K. Connor

Naringenin: A weakly estrogenic bioflavonoid that exhibits antiestrogenic activity

Treatment of immature 21-day-old female Sprague-Dawley rats with 17 beta-estradiol (E2) (0.5 microgram/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 microgram/rat) plus naringenin (30 mg/rat); there was 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 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (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 E2-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 E2-induced 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 cytosol. 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 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1-1000 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 cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 microM naringenin plus 1 nM E2, naringenin inhibited E2-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.

Methods for xenoestrogen testing

Research in our laboratories has focused on development of a battery of in vivo and in vitro bioassays for determining estrogenic activity and potency of different classes of natural and synthetic industrial-derived estrogenic compounds (xenoestrogens) including food/beverage extracts, phytoestrogens, phenolic compounds, organochlorine pesticides and pollutants. For many of the weak estrogenic compounds, their activity as estrogen receptor (ER) agonists or antagonists is dependent on the gene/gene promoter, cell context and expression of ER(alpha) or ER(beta) isoform. For example, extracts of red wine, bound to the ER, exhibited estrogenic activity in T47D, MCF-7 (breast) and Hep G2 (liver) human cancer cell lines, whereas reconstituted organochlorine pesticide residues found in food were active only in Hep G2 cells that transiently expressed ER(alpha) or ER(beta). The relative potencies of red wine extracts versus reconstituted organochlorine pesticides were assay-dependent; however, estrogen equivalent daily intakes from a glass of red wine (approximately 0.5-2 microg estrogen equivalents/day) were at least 10(3) higher than observed for the reconstituted organochlorine pesticide mixtures. Risk assessment of xenoestrogens and other synthetic chemicals which modulate endocrine responses must take into account high dietary levels of natural products in food, drugs and health food store extracts which also modulate endocrine responses.

An enzyme-linked immunosorbant assay (ELISA) specific for antibodies to TNP-LPS detects alterations in serum immunoglobulins and isotype switching in C57BL/6 and DBA/2 mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds

Abstract An enzyme-linked immunosorbant assay (ELISA) was developed to detect IgM and IgG antibodies specific for trinitrophenyl-lipopolysaccharide (TNP-LPS). Treatment of C57BL/6 and DBA/2 mice with 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) and other aryl hydrocarbon (Ah) receptor agonists followed by immunization with TNP-LPS resulted in a dose-dependent decrease in serum IgM which paralleled the decrease in the splenic PFC response. The ED 50 values for the IgM and splenic PFCs in C57BL/6 mice for 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (pentaCB) and 3,3′,4,4′,5,5′-hexaCB were 2.8 and 1.6, 11 and 14, and 25 and 20 μg/kg, respectively; in the less Ah-responsive DBA/2 mice, the ED 50 values were 8.5 and 10, 61 and 69, and 73 and 71 μg/kg, respectively. In addition, treatment of C57BL/6 mice with TCDD resulted in alterations of serum IgG relative to IgM and a delay of isotype switching was observed after immunization and boosting with TNP-LPS. This ELISA may prove to be a useful tool in monitoring immune function during long-term exposure of mice to TCDD and related compounds and exploring the mechanism of Ah receptor-mediated immunosuppression.