Biochemical and molecular mechanisms

DIFFERENTIAL RESPONSIVENESS OF THE AH RECEPTOR-DEPENDENT GENE
EXPRESSION INDUCED BY HAHS AND PAHS
Bin Zhao, Jessica E. Bohonowych and Michael S. Denison
Department of Environmental Toxicology, University of California, Davis, CA, USA
Introduction
The Ah receptor (AhR) is a basic helix-loop-helix PAS-containing transcription factor which activates gene
expression in a ligand-dependent manner.1,2 The best-characterized high affinity ligands for the AhR include avariety of halogenated aromatic hydrocarbons (HAHs), such as the polychlorinated dibenzo-p-dioxin, dibenzofurans,
and biphenyls and polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene, beta-naphthoflavone(BNF) and dibenz(a,h)anthracene.3 Recently, we and others have demonstrated that the AhR can bind and beactivated by a wide variety of chemicals that are structurally distinct from the classical HAH and PAH ligands for theAhR.3,4 Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), the prototypical and most potent HAH
AhR ligand, produces a wide variety of species- and tissue-specific toxic and biological responses, such as tumor
promotion, immuno-, hepato- and dermal toxicity, lethality, wasting, endocrine disruption, birth defects, persistentoxidative stress and induction of numerous enzymes, including that of microsomal cytochrome P4501A1 (CYP1A1).
Biochemical, genetic and knockout animal studies have demonstrated that the majority of the above toxic andbiological effects of TCDD and related dioxin-like HAHs are mediated by the AhR.1,2,5,6 Although both HAHs andPAHs can bind to the AhR with relatively high affinity, only HAHs produce TCDD-like AhR-dependent spectrum oftoxicity, PAHs rarely produce TCDD-like adverse effects even though they can produce many of the same biologicaleffects, albeit more transiently. The greater toxicological potency of HAHs compared to PAHs has been
hypothesized to result from their significantly higher AhR binding affinity, which is expected to result in decreased
HAH ligand dissociation from AhR and also to their resistance of HAHs to metabolism. The exact molecularmechanisms responsible for AhR-dependent toxicity and the inability of PAHs to produce these effects are not clear.
Toxicity in vivo results from a combination of biological, biochemical, pharmacodynamic and pharmacokinetic
events, and given that AhR functions as a transcription factor, it is clear that the ability of chemicals to produce
AhR-dependent toxicity appears to be primarily driven by persistent activation of AhR-dependent gene expression intarget cells. Therefore, we examined the similarities and differences between the ability of HAHs and PAHs to bindto and transform the AhR and induce AhR-dependent gene expression using a variety of in vitro and in vivo
bioassays in order to attempt to examine this difference in toxic/biological potency of AhR ligands.
Materials and Methods
Chemicals: TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and [3H]TCDD were generously provided by Dr. Steven
Safe (Texas A&M University, College Station, TX). BNF and DMSO were from purchased from Aldrich (St. Louis,MO) and [3H]BNF was from Dr. Mark Hahn (Woods Hole Oceanographic Institute). Cell culture reagents and media
were purchased from Gibco/BRL (Grand Island, NY) and G418 was from Gemini Bio-Products (Woodland, CA).
Cell culture and enhanced green fluorescent protein (EGFP) bioassay: The stably transfected H4G1.1c2 cells that
contained the EGFP gene under the control of DREs were maintained and assayed as previously described.7,8,9

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AhR ligand binding: The AhR [3H]TCDD or [3H]BNF ligand binding and dissociation experiments were carried out
as described previously in detail by using the hepatic cytosol from various species (rat, mouse, guinea pig, andhamster).10
Results and Discussion

