Biomarkers of PAH exposure in oyster toadfish (Opsanis tau) from the Elizabeth River, Virginia

Environmental Sciences, 2, 3 (1993) 161-177MYU, Tokyo

ES034

Biomarkers of PAH Exposure in Oyster Toadfish(Opsanis tau) from the Elizabeth River, Virginia

Tracy K. Collier!, John E. Stein!, Anders Goks0yrZ,

Mark S. Myers!, Jay W. Gooch3*, Robert J. Huggett4

and Usha Varanasi!

'Environmental Conservation Division, Northwest Fisheries Center,National Marine Fisheries Service, National Oceanic and Atmospheric Administration,

2725 Montlake Blvd. E., Seattle, WA 98112, U.S.A.2Laboratory of Marine Molecular Biology, University of Bergen, RIB N-5020, Bergen, Norway

3Chesapeake Biological Laboratory, University of Maryland, P.O. Box 38,Solomons, MD 20688, U.S.A.

4Division of Chemistry and Toxicology, School of Marine Science,Virginia Institute of Marine Science, The College of William and Mary,

P.O. Box 1346, Gloucester Point, VA 23062, U.S.A.

(Received April 6, 1993; accepted June 7, 1993)

Key words: cytochrome P450 lA, DNA adducts, bile metabolites, PAR, biomarkers, fish

Surficial sediments of the Elizabeth River, Virginia, USA show a pronouncedgradient of polycyclic aromatic hydrocarbon (PAH) contamination. Oystertoadfish (Opsanis tau), which live in the Elizabeth River, are bottom-dwelling fishwith limited migratory movement. This presents an opportunity to evaluatebiomarkers of PAH exposure in a feral fish. The biomarkers measured in the pre­sent study were levels of fluorescent aromatic compounds (FACs) in bile, whicharise largely from metabolism of PAHs; hepatic levels of hydrophobic xenobiotic­DNA adducts, detected by 32P-postlabeling; and hepatic monooxygenase activitiescatalyzed by cytochrome P450 1A (CYP1A), an enzyme known to be readily in­duced in fish exposed to PAHs and many other organic xenobiotic compounds.Toadfish were sampled from six sites in and adjacent to the Elizabeth River, withlevels of PAHs in the sediments at the capture sites ranging from less than 10 toalmost 100,000 ppb on a dry weight basis. Levels of biliary FACs and hepaticxenobiotic-DNA adducts were highly correlated with levels of PAHs in sediments,

*Current address: Environmental Science Department, The Procter and Gamble Co., Cincinnati, OR45217, U.S.A.

161

•

1. Introduction

showing the usefulness of these measures for assessing PAH exposure in feral fish.Hepatic CYPIA-associated monooxygenase activities, however, were near or belowlimits of detection and showed no differences with respect to site of capture.Immunochemical assay of hepatic CYPIA, whether by Western blot or im­munohistochemical localization, corroborated the finding of weak expression ofCYPIA in the fish sampled in this study. An interesting finding was the inability todetect any expression of CYPIA in normal liver parenchyma (hepatocytes), which isin contrast to other fish species studied thus far.

Polycyclic aromatic hydrocarbons (PAHs) can elicit a wide variety of seriouseffects in fish, including induction of neoplasia and reproductive dysfunction. Theseeffects have been noted in a large number of laboratory studies, with some cor­roboration in field studies,o-3) Establishing causal relationships between PAH ex­posure and serious biological effects in field studies has been hampered by the lackof methods for directly determining PAH exposure in fish, in that the extensivemetabolism of PAHs by fish makes standard chemical analyses of fish tissues forlevels of parent PAHs of little value. (4) Thus, many investigators studying possiblebiological impacts of environmental PAH contamination have had to rely onanalyses of PAHs in sediments or water at the site of capture. However, becausemany fish species are highly mobile, it is desirable to be able to assess the PAH ex­posure of individual fish, rather than measuring PAHs in the environment fromwhere the fish are captured.

In recent years, our knowledge of the mechanisms and pathways of PAHmetabolism(4) has prompted the development of methods for assessing PAH ex­posure in individual fish. One of these methods, the measurement of fluorescentaromatic compounds (FACs) in bile, (5-7) relies on the fact that many fluorescentmetabolites of PAHs are primarily excreted via the hepatobiliary system in fish. Inaddition, electrophilic intermediates of high-molecular-weight PAHs can formcovalent adducts with nucleophilic centers within biological macromolecules, in­cluding hepatic DNA. A method utilizing 32p postlabeling of deoxynucleotides andchromatographic separation of adducted nucleotides from normal nucleotides ishighly sensitive for detecting bulky hydrophobic xenobiotic-DNA adducts, such asthose formed from PAHs. (8) Laboratory studies with fish have shown that levels ofboth biliary FACs and hepatic xenobiotic-DNA adducts are highly dose-responsiveto PAH exposure, and the formation of xenobiotic-DNA adducts additionally pro­vides an indication of genotoxic exposure.(9,lO)

It is also well known that hepatic cytochrome P450 lA (CYPIA), together withthe associated monooxygenase activities aryl hydrocarbon hydroxylase (AHH) and

Tracy K. Collier et al.Environmental Sciences, 2, 3 (1993) 161-177162

ethoxyresorufin-O-deethylase (EROD), can be rapidly induced in many fish speciesafter exposure to a wide variety of organic contaminantsy1,12) Similar to biliaryFACs and hepatic DNA-xenobiotic adducts, hepatic CYP1A has been shown to bedose-responsive in fish exposed to PAHs in laboratory studiesYO,13-1S) Thus the in­duction of CYP1A represents an early biological effect which may be of use inestimating PAH exposure of fish sampled in field studies.

