Molokhia (Corchorus olitorius L.) extract suppresses transformation of the aryl hydrocarbon receptor induced by dioxins
Introduction
Dioxins, widespread environmental contaminants, enter the body mainly through diet. Among dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener and causes various toxicological effects such as tumor-promotion, teratogenicity, immunotoxicity, wasting syndrome, lethality, and so on (Hankinson, 1995, Grassman et al., 1998, Tuomisto et al., 1999, Ito et al., 2002). Most of these toxicological effects are expressed through interaction between dioxins and the aryl hydrocarbon receptor (AhR) (Fernandez-Salguero et al., 1996), which is a member of the basic helix–loop–helix (bHLH) protein and belongs to the Per-Arnt-Sim (PAS) family. Although the AhR is at the moment an orphan receptor, it is known that halogenated and polycyclic aromatic hydrocarbons (HAHs and PAHs) such as dioxins, β-naphthoflavone (Sinal et al., 1999), benzo[a]pyrene (Saeki et al., 2003), and 3-methylcholanthrene (MC) (Bhagavatula, 2000) bind to the AhR as ligands. In the absence of ligands, the AhR stably exists in cytosol forming a complex with a dimer of 90-kDa heat shock protein (hsp90) (Chen and Perdew, 1994), AhR-associated protein (ARA9, also termed as XAP2 or AIP) (Ma and Whitlock, 1997, Meyer et al., 1998), and p23 (Premnath et al., 2003). Ligand binding to the AhR results in its transformation to a DNA binding form by series of processes: A conformational change in the receptor protein, its translocation into the nucleus, dissociation of hsp90, and heterodimerization with another bHLH/PAS family protein, AhR nuclear translocator (Arnt) (Denison et al., 1986, Elferink et al., 1990). This heterodimer binds to dioxin response element (DRE), which is a cis-acting element found in the 5′ regulatory regions of dioxin-responsive genes, and regulates the expression of target genes encoding the cytochrome P450 (CYP) 1A and 1B subfamilies (Hyunsung et al., 1996), UDP-glucuronosyltransferase (Iyanagi et al., 1986), glutathione S-transferase (Rushmore et al., 1990) and so on.
Regarding the antagonists of the AhR, it has been reported that certain synthetic flavonoids, e.g., α-naphthoflavone (Gasiewicz and Rucci, 1991), 3′-methoxy-4′-nitroflavone (Nazarenko et al., 2001), and 3′,4′-dimethoxyflavone (Lee and Safe, 2000) suppress AhR transformation. It has also been reported that natural flavonoids, e.g., galangin, kaempferol (Ciolino et al., 1999, Ashida et al., 2000), and (−)-epigallocatechin 3-gallate (Williams et al., 2000, Fukuda et al., 2004a) show the antagonistic effect on AhR transformation, and other phytochemicals such as curcumin (Ciolino et al., 1998), resveratrol (Casper et al., 1999), and lutein (Fukuda et al., 2004a) suppress AhR transformation and/or CYP1A1 expression in vitro. Moreover, chlorophylls and dietary fibers may interact with dioxins and inhibit absorption of dioxins from small intestine (Morita et al., 1995, Morita et al., 2001). These reports indicate that an intake of vegetables, fruits, and tea have a possibility of reducing AhR-dependent biological effects caused by dioxins, since these foodstuffs abundantly contain various natural antagonists. Indeed, green tea extract (Williams et al., 2000, Fukuda et al., 2004a), black tea extract (Fukuda et al., 2004c), and propolis extract (Park et al., 2004) suppress AhR transformation and/or expression of CYP1A1. From findings in our preliminary report (Park et al., 2004), it has been demonstrated that certain vegetable extracts also suppressed AhR transformation using a gel retardation AhR binding assay in vitro. However, the effective foodstuffs suppressing AhR transformation in vivo are not clear yet.
In this study, we examined suppressive effect of ethanolic extract from 41 kinds of vegetables and fruits on AhR transformation in cell-free system using a quantitative measurement based on an enzyme-linked immunosorbent assay (ELISA) combining with southwestern chemistry technique (SW-ELISA) (Fukuda et al., 2004b). This method can evaluate AhR transformation as a binding of transformed AhR:Arnt complex to DRE. Among the extracts tested here, molokhia (Egyptian spinach) extract showed the strongest suppressive effect on AhR transformation. Moreover, molokhia extract also suppressed translocation of the AhR into the nucleus and subsequent binding of the AhR to DRE both in the cultured cell lineages and animal experiments.
Section snippets
Materials
TCDD and MC were obtained from AccuStandard (New Haven, CT) and Sigma–Aldrich Co. (St Louis, MO), respectively. Anti-fluorescein isothiocyanate (FITC) rabbit IgG, anti-Arnt goat IgG, anti-CYP1A1 goat IgG, and anti-AhR mouse IgG were from DakoCytomation (Kyoto, Japan), Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Daiichi Pure Chemicals Ltd. (Tokyo, Japan), and Affinity BioReagents (Golden, CO), respectively. Biotinylated secondary antibody was from Jackson Immuno Research Lab. (West Grove,
Suppressive effect of the ethanolic extract from vegetables and fruits on AhR transformation in cell-free system
In this study, we used a newly developed SW-ELISA for estimation of AhR transformation. This method is able to measure DNA binding activity of transformed AhR quantitatively as the same principal as a gel retardation AhR binding (GRAB) assay (Fukuda et al., 2004b). We, first, surveyed suppressive effect of the ethanolic extract from 41 kinds of vegetables and fruits on 1 nM TCDD-induced AhR transformation by SW-ELISA in cell-free system using the rat liver cytosolic fraction. The dose-response
Discussion
The AhR plays an important role in most of the diverse biochemical responses by dioxins, and AhR-deficient mice are resistant to dioxin-induced toxicity (Fernandez-Salguero et al., 1996). The AhR distributes in various tissues, especially liver and lung (Carver et al., 1994), and the liver is one of the major organs for detoxification of xenobiotics. AhR transformation results in expression of some detoxification enzymes such as CYP1A and 1B subfamilies (Hyunsung et al., 1996),
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