The Drug Salicylamide Is an Antagonist of the Aryl Hydrocarbon Receptor That Inhibits Signal Transduction Induced by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin

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[CANCER RESEARCH 64, 429–434, January 1, 2004] The Drug Salicylamide Is an Antagonist of the Aryl Hydrocarbon Receptor That Inhibits Signal Transduction Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Christopher J. MacDonald, Henry P. Ciolino, and Grace Chao Yeh Cellular Defense and Carcinogenesis Section, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, Maryland

ABSTRACT (a)pyrene, and heterocyclic amines such as 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine. These classes of xenobiotics are all 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environ- suspected human carcinogens. For example, TCDD, a widespread mental contaminant, that has been linked with a variety of deleterious environmental contaminant generated by a variety of industrial pro- effects on human health, including increased cancer rates and reproductive anomalies. The detrimental effects of TCDD are mediated via the aryl cesses, has been linked to an increase in many types of human hydrocarbon receptor (AhR), a transcription factor that regulates the cancers, including hepatocellular cancer (3, 4). In animal models, expression of the carcinogen-activating enzymes cytochromes P-450 TCDD has been shown to cause acute toxicity (5), increased oxidative (CYP) 1A1, 1A2, and 1B1. In the present study, we examined the ability stress (6), impaired ovulation (7), and immunotoxicity (8). These of synthetic derivatives of salicylic acid to affect TCDD-stimulated AhR- effects in animal models are believed to closely reflect the deleterious mediated signal transduction in human hepatoma HepG2 cells. Salicyl- effects of TCDD in humans (9). TCDD has the highest affinity for the amide (SAL), an analgesic drug, caused a potent and long-lasting inhibi- AhR of any known compound, and is considered the prototypical AhR tion of TCDD-induced CYP enzyme activity. Acetylsalicylic acid (aspirin) ligand. Its interaction with the AhR is essential to its toxicity, as and the naturally occurring phytochemical salicylic acid had no effect on demonstrated in AhR-knockout mice, which are impervious to TCDD CYP activity. SAL inhibited the increase in CYP1A1, -1A2, and -1B1 exposure (10). mRNA levels that occurs on exposure to TCDD. TCDD-induced transcription of these genes was also inhibited by SAL, but not by aspirin or Because of the central role of the AhR in the toxicity of TCDD and salicylic acid, as demonstrated by luciferase reporter assays. The tran- genotoxicity of numerous xenobiotics, the development of inhibitors scription of the CYP1 family of genes is regulated by the interaction of of AhR-mediated signal transduction has been an important goal of TCDD-activated AhR with the xenobiotic-responsive element present in chemoprevention science. In the present study, we have examined the the promoter regions of these genes. As shown by electrophoretic mobility effects of the salicylates on the AhR and the metabolic pathway it shift assay, SAL completely blocked the binding of TCDD-activated AhR regulates. Salicylic acid was first identified in willow bark extracts as to the xenobiotic responsive element. Also, SAL substantially blocked the an active anti-inflammatory compound more than 200 years ago, and binding of TCDD to the cytosolic AhR. These results demonstrate that its synthetic derivative, acetylsalicylic acid (aspirin), has be used to SAL, a commonly used analgesic, is a potent inhibitor of AhR-mediated treat a number of human maladies for over a century. Recent studies signal transduction, and may be an effective agent in the prevention of have demonstrated that salicylate usage is associated with a reduction TCDD-associated disease. of incidence of several