The current study represents an investigation of the responses of each of thesethree biomarkers in a feral fish species sampled from sites contaminated to differentdegrees with P AHs. Because the specific question to be answered was whether thesebiomarkers of PAH exposure would reflect the PAH contamination at the site ofcapture, it was desirable to evaluate them at sites with a gradient of PAH contamina­tion, and in a fish species with limited migratory patterns. Oyster toadfish living inand near the Elizabeth River , VA, USA meet these criteria. Surficial sediments inthe Elizabeth River show a defined gradient of contamination by P AHs, with sedi­ment levels ranging from less than 10 to almost 100,000 ppb on a dry weightbasis. (16-18) Oyster toadfish are common bottom-dwelling fish in this area, and they aresluggish swimmers which do not appear to migrate extensively.(19-21) Accordingly,the successful testing of biomarkers of PAH exposure in this situation should pro­vide a useful validation to support their use in determining the PAH exposurehistory of individual fish.

Because of the large range of PAH levels in sediments from the Elizabeth River,this study also presented an opportunity to further evaluate a recently developedHPLC/fluorescence screening method for estimating levels of total PAHs in sedi­ment. (22) Such measurements are increasingly needed because of the comparativelylong time required for standard chemical analyses, and the demand by resource andregulatory agencies for rapid assessment of possible environmental contamination.

r et al.

. fish.lelowIture.

I

im- 'T)ll ofity to

I

ich is I;,1

:riousrhese: cor­f! ex­~lack

:nsive~s forssiblelyoncauseH ex­from

Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 163

PARf! ex­:scent:scent;h.lnforms, in­s and:les islch asels ofmsiver pro-

. with

) and

2. Methods

2.1 Sampling of sediment and /ishOyster toadfish were captured by trawling and in fish traps in October at several

sites in the Elizabeth River, VA and at one site in the York River, VA, USA (Fig. 1).One oyster toadfish was also captured by trawling from Raritan Bay, NY, USA inOctober. Many of the animals were noted as being mature (i.e., females were egg­bearing and males had developed testes). Unfortunately, because the gonads of thefish sampled in this study were not examined histologically, their specific reproduc­tive status is not known. It had been anticipated that the animals sampled in thisstudy would not be in spawning condition, based on a report that spawning activityof toadfish in Chesapeake Bay peaks in June and JUly.(19) However, there has alsobeen a report of eggbearing female oyster toadfish being found as late as October 25in Chesapeake Bay. (20) The fish were necropsied on board immediately after capture,and liver and bile were removed and frozen in liquid nitrogen. These samples werestored at - 80° until analysis. Some liver samples were also taken and fixed in 10%

2.2 Analyses of sedimentSediment samples were screened for the presence of PAHs by means of a recent­

ly developed technique which involves sonic extraction followed by size-exclusion

neutral buffered formalin for performing immunohistochemical localization ofCYP1A. Sediment samples for PAH screening were obtained by Smith-MacIntyregrab and subsamples of the sediment samples were analyzed by Van Veld et al.(16)Sediment samples were stored at -20 0 until analysis.

Tracy K. Collier et al.

Fig. 1. Sampling sites in and adjacent to the Elizabeth River, Virginia.

Environmental Sciences, 2, 3 (1993) 161-177164

lier et al. Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 165

ation of:acIntyreI et al. (16)

a recent­~xclusion

high-pressure liquid chromatographyYZ) The eluate was monitored fluorimetrically

at excitation and emission wavelengths appropriate for 4- to 6-ring aromatic com­pounds (i.e., 380 nm excitation, 430 nm emission), and the results were quantitatedbased on fluorescence of a standard solution of benzo[a]pyrene(BaP). Analyses ofsediment for total PAHs were previously reported by Van Veld et al. (16) Those data,which were obtained using Soxhlet extraction, gel permeation chromatographycleanup, and capillary gas chromatography, are used here for statistical comparisonto biomarker results.

2.3 Analyses of biomarkers2.3.1 FACs in bile

Bile samples were analyzed for FACs according to the method of Krahn et aI., (5)

with minor modification yO) Excitation and emission wavelengths used were ap­propriate for 4- to 6-ring aromatic compounds, (Z3) and the results were quantitatedbased on fluorescence of a standard solution of BaP. Total biliary protein was esti­mated according to the method of Lowry et al., (Z4) using bovine serum albumin asthe standard. Values were reported in terms of protein to reduce variability due tofluctuation in biliary water content.(lO)2.3.2 Hepatic xenobiotic-DNA adducts

Levels of xenobiotic-DNA adducts were measured by means of 3Zp postlabelingassay with adduct enrichment by n-butanol extraction, as described by Stein et al.(Z5)Adduct levels are reported as nanomoles of adducts per mole of nucleotides.2.3.3 Hepatic cytochrome P450

Hepatic microsomal AHH activities were measured as described by Collier etal.,(Z6) using 14C_BaP as the primary substrate. Hepatic microsomal EROD activitieswere measured according to a spectrophotometric method, (Z7) using the extinctioncoefficient for resorufin of 73 mM-1cm-). Immunochemical analyses for thepresence of microsomal proteins cross-reacting to anti-cod CYPIA(Z8) and to anti­rainbow trout CYPcon [a constitutive form, most probably CYP3] were performedby means of SDS-PAGE and Western blotting, as described by Goks0yr et al.(Z8) Im­munohistochemicallocalization of cytochrome CYPIA in formalin-fixed liver sec­tions was carried out according to methods described by HUS0Y et al.(Z9) and Myerset al.(30) Briefly, liver sections cut to 4-5,um were deparaffinized and hydrated, andendogenous peroxidase activity was blocked with HzOz. Sections were incubatedovernight with polyclonal rabbit anti-cod P450 lA as the primary antibody, andavidin-biotin techniques were used to visualize cross-reacting areas, with DAB­NiCh peroxidase as the chromogenic reactant. Both positive and negative controltissue sections (from English sole) were assayed immunohistochemically at the sametime (results not shown).

