J BIOCHEM MOLECULAR TOXICOLOGYVolume 15, Number 4, 2001

Activation of the Ah Receptor Signaling Pathwayby ProstaglandinsShawn D. Seidel, Greg M. Winters, William J. Rogers, Michael H. Ziccardi,Violet Li, Bart Keser, and Michael S. DenisonDepartment of Environmental Toxicology, University of California, Davis, CA 95616-8588, US; E-mail: msdenison@ucdavis.edu

Received 14 March 2001; revised 6 May 2001; accepted 12 May 2001

ABSTRACT: The aryl hydrocarbon receptor (AhR)is a ligand-dependent transcription factor that medi-ates many of the biological and toxicological actionsof a diverse range of chemicals, including the en-vironmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although no endogenousphysiological ligand for the AhR has yet been de-scribed, numerous studies support the existence ofsuch a ligand(s). Here we have examined the abilityof prostaglandins and related chemicals to activate theAhR signaling system. Using two AhR-based bioas-say systems we report that relatively high concentra-tions of several prostaglandins (namely, PGB3, PGD3,PGF3a , PGG2, PGH1, and PGH2) can not only stim-ulate AhR transformation and DNA binding in vitro,but also induce AhR-dependent reporter gene expres-sion in mouse hepatoma cells in culture. PGG2 alsoinduced AhR-dependent reporter gene expression to alevel three-to four fold greater than that observed witha maximal inducing dose of TCDD. Sucrose gradientligand binding analysis revealed that PGG2 could com-petitively displace [3H]TCDD from the AhR. Overall,our results demonstrate that selected prostaglandinsare weak agonists for the AhR and they represent astructurally distinct and novel class of activator of theAhR signal transduction pathway. C© 2001 John Wiley& Sons, Inc. J Biochem Mol Toxicol 15:187–196, 2001

Correspondence to: Dr. Michael S. Denison.Present Address of Shawn D. Seidel: Department of Neuro-

science, 4447 Boyce Hall, University of California, Riverside, CA92521, US.

Contract Grant Sponsor: National Institutes of EnvironmentalHealth Sciences.

Contract Grant Number: ES07685, ES05707Contract Grant Sponsor: Environmental Toxicology Training.Contract Grant Number: ES07059Contract Grant Sponsor: Superfund Basic Research.Contract Grant Number: ES04699Contract Grant Sponsor: California Agriculture Experiment

Station.c© 2001 John Wiley & Sons, Inc.

KEYWORDS: Ah Receptor; Prostaglandins; TCDD;Prostaglandin G2

INTRODUCTION

The Ah receptor (AhR) is a ligand-dependent tran-scription factor which regulates many of the toxic andbiological effects of a variety of hazardous chemicals,including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD,dioxin). Exposure to TCDD, the prototypical and mostpotent AhR ligand, results in a wide variety of species-and tissue-specific toxic and biological responses, manyof which have been shown to be AhR-dependent [1–3].The induction of gene expression, most notably thatof CYP1A1, is one such response that has been usedas the model to define the mechanism of TCDD-and AhR-dependent action [2–4]. Following ligand(TCDD) binding, the cytosolic TCDD:AhR complexundergoes transformation†, during which it translo-cates into the nucleus and dissociates from twomolecules of hsp90 (a heat shock protein of 90 kDa)and at least one immunophilin-like protein [5–7].Once in the nucleus, the dimerization of the lig-anded AhR with the ARNT (AhR nuclear transloca-tor) protein, converts the AhR complex into its highaffinity DNA binding form [2,3,8–11]. Binding of thetransformed heteromeric AhR complex to its spe-cific DNA recognition site, the dioxin responsive el-ement (DRE), leads to alterations in chromatin struc-ture, increased promoter accessibility and increasedrates of CYP1A1 gene transcription [3,4]. DREs havealso been identified in the upstream region of otherTCDD-inducible genes, including CYP1A2/1B1, UDP-glucuronosyltransferase (UGT∗01) and glutathione S-transferase Ya [reviewed in 3], and they are responsible

†In this report, we have defined transformation as the processby which the TCDD:AhR complex is converted into a form which canbind to DNA with high affinity.

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for conferring TCDD- and AhR-responsiveness uponthese genes.

