BIOANALYSISRECOMBINANT CELL LINES FOR THE DETECTION OF DIOXINSAND AH RECEPTOR LIGANDS - NOT ALL ASSAYS ARE CREATED
EQUAL
Dalho Han, Scott R. Nagy and Michael S. Denison
Department of Environmental Toxicology, University of California, Davis, CA USA

Introduction
Halogenated aromatic hydrocarbons (HAHs), such as the polyhalogenated dibenzo-p-dioxins,biphenyls and dibenzofurans, represent a large group of compounds which because of their ubiquitous
distribution, resistance to biological and chemical degradation, high toxicity and potential forbioaccumulation/biomagnification, can have a significant impact on the health and well being of
human and animals. The worldwide distribution of HAHs and their presence in a variety of wildlife,
domestic and human tissues as well as in food, water, and soil samples, jas resulted in the developmentof numerous methods for their detection and quantitation. Although chemical extraction procedures
coupled with high resolution gas chromatography-mass spectrometry is considered the gold standard
for the identification and quantitation of individual HAH congeners, the high cost and time consuming
nature of these methods has led to the development of alternative assay methodologies. Several rapid
and inexpensive screening bioassays capable of detecting and estimating the relative potency ofcomplex mixtures of HAHs have been developed by our lab and others1. These bioanalytical methods
are based on the ability of the compounds to be specifically recognized and bound by antibodies(immunoassays) or their ability to transduce a specific biological response in vitro or in cells in culture(bioassays). These bioassays are based on the mechanism of action of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD, dioxin) and related HAHs and utilize the Ah receptor (AhR), a ligand-dependent factor
which mediates the effects of these chemicals2. One cell bioassay (chemically activated Luciferase
expression (CALUX)) utilizes a recombinant cell line (mouse or rat liver cells) that contain a stably
integrated AhR-responsive luciferase reporter gene plasmid (pGudLuc1.1) and takes advantage of theability of the AhR to activate gene expression. Exposure of these cells to extracts containing TCDD
and/or related HAHs results in the induction of luciferase expression in a time-, dose- and chemical
specific manner1,3,4. Although this particular CALUX bioassay system provides a avenue by which todetect dioxins and related AhR ligands, it has a limitation with regards to the transient nature of themeasured induction response from this specific plasmid (garrison). The instability of the luciferase
gene in this specific plasmid results from the targeting of the expressed luciferase to peroxisomes,
where it is subsequently degraded. This results in a reduced reporter gene signal at all times later than4-6 hours after chemical treatment and the maximal fold induction would be dramatically reduced if theinduction response were examined at later times that are commonly used3. Here we have compared thetime and dose response curves for TCDD using the pGudLuc1.1-CALUX cell bioassay and two newrecombinant cell lines that contain other stably transfected reporter gene plasmids (i.e. they contain thegenes for a mutated form of firefly luciferase or that of enhanced green fluorescent protein (EGFP)).
Materials and Methods
Construction of the pGudLuc6.1 Expression Vector and Stable Transfection
The DRE driven, TCDD responsive reporter plasmid pGudLuc6.1 was constructed by subcloning

