Topoisomerase poisoning by genistein in the intestine of rats.

The isoflavone genistein has been shown to act as topoisomerase II poison in various cell lines. Here, we address the question whether genistein is able to affect topoisomerase II in vivo. Juvenile male Wistar rats received either a single dose of genistein subcutaneously (s.c.; 10 mg/kg BW) or a lifelong isoflavone-rich diet encompassing in utero, lactation phase and 10 days of oral consumption, whereas genistein was mainly taken up as glycosides (25-50 mg/kg BW). The effects on the level of covalent topoisomerase II-DNA-complexes in the duodenum and colon were measured using the "Isolation of in vivo complexes of enzyme to DNA" (ICE)-bioassay. Simultaneously, serum as well as tissue concentrations of genistein and its metabolites were quantified by LC-MS. Genistein (s.c.) significantly increased the amount of covalent topoisomerase IIα and ß-DNA complexes in the gut, showing more persistent effects in the colon than in the duodenum. In case of a lifelong dietary isoflavone exposure, no effects on the stabilization of cleavage complexes was observed, except a slight increase of topoisomerase IIα-DNA-complexes in the colon. The differences between the exposure routes might be attributed to the higher serum concentration of the genistein aglycon after subcutaneous treatment probably due to circumvention of first-pass metabolism compared to oral consumption of an isoflavone-rich diet. These data indicate that subcutaneously administrated genistein clearly possesses topoisomerase poisoning properties in vivo, whereas an isoflavone-rich diet containing genistein only caused a slight effect which relevance has to be clarified in further studies.

Proliferative and estrogenic sensitivity of the mammary gland are modulated by isoflavones during distinct periods of adolescence.

Isoflavone (ISO) exposure during adolescence has been demonstrated to modulate the estrogenic and proliferative sensitivity of the adult breast tissue. In this study, we investigated whether ISO exposure restricted to the period of puberty is sufficient to result in similar effects. Female rats were divided into three groups receiving either lifelong an ISO-free diet (IDD) or an ISO-rich diet (ISD), or an ISD from postnatal day (PND) 30 to PND 60 covering the time period of puberty (pISD). Serum concentrations of ISO and metabolites were determined at PND 50 and 80. At PND 50, ISD animals had significant higher equol serum levels than pISD animals. Onset of puberty occurred significantly earlier in the pISD and ISD group compared to the animals fed IDD. Cycle length was shortest in pISD group. To determine estrogen sensitivity of the adult breast tissue, adult rats were ovariectomized and subcutaneously treated either with estradiol (E(2)) or with genistein (GEN) for 3 days (PND 77-80). Analysis of Ki-67 and proliferating cell nuclear antigen (PCNA) expression showed a reduced proliferative response of the breast to E(2) in pISD and ISD animals compared to IDD group, while the induction of progesterone receptor (PR) was higher in both IDD and pISD compared to ISD fed rats. Our results demonstrate that ISO exposure during puberty is sufficient to reduce the proliferative response of the adult mammary gland but not to reduce the response of classical E(2) sensitive genes like the PR. In summary, our results demonstrate that animals exposed during different periods of their adolescence to ISO differ in several physiological aspects. In addition, the detected differences in the serum equol levels between pISD and ISD rats and the detected differences in the estrogen sensitivity of the breast clearly underline this assumption.

In utero and postnatal exposure to isoflavones results in a reduced responsivity of the mammary gland towards estradiol.

SCOPE: Exposure scenarios during different stages of development of an organism are discussed to trigger adverse and beneficial effects of isoflavones (ISO). The aim of this study was to investigate how in utero and postnatal ISO exposure modulates the estrogen sensitivity of the mammary gland and to identify the underlying molecular mechanisms. METHODS AND RESULTS: Therefore, rats were exposed to either ISO-free (IDD), ISO-rich (IRD) or genistein-rich diet (GRD), up to young adulthood. Proliferative activity (PCNA expression) in the mammary gland at different ages and the estrogen sensitivity of the mammary gland to estradiol (E2) or genistein (GEN) in adult ovariectomized animals was determined and compared with different treatments. Treatment with E2 resulted in a significant lower proliferative and estrogenic response of the mammary gland in IRD and GRD compared with IDD. This correlates to a change in the gene expression pattern and a decrease in the ratio of estrogen receptor alpha (ERα) beta (ERß CONCLUSIONS: Our results provide evidence that in utero and postnatal exposure to a diet rich in ISO but also to GEN reduces the sensitivity of the mammary gland toward estrogens and support the hypothesis that in utero and postnatal ISO exposure reduces the risk to develop breast cancer.