The best characterized high affinity ligands forthe AhR include a variety of toxic and carcinogenichalogenated aromatic hydrocarbons (HAHs) such aspolychlorinated dibenzo-p-dioxins, dibenzofurans andbiphenyls, polycyclic aromatic hydrocarbons [PAHs)such as benzo(a)pyrene, 3-methylcholanthrene anddibenz(a,h)anthracene, a variety of indole-containingcompounds and heterocyclic amines [12–13]. More re-cently, several classes of naturally-occurring exogenouschemicals which can act as AhR ligands/activators havealso been identified [13–18]. Although the ability ofxenobiotics to bind to and activate the AhR is well doc-umented, a high affinity endogenous physiological lig-and for the AhR remains to be identified. The existenceof a ligand has been suggested both from studies in cellsin culture, where induction of AhR-dependent gene ex-pression has been observed in the absence of added ex-ogenous ligand [19–21], and indirectly from the occur-rence of developmental defects in AhR knockout mice[22–24]. Interestingly, the identification of tryptamine,indole acetic acid, bilirubin and biliverdin as weak en-dogenous ligands for AhR [15–18] and the recent iden-tification of lipoxinA4 as a more potent AhR ligand [25]not only indicates that the AhR can be activated byphysiological substances, but that the AhR signalingsystem can be stimulated by chemicals with physicaland chemical properties distinct from that of knownhigh affinity AhR ligands. However, the existence of anendogenous ligand which can activate the AhR at nor-mal physiological concentrations and the endogenousrole(s) of the AhR signaling pathway remain unknown.

Numerous studies have demonstrated that TCDDalters expression of a variety of gene products [re-viewed in 3]. Given that many, if not all, AhR ligandsare substrates for, or are metabolized by, one or moreof these induced enzymes, we have begun to exam-ine these TCDD-induced gene products with respect totheir substrate specificity in an attempt to identify en-dogenous activators of the AhR pathway. Recently, weand others have reported that TCDD induces expres-sion of the prostaglandin endoperoxide H2-synthase-2gene (PGHS-2) [26,27]. In addition, other studies havenot only shown that TCDD can alter arachidonic acid(AA) metabolism and that AA is a substrate for CYP1A1[28–32], but they have suggested that AA may alsoplay a role in stress-induced expression of CYP1A1[20]. More recently, a role for sphingolipids in CYP1A1induction by 3-methylcholanthrene was reported [33].Given the relationship between AA, prostaglandinsand CYP1A1, and the identification of the AA metabo-lite lipoxinA4 as an AhR ligand [25] we utilized twoAhR-dependent bioassay systems [18,34] to examinethe ability of prostaglandins and related chemicals to

activate the AhR signaling system in vitro and in cellsin culture. Here we report the identification of severalprostaglandins that can stimulate both AhR transfor-mation and DNA binding, and AhR-dependent geneexpression.

EXPERIMENTAL PROCEDURES

Materials

TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF)and [3H]TCDD (specific activity 21 Ci/mmole) were ob-tained from Dr. Steve Safe (Texas A&M University) andwas handled and disposed of as previously described[34]. All of the indicated prostaglandins (95–98% pu-rity) were purchased from Cayman Chemical Com-pany (Ann Arbor, MI), [g 32P]ATP (6,000 Ci/mmol) waspurchased from Amersham (Arlington Heights, IL),and luciferase lysis buffer and reagent from Promega(Madison, WI). DMSO was obtained from Aldrich(St. Louis, MO) and n-hexane from Mallinckrodt(Phillipsburg, NJ).

Animals and Preparation of Cytosol

Male Hartley guinea pigs (250–300 g) wereobtained from Charles River Breeding Laboratories(Wilmington, DE), were allowed free access to food andwater, and exposed to 12 h of light and 12 h of dark.Liver cytosol was prepared as previously described [35]in HEDG buffer (25 mM HEPES, pH 7.5, 1 mM EDTA,1 mM DTT, and 10% (v/v) glycerol and stored until useat −80◦C. Protein concentrations were determined bythe method of Bradford [36] using bovine serum albu-min as the standard.

