BIOANALITICAL-POSTERAPPLICATION OF THE LUCIFERASE RECOMBINANT CELLCULTURE BIOASSAY FOR DETECTION OF DIOXIN-LIKE ACTIVITY
OF PAHS IN THE SERUM OF WILDLIFE
Michael H. Ziccardi '", lan A. Gardner" and Michael S. Denison ^Wildlife Health Cenler, School of Veterinary Medicine'; Departmeni of Medicine andEpidemiology, School of Veterinary Medicine";and Department of Environmenlal Toxicology,
University of Califomia, Davis California USA''

Introduction
Halogenated and non-halogenated polycyclic aromatic hydrocarbons (HAHs/PAHs) have long
been recognized as significanl and widespread pollutants in the environment. In mammaliansyslems, exposure lo such xenobiotics has produced a variety of species- and tissue-specific toxic
and biologic effecis. The majority of these effecis are mediated by the Ah recepior (AhR), andnon-mortalily-related effecis include tumor promotion, birth defects, hepatotoxicity,immunotoxicity, dermal toxicity, and alterations in endocrine homeostasis, as well as induction ofmany metabolic enzymes such as the cytochrome P450 (CYP) system '.
Because ofthe potential for high morbidity and mortality associated with exposure to thesecompounds, many analytical techniques have been developed to detect their presence in biologicalsamples. These procedures often rely on very accurate methods, such as gas chromatography,
high-pressure liquid chromatography or olher methods; however, their use also has limitations,such as high analysis cosls, lack of rapidity and the need for large sample volumes. All of thesefactors negatively affect their utility for rapid screening and analysis of large numbers of samples.
To better assess biological responses of such compounds in complex systems, much research effort
has been directed toward the development of bioassays. Techniques that examine in vivo
responses in tissues (such as measurement of CYPI Al induction by the quantification ofelhoxyresorufin-o-deethylase (EROD) activity, aryl hydrocarbon hydroxylase (AHH) activity or
CYPlAl mRNA levels) have been used widely lo determine exposure of individuals '"^. However,this approach has several disadvantages, including species differences in response lo HAHs/PAHs', their requiring eiiher the euthanasia ofthe animal or the use of invasive surgical techniques forsample colleclion, and that many CYPI Al-inducing chemicals also acl as substrates for thisactivity, thus competitively inhibiting response al high concentrations ''. Olher methods that use
detection of enzymatic activity, Ah receptor binding or the induction of reporter genes have been
more recenlly used to evaluate exposure of animals or environments to HAHs/PAHs. The H411E
rat hepatoma cell bioassay ' has been used to delect HAHs/PAHs and provides an estimate ofthebiological potency (expressed as 2,3,7,8- TCDD equivalents or TEQs) of compounds, however,since this melhod also measures increases in EROD aclivily, il has many ofthe same limitations
described above. The application of recombinant reporter plasmids, such as the firefly luciferase(Luc) gene ' has proven to be a very effective melhod to detect these chemicals and to provide anapproach to estimate TEQs (Ihrough calculation of induction equivalency factors, or I-EFs) in avariely of compounds ^. The recombinant cell culture luciferase bioassay system has been found