Table 1

Relative fluorescence of extracts of sediments collected from five sites in the Elizabeth River and areference site in the- York River. a

3.2 Analyses of biomarkers3.2.1 FACs in bile

Levels of FACs in bile followed the same gradient as concentrations of PAHs insediments at each of the stations, with lowest values found at the station at themouth of the York River and at the station nearest the mouth of the ElizabethRiver, and the highest levels of FACs in the bile of oyster toadfish from Station 5(Table 2). ANOVA of the log-transformed FAC data showed that Stations 2 and 3could not be differentiated (p>0.05) from each other, nor could Stations 4,5, or 6.However, levels of FACs in bile were significantly different among other sites.Regression of the FAC values against the concentrations of PAHs in sediments atthe capture sites showed a very strong correlation (p < 0.001, r2 = 0.95; Fig. 3).

3.1 Sediment chemistryThe levels of fluorescent 4-5-ring PAHs, determined using a rapid HPLC screen­

ing method, showed a clear gradient of contamination in surficial sediment samples(Table 1), with the lowest fluorescence values measured in the extract of the sedi­ment sample collected from the mouth of the York River (Station 1). Levels offluorescence in sediment extracts increased progressively upstream from the mouthof the Elizabeth River to Station 5, a site near an old creosote plant. Decreasedlevels of fluorescence were measured in the sediment extract from Station 6, relativeto Station 5. Moreover, a wide range of values was seen, with sediment samplesfrom the relatively uncontaminated York River (Station 1) showing fluorescenceequivalent to only 1 picogram BaP per gram sediment, compared to 88 nanogramsper gram at Station 5. The values obtained using the screening technique were veryclosely correlated (p < 0.001, r2 = 0.85) with previously published(16) results ofGC/FID analyses of PAHs in these same sediment samples (Fig. 2). Because thescreening technique is rapid compared to more detailed chemical analysis ofaromatic hydrocarbons in sediments, (22) these results provide additional impetus touse this screening method for prioritizing more detailed analyses of large numbersof samples, or even in lieu of detailed chemical analyses.

6

8.9

5

88

Tracy K. Collier et al.

4

10

3

1.3

2

0.480.001

Environmental Sciences, 2, 3 (1993) 161-177

StationRelativefluorescenceb

3. Results

166

'Sites as shown in Fig. 1.

bng benzo[a]pyrene equivalents/ g wet sediment; single determination per sample.

ier et al. Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 167

Fig. 2. Relationship between levels of PAHs in surficial sediment measured by means of capillary gaschromatography(l6) and levels of fluorescence in organic extracts measured by means of HPLC/fluorescence (380 nm excitation/430 nm emission), and quantitated using a benzo[a]pyrene (BaP) stan­dard.

100 5 •

100000

3•

100 1000 10000

PAHs in sediment(ng/g dry wt)

r2=O.85p<O.001

10

Z'3:-Q)Q) 10u3:

5iEUClt/l--Q)t/l

1... -or:::::::IJ!!-as->Q)'-> ::::I .1.- C"1OQ)'Q)D.D:as

III.01Cl.s

.0011

icreen­lmplese sedi­vels ofmouthTeasedelativelmples:scence)gramsre verylIlts oflse thevsis ofetus toImbers

er and a

6

8.9

Table 2

Levels of fluorescent aromatic compounds (FACs) in bile and levels of xenobiotic-ONA adducts(Adducts) and activities of ethoxyresorufin-O-deethylase (EROO) in liver of oyster toadfish from fivesites in the Elizabeth River and a reference site (Station 1) in the York River.'

Station 2 3 4 5 6

FACsb 120±47(3)' 320±61(5) 490 ± 75(10) 1200± 180(8) 1600 ± 260(10) 1200 ± 310(7)Adducts 5.3±0.56(5) 14±4.1(5) 37 ± 11(5) 47 ± 8.3(5) 140±20(5) 96±29(5)EROO 0.15±0.1O(6) NOd NO NO 0.13 ±0.05(4) 0.15 ±0.07(6)

AHsinat the

izabethation 5Zand 35, or 6.r sites.lents atig. 3).

'Sites as shown in Fig. 1. Values are mean±SE(n).bUnits for the different measures were as follows:

FACs-ng benzo[a]pyrene equivalents/mg biliary proteinAdducts-nmoles adducts / mole nucleotides

EROO-nmoles resorufin formed/ mg microsomal protein/ min.'Values at different stations that have a common underline were not significantly different after statisticalanalysis of the log-transformed data by means of ANOVA and Fisher's protected least significantdifference test.

dNO-not determined. Hepatic aryl hydrocarbon hydroxylase activities, which are also primarily catalyz­ed by CYPIA in fish, were measured in 3-5 fish from each site and were always below the limit of detec­tion of 0.005 nmoles benzo[a]pyrene metabolized/mg microsomal protein/min.

Fig. 4. Relationship between levels of DNA-xenobiotic adducts in liver of oyster toadfish measuredand levels of PAHs in sediments (measured by means of capillary gas chromatography(l6»).

Fig. 3. Relationship between levels of fluorescent aromatic compounds (FACs) in bile of oystertoadfish and levels of PAHs in sediments (measured by means of capillary gas chromatography(16»).

5

100000

100000

Tracy K. Collier et al.

r2=O.95p<O.001

r2=O.97p<O.001

100 1000 10000PAHs in sediment

(ng/g dry wt)

100 1000 10000

PAHs in sediment(ng/g dry wt)

10

101+-""-'~TrnJ"~-"""""""nr--'-~rnTT~-""'C"TTr""--'-r-r-TTTTTI

1

1000

~-60''g ~ 100.~ .Q--00:DE0::::cOQ)E>:< C 10<C-ZC

C 10000:§o...c.~

.!2~:D 1000.- 0).Q EC __.- I/)1/)­OC<C~LLas

.~ 100:::JC"Q)

Q.asm0)C- 10 +-.--r-r"""'''''---'--'-'''''''''',...--.-..,...,.-rrnnr--.-..,...,..,..,.."nr--.-..,...,........."

1

Environmental Sciences, 2, 3 (1993) 161-177168

lier et al. Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 169

)f oysterhy06)).