Gel Retardation of AhR Binding(GRAB) Assay

Analysis of the ability of test chemicals to induceAhR transformation in vitro and DNA binding in vitrowas performed using guinea pig hepatic cytosolic AhRas previously described [19]. Guinea pig cytosolic AhRcomplexes were used as the preferred GRAB screeningprocedure given the high efficiency of this AhR to trans-form into its DNA binding in vitro, as compared to thatfrom other species [11] thus resulting in increased signaland sensitivity of the GRAB assay. Briefly, guinea pigcytosol (8 mg/mL) was incubated with carrier solvent(DMSO, water, or ethanol at 20 mL/mL) 20 nM TCDD or100 mM of the indicated prostaglandin for 2 h at 20◦C.The cytosol was subsequently incubated with [32P]-labeled DRE and the ligand:AhR:[32P]DRE complex

Volume 15, Number 4, 2001 PROSTAGLANDINS AS AhR AGONISTS 189

resolved by gel retardation analysis [18]. The amountof AhR:DRE complex was determined by measuringthe amount of induced protein-DNA complex in thechemical-treated lane minus the amount present inthe same position in an appropriate solvent carrierlane using a Molecular Dynamics phosphorimager. Theamount of induced AhR:DRE complex was expressedas a percentage of that induced by TCDD.

Cell Culture, Chemical Treatment,and Luciferase Measurement

Analysis of the ability of a test chemicals to induceAhR-dependent gene expression was carried out usingH1L1.1c2 cells, a mouse hepatoma cell (Hepa1c1c7) linethat contains a stably integrated DRE-driven firefly lu-ciferase reporter gene [34]. These recombinant mousecells were used as the preferred gene expression screen-ing procedure given their high degree sensitivity andresponsiveness compared to other cell lines we have de-veloped [34] and these aspects result in lower detectionlimits and increase signal to noise ratio for gene AhR-dependent expression. We have observed a high degreeof correlation between the induction of luciferase andCYP1A1 in this cell line (Denison, unpublished results).These cells were grown and maintained as we havepreviously described in detail [34]. H1L1.1c2 cells weregrown to confluence in 96-well plates and were treatedwith TCDD (1 nM), prostaglandins (indicated concen-tration) or carrier solvent (DMSO, water or ethanol at20 mL/mL) for 4 h at 37◦C. No visible toxicity was ob-served in any of the treated cells. After incubation, cellswere washed with PBS and lysed using 25 mL cell cul-ture lysis buffers (Promega) with shaking for 20 min atroom temperature. Luciferase activity was measuredwith a Dynatech ML 3000 Microplate Luminometer us-ing stabilized luciferase reagent (Promega) and proteinconcentrations determined using fluorescamine [37]with bovine serum albumin as a standard. Luciferaseactivity was expressed as a percentage of the activityinduced by TCDD.

Sucrose Gradient Centrifugation

Measurement of AhR ligand binding was carriedout using a modification of the sucrose gradient cen-trifugation assay as we have previously described [38].In this assay, guinea pig hepatic cytosol (5 mg pro-tein/mL) was incubated with 5 nM [3H]TCDD in thepresence of DMSO (10 mL/mL), 1 mM TCDF, or 100 mMPGG2 for 1 h at 4◦C. After treatment with dextran-coated charcoal to remove unbound and loosely boundradioligand, samples were subjected to centrifugationin linear 10–30% sucrose (v/v) gradients at 65,000 rpm

in a Beckman VTi65.2 rotor for 2 h at 4◦C. Followingcentrifugation, gradients were fractionated and theradioactivity present in each fraction was determinedby liquid scintillation. Specific binding of [3H]TCDDto the AhR was computed by subtracting the amountof [3H]TCDD bound in the presence of TCDF or PGG2from the amount of [3H]TCDD bound in the absence ofcompetitor.

RESULTS

To examine the possibility that prostaglandins maybe AhR agonists, we examined the ability of numerousprostaglandins to stimulate transformation and DNAbinding of guinea pig hepatic cytosolic AhR in vitro.The GRAB assay results in Figure 1, reveal that incu-bation of hepatic cytosol with relatively high concen-trations (100 mM) of several prostaglandins, namelyPGA3, PGB3, PGD3, PGE3, PGF3a, PGG2, PGH1, PGH2,and PGK2 resulted in formation of an induced protein-DNA complex with a mobility comparable to that in-duced by TCDD. Although these results provide noquantitative information as to the relative potency ofthese prostaglandins compared to TCDD, the reducedamount of induced protein-DNA complex from eachat 100 mM (∼5,000-fold more than that of TCDD inthe same experiment) suggests that they are signif-icantly weaker agonists. It should be noted that al-though these chemicals are used at relatively highconcentrations (100 mM) these concentrations are stillwithin the solubility range of these chemicals in buffer(0.23–28 mM, depending on the specific prostaglandin(Cayman Chemical Product Information)).