TCDD and BNF dose response experiments in H4G1.1c2 cells for 24 hours revealed that both TCDD and BNF
induced AhR dependent GFP expression to a comparable maximum activity in a dose dependent manner,
although BNF was at least 1,000-fold lower in inducing potency. This difference in relative inducing potency
for BNF and TCDD is significantly greater than their relative ligand binding affinities. The greater AhR
binding affinity of TCDD compared to BNF has been suggested to contribute to the relative differences in toxic
potency, in that it may allow BNF to more readily dissociate from the AhR, leading to more rapid inactivationof BNF-occupied AhR and hence more transient induction. However, our ligand dissociation experiments
clearly demonstrate that binding of either [3H]TCDD or [3H]BNF to cytosolic AhR from various species was
essentially irreversible in the window of the tested time (24 hours) indicating that the dissociation hypothesis withthese ligands and species is incorrect and that differences in HAHs and PAHs persistence of gene expression resultsfrom other factors.
Another possibility is that the toxicity of a given AhR ligand is simply based on its metabolic persistence,
rather than its relative affinity for the AhR. To test this hypothesis, the EGFP assay provides us with a uniqueavenue in which to easily examine ligand-induced AhR-dependent gene expression in a “real-time” manner foran extended period of time in the same cells which can not be accomplished by some other systems.8 Whileinduction of EGFP reporter gene expression in stably transfected rat hepatoma (H4IIe) cells by a single dose of1 nM TCDD was maximal at 5 days and remained elevated for up to 10 days, induction by 0.1 µM BNF wasmaximal by 2 days and it decreased to background by day 10 (Figure 1A). Interestingly, the induction of EGFP
expression by 0.1 µM BNF reached the same maximal level as TCDD at 1 day, while less induction level wasobserved for BNF after 2 days. If the persistence of activation is simply due to metabolism of BNF, then initialtreatment of cells with increasing concentrations should result in greater levels of EGFP accumulation andpersistence of induction. In fact, BNF at higher concentrations (1 and 10 µM) induced progressively higher andmore persistent levels of EGFP expression. It should be noted that because EGFP is extremely stable (T1/2 >23h), reporter gene signal output at any time point is the combination of newly expressed EGFP, but also existing
EGFP that will be very slowly lost. These results demonstrated that decreased PAH induction responsecompared to that induced by TCDD at later time points is very likely due to metabolism/inactivation of BNF,
which progressively results in lower levels of EGFP expression over time. Another avenue in which to test themetabolism theory is to examine the effects of daily treatment of cells to BNF on the induction responsecompared to TCDD. When cells were repeatedly treated daily with 1 µM BNF, a relatively persistent EGFP
expression was observed that was fairly comparable to that obtained with a single treatment with 1 nM TCDD
(Figure 1B). These results demonstrate that chronic exposure of cells to BNF results in persistent induction ofAhR-dependent gene expression and further support the hypothesis that the transient induction response withBNF was primarily due to its metabolism. Relatively persistent binding of both ligands and the relatively rapid
turnover of occupied AhR in treated cells are inconsistent with ligand dissociation being responsible forpersistent gene activation. While AhR binding affinity is an important consideration when lower than saturating
concentrations of chemicals are present in a response cell, our results are consistent with the hypothesis that thegreater persistence of HAH-inducible gene expression is more dependent upon the metabolic stability of theHAH ligand rather than its affinity for and persistence in binding to the AhR. While it is possible that there aredifferences in the ability of HAH- versus PAH-bound AhR complexes to bind to and activate gene expression,these differences remain to be identified. Overall, we propose that the toxicity of HAHs results from chronic

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activation of AhR-responsive genes as a result of continual activation of newly synthesized AhR, rather thanpersistent activation of the existing pool of AhR in a given cell. In this scenario, PAHs and HAHs activate
AhR-dependent gene expression exactly the same, but as CYP1A enzymes are induced by the AhR, they (andother cellular enzymes) subsequently begin to metabolize the PAH reducing its concentration over time; theHAH is unaffected. As new AhR is synthesized it can be bound and activated by the persistent HAH andremaining PAH, but over time there will be less PAH in the cell and progressively less nascent AhR is bound by
the PAH and the induction response is attenuated. The results described here would suggest thatmechanisms/events that maintain relatively high cellular concentrations of PAHs would result in
AhR-dependent toxicity. In fact, such an effect has been observed in fish in vivo. For example, chronic dietary
exposure of catfish to relatively high levels of BNF resulted in TCDD-like toxic effects, presumably due to thefact that relatively high body burdens of BNF could be maintained due to the typically low rates of metabolism
found in fish.11 Similarly, work by DiGuilio and coworkers demonstrated that in fish in which PAH metabolism
by CYPs was inhibited by chemicals or CYP-morpholinos PAHs could produce TCDD-like toxicity.12,13 Our
results coupled with these data strongly suggest that the lack of AhR-dependent toxicity by PAHs, compared toHAHs, is primarily due to their metabolic lability which results in lower AhR-dependent gene expression overtime. Thus, environmental exposures that result in inhibition and/or down-regulation of PAH metabolism has
the potential to allow previously nontoxic AhR ligands to produce adverse effects.
Acknowledgements
This work was supported by the National Institutes of Environmental Health Science (ES012498, ES04699,ES05707), the American Taxpayers and the California Agricultural Experiment Station.
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A
3000
TCDD 1 nM
BNF 0.1 uM
BNF
1 uM
BNF 10 uM

GFP Activity (RFU)

2500

200015001000

5000012345678910Time (Days)B

TCDD_Single
BNF_Single
TCDD_Multiple
BNF_Multiple

GFP Activity (RFU)

4000

3000

2000

1000

001234567Time (Days)

Figure 1. Time-dependent EGFP reporter gene expression in rat H4G1.1c2 cells. Single
treatment of the indicated concentrations of TCDD (1 nM) or BNF (0.1, 1 and 10 μM)(A) or single treatment (1 nM TCDD or 1 μM BNF) and multiple treatment that iscarried out by changing the medium contained indicated concentration of compounddaily (B) was incubated with H4G1.1c2 cells at 33ºC, respectively, and AhR-dependent
EGFP induction over time was measured. Values are expressed as relative fluorescence
units and represent the mean ± SD of triplicate determinations.

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