types of cancer (11–13), and animal studies have shown that salicylates are potent inhibitors of chemically in- INTRODUCTION duced carcinogenesis (14, 15). We have, therefore, examined the effects of salicylates on the AhR function in HepG2 cells, a well- The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed differentiated human liver cancer cell line that has been extensively cytosolic protein that is a member of the basic helix–loop–helix used as a model system to study the AhR (16–19). We demonstrated Per-Arnt-Sim super family of transcriptional regulatory proteins (1, that the synthetic salicylate, salicylamide (SAL), is a potent and 2). On binding the ligand, the “activated” AhR translocates to the long-lasting inhibitor of TCDD-induced signal transduction mediated nucleus, in which it forms a heterodimer with its partner, the aryl by the AhR. hydrocarbon nuclear translocator. Together, they form a transcription factor that binds to the xenobiotic-responsive element (XRE) present in the promoter region of a number of genes. The best characterized MATERIALS AND METHODS molecular response to AhR activation is the transcriptional induction Materials. Human hepatoma HepG2 cells were from American Type Cul- of CYP1A1, CYP1A2, and CYP1B1, the genes that encode the 1A1, ture Collection (Manassas, VA). RPMI 1640, TRIzol, and LipofectAMINE 1A2, and 1B1 enzyme isoforms, respectively, of cytochrome P-450 were from Life Technologies, Inc. (Grand Island, NY). Glutamine and fetal (CYP). These enzymes catalyze the epoxidation of certain classes of bovine serum were from BioFluids (Rockville, MD). Polydeoxyinosinic-de- xenobiotics, resulting in the generation of highly reactive electrophilic oxycytidylic acid, protease inhibitors, ethoxyresorufin, acetylsalicylic acid metabolites. These metabolites can covalently bind specific residues (aspirin), salicylic acid and SAL were from Sigma (St. Louis, MO). [32P]dATP 32 of DNA, causing DNA adduct formation that may result in mutation and [ P]dCTP were from DuPont-NEN (Boston, MA). TCDD was from the 3 and subsequent cellular transformation. The AhR and the genes that it Midwest Research Institute (Kansas City, MO). [ H]TCDD was from Chem- syn Science Labs, Lanexa, KS. The Omniscript kit was from Qiagen (Valencia, regulates are, therefore, central in mediating the genotoxicity of many CA). Tris-borate gels, Tris-borate running buffer, and high-density sample environmental contaminants. buffer were from Invitrogen (Carlsbad, CA). Ligands of the AhR include several classes of environmental car- Cell Culture. HepG2 human hepatoma cells were grown with RPMI 1640 cinogens, including polyhalogenated biphenyls such as 2,3,7,8-tetra- supplemented with 2 mM glutamine and 10% fetal bovine serum in a 5% CO2 chlorodibenzo-p-dioxin (TCDD), aryl hydrocarbons such as benzo- humidified incubator at 37°C. For all of the treatment compounds, DMSO was used as the vehicle and did not exceed 0.1%. Received 4/10/03; revised 9/23/03; accepted 9/29/03. Carcinogen Activation Capacity in HepG2 Cells. The ability of SAL to The costs of publication of this article were defrayed in part by the payment of page affect CYP enzyme activity was evaluated in intact cells by measurement of charges. This article must therefore be hereby marked advertisement in accordance with ethoxyresorufin-O-deethylase (EROD) activity. In 24-well plates, HepG2 cells 18 U.S.C. Section 1734 solely to indicate this fact. were incubated with increasing concentrations of salicylates in the presence of Requests for reprints: Grace Chao Yeh, Basic Research Laboratory, Building 538/ Room 141, National Cancer Institute-Frederick, Frederick, MD 21702-1201. Phone: 1nM TCDD for 24 h, for dose-response experiments. For time course exper- (301) 846-5369; Fax: (301) 846-6395; Email: [email protected]. iments, cells were incubated with increasing concentrations of SAL, or with 429