Oral treatment with genistein reduces the expression of molecular and biochemical markers of inflammation in a rat model of chronic TNBS-induced colitis.

BACKGROUND: Inflammatory bowel disease (IBD) in humans has a high incidence in Europe and the USA, whereas in East Asia, incidence has been historically low. The risk of IBD appears to increase in Asian immigrants adopting western lifestyles, suggesting a strong link of environmental/dietary factors in the development of IBD. Exposure to high levels of isoflavones such as genistein (Gen) in traditional East Asian diets has been associated with a decreased risk of developing breast cancer and may also be beneficial for the prevention of IBD. AIM: In this study, the effect of orally administered genistein on the inflammatory response in the TNBS-induced chronic colitis rat model was investigated. METHODS: Eighteen male Wistar rats, aged 12 weeks, were randomized to one of three groups (n = 6). Two groups received a 2,4,6-trinitrobenzenesulfonic acid (TNBS) enema, then were treated daily by oral gavage with either Gen (100 mg/kg b.w.) or vehicle, for 14 days. The last group served as a control group, not receiving the TNBS enema. At the end of the 14 days, animals were killed and tissues collected. Molecular and biochemical inflammatory markers in the colon, specifically cyclooxygenase-2 (COX-2) and myeloperoxidase (MPO), were analyzed. In addition, to assess the efficacy of Gen treatment, relative wet weights of the accessory sexual organs, specifically prostate and the seminal vesicle, were compared between the groups treated or not with Gen. RESULTS: Wet weights of both prostates and seminal vesicles were significantly (P < 0.01) reduced upon Gen administration. In the colon, expression of COX-2 mRNA and protein was reduced (P < 0.05) in the Gen treatment group, as compared to the control group, whereas there was no significant inhibitory effect of Gen on the expression of proliferating cell nuclear antigen. In Gen treated animals colon wet weight was not altered, however a decrease in MPO activity (P < 0.01) was seen. CONCLUSION: These results may provide evidence that oral administration of Gen exerts beneficial anti-inflammatory effects in a rodent model of TNBS-induced chronic colitis. While the sample size of this study was small, it nevertheless might encourage the realization of larger blinded randomized controlled studies for the proof of concept.

In utero and postnatal exposure to a phytoestrogen-enriched diet increases parameters of acute inflammation in a rat model of TNBS-induced colitis.

Inflammatory bowel disease (IBD) is very common in Europe and USA. Its incidence in East Asia has been traditionally low, albeit the risk of IBD increases in Asian immigrants adopting western lifestyles, suggesting a strong role of environmental/dietary factors in IBD. A lifelong exposure to phytoestrogen-rich diets has been associated with a decreased risk of developing breast cancer and might also be protective against IBD. We studied the influence of in utero and postnatal exposure to a phytoestrogen (PE)-rich diet on acute inflammation in an animal model of TNBS-induced colitis. Wistar rats were exposed in utero and postnatally to high (genistein: 240 microg/g feed; daidzein: 232 microg/g feed) or very low levels (genistein and daidzein <10 microg/g feed) of phytoestrogen isoflavones fed to pregnant dams with the diet and throughout nursing. After weaning, the offspring had free access to these diets. At the age of 11 weeks, colitis was induced with an enema of TNBS. After 3 days, animals were sacrificed and tissues were collected for histological evaluation and analysis of molecular markers of inflammation. Animals kept on a PE-rich diet (PRD) had higher colon weights than animals on low PE-levels (PDD), suggesting enhanced acute inflammation by phytoestrogens. This result was supported by histological findings and by analysis of myeloperoxidase activity. Interestingly, relative mRNA and protein expression of cyclooxygenase-2 (COX-2) were modulated in rats on PRD, providing evidence that COX-2, the inducible isoform of the enzyme, is involved in the management of colonic inflammation. Our results suggest that early-in-life exposure to PE might not protect against the development of IBD but enhances the extent of acute inflammation.