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1754 Biol. Pharm. Bull. 26(12) 1754—1760 (2003) Vol. 26, No. 12 Screening of the Inhibitory Effect of Vegetable Constituents on the Aryl Hydrocarbon Receptor-Mediated Activity Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin ,a a a b a Yoshiaki AMAKURA,* Tomoaki TSUTSUMI, Kumiko SASAKI, Takashi YOSHIDA, and Tamio MAITANI a Division of Foods, National Institute of Health Sciences; 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–8501, Japan: and b Faculty of Pharmaceutical Sciences, Okayama University; 1–1–1 Tsushima, Okayama 700–8530, Japan. Received June 12, 2003; accepted August 22, 2003 The aryl hydrocarbon receptor (AhR) is a ligand-activated nuclear transcription factor that mediates re- sponses to environmental contaminants such as dioxins, which have many adverse health effects. We performed a preliminary screening of the inhibitory effects of vegetable constituents on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of AhR using the AhR-based bioassay for dioxins, the Ah-Immunoassay. Ninety veg- etable constituents including flavonoids, tannins, saponins, terpenes, etc., were assayed in vitro. Among them, flavones, flavonols, anthraquinones, piperine, coumestrol, brevifolincarboxylic acid, and resveratrol showed marked inhibitory effects on AhR-based bioassay activation by TCDD, and their effects were dose dependent. Curcumin, carnosol, and capsaicin also inhibited the activation of AhR in this assay, although to a lesser degree. These results suggest that several vegetable constituents might play a role in protection against dioxin toxicity. Key words vegetable constituent; aryl hydrocarbon receptor; dioxin; food; Ah-immunoassay The toxic expression system of dioxins is generally agreed by TCDD by applying AhR-based bioassays for dioxins and on based on various studies.1—3) Briefly, potent dioxins showed that inhibitory effects were exerted by extracts of known as xenobiotic ligands, most notably 2,3,7,8-tetra- sage, green leafy vegetables, and citrus.13) In addition, the in- chlorodibenzo-p-dioxin (TCDD), enter cells and specifically teraction of vegetable constituents with AhR was determined bind to the aryl hydrocarbon receptor (AhR). AhR is located using an in vitro reporter gene assay.14) The present study in the cytoplasm as one component of a complex composed was performed to evaluate the inhibitory activity of 90 veg- of two molecules of a 90-kDa heat shock protein. After lig- etable constituents against TCDD-induced activation of AhR and binding, AhR dissociates from the complex and translo- using an AhR-based bioassay for dioxins, the Ah-immunoas- cates to the nucleus and dimerizes with the AhR nuclear say (Ah-I), as an experimental model. translocator protein (ARNT). AhR-ARNT heterodimers act as transcriptional activators by binding to specific DNA se- MATERIALS AND METHODS quences termed xenobiotic-response elements (XREs) in the enhancer regions of proteins involved in xenobiotic metabo- Samples and Reagents Dimethyl sulfoxide (DMSO) lism and function as ligand-activated transcription factors and TCDD (98%) were purchased from Wako Pure Chemical mediating the pleiotropic biological responses to dioxins. Industries, Ltd. (Osaka, Japan). The following compounds These biological responses include teratogenesis, carcinogen- were isolated from the species indicated in parentheses15—17): esis, epithelial hyperplasia, and induction of drug-metaboliz- Agrimoniin (Agrimonia japonica); brevifolincarboxylic acid ing enzymes. (Phyllanthus flexuosus); gemin A, pedunculagin, and tellima- AhR activation caused by the binding of dioxins is as- grandin I (Geum japonicum); geraniin (Geranium thun- sumed to be one of the first and key steps in the development bergii); procyanidins B2 and C1 (Theobroma cacao); and ca- of dioxin toxicity. Accordingly, the inhibition of AhR activa- suarinin and procyanidin B4 (Cowania mexicana). Pentagal- tion would be expected to provide protection against such loylglucose was obtained by methanolysis of tannic acid.18) toxicity. The main route of dioxin contamination in humans Naringin 49-O-a-glucose was isolated from a natural food appears to be through food,4—7) and therefore if factors that additive, enzymatically modified naringin.19) Their purities inhibit AhR activation are present in our daily diet, they were greater than 90% as determined by HPLC. The other might play a role in protection against dioxin toxicity, conse- compounds tested were purchased from Funakoshi (Tokyo, quently leading to a lower risk of dioxins to human health. Japan), Wako, Tokyo Kasei (Tokyo, Japan), Sigma-Aldrich AhR ligands such as dioxins are artificial products that (St. Louis, MO, U.S.A.), and Nakahara Science (Gifu, have recently appeared, and AhR might primarily function as Japan). Most chemicals were of the highest grade commer- a regulatory receptor for exogenous natural products such as cially available and used without further purification (mostly vegetable constituents. Recently, several