ANA II

Application of the CALUX bioassay system for the detection ofdioxin-like chemicals (Ah receptor ligands) in whole serum samples
and in extracts from commercial and consumer products.
M.S. Denison. W.J. Rogers and M.Fair
Department of Environmental Toxicology, University of Califomia, Davis. CA, 95616, USA.
M.Ziccardi
Wildlife Health Center, Institute of Toxicology and Environmenlal Health, University ofCalifomia, Davis, CA, 95616, USA .
G. Clark
Xenobiotic Detection Systems, Inc., 310 Alcona Ave., Durham, NC, 27703, USA.
A.J. Murk and A. BrouwerDepartmeni of Toxicology, Agricultural University, PO Box 8000, 6700 EA Wageningen, The
Ncdicrlands

1. Abstract
Halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD), are a diverse group of widespread and persistent environmenlal contaminants which
produce a wide variety of species- and tissue-specific toxic and biological effects, many of which
are mediated by the Ah recepior (AhR). Using a recenlly developed recombinant cell line which
responds to TCDD and related chemicals with the AhR-dependent induction of firefly luciferase,
we have developed a chemical-activated luciferase gene expression (CALUX) cell bioassay system
for the detection of TCDD-like chemicals. Wc have utilized this bioassay as a screening tool not
only lo determine the widespread nature of these chemicals in industrial, commercial, consumer
and environmenlal samples, bul also for the detection of TCDD-like chemicals in whole semm
samples. Here we repon the identification of CALUX-posiiive compounds preseni in DMSO
extracts from a wide variety of commercial and consumer plastic, mbber and paper products andwe have now optimized the bioassay system or detection of TCDD in small (25-50 ^.1) aliquots ofwhole semm. Although the identity of the specific chemical(.s) responsible for the positive
CALUX signals currcntiy remain to be detennined as well as their toxicological and biologicalimpact, our data demon.slrale the widespread disU'ibution of chemicals in everyday producis thatcan specifically acdvate gene expression in an AhR-dependcnl manner. In addilion, thc availablililyof a rapid and small volume semm bioassay for TCDD-like chemicals provides an avenue forscreening of samples were limited blood volumes are available (e.g. endangered species) as well aslarge scale screening of populations for epidemiological studies.
2. Introduction
Polychlorinated diaromatic hydrocarbons (PCDHs) arc a diverse group of widespreadenvironmenlal contaminants, which include polychlorinated biphenyls (PCBs), dibenzofurans(PCDFs), dibenzo-p-dioxins (PCDDs) as well as many other subclasses of PCDHs and polycyclicaromatic hydrocarbons (PAHs).l"3) Some PCDHs are not only lipophilic and persistent in theenvironment bul ihey are known to bioaccumulate and biomagnify in the food chain.L2) Exposure