3.2.2 Xenobiotic-DNA adductsThe levels of xenobiotic-DNA adducts in the livers of the oyster toadfish sampl­

ed from the Elizabeth River followed the gradient of PAH contamination in thesediments at the capture sites very closely (p < 0.001, r2 = 0.97; Table 2, Fig. 4).ANOVA of the log-transformed adduct data also showed clear site differences, withStations 5 and 6 being different from all other sites, Stations 3 and 4 being differentfrom all other sites, and Station 2 and Station 1 each being different from all othersites (Table 2).3.2.3 Cytochrome P450

There were no differences between sites for any measurements of CYP1A in thisstudy. Hepatic AHH activities were undetected « 5 pmoles BaP metabolized perminute per mg microsomal protein) in all samples analyzed, and hepatic EROD ac­tivities were very near the limit of detection for the spectrophotometric method used(Table 2), even though spectral analyses for total CYP showed levels of around0.45 nmoles/mg microsomal protein, with no evidence of degradation tocytochrome P420 (data not shown). Immunochemical analysis by means of SDS­PAGE and Western blot of a hepatic microsomal suspension from the toadfish cap­tured in Raritan Bay showed a faint cross-reaction between an antibody to codCYP1A and a protein with an estimated molecular weight of 57.4 KD (Fig. 5(a».

2 3

A

4 5 2 3

B

4 5

measured

Fig. 5. Western blots of hepatic microsomal suspension of an oyster toadfish captured in Raritan Bay,New York. A) Rabbit anti-cod CYPIA IgG used for immunoassay. Lanes 1, 2, 3, and 4 contained 2.5,5,10, and 20llg, respectively, of microsomal protein, and Lane 5 contained a prestained low-molecular­weight standard. B) Rabbit anti-trout CYPcon IgG used for immunoassay. Lane 1 contained a prestain­ed low-molecular-weight standard, and Lanes 2,3,4, and 5 contained 2.5,5, 10, and 20 Ilg, respectively,of microsbmal protein.

Fig. 6. Immunohistochemical localization of CYPIA protein in liver of an oyster toadfish from Station5 in the Elizabeth River. Primary antibody was rabbit anti-cod CYPIA IgG, developed using an avidin­biotin peroxidase method. A) Note complete absence of staining of the liver parenchyma (hepatocytes),with arrows pointing to bile preductules. B) Note faint staining of the exocrine pancreatic ducts (smallerarrows) and blood vessel endothelium (larger arrow). Similar staining patterns were observed in liver ofanother toadfish from this site, and livers of two toadfish from Station I, the least-contaminated site.

This same microsomal suspension also contained apparently higher levels (based onintensity of staining) of a CYP (MW 52 KD) which cross-reacted with an antibodyto CYPcon from rainbow trout (Fig. 5(b». Immunohistochemical analysis (Fig. 6)of liver from toadfish from Station 5 did not show any increased staining by anti­cod CYPIA compared to toadfish from Station 1. There was no discernible stainingof the liver parenchyma (e.g., hepatocytes) in any of the toadfish examined (Fig.6(a». Minor staining was observed (Fig. 6(b» in the intrahepatic exocrine pan­creatic ducts, endothelium of blood vessels associated with the exocrine pancreas,and occasionally in the bile canaliculi and epithelium of the biliary preductules andductules.

Tracy K. Collier et al.

A

B

Environmental Sciences, 2, 3 (1993) 161-177170

ier et al. Environmental Sciences, 2,3 (1993) 161-177

4. Discussion

Tracy K. Collier et al. 171

StationI avidin­ocytes),(smallerliver of

:ed site.

,ed on:ibody=<'ig. 6){anti­ainingI (Fig.~ pan­creas,~s and

The present results demonstrate the utility of the bile fluorescence method fordetermining exposure of fish to PAHs in field studies. Levels of biliary FACs ofoyster toadfish were highly correlated with PAH contamination of the surficialsediments, over a gradient of PAH concentrations spanning four orders ofmagnitude. Other studies utilizing flatfish from Puget Sound, WA have not alwaysshown such a consistent relationship between levels of FACs in bile and levels ofPAHs in sediments at the site of capture. (6,25) Because levels of FACs in bile areshown to decrease rapidly after exposure of fish to PAHs,(lO) it can be hypothesizedthat the lower degree of correlation reported previously may be at least partially dueto movement of fish through areas of differing levels of sediment contamination.Thus, the lack of extensive migration reported for oyster toadfish could be a signifi­cant factor in the excellent correlations found in this study between levels of sedi­ment PAHs and biliary FACs. Because GC/MS analysis has shown the presence ofnumerous metabolites of PAHs in the bile of fish from urban areas(?) and fish expos­ed to petroleum following the EXXON Valdez oil spill,(31) it seems clear that the bilefluorescence method is one of the best methods for determining recent PAH ex­posure in organisms that rapidly metabolize PAHs. Moreover, an additionalstrength of this method is that by changing excitation/emission wavelengths,analyses can be adapted for assessment of different types of PAH exposure, such asfrom urban runoff or from petroleum exposure following an oil Spill.(31,32)

Levels of xenobiotic-DNA adducts in liver of toadfish were also highly cor­related with levels of PAHs in surficial sediments at the site of capture. This findingis consistent with previous studies showing that the levels of xenobiotic-DNA ad­ducts in fish liver are indicators of exposure to genotoxic compounds, such as high­molecular-weight PAHs. (9,25,33) Additionally, a recent study has shown that thelevels of hepatic DNA adducts in English sole exposed to benzo[a]pyrene increasewith multiple doses and that a substantial portion (32% of maximum levels) washighly persistent. (34) These results suggest that hepatic DNA adducts may also serveas indicators of cumulative PAH exposure. Thus, given that toadfish are not highlymobile, the measurement of xenobiotic-DNA adducts in these fish should provide auseful measure of the genotoxic potential of sediments from a given area. It is im­portant to note that the measurement of xenobiotic-DNA adducts provides an in­tegration of several biological processes involved in determining genotoxicity, in­cluding uptake, activation, and detoxication of xenobiotic compounds.