To examine the ability of each chemical to induceAhR-dependent expression, we utilized a recombinantmouse hepatoma cell line (H1L1.1c2) which contains astably integrated DRE-luciferase reporter plasmid [34].These cells respond to AhR agonists with the inductionof firefly luciferase in a time-, dose- and AhR-dependentmanner [18,34]. Incubation of H1L1.1c2 cells with100 mM of the indicated prostaglandins for 4 h revealedthat several prostaglandins (B3, D3, F3a, G2, H1, H2,and K1) induced luciferase activity significantly abovebackground (ethanol alone), with prostaglandin G2 in-ducing luciferase activity significantly greater (aboutfourfold) than that induced by 1 nM TCDD (Figure 2).The structures of the inducing chemicals are shown inFigure 3. It should be noted that some prostaglandins(A3, K2, and E3) were positive in the GRAB assayyet were unable to induce gene expression in the lu-ciferase assay (compare Figures 1B and 2). These resultsare comparable to those in our previous studies whichdemonstrated that activation of the AhR by a chem-ical in vitro does not necessarily correlate with its

190 SEIDEL ET AL. Volume 15, Number 4, 2001

FIGURE 1. Prostaglandin-induced DNA binding of the guinea pig hepatic cytosolic AhR complex in vitro. Guinea pig hepatic cytosol (8 mg/mL)was incubated with control solvent (DMSO or ethanol (20 mL/mL)), 20 nM TCDD or 100 mM of the indicated prostaglandin in DMSO or ethanolfor 2 h at 20◦C. Aliquots were incubated with [32P]lDRE oligonucleotide and inducible AhR:[32P]DRE complexes were resolved by gel retardationanalysis as described in the Material and Methods. (A) Typical gel retardation assay results for these experiments, with the arrow indicating theposition of the TCDD-inducible protein-DNA complex. Only the protein-DNA complexes are shown. (B) Phosphoimager quantitation of theamount of inducible AhR:DRE complex formed in these experiments. Values represent the mean ±SD of at least triplicate determinations andare expressed as a percent of TCDD.

Volume 15, Number 4, 2001 PROSTAGLANDINS AS AhR AGONISTS 191

FIGURE 2. Induction of firefly luciferase activity in stably trans-fected mouse hepatoma (HlL1.1c2), cells by prostaglandins. Conflu-ent 96 well plates containing H1L1.1c2 cells were incubated withcontrol solvent (DMSO, water, or ethanol (10 mL/mL final concen-tration)), 1 nM TCDD 50 or 100 mM of the indicated prostaglandinin solvent (all prostaglandins were in ethanol except PGI2 (water)and PGK2 (DMSO)) for 4 h at 37◦C. Luciferase activity in cell lysateswas determined as described in the Materials and Methods. Valuesrepresent the mean ±SD of at least triplicate determinations and areexpressed as a percent of TCDD.

FIGURE 3. Structures of the AhR active prostaglandins and lipidsidentified in these studies.

ability to induce AhR-dependent gene expression in in-tact cells [39]. Some of these differences could also resultfrom the species-specific differences between these twoassays as has been observed with a very limited num-ber of chemicals [34]. However, since the overall ob-jective of these initial studies was the detection of

novel AhR agonists, use of the most sensitive screen-ing bioassays (i.e., the guinea pig GRAB assay andthe mouse luciferase bioassay), were preferred evengiven the differences in species between these bioas-says. Any apparent species differences, such as thoseobserved above, can be further examined in detail.To determine the relative potency of the inducingprostaglandins, dose response curves for luciferase in-duction were performed with each chemical. As shownin Figure 4, these prostaglandins were relatively weakinducers, (requiring between 1–10 mM in order to in-duce luciferase gene expression above background);PGG2 induced luciferase reporter gene expression ata concentration less than 1 mM. Although PGG2 wasa more potent inducer than the other prostaglandinsexamined, it was still ∼100,000-fold lower in inducingpotency than the TCDD (Figure 4). These dose-responseresults also clearly demonstrate that PGG2 induces re-porter gene expression several fold greater than that ofa maximal inducing concentrations of TCDD.