Ligand-Binding Assay. AhR ligand binding was measured according to the methods of Raha et al. (26). Cytosolic protein (1.5 mg/500 ␮l) was incubated with 20 nM [3H]TCDD in the presence of DMSO (5 ␮l/500 ␮l), 5 mM SAL, or 10 ␮M TCDD (unlabeled) for2hat4°C. Samples were centri- fuged in linear gradients of 5–30% sucrose (v/v) in 12 ml of Beckman Quick-Seal rotor tubes at 65,000 rpm for2hat4°C in a Beckman VTI-65–1 rotor. Fractions (ϳ500 ␮l each) of the gradients were collected, and radioac- tivity was determined by scintillation counting. Statistical Analysis. Statistical analyses were performed with STATVIEW Statistical Analysis Software (SAS Institute, San Francisco, CA). Differences between group mean values were determined by a one-factor ANOVA, fol- lowed by Fisher’s protected least significant difference post hoc analysis for pairwise comparison of means. A two-sided P value of Ͻ0.05 was considered significant. CYP mRNA levels in Figs. 3 and 4 are shown on a linear scale.

Fig. 1. Structures of salicylic acid, aspirin, and salicylamide (SAL). RESULTS

other test compounds at concentrations that inhibit 50% (IC50), in the presence Effect of Salicylates on TCDD-Induced CYP Enzyme Activity. of1nM TCDD for various times from 6 to 144 h. Medium was decanted, and In this study, we examined three salicylates the structures of which cells were washed once with PBS. Medium containing 5 ␮M ethoxyresorufin are illustrated in Fig. 1. The cellular capacity for enzymatic acti- was added. Increasing fluorescence as a result of the conversion of vation of procarcinogens was measured by EROD assay. Treatment ethoxyresorufin to resorufin by CYP enzymes was measured using a CytoFluor of HepG2 cells with 1 nM TCDD resulted in an increase in multiwell plate reader (Applied Biosystems, Foster City, CA), with an exci- EROD-specific activity from nondetectable levels to 17 pmol/min/ tation of 530 nm and emission at 590 nm. The reaction was allowed to run for 30 min. well in controls (Fig. 2A). The addition of SAL caused a concen- Reverse Transcription-PCR. In 6-well plates, cells were treated with various concentrations of SAL alone, or with 100 pM TCDD, for 8 h. Cells were washed twice with PBS, and total RNA was isolated with TRIzol reagent per manufacturer’s instructions. cDNA was synthesized from 2 ␮g of total RNA using an Omniscript kit according to the manufacturer’s instructions. Semiquantitative reverse transcription-PCR for cytochrome P4501A1 (CYP1A1), cytochrome P4501A2 (CYP1A2), and cytochrome P4501B1 (CYP1B1) expression was performed as described previously (20). Primer sequences and conditions for CYP1A1 and CYP1B1 were as described by Dohr et al. (21), and for cytochrome P4501A2 (CYP1A2) as described by Chung and Bresnick (22). Primers for glyceraldehyde-3- phosphate dehydrogenase were from Clontech (Palo Alto, CA). The opti- mum cycle number that fell within the exponential range of response for CYP1A1 (23 cycles), CYP1A2 (28 cycles), CYP1B1 (30 cycles) and glyceraldehyde-3-phosphate dehydrogenase (17 cycles) was used. The level of CYP mRNA was normalized to the level of glyceraldehyde-3- phosphate dehydrogenase mRNA. Luciferase Assay. HepG2 cells were seeded onto 6-well plates at 400,000 cells/well. After 24 h, the cells were transiently transfected with a luciferase reporter vector (1.5 ␮g/well) containing three repeats of the XRE and a ␤-galactosidase vector (pCMV-␤-gal; 0.6 ␮g/well) with the use of Lipo- fectAMINE. Transfected cells were treated with various concentrations of salicylates in the presence of 10 nM TCDD. Luciferase activity was measured using a Microlumat LB 96P luminometer (EG&G Berthold). ␤-galactosidase activity was determined by the method of Rosenthal (23). Electrophoretic Mobility Shift Assay. HepG2 cells were incubated with DMSO (0.1%), 10 nM TCDD alone, or 10 nM TCDD with 1 mM SAL for 2 h at 37°C. Nuclear protein was isolated, and electrophoretic mobility shift assay was performed by the method of Denison et al. (24). Synthetic oligonucleo- tides containing the AhR-binding site of the XRE (forward primer, 5Ј-GAT CTG AGC TCG GAG TTG CGT GAG AAG AGC CG-3Ј; reverse primer, 5Ј-CGG CTC TTC TCA CGC AAC TCC GAG CTC AG-3Ј; Ref. 25) were labeled with [32P]dCTP. Binding reactions were performed for 25 min (10-min preincubation without probe ϩ 15-min incubation with probe) at room tem- perature and contained 10 ␮g of nuclear protein, 2 ␮g of polydeoxyinossinic deoxycytidylic acid and labeled probe (ϳ50,000 cpm) in a final volume of 20 ␮ l of binding buffer [25 mM Tris-HCl (pH 7.9), 50 mM KCl, 1 mM MgCl2, 1.5 mM EDTA, 0.5 mM DTT, 5% (v/v) glycerol]. To determine the specificity of binding to the oligonucleotide, excess unlabeled specific probe was incubated with the nuclear extract of TCDD-treated cells for 15 min. DNA–protein complexes were separated under nondenaturing conditions on a 6% (w/v) Fig. 2. Effect of salicylates on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced polyacrylamide gel with 0.5ϫ Tris/borate/EDTA running buffer. The gels cytochrome P-450 (CYP) activity in HepG2 cells. CYP activity was determined in intact cells by ethoxyresorufin-O-deethylase (EROD) assay. A, cells were treated with 1 nM were dried, and the amount of labeled probe present in the DNA–protein TCDD and the indicated concentrations of salicylate for 24 h. B, cells were treated with complexes was determined with a Bio-Rad GS-363 molecular imaging system. TCDD and salicylamide (SAL) for the times indicated. n ϭ 4 Ϯ SE. 430

