chemically derived total PAH concentration (Chem PAHs) to RGS estimates of benzo(a)pyrene equivalents (RGS BaPEQs) 3 = 0.564 P < 0.001. (B) Comparison of chemically &rived TEQs (Chem TEQ) to P450 RGS estimated TEQs (RGS TEQ) ? = 0.564P < 0.001. Best fit regression lines are shown with 95% confidence intervals.

magnitude of the valuesdiffered.RGSTEQswere16 to lo4 times higherthanChemTEQs(TableI). Similarly,RGSBaPEQswere10 to Iti timeshigherthanChemPAHs. The levelsof inductionandthe resultantestimates of RGSTEQ and RGS BaPEQdecreasedwith distancefrom shore(Table I). Thispatternalsoheldfor ChemTEQ andChemPAHs, with those samples with the highestconcentrations beingthe closestto shore (Site 1).Threesamples exceedingecologicalscreeninglevelswere foundclosestto shore.One sampleexceededthe effectsrangelow (ER-L) screeninglevels for ChemPAHs, includingtotal PAHs, low andhigh molecularweight PAHs, andnine individual PAHs (10).Two additionalsamples from site1 hadcontaminantconcentrationsexceedingecologicalscreeninglevels.Thesesamples with

SCIENTIFIC

MEETINGS

ChemTEQs of 68.24pg/g and901.27pg/g exceededthe low and high risk to aquaticlife screeningvaluesrespectively(11). Whenusingthe RGSassayasa screeningmethod,the samples with the highestresponses or inductionlevelsmight be “chosen” asthe samplesexpectedto containthe highestcontaminantconcentrations.In this study,this wasthe case,andthe threesamples with the highestcontaminantconcentrationswould have been detected.However, there are two importantpoints: 1) induction washigherthan that of the positivecontrols(aroundlOO-fold)in eight samples(129- to 316-fold), and 2) the sampleswith the highestoverall inductionlevelswere not necessarilythe samples with the highestdioxin or PAH concentrations. Additionally, some samplesexhibited significantRGS induction,althoughvery low levelsof PAHs, dioxins,or furansweredetected.Theseresponses could be consideredfalse positives.A false negativecould be identified only if arbitrary limits were set on the numberof samplesfurther characterized.For example,if budgetconstraints limited the numberof samplesthat could be chemicallycharacterized to five samplesand we choseto characterizeonly those sampleswith the highestRGS response,the samplewith the highestconcentrationof PAHs would not have been detected, resultingin a falsenegative. Onepotentialexplanationfor the variableRGSresponse in this study is inductionby othercompoundsnot measured by GC/MS. Other chemicalsthat may inducethe RGS systemincludeplanar PCBsaswell assomedioxin congeners not measured in thisstudy. The contributionof PCBsto inductionis unlikely sinceprevious samplingeventsat the samesitesfound total PCB concentrations belowthe 10rig/g RGSdetectionlimit. Another explanationsuggeststhat in tropical environments, P450 activity is higher in somefishesand may be relatedto a herbivorousdiet (12). This suggests further consideration of the presence of naturallyoccurringtoxins suchasciguatoxinsor other allelochemicals potentiallyinducingthe RGSassayor interfering with cell function. Furthermore,coralreefenvironments mayhave different sedimentcharacteristicsdue to physicaland chemical factors that may affect bioavailability. The RGS assaydetects compoundsthat may or may not be specificallymeasured by GC/MS andassesses the synergisticand/orantagonistic effectsof the constituentsin the mixture. This study found significantco-variancebetweenChemTEQ andRGSTEQ aswell asbetweenChemPAHs andRGSBaPEQs. However,therewasa high level of variability (morethan35%of values fall outsidethe 95% CI); and of the sampleswith the highestRGS response(greaterthan positive controls),a high percentage(5 of 8) couldbe identifiedasfalsepositives.Qualitatively, the RGSassayrevealeda contaminantgradienton a scale of hundredsof meters.We concludethat this methodis usefulas a broadareaassessment tool for screeningpurposes, providedthat data areinterpretedcarefully. Useof thisscreeningmethodis substantiallycomplicated by the presenceof multiple compounds, as at JohnstonAtoll. This may includenaturalor other anthropogeniccompounds that werenot chemicallymeasured in the GC/MS analysis.While the response to thesevariouscompounds may becomplex,thevalueof theRGS assayis that it gives an integratedandmorebiologicallyrelevant response thanchemistryalone.Additionally, given theexpense of GC/MS analysisof dioxin congeners (>$lOOO/sample), useof the

ECOLOGY

ontaminant

concentrations

in sediment

samples from

030 064 074 076 086 088 095 104 108 120” 217 225b 233 234 238 242 248 249 254 256’ 257 307 013 027 043 118 243 038 124 105 119 Control”

Control Control Control Control

EVOLUTION

Table

I

the west end of Johnston

Atoll

Site

Chem EQ e&

J-EQe&

1’

0.01 0.95 0.40 0.02 2.04 7.29 OSXI 13.63 0.00 2.35 0.04 68.27 0.78 4.44 6.43 2.38 0.05 1.26 9.13 901.29 5.87 2.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2OOO.W 2000.00 2OcQOa 2000.00 2000.00 2000.00

2400 18200 1700 1800 7800 31200 1200 6400 1100 13200 3400 21300 4400 30700 7400 5200 5500 7300 18700 14700 12800 2600 700 600 300 200 200 200 400 200 200

1

1

:2 3 3 3 3 4 4 4 4

305

compared

RGS

Sample

Number

AND

to responses Chem

Pm n&3 21.7* 158.5* 1.3* 77.1* 937.9 1125.0 11.6* 8.0* 2.1* 9711.0 151.7* 64.9* 124.5* 1988.0 41.5 343.2* 174.8 nd 906.3 50.5* 2383 21.4* 2.4* 1.4* 2.8* 2.4* 1.4* 3.2* 2.4* 0.8* nd

generated

by the RGS assay RGS

BeEQrig/p

Induction

2000 15200 1400 1500 6500 26000 1000 5300 900 11000 2800 17700 3700 25600 6100 4400 4600 6100 15600 12300 10600 2200 600 500 300 loo 200 200 400 400 200

25 183 17 18 79 316 12 64 11 133 34 218 45 308 74 53 55 73 188 147 129 26 6 3 2 2 2 5 2 2 79 101 90 77 81 137

’ Site 1 is adjacent to the former herbicide orange storage site and born pits on Johnston Island. ’ Sites 3 and 4 are across the shipping channel from site 1, approximately iCOO m from shore. ’ Exceeds screening level for PAHs (Effects Range-Low for total PAHs = 4022 rig/g). b Exceeds low risk screening limit for TCDD (60 pg/g). ’ Exceeds high risk screening limit for TCDD (100 pg/g). d Positive Controls. [PAHs] below quantifiable limit. nd-none detected.

l

RGS assay ($150/sample) shows potential for possible cost savings through incremental, phased use in combination with GUMS methods. As site specific complexities and relationships between the two methods and the matrix are further defined, the understanding of and confidence in the RGS results can improve. This can lead to reduced need for GUMS analyses and increased emphasis on RGS results. Annual long-term monitoring, which may span decades, lends itself to such incremental cost savings

through the development of large data sets and cumulative experience gained over time. This research supported by Army Research Office grant DAAG 55-98-l-0304 for the Johnston Atoll Reef Study.

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