The aforementioned results imply that PGG2 is aligand for the AhR, but they do not directly confirmthis. Accordingly, the ability of PGG2 to competitively

FIGURE 4. Dose-dependent induction of luciferase activity inH1L1.1c2 cells by TCDD and prostaglandins. Confluent plates ofH1L1.1c2 cells were incubated with DMSO (10 mL/mL final con-centration) or increasing concentrations of TCDD or the indicatedprostaglandins for 4 h at 37◦C. Luciferase activity in cell lysates wasdetermined as described in the Materials and Methods. Values rep-resent the mean ±SD of at least triplicate determinations and are ex-pressed as a percent of TCDD. The lowest dose for each prostaglandinthat resulted in the induction of luciferase to a level that was signif-icantly greater than that of DMSO (at P< 0.05 (*) as determined bythe Student’s t-test was: PGB3–10 mM, PGD3–1 mM, PGF3a–10 mM,PGG2–100 nM, PGH1–1 mM, and PGH2–100 mM.

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FIGURE 5. PGG2 competes with [3H]TCDD for binding to the cy-tosolic AhR. Guinea pig hepatic cytosol (5 mg protein/mL) was incu-bated with 5 nM [3H]TCDD in the absence (◦) or presence of 1 mMTCDF (•) or 100 mM PGG2 (N). Aliquots (300 mL) of each incubationwas analyzed by sucrose density centrifugation as described in theMaterials and Methods. The amount of total [3H]TCDD specific bind-ing activity (mean± SD) in these experiments was 121± 9 fmoles/mgprotein (n = 3), of which 27.5± 6.5 percent was displaced by PGG2.

bind to the AhR was determined using sucrose densitygradient analysis. Ligand binding analysis of cytosol in-cubated with 5 nM [3H]TCDD resulted in the formationof the typical 8–10S AhR binding peak (Figure 5), whichwas competitively eliminated by a 200-fold molar ex-cess of unlabeled TCDF (another high affinity AhR lig-and). PGG2 at a concentration of 100 mM competitivelydisplaced 27.5± 6.5 percent of [3H]TCDD specific bind-ing to the AhR. Not only do these results demonstratethat PGG2 is a competitive ligand for the AhR, albeit aweak one, they are consistent with the reduced amountof induced protein-DNA complex formed in the pres-ence of 100 mM PGG2 (Figure 1).

The results of our studies are consistent withthe ability of the prostaglandins to bind to and acti-vate the AhR and AhR signal transduction pathway.However, given the relatively high concentration ofeach prostaglandin necessary for induction and thatthe purity of each was only 95–98%, we were con-cerned as to whether the induction was due to acontaminant in the prostaglandin preparation ratherthan the prostaglandin itself. Although analysis ofHPLC-purified PGG2 from these preparations wouldbe optimal to confirm its identity as an inducer, thiswas not feasible given the extreme lability of PGG2.Accordingly, we examined the possibility that thesepreparations contained a AhR active contaminant. Be-cause previous studies in our laboratory indicated

that silica gel can contain Ah agonists [39,40], cou-pled with the fact that some of the prostaglandinswe obtained from Cayman Chemical were purifiedusing acid-washed silica gel chromatography, weexamined whether the high level of induction we ob-served with PGG2 was due to a silica gel contami-nant(s). For these experiments, we used a sample ofthe acid washed silica used by Cayman Chemical forthe prostaglandin purification procedure, as well astheir specific sample purification and elution proto-col. Using the Cayman Chemical protocol for the isola-tion of PGG2, chromatography elution fractions werecollected and analyzed using the GRAB and DRE-luciferase bioassays. Although our results reveal thatthe initial wash fraction (90% n-hexane:10% ethyl ac-etate) of the acid-washed silica column contained sig-nificant activity (60.5 and 51% of that obtained using1 nM TCDD, in the GRAB and DRE-luciferase bioas-says, respectively (Figure 6)), the elution fraction thatwould contain PGG2 (30% ethyl acetate:70% hexane)produced only 13.7 and 7% of the response induced by1 nM TCDD in the GRAB and DRE-luciferase bioas-says, respectively. Although a small amount of the

FIGURE 6. GRAB and DRE-dependent luciferase analysis of AhRactivity in extracts from the acid washed silica used to purify PGG2.Acid-washed silica was obtained from Cayman Chemical and frac-tions were collected from elutions based on the Cayman PGG2 pu-rification protocol. A 1 mL acid-washed silica column was elutedtwo times with 3 mL 90% n-hexane:10% ethyl acetate, 3 mL 80%n-hexane:20% ethyl acetate and finally with 3 mL 70% n-hexane:30%ethyl acetate (it is in this fraction that PGG2 normally elutes). One mLfractions were collected, blown down under nitrogen, the residue re-maining resuspended in 1 mL DMSO and an aliquot was analyzedusing GRAB and luciferase bioassays. Values represent the mean±SD of triplicate determinations and are expressed as a percent ofTCDD.