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to be extremely sensitive in directly delecting AhR-dependent potential of a variety of pure HAH
compounds'". However, the determination of luciferase induction associated with PAH
compounds has been done in only a few studies " ' " and very little work has yel been done lo
characterize and optimize this melhod for the direcl analysis of small volumes of whole serum '^Therefore, the aims ofthis sludy vvere lo determine the induction potential (described in relation toTCDD) for a variety of PAH compounds, and lo use this melhod to analyze samples from selectedwildlife populations with differing known exposure lo pollulanls containing high levels of PAHs,
namely oil spill events.
Experimental Methods
Chemicals: All aromatic hydrocarbons were considered hazardous and appropriate personal
protective methods and materials were used in all experimenls. 2,3,7,8-Telrachlorodibenzo-pdioxin (TCDD) was obtained from Dr. S. Safe (Texas A&M University). Monocyclic and PAH
compounds used in this sludy are specified in Table 1 and were purchased from Accustandards Co.
(St. Louis, MO) and diluted in methanol (MeOH).
Serum. Serum samples were collected from a variety of wildlife species (under appropriatelACUC approval) wilh differing exposure lo PAHs. Free-ranging common murres (Uria aalge)
were sampled during rouiine population surveys in the coastal California waters, as vvell as during
several oil spill evenls that occurred in Califomia during 1997. Northem sea otters (Enhydralutris) were collected eiiher as part of larger health assessment projecls or from animals
undergoing rehabilitation during the E.xxon Valdez oil spill.
Cell Line. Recombinant HlL].lc2 cells were used in these studies. These cells respond to AhR
agonists with the induction of firefly luciferase '. Treatment of cells in the 96-well microplate
format was previously described '^ Dose-response experiments for PAHs and TCDD wereconducted using ten-fold serial dilutions (v/v) ofthe inducing products in DMSO or methanol.
Analysis ofall serum samples was done by addilion of 75 pl of eiiher control seruni (containingDMSO, or TCDD at a 1% final concentration) or sample serum al a 50% dilution in media. Aftera three-hour induction period, cells were analyzed as previously described '^ and expressed as
relative lighl units (RLUs) per mg protein.
Data Analysis. Median (EC50) and 20%(EC2o) effective concenlralions of maximal TCDD
induction, and induction equivalency factors for each (I-EF50 and I-EF20) were calculated by fitting
dose-response data to a four-parameter Hill model by leasl squares (Sigma Plol; SPSS, Chicago,IL). Spearman's rank order correlation comparing EC and 1-EF values from other published dalawere calculated using Statistica (Statsoft, Tulsa, OK).
Results and Discussion
Ofthe sixteen PAH evaluated, no statistically significant luciferase induction was observed forcells treated wilh monocyclic, 2- or 3-ringed compounds (all P values > 0.05), mosl likely due tothe compound's inability to bind to and activate the AhR "'''. In contrast, mosl four- to six-ring
siruclures lested (wilh the exception of benzo(g,h,i)perylene) showed high levels of induction, andhad relatively low EC50 and EC20 values, and relatively high corresponding I-EF50 and I-EF20values (Table 2). Calculated ECjoand 1-EF values correlated somewhat with values determinedusing a ral cell line "; Spearman r = 0.71, P = .11) and a human cell line ''; Spearman r = 0.84, PORGANOHALOGEN COMPOUNDS
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kh= .04), and improved using the EC20 and I-EF20 values (Willett: Spearman r = 0.77, P = .07; Jones:Spearman r = 0.93, P = .003). Induction equivalencies determined for each ofthe individual
congeners were from 9.3 lo 144 times grealer than that reported in the human cell line, and 0.2 to
7.5 limes grealer than with the ral luciferase bioassay using the I-EF50 values, and 10.3 to 1,350
times greater and 0.2 to 501 times greater, respectively, using the I-EF20 data. These differences
are most likely attributable lo the reduced incubation lime (three hours) compared to these olher
assays (16 to 24 hours). Since reduction ofthe incubation period would decrease PAH metabolism
and increase its inducing potency relative lo TCDD, our estimated I-EF values would be greaterthan those where there was significant PAH metabolism. These data support the fact that theluciferase bioassay melhod is a sensitive technique lo delect PAH compounds. Furthermore, ourmore rapid analytical technique suggests that previous cell bioassay analyses may haveunderestimated the induction potency of PAHs.
Using this informalion, the bioassay was evaluated whelher it could delect PAH exposure inwildlife species, specifically that due to exposure lo a marine petroleum spill. Statisticallysignificant differences were noted between oiled and unoiled common murres and Northem sea
otter populations (P < 0.05). While it is understood that this assay is not analytically specific (inthat it cannot differentiate petroleum exposure from olher CYPI Al-inducing compounds such as
HAHs and olher AhR ligands), its use as a rapid screening lool for samples from large numbers ofanimals can prove extremely beneficial for decision making during spill evenls. Should
quantitation of true petroleum exposure be necessary, il can allow for significanl cosl reduction inorder to identify those to be further characterized by GC/MS. This bioassay can also allow forlarge-scale assessment of toxin loads in wildlife populations exclusive of oil spill events by the
analysis of eiiher small amounts of serum collected during an ongoing effort, or throughretrospective serological surveys of banked samples. Thus, Ihrough the abilily lo analyze large
numbers of samples in an effective and sensitive manner, this melhod has the potential to be a very
powerful lool for the epidemiological assessment of wildlife population and ecosystem health.
Acknowledgments
This work was supported by the US National Institutes of Environmental Health Sciences(ES07685 and ES04699) and the Califomia Departmeni of Fish and Game's Oil Spill Response
Trusl Fund through the Oiled Wildlife Care Network al the Wildlife Heallh Center, School of
Veterinary Medicine, University of Califomia, Davis.
References
1. Whitlock, J.P. (1999) Ann Rev Pharm Tox 39, 103-25.
2. Burke, M.D. and Mayer, R.T. (1974) Drug Metab Disp 2, 583-8.
3. Nebert, D.W. and Gelboin, H.V. (1968) J Biol Chem 243, 6242-9.
4. Vanden Heuvel, J.P., Clark, G.C, Thompson, C.L., McCoy, Z., Miller, C.R., Lucier, G.W. andBell, D.A. (1993) Carcinogenesis 14, 2003-6.
5. Denison, M.S. and Wilkinson, CF. (1985) Eur J Biochem 147, 429-35.
6. Kennedy, S.W., Lorenzen, A., James, CA. and Collins, B.T. (1993) Anal Biochem 211,102-12.
7. Bradlaw, J.A. and Casteriine, J.L. (1979) J Assoc Off Anal Chem 62, 904-16.
8. Denison, M.S., El-Fouly, M.H., Aarts, J.M.M.J.G., Brouwer, A., Richter, C. and Giesy, J.P.
(1993) Organohalogen Compounds 13, 365-8.
9. Garrison, P.M., Tullis, K., Aarts, J.M., Brouwer, A., Giesy, J.P. and Denison, M.S. (1996) FundAppl Tox 30, 194-203.

