ANA/BIOAIn Vitro Recombinant Receptor/Reporter Gene Assays forAssessing the Estrogenic and Dioxin-Like Activityof Compounds and Complex Mixtures
p. Balaguerl, M. Denison^ and T. Zachar9wsk|3
11NSERM U58, 60 Rue de Navacelles, 34090 Montpellier, France, 2Department ofEnvironmental Toxicology, University of California, Davis, CA, USA 95616 and^Department of Pharmacology and Toxicology, University of Western Ontario, London,Ontario, Canada N6A 5C1.

Abstract
In vitro recombinant receptor/reporter gene assays have been developed to assess
and rank the potency of substances and complex mixtures suspected of possessing
estrogenic and/or dioxin-like activity. The assays take advantage of the receptor-mediatedmechanism of action of estrogenic and dioxin-like compounds. The Environmental
Estrogen Bioassay (E2 Bioassay) uses a Gal4-human estrogen receptor chimeric construct(Gal4-HEG0) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) which can betranseintly transfected into any established cell line and has been stably transfected intoHeLa cells. The E2 Bioassay exhibits 40- to 50-fold induction following treatment with 1 nM
17p-estradiol (E2) and has a detection limit of 5 pg of E2/ml. The Ah receptor (AhR)
Bioassay uses Hepa 1c1c7 wild type cells stably or transiently transfected with a DREregulated luciferase reporter gene and the endogenously expressed AhR and Ah receptor
nuclear translocator (ARNT). This bioassay has a detection limit of 16 pg of TCDD/ml andexhibits approximately 10-fold induction in luciferase activity following treatment with 1 nM
TCDD. The utility of the assays are demonstrated by examining the activity of pulp andpaper mill black liquor, chloro-S-triazine-derived compounds and naringenin, a plant
bioflavonoid.
Introduction
Several in vivo and in vitro assays have been developed to assess the estrogenic or
dioxin-like activity of individual compounds or complex mixtures. These assays use avariety of endpoints including increased in target organ weight'', enzyme and recombinant
reporter gene induction^'^'^'^'^'^', ligand binding ^•^•'^°\ increased protein expression"''''and cell proliferation or differentiation' •''''. Advantages of in vitro bioassays include costeffectiveness, accurate prediction of potential toxic impact, the capability of handling large
sample numbers and they also provide an indication of the possitile mechanism of action.
Moreover, they account for synergistic, antagonistic and additive interactions which may
occur between compounds within complex mixtures that are not considered by using

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conventional chemical residue analysis.
In this study, we demonstrate the utility ofrecombinant receptor/reporter gene bioassays by detecting the presence of estrogenic anddioxin-like acfivity in pulp and paper mill black liquor, a recovered by-product in the pulping
industry and assessing the estrogenic potency of naringenin, a plant bioflavonoid and two
commonly used triazine herbicides, atrazine and simazine.
Materials and Methods
Materials. Black liquor, a recovered by-product in the pulping industry, was obtained from amodernized bleached kraft mill located in southern Ontario (supplied by Dr. K. Burnison,
National Waters Research Institute, Burlington, Ontario). Approximately 3% of black liquor
is lost to the sewer system which carries the waste to the primary and secondary treatmentfacilities of the mill before the final effluent is discharged to the receiving waters. All otherchemicals were of the highest quality available from commercial sources.
Reporter Gene Assays. The MCF-7 human breast cancer cells, Hepa 1c1c7 wild type
mouse hepatoma cells and stably transfected HeLa cells were maintained as previously
described'"''^'. Cells were transiently transfected using the calcium phosphate coprecipitation technique'^* and reporter gene assays were performed as described in Brasieret a l ^ ^ or using the Luciferase Assay System (Promega). The stable transfectants wereprepared as previously described^'®'.
Results
TCDD treatment of Hepa 1c1c7 wild type cells transiently transfected with a DREregulated reporter gene (pGudLucl.1) exhibited a dose dependent increase in luciferase
activity (Figure 1). Based on these results, the AhR bioassay has a detection limit of 16 pgof TCDD/ml and a 10-fold maximum induction. Cells treated with serially diluted black
liquor also exhibited a dose dependent induction in reporter gene activity.

-13 -12 -11 -10 -9 -8Log [ TCDD] (M)T"C- 6 - 5 - 4 -3 -2
DILUTION FACTORFigure 1. Effect of TCDD and pulp and paper mill black liquor on the induction of a DREregulated luciferase reporter gene transiently transfected into Hepa 1c1c7 wild
type cells.

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100"
1•

NARINGENIN•ATRAZINEO SIMAZINE

8060-y^40-H
U<tu70 •t/5<Be;tu

100

U80-l-l60-mh

1 1 1 1 1 1 1 1 1C -fi-7-6-S- Log [NARINGENIN] (M)1 1 •-8-7-6-5- Log Concentration (M)17P-ESTRADIO•^*-= ^BLACK LIQUOR

4020I-13-12-11-10 -9 -8-Log[E2] (M)•-6 -5 -4 -3
DILUTION FACTOR

Figure 2. Effect of 17p-estradiol (E2), pulp and paper mill black liquor, atrazine, simazine
and naringenin on the induction of a Gal4-regulated luciferase reporter gene,
17m5-G-Luc.
Figure 2 summarizes the the ability of E2, black liquor, atrazine, simazine andnaringenin to induce Gal4-estrogen receptor chimera-mediated induction of the Gal4regulated reporter gene, 17m5-G-Luc. E2 (1 nM) maximally induced luciferase activity 40to 50-fold, exhibiting an EC50 of 20 pM. Black liquor and naringenin also exhibited
significant estrogenic activity. In contrast, atrazine and simazine did not induce luciferase
activity, at concentrations as high as 10 pM.
Discussion
This report demonstrates the utility of in vitro recombinant receptor/reporter genebioassays to detect and assess the estrogenic and dioxin-like activity of compounds andcomplex mixtures. Several in vivo and in vitro bioassays have been previously usedhowever, most exhibit modest responsiveness and can be induced tiy a number ofdivergent mechanism which may lead to false-positive results. Moreover, many of theendpoints that are monitored are laborious and time consuming which limits the number ofsamples that can be examined in a timely manner. The E2 Bioassay and AhR bioassays
address these limitations and provides a sensitive method which is amenable toautomation.