In the current study we did not detect any alterations in CYP1A-associated en­zyme activity in hepatic microsomes of oyster toadfish sampled from the differentsites in and adjacent to the Elizabeth River. Activities were consistently very low,with AHH activities being undetectable using a radiometric method, and EROD ac­tivities being very near the detection limit for the spectrophotometric method used.The CYP1A enzyme system in fish is generally acknowledged to be inducible by a

wide range of aromatic compounds, including PAHs,(11,12) and Van Veld et al.(16)clearly demonstrated induction of CYP1A in both hepatic and intestinalmicrosomes of spot (Leiostomus xanthurus) captured at the same time and at thesame sites as the oyster toadfish in the current study. Thus the inability to detectchanges in hepatic CYP1A-associated enzyme activity in oyster toadfish exposed tosuch a clear gradient of PAH was a surprising result of the current study, especiallyconsidering that Guobaitis et al. (35) had previously shown induction of hepatic AHHactivity in oyster toadfish by 3-methylcholanthrene (3MC) and j1-naphthoflavone,classic inducers of CYP1A in fish. Additionally, Milling and Maddock(36) showed anapparent induction of CYP1A in oyster toadfish exposed to 3MC, as evinced by in­creased mutagenicity of BaP mediated by liver 9000 g supernatants in theSalmonella assay. Interestingly, Milling and Maddock(36) found no apparent effectof treatment with Aroclor 1254, a PCB mixture shown to induce CYP1A activitiesin many other fish speciesyo,12) The current result might be partially due to the sex­ual maturity of many of the sampled fish, as gonadal maturation and spawning canhave substantial effects on levels and activities of CYP1A in many fish species, oftenresulting in profoundly depressed hepatic AHH and EROD activities.(2,37,38)However, these enzyme activities are generally still inducible by exposure of spawn­ing fish to organic contaminants, (26,39-41) and the depression of CYP1A is usuallymore pronounced in female fish than in males, as estradiol has a suppressive effectwhereas testosterone can have a stimulatory effect on CYP1A. (12) Nonetheless, wedid not see any induction in any of the animals we examined, including males andthose females which were not noted as being mature. In the studies by Guobaitis etal. (35) and Milling and Maddock, (36) the sexual maturities of the toadfish studied werenot given. Further study of the CYP1A system in the oyster toadfish, including howit may be affected by spawning status and levels of steroid hormones, is essential ifthe current result is to be adequately explained. We have considered other plausiblereasons for the failure to detect induced CYP1A at the more contaminated sites. Itis unlikely that the failure to detect changes in CYP1A-associated catalytic activitywas due to degradation of the protein during sample handling, because 1) im­munochemical analysis by Western blot showed very low levels of proteins cross­reacting to an anticod CYP1A but apparently higher levels of protein cross-reactingto an antitrout CYPcon [most likely a member of the CYP3 family(42)]; 2) there wasno discernible staining of hepatocytes in toadfish from either the most or leastcontaminated site when liver sections were analyzed by means of im­munohistochemistry using an anticod CYP 1A as the primary antibody; and 3) spec­tral analyses of total CYP showed an abundant CYP pool (approx. 0.45nmoles / mg) and little evidence of degradation to cytochrome P420 (data notshown); and 4) analyses (by Bruce Woodin and John Stegeman of Woods HoleOceanographic Institute) of some of these same samples, using monoclonal an­tibodies (MAb 1-12-3) to CYP1A isolated from the scup, showed very low cross­reactivity and no site differences. We have recently analyzed hepatic CYP1A [bothcatalytically and immunochemically, using a recently developed ELISA(43)] in oyster

172 Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al.

Collier et al. Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 173

d et al. (16)

intestinalmd at theto detect

xposed toespeciallyaticAHHloflavone,howed anced by in­:s in the:ent effectactivities

o the sex­lining can:ies,oftenties. (2.37,38)

:)f spawn­is usuallysive effectleless, wenales andlobaitis etdied were:ding howssential ifplausibled sites. Itlc activityse 1) im­ins cross­s-reactingthere wast or least

of im­d 3) spec­'ox. 0.45:data notods Holelonal an­:)w cross­1A [bothin oyster

toadfish captured from other sites in Chesapeake Bay in April, 1990 (before spawn­ing), and found higher levels of CYP1A and both AHH and EROD activities intoadfish from a contaminated site, compared to CYP1A levels in fish from a lesscontaminated site, although levels and activities of CYP 1A were still very low com­pared to those in most other bottom-dwelling species we have examined (Collier, un­published data), and were substantially lower than the AHH activities reported evenfor control toadfish by Guobaitis et al.(35) The large difference between our resultsand the findings of Guobaitis et al. (35) also raises the question of whether there arestrain or population differences in the capabilities of oyster toadfish to respond to in­ducers of CYP1A.

The weak expression of hepatic CYP1A in these fish, especially in hepatocytes,is interesting, especially in view of the presence in these same animals of substantiallevels of biliary FACs and hepatic xenobiotic-DNA adducts. It is possible that theFACs measured in bile were derived from metabolism at sites other than the liverand were transported through the liver to the gall bladder. It is known, for example,that the toadfish intestine is capable of substantial qtetabolism of BaP, and thatBaP metabolites are found in the portal blood system of BaP-exposed animals.(44)Another possibility is that the levels of FACs and of xenobiotic-DNA adducts seenwere the result of metabolism of PAHs by either the exocrine pancreas or biliarycells, which did show some staining for CYP1A when examined im­munohistochemically. It is also intriguing to note that this pattern of im­munohistochemical staining for CYP1A (e.g., no staining of the hepatic paren­chyma or hepatocytes) has not been previously reported in any other fish species.Rainbow trout, Oncorhynchus mykiss;(45) scup, Stenotomus chrysops; (46) mum­michog, Fundulus heteroclitus;(47) Atlantic cod, Gadus morhua;(29) and winterflounder, Pleuronectes americanus(48) each showed readily discernible staining ofhepatocytes when probed with various antibodies to CYP1A derived from fish.Most of these species are also shown to develop liver tumors arising fromhepatocytes (e.g., hepatocellular neoplasms) when exposed to chemical con­taminants, whereas oyster toadfish appear to be highly resistant to such lesions, asno cases of hepatocyte-derived tumors have been observed in this species, evenwhen sampled from highly contaminated environments.(49.50) It is interesting tospeculate that the lack of expression of CYP1A in hepatocytes of toadfish may con­fer some resistance to chemically induced hepatocellular neoplasia. One approachto use in testing this hypothesis would be to determine the amounts of xenobiotic­DNA adducts in different cell types in the liver of oyster toadfish exposed to PAHs.

