Pergamon OOM-2952(95)OMl-G Biochemical PhwmacoJogy, Vol. 50, No.9, pp. 1485-1493. 1995. Copyright 0 1995 Elseviet Science Inc. Printed b Gnat Britain. All lights rcstrvcd ocw295295Y$9.50+ 0.00 NARINGENIN: A WEAKLY ESTROGENIC BIOFLAVONOID THAT EXHIBITS ANTIESTROGENIC ACTIVITY MARY F. RUH,* TIMOTHY ZACHAREWSKI,-/ KEVIN CONNOR,+ JAMES HOWELL,3 ICHEN CHEN$ and STEPHEN SAFE$$ *Department of Pharmacological and Physiological Science, Saint Louis University, School of Medicine, St. Louis. MO 63104, U.S.A. fDepartment of Pharmacology and Toxicology, University of Western Ontario, London, Ontario N6A 5C1. Canada; $Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4464, U.S.A. (Received 20 February 1995; accepted 2 June 1995) Abstract-Treatment of immature 21-day-old female SpragueDawley rats with 17/%estradiol(E2) (OS @at) caused a sig,nificant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mglrat, the bioflavonoid naringenin did not cause a significant increase in amy of these EZinduced responses. However, in rats cotreated with E2 (0.5 @rat) plus naringenin (30 murat). there WBS a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a ‘*P-lab&d estrogen responsive element (ERE) or progesterone respon- sive elemenl: (PRE) was determined using gel electrophoretic band shift assays. Incubation of [32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from EZ-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the EZinduced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine eytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-. or naringenin plus E2-treated cells, The results of in vitro studies showed that I nM E2 increased (3- to Cfold) the growth of MCF-7 human breast cancer cells, whereas l-1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced txll growth. In MCF-7 cells transiently transfected with apS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 pM naringenin plus I nM E2, naringenin inhibited M-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells. Key words: icstradiol; naringenin; estrogenicity; antiestrogenicity Several studies indicate that there is a decreased risk for different types of cancer among vegetarians [l-8], and this observation has spurred research on various stmc- tural classes of ph:ytochemicals that may contribute to the observed anticarcinogenic effects. Bioflavonoids are major constituents of plants and vegetables [9], and sev- eral of these compounds elicit responses that may pro- vide some protective effects [lo]. In areas, such as Japan and China where dietary consumption of bioflavonoids is high, there is a decreased incidence of breast cancer in women, and this corresponds to the inhibition of mam- mary cancer cell or tumor growth by some bioflavonoids in both in vitro and in vitro models [1 l-171. It has also been reported that different structural classes of biofla- vonoids bind to the .ER” and induce a diverse spectrum of estrogen-induced responses [ 12, 18-281. For example, bioflavonoids, sue h as flavone, apigenin, daidzen, 9 Corresponding author. Tel. (409) 845-5988; FAX (409) 862-4929. ‘I Abbreviations: CAT, chloramphenicol acetyl transferase; E2,17&estradiol; ER, estrogen receptor: ERE, estrogen respon- sive element; PR. progesterone receptor; and PRE, progesterone responsive element. kacmpferol, and quercetin, induce proliferation of estro- gen-responsive MCF-7 human breast cancer cells and induce estrogen-inducible genes and constructs derived from estrogen-induced genes [26, 271. Moreover, it has been suggested “that dietary flavonoids have the potcn- tial to contribute to the growth of estrogen-dependent tumors in postmenopausal women, where 17gestradiol is limiting” [27]. However, it has also been shown that most biofla- vonoids are weak ER agonists, and therefore a sub-ef- fective (estrogenic) dose of these phytoestrogens may exhibit partial antiestrogenic activity. Markaverich et al. [12] have reported previously that the bioflavonoids quercetin and luteolin inhibit EZinduced proliferation of MCF-7 human breast cancer cells and uterine wet weight in immature 21-day-old female rats. This study utilizes naringenin, a plant bioflavonoid, as a model for investi- gating the estrogenic activity of bioflavonoids in the fe- male rat uterus and MCF-7 human breast cancer cell line. Since naringenin is reported to be a relatively weak estrogen, we hypothesize that this compound may also exhibit partial antiestrogenic activity. The results of cotreatment studies (E2 plus naringenin) demonstrated that naringenin inhibits several estrogen-induced re- sponses in the rat uterus and in MCF-7 cells in culture. 1485