Soy and the soy isoflavone genistein promote adipose tissue development in male mice on a low-fat diet.

PURPOSE: Several nutrients act as phytoestrogens, being anti-adipogenic when consumed with a fat-rich diet. Their effect on a low-fat diet (LFD) background is unknown. We tested soy and genistein effects on adipose tissue in LFD-fed mice and genistein activity in the 3T3-L1 adipogenesis model. METHODS: C57BL/6 J male mice were fed an 8.5% soy-supplemented LFD (SS-LFD) or a soy-free LFD (SF-LFD) for 147 days. Groups of 3-week-old (pubertal) and 6-week-old (adult) mice on the SF-LFD were also treated with 17ß-estradiol (E2, 5 µg/kg/day) ip or pure genistein (5 mg/kg/day) by gavage for 15 days. Body fat deposition and gene expression profiles were evaluated. E2 and genistein effects on ERα, ERß and PPARγ transcriptional activities were characterized in ERα- or ERß-transfected 3T3L1 cells during differentiation, by the use of reporter plasmids. RESULTS: The SS-LFD group increased fat mass compared with the SF-LFD group. Genistein alone increased while E2 decreased fat pads in the 15-day-treated mice. In visceral fat, genistein differentially regulated 13 metabolic pathways compared to E2. PPARγ-controlled genes were downregulated by E2, while they were upregulated by genistein. In 3T3-L1 cells, genistein activated ERß-driven transcription, differentiation and lipid accumulation, while inhibited ERα-driven transcription, without effects on lipid accumulation. E2 activated both ERs only in preadipocytes. In differentiated untransfected cells, genistein inhibited PPARγ, while activated PPARγ in the presence of ERß. CONCLUSIONS: Soy and genistein at nutritional doses induce fat development in LFD-fed mice and adipogenesis in 3T3-L1 cells, with a mechanism that involves, at least in vitro, ERß and is dependent on cell differentiation stage.

Genistein is an efficient estrogen in the whole-body throughout mouse development.

The widespread use of diets containing estrogenic compounds raises questions on how relevant the presence of phytoestrogens may be, in order to allow a correct development of the reproductive ability and sexual maturity in humans and animals. The isoflavone genistein is the most estrogenically active molecule present in soy. Here we show that genistein, through an estrogen receptor (ER)-mediated action, modulates gene expression in the whole body of male mice in a dose- and time-dependent manner, at all ages. By luciferase bioassays, we show that genistein-induced ER activation is present in reproductive and nonreproductive organs of the transgenic mice Estrogen Responsive Element (ERE)-tK-LUC, although to an extent that is lower than what observed with the administration of estradiol. Peak activity was registered at genistein doses of 500-5000 microg/kg, at 12 h from the administration by gavage. In the liver, ER-alpha and ER-beta messenger RNAs and two target genes, CYP17 and the progesterone receptor, were modulated by genistein. CYP17 and PR time-dependent induction was similar to that of luciferase. ER-alpha protein level followed an opposite regulation by genistein and estradiol. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in reproductive and nonreproductive tissues of the pups, with maximal upregulation at 16-24 h. We also followed responsiveness to genistein in the testis, from early development to adult age. Testis are well responsive to genistein as well as to estradiol already at day 14.5 of fetal development, as determined by exposing organotypic cultures from mouse fetus testis. Ovaries were not responsive under the same conditions. Activation of luciferase correlates with an activation of cell proliferation in testis, but not in the ovaries. Prolonged exposure (15 days) to genistein also decreases prostate weight like estradiol. In conclusion, our results show that genistein affects reproductive and nonreproductive organs of male mice in a dose- and time-dependent manner, at all developmental ages.

MAK-4 and -5 supplemented diet inhibits liver carcinogenesis in mice.