5. Summary

This study of oyster toadfish in the Elizabeth River presented an excellent oppor­tunity to field-test biomarkers of PAH exposure and effects in a feral fish species ex­posed to a well-defined gradient of PAH contamination. Whereas biliary levels of

References

Acknowledgements

The anti-trout CYPcon polyclonals were a generous gift from Malin Celander ofthe University of Goteborg. We are very grateful to several members of the En­vironmental Conservation Division staff, including Dr. William Reichert for theanalyses of xenobiotic-DNA adducts, Dr. Margaret Krahn for the sediment screen­ing analyses, Tom Hom for the analyses of biliary FACs, and Bich-Thuy Eberhart,Maryjean Willis, and Ethel Blood for technical assistance in assays of CYPIA. Fi­nally, we thank Dr. Ernest Warriner of the Virginia Institute of Marine Science forhis valuable efforts in helping to organize this multi-institutional effort.

Tracy K. Collier et al.Environmental Sciences, 2, 3 (1993) 161-177

FACs (primarily metabolites of PAHs) and levels of hydrophobic xenobiotic-DNAadducts in liver of oyster toadfish closely followed the trends of PAH contamina­tion of the sediment, CYPIA-associated monooxygenase activities were very lowand not different between sites. Moreover, immunochemical and im­munohistochemical analyses showed very low expression of CYPIA in toadfishliver. These results point to the importance of not relying on any single biomarkerof contaminant exposure or effects in conducting field surveys in the aquatic environ­ment. Both biliary FACs and hepatic xenobiotic-DNA adducts arise largely from ex­posure of fish to PAHs; given the clear PAH gradient in the sediments of theElizabeth River and the comparatively nonmigratory nature of toadfish, theseresults provide additional assurance of the utility of these biomarkers for assessingPAH exposure and disposition in individual fish. However, the induction ofCYP lA is a common physiological effect resulting from PAH exposure of fish, andthe failure to detect any induction of this protein in these exposed fish was not ex­pected. Further study is necessary to explain this result.

Baumann, P. C. (1989): PAH, metabolites, and neoplasia in feral fish populations. In: D. Varanasi,Ed., Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. CRC Press,Inc., Boca Raton, FL, pp. 269-290.

2 Johnson, L. L., Casillas, E., Collier, T. K., McCain, B. B. and Varanasi, D. (1988): Contaminanteffects on ovarian development in English sole (Parophrys vetulus) from Puget Sound, Washington.Can. J. Fish. Aquat. Sci. 45: 2133-2146.

3 Myers, M. S., Rhodes, L. D. and McCain, B. B. (1987): Pathologic anatomy and patterns of occur­rence of hepatic neoplasms, putative preneoplastic lesions, and other idiopathic hepatic conditions inEnglish sole (Parophrys vetulus) from Puget Sound, Washington. JNCI78: 333-363.

4 Varanasi, D., Stein, J. E. and Nishimoto, M. (1989): Biotransformation and disposition ofpolycyclic aromatic hydrocarbons (PAH) in fish. In: D. Varanasi, Ed., Metabolism of polycyclicaromatic hydrocarbons in the aquatic environment. CRC Press Inc., Boca Raton, FL, pp. 93-150.

5 Krahn, M. M., Myers, M. S., Burrows, D. G. and Malins, D. C. (1984): Determination ofmetabolites of xenobiotics in the bile of fish from polluted waterways. Xenobiotica 14: 633-646.

6 Krahn, M. M., Rhodes, L. D., Myers, M. S, Moore, L. K., Macleod, W. D. Jr. and Malins, D. C.(1986): Associations between metabolites of aromatic compounds in bile and the occurrence of

174

:ollier et al. Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 175

"tic-DNAmtamina­very lowand im­I toadfish,iomarker~ environ­rfrom ex­Its of theIsh, theseassessing

llction of'fish, andas not ex-

:lander off the En­rt for thent screen­Eberhart,rpIA. Fi­~ience for

J. Varanasi,eRC Press,

ontaminantVashington.

as of occur­anditions in

position off polycyclicpp.93-150.aination of633-646.alins, D. C.currence of

hepatic lesions in English sole (Parophrys vetulus) from polluted sites in Puget Sound, Washington.Arch. Environ. Contam. Toxicol. 15: 61-67.

7 Krahn, M. M., Burrows, D. G., MacLeod, W. D. Jr. and Malins, D. C. (1987): Determination of in­dividual metabolites of aromatic compounds in hydrolyzed bile of English sole (Parophrys vetulus)from polluted sites in Puget Sound, Washington. Arch. Environ. Contam. Toxicol. 16: 511-522.

8 Gupta, R. C. and Randerath, K. (1988): Analysis of DNA adducts by 32P-postlabeling and thin layerchromatography. In: E. C. Friedberg and P. H. Hanawalt, Eds., DNA Repair. Marcel Dekker, NewYork, pp. 399-418.

9 Varanasi, U. (1989): Formation and persistence of benzo(a)pyrene-diolepoxide-DNA adducts in liverof English sole (Parophrys vetulus). Chem.-Biol. Interact. 69: 203-216.

10 Collier, T. K. and Varanasi, U. (1991): Hepatic activities of xenobiotic metabolizing enzymes andbiliary levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental con­taminants. Arch. Environ. Contam. Toxicol. 20: 462-473.