Volume 15, Number 4, 2001 PROSTAGLANDINS AS AhR AGONISTS 193

signal obtained in our analysis could derive from theacidic washed silica gel in the PGG2 preparation, thischemical(s) is not responsible for the extremely highlevel of induction observed with PGG2 incubation. Thefact that all of the prostaglandins used in our studieswere eluted from acid-washed silica columns yet onlya few were active in our bioassays, also argues that thehigh AhR-dependent activity we observed is not sim-ply due to an AhR-active silica gel contaminant(s).

DISCUSSION

Here we have demonstrated that several highlypurified prostaglandins (PGB3, D3, F3a, G2, H1, andH2) act as AhR agonists by their ability to stimulateAhR transformation and DNA binding in vitro and in-duce AhR-dependent gene expression in intact cells inculture. Although AhR activation required relativelyhigh concentrations of prostaglandins (100 mM), farabove that which would be found normally, severalprostaglandins were active at 1–10 mM. The ability ofmany of these chemicals to transform the AhR into itsDNA binding form in vitro and their ability to acti-vate gene expression in intact cells suggests that thesechemicals can act as AhR ligands. In fact, our ligandbinding results demonstrate that PGG2 is an AhR lig-and, albeit weak. However, the ability of PGG2 to in-duce luciferase activity three-to five fold greater thanthat observed with a maximal inducing dose of TCDD,suggests the involvement of factors in addition to theAhR. Although the mechanism by which the potentia-tion of induction of gene expression by PGG2, and otherprostaglandins remains to be elucidated, it is possi-ble that the prostaglandins could be metabolically con-verted into a more potent (AhR active) form(s) in intactcells. Alternatively, in addition to directly activating theAhR, the prostaglandins could activate additional cel-lular signal transduction pathways that augment theinduction response. In fact, support for the latter hy-pothesis comes from the observation that activationof the protein kinase C signaling pathway results ina synergistic increase in AhR-dependent gene expres-sion, respectively [41–44]. The specific cellular signalingpathway(s) affected by PGG2 that is responsible for thisdramatic increase in AhR-dependent gene expression iscurrently being examined.

Dose–response studies indicate that the activeprostaglandins are relatively weak activators of theAhR pathway, however, it is not clear how much ofeach inducer actually entered the cell. At physiologicalpH, prostaglandins are charged and don’t readily passacross biological membranes without the aid of a mem-brane transport protein [44,45]. However, there are sev-eral factors that would be expected to reduce the overall

amount of prostaglandin that would reach and activatethe AhR in the treated cells. First, exposure of cells to rel-atively high concentrations of prostaglandins is knownto reduce prostaglandin transporter efficiency [46] andthis would slow cellular uptake of the prostaglandins.Second, the total amount of prostaglandin available forabsorption/transport would be expected to be signifi-cantly less than the added concentration because of theloss due to binding to serum proteins and lipoproteinspresent in the media. Third, the limited stability/half-life of some of the prostaglandins would also result inreduced concentrations with regards to time of incuba-tion (i.e., PGG2 is rapidly metabolized in the intact cell[47] and has a half-life of approximately 10 min in aque-ous solution (Cayman Chemical Company TechnicalLiterature). According, it is likely that these chemicalsare actually significantly more potent in the intact cellthan our incubation concentration values suggest.

