Molokhia (Corchorus olitorius L.) extract suppresses transformation of the aryl hydrocarbon receptor induced by dioxins

https://doi.org/10.1016/j.fct.2005.07.007Get rights and content

Abstract

Dioxins enter the body mainly through diet and cause the various toxicological effects by binding to the cytosolic aryl hydrocarbon receptor (AhR) followed by its transformation. In recent reports, it has been shown that certain natural compounds suppress AhR transformation in vitro. In this study, we demonstrated that ethanolic extract from molokhia, known as Egyptian spinach, showed the strongest suppressive effect on AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cell-free system using rat hepatic cytosol among 41 kinds of extracts from vegetables and fruits. The molokhia extract also suppressed TCDD-induced AhR transformation in mouse hepatoma Hepa-1c1c7 cells and in intestinal permeability system constructed with human colon adenocarcinoma Caco-2 cells and human hepatoma HepG2 cells. Moreover, oral administration of the molokhia extract (100 mg/kg body weight) decreased 3-methylcholanthrene-induced AhR transformation to the control level by inhibiting translocation of the AhR from cytosol into the nucleus in the liver of rats. The molokhia extract-administered rat liver showed a tolerance to TCDD-induced AhR transformation by ex vivo experiment. These results indicate that molokhia is an attractive food for isolation and identification of a natural antagonist for the AhR.

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),

References (40)

  • J.E. Lee et al.

    Involvement of a post-transcriptional mechanism in the inhibition of CYP1A1 expression by resveratrol in breast cancer cells

    Biochemical Pharmacology

    (2001)
  • Q. Ma et al.

    A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Journal of Biological Chemistry

    (1997)
  • J.T. Tuomisto et al.

    TCDD-Induced anorexia and wasting syndrome in rats: Effects of diet-induced obesity and nutrition

    Pharmacology Biochemistry and Behavior

    (1999)
  • S.N. Williams et al.

    Comparative studies on the effects of green tea extracts and individual tea catechins on human CYP1A gene expression

    Chemico-Biological Interactions

    (2000)
  • Y. Amakura et al.

    Screening of the inhibitory effect of vegetable constituents on the aryl hydrocarbon receptor-mediated activity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Biological and Pharmaceutical Bulletin

    (2003)
  • K. Azuma et al.

    Phenolic antioxidants from the leaves of Corchorus olitorius L

    Journal of Agricultural and Food Chemistry

    (1999)
  • M. Bhagavatula

    Persistent expression of 3-methylcholanthrene-inducible cytochromes P4501A in rat hepatic and extrahepatic tissues

    Journal of Pharmacology and Experimental Therapeutics

    (2000)
  • L.A. Carver et al.

    Tissue specific expression of the rat Ah-receptor and ARNT mRNAs

    Nucleic Acids Research

    (1994)
  • R.F. Casper et al.

    Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity

    Molecular Pharmacology

    (1999)
  • H.P. Ciolino et al.

    Dietary flavonols quercetin and kaempferol are ligands of the aryl hydrocarbon receptor that affect CYP1A1 transcription differentially

    Biochemical Journal

    (1999)
  • Cited by (36)

    • Green and novel approach for enhancing flame retardancy, UV protection and mechanical properties of fabrics utilized in historical textile fabrics conservation

      2022, Progress in Organic Coatings
      Citation Excerpt :

      However, after coating the L textile fabrics with green coating layer (CH-ME40) in L-CH-ME40 sample significant inhibition for bacterial growth was noticed achieving 11 mm of clear inhibition zone (Fig. S2). This positive action in inhibition of bacterial growth was attributed to ME which contained various anti-inflammatory and antioxidants compounds [15–18,36]. Renewable, cost-effective and green flame retardant, UV protective and reinforcement textile coating was developed.

    • Glabridin inhibits dexamethasone-induced muscle atrophy

      2019, Archives of Biochemistry and Biophysics
    • Health-promoting properties of Corchorus leaves: A review

      2019, Journal of Herbal Medicine
      Citation Excerpt :

      Most of these responses are expressed through interaction between dioxins and aryl hydrocarbon receptor (AhR) and its subsequent transformation (Fernandez-Salguero et al., 1996). An In vitro study on rats revealed that an ethanol extract of C. olitorius could effectively suppress transformation of AhR due to the presence of AhR antagonistic bioagents (Nishiumi et al., 2006). Such protective effects of C.olitorius leaves have been investigated in detail and presented in Table S5 (Supplementary on-line data).

    View all citing articles on Scopus
    View full text