11 Goksoyr, A. and Forlin, L. (1992): The cytochrome P-450 system in fish, aquatic toxicology and en­vironmental monitoring. Aquat. Toxicol. 22: 287-312.

12 Buhler, D. R. and Williams, D. E. (1989): Enzymes involved in metabolism of PAH by fishes andother aquatic animals: oxidative enzymes (or phase I enzymes). In: U. Varanasi, Ed., Metabolism ofpolycyclic aromatic hydrocarbons in the aquatic environment. CRC Press, Inc., Boca Raton, FL, pp.151-184.

13 Goddard, K. A., Schultz, R. J. and Stegeman, J. J. (1987): Uptake, toxicity, and distribution ofbenzo(a)pyrene and monooxygenase induction in the topminnows Poeciliopsis monacha andPoeciliopsis lucida. 15: 49-455.

14 McKee, M. J., Hendricks, A. C. and Ebel, R. E. (1983): Effects of naphthalene on benzo[a]pyrenehydroxylase and cytochrome P-450 in Fundulus heteroclitus. Aquat. Toxicol. 3: 103-114.

15 Statham, C. N., Elcombe, C. R., Szyka, S. P. and Lech, 1. J. (1978): Effect of polycyclic aromatichydrocarbons on hepatic microsomal enzymes and disposition of methylnaphthalene in rainbowtrout in vivo. Xenobiotica 8: 65-71.

16 Van Veld, P. A., Westbrook, D. J., Woodin, B. R., Hale, R. C., Smith, C. L., Huggett, R. J. andStegeman, J. J. (1990): Induced cytochrome P-450 in intestine and liver of spot (Leiostomus xan­thurus) from a polycyclic aromatic hydrocarbon contaminated environment. Aquat. Toxicol. 17:119-132.

17 Huggett, R. J., Bender, M. E. and Unger, M. A. (1987): Polynuclear aromatic hydrocarbons in theElizabeth River, Virginia. In: K. Dickson, A. Maki and W. Brungs, Eds., Fate and Effects of Sedi­ment-Bound Chemicals in Aquatic Ecosystems. Pergamon Press, Elmsford, NY, pp. 327-341.

18 Bieri, R. H., Hein, C., Huggett, R. J., Shou, P., Slone, H., Smith, C. and Su, C. (1986): Polycyclicaromatic hydrocarbons in surface sediments from the Elizabeth River subestuary. Inti. J. Environ.Anal. Chem. 26: 97-113.

19 Gray, G.-A. and Winn, H. E. (1961): Reproductive ecology and sound production of the toadfish,Opsanis tau. Ecology 42: 274-282.

20 Hildebrand, S. F. and Schroeder, W. C. (1928): Fishes of Chesapeake Bay. Bull. U. S. Bur. Fish. 43:1-366.

21 Markle, D. F. (1976): The seasonality of availability and movements of fishes in the channel of theYork River, Virginia. Chesapeake Sci. 17: 50-55.

22 Krahn, M. M., Ylitalo, G. M., Joss, J. and Chan, S.-L. (1991): Rapid, semi-quantitative screening ofsediments for aromatic compounds using sonic extraction and HPLC/fluorescence analysis. Mar. En­viron. Res. 31: 175-196.

23 Krahn, M. M., Schnell, J. V., Uyeda, M. Y. and MacLeod, W. D. Jr. (1981): Determination of mix­tures of benzo[a]pyrene, 2,6-dimethylnaphthalene and their metabolites by high-performance liquidchromatography with fluorescence detection. Anal. Biochem. 113: 27-33.

24 Lowry, O. H., Rosebrough, N. T., Farr, A. L. and Randall, R. J. (1951): Protein measurement withthe Folin phenol reagent. J. Bioi. Chem. 193: 265-275.

25 Stein, J. E., Collier, T. K., Reichert, W. L., Casillas, E., Hom, T. and Varanasi, U. (1992): Bioin­dicators of contaminant exposure and sublethal effects: Studies with benthic fish in Puget Sound,Washington. Environ. Toxicol. Chem. 11: 701-714.

26 Collier, T. K., Stein, J. E., Wallace, R. J. and Varanasi, U. (1986): Xenobiotic metabolizing enzymesin spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marinesediments from contaminated and reference areas. Compo Biochem. Physiol. 84C: 291-298.

27 Klotz, A. V., Stegeman, J. J. and Walsh, C. (1984): An alternative 7-ethoxyresorufin-O deethylase ac­tivity assay: A continuous visible spectrophotometric method for measurement of cytochrome P-450monooxygenase activity. Anal. Biochem. 140: 138-145.

28 GokS0yr, A., Andersson, T., Hansson, T., Klungsoyr, J., Zhang, Y. and Forlin, L. (1987): Speciescharacteristics of the hepatic xenobiotic and steroid biotransformation systems of two teleost fish,Atlantic cod (Gadus morhua) and rainbow trout (Salmo gairdneri). Toxicol. Appl. Pharmacol. 89:347-360.

29 Hus0Y, A.-M., Willis, M. L., Myers, M. S., Collier, T. K., Celander, M. and Goks0yr, A. (1993): Im­munohistochemicallocalization of CYPIA and CYP3A isozymes in hepatic and extra hepatic tissuesof Atlantic cod. Submitted.

30 Myers, M. S., Willis, M. L., Hus0Y, A.-M., Goks0yr, A. and Collier, T. K. (1993): Im­munohistochemicallocalization of cytochrome P450 lA in contaminant-associated hepatic legions ofEnglish sole (Pleuronectes vetulus). Mar. Environ. Res. Submitted.

31 Krahn, M. M., Burrows, D. G., Ylitalo, G. M., Brown, D. W., Wigren, C. A., Collier, T. K., Chan,S.-L. and Varanasi, U. (1992): Mass spectrometric determination of metabolites of aromatic com­pounds in bile of fish captured from Prince William Sound, Alaska, after the EXXON Valdez oilspill. Environ. Sci. Technol. 26: 116-126.