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the 1810 bp HindIII fragment excised from the plasmid pGudLuc1.1 into the HindIII site upstream ofthe luciferase gene in the plasmid pGL3-basic (Promega). This fragment contains four functional DREs
from the murine CYP1A1 gene that confer TCDD responsiveness upon a MMTV promoter andadjacent luciferase gene. Mouse hepatoma (hepa1c1c7) cells were stably co transfected with thisplasmid and pSV2neo using polybrene and responsive clones isolated as previously described 3. This
resulted in the isolation of a TCDD-responsive stable cell line (i.e. H1L6.1c3).
Cell Cultures and Chemical Treatment
Mouse hepatoma (Hepa1c1c7) cells that had been previously stably transfected with pGudLuc1.1(designated as H1L1.1c2 cells) or pGreen1.1 (designated as H1G1.1c3 cells) and the H1L6.1c3 cells
were grown as previously described3,5. For the luciferase induction studies, H1L1.1c2 and H1L6.1c3cells were grown in 24 well plates and incubated with the indicated concentration of TCDD for theindicated time and luciferase activity in aliquots of cell lysates determined as previously describedusing an Anthos Lucy 2 microplate luminometer3. For GFP induction studies, H1G1.1c3 cells wereplated into clear-bottomed black 96 well microplates and incubated with the indicated concentration ofTCDD for the indicated time and GFP activity in intact cells was measured (without the removal ofmedia) using a Fluostar microtiter plate fluorometer with excitation and emission wavelengths of 485
nm (25 nm bandwidth) 515 nm (10 nm bandwidth), respectively5. All samples were run in triplicate
and the reporter gene activity present in wells containing DMSO only were subtracted from the reporter
gene activity in all treated samples. Reporter gene activity was normalized to sample protein
concentration, determined using the fluorescamine protein assay and bovine serum albumin as thestandard5. Values are presented as the mean ± SD and are expressed as a percent of the maximal TCDD
activity observed in each cell line.
Results and Discussion
The time course of induction of luciferase and EGFP by 1 nM TCDD in the three recombinant celllines is shown in figure 1. Similar to what we have observed previously, the time course of induction
with pGudLuc1.1-containing H1L1.1c2 cells is transient, with maximal activity observed at 4-6 hours
after treatment with TCDD 3. In contrast, expression of luciferase from luciferase from thepGudLuc6.1-containing H1L6.1c3 cells and the pGreen1.1-containing H1G1.1c3 cells progressively
increases with time, with luciferase activity increasing at a significant faster than that of EGFP. The
difference between the results obtained with the cell lines containing the two different luciferase genes
was not unexpected. The relatively rapid loss of luciferase activity in H1L1.1c2 cells derives from thefact that the firefly luciferase gene that was used to create this responsive cell line (present in the pGL2
vector from Promega) results in the production of firefly luciferase that contains a peroxisomal
targeting sequence. This sequence targets the final luciferase protein to the peroxisome where it islikely that peroxisomal enzymes are responsible for the subsequent loss in activity. The luciferase gene
expressed in the H1L6.1c3 cells (from the pGudLuc6.1 vector) contains several mutated residues that
result in and increase its stability in mammalian cells (this gene is contained in the pGL3 vector fromPromega). These mutations have deleted the peroxisomal targeting sequence such that the resulting
luciferase protein now accumulates in cytosol, and this and other minor amino acid mutations havesignificantly stablized the luciferase protein. This increased stability is readily apparent in figure 1.
EGFP expression in H1G1.1c3 cells also progressively increases with time, although the induction
occurs at a slower rate than that in H1L6.1c3 cells. This slow rate of induction has been previously
observed and results both from a slow rate of formation of the final correctly folded fluorescent EGFP
protein, but also the relatively lower sensitivity of a fluorescent assay as compared to a luminescentassay. >From an instrumental analysis point of view, luminescence is a significantly more sensitive

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BIOANALYSISFigure 1. Time course of induction of reporter gene expression in three different stably transfected
TCDD-responsive cells lines.

Figure 2. TCDD dose response curves for the induction of reporter gene expression in three different
stably transfected TCDD-responsive cells lines.

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technique than that of fluorescence and this could explain why luciferase activity is detected at a muchearlier time than that of EGFP. Dose response analysis with each cell line at more optimal analysis
times (4 hours for H1L1.1c2 cells and 24 hours for H1L6.1c3 and H1G1.1c3 cells) reveal similar
relative dose response curves and EC 50s for H1L6.1c3 and H1G1.1c3 cells, while the EC50 for TCDD in
the H1L1.1c2 cells was about 5-6-fold less potent (figure 2). The reason for the lower potency in theH1L1.1c2 cells is unclear at this time but may be related to the relatively greater instability of theluciferase protein in these cells and/or an effect resulting from elements at the specific site of genomic
integration of the plasmid. Although our results demonstrate differences in the response of these cells
to TCDD, each cell line has specific advantages that can be used when identifying and characterizing
AhR ligands. The H1L1.1c2 cells would be most advantageous when examining ligands that induce
AhR-dependent gene expression in a transient manner, such as that observed with prostaglandins6,
where the induction response is over by 6 hours. The other cell lines would be less appropriate for thattype of response. We believe that the H1L6.1c3 and H1G1.1c3 cells would be more appropriate forscreening of samples for HAHs or more stable AhR ligand screening analysis since the time ofinduction would be less critical. It has been reported that increasing the time of induction also allows
metabolically less stable AhR ligands present in a sample extract (i.e. PAHs) to be degraded and thus
not contribute to the overall induction response 7. H1L1.1c2 cells would be less appropriate for HAH
analysis since samples would optimally have to be examined within 4-6 hours after treatment, a periodof time where metabolically less stable contaminants could also activate the AhR. This could result infalse positives and/or overestimation of activity. H1G1.1c3 cells can also be used to examine increases
in EGFP in real time in intact cells and as such, the same cells can be followed for extended periods toexamine the relative potency of a given ligand or extract. Overall, although the recombinant cells
bioassay systems we have developed can be used to identify and characterize AhR ligands, each of theassays have characteristics that make them appropriate for some application and not others. The choiceof which cell line to use will certainly be dependent on the specific questions and issues beingexamined.
Acknowledgments.
This work was supported by the National Institute of Environmental Health Sciences (ES07685,04699, 05707) and an NIEHS Environmental Toxicology Training Grant (T32ES07059).
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