Toxicology P231

CHARACTERIZATION OF THE CALUX AND GRAB BIOASSAYS FORSENSITIVITY AND SPECIFICITY IN DETECTION OFPHARMACOLOGICAL AGENTS THAT ACTIVATE THE Ah RECEPTOR
SIGNALING SYSTEM
George C. Clark1, David J. Brown1, Shawn. D. Seidel2 Dorothy Phelan2and Michael S. Denison2
1Xenobiotic Detection Systems Inc., Durham NC, and2Department of Environmental Toxicology, University of California, Davis, CA.

Introduction:
The Ah receptor (AhR) is a ligand dependent transcription factor that mediates themajority of the biochemical and toxicological effects of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD, dioxin) and related chemicals. The understanding of the mechanistic
basis by which these chemicals express their toxic effects presents the opportunity todevelop rapid screening methods for these environmental toxins.
We and others have used various aspects of the AhR-dependent mechanism of action
to develop rapid and sensitive bioassays for the detection of HAHs and other AhR
ligands. Two AhR-based assays which have gained widespread use include
measurement of the chemical-dependent activation of: 1) gene expression in cells thathave an intact and complete Ahr signaling pathway ("in cello") and 2) AhR binding andthe resulting DNA binding that occurs in a cell free system (in vitro). The two variationsof these assays investigated in the present studies include the Chemically Activated
LUciferase eXpression (CALUX bioassay) using a cell based system, and the Gel
Retardation of AhR DNA Binding (GRAB bioassay) using an in vitro cell free system.
Activation of these bioassay systems occurs in a time-, dose-, chemical- and AhRdependent manner. Given the potential utilization of these AhR-based bioassays forlarge scale chemical and environmental screening for TCDD and related HAHs, thestudies described here were carried out in order to assess the advantages and limitationsof each assay system.

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Materials and Methods:
Chemicals. A variety of chemicals, pharmacological agents, and extracts of newspaper
were used in these investigations. A brief list abbreviations and chemicals used follows:
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; IDAZ, Idazoxan; PHEN, Phentolamine;
EPI, Epinephrine; GUAN, Guanabenz; NP, Newspaper; TCDF, 2,3,7,8tetrachlorodibenzofuran; PCB, 3,3',4,4',5-Pentachlorobiphenyl; 3MC, 3Methylcholanthrene; BNF, b-Naphthoflavone; ANF, a-Naphthoflavone.
CALUX bioassay: CALUX analysis was carried out as described in detail in Garrison
et al. (Fund. Appl. Toxicol. 30, 194-203 (1996)). Briefly, chemicals or extracts wereapplied to monolayers of stably transfected cells in DMSO and incubated at 37 C in ahumidified CO2 atmosphere for 4 hours. After 4 hours cells were lysed and luciferase
activity quantified with luciferase reagent (Promega) in a luminometer.\
GRAB bioassay: GRAB analysis was carried out using guinea pig hepatic cytosol asdescribed in Bank et al. (1995). Briefly, guinea pig hepatic cytosol was incubated withchemical or extracts in DMSO and allowed to bind to Dioxin Response Elements(DREs) labeled with radioactive 32P in the presence of excess nopnspecific DNA.
Specific binding was detected by electrophoresis in polyacrylamide gels and quantitationof specific AhR:32P-DRE complexes.
Results:
Dose dependent responses of the bioassays to TCDD.
The sensitivity of the CALUX and GRAB assays were compared by their relative
response to various doses of TCDD. Shown in Figure 1 is the dose dependent
response to TCDD of the two assays. The CALUX assay was approximately 10-foldmore sensitive in detecting TCDD than the GRAB assay with the shapes of the dosereponse curves being similar. The increased sensitivity of the CALUX assay may be aresult of the amplification of signal due to the mechanistic basis by which the cells cells
can respond to these toxins, producing more luciferase on a mole basis than measured
by only binding to the receptor.

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Induction Response (percent of TCDD)

120

100

80

60

40

20010 -12

10 -11

10 -10

10 -9

10 -8

10 -7

TCDD Concentration (M)Figure 1. Dose-dependent activation of the CALUX (closed circles) and GRAB (opencircles) bioassays by TCDD.
The GRAB assay estimates binding to both TCDD and specific DRE sequences without
an amplification step based upon the mechanism of activation.
Response of the CALUX and GRAB bioassays to pharamcological agents andenvironmental extracts.
We have identified several pharmacologically-relevant chemicals including imidazoline
receptor ligands (idazoxan, guanabenz and epinephrine) and benzimidazole drugs(omeprazole, albendazole and thiabendazole) which induced luciferase activity in several
different CALUX cell lines. Although some of the imidazoline receptor ligands werealso potent activators of AhR transformation and DNA binding in vitro, others wereessentially inactive; all of the benzimidazoles were significantly less potent in the in vitro
bioassay.

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In contrast, the GRAB bioassay responded indiscriminatly to a number of extracts ofnewspapers and other common house-hold products. The CALUX assay displayed
greater specificity in responding to these extracts.
Conclusions:
Our results reveal a significant discrepancy between the ability of a chemical to activate
the AhR in vitro and to activate the Ah receptor system in intact cells. The cell basedsystem (CALUX) is more sensitive and yet more discriminating in detecting AhR
agonists.
The in vitro GRAB analysis may detect more activators of the AhR since chemicals areadded directly to the incubation mixture and pharmacokinetics of penetrating the cell are
circumvented. However, activation alone apparently generates a number of false
positive results which are problematic for a screening assay for risk assessment
purposes of environmental hazards.
Overall, our results demonstrate the usefulness of the CALUX assay and cell based
systems for the detection and quantiation of the AhR agonists. In vitro assays may
prove to be useful as environmental screening methods, however a high frequency offalse positive results are of concern.
References:
1. Garrison, et al. Fund. Appl. Toxicol. 30, 194-203 (1996).
2. Bank et al. Arch. Biochem. Biophys. 317, 439-448 (1995).
Acknowledgements:XDS acknowledges NIEHS for support of this research from NIH
grant SBIR2 R44 ES 08372-02; Validation Of A Bioassay For Dioxin-Like Toxicantsand ES07865 (MSD).

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