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10. Sanderson, J.T., Aarts, J.M., Brouwer, A., Froese, K.L., Denison, M.S. and Giesy, J.P. (1996)
Tox Appl Pharm, 137,316-25.
11. Willett, K.L., Gardinali, P.R., Sericano, J.L., Wade, T.L. and Safe, S.H. (1997) Arch Env
Contam Tox 32, 442-8.
12. Jones, J.M. and Anderson, J.W. (1999) Env Tox Pharm 7, 19-26.
13. Ziccardi, M.H., Gardner, I.A. and Denison, M.S. (2000) Tox Sci 54, 183-93.
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Table I: Calculated median (EC50) and 20% maximal (EC20) luciferase induction, andcorresponding induction equivalency factors (I-EF50 and I-EF20) for polycyclic aromatichydrocarbons analyzed using the luciferase bioassay.'
ChemicalI-EF2o(M)EC5o(M)
I-EF5o(M)EC2o(M)
TCDD
1.22x10"3.46x10"1
1.08x10-'
Chrysene
1.12x10"°1.82x10"'1.90x10"'7.53x10"'
Benzo(a)pyrene
1.62x10"*3.89x10"'8.89x10"^1.87x10"^Benzo(b)fluoranthene
6.52x10"'2.76x10"'1.25x10"1.87x10"^
Benzo(k)fluoranthene
6.54x10"''3.30x10"'
1.05x10"'2.88x10"^lndeno(l,2,3-cd)pyrene
4.23x10"*1.31x10"'2.64x10"1.56x10"^
Dibenz(a,h)anthracene
7.82x10"*6.39x10''5.41x10"PAHs that did nol produce luciferase activity: benzo(g,h,i)perylene, naphthalene, 2melhylnaphlhalene, acenaphthylene, acenaphthene, Fiuorene, anthracene, phenanthrene, pyrene

7.00 -'="•5 6.00a.
" 5.00iss '••°°IA

3.00<—000Lucif(« 2.00
a.....^_..
Sea WorldCOML

mFarallones 1.
CO.Mli•1

CA Spill COMIJ

AK Otters

EVOS Otters

Figure 1: Luciferase activity in serum from common murres (COMU) from Sea World(n=23), the Farallones islands, (n=20), and during a 1997 oil spill in California (n=94),
and sea otters from southeast Alaska (n=46) and the Exxon Valdez oil spill (n=16)

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