32 Collier, T. K., Krahn, M. M., Krone, C. A., Johnson, L. L., Myers, M. S., Chan, S.-L. andVaranasi, U. (1993). Oil exposure and effects in subtidal fish following the EXXON Valdez oil spill.In Proceediings, 1993 International Oil Spill Conference. Amer. Petrol. Inst., Tampa Bay, FL, pp.301-305.

33 Varanasi, U., Reichert, W. L. and Stein, J. E. (1989): 32P-Postlabeling analysis of DNA adducts inliver of wild English sole (Parophrys velulus) and winter flounder (Pseudopleuronectes americanus).Cane. Res. 49: 1171-1177.

34 Stein, J. E., Reichert, W. L., French, B. and Varanasi, U. (1993): 32P-Postlabeling analysis of DNAadduct formation and persistence in English sole (Pleuronectes vetulus) exposed to benzo[a]pyreneand 7H-dibenzo[c,g]carbazole. Chem.-Biol. Interact. 88: 55-69.

35 Guobaitis, R. J.; Ellingham, T. J. and Maddock, M. B. (1986): The effects of pretreatment withcytochrome P-450 inducers and preincubation with a cytochrome P-450 effector on the mutagenicityof genotoxic carcinogens mediated by hepatic and renal S9 from two species of marine fish. Mutat.Res. 164: 59-70.

36 Milling, D. M. and Maddock, M. B. (1986): Activation and detoxication of promutagens by toadfish(Opsanus tau) hepatic postmitochondrial fractions in the Salmonella assay. Mutat. Res. 164: 81-89.

37 Johnson, L. J., Stein, J. E., Collier, T. K., Casillas, E. and Varanasi, U. (1993): Indicators ofreproductive development in prespawning female winter flounder (Pleuronectes americanus) from ur­ban and non-urban estuaries on the Northeast coast of the United States. Sci. Total Environ. inpress.

38 Goks0yr, A. and Larsen, H. E. (1992): The cytochrome P-450 system of Atlantic salmon (Salmosalar): 1. Basal properties and induction of P450 lAI in liver of immature and mature fish. FishPhysiol. Biochem. 9: 339-349.

39 Payne, J. F. and Fancey, L. L. (1982): Effect of long term exposure to petroleum on mixed functionoxygenases in fish: further support for use of the enzyme system in biological monitoring.Chemosphere 11: 207-213.

40 Payne, J. F., Fancey, L. L., Rahimtula, A. D. and Porter, E. L. (1987): Review and perspective onthe use of mixed-function oxygenase enzymes in biological monitoring. Compo Biochem. Physiol.

176 Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al.

~. Collier et al. Environmental Sciences, 2, 3 (1993) 161-177 Tracy K. Collier et al. 177

(1992): Bioin­Puget Sound,

,Iizing enzymescts of marine91-298.I deethylase ac­lchrome P-450

[1987): Speciesvo teleost fish,'harmacol. 89:

A. (1993): 1m­hepatic tissues

:. (1993): Im­latic legions of

., T. K., Chan,aromatic com­ON Valdez oil

lan, S.-1. andraldez oil spill.1 Bay, FL, pp.

NA adducts in'S americanus).

lalysis of DNAlenzo[a]pyrene

:reatment withe mutagenicityrre fish. Mutat.

ens by toadfish~S. 164: 81-89.Indicators of

anus) from ur­al Environ. in

almon (Salmolure fish. Fish

nixed functiontl monitoring.

perspective on:hem. Physiol.

86C: 233-245.

41 Walton, D. G., Fancey, 1. L., Green, J. M., Kiceniuk, J. W. and Penrose, W. R. (1983): Seasonalchanges in aryl hydrocarbon hydroxylase activity of a marine fish Tautogolabrus adspersus(Walbaum) with and without petroleum exposure. Compo Biochem. Physiol. 76C: 247-253.

42 Stegeman, J. J. (1993): Cytochrome P450 forms in fish. In: J. B. Schenkman and H. Greim, Eds.,Cytochrome P450. Springer-Verlag, Berlin, pp. 279-291.

43 Goksoyr, A. (1991): A semi-quantitative cytochrome P-450 1A1-ELISA: a simple method for study­ing the monooxygenase induction response in environmental monitoring and ecotoxicological testingof fish. Sci. Total Environ. 101: 253-261.

44 Van Veld, P. A., Patton, 1. S. and Lee, R. F. (1988): Effect of preexposure to dietary benzo[a]pyrene(BP) on the first-pass metabolism of BP by the intestine of toadflsh (Opsanus tau): In vivo studies us­ing portal vein-catheterized fish. Toxicol. Appl. Pharmacol. 92: 255265.

45 Lorenzana, R. M., Hedstrom, O. R., Gallagher, J. A. and Buhler, D. R. (1989): Cytochrome P450isozyme distribution in normal and tumor bearing hepatic tissue from rainbow trout (Salmo gaird­neri). Exper. Molec. Pathol. 50: 348-361.

46 Smolowitz, R. M., H. M. E and S. J. J. (1991): Immunohistochemical localization of cytochromeP450 1A1 induced by 3,3' ,4,4'-tetrachlorobiphenyl and by 2,3,7, 8-tetrachlorodibenzofuran in liveran extrahepatic tissues of the teleost Stenotomus chrysops (scup). Drug. Metab. Disp. 19: 113-123.

47 Yan Veld, P. A., Vogelbein, W. K., Smolowitz, R., Woodin, B. R. and Stegeman, J. 1. (1992):Cytochrome P450 1A1 in hepatic lesions of a teleost fish (Fundulus heteroclitus) collected from apolycyclic aromatic hydrocarbon-contaminated site. Carcinogenesis 13: 505-507.

48 Smolowitz, R. M., Moore, M. J. and Stegeman, J. J. (1989): Cellular distribution of cytochromeP450E in winter flounder with degenerative and neoplastic disease. Mar. Environ. Res. 28: 441-446.

49 Hargiss, W. (1989). Personal communication.50 Vogelbein, W. K. (1992). Personal communication.