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Species-Specific Differences in Ah Receptor Ligand Binding andTranscriptional Activation: Implications for Bioassays for the Detection ofDioxin-Like Chemicals
M. S. Denison^. P. M. G a r r i s o n i , J.M.M.J.G. Aarts2, K. Tullis^, J.A.C.
Schalk^, M.A.Cox2 and A. Brouwer^ IDepartment of Environmental Toxicology,
University of California, Davis, CA 95616 and 2Department of Toxicology, Agricultural
University, Wageningen, The Netheriands

1. Introduction
Polychlorinated diaromatic hydrocarbons (PCDHs) are a diverse group of widespreadenvironmental contaminants, which include polychlorinated biphenyls (PCBs),dibenzofurans (PCDFs), dibenzo-/>dioxins (PCDDs) as well as many other subclasses of
PCDHs and polycyclic aromatic hydrocarbons (PAHs).i) Some PCDHs are not only
lipophilic and persistent in the environment but they are known to bioaccumulate andbiomagnify in the food chain.2) Exposure to specific PCDHs results in a wide variety ofspecies- and tissue-specific effects, such as tumor promotion, lethality, birth defects,
hepatotoxicity, immunotoxicity, dermal toxicily, alterations in endocrine homeostasis andinduction of numerous enzymes, including that of cytochrome P4501A1.1-5) Induction ofcytochrome P450IA1 by P(5DHS is mediated by the Ah receptor (AhR) complex to whichthese chemicals bind with high affinity.2-5) Following ligand (PCDH) binding, the AhR
complex is transformed into its DNA binding form and PCDH:AhR complexes accumulates
within the nucleus.^.s.s) The binding of transformed PCDH:AhR complexes to its specific
DNA recognition site (the dioxin responsive element (DRE)) adjacent to the P450IA1 geneis necessary for its transcription activation.5.6) Since quantitative structure-activityrelationship (QSAR) studies also support a role for the AhR in mediating the toxicity ofthese chemicals'-"), the induction of P4501A1 and its monooxygenase activities (7ethoxyresorufin O-deethylase (EROD)) has been one response that has been used as abioassay to evaluate the relative biological/toxicological potency of PCDHs and complex
mixtures containing these chemicals.'i'^.^.s)
In the environment, including some hazardous waste sites, PCDHs are present ascomplex mixtures. Samples from these sites may theoretically contain up to 209 different
PCBs, 135 different PCDFs, and 75 different PCDDs isomers and congeners and manyother related chemicals with vastly different chemical, physical, and toxicologicalproperties.''3) Currently, the toxicological potency of complex mixtures of contaminants is
assessed by the addition of the toxic equivalent factor (TEF) multiplied by theconcentration of the individual compounds present in the mixture. TEF values have been
determined for many PCDHs by measuring their ability to induce EROD activity in cell
culture or a toxic endpoint'^^.s) and then expressing the magnitude of the response interms relative to that observed with the most potent PCDH, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), as toxic equivalents. The specific TEF values used in these analysis vary
widely relative to the specific species and endpoint examined.''^.s) Although additivemodels to assess toxicological potency of mixtures of PCDHs based on TEF values doORGANOHALOGEN COMPOUNDS
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take into account the biological effects of the compounds, however, they do not account forinteractive effects (antagonism, synergism'^^.s)) between or among active or inactive
PCDHs or other xenobiotics. These points are significant, expecially since species- andtissue-differences in PCDH responsiveness (particularly to that of TCDD) have beenobserved.2-3'9) Although the AhR appears to mediate these responses in all species, it is
not identical.^O''^^) We recently observed species-specific antagonism of TCDD-inducible,AhR-dependent gene expression by several "nonAhR" di-ortho-substituted PCBs (such as2,2',5,5'-tetrachlorobiphenyl (TCB)) (Aarts et al., submitted) and of hepatic AhR
transformation and DNA binding by a-napthoflavone (ANF) an AhR antagonist.'^) Theseobservations are of particular importance, given that PCDHs/PAHs exist in theenvironment as complex mixtures and that interactions between chemicals within themixture could not only differentially affect the overall biological/toxicological potency of themixture to various species, but" also measurements using available AhR-dependent
bioassays. Here we liave examined the apparent species differences in AhR antagonism
by examining the effect of TCB and ANF on AhR functionality (AhR ligand binding,transformation, DNA binding and transcriptional activation).
2. Materials and Methods
Preparation o1 Cvtoso): Hepatic cytosol from male Hartley guinea pigs {250-300g,Michigan Department of Public Health, Lansing, Ml USA), and male Sprague-Dawley rats
(200g, Charies River Laboratories, Wilmington, DE USA) and from male C57BLy6N mice
(20g, Charies River Breeding Laboratories) was prepared as described.8.13)
AhR Ligand and DNA Binding Analvsis: AhR ligand binding in hepatic cytosol wasdetermined by sucrose density centrifugation using [3HJTCDD and DNA binding wascarried out using gel retardation analysis.'^lLuciferase Expression Assav: Continuous cell lines (Hepal (HlL1.1c2), HepG2(HG2L1.1c3), GPC16 (G16L1.1c8) and H4IIE (H4L1.lc4)) which have been stablytransfected with the HAH-inducible reporter plasmid pGudLucl.1 (Garrison et al.,submitted) were used in these studies. pGudLucl.1 contains the firefly luciferase gene
under PCDH-inducible control of four dioxin responsive elements. Exposure of these cells
to TCDD and related AhR agonists results in induction of firefly luciferase activity in a time, dose- and AhR-dependent manner. Cells were incubated with the indicated chemical for
24 h and, after incubation, cells were lysed and luciferase activity of the cleared lysate wasdetermined using the Promega luciferase assay system.
3. Results
We and others have previously demonstrated that ANF can act as an antagonist to the rat
AhR.) However, preliminary studies revealed that ANF was not a very effective antagonistof the guinea pig AhR complex (Denison, unpublished observations). In order tocharacterize this in greater detail, we incubated rat and guinea pig hepatic cytosol withTCDD (5nM) alone or increasing concentrations of ANF in the absence or presence ofTCDD for 2 h at 20°C followed by measurement of the amount of AhR that wastransformed into its DNA binding form. These results demonstrate that although ANF is aweak partial agonist in rat (it weakly induces AhR transformation and DNA binding) it
antagonized the action of TCDD (i.e. ANF reduced the amount of TCDD-inducible proteinDNA complex formation by 70%) (Fig 1). In contrast, ANF was a significantly better AhR
agonist of the guinea pig AhR, inducing complex formation to about 60% of that induced
by TCDD, and it appeared to only slightly antagonize TCDD-inducible protein-DNA

