Wirksamkeit und Sicherheit von Flavanonen als Alternative zur postmenopausalen Hormontherapie
Final Report Abstract
Hops extract and its main active constituent 8-prenylnaringenin (8-PN) are food supplements supposedly alleviating menopausal complaints such as hot flashes and mood swings and preventing osteoporosis. In this project we aimed to assess the effectiveness as well as the safety of 8-PN, its parent compound naringenin (NAR) and another prenylated naringenin 6-(1.1-dimethylallyl)naringenin (6-DMAN). Larger scale synthesis of 8-PN and 6-DMAN by our collaborators at the lab of Peter Metz enabled us to test the efficacy and safety of these phytoestrogens in animal experiments using ovariectomized Lewis rats as our model. Initially the affinity of all test substances as well as E2 to both estrogen receptors, ER alpha and ER beta was established by performing a cell free fluorescence polarization based ligand binding assay. Two short term experiments lasting three days and one long term animal experiment lasting eight weeks using female ovariectomized young adult rats were performed. The first three-day uterotrophic assay aimed at characterizing the effects of the test substances on the uterus and the mammary gland with a focus on proliferation, which is an essential safety concern. The second 3-day experiment investigated the impact of PAH induced AHR signaling on the effects of 8-PN and of estradiol. In a longer-term experiment hops extract containing 8-PN as one of its main active constituents and E2-benzoate were administered orally to investigate long term protective effects of the extract on E2 depletion induced bone loss and to observe the metabolism of its main active compounds, including 8-PN. Numerous physiological endpoints like uterus wet weight, vaginal epithelial height, mammary gland terminal endbud formation, osteoporosis related bone parameters were assessed. Protein analysis using immunohistochemistry was performed in the same organs and expression of a number of E2 regulated genes, some of them newly discovered by us using microarray analysis, was tested. In addition, several in vitro experiments were performed. The impact on gene expression in raphe nuclei derived neuronal cells which we had previously stably transfected with ERβ was assessed in undifferentiated and differentiated cells. Proliferation assays and cell cycle analysis was performed using MCF-7 breast cancer cells. ERα/β-Heterodimer-mediated reporter gene expression and expression of estrogen responsive genes were assessed in the human U2OS osteosarcoma cells. Metabolism and the metabolite profile of all three test substances were determined by incubating them with rat and human liver microsomes. 8-PN is a unique phytoestrogen, showing a surprisingly strong affinity to both ERs with a clear preference for ER alpha. 6-DMAN is also a fairly strong ligand to both ERs while NAR itself has only a very slight affinity to ERs. Results obtained from our in vivo and in vitro studies largely correlate with this finding. 8-PN has the strongest effects, being slightly uterotrophic and also slightly promoting formation of terminal endbuds in the mammary gland in short term experiments where it was used as a pure substance. Long term administration of 8-PN containing hops extract had no effect on uterine proliferation. Nevertheless, it does show some osteoprotective effects. 8-PN also has the most pronounced impact on raphe nucleus cell gene expression. 6-DMAN on the other hand has much weaker effects and possesses some tissue specificity. While weak effects on uterine proliferation and gene expression as well as on vagina tissue have been observed, it appears to not have any effects on the formation of mammary gland terminal endbuds and raphe nuclei neurons. NAR has very low to no estrogenic effect, corresponding with very weak affinity to both ERs. In general activity of the tested naringenins correlates with the affinity to both ERs. The moderate effects of all tested substances in vivo and the low probability of widespread commercial use do not warrant further investigation of these nevertheless interesting and in the case of 8-PN very strong phytoestrogens.
Publications
- Effect of 17β-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line. J Cell Physiol. 2012 Oct;227(10):3434-45
Amer DA, Jähne M, Weigt C, Kretzschmar G, Vollmer G
(See online at https://doi.org/10.1002/jcp.24044) - Hop extracts and hop substances in treatment of menopausal complaints. Planta Med. 2013 May;79(7):576-9
Keiler AM, Zierau O, Kretzschmar G
(See online at https://doi.org/10.1055/s-0032-1328330) - Assessment of the proliferative capacity of the flavanones 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland. Mol Cell Endocrinol. 2014 Jul 5;392(1-2):125-35
Helle J, Kräker K, Bader MI, Keiler AM, Zierau O, Vollmer G, Welsh J, Kretzschmar G
(See online at https://doi.org/10.1016/j.mce.2014.05.014) - Assessment of the effects of naringenin-type flavanones in uterus and vagina. J Steroid Biochem Mol Biol. 2015 Jan;145:49-57
Keiler AM, Dörfelt P, Chatterjee N, Helle J, Bader MI, Vollmer G, Kretzschmar G, Kuhlee F, Thieme D, Zierau O
(See online at https://doi.org/10.1016/j.jsbmb.2014.10.001) - Cross-Talk in the Female Rat Mammary Gland: Influence of Aryl Hydrocarbon Receptor on Estrogen Receptor Signaling. Environ Health Perspect. 2016 May;124(5):601-10
Helle J, Bader MI, Keiler AM, Zierau O, Vollmer G, Chittur SV, Tenniswood M, Kretzschmar G
(See online at https://doi.org/10.1289/ehp.1509680) - A standardized Humulus lupulus (L.) ethanol extract partially prevents ovariectomy-induced bone loss in the rat without induction of adverse effects in the uterus. Phytomedicine. 2017 Oct 15;34:50–8
Keiler AM, Helle J, Bader MI, Ehrhardt T, Nestler, K, Kretzschmar G, Bernhardt R, Vollmer G, Nikolic D, Bolton JL, Pauli GF, Chen SN, Dietz BM, vanBreemen RB, Zierau O
(See online at https://doi.org/10.1016/j.phymed.2017.08.001) - Effects of the aryl hydrocarbon receptor agonist 3-methylcholanthrene on the 17β-estradiol regulated mRNA transcriptome of the rat uterus. J Steroid Biochem Mol Biol. 2017 Jul;171:133-143
Helle J, Keiler AM, Zierau O, Dörfelt P, Vollmer G, Lehmann L, Chittur SV, Tenniswood M, Welsh J, Kretzschmar G
(See online at https://doi.org/10.1016/j.jsbmb.2017.03.004)