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to TCDD-like chemicals produces a wide variety of species- and tissue-specific toxic and biologicaleffects, many of which are mediated by the Ah recepior (AhR). The AhR is a ligand DNA
regulatory protein which activates gene expression in a ligand (TCDD/PCDH)-depcndcnt manner.
Following ligand binding, the AhR complex is transformed inlo ils DNA binding form, AhRcomplexes accumulate wilhin the nucleus^-S) and thc binding of the PCDH:AhR complex to its
specific DNA recognition site (the dioxin responsive clement) stimulates expression of the adjacentgene.'''5) We have recenlly developed a novel recombinant cell line which responds to TCDD andrelaled chemicals (PCDHs and PAHs) wilh the induction of firefly luciferase and this bioassay, likethe induction of EROD activity in rat hepatoma (H4IIE) cells, has been used has been used for thedetection of TCDD-like chemicals in complex mixtures of chemicals from a variety ofmatrices.L6.10) Given the broad applicability of this assay and the documented widespread
distribution of PCDHs and PAHs in a wide variely of products, we have begun screening for lhe
presence of PCDH-like chemicals in industrial, commercial, consumer and environmental samples
as well as optimization of the bioassay syslcm for direcl detection of these chemicals in biologicalmaterials (e.g., whole semm).
3. Materials and Methods
Sample Pneparatlon: The indicated samples (Figure 1) were finely chopped up with scissors andextracted ovemight (-16 hours) in glass tubes wilh DMSO (1.5 nX of DMSO/mg of material for all
samples except paper products which were extracted with 10 nl/mg of DMSO). DMSO wascollected from each .sample and stored in a tenon-capped vial until use.
Lucifcra.sc F.xpre.s.sion Assay: A mouse hepatoma (Hcpalclc7) cell line sutbly transfected with thc
HAH-inducible reporter pla.smid pGudLucl.l '") (HlLl.lc2) were used in these studies. The
pGudLucI.l vector contains the firefly lucifcra.se gene under PCDH-induciblc conlrol of fourdioxin responsive elements and exposure of these cells lo TCDD and related AhR agonists resulls
in induction of firefly luciferase activity in a time-, dose- and AhR-dependent manner.'W Cells,grown in 96 well microplates, were incubated with 2.5 jil of extract for 4 hours followed by lysis
in the wells and automatic measurement of luciferase activity (using the Promega luciferase as.saysyslem) in a Dynatech ML-300() luminescence plate reader. Activity was corrected for the amounlof luminescence present in the DMSO-Ux;ated sample wells.
4. Results
Wc have previously demonstrated that the recombinant mouse hepatoma cell line, HlLl.lc2,
responds to dioxin-like chemicals in a dose-, lime- and AhR-dcpcndcnt manner with the inductionof firefly luciferase. The induction was specific for PCDHs and PAHs which are known lo exert
their action on gene expression via the AhR. Given the rapidity, inexpensive and sensitive natureof this a.s,say (minimal detection limits of ~ 10 ppb), as well as its insensilivity to subsU'ate inhibition(as observed with P4501A1^^) this bioassay provides an avenue for thc large scale screening ofsamples.
Commercial/Consumer Samples: Analysis of DMSO extfacLs from a variety of plastic, rubber andpaper products using the CALUX assay syslcm has revealed the presence of AhR ligands in manyof these samples. Specifically, DMSO extracts from every paper product examined (Figure 1)
induced lucifcra.se activity (wilh maximal induction to -25% of that ob.served with TCDD).
Although the highest level of induction from these sample cxtfacts was observed u.sing prinlednewspaper, it was unclear whether the activity we ob.served was due lo the paper and or the ink
used in the printing process. Consequently, we carried oul addilional experiments utilizing DMSO
extracts of ink-free newspaper and compared the results to that obtained using extracts from inked
paper. These results (data not shown) clearly demonsU'ale that the majority of the activity observed
wilh the newspaper extracts resulls from the ink used in the printing process, allhough low levelsof activity were observed wilh a DMSO extract of ink-free paper itself. To date, allORGANOHALOGEN COMPOUNDS
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ANAM>o^—> X
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Figure 1. Induction of lucifera.se activity by DMSO exUracts of the indicated producis. HlLl.lc2
cells grown in 96 well microplates were incubated with an aliquot of a DMSO extract of theindicated producis for 4 hours followed by measurement of lucifcra.se activity. Values represent
the mean ± SD of at leasl three determinations and were corrected for background luminescenceobserved widi DMSO alone.
DMSO exU'acts of paper products we have examined have resulted in a positive CALUX response,
alUiough the magnimde of the signal was varied. We have also screened and observed the presenceof CALUX-posilive chemicals in a variely of rubber and plastic producis, including:, rubber
stoppers and 0-rings, sneaker soles, a variety of mbber/silicone tubing, PVC pipe, mbber bands,
baby pacifiers and nipples and other products. It should be noted that although this assay can beused lo detect TCDD and relaled PCDHs il also can detect a variety of less/non-toxic PAHs as well.
Thus, the identification of a positive CALUX response from a given sample does nol necessarily
indicate the presence of TCDD or the more toxic PCDHs. In fact, .several lines of evidence suggesl
that the majority of the CALUX positive samples actually are nol due to dioxins themselves.
Measurement of the dioxin-like activity of these extracts using an in vitro AhR ligand or DNA
binding assay would indicate that these extracts should be signficantly more active in the cellbioassay than they are. This decreased CALUX response is very likely the result of metabolism of282

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the inducing chemicals by the degradation enzymes present in these cells, something that is morecharacteristic of PAHs inducers ralher than mosl PCDHs. In addilion, preliminary eli.sa assays
using an antibody against TCDD (and which cross-reacts with other dioxins) would support theseconclusions. Although the identity ofthe sptecific chemical(s) responsible for the positive CALUX
signals currenlly remain lo be determined as well as their toxicological and biological impact, ourdala demonstrate the widespread disuibulion of chemicals in everyday products that can specificallyactivate gene expression in an AhR-dependent manner. Experiments are in progress to identify theCLAUX positive chemicals in die most active samples.

TCDD in Serum
TCDD in DMSO

5 -3_la:^^4 ->^• >•+3o<0)3(/)
n2 -"(J31 -,0-14,Q-13,QTCDD Concentration in Serum (M)Figure 3. Dose-dependent induction of luciferase activity in HlLl.lc2 cells by TCDD added toeach well in DMSO or in whole semm. HlLl.lc2 cells were grown overnight in 96 well
microplates followed by the addilion of TCDD in DMSO (added to 100 |il of media present in eachwell) or by Uie addition of 50 |J1 of whole semm (containing the indiclaicd TCDD concentration) to50 ^1 of media present in each well. Cells were incubated for 4 h al 37^0 followed by analysis ofluciferase activity as described in Figure 1. Values represent the mean ± SD of al least threedelerminauons.
Semm Bioas,say: Measurement of TCDD-like chemicals in biological materials (particularly bloodand serum) has required solvent extraction of the material, followed by concentration andsubsequent analysis by biological/analytical methodologies. The availability of a bioassay which
can directly detect and provide a relative measurement of TCDD-like chemicals in whole semm
would be a significanl advance relative to large .scale .screening of blood samples. The sensitivity ofthe CALUX bioassay, combined with our modification of the bioxs.say syslem which allows direct
growth of cells and sub.sequenl analysis of activity in 96 well microplates, provides an avenue lodevelop a rapid screening as.say for small volumes of .semm. Utilizing this syslem, we have now
optimized the CALUX bioas.say for direct mea.suremenl of TCDD-like chemicals preseni in small

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volumes of samples. Semm was .spiked with TCDD al varying concentrations and added lo 96
well microplates containing HlLl.lc2 cells and after 4 hours luciferase activity was measured(Figure 2). For comparison, induction was also carried out by directly adding TCDD, in DMSO,
to the sample wells. As depicted in Figure 2, dose dependent induction of lucifera.se activity wasobserved by bodi mediods with die dose-response curve for induction by TCDD in semm requiring
approximately a 10-fold greater concentration for a similar induction njsponse. This is likely due lothe decreased availability of TCDD to the cells because of serum protein binding. TTiese results
demonstrate dial the CALUX syslem can be used to directly detect TCDD preseni in whole semm
samples and can be u.sed to provide a relatively accurate estimation of TCDD-like activity in asample. In addilion, we have also determined dial the optimal whole semm volume for theseanalysis is only 50 |il (mixed wilh 50 ^l of cullure media) and the results present in Figure 2 wereobtained using 50 nl of fetal calf semm containing die indicated TCDD concentration. Overall, lherapidity and sensitivity of the CALUX assay, combined with thc low volume of sample needed foranalysis provides us with a unique a.ssay syslem for the direct analysis of very small volumes ofsemm samples for the presence of TCDD-like chemicals. This modificadon of the CALUX a.ssay
nol only has applications for the screening of samples where only small volumes of blood areavailable (e.g., endangered and/or small species), but it will allow for large scale screening of
populations for epidemiology studies.
This work was supported by the US Nalional Institutes of Environmental Heallh Sciences(ES07685 and ES04699), the Califronia Depariment of Fish and Game and the Dutch Technology
FoundaUon (STW; WBI22.2823).
5. References
1. Giesy, J. P., Ludwig, J. P. and Tillitt, D. E. (1994b) Dioxins, dibenzofurans, PCBs andcolonial, fish-eating waler birds. In Dioxins and Health (A. Schecter, Ed.), pp. 249-307,
Plenum Press. New York.
2. Safe, S. H. (1994) Polychlorinated biphenyls (PCB.s): environmental impacl, biochemical andloxicrespon.sesand implications forri.ska.ssessmeni. Cril. Rev. Toxicol. 24, 87-149.
3. Poland, A. and Knutson, J.C. (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and related
halogenated aromatic hydrocarbons: examination of thc mechanism of toxicity, Ann. Rev.
Pharmacol.Toxicol. 22, 517-542.
4. Whitlock, J.P., Jr. (1986) The regulation of cytochrome p-450 gene expression, Ann. Rev.
Pharmacol. Toxicol. 26, 333-369.
5. Denison, M.S., Fisher, J.M. and Whitlock, J.P., Jr. (1988) The DNA recognition site for thedioxin-Ahreceptorcomplex: nucleotide sequence and functional analysis, J. Biol. Chem. 263,17721-17724.
6. Casteriine, J. L., Bradlaw, J. A., Puma, B. J. and Ku., Y. (1983) Screening of fresh water
fish extracts for enzyme-inducing substances by an aryl hydrocarbons hydroxylase induction
bioassay technique. J. Assoc. Off. Anal. Chem. 66:1136-1139.
7. Tillill, D.E., D.A. Ankley, D.A. Verbmggc, J.P. Giesy, J.P. Ludwig, and T.J. Kubiak.
1991. H4IIE rat hepatoma cell bioassay-derived 2,3,7,8-lclrachlorodibenzo-p-dioxin
equivalenLs in colonialfish-eatingwater birds from the Greal Lakes. Arch. Environ. Toxicol.
21,91-101.
8. Murk, A.J., Jonas, A., Brouwer, A., Leonards, P.E.G. and Denison, M.S. (1996)
Applicalion ofthe CALUX (chemical activated luciferase gene expression) assay for measuring
TCDD-equivalents in sediment, pore water and blood plasma samples, presented al Dioxin 96.
9. Murk, A.J., Luil, R., Spenkelink, A., Leonards, P.E.G., van Hallum, B. and Smil, M.
(1996) Monitoring exposure and effects of polyhalogenated aromatic hyrdocarbons (PHAHs)
in European otters (Lutra lutra), presented at Dioxin 96.
10. Garrison, P.M., Aarts, J.M.M.J.G., Brouwer, A. and Giesy, J.P. and Denison, M.S. (1996)
Ah receptor-mediated gene expression: production of a recombinant mouse hepatoma cellbioassay system for detection of 2,3,7,8-tcirachlorodibenzo-p-dioxin-like chemicals. Fund.
Appl. Toxicol. 30. 194-203.

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