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complex formation (Fig. 1). These results were consistent with ligand binding analysis(data not shown), which revealed that ANF effectively competed with TCDD for binding tothe rat AhR complex; no competition was observed with the guinea pig AhR complex.

Figure 1. Dose-dependent effect of a_ c
napthoflavone (ANF) on guinea pig9= »and rat cytosolic AhR transformation C ™
and DNA binding. Cytosol was -» C£DD
incubatedwithi n c r e a s i n g o ,0 P
^ oconcentrations of ANF alone (0,0) or
1 a
TCDD and increasing concentrations of I E c
ANF ( , ) and the amount of inducible ,I oO V"
protein-DNAcomplex
formeddetermined by gel retardation analysis.
Values represent the mean of at least
three determinations.
ANFoaoConcentration(M)• G16U.1C8• H4L1.1C4•
HlL(.lc2
Q HG2L1.1C31 ^IJcontrol

TCDO

1 , Jjfr^WM
TCB

TCODITCBFigure 2. TCB antagonizes TCDD induction of luciferase activity in a species-specificmanner. Cells were incubated with DMSO, TCDD (InM), TCB (5nM) or TCDD and TCB
for 24 h. Values represent the mean ± SE of at least three determinations.
In order to characterize the .apparent species-specific nature of TCB antagonism, we haveexamined the effect of TCB on the ability of TCDD to induce luciferase activity in stablytransfected cell lines from various species (guinea pig Gl6L1.lc8, rat H4L1.1c4, mouse
H1L1.1C2 and human HG2L1.1c3 cell lines); Exposure to TCDD (5 nM) alone induced
luciferase activity in all cell lines while TCB (5 nM) had no significant effect (Fig. 2).
Although induction of luciferase activity by TCDD in guinea pig and human cells was notaffected by co-administration of TCDD and TCB (Fig. 5), significantly less luciferase
induction was observed in the rat and mouse cell lines (47% and 86%, respectively).
Since the DNA regulatory elements controlling expression of the luciferase gene are
identical in each of the stably transfected cell lines, the differences in responsiveness ofthe cell lines are the result of species differences in the AhR, its associated proteins and/or

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trans-acting factors present in each cell type. Ligand binding analysis (data not shown)has demonstrated that this species difference in antagonism is due to the differential
ability of TCB to competitively inhibit TCDD binding to the mouse and rat AhRs but not to
the guinea pig and human AhRs. These results also demonstrate that the ligand binding
specificity of the AhR is not identical among species.
4. Conclusions
Our results demonstrate a significant species-specific difference in the ability of an AhR
ligand (ANF) and a previously documented "nonAhR" ligand (TCB) to act as an AhR
antagonists. Additional experiments have demonstrated lhat the differential antagonism iscorrelated with the ability of the TCB or ANF to bind to the AhR. In those species in whichthese chemicals are not antagonistic, they fails to compete with TCDD binding, and, as in
the case of ANF and guinea pig AhR, they can act as an AhR ligand. These results
demonstrate distinct species differences in the ligand binding specificity of the AhR. Inaddition, they raise some interesting questions regarding analysis of the biological effectsof complex mixtures using AhR-dependent bioassay systems, in that the presence of highlevels of TCB in a complex mixture would affect its biological activity in guinea pig or
human cells significantly less than that which would be observed in rat or mouse. The
presence of AhR antagonists in sample extracts could be detected utilizing a combinationof both in vitro (ligand and DNA binding assays) and in vivo (cell based) bioassays inwhich a known amount of inducer (TCDD) is mixed with the test mixture and the relative
potency of the response compared to the inducer alone. The presence of AhR antagonists
in complex chemical mixtures and the observed species differences in AhR ligand binding
would complicate detection of dioxin-like chemicals using AhR-based bioassays.
Accurate estimation of the potency of unknown sample extracts containing dioxin-likechemicals should involve multiple species bioassay systems (both in vivo and in vitro).
This work was supported by a NIEHS Superfund Basic Research grants (ES04911 andES04699) and the Dutch Technology Foundation (AB and MSD).

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