In this study we examine the effect of the phytoestrogen genistein on DNA methylation. DNA methylation is thought to inhibit transcription of genes by regulating alterations in chromatin structure. Estrogenic compounds have been reported to regulate DNA methylation in a small number of studies. Additionally, phytoestrogens are believed to affect progression of some human diseases, such as estrogen-dependent cancers, osteoporosis and cardiovascular disease. Specifically, our working hypothesis is that certain soy phytoestrogens, such as genistein, may be involved in preventing the development of certain prostate and mammary cancers by maintaining a protective DNA methylation profile. The objective of the present study is to use mouse differential methylation hybridization (DMH) arrays to test for changes in the methylation status of the cytosine guanine dinucleotide (CpG) islands in the mouse genome by examining how these methylation patterns are affected by genistein. Male mice were fed a casein-based diet (control) or the same diet containing 300 mg genistein/kg according to one of four regimens: control diet for 4 wk, genistein diet for 4 wk, control diet for 2 wk followed by genistein diet for 2 wk and genistein diet for 2 wk followed by control diet for 2 wk. DNA from liver, brain and prostate were then screened with DMH arrays. Clones with methylation differences were sequenced and compared with known sequences. In conclusion, consumption of genistein diet was positively correlated with changes in prostate DNA methylation at CpG islands of specific mouse genes.

The bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells. Dihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration. Exposure of Hon and Mag (1–10 μM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 μM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production. This study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.

Previous evidence suggests soy genistein may be protective against prostate cancer, but whether this protection involves an estrogen receptor (ER)-dependent mechanism is unknown. To test the hypothesis that phytoestrogens may act through ERα or ERβ to play a protective role against prostate cancer, we bred transgenic mice lacking functional ERα or ERβ with transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Dietary genistein reduced the incidence of cancer in ER wild-type (WT)/transgenic adenocarcinoma of mouse prostate mice but not in ERα knockout (KO) or ERβKO mice. Cancer incidence was 70% in ERWT mice fed the control diet compared with 47% in ERWT mice fed low-dose genistein (300 mg/kg) and 32% on the high-dose genistein (750 mg/kg). Surprisingly, genistein only affected the well differentiated carcinoma (WDC) incidence but had no effect on poorly differentiated carcinoma (PDC). No dietary effects have been observed in either of the ERKO animals. We observed a very strong genotypic influence on PDC incidence, a protective effect in ERαKO (only 5% developed PDC), compared with 19% in the ERWT, and an increase in the incidence of PDC in ERβKO mice to 41%. Interestingly, immunohistochemical analysis showed ERα expression changing from nonnuclear in WDC to nuclear in PDC, with little change in ERβ location or expression. In conclusion, genistein is able to inhibit WDC in the presence of both ERs, but the effect of estrogen signaling on PDC is dominant over any dietary treatment, suggesting that improved differential targeting of ERα vs. ERβ would result in prevention of advanced prostate cancer.

Dietary supplements containing concentrates of plant-derived estrogens are being increasingly used by consumers as alternatives for hormone replacement therapy, for treatment of menopausal symptoms, and as cancer preventives. The effect of dietary genistein on dimethylbenz[a]anthracene (DMBA)-induced mammary tumor development was investigated in wild-type (ER alpha WT) and estrogen receptor-alpha knockout (ER alpha KO) mice. ER alpha WT and ER alpha KO mice were fed a casein-based diet containing 0 or 1 g genistein/kg diet from weaning. Tumors were induced by oral administration of DMBA and subscapular implantation of medroxyprogesterone acetate. No tumors were observed in ER alpha KO mice. In ER alpha WT mice, dietary intake of genistein influenced tumor development, enhancing anaplasia of mammary cancer. Mice consuming genistein expressed malignant mammary adenocarcinoma, whereas benign adenomas were observed in mice fed the control diet. Dietary intake was also influenced by genistein, with ER alpha WT and ER alpha KO mice fed genistein consuming less food (p < 0.0001) and subsequently weighing less than mice fed the control diet (p < 0.0001). Significant differences in food intake by genotype were also observed (p = 0.0017), with ER alpha KO mice consuming less than ER alpha WT mice. Overall, this study found no protective effect of genistein on DMBA-induced mammary tumors in mice and suggests a potential adverse effect on tumor development when high levels of genistein are consumed.