Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA)
Introduction
Nordihydroguaiaretic acid (NDGA) is a lignan found in the leaves and twigs of the shrub Larrea tridentata Lambert et al., 2002, Roed-Petersen and Hjorth, 1976. It has antioxidative activity and is used commercially as a food additive to preserve fats and butter, although it has been banned in some countries, including the U.S.A., since it was shown to induce cystic nephropathy in the rat Evan and Gardner, 1979, Gardner et al., 1987. Recently it has been found to inhibit cancer cell growth in vitro and to have cancer-preventive activity in animal models Hofmanova et al., 2002, McDonald et al., 2001, Nakadate et al., 1989, Park et al., 1998.
Some naturally occurring compounds present in plants have been found to possess estrogenic properties; they are referred to as phytoestrogens Benassayag et al., 2002, Brzezinski and Debi, 1999, Jefferson and Newbold, 2000, Knight and Eden, 1995, Murkies et al., 1998. The majority of them belongs to the flavonoid family (coumestans, prenylated flavonoids and isoflavones), and are present in many plants. A class of non-flavonoid phytoestrogens, lignans, has also been identified. They are thought not to be intrinsically estrogenic, but are converted to estrogenic compounds, such as enterolactone and enterodiol, by the gut microflora. Lignan-type phytoestrogens bind to estrogen receptors (ERs) with very low affinity, and high concentrations are required to manifest ER-mediated actions, even in vitro.
In the present study, we showed that a lignan, NDGA, has a relatively high binding affinity to both ERα and ERβ and is able to activate ER-mediated actions. Moreover, our data indicate that NDGA possesses a SERM-like activity, preferentially inducing ERα mediated transcription while showing mixed agonism/antagonism of ERβ mediated action.
Section snippets
Chemicals
Nordihydroguaiaretic acid (NDGA), 17β-estradiol (E2) and an estrogen antagonist, 4-hydroxytamoxifen (TAM) were purchased from Sigma Chemicals, St. Louis, MO, U.S.A. They were dissolved in ethanol to give stock solutions.
Cell culture
An estrogen-responsive cell line with growth hormone-secreting ability, MtT/E-2, was maintained in DME/F12 mixed medium (Sigma Chemicals) containing penicillin and streptomycin with 8% horse serum (Gibco/Invitrogen Corp., Carlsbad, CA, USA) and 2% fetal bovine serum
Ligand binding for ERs
The binding affinity of NDGA for recombinant human ERs α and β was determined by competitive binding analysis (Fig. 1). For both types of ER, the concentrations needed to produce 50% inhibition of 3H-E2 binding were 3 × 10−9 M for E2 and 3 × 10−5 M for NDGA. Thus, the affinity of NDGA was essentially1/10,000 of that of E2 for both receptors.
Estrogen responses in MtT/E-2 cells
The estrogenic activity of NDGA was examined using estrogen-responsive growth assay in MtT/E-2 cells (Fig. 2A). Increased growth of the cells was observed
Discussion
Phytoestrogen is a general term to define classes of estrogenic compounds that are of plant origin. Such compounds include flavonoids (kaempferol and quercetin), isoflavonoids (genistein, daidzein, formonetin and equol), lignans (enterolactone and enterodiol), mycotoxins (zearelenol) and stilbenes (resveratrol) Benassayag et al., 2002, Brzezinski and Debi, 1999, Jefferson and Newbold, 2000, Knight and Eden, 1995, Murkies et al., 1998. In general, plant lignans such as pinoresinol,
Acknowledgements
We thank Mr. Y. Mizuno for his expert technical assistance. This work was supported in part by Grant-in-Aid (H13-Seikatsu) from the Ministry of Health, Labor and Welfare, Japan and a Grant-in-Aid (#14042241) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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