investigations have .98%). The Ah-I kit was obtained from Kubota (Osaka, studied the effects of dietary components such as flavonoids, Japan). etc. on the binding of TCDD to AhR, and it was found that Evaluation Using AhR-Based Bioassay To evaluate the these components exert antagonistic activity.8—12) However, AhR-based activity, the Ah-I was applied as an experimental fundamental studies on the relationship between the AhR model. The Ah-I method is a receptor-binding assay using pathway and food constituents are still limited. cytosol containing AhR extracted from mammalian liver Based on this background information, we reported the cells and immunologically measures the dioxin level utilizing screening for the inhibitory effects on the activation of AhR an antigen-antibody reaction. This is a technique to detect ∗ To whom correspondence should be addressed. e-mail: [email protected] © 2003 Pharmaceutical Society of Japan December 2003 1755 the reactivity of AhR with dioxins and dioxin-like com- fluenced by glycosidation in the B-ring. pounds on an ELISA plate without using living cells and is Among the flavanones, hesperetin and naringenin inhibited useful for screening the biological toxicity of dioxins. There- the activation of AhR induced by TCDD with EC70 values of fore the Ah-I is used as a simple dioxin-monitoring 14.6 and 27.7 m M, respectively. The flavanone glycosides method.20,21) The Ah-I was employed as follows. The cytosol (naringin, hesperidin, naringin 49-O-a-glucoside, taxifolin, (200 ml) was added to the sample, which as dissolved in and fustin) did not inhibit the activity at the EC70 level. The DMSO at final concentrations of 0.5—50 m M, or to DMSO tendency of glycosides to weaken these activities was similar alone as the control, the mixture was preincubated for to those of flavones and flavonols. 20 min, and then incubated with 5 nM TCDD (final concen- The isoflavones mostly did not show an inhibitory effect tration of 0.025 nM) for 2 h at 30 °C (the final DMSO concen- on activation of AhR induced by TCDD in bioassay at the tration was 1% in culture medium). After incubation, the for- EC70 level, and they slightly inhibited the activity at high mation of the AhR-TCDD complex was determined using an concentrations of 25—50 m M (ca. 10—20% inhibition). Ah-I kit, and the absorbance was determined at 405 nm using Genistein had a slight inhibitory effect at the EC70 level of a microplate reader. The AhR activity was calculated as: 40.8 m M and equol, which is regarded as the final metabolite {12[(A2B)2(C2D)]/(A2B)}3100, where A is the ab- of isoflavones, also exhibited inhibition of activity at sorbance of the control with TCDD added, B is the ab- 41.2 m M. Although most isoflavone themselves have low in- sorbance of the control with DMSO added, C is the ab- hibitory effects less than the EC70 level, it is interesting that sorbance of the sample solution with TCDD added, and D is their metabolite has an inhibitory effect at the EC70 level. the absorbance of the sample solution with DMSO added. Based on these results in flavonoids, the inhibitory effect on All experiments were carried out in duplicate or triplicate. TCDD-induced activation of AhR in flavonoids is generally The values obtained with DMSO alone were considered as in the following order: flavones5flavonols..flavanones. 100% of the control value. To define the potency of each of isoflavones5flavonoid glycosides. the compounds tested, AhR activity was expressed as con- Among the remaining compounds tested, anthraquinones centration–response curves. The concentrations producing showed remarkable inhibition of AhR activation comparable AhR activity equal to 70% of the maximal response to to that of flavones and flavonols. Aloe-emodin and emodin TCDD in controls were calculated and expressed as the EC70 had the strongest potencies among the tested samples at the value. EC70 levels of 0.5 and 0.6 m M, respectively. In addition, alizarin showed a marked inhibitory effect at 3.2 m M. RESULTS AND DISCUSSION Piperine, resveratrol, brevifolincarboxylic acid, coume- strol, and shikonin inhibited activation in this assay (EC70 The chemical structures of the tested compounds are values were 2.7, 3.9, 3.9, 5.6, and 5.6 m M, respectively). Cap- shown in Fig. 1. We used the Ah-I as a model in our prelimi- saicin, carnosol, and curcumin had EC70 values of 28.1, 30.7, nary experiments and evaluated the influence of individual and 35.4 m M, respectively. Among them, resveratrol and cur- vegetable constituents on the AhR pathway induced by cumin have been reported to show antagonist activity on TCDD. Figure 2 shows the dose–response curves plotted on a AhR.10,12) As they also had inhibitory effects in this present log scale for some individual samples. Most showed an in- assay system, it is suggested that they are candidate prophy- hibitory effect on AhR activation at high concentrations lactic agents for the prevention of dioxin toxicity.

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