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796

Ah
Receptor

TCDD
789

ARNT

B

4xDRE

MMTV

Luciferase

4 X (TTGCGTGAGA)

DA/B

H—h-h185595250Estrogen
Receptor

E2881i^Gal4148 250

595

E2D

17m5GlobGal4-Estrogen
Receptor Chimera

Luciferase

5 X (CGGAGGACTGTCCTCCG)
Figure 3. Components of the AhR and E2 Bioassays. A - Endogenously expressed AhR andARNT, B - the DRE-regulated reporter gene, pGudlucl.l, C - The Gal4 - humanestrogen receptor chimera, D - the Gal4-regulated reporter gene (17m5-G-Luc).
The AhR Bioassay uses Hepa 1c1c7 wild type cells which endogenously express
AhR and ARNT and a transiently transfected DRE-regulated reporter gene (pGudlucl.l) toassess the dioxin-like activity of a compound or complex mixture. In addition, a stablytransfected cell line has also be prepared. The advantage of this bioassay is that inductionof luciferase activity can only occur through the AhR; therefore, other mechanisms such asmRNA stability or oxidative stress, which have been reported to influence AHH activity are

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excluded. Consequently, the specificity and diagnostic nature of this bioassay is improved
which is an important consideration when examining complex environmental and industrial
samples. Induction of pGudlucl.l activity following treatment of transiently transfected
Hepa 1c1c7 wild type cells demonstrates that black liquor possess ligands with dioxin-like
activity. However, previous studies have shown that this induction is not due topolychlorinated dibenzo-p-dioxins or dibenzofurans and therefore, further research isnecessary to identify the inducing agent '^'.
The central component of the E2 Bioassay is the Gal4-human estrogen receptor
chimera (Gal4-HEG0) which is composed of the Gal4 DNA binding domain linked to theligand binding domain of the ER (i.e. domains D, E and F, see Fig. 3c). Gal4 is a DNA
binding yeast transcription factor involved in the regulation of galactose utilization and has
no known mammalian homologue. It recognizes and binds to a specific response element
referred to as a 17mer (Fig. 3d) which, in isolation, is sufficient to confer Gal4-dependent
induction to a recombinant reporter gene. The Gal4-regulated reporter (17m5-G-Luc)
consists of five tandem 17mer response elements upstream of the rabbit p-globin promoter
linked to the firefly luciferase cDNA reporter gene. Luciferase activity is easily measured
and provides a rapid, sensitive, non-radioactive and non-toxic assay to monitor theinduction potency ot compounds or complex mixtures '^'. Since there is no known
mammalian equivalent to Gal4 and reporter gene induction is strictly regulated by the17mer response elements, any increases in 17m5-G-Luc activity can only occur via Gal4HEGO, thus demonstrating that the sample possesses estrogenic activity which is mediatedby the estrogen receptor.
Recent studies have suggested that black liquor, atrazine, simazine and naringenin
19 20 21i

possess estrogenic activity'. The results presented in Figure 2 clearly demonstratethat black liquor and naringenin possess estrogenic activity that is mediated by the estrogen
receptor. In contrast, no induction in luciferase activity was observed in the E2 Bioassay
following treatment of the cells with atrazine or simazine, thus the estrogenic effects elicitedby these compounds are not ER-mediated. However, these results do not exclude that apossible interaction could occur between chloro-S-triazines and ER-mediated responses.
This study demonstrates the utility of recombinant receptor/reporter gene bioassays
in the assessment of compounds and complex mixtures which are suspected of possessing
estrogenic and dioxin-like activity. The assays can be used: (i) to complement conventional
chemical analysis by indicating the presence of compounds that may not be detected by
GC-MS analysis due to the lack of appropriate standards, (ii) in the assessment of thepotential toxic potency of the sample, (iii) to indicate possible mechanisms of action, (iv) as
economical monitoring systems to assess remediation efforts when detailed chemical
residue analysis is not required, (v) as a screen for identify complex mixture fractions thatpossess activity during the isolation and identification of the active agents and (vi) in
determining toxic equivalency factors for environmental estrogens and dioxin-likecompounds. Moreover, the availability of stable transfectant cell lines should facilitate theincorporation of these assays into most laboratories.
Acknowledgements
This work has been supported by funds from: (i) INSERM to PB, (ii) the Dutch
Technology Foundation and NIEHS Superfund Basic Research Program (ES04699 andES04911) to MD and (iii) the Medical Research Council of Canada, the Canadian Networkof Toxicology Centres and the University of Western Ontario (Acedemic Development Fundand the Vice-President's (Research) Special Competition to TZ. TZ is supported by a
PMAC-HRF/MRC Research Award in Medicine and travel funds for the presentation were
provided by the Health Research Foundation.

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