Table 1 The effect of salicylamide on cytochrome (CYP)-activity, after induction was no significant effect on microsomal EROD activity (P ϭ 0.70; HepG2 cells were incubated with 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) Table 1), whereas ␣-naphthoflavone completely abolished EROD for 24 h. Cells were washed, and competitors (10 ␮M ␣-naphthoflavone or 250 ␮M salicylamide) were added. Ethoxyresorufin-O-deethylase (EROD) activity was measured activity. in intact cells 2 h later. Microsomes from TCDD-treated cells were incubated with DMSO Effect of SAL on CYP1A1, -1A2, and -1B1 mRNA Levels. (control) or competitors, and EROD activity was measured. Experiments were performed in quadruplicate two times. TCDD caused a 13.3-, 7.5-, and 2.8-fold increase in CYP1A1, -1A2, and -1B1 expression in HepG2 cells, respectively, compared with Treatment Activitya DMSO controls. SAL cotreatment inhibited the TCDD-induced in- Intact cells Control 20.16 Ϯ 0.49 crease of all three CYP isozymes in a concentration-dependent man- ␣-Naphthoflavone 2.05 Ϯ 0.08 ner (Fig. 3A). SAL treatment alone had no effect on basal CYP Salicylamide 19.15 Ϯ 0.22 Microsomes expression (Fig. 3B). Aspirin did not have an effect on TCDD-induced Control 2.22 Ϯ 0.06 CYP mRNA (data not shown). ␣-Naphthoflavone Not detected Salicylamide 2.18 Ϯ 0.06 Effect of Salicylates on XRE-Controlled Luciferase Reporter a Activity for intact cells is in units of pmol/min/100 k cells; activity for microsomes Activity. HepG2 cells were transiently transfected with a lucifer- is in units of pmol/min/2.5 ␮g. ase reporter vector containing multiple copies of the XRE. TCDD induced luciferase activity by ϳ45-fold above control levels. The addition of SAL resulted in a concentration-dependent decrease of tration-dependent inhibition of EROD activity, with an interpo- luciferase activity (Fig. 4). Aspirin and salicylic acid had no effect ϳ ␮ lated IC50 of 170 M. SAL at 1 mM suppressed EROD levels to (Fig. 4). 10% of cells treated with TCDD alone. In contrast, both aspirin and Effect of SAL on AhR Activation. The effect of SAL on TCDD- salicylic acid had no inhibitory effect at any concentration (Fig. induced activation of the AhR was measured by electrophoretic 2A). TCDD caused a persistent increase in EROD activity in mobility shift assay. TCDD (10 nM) caused a significant increase HepG2 cells that remained at maximal levels for 6 days (Fig. 2B). in AhR translocation to the nucleus and in binding to the XRE. SAL, added along with TCDD, caused an inhibition of enzyme This was completely suppressed in cells cotreated with 1 mM SAL activity that persisted throughout the 6-day time course, regardless of the concentration used (Fig. 2B). (Fig. 5). The specificity of the bandshift was confirmed by treat- The inhibitory effect of SAL was further examined to test whether ment of nuclear extracts with excess unlabeled XRE and by incu- this was due to a direct effect on CYP enzyme activity. The addition bation with an antibody to the AhR, which abolished AhR-XRE of SAL (250 ␮M) to HepG2 cells that were first incubated with TCDD binding (data not shown), as we have demonstrated previously (to induce CYP activity) did not have a significant effect on EROD (27). activity (P ϭ 0.16), whereas 10 ␮M ␣-naphthoflavone (a positive Effect of SAL on AhR Ligand Binding. Competitive binding control) inhibited EROD activity by 84%, compared with controls assays were performed using the cytosolic fraction from HepG2 cells. 3 (Table 1). SAL (250 ␮M) was also added to microsomes isolated from Binding of [ H]-TCDD to the AhR was competitively inhibited by TCDD-treated HepG2 cells, and EROD activity was assayed. There excess unlabeled TCDD or SAL (Fig. 6).