Prostaglandins can be produced from three dif-ferent precursors, di-homo-g -linolenic acid, AA, andeicosapentaenoic acid, and which precursor is used de-termines which type of prostaglandin is made, series1, 2, or 3, respectively [48]. Of the three different seriesof prostaglandins, the 2-series is the most biologicallyactive and abundant. Although the same enzymes pro-duce all three types of prostaglandins, the preferredsubstrate of PGHS is AA [48]. In our analysis the two2-series prostaglandins, PGG2 and PGH2, showed thegreatest potency, although they have short half-lives,whereas three other prostaglandins (PGB3, PGD3, andPGF3a) are members of the 3-series prostaglandins,which, in general, exhibited little or no biological ac-tivity and occur at low concentrations [48]. The resultsof our studies may suggest a biological role for these3-series prostaglandins and that of PGG2 and/or theirmetabolites. The structural components of the induc-ing prostaglandins necessary for activation of the AhRsignaling pathway remain to be determined.

Prostaglandins are considered local hormonessince they are not synthesized in one central organ andtheir concentrations in the blood are usually belowthe nM range [49]. Dose–response relationship studiesusing luciferase bioassay reveal that these chemicals,with the exception of PGG2, are relatively weak AhRagonists that induce AhR activation only at concen-trations of 10 mM or greater, which is much higherthan is seen in blood. However, since prostaglandinsare local hormones, concentrations may actually reach5–10 mM in the proximity of hepatocytes due tonon-parenchymal liver cells secreting these chemicalsinto the narrow space of Disse [50]. At these concen-trations it is possible for selected prostaglandins, ora combination of prostaglandins, to activate the AhR.Prostaglandins, after synthesis, are generally releasedfrom the cell and either act on the parent cell or

194 SEIDEL ET AL. Volume 15, Number 4, 2001

neighboring cells in an autocrine/paracrine fashion[51]. The prostaglandins then bind to their respectivecell surface receptors which then cause second messen-ger production in the cell [55]. However, since recentstudies have shown that eiconsanoids (leukotriene B4and a PGJ2 derivative) are also ligands for the nuclearperoxisome proliferator-activated receptors [52,53],these ligands can not only activate cell surface signaltransduction pathways, but they can also enter cellsand directly activate intracellular receptors.

From a physiological point of view, there is ev-idence for a role for the AhR in modulating AAand prostaglandin synthesis and metabolism. TCDDbeen reported to induce P450-dependent metabolismof AA, with some of the oxidative metabolites beingbiologically active [31,54]. TCDD has not only beenshown to induce the expression of PGHS-2 [26,27],but when Hepa1c1c7 cells and some of its derivedAhR signaling mutants were examined (both the Arnt-defective and low AhR cell lines), observed differencesin prostaglandin synthesis indirectly suggested a rolefor a functional AhR signaling pathway in the regu-lation of prostaglandins [27]. Given the critical role ofthese chemicals in mediating changes in intermediarymetabolism, cell growth, fluid homeostasis, alterationsin hormone synthesis, and alterations in intracellularcalcium, it has also been postulated that the alterationsin AA metabolism and prostaglandin synthesis mayplay a role in the toxicity of TCDD [27]. In fact, it hasbeen reported that administration of fish oil rich in v-3fatty acids reduces TCDD toxicity in rats [55]. The pos-sibility of a prostaglandin, prostaglandin metabolite orother biological lipid being a more effective activatorof AhR-dependent gene expression, and the observedrole of the AhR in regulating PGHS-2 expression sug-gests a self-regulatory mechanism for prostaglandinmetabolism and synthesis. These data also raises thepossibility that an unknown lipid and/or steroid couldbe an endogenous regulator of the AhR. Support forthis latter class of chemicals comes from recent workdemonstrating the ability of 17a-ketocholesterol to bindto and antagonize the AhR when present at physiolog-ical levels [56].

Overall, our analysis has revealed that severalprostaglandins are activators of the AhR signaling path-way. Given their inability to activate other cellular sig-naling pathways and the greater potency of these chem-icals in intact cells, it is likely that metabolites of theselong chained lipids and/or related products may be en-dogenous ligands for the AhR. The results presentedhere not only have identified these chemicals as addi-tional members of a novel group of arachidonic/fattyacid AhR ligands (265), but they further strengthen ourfindings that the AhR can be activated by a wide vari-ety of structurally dissimilar chemicals. Thus, it is very

likely that the promiscuous ligand binding specificityof the AhR will allow it to be activated by a varietyof distinct endogenous chemicals. Defining the spec-trum of chemicals which can bind to and activate theAhR should provide further insights into its endoge-nous ligands and physiological role in organisms.

ACKNOWLEDGMENTS

We thank Drs. Robert Rice and Reen Wu for criticalevaluation of this manuscript.

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