BACKGROUND: Maharishi Amrit Kalash (MAK) is an herbal formulation composed of two herbal mixtures, MAK-4 and MAK-5. These preparations are part of a natural health care system from India, known as Maharishi Ayur-Veda. MAK-4 and MAK-5 are each composed of different herbs and are said to have maximum benefit when used in combination. This investigation evaluated the cancer inhibiting effects of MAK-4 and MAK-5, in vitro and in vivo. METHODS: In vitro assays: Aqueous extracts of MAK-4 and MAK-5 were tested for effects on ras induced cell transformation in the Rat 6 cell line assessed by focus formation assay. In vivo assays: Urethane-treated mice were put on a standard pellet diet or a diet supplemented with MAK-4, MAK-5 or both. At 36 weeks, livers were examined for tumors, sera for oxygen radical absorbance capacity (ORAC), and liver homogenates for enzyme activities of glutathione peroxidase (GPX), glutathione-S-transferase (GST), and NAD(P)H: quinone reductase (QR). Liver fragments of MAK-fed mice were analyzed for connexin (cx) protein expression. RESULTS: MAK-5 and a combination of MAK-5 plus MAK-4, inhibited ras-induced cell transformation. In MAK-4, MAK-5 and MAK4+5-treated mice we observed a 35%, 27% and 46% reduction in the development of urethane-induced liver nodules respectively. MAK-4 and MAK4+5-treated mice had a significantly higher ORAC value (P < 0.05) compared to controls (200.2 +/- 33.7 and 191.6 +/- 32.2 vs. 152.2 +/- 15.7 ORAC units, respectively). The urethane-treated MAK-4, MAK-5 and MAK4+5-fed mice had significantly higher activities of liver cytosolic enzymes compared to the urethane-treated controls and to untreated mice: GPX(0.23 +/- 0.08, 0.21 +/- 0.05, 0.25 +/- 0.04, 0.20 +/- 0.05, 0.21 +/- 0.03 U/mg protein, respectively), GST (2.0 +/- 0.4, 2.0 +/- 0.6, 2.1 +/- 0.3, 1.7 +/- 0.2, 1.7 +/- 0.2 U/mg protein, respectively) and QR (0.13 +/- 0.02, 0.12 +/- 0.06, 0.15 +/- 0.03, 0.1 +/- 0.04, 0.11 +/- 0.03 U/mg protein, respectively). Livers of MAK-treated mice showed a time-dependent increased expression of cx32. CONCLUSION: Our results show that a MAK-supplemented diet inhibits liver carcinogenesis in urethane-treated mice. The prevention of excessive oxidative damage and the up-regulation of connexin expression are two of the possible effects of these products.

Genistein accumulates in body depots and is mobilized during fasting, reaching estrogenic levels in serum that counter the hormonal actions of estradiol and organochlorines.

Isoflavones are important dietary compounds that are consumed with the daily diet and elicit important biological actions. Here we report on the ability of genistein to partially accumulate in body depots of male mice, be released following fasting, and modulate the actions of estradiol and environmental estrogens in reproductive and nonreproductive target organs of estrogen-reporter mice (ERE-tK-luciferase). After the consumption of 50 mg/kg/day for 3 days, genistein accumulates in body compartments where it remains at functionally active levels for at least 15 days. Following 48 h of fasting, its concentration increased in serum from 99 +/- 13 to 163 +/- 17 nM. These levels are sufficient to exert an estrogenic effect in the testis and liver, as revealed by a twofold increase in luciferase gene expression. beta-Benzene-hexachloride (betaBHC) given at the concentration of 100 mg/kg/day for 3 days also accumulates in the body and is released by fasting, reaching serum levels of 176 +/- 33 nM, upregulating the luciferase gene in the liver and inhibiting its expression in the testis. When genistein was given in combination with betaBHC at doses sufficient to induce accumulation of both in body depots, the genistein mobilized by fasting reversed the action of the mobilized betaBHC in the testis. Acute administration of nutritional doses of genistein inhibited the action of estradiol and reversed the antiestrogenic action of o,p'-DDT: 1,1,1,-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl)ethane in the liver and the antiestrogenic action of betaBHC in the testis. Genistein had an additive effect with the ER agonist p,p'-DDT: 1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane in the liver. The observed effects may be relevant to a protective action of phytoestrogens against estrogen receptor-interacting pollutants as well as the dietary modulation of estradiol action.