Fig. 3. Effect of salicylamide (SAL) on cytochrome P-450 (CYP) mRNA levels. Cells were treated with 100 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the indicated concentration of SAL (A) or with SAL alone (B) for 8 h. Levels of cytochrome P4501A1 (CYP1A1), cytochrome P4501A2 (CYP1A2), and cytochrome P4501B1 (CYP1B1) mRNA were measured by reverse transcription-PCR and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). n ϭ 3 Ϯ SE. 431

DISCUSSION TCDD is a major environmental contaminant, generated by a variety of industrial processes. There are numerous thermal sources of TCDD, such as municipal, hazardous and medical waste incinerators, diesel engine emissions, fires, and residential heating. The manufac- ture of chlorinated chemicals, wood pulp, and paper also release TCDD (28). Major industrial accidents, including those in Nitro, West Virginia (1949) and Seveso, Italy (1976), resulted in the release of huge amounts of TCDD, causing acute exposure to both workers and residents in the surrounding areas. Because of the chemical stability of TCDD and other related dioxins, there is persistent contamination worldwide. The primary source of human exposure to TCDD is food, particularly dairy products, meat, and fish (29). TCDD is detectable in human serum and is known to accumulate in adipose tissue of humans

Fig. 6. Competition with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding the aryl hydrocarbon receptor (AhR). Cytosolic fractions of HepG2 cells were incubated 3 with 20 nM [ H]-TCDD in the presence of DMSO (f),1(‚),2(Ⅺ), or 5 mM salicylamide (SAL; F), or 10 ␮M unlabeled TCDD (᭜)for2hat4°C. The specific binding was measured by sucrose density gradient centrifugation. Results are representative of two separate experiments.

and animals (30). It also reaches significant levels in human breast milk, providing a pathway for infant exposure (29). The toxicity of TCDD and other dioxins have, as a result, been intensely studied. Animal experimentation has demonstrated that, based on the minimal effective carcinogenic dose, TCDD is the most potent animal carcinogen yet discovered (31). There is mounting experimental evidence suggesting that TCDD may affect human fer- tility, because it is associated with anovulation and dysfunctional spermatogenesis in primates and other animals (32–35). Developmen- tal anomalies in infants may arise as a result of exposure via breast milk (36). Epidemiological studies have indicated that TCDD expo- Fig. 4. Effect of salicylates on xenobiotic-responsive element (XRE)-controlled transcription. Cells transfected with an XRE-controlled luciferase reporter vector and a vector sure is associated with an increase in many types of cancer, including expressing ␤-galactosidase were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; lung, liver, and rectal cancers (3). TCDD has also been linked to 10 nM) for 24 h in the presence of increasing concentrations of salicylate. The amount of increased ischemic heart disease (37, 38). luciferase was normalized to ␤-galactosidase levels. n ϭ 3 Ϯ SE. Given the ubiquity of TCDD contamination and its adverse impact on human health, the mechanisms of TCDD toxicity are of great interest. TCDD does not bind DNA, which indicates that it is not directly genotoxic. The primary mechanism of biological damage by TCDD has been reported to be the generation of reactive oxygen species (39–41). This occurs as a consequence of CYP enzyme activity. A postulated mechanism for this is the auto-oxidation of the cytochrome P-450 mono-oxygenase complex, which generates superoxide (40). Superoxide can spontaneously dismutate, liberating hy- drogen peroxide as a byproduct, which, in the presence of transition metals, can produce hydroxy radicals, leading to indiscriminate damage to cellular biomolecules, including DNA. This hypothesis is consistent with the observation that the deleterious effects of TCDD are dependent on the AhR. The basal expression of CYP is very low. Only after ligands of the receptor, such as TCDD, activate the AhR, does the expression of CYP1A1 (as well as CYP1A2 and CYP1B1) increase. This AhR-dependent mechanism has been demonstrated in AhR-null mice, which do not up-regulate CYP expression when challenged with TCDD and are unaffected by TCDD, even at doses that are 10 times higher than that which induces severe toxic and pathological effects in wild-type mice (10). Inhibition of AhR-mediated signal transduction would, therefore, Fig. 5. Effect of salicylamide (SAL) on aryl hydrocarbon receptor (AhR) activation. prevent TCDD-induced biological damage. We, and others, have HepG2 cells were incubated with DMSO or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin described natural and synthetic compounds that act in this regard, e.g., (TCDD) with or without 1 mM SAL for 2 h. Nuclear protein was isolated, and activated ␣ AhR in 10 ␮g of protein was measured by electrophoretic mobility shift assay. Results are the synthetic flavonoid -naphthoflavone acts antagonistically by representative of two separate experiments. competing with TCDD for binding of the AhR (42). However, by 432

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. SAL INHIBITS AhR FUNCTION AND CYP1 EXPRESSION themselves, these compounds act as agonists, activating the AhR, ing the activation of the AhR to its DNA-binding form. This could resulting in an increase in CYP1A1 expression. Our laboratory has occur as a result of inhibiting the binding of the ligand to the receptor, recently characterized several naturally occurring phytochemicals that which was confirmed by ligand-binding assay. We demonstrated that act in this dual manner (43–45). The AhR is a promising molecular SAL substantially reduced the binding of TCDD to the cytosolic AhR target, and a pure antagonist of the AhR would be potentially thera- (Fig. 6). Although the inhibition was not completely blocked, this was 3 peutic for the prevention of human diseases that result from both acute most likely caused by the high levels (20 nM)of[H]-TCDD neces- and chronic TCDD exposure. In the present study, we have examined sary to elicit detectable binding in this assay system (46). three members of the salicylate family for their effects on the AhR. This is the first demonstration of SAL as a potent inhibitor of the These include salicylic acid, acetylsalicylic acid (aspirin), and SAL, a AhR that, as a result, can block signal transduction initiated by TCDD natural phytochemical and two synthetic derivatives of salicylic acid, exposure. It is, furthermore, a pure antagonist of the AhR, unlike most respectively (for structures see Fig. 1). These compounds have im- other AhR inhibitors that have been characterized in this laboratory portant advantages, being relatively inexpensive, and more impor- and others. Unlike several other compounds that directly bind and tantly, they have already been used extensively in humans. Further- inhibit CYP enzyme activity, it is exceptionally long-lasting. Finally, more, SAL is commercially available in many pain remedy SAL has already been extensively used in humans; therefore, its safety preparations and is often prescribed for patients with aspirin sensi- and pharmacokinetics are already well established. SAL may, there- tivity. fore, serve as a promising chemopreventive compound that targets the There was a concentration-dependent decrease in TCDD-induced AhR. CYP enzyme activity, as measured by EROD assay, in HepG2 cells cotreated with SAL, but not in HepG2 cells cotreated with aspirin or salicylic acid (Fig. 2A). There is a sustained increase in EROD activity REFERENCES upon the addition of TCDD, because, unlike other ligands of the AhR 1. Hankinson, O. The aryl hydrocarbon receptor complex. Annu. Rev. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. The Drug Salicylamide Is an Antagonist of the Aryl Hydrocarbon Receptor That Inhibits Signal Transduction Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Christopher J. MacDonald, Henry P. Ciolino and Grace Chao Yeh

Cancer Res 2004;64:429-434.

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