ANALYSIS

Direct Detection of Dioxins and Related Chemicals in Small Quantities of
Human Serum Using a Recombinant Cell Bioassay
Michael Ziccardi'. William J. Rogers^ Arnold J. Schecter\ Olaf Papke" and Michael S. Denison"'Department of Medicine and Epidemiology, University of California, Davis, CA 95616 US A
^Department of Environmental Toxicology, University of Califomia, Davis CA 95616 USA^College of Medicine, State University of New York, Binghamton, NY 13903 USA''ERGO Forschungsgesellschaft mbH, Hamburg, Germany

Introduction
Halogenated and non-halogenated aromatic hydrocarbons, such as polychlorinated dibenzo-pdioxins, biphenyls, benzo(a)pyrene and related chemicals have been recognized as significant andwidespread pollutants in the environment. In mammalian systems, exposure to such xenobiotics
have been observed to produce a variety of species- and tissue-specific toxic and biologic effects,
including tumor production, lethality, birth defects, organ toxicity and induction of many metabolicenzymes, including the cytochrome P450 (CYP) system'^ Induction of these enzymes is thought
to be mediated by the Ah receptor (AhR), a soluble intracellular protein which strongly binds thesechemicals and, through translocation and DNA binding, causes increased transcription of CYPI A1
enzymes'^'. This induction, consequently, has been one response that has been used not only as abioassay to evaluate the relative biological/toxicological potency of HAHs and complex mixturescontaining these chemicals, but, through implication, as a biomarker of exposure to xenobiotics'.
While the quantification of CYP enzymes may act as an excellent "bioassay of effect" in somespecies, because of differing intra- and interspecies response to various concentrations ofxenobiotics'' and inhibition of CYP enzymatic activity by many AhR inducers , it is difficult to assess
exposure using the resultant data. Additionally, analytic methods that are routinely employed todetermine pollutant exposures often require both prior knowledge of exposure chemical class as wellas significant amounts of biologic tissue for analysis. In order to accurately assess such exposureusing easily obtainable samples, we have modified a specific "bioassay of exposure" forenvironmental samples in order to detect these substances in the semm of vertebrates. As described
previously, this assay uses the AhR-dependent mechanism of action (CYPlAl induction) in cell
culture combined with a recombinant luciferase gene vector as an indicator to quantify TCDD-likeligand present in samples through measurement of chemically-induced luminescence^'.
While this assay works well for samples that contain high levels of AhR ligands and has been
optimized for this use, at levels normally associated with chronic or sub-acute exposures (as well asexposure to those chemicals not associated with extremely strong induction), direct analysis of semm
may not be adequate to separate tme xenobiotic exposure from "background" (i.e. non ligand-ORGANOHALOGEN COMPOUNDS
Vol. 31(1997)

215

Dioxin '97, Indianapolis, Indiana, USA
dependent) induction of cells. In order to improve both precision and validity of the assay, two
additional optimization steps have recentiy been incorporated into the protocol. First, by firstextracting semm in a simple liquid-phase manner, the sensitivity of the assay improves dramatically,into the picomolar range for TCDD. Secondly, through the parallel analysis of samples with a potent
Ah receptor antagonist (4-amino-3-methoxyflavone or AMF), ligand independent luciferase activity
can be easily corrected for.
Here, we describe the methods we employ to analyze samples for AhR ligands and present
preliminary data on samples from a human population.
Experimental Methods
Seruni. Semm samples were collected from a variety of human subjects with HAH exposure as partof a larger baseline exposure trial.
Cell Line. Mouse hepatoma (hepalclc7) cells were stably transfected with the HAH-inducible
luciferase expression vector pGudLucl.l (HlLl.lc2) as described'. This vector contains theluciferase gene under HAH-inducible control of four dioxin responsive elements such that, when
exposed to AhR ligands, induction of luciferase occurs in a time-, dose- and AhR-dependent manner.
Sample preparation. Semm samples were mixed twice with an equal volume of acetone (toprecipitate semm proteins), centrifuged and the resultant supematants were collected. The extractant
was blown to dryness under N^ and then resuspended in DMSO.
Induction Protocol. Plates of stable cell clones were Irypsinized and resuspended in 10 ml alphaMEM. One-hundred nl of this cell suspension was aliquoted into all wells of a sterile 96-well
microtiter plate. Plates were incubated for 24 hours prior to ligand exposure, allowing cells in all
wells to retum to confluence. Cells were then induced by the addition of culture media containingextractant (50:1), as well as addition culture media containing extractant as well as the AhR
antagonist AMF (50:1). All inductions were done in triplicate. After a 4 hour induction period, cells
were washed with PBS, then lysed. Subsequent luciferase activity was determined using thePromega luciferase assay system in a Dynatech ML300 automated microplate luminometer.
Proiein Correction. In order to correct for cell number per well, protein concentrations weredetermined in triplicate using a modification of a previously-developed method'". Briefly,
fluorescamine in acetone was added to all wells, including a standard curve of 0 to 75 pg bovine
semm albumin assayed in triplicate. Subsequent fluorescence was quantified using a multiplate
fluorometer at 400 nm excitation and 460 nm emission wavelengths.
Data Atialysis. All samples were expressed as relative light units (RLUs) per mg protein.
Results and Discussion
We have previously shown that this luciferase-based bioassay works in a dose-, time- and AhRdependent manner for the screening of chemicals, natural and manufactured products'. Also, we216

ORGANOHALOGEN COMPOUNDS
Vol. 31 (1997)

ANALYSIS
have shown the utility ofthis bioassay in analysis of HAHs in sediment, milk and whole semm"",however, at the physiologic levels that HAHs are seen in vivo in most samples, the lower detection
limits ofthe assay may not be sufficient. Although, when this assay is coupled with a simple liquidphase sample extraction technique and the elimination of non-ligand dependent induction throughthe use of an AhR antagonist, sufficient .sensitivity for lower level exposures may be seen.
As an example of these techniques, semm samples collected from HAH-exposed humans wereextracted with acetone and analyzed using the bioassay. As is seen in Figure 1, when results arecompared with TCDD as a positive control, most are seen to be positive and, in fact, some havefairly high levels of activity. Using this screening infomiation, further analyses, including theanalyses of serial dilutions of positive extracts in order to calculate relative TEQs in the semm, as
well as incorporating additional analytic measures for specific classes of compounds (via GC/MS),
can be conducted in a time- and cost-effective manner.
Although this assay, as described, does not differentiate between HAHs, PAHs and other inducing
chemicals, the coupling ofthis extraction procedure with our recently-optimized acid denaturation,
which inactivates non-HAH chemicals'-' will allow direct estimation of HAH-equivalents in the.sesamples. These data presented represent a fraction of the total number of blood samples that we havebegun to analyze. Since GC/MS analysis has already been carried out on each of these samples,
determination of HAH TEQs in each of these using the bioassay will provide direct TEQ comparisonof data from this biologically-based system with that from instmmental analysis.

IK.Tiracted Scrum Sample

D E x i r a c i e d Seium Sample + A n i a g o n i s t

5IDMSO

InM

1416

1718

1920

21

22

2.1

24:f

26

28

Figure 1: Induction of luciferase activity by DMSO extracts of human serum samples. HlLl.lc2
cells grown in 96-well microtiter plates were incubated with an aliquot of acetone-extracted serum
both in and not in the presence of <m Ah receptor antagonist, 4-amino-3-methoxynavone for 4 hours,
followed by measurement of luciferase activity. Values represent the mean ± SD of triplicate
determinations.

ORGANOHALOGEN COMPOUNDS
Vol. 31 (1997)

217

Dioxin '97, Indianapolis, Indiana, USA
While these additional techniques increase both our analysis time and cost, when compared with theextensive and expensive clean-up procedures necessary (i.e. GC/MS), to the lower sensitivity (i.e.
fluorescent/spectrophotometric methods) or to the requirement of difficultly-acquired tissues (i.e.
EROD) of other methods, this bioassay provides us with a sensitive and relatively rapid procedure
for large-scale screening of samples from human populations for HAHs. As such, this bioassay
complements conventional chemical analytical procedures by low cost screening of samples in order
to identify those to be further characterized by GC/MS.
Acknowledgments
This work was supported by tiie US National Institutes of Environmental Health Sciences (ES07685and ES04699), the University of California - School of Veterinary Medicine (Robert CessnaFellowship) and the Dutch Technology Foundation (STW; WBI22.2823).
Literature Cited
(1)
(2)

(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)

(12)
(13)

Whitlock, J. P. Ann Rev. Pharmacol. ToxicoL 1990, 30, 251 -277.
Nelson, D. R.; Kamataki, T.; Waxman, D. J.; Guengerich, F. P.; Estrabrook, R. W.;
Feyereisen, R.; Gonzalez, F. J.; Coon, M. J.; Gunsalus, L C ; Gotoh, O.; Okuda, K.;
Nebert, D. W. DNA Cell Biol. 1991, 12, 1-51.
Guengerich, F. P. Principles and Methods of Toxicology, 3rd Edition; Raven Press Ltd.;New York, 1994; pp. 1259-1313.
Denison, M. S.; Wilkinson, C. F. Eur. J. Biochem. 1985, 147, 429-435.
Denison, M. S.; Fisher, J. M.; Whidock, J. P. J. BioL Chem. 1988, 263, 17221-17224.
Fisher, J. M.; Jones, K. W.; Whitlock, J. P. Molec, Carcinogen. 1989, 1, 216-221.
Denison, M. S. Chemosphere 1991, 23, 1825-1830.
Denison, M. S.; El-Fouly, M. H.; Aarts, J. M. M. J. G.; Brouwer, A.; Richter, C ; Giesy,J. P. Proc. Int Dioxin Conf 1993, 13, 365-368.
Garrison, P. M.;Tullis, K.; Aarts, J. M. M. J. G.; Brouwer, A.; Giesy, J. P.; Denison, M.
S. Fund AppL Toxicol. 1996, 30, 194-203.
Kennedy, S. W.; Jones, S. P.; Bastein, L. J. AnaL Biochem 1995, 226, 362-370.
Aarts, J.M.M.J.G.; Cenijn, P. H.; Blankvoort, B. M. G.; Murk, A. J.; Brouwer, A.;
Bovee, T. F. H.; Tragg, W. A.; Hoogenboom, L. A. P.; Patandin, S,; Weisglas-Kupems,N.; Sauer, P. J. J.; Denison, M. S. Organohalogen Compounds 1996, 27, 285-290.
Denison, M. S.; Rogers, W. J.; Fair, M.; Ziccardi, M.; Clark, G.; Murk, A. J.; Brouwer,
A. Organohalogen Compounds 1996, 27, 280-284.
Murk, A. J.; Jonas, J. A.; Brouwer, A.; Leonards, P. E. G.; Denison, M. S.
Organohalogen Compounds 1996, 27, 291-296.

218

ORGANOHALOGEN COMPOUNDS